Split (1)

train · 13.2k rows

text stringlengths 645 543k	pmid stringlengths 1 8	accession_id stringlengths 9 11	license stringclasses 8 values	last_updated stringdate 2021-01-04 15:57:02 2024-12-12 23:16:03	retracted stringclasses 1 value	citation stringlengths 16 77
==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2013/210604Research ArticleInvolvement of Nrf2-Mediated Upregulation of Heme Oxygenase-1 in Mollugin-Induced Growth Inhibition and Apoptosis in Human Oral Cancer Cells Lee Young-Man 1 Auh Q-Schick 2 Lee Deok-Won 3 Kim Jun-Yeol 1 Jung Ha-Jin 1 Lee Seung-Ho 4 Kim Eun-Cheol 1 1Department of Maxillofacial Tissue Regeneration and Research Center for Tooth & Periodontal Regeneration, School of Dentistry, Kyung Hee University, 1 Heogi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea2Department of Oral Medicine, School of Dentistry, Kyung Hee University, Heogi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea3Department of Oral and Maxillofacial Surgery, School of Dentistry, Kyung Hee University, Heogi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea4College of Pharmacy, College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of KoreaEun-Cheol Kim: eckim@khu.ac.krAcademic Editor: George Perry 2013 2 5 2013 2013 21060412 2 2013 3 4 2013 5 4 2013 Copyright © 2013 Young-Man Lee et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Although previous studies have shown that mollugin, a bioactive phytochemical isolated from Rubia cordifolia L. (Rubiaceae), exhibits antitumor effects, its biological activity in oral cancer has not been reported. We thus investigated the effects and putative mechanism of apoptosis induced by mollugin in human oral squamous cell carcinoma cells (OSCCs). Results show that mollugin induces cell death in a dose-dependent manner in primary and metastatic OSCCs. Mollugin-induced cell death involved apoptosis, characterized by the appearance of nuclear shrinkage, flow cytometric analysis of sub-G1 phase arrest, and annexin V-FITC and propidium iodide staining. Western blot analysis and RT-PCR revealed that mollugin suppressed activation of NF-κB and NF-κB-dependent gene products involved in antiapoptosis (Bcl-2 and Bcl-xl), invasion (MMP-9 and ICAM-1), and angiogenesis (FGF-2 and VEGF). Furthermore, mollugin induced the activation of p38, ERK, and JNK and the expression of heme oxygenase-1 (HO-1) and nuclear factor E2–related factor 2 (Nrf2). Mollugin-induced growth inhibition and apoptosis of HO-1 were reversed by an HO-1 inhibitor and Nrf2 siRNA. Collectively, this is the first report to demonstrate the effectiveness of mollugin as a candidate for a chemotherapeutic agent in OSCCs via the upregulation of the HO-1 and Nrf2 pathways and the downregulation of NF-κB. ==== Body 1. Introduction Oral squamous cell carcinoma (OSCC) represents the fifth most common cancer worldwide and is a significant cause of cancer morbidity and mortality. Each year, approximately 300,000 new cases are diagnosed with only a 50% survival rate over 5 years. Common treatments, including surgery, radiation therapy, and chemotherapy, have very low success rates [1]. Two of the most frequently used chemotherapeutic drugs, 5-fluorouracil and cisplatin, cause side effects such as bone marrow suppression, gastrointestinal toxicity, and renal damage, which remain problems that need to be resolved [2]. To overcome such side effects and limitations, the direction of present research into novel antitumor agents has turned to natural products, especially plants used in traditional medicine [3]. Previously, we demonstrated that highly purified sulfur [4] and herbal medicines, such as Caesalpinia sappan [5], Coptidis rhizoma [6], and verticinone [7], exerted antitumor effects on oral cancer cells in vitro. Moreover, we showed that a single compound isolated from Caesalpinia sappan heartwood, isoliquiritigenin 2′-methyl ether (ILME), had antioral cancer effects involving mitogen-activated protein kinases (MAPKs) and the nuclear factor-κB (NF-κB) pathway [8]. In addition, we reported that a flavonoid extracted from Caesalpinia sappan, sappanchalcone, suppressed oral cancer cell growth and induced apoptosis through activation of p53-dependent mitochondrial MAPK and NF-κB signaling [9]. The roots of Rubia cordifolia L. have been widely used as a traditional herbal medicine in Korea to treat cough, bladder and kidney stones, joint inflammation, uterine hemorrhage, and uteritis [10]. In addition, this plant has been used in traditional Chinese medicine for the treatment of arthritis, dysmenorrheal, hematorrhea, hemostasis, and psoriasis [11, 12]. Among the bioactive components from Rubia cordifolia, mollugin (C17H16O4; methyl 2,2-dimethyl-6-hydroxy-2H-naphtho[1,2-b]pyran-5-carboxylate) has been reported to have antitumor and anti-inflammatory activities, and neuroprotective and apoptotic effects [13–16]. A recent study demonstrated that mollugin induced apoptosis through endoplasmic reticulum stress-mediated activation of c-Jun N-terminal kinase (JNK) and the mitochondria-dependent caspase cascade, regulated by Bcl-xL in human Jurkat T cells [14]. Moreover, mollugin inhibited proliferation and induced apoptosis by suppressing fatty acid synthase in HER2-overexpressing human breast and ovarian cancer cells [17]. Heme oxygenase-1 (HO-1) is an inducible cytoprotective enzyme that catalyzes the initial rate-limiting reaction in heme catabolism. Previously, we reported that the HO-1 pathway plays a key role in the adaptation of cells to stressful conditions and the recovery of dental pulp cells and periodontal ligament cells from injury [18–20]. Despite its cytoprotective properties, recent evidence suggests a role for HO-1 in promoting cancer [21]. HO-1 is overexpressed in various types of cancer and is further induced by radiation and chemotherapy [22, 23]. Regarding the mechanisms of HO-1 induction, several studies have suggested the involvement of MAPK and NF-κB pathways, as well as nuclear factor erythroid 2–related factor 2 (Nrf2) [18, 24]. However, the precise cellular mechanisms of mollugin on OSCCs are not completely understood. The aim of this study was to investigate the chemotherapeutic effect of mollugin on human primary and metastatic OSCCs in vitro. In addition, we further explored whether the effect of mollugin is related to Nrf2 activation and HO-1 expression. 2. Materials and Methods 2.1. Reagents Mollugin was isolated from root of heartwood of Rubia cordifolia L. as described previously [13]. Antibody against NF-κB p65, IκB, Nrf2, Bcl-xL, Bcl-2, p53, p21, or phosphorylated isoforms of IκB and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Delaware Avenue, CA). ERK, JNK, p38 or phosphorylation of ERK, JNK, and p38 were purchased from Cell signaling, Inc. (Beverly, MA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL (Grand Island, NY). All other chemicals were obtained from Sigma (St. Louis, MO), unless indicated otherwise. 2.2. Cell Culture The cell line HNSCC4 (HN4), from a primary OSCCs, and cell line HNSCC12 (HN12), from a metastatic carcinoma of the OSCCs [25], were derived in the laboratory of Dr. John F. Ensley (Wayne State University). Cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Cells were dissociated with 0.25% trypsin just before transferring for experiments and counted using a hemocytometer. Human keratinocyte cell line HaCaT (nontransformed human cell line) were incubated in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 50 μg/mL streptomycin, and 50 μg/mL penicillin at 37°C in 5% CO2. Human gingival fibroblasts (HGFs) cell line by transfection with the E6/E7 open reading frames of HPV type 16 was cultured according to the following protocol, as reported by our study [26]. Briefly, HGFs were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C. 2.3. Antiproliferative Assay Cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded in flat-bottomed 96-well plates, at 1 × 105 cell/well 24 h prior to treatment. The cells were treated for various lengths of time with the agents indicated. Then 25 μL of 5 mg/mL MTT was added to each well. After 4 h incubation at 37°C, 100 μL of lysing buffer was added. The buffer consisted of 20% w/v of sodium dodecyl sulfate in 0.1% of HCl solution. The plates were incubated for a further analysis on an ELISA reader at 570 nm. The same experiment was repeated in three separate cultures, and the data were presented as the means ± SD of five observations. 2.4. Flow Cytometry 2.4.1. Propidium Iodide (PI) Staining Cells were seeded at 5 × 105cells/well in six-well plates. After 24 h, cells were treated with mollugin the 3 days. After treatment, cells were harvested and pelleted by centrifugation (400 ×g, 4°C, 5 min). The cells were fixed with cold 75% ethanol for 24 h and then stained with PI solution, consisting of 45 mg/mL PI, 10 mg/mL RNase A, and 0.1% Triton X-100. After incubation in the dark at 4°C for 1 h, fluorescence-activated cells were sorted using the FACScan flow cytometer, and the data were analyzed using Cellfit Analysis Software. 2.5. Fluorescein-Isothiocyanate-(FITC-) Annexin V and Propidium Iodide (PI) Double Staining Cells (5 × 105) were seeded in 6-well plates, incubated for 24 h, and then treated with or without mollugin, and the incubation was continued for 3 days. After treatment, the cell pellet was prepared in a FACStar tube containing annexin V-FITC solution and incubated in 5% CO2 at 37°C. The PI solution (without NP 40) was then added, and the ratio of PI-positive and annexin V-positive cells was measured using the flow cytometer. 2.6. Morphological Analysis of Apoptosis by Staining with 4′,6-Diamino-2-Phenylindole Dihydrochloride (DAPI) To confirm that the nuclei underwent morphological changes, the cells were cultured in 60 mm dishes overnight and then washed twice in DMEM. The cells were then treated with mollugin and fixed in 4% paraformaldehyde, after which they were incubated in 1 μg/mL DAPI solution for 30 min in the dark. The cells were then examined using a fluorescence microscope (Zeiss, Oberköchen, Germany). 2.7. Western Blot Analysis Western blot analysis was performed by lysing cells in 20 mM Tris-HCl buffer (pH 7.4) containing protease inhibitor mixture (0.1 mM phenylmethanesulfonyl fluoride, 5 mg/mL aprotinin, 5 mg/mL pepstatin A, and 1 mg/mL chymostatin). Protein concentration was determined using the Lowry protein assay kit (P5626; Sigma). An equal amount of protein for each sample was resolved using 7% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% skim milk and sequentially incubated with primary antibody (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated secondary antibody followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ). 2.8. Isolation of RNA and RT-PCR Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. It was then reverse-transcribed using AccuPower RT premix (Bioneer, Daejeon, Korea). The PCR amplification of the resulting cDNA samples was performed using a GeneAmp PCR System 2400 thermal cycler (PerkinElmer, Wellesley, MA, USA). The primers information is summarized in Table 1. The following PCR conditions were used: 34 cycles of denaturation at 95°C for 30 s, primer annealing at 60°C for 30 s, and extension at 72°C for 30 s. The PCR products were resolved on 1-2% agarose gels and stained with ethidium bromide. 2.9. HO-1 and Nrf2 siRNA Transfection The target sequence for human HO-1 siRNA was 5′-AACUUUCAGAAGGGCCAGGUGTT-3′ (forward) and 5′-CACCUGGCCCUUCUGAAAGUUTT-3′ (reverse). Transfection of Nrf2 siRNA was performed using the target sequence 5′-AAGAGUAUGAGCUGGAAAAACTT-3′ (forward) and 5′-GUUUUUCCAGCUCAUACUCUUTT-3′ (reverse). Cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacture's instruction. Silencer Negative Control siRNA (Invitrogen) was introduced into cells by the same method. Following transfection, cells were cultured in six-well plates at 37°C until required. 2.10. Statistical Analysis Data are expressed as the mean ± standard deviation. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences (P < 0.05) between the means of control and sulfur-treated cells were analyzed using Dunnett's test. 3. Results 3.1. Effect of Mollugin on HN4 and HN12 Cell Viability To investigate the effect of mollugin on the proliferation of human OSCCs, the MTT assay was used. As shown in Figure 1, mollugin inhibited cellular proliferation in a dose- and time-dependent manner. Moreover, mollugin displayed higher cytotoxicity in primary OSCC HN4 cells compared with metastatic OSCC HN12 cells, although the overall trend of the effects of mollugin on HN4 and HN12 cells was similar. Furthermore, we tested whether mollugin has any toxic effect on nonneoplastic human skin keratinocytes (HaCaT) and gingival fibroblasts (GF). Interestingly, we did not find significant cytotoxicity or cell death by mollugin on HaCaT and GF (Figures 1(c) and 1(d)). 3.2. Effect of Mollugin on HN4 and HN12 Cell Apoptosis To determine whether the cause of cell death was apoptosis, HN4 and HN12 cells were exposed to 40 μM mollugin for 3 days. This concentration was used because it reproducibly induced 50% growth inhibition in HN4 and HN12 cells cultured for 3 days. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), a fluorescent DNA-binding dye (Figure 2(a)). DAPI staining showed that the control nuclei were large and round without condensation or fragmentation, whereas most of the nuclei of mollugin-treated cells were condensed and fragmented, as is typical during apoptosis. The onset of apoptotic cell death by mollugin was confirmed by flow cytometry of HN4 and HN12 cells (Figure 2(b)). Without treatment, only 3.88% HN4 and 2.80% HN12 cells were in the sub-G1 region; however, mollugin treatment increased the proportions of cells in the sub-G1 region to 20.26% and 31.46%, respectively. To determine whether the growth inhibitory effect of mollugin is associated with cell death, annexin V-propidium iodide (PI) double staining of HN4 and HN12 cells and flow cytometry was performed (Figure 2(c)). Treatment of cells with mollugin for 3 days increased the number of annexin V+/PI+ stained cells. These results demonstrate that the inhibition of cell growth by mollugin was caused by the induction of apoptosis. 3.3. Effect of Mollugin on NF-κB Activation and Expression of NF-κB-Regulated Genes Since NF-κB transcription factors and their upstream activating molecules are attractive targets for cancer therapeutics, we evaluated the effect of mollugin on NF-κB activation by measuring the phosphorylation and degradation of IκBα and NF-κB p65. As shown in Figure 3(a), treatment of HN4 and HN12 cells with mollugin decreased the steady-state levels of phosphorylated IκBα and nuclear NF-κB p65. To clarify the molecular mechanism by which mollugin induces apoptosis in primary and metastatic OSCCs, we determined the levels of NF-κB-regulated gene products involved in apoptosis and antiapoptosis (Figure 3(b)). Mollugin treatment upregulated p53 and p21 expression in both HN4 and HN12 cells in a dose-dependent manner. In contrast, the expression of antiapoptotic Bcl-2 and Bcl-xl proteins was dose-dependently reduced by mollugin treatment in HN4 and HN12 cells. Next, we investigated whether mollugin could modulate levels of NF-κB-regulated gene products involved in invasion (ICAM-1 and MMP-9) and angiogenesis (VEGF and FGF-2) of OSCCs. The results show that mollugin downregulated the expression of NF-κB-regulated gene products, including MMP-9, ICAM-1, VEGF, and FGF-2 mRNA in a dose-dependent manner (Figure 3(c)). 3.4. Effect of Mollugin on MAPK and Nrf2 Activation To clarify the mechanism underlying the apoptosis, we assessed the effect of mollugin on Nrf2 and NF-κB upstream pathways. Treatment with mollugin (40 μM) increased levels of phosphorylated (activated) ERK, p38, and JNK; the phosphorylated proteins were easily detectable by Western blot after 30 or 60 min. In contrast, mollugin did not affect total p38, ERK, or JNK levels (Figure 4(a)). Since the stress response element/Nrf2 transcription factor pathway is important for HO-1 expression [23], we next determined whether Nrf2 signaling is involved in mollugin-induced HO-1 expression and apoptosis. Initially, Nrf2 activation was assessed by the accumulation of Nrf2 in the nucleus. Treatment with mollugin resulted in an accumulation of Nrf2 in the nucleus in primary and metastatic OSCCs (Figure 4(b)). To determine whether mollugin induces expression of antioxidant genes, HO-1 expression in oral cancer cells and the change in the pattern of HO-1 expression were evaluated. HO-1 protein expression in primary and metastatic OSCCs increased following mollugin treatment in a time-dependent manner (Figure 4(c)). To confirm the involvement of MAPK pathways in the mollugin-driven activation of NF-κB and Nrf2, inhibitors of ERK (PD98059), JNK (SP6100126), and p38 (SB203580) MAPK signaling were used. Mollugin-induced NF-κB, Nrf2, and HO-1 expression was effectively inhibited by PD98059, SP600126, and SB203580 (Figure 4(d)), suggesting that p38, ERK1/2, and JNK play important roles in activating NF-κB, Nrf2, and HO-1 in HN4 and HN12 cells. 3.5. Involvement of the HO-1 Pathway in Mollugin-Induced Apoptosis To examine the cytotoxic potential of tin protoporphyrin (SnPP), an HO-1 inhibitor, its effect on viability was initially measured by the MTT assay (Figure 5(a)). Although, 10 or 20 μM SnPP had no cytotoxicity effect for 3 h, we found that SnPP exhibited slight cytotoxicity on HN4 and HN12 cells at 16 h. Based on these findings, we chose to use SnPP for 3 h. To determine whether the induction of HO-1 plays a role in mollugin-induced growth inhibition and apoptosis in OSCCs, cells were treated with SnPP, an HO-1 inhibitor, for 3 h before incubation with mollugin for 3 days. MTT assays showed that mollugin-induced cytotoxicity and apoptosis in HN4 and HN12 cells were dose-dependently reversed by pretreatment with SnPP (Figures 5(a) and 5(b)). In addition, SnPP treatment abolished the induction of p53 and p21WAF1/CIP1 expression by mollugin, but reversed the expression of Bcl-2 and Bcl-xl (Figure 5(c)). Additionally, we used a siRNA-expressing plasmid to induce HO-1 gene silencing. HN4 and HN12 cells were transfected with an HO-1-targeting siRNA expression vector (Figure 6). The siRNA approach resulted in high silencing efficacies for HO-1, which was confirmed by Western blot analysis (Figure 6(c)). Silencing of HO-1 significantly attenuated mollugin-induced cytotoxicity (Figure 6(a)) and apoptosis (Figure 6(b)). Moreover HO-1 RNA blocked mollugin-induced Nrf2, p53, and p21 upregulation as well as Bcl-2 and Bcl-xl downregulation (Figure 6(c)). 3.6. Involvement of the Nrf2 Pathway in Mollugin-Induced HO-1 Induction and Apoptosis To investigate whether mollugin-induced apoptosis and HO-1 expression were mediated by Nrf2 activation, a specific small interfering RNA (siRNA) against Nrf2 was used. As shown in Figure 7(c), transfection with Nrf2 siRNA for 5 h in HN4 and HN12 cells blocked mollugin-induced HO-1 and Nrf2 expression. However, Nrf2 siRNA did not affect the mollugin-induced p65 expression. Transfection with Nrf2 siRNA inhibited mollugin-induced cytotoxicity and apoptosis in HN4 and HN12 cells (Figures 7(a) and 7(b)). Moreover, transfection with Nrf2 siRNA in HN4 and HN12 cells reduced mollugin-induced apoptosis proteins (p53 and p21), but reversed antiapoptosis proteins (Bcl-2 and Bcl-xl) (Figure 7(c)). 4. Discussion This study is to our knowledge the first to demonstrate that mollugin directly induces growth inhibition and apoptosis in primary and metastatic OSCCs and to address the molecular basis of this effect. With regard to antitumor activity, mollugin has been shown to exert cytotoxic effects on human colon cancer (Col2) cells (IC50 = 12.3 μM; [11]), human liver carcinoma (HepG2) cells (IC50 value = 60.2 μM; [10]), and basal HER2-expressing human breast cancer cells (IC50 value = 58 μM; [17]). In the present study, we found that mollugin (10–80 μM) resulted in significant growth inhibition, with an average IC50 value of 46.3 μM in metastatic OSCCs (HN12) and 43.9 μM in primary OSCCs (HN4) after 3 days. These antiproliferative properties of mollugin are consistent with previous reports in human acute leukemia Jurkat T cells [14] and HER2-overexpressing breast and ovarian cancer cell lines [17]. Moreover, we demonstrated that mollugin induced OSCC cell death by triggering apoptosis due to the presence of several apoptotic characteristics, including sub-G1 phase accumulation, increase in annexin+/PI+ cells, and DNA fragmentation. NF-κB signaling is a ubiquitous pathway in cell proliferation, survival, and apoptosis. Normally, NF-κB proteins are inhibited by binding to IκB proteins in the cytoplasm. Stress factors cause degradation of IκB proteins through the ubiquitin-proteosomal pathway, and NF-κB is released to bind to the DNA of promoter regions of specific genes that increases their transcription rate [27, 28]. To explore the mechanism underlying mollugin-induced apoptosis in OSCCs, we examined NF-κB activation. In agreement with previous findings that mollugin suppressed NF-κB activation in HER2-overexpressing SK-BR-3 breast cancer cells [17], our results indicate that mollugin significantly inhibited NF-κB activation by reducing the level of phospho-IκBα, leading to blockage of the IκBα pathway in NF-κB activation in OSCCs. In addition, it was reported that mollugin significantly suppressed TNF-α-induced NF-κB transcriptional activation in HT-29 human colonic epithelial cells [13]. Genes involved in the proliferation, apoptosis, antiapoptosis, angiogenesis, and metastasis of cancer are regulated by NF-κB [29]. Constitutive activation of NF-κB negatively regulates the proapoptotic functions of p53 by inducing the expression of an array of antiapoptotic genes, including Bcl-2 and Bcl-xL [30]. In our study, treatment with mollugin induced the downregulation of antiapoptotic Bcl-2 and Bcl-xL levels as well as the upregulation of apoptotic p53 and p21 levels in OSCCs. Another hallmark of OSCC behavior is their propensity for local invasion and metastasis. MMP-9 is frequently overexpressed in OSCCs and is dependent upon NF-κB for expression [31]. Adhesion molecules such as ICAM-1 are regulated by NF-κB and are essential for the adhesion of tumor cells to endothelial cells and thus mediate tumor cell metastasis [32]. Angiogenesis is important for tumor growth and progression. The development of angiogenesis is stimulated by cytokines and growth factors, and the expression of these cytokines and growth factors is correlated with the pathological neovascularization circumstances. Among these angiogenic factors, VEGF and FGF-2 are important angiogenic factors and essential for cancers [33]. In this study, we found that mollugin inhibited the expression of NF-κB-regulated gene products involved in invasion (MMP-9 and ICAM-1) and angiogenesis (FGF-2 and VEGF). Thus, our results demonstrate that mollugin inhibits NF-κB, leading to suppression of proliferation, invasion, and angiogenesis in OSCCs. The MAPKs are proline-directed Ser/Thr protein kinases that regulate many cellular processes, including cell proliferation and death. JNK and p38 are activated by cellular stress and are associated with apoptosis [34]. The involvement of ERK in the induction of apoptosis by quercetin, resveratrol, and taxol has been reported [35, 36]. In the present study, phosphorylation of p38, ERK, and JNK was detected in mollugin-treated OSCCs, which is consistent with our previous reports that sappanchalcone may exert its effect on OSCCs through p38, ERK, and JNK activation [9]. Therefore, NF-κB regulation downstream of the MAPK (ERK1/2, p38, and JNK) pathways may be involved in mollugin-induced apoptosis in OSCCs. However, mollugin activated p38 MAPK in hippocampal and microglial cells [15] and ERK in HER2-overexpressing breast cancer cells [17]. Therefore, MAPK can be differentially activated, and its involvement in apoptosis is highly context and model dependent. The role of HO-1 in tumor development is still not understood completely. Some recent reports demonstrate discordant or even completely contrasting results. Nevertheless, accumulating evidence indicates that HO-1 is expressed or overexpressed in a wide variety of human tumors and plays a critical role in the progression of neoplastic diseases [37]. For example, it has been shown that increased expression of HO-1 is associated with a higher rate of proliferation of various tumor cells [37, 38], although opposite effects have been observed in breast cancer cells [39]. Furthermore, the increased basal level of HO-1 expression in tumor cells can be further elevated by chemotherapeutics or phytochemicals, such as curcumin, carnosol, and ILME [8, 22, 23]. Our results show that mollugin upregulated HO-1 expression in a time- and dose-dependent manner (data not shown), consistent with results in hippocampal and microglial cells [15]. We found that growth inhibition and apoptosis of OSCCs by mollugin was reversed in the presence of an HO-1 inhibitor. In addition, mollugin induced downregulation of antiapoptotic Bcl-2 and Bcl-xL levels, while upregulation of apoptotic p53 and p21 levels was reversed by SnPP in a dose-dependent manner. These results suggest that the growth inhibition and apoptosis-inducing effect of mollugin is mediated via HO-1 expression. Regarding HO-1, Nrf-2 is a relatively well-known transcription factor [40]. In response to diverse HO-1 inducers, Nrf2 translocates from the cytosol to the nucleus where it binds to the antioxidant response element in the promoter region of the HO-1 gene [41]. In our study, mollugin also induced the translocation of Nrf-2 into the nucleus, as evidenced by Western blot. Furthermore, the results of this study demonstrate a correlation between apoptotic gene upregulation and the suppression of antiapoptotic genes of OSCCs to undergo mollugin-induced growth inhibition and apoptosis when Nrf-2 is knocked down. Based on these observations, it is reasonable to conclude that Nrf2 activation mediates mollugin-induced growth inhibition and apoptosis in OSCCs. Redox-sensitive transcription factor (NF-κB, AP-1, and Nrf2) pathways are known to be important molecular targets in chemoprevention. Activation of Nrf2 and NF-κB involves regulation of protein kinases, which may induce their nuclear translocation [42]. It was previously shown that mollugin induced apoptosis via JNK in human Jurkat T cells [14]. In this regard, specific protein kinase inhibitors of PI3 K and ERK repressed Nrf2 phosphorylation and nuclear translocation of Nrf2 and NF-κB in HepG2 cells, as previously reported in different cell types [43, 44]. The present results also suggest that the MAPK pathway plays a role in the mollugin-induced nuclear translocation of Nrf2 and NF-κB in HN4 and HN12 cells. A variety of anticarcinogenic phytochemicals suppress NF-κB signalling and activate the Nrf2-ARE pathway [45], suggesting that the suppression of NF-κB signalling and the activation of the Nrf2-ARE pathway may crosstalk with each other. Indeed, inhibition of NF-κB activation by phospholipid hydroperoxide glutathione peroxidase and 15-lipoxygenase is concomitant to upregulation of HO-1, probably via Nrf2 activation [46]. Thus, the findings support that the participation of NF-κB p65 in the negative regulation of Nrf2-ARE signalling, providing a new insight into a possible role of NF-κB in suppressing the expression of anti-inflammatory or antitumour genes [47]. Our results suggest that MAPK singnaling event mediates the link between NF-κB and Nrf2. Although the NF-κB pathway was inhibited by mollugin exposure, Nrf2 siRNA did not seem to participate in the translocation of NF-κB, suggesting that NF-κB and Nrf2 pathways might act in parallel in our in vitro model. A summary of our results is shown in Figure 8. Colectivelly, our results demonstrate for the first time that mollugin inhibits cell growth and induces apoptosis in OSCCs through the NF-κB/MAPK/Nrf2/HO-1 signaling pathway. Therefore, mollugin may have potential as a chemotherapeutic agent for OSCCs. The activities of mollugin against multiple antitumor targets should be studied intensively for further clinical application either as a natural agent alone or in combination with other commonly used chemotherapeutics. Further investigation of mollugin in animal models of OSCCS will contribute to additional understanding of its in vivo activity toward malignant cells and its potential toxicity toward normal tissues. Authors' Contribution Y.-M. Lee and Q.-S. Auh contributed equally to this work as 1st author. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (no. 2012R1A5A2051384). Figure 1 Effects of mollugin on cell viability in primary (HN4, (a)), metastatic oral cancer cells (HN12, (b)), skin keratinocytes (HaCaT, (c)), and gingival fibroblasts (d) as measured by MTT assay. Statistically significant difference as compared to control, P < 0.05. The same experiment was repeated in three separate cultures, and the data were presented as the means ± SD of five observations. Figure 2 Confirmation of mollugin-induced apoptosis by DAPI staining (a), cell cycle analysis (b), and annexin V-PI staining (c) in OSCCs. Cells were incubated with 40 μM mollugin for 3 days. Data are presented as mean ± SD from triplicate determinations (b, c). Results are representative from three independent experiments (a)–(c). Figure 3 Effects of mollugin on activation of NF-κB (a), and expression of NF-κB-regulated gene products involved in apoptosis and antiapoptosis (b), metastasis, and angiogenesis (c) in OSCCs. Cells were treated with 40 μM mollugin for indicated times (a) or 3 days (b, c). The figure is representative of 3 independent experiments. Figure 4 Effect of mollugin on phosphorylation of MAPK (a), activation of Nrf2 (b), and expression of HO-1 (c) in OSCCs. Effects of MAP kinase inhibitors on mollugin-induced activation of NF-κB, Nrf2, and HO-1 (d). Cells were treated with 40 μM mollugin for indicated times (a)–(c). Cells were pretreated with the p38 inhibitor SB203580 (20 μM/L), the ERK inhibitor PD98059 (20 μM/L), or the JNK inhibitor SP600125 (20 μM/L) for 1 hour and treated with 40 μM mollugin for 30 min (MAPK, Nrf2, and NF-κB) or 3 days (HO-1). The results are representative of three independent experiments. Figure 5 Effects of HO-1 inhibitor (SnPP) on cytotoxicity (a), mollugin-induced growth inhibition (b), apoptosis (c), and apoptosis-related proteins expression (d) in OSCCs. Cells were pretreated with SnPP for 3 h and treated for 3 days with mollugin (b)–(d). Cell viability was examined by MTT assay (a), and apoptosis was examined by flow cytometry (b), respectively. The same experiment was repeated in three separate cultures, and the data were presented as the means ± SD of five observations (a, b). Data are presented as mean values from triplicate determinations (c). Western data show one representative result of three independent experiments (d). Statistically significant difference as compared to control, P < 0.05. #Statistically significant difference as compared to mollugin, P < 0.05. Figure 6 Effects of HO-1 siRNA on mollugin-induced growth inhibition (a), apoptosis (b), and apoptosis-related proteins expression (c) in OSCCs. Cells were pretreated with HO siRNA (30 nM) for 5 h and treated for 3 days with mollugin (a)–(c). Cell viability was examined by MTT assay (a), and apoptosis was examined by flow cytometry (b), respectively. The same experiment was repeated in three separate cultures, and the data were presented as the means ± SD of five observations (a). Data are presented as mean values from triplicate determinations (b). Western data show one representative result of three independent experiments (c). Statistically significant difference as compared to control, P < 0.05. #Statistically significant difference as compared to mollugin, P < 0.05. Figure 7 Effect of Nrf2 siRNA on mollugin-induced growth inhibition (a), apoptosis (b), and apoptosis-related proteins expression (c). Cells were treated with 40 μM mollugin for indicated times (a). Cells were pretreated with Nrf2 siRNA (250 nM) for 5 h and treated for 3 days with mollugin 40 μm (a)–(c). Statistically significant difference as compared to control, P < 0.05. #Statistically significant difference as compared to mollugin, P < 0.05. Data are representative of 3 independent experiments. Figure 8 Schematic diagram illustrating the Nrf2 pathway and other signaling pathways triggered by exposure to mollugin in in OSCCs. Mollugin treatment leads to the activation of the cellular stress-dependent signaling pathways mediated by MAPK activation (ERK, JNK, and p38) and subsequent transcription factor activation (NF-κB and Nrf2). Ultimately, these transcription factors result in altered gene expression to produce cytoprotective gene HO-1, allowing lead to apoptosis. Table 1 Reverse transcriptase-polymerase chain reaction primers. Gene Sequence 5′-3′ Size MMP-9 Forward: GTTGGGGAGGGCTGTCCGTGA 911 bp Reverse: CGTGGCGCTATCCAGCTCACC ICAM-1 Forward: CGCCCGATTGCTTTAGCTTG 320 bp Reverse: CGACTCACCTGGGAACAGAG VEGF Forward: TCAAGGTTGGCGGAAGTGAGG 443 bp Reverse: CCCTTGCATCAGTAGGCTTCA FGF2 Forward: GCCTCATTTCCATTTCGTGG 224 bp Reverse: CGGGGGTACTGGTTTACAG β-actin Forward: CATGGATGATGATATCGCCGC 319 bp Reverse: ACATGATCTGGGTCATCTTCT ==== Refs 1 Schliephake H Prognostic relevance of molecular markers of oral cancer—a review International Journal of Oral and Maxillofacial Surgery 2003 32 3 233 245 2-s2.0-0037864294 12767868 2 Yamachika E Habte T Oda D Artemisinin: an alternative treatment for oral squamous cell carcinoma Anticancer Research 2004 24 4 2153 2160 2-s2.0-4444240287 15330155 3 McCann J Texas center studies research alternative treatments Journal of the National Cancer Institute 1997 89 20 1485 1486 2-s2.0-0031572517 9337344 4 Lee J Lee HJ Park JD Anti-cancer activity of highly purified sulfur in immortalized and malignant human oral keratinocytes Toxicology in Vitro 2008 22 1 87 95 2-s2.0-37249033214 17920232 5 Kim EC Hwang YS Lee HJ Caesalpinia sappan induces cell death by increasing the expression of p53 and p21WAF1/CIP1 in head and neck cancer cells The American Journal of Chinese Medicine 2005 33 3 405 414 2-s2.0-21344475106 16047558 6 Lee HJ Son DH Lee SK Extract of Coptidis rhizoma induces cytochrome-c dependent apoptosis in immortalized and malignant human oral keratinocytes Phytotherapy Research 2006 20 9 773 779 2-s2.0-33748809213 16807885 7 Yun YG Jeon BH Lee JH Verticinone induces cell cycle arrest and apoptosis in immortalized and malignant human oral keratinocytes Phytotherapy Research 2008 22 3 416 423 2-s2.0-40849094364 18058993 8 Lee YM Jeong GS Lim HD An RB Kim YC Kim EC Isoliquiritigenin 2′-methyl ether induces growth inhibition and apoptosis in oral cancer cells via heme oxygenase-1 Toxicology in Vitro 2010 24 3 776 782 2-s2.0-77649180814 20040371 9 Lee YM Kim YC Kim EC Mechanism of sappanchalcone-induced growth inhibition and apoptosis in human oral cancer cells Toxicology in Vitro 2011 25 8 1782 1788 21963806 10 Son JK Jung SJ Woo MHMH Anticancer constituents from the roots of Rubia cordifolia L. Chemical Pharmacology Bulletin 2008 56 2 213 216 11 Chang LC Chavez D Gills JJ Fong HHS Pezzuto JM Kinghorn AD Rubiasins A-C, new anthracene derivatives from the roots and stems of Rubia cordifolia Tetrahedron Letters 2000 41 37 7157 7162 2-s2.0-0034601645 12 Tse WP Cheng CHK Che CT Zhao M Lin ZX Induction of apoptosis underlies the Radix Rubiae-mediated anti-proliferative action on human epidermal keratinocytes: implications for psoriasis treatment International Journal of Molecular Medicine 2007 20 5 663 672 2-s2.0-38449102690 17912459 13 Kim KJ Lee JS Kwak MK Anti-inflammatory action of mollugin and its synthetic derivatives in HT-29 human colonic epithelial cells is mediated through inhibition of NF-κ B activation European Journal of Pharmacology 2009 622 1-3 52 57 2-s2.0-70349883924 19765578 14 Kim SM Park HS Jun DY Mollugin induces apoptosis in human Jurkat T cells through endoplasmic reticulum stress-mediated activation of JNK and caspase-12 and subsequent activation of mitochondria-dependent caspase cascade regulated by Bcl-xL Toxicology and Applied Pharmacology 2009 241 2 210 220 2-s2.0-70350221910 19716835 15 Jeong GS Lee DS Kim DC Neuroprotective and anti-inflammatory effects of mollugin via up-regulation of heme oxygenase-1 in mouse hippocampal and microglial cells European Journal of Pharmacology 2011 654 3 226 234 2-s2.0-79951674708 21236252 16 Jun DY Han CR Choi MS Bae MA Woo MH Kim YH Effect of mollugin on apoptosis and adipogenesis of 3T3-L1 preadipocytes Phytotherapy Research 2011 25 5 724 731 2-s2.0-79955545476 21077262 17 Do MT Hwang YP Kim HG Na M Jeong HG Mollugin inhibits proliferation and induces apoptosis by suppressing fatty acid synthase in HER2-overexpressing cancer cells through modulation of a HER2/Akt/SREBP-1c signaling Journal of Cell Physiology 2012 228 5 1087 1097 18 Lee SK Choi HI Yang YS Nitric oxide modulates osteoblastic differentiation with heme oxygenase-1 via the mitogen activated protein kinase and nuclear factor-κ B pathways in human periodontal ligament cells Biological and Pharmaceutical Bulletin 2009 32 8 1328 1334 2-s2.0-69149091631 19652369 19 Lee SK Pi SH Kim SH Substance P regulates macrophage inflammatory protein 3α /chemokine C-C ligand 20 (CCL20) with heme oxygenase-1 in human periodontal ligament cells Clinical and Experimental Immunology 2007 150 3 567 575 2-s2.0-36048948249 17924972 20 Pi SH Kim SC Kim HT Lee HJ Lee SK Kim EC Defense mechanism of heme oxygenase-1 against cytotoxic and receptor activator of nuclear factor-κ B ligand inducing effects of hydrogen peroxide in human periodontal ligament cells Journal of Periodontal Research 2007 42 4 331 339 2-s2.0-34250173125 17559630 21 Poss KD Tonegawa S Reduced stress defense in heme oxygenase 1-deficient cells Proceedings of the National Academy of Sciences of the United States of America 1997 94 20 10925 10930 2-s2.0-0030886054 9380736 22 Balogun E Hoque M Gong P Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element Biochemical Journal 2003 371 3 887 895 2-s2.0-0037569694 12570874 23 Martin D Rojo AI Salinas M Regulation of heme Oxygenase-1 expression through the phosphatidylinositol 3-kinase/akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol The Journal of Biological Chemistry 2004 279 10 8919 8929 2-s2.0-1542335553 14688281 24 Anwar AA Li FYL Leake DS Ishii T Mann GE Siow RCM Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2 Free Radical Biology and Medicine 2005 39 2 227 236 2-s2.0-20444456655 15964514 25 Cardinali M Pietraszkiewicz H Ensley JF Robbins KC Tyrosine phosphorylation as a marker for aberrantly regulated growth-promoting pathways in cell lines derived from head and neck malignancies International Journal of Cancer 1995 61 1 98 103 2-s2.0-0028947575 7705939 26 Pi SH Lee SK Hwang YS Choi MG Lee SK Kim EC Differential expression of periodontal ligament-specific markers and osteogenic differentiation in human papilloma virus 16-immortalized human gingival fibroblasts and periodontal ligament cells Journal of Periodontal Research 2007 42 2 104 113 2-s2.0-33847082493 17305867 27 Ghosh S Karin M Missing pieces in the NF-κ B puzzle Cell 2002 109 S81 S96 11983155 28 Suh Y Afaq F Johnson JJ Mukhtar H A plant flavonoid fisetin induces apoptosis in colon cancer cells by inhibition of COX2 and Wnt/EGFR/NF-κ B-signaling pathways Carcinogenesis 2009 30 2 300 307 2-s2.0-60149112516 19037088 29 Aggarwal BB Nuclear factor κ B, the enemy within Cancer Cell 2004 6 3 203 208 15380510 30 Sethi G Sung B Aggarwal BB Nuclear factor-κ B activation: from bench to bedside Experimental Biology and Medicine 2008 233 1 21 31 2-s2.0-37549004392 18156302 31 Bond M Fabunmi RP Baker AH Newby AC Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-κ B FEBS Letters 1998 435 1 29 34 2-s2.0-0032508674 9755853 32 Lawson C Ainsworth M Yacoub M Rose M Ligation of ICAM-1 on endothelial cells leads to expression of VCAM-1 via a nuclear factor-κ B-independent mechanism Journal of Immunology 1999 162 5 2990 2996 2-s2.0-0033104737 33 Kerbel RS Tumor angiogenesis: past, present and the near future Carcinogenesis 2000 21 3 505 515 2-s2.0-0034092011 10688871 34 Xiao D Choi S Johnson DE Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2 Oncogene 2004 23 33 5594 5606 2-s2.0-3342895063 15184882 35 She QB Bode AM Ma WY Chen NY Dong Z Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase Cancer Research 2001 61 4 1604 1610 2-s2.0-0035866404 11245472 36 Brozovic A Fritz G Christmann M Long-term activation of SAPK/JNK, p38 kinase and fas-L expression by cisplatin is attenuated in human carcinoma cells that acquired drug resistance International Journal of Cancer 2004 112 6 974 985 2-s2.0-8644269825 15386344 37 Jozkowicz A Was H Dulak J Heme oxygenase-1 in tumors: is it a false friend? Antioxidants and Redox Signaling 2007 9 12 2099 2117 2-s2.0-35848942608 17822372 38 Berberat PO Dambrauskas Z Gulbinas A Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment Clinical Cancer Research 2005 11 10 3790 3798 2-s2.0-21044452445 15897578 39 Hill M Pereira V Chauveau C Heme oxygenase-1 inhibits rat and human breast cancer cell proliferation: mutual cross inhibition with indoleamine 2,3-dioxygenase FASEB Journal 2005 19 14 1957 1968 2-s2.0-28744447937 16319139 40 Alam J Cook JL Transcriptional regulation of the heme oxygenase-1 gene via the stress response element pathway Current Pharmaceutical Design 2003 9 30 2499 2511 2-s2.0-0141885079 14529549 41 Ishii T Itoh K Takahashi S Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages The Journal of Biological Chemistry 2000 275 21 16023 16029 2-s2.0-0034717329 10821856 42 Pi J Bai Y Reece JM Molecular mechanism of human Nrf2 activation and degradation: role of sequential phosphorylation by protein kinase CK2 Free Radical Biology and Medicine 2007 42 12 1797 1806 2-s2.0-34248593492 17512459 43 Nguyen T Sherratt PJ Huang HC Yang CS Pickett CB Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element: degradation of Nrf2 by the 26 S proteasome The Journal of Biological Chemistry 2003 278 7 4536 4541 2-s2.0-0013282861 12446695 44 Sethi G Sung B Aggarwal BB Nuclear factor-κ B activation: from bench to bedside Experimental Biology and Medicine 2006 6 5 515 524 45 Lim JH Park HS Choi JK Lee IS Hyun JC Isoorientin induces Nrf2 pathway-driven antioxidant response through phosphatidylinositol 3-kinase signaling Archives of Pharmacal Research 2007 30 12 1590 1598 2-s2.0-38149000858 18254247 46 Li W Khor TO Xu C Activation of Nrf2-antioxidant signaling attenuates NFκ B-inflammatory response and elicits apoptosis Biochemical Pharmacology 2008 76 11 1485 1489 2-s2.0-55949120110 18694732 47 Banning A Brigelius-Flohé R NF-κ B, Nrf2, and HO-1 interplay in redox-regulated VCAM-1 expression Antioxidants and Redox Signaling 2005 7 7-8 889 899 2-s2.0-22044433408 15998244	23738323	PMC3659465	CC BY	2021-01-05 10:05:04	yes	Biomed Res Int. 2013 May 2; 2013:210604
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23704922PONE-D-12-3120810.1371/journal.pone.0063588Research ArticleBiologyImmunologyImmune SystemCytokinesMolecular cell biologySignal transductionSignaling in cellular processesSTAT signaling familyMedicineGastroenterology and HepatologyGastrointestinal CancersOncologyCancers and NeoplasmsGastrointestinal TumorsGastric CancerIL-26 Promotes the Proliferation and Survival of Human Gastric Cancer Cells by Regulating the Balance of STAT1 and STAT3 Activation IL-26 Promotes Human Gastric CancerYou Wei 1 2 Tang Qiyun 3 Zhang Chuanyong 1 2 Wu Jindao 1 2 Gu Chunrong 1 2 Wu Zhengshan 1 2 * Li Xiangcheng 1 2 * 1 Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, P.R. China 2 Key laboratory of living donor liver transplantation of ministry of health, Nanjing, Jiangsu Province, P.R. China 3 Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, P.R. China Pizzo Salvatore V. Editor Duke University Medical Center, United States of America * E-mail: Lixc2012ac@yahoo.cn (XL); Wuzs@163.com.cn (ZW)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: WY ZW XL. Performed the experiments: WY QT CZ JW CG. Analyzed the data: WY QT. Contributed reagents/materials/analysis tools: CA JW. Wrote the paper: WY XL. 2013 21 5 2013 8 5 e6358811 10 2012 2 4 2013 © 2013 You et al2013You et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Interleukin-26 (IL-26) is one of the cytokines secreted by Th17 cells whose role in human tumors remains unknown. Here, we investigated the expression and potential role of IL-26 in human gastric cancer (GC). The expression of IL-26 and related molecules such as IL-20R1, STAT1 and STAT3 was examined by real-time PCR and immunohistochemisty. The effects of IL-26 on cell proliferation and cisplatin-induced apoptosis were analyzed by BrdU cooperation assay and PI-Annexin V co-staining, respectively. Lentiviral mediated siRNA was used to explore its mechanism of action, and IL-26 related signaling was analyzed by western blotting. Human GC tissues showed increased levels of IL-26 and its related molecules and activation of STAT3 signaling, whereas STAT1 activation did not differ significantly between GC and normal gastric tissues. Moreover, IL-26 was primarily produced by Th17 and NK cells. IL-26 promoted the proliferation and survival of MKN45 and SGC-7901 gastric cancer cells in a dose-dependent manner. Furthermore, IL-20R2 and IL-10R1, which are two essential receptors for IL-26 signaling, were expressed in both cell lines. IL-26 activated STAT1 and STAT3 signaling; however, the upregulation of the expression of Bcl-2, Bcl-xl and c-myc indicated that the effect of IL-26 is mediated by STAT3 activation. Knockdown of STAT1 and STAT3 expression suggested that the proliferative and anti-apoptotic effects of IL-26 are mediated by the modulation of STAT1/STAT3 activation. In summary, elevated levels of IL-26 in human GC promote proliferation and survival by modulating STAT1/STAT3 signaling. This work was supported by grants from the National Natural Science Foundation (81170415 for XC) and Jiangsu Province’s Key Provincial Talents Program (RC2011079 for QT and RC2007056 for XC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Gastric cancer (GC) is the second most common cause of cancer-related death in the world. GC is difficult to cure even in Western countries because it is often not detected until the advanced stages of the disease [1]. Although a number of factors are associated with the development and progression of GC, a link between chronic gastric inflammation such as atrophic gastritis induced by Helicobacter pylori and the risk of GC has become evident in recent years [2]. Chronic inflammation leading to GC is a long and complicated process that occurs over many years and is characterized by inflammatory damage to the gastric mucosa, cytokine-induced DNA synthesis and cell proliferation, hyperplasia and carcinogenesis [3]. The association between chronic inflammation and the immune system has been well studied, and lymphocytes are the main mediators of inflammation-promoted carcinogenesis [4]. Th17 cells are a novel type of T lymphocytes that express RORγT and secrete various cytokines including IL-17A, IL-17F, IL-21, IL-22 and IL-26. The differentiation of Th17 cells is regulated by several cytokines including IL-1β, IL-6, IL-23, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β) [5], [6]. Recent clinical studies showed that Th17 cells may be closely related to H. pylori associated pathology and carcinogenesis of GC [7], [8], [9], [10]. Although several Th17 related cytokines have been studied, little is known about interleukin-26 (IL-26) in relation to gastric tumors. IL-26 is a secreted protein that functions either as a monomer or a homodimer. It was originally described by Knappe et al. [11] under the name of AK155. IL-26 has weak but significant sequence homology to IL-10, and its encoded protein is therefore a member of the IL-10 family of cytokines, which mostly belong to the class-2 cytokine family. IL-26 can be secreted by primary T cells, NK cells and T cell clones and is usually co-expressed with other important IL-10-related cytokines such as IL-22 [12], [13]. IL-26 binds to a distinct cell surface receptor complex consisting of the IL-20R1 and IL-10R2 chains, and its functional activities are different from those mediated by IL-10. IL-20R1 functions as the specific ligand-binding chain for IL-26, and IL-10R2 is an essential second chain to complete assembly of the active receptor complex. Neutralizing antibodies against either the IL-20R1 or IL-10R2 chains can block induction of IL-26 signaling [12]. Once fully assembled, the receptor complex undergoes a conformational change(s) that induces activation of the receptor-associated Janus tyrosine kinases, Jak1 and Tyk2, and subsequent transient docking and phosphorylation of the STAT proteins, STAT1 and STAT3 [14], [15]. As a Th17 related cytokine, the role of IL-26 in tumors has not been investigated. Here, we examined the potential involvement of IL-26 in human GC for the first time and explored its pro-survival and proliferative effects in vitro. Materials and Methods Patients The present study included 60 patients with GC who underwent surgery from 2006 to 2009 at the First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu province (Table 1). All experiments were conducted according to the principles expressed in the Declaration of Helsinki. Written informed consents for gene expression analyses in tissues were obtained from all patients prior to surgery or endoscopic examination. The study protocol and consent procedures were approved by the ethics committee of the First Affiliated Hospital of Nanjing Medical University. The diagnosis and staging of gastric cancer was performed according to the AJCC (TNM) Staging System. 10.1371/journal.pone.0063588.t001Table 1 Clinical Characteristics of 60 GC patients. Patient Demographics IL-26 positive IL-26 negative P valuea,b Age, y (range) 56(32,85) ND ND Sex 0.606 Male 41 23 18 Female 19 12 7 Main location 0.455 U 34 15 19 ML 26 14 12 Size, cm (range) 0.0015b >5 27 23 4 <5 33 15 18 Depth of invasion 0.095 T1/T2 38 24 14 T3/T4 22 9 13 Lauren classification 0.726 Intestinal 28 11 17 Diffuse 32 14 18 Lymph node metastasis 0.316 Positive 39 22 17 Negative 21 9 12 H.pylori infection 0.004c Positive 49 39 10 Negative 11 4 7 Stage 0.766 I 12 7 5 II 15 9 6 III 13 6 7 IV 20 9 11 ND = Not Determined. a By Chi-square test. b P<0.05. c P<0.01. Quantitative Real-time PCR Reverse transcription reactions were performed using the SuperScript First-Strand Synthesis System (Invitrogen, CA) after treatment of RNA templates with DNase I to avoid genomic DNA contamination. To determine the relative level of cDNA in the reverse transcribed samples, real-time PCR was performed with the Roche LightCycler480 (Roche Diagnostics IN, USA) using primers for IL-26 as follows: forward, AAGCAACGATTCCAGAAGACC and reverse, AAGTCCTCCACAAAGCGTATTTT, with an amplified length of 175 bp. GAPDH was used as a control with the following primers: forward, AAGGTGAAGGTCGGAGTCAAC; reverse, GGGGTCATTGATGGCAACAATA; amplified length, 102 bp. The real-time PCR reaction was performed according to the instructions included in the SYBR® Premix Ex Taq™ kit (Takara, Japan). Data were normalized to the GAPDH levels in the samples. ELISA IL-26 concentration in the serum of GC patients and healthy controls was measured using commercially available sandwich ELISA kits (Biotang Inc., MA ). Western Blot Analysis Proteins were extracted from MKN45, SGC7901 and their STAT1 and STAT3 siRNA modified cell lines, and quantified using a protein assay (Bio-Rad Laboratories, CA). Protein samples (30 µg) were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed using antibodies against IL-26, IL-20R1, IL-10R2, p-STAT3(S727), STAT3, p-STAT1(Y701), STAT1, Bcl-2, Bcl-xl, c-Myc and α-Tubulin (all purchased from Abcam, MN). The results were visualized with a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system, Thermo Scientific, IL) and exposure to autoradiography film (Kodak XAR film). Immunohistochemistry (IHC) Tissues were collected and fixed in 4% paraformaldehyde overnight at 4°C, processed, and cut into 5 µm thick sections. Immunohistochemical staining of sections was performed with antibodies against IL-26, IL-20R1, p-STAT3(S727), and p-STAT1(Y701) using techniques described previously [16]. BrdU Cell Proliferation Assay Cultured cells were plated at a density of 1×104 cells/well in 96-well plates. The cell proliferation at 0, 12, 24, 48, 60 and 72 h was evaluated using the BrdU cell proliferation assay kit. BrdU solution (Cell signaling Technology, MA) was added to each well according to the manufacturer’s instructions for 12 h. The cell proliferation rate was determined by measuring the absorbance at 450 nm using a computer controlled microplate-reader. Isolation and Culture of Human GC Infiltrated Leukocytes Fresh tumor tissues were washed twice in RPMI 1640. Fatty, connective, and necrotic tissue was removed. Tissues were minced into 1–2 mm pieces in RPMI 1640, transferred into 15 or 50 ml conical tubes, and incubated with triple enzyme digestion medium containing DNase (30 U/mL), hyaluronidase (0.1 mg/mL), and collagenase (1 mg/mL) for 2 h at room temperature with gentle shaking. Tissues were resuspended in 10 mL RPMI 1640 and filtered through a 70-µm cell strainer (BD Pharmigen). Cells trapped by the strainer were placed into individual wells containing 1 mL of T-cell growth medium in a 24-well plate for further detection by flow cytometry. Th17 and NK Cell Isolation, Stimulation, and Flow Cytometric Analysis Th17 cells were obtained by in vitro stimulation of CD4 positive peripheral blood mononuclear cells (PBMCs), which were isolated by flow cytometry under the conditions (20 ng/mL IL-1β, 20 ng/mL IL-6, 20 ng/mL IL-23, 5 ng/mL TGF-β, 5 µg/mL anti–IL-12, and 5 µg/mL anti–IL-4) described previously [17]. NK cells were obtained using an NK cell isolation kit purchased from Miltenyi Biotec (Cat. 130-092-657). Th17 and NK cells were maintained in vitro and stimulated by 100 ng/mL LPS and H. pylori lysates, respectively, and analyzed by intracellular cytokine staining. For intracellular cytokine staining, cells were stimulated at 37°C for 5 h with a Leukocyte Activation Cocktail (BD Pharmingen). Cells were then stained with surface markers, fixed, and permeabilized with IntraPre Reagent (Beckman Coulter), and finally stained with intracellular markers. Data were acquired on a FACSVantage SE and analyzed with CellQuest software. Fluorochrome-conjugated mAbs against IL-26, CD3, CD4, CD8, CD16, CD56 and RORγT were purchased from BD Pharmingen. Cisplatin Induced Apoptosis and Flow Cytometric Analysis The analyzed cells were cultured in complete medium with 1 µg/mL cisplatin for 24 h. Cells were further analyzed by flow cytometry (FACSCalibur™; BD Biosciences, NJ) using a PI/Annexin staining kit (BD Biosciences). siRNA Design and Lentivirus Production and Transduction The siRNAs targeting STAT1 and STAT3 were designed and synthesized by Genscript Co. (Nanjing, China) as follows: siRNA targeting STAT1, GGACAAGGTTATGTGTATA; and siRNA targeting STAT3, GGACATCAGCGGTAAGACC. The synthesized siRNAs were subcloned into the lentiviral vector pLL3.7 by HapI and XhoI (New England Biolab, United Kingdom) together with control siRNA and named pLL3.7-cs, pLL3.7-STAT1-SiRNA and pLL3.7-STAT3-SiRNA. Recombinant lentivirus was generated from 293T cells [16]. The human GC cell lines MKN45 and SGC7901 were purchased from Kaiji Biotech Co (Nanjing, China) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 U/mL penicillin and 100 U/mL streptomycin (Gibco, CA), and transduced with lentivirus using polybrene (8 µg/mL). Statistical Analysis The results are expressed as mean ± SD of at three independent experiments. Comparisons between groups were performed with the Mann-Whitney U test. The correlation of IL-26 expression and various clinical characteristics was analyzed using the Chi-square test. P values <0.05 were considered as statistically significant. Results 1. Increased Activation of IL-26 and Related STAT3 Signaling in Human Gastric Cancer IL-26 expression in human gastric cancer was examined in 60 fresh tumor tissues and paired adjacent normal stomach tissues from 60 GC patients. Real-time PCR analysis showed significant over-expression of IL-26 mRNA in human GC compared to normal gastric tissues (P = 0.004, by Mann-Whitney U test) (Figure 1A). Serum IL-26 levels were also significantly higher in GC patients than in healthy controls (P = 0.0064, by Mann-Whitney U test) (Figure 1B). Furthermore, analysis of IL-26 protein expression in 60 paraffin embedded tissue samples showed positive expression in 47 GC samples, and weakly positive (n = 14) or negative (n = 46) expression in adjacent normal tissues. The IL-26 positive cells were mainly located in non-parenchymal tissues, which may consist of immune cells surrounding and infiltrating into cancer or normal gastric tissues. Furthermore, quantification of the IHC staining results with Image-pro plus (ver 5.0) in five random fields per slide showed that IL-26 expression was significantly higher in GC tissues than in adjacent normal tissues (P = 0.0087, by Mann-Whitney U test). Detection and quantification of the expression of IL-20R1, a receptor of IL-26, showed that it was expressed at significantly higher levels in human GC than in normal tissues (P = 0.0041, by Mann-Whitney U test). The activation status of STAT1 and STAT3, which are important downstream signaling factors of IL-26, was investigated by IHC using specific antibodies against the phosphorylated residues. A significant increase of p-STAT3 (S727) was detected in GC tissues, while no statistically significant differences in p-STAT1 (Y701) levels were detected between human GC tissues and normal tissues (P = 0.0094 for STAT3 and P = 0.392 for STAT1, by Mann-Whitney U test) (Figure 1C, D). 10.1371/journal.pone.0063588.g001Figure 1 IL-26 and related STAT3 signaling is activated in human gastric cancer cells. A: IL-26 mRNA expression in human gastric cancer (GC) (n = 60) and adjacent normal tissues (n = 60) detected by Real-time PCR. Data in A represent mRNA expression relative to that of GAPDH, expressed as the mean ± SD. B: Detection of IL-26 in human sera of healthy controls (n = 30) and GC patients (n = 60) by ELISA. C: Representative image of the expression and distribution of IL-26, IL-20R1 p-STAT3(S727) and p-STAT1(Y701) in human GC tissues, normal stomach tissues and negative controls stained with isotype IgG. The micrographs at higher magnification show the specific localization of the different markers. D. Average integrated optical density (IOD) was obtained by analyzing five fields for each slide evaluated by image-pro plus software (ver5.0) for IHC staining of IL-26, IL-20R1 p-STAT3(S727) and p-STAT1(Y701) in human GC and normal gastric tissues. Statistical analyses in figures A, B and D were performed using the Mann-Whitney U test. Analysis of the correlation between IL-26 expression and clinicopathological features showed statistically significant associations between IL-26 expression and tumor size and H. pylori infection (Table 1). 2. Th17 and NK Cells are the Main Cellular Sources of IL-26 in Human Gastric Cancer The cellular sources of IL-26 are primary T cells, natural killer (NK) cells and T cell clones after stimulation with specific antigens or mitogenic lectins [14]. Therefore, to define the source of IL-26 in human GC, tumor infiltrating leukocytes (TILs) were isolated from 20 fresh human GC tissue samples and co-stained with different markers. IL-26 expression was significantly higher in CD3 positive cells (T cells) from human GC TILs than in T cells derived from peripheral blood mono-nuclear cells (PBMCs) (16.52±9.32% for T-TILs vs. 3.78±2.91% for T-PBMCs, P<0.0001) which was consistent with the results of real-time PCR and IHC (Figure 2a1, a2). Among the total number of T cells, 13.5±3.71% of CD4 positive T cells expressed IL-26 while only 3.21±2.61% of CD8 T cells were positive for IL-26 (Figure 2a3, a4). Furthermore, co-staining of IL-26 with CD4 and RORγt, a marker for Th17 cells, was observed, with 53.21±16.82% of Th17 cells expressing IL-26 in human gastric cancer samples (Figure 2a5, a6). The expression of IL-26 in NK cells, another frequently reported cellular source of IL-26, was analyzed by measuring the number of CD3-,CD16+,CD56+ cells co-staining with IL-26, which showed that 43.81±19.44% of total NK cells secrete IL-26 (Figure 2a7,a8). Taken together, our results indicated that the main cellular sources of IL-26 in human GC were Th17 and NK cells and the percentage of each component have been summarized in a pie chart (Figure 2a9 and a10). 10.1371/journal.pone.0063588.g002Figure 2 Th17 and NK cells are the main sources of IL-26 in human gastric cancer. a1–a8: A representative case of IL-26 expression in human gastric cancer (GC) tumor infiltrating leukocytes (n = 20) detected by flow cytometry after co-staining with antibodies against CD4, CD8, RORγT, and CD3+CD16+CD56. a9. Comparison of the percentage of IL-26 positive cells in CD3 positive T cells between PBMCs as controls, CD3, CD4, CD8 positive, Th17 and NK cells derived from GC tissues. The experiment was performed in triplicate. a10: A pie chart for the percentage of various components contribute to IL-26 production. b1–b4: Representative CD4+ T cells, in vitro stimulated Th17 cells and isolated NK cells examined by flow cytometry. c1–c8: Flow cytometric analysis of IL-26 expression in Th17 and NK cells stimulated with LPS and H.Pylori lysates. c9: Comparison of IL-26 expression in each group. The experiments were performed in triplicate. Statistical analyses in figure a9 and c9 were performed using the Mann-Whitney U test and compared to the control group. P<0.01. To further investigate the secretion of IL-26 in Th17 and NK cells, PBMCs were collected from 20 healthy volunteers and stimulated under Th17 polarizing conditions. The other main source of IL-26, NK cells, was obtained from PBMCs using a commercial human NK isolation kit. The characteristics of induced or isolated Th17 and NK cells were verified by intracellular cytokine staining and further analyzed by flow cytometry (Figure 2b1, b2). Stimulation of Th17 and NK cells with LPS and H.Pylori lysates resulted in a significant increase in the secretion of IL-26, indicating that IL-26 is a vital cytokine response in gastric cancer (Figure 2C). 3. In vitro Proliferative and Anti-apoptotic Effects of IL-26 The effect of IL-26 was further investigated with a series of in vitro studies performed in two human GC cell lines, MKN45 and SGC-7901. The effect of IL-26 on cell proliferation was assessed with the BrdU incooperation assay in cells treated with different concentrations of IL-26 (1, 10 and 50 ng/mL). No significant differences in cell proliferation were detected between untreated cells and those treated with 1 ng/mL IL-26. However, the rate of cell proliferation increased significantly in response to 10 and 50 ng/mL IL-26 compared to that in untreated cells (Figure 3A). Furthermore, assessment of the anti-apoptotic effect of IL-26 by PI-AnnexinV co-staining in cells treated with the chemotherapy drug cisplatin for various time spots showed that IL-26 had a significant anti-apoptotic effect even at a dose of 1 ng/mL. The anti-apoptotic effect increased with increasing concentrations of IL-26 to 10 and 50 ng/mL (Figure 3B1, B2). These results indicated that IL-26 has proliferative and anti-apoptotic effects on human GC cell lines, which partially explains the association between IL-26 expression and different clinical characteristics observed in clinical studies. However, the underlying molecular mechanism needs to be analyzed further. 10.1371/journal.pone.0063588.g003Figure 3 Proliferative and anti-apoptotic effects of IL-26 in human gastric cancer cell lines in vitro. A: Growth curve of the gastric cancer (GC) cell lines MKN45 and SGC7901 in the presence or absence of IL-26 at the indicated concentrations as determined by the BrdU cooperation assay. The experiment was performed in triplicate. B1: Analysis of cisplatin (1 µg/ml) induced apoptosis in MKN45 and SGC7901 cells and in cells treated with the indicated concentrations of IL-26 for 24 h. Cells were analyzed by PI-Annexin V co-staining. Untreated MKN45 and SGC7901 cells were used as controls. B2: Comparison of the percentage of apoptotic cells in various time spot in each group (experiments were performed in triplicate). Statistical analyses in figures A and B2 were performed with Two way ANOVA and compared to the control group. P<0.01. 4. The Proliferative and Anti-apoptotic Effects of IL-26 are Associated with STAT3 Activation Previous studies have shown that binding of IL-26 to its receptor complex can induce rapid activation of STAT3 and to a lesser degree, STAT1 [12]. Therefore, we first determined the expression of the IL-26 receptor complex (IL-20R1 and IL-10R2) in MKN45 and SGC7901 cells and verified that both receptors were present in GC cell lines to confirm that transduction of IL-26 signals was possible (Figure 4A). We then examined the activation of STAT1 and STAT3 signaling and showed that phosphorylation of the S727 residue of STAT3 and Y701 of STAT1 increased in response to 10 ng/mL IL-26. IFN-γ (10 ng/mL) and IL-6 (30 ng/mL) were used as activation controls for STAT1 and STAT3, respectively (Figure 4B). Based on previous studies showing that STAT1 and STAT3 signaling pathways have opposite effects on tumorigenesis [18], we investigated downstream targets of STAT1 and STAT3 to further elucidate the mechanisms underlying the proliferative and pro-survival effects of IL-26 mediated by STAT1 and STAT3 signaling in GC cell lines. Our results showed that IL-26 stimulation upregulated the expression of Bcl-2, Bcl-xl and c-myc, suggesting that IL-26 has a stronger effect on STAT3 activation than on STAT1 signaling directly or indirectly (Figure 4B). This result also contributes to our understanding of the pro-survival and proliferative effect of IL-26 in human GC cell lines. 10.1371/journal.pone.0063588.g004Figure 4 The proliferative and anti-apoptotic effects of IL-26 are mediated by STAT3 activation. A: Expression of IL-20R1 and IL-10R2 in MKN45, SGC7901, gastric cancer and normal gastric tissue detected by western-blotting. B: MKN45 and SGC7901 cells were stimulated or not with IL-26 (10 ng/ml), IL-6 (30 ng/ml) and IFN-γ (10 ng/ml), proteins were extracted, and analyzed by western blotting for STAT3 and STAT1 activation (phosphorylated S727 for STAT3 and Y701 for STAT1) and downstream protein expression of Bcl-2, Bcl-xl and c-myc. 5. The Tumor Promoting Effect of IL-26 is Dependent on the STAT1/STAT3 Balance The effect of IL-26 on the balance between STAT1 and STAT3 activation was investigated using lentiviral mediated siRNA silencing of STAT1 and STAT3 in MKN45 and SGC-7901 cells. The corresponding siRNAs effectively down-regulated the expression of STAT1 and STAT3 in both cell lines (Figure 5A). Cell proliferation was assessed by BrdU cooperation assay in control siRNA transfected cells and STAT1 and STAT3 siRNA treated GC cell lines grown in media containing 10 ng/mL IL-26. Knockdown of STAT3 expression significantly inhibited the growth of MKN45 and SGC-7901 cells, whereas STAT1 knockdown had no effect on cell proliferation (Figure 5B1, B2). Cisplatin induced apoptosis was assessed by PI-Annexin V co-staining in the same cells maintained in media with 10 ng/mL IL-26, and the results showed a significant decrease in the pro-survival effect of IL-26 in STAT3 knockdown cells, whereas no statistically significant changes in the rate of apoptosis were detected in response to STAT1 silencing (Figure 5C1, C2). These results indicated that IL-26 has pro-survival and proliferative effects on human GC cell lines mediated at least partially by STAT1/STAT3 signaling. 10.1371/journal.pone.0063588.g005Figure 5 The tumor promoting effect of IL-26 is dependent on STAT1/STAT3 balance. A: Expression of STAT1 and STAT3 in MKN45 and SGC7901 cells transfected with control siRNA (indicated as MKN45cs and SGC7901cs) or specific siRNAs targeting STAT1 and STAT3 as determined by western blotting. B: Cell growth of MKN45cs and SGC7901cs and STAT1 and STAT3 siRNA treated cells as determined by BrdU cooperation assay. Experiments were performed in triplicate. C1: Cisplatin (1 µg/ml) induced apoptosis of MKN45cs, SGC7901cs and STAT1 and STAT3 siRNA treated cells as determined by PI-Annexin V co-staining. B2: Comparison of the percentage of apoptosis in each group (experiments performed in triplicate). Statistical analyses in figures B and c2 were performed with the Mann-Whitney U test and compared to the control group. **P<0.01. Discussion Previous studies showed that IL-26 is co-expressed with another important IL-10-related cytokine, IL-22, which is also secreted by Th17 cells [19], [20]. However, the expression and the role of IL-26 in human cancer have not been investigated in detail. Here, we examined for the first time the expression of IL-26 in human GC tissues and showed that IL-26 and related molecules such as IL-20R1 are overexpressed in human GC tissues. IL-26 is co-expressed with IFN-γ and IL-22 in human Th1 clones, but not in Th2 clones. Furthermore, IL-26 is co-expressed with IL-17 and IL-22 by Th17 cells, an important subset of CD4+ T-helper cells that is distinct from Th1 and Th2 cells [12], [21]. In the present study, IL-26 was found to be produced primarily by CD4 positive T helper cells in human GC, among which almost half of the Th17 cells secreted IL-26. We examined another possible cellular source, NK cells, which are reported to be related to tumor volume and dissemination in human GC [22], [23] and showed that NK cells were also important sources of IL-26 production. Our results are supported by a recent study in which a novel subset of CD56+ NKp44+ NK cells was found to co-expresses IL-22 and IL-26, especially in response to treatment with IL-23 [24]. Hughes et al. described a different subset of immature NK cells that do not express CD56 or NKp44 but express CD117 and CD161 and constitutively express IL-22 and IL-26 [25]. Moreover, our results showed for the first time that IL-26 promotes the proliferation of human GC cells by affecting the balance of STAT1 and STAT3 activation, thus providing new evidence of the relationship between NK cells and tumor volume in human GC. IL-26 has been reported to signal via a heterodimeric receptor complex composed of the IL-20R1 and IL-10R2 chains. IL-20R1 functions as the specific ligand-binding chain for IL-26, and IL-10R2 functions as an essential second chain to complete the assembly of the active receptor complex. Neutralizing antibodies against either the IL-20R1 or IL-10R2 chains are capable of blocking IL-26 signaling. Once fully assembled, the receptor complex undergoes a conformational change(s) that induces the activation of the receptor-associated Janus tyrosine kinases, Jak1 and Tyk2, and the subsequent transient docking and phosphorylation of the STAT proteins, STAT1 and STAT3 [26], [27]. As one of the downstream signaling factors of IL-26, STAT3 is most often associated with tumorigenesis and it is also considered as an oncogene. STAT3, which is considered a point of convergence for numerous oncogenic signaling pathways, is constitutively activated both in tumor cells and in immune cells in the tumor microenvironment [28], [29], [30]. In particular, STAT3 activation has been reported in nearly 70% of solid and hematological tumors, including multiple myeloma, several lymphomas and leukemia, breast cancer, head and neck cancer, prostate cancer, ovarian carcinoma, melanoma, renal carcinoma, colorectal carcinoma and thymic epithelial tumors [31]. STAT3 is known to promote cell proliferation and angiogenesis and play a role in tumor immune-escape, but it also impairs the invasiveness and metastatic potential of tumors. Our results showed that STAT3 is activated in human GC tissues and its activation may be associated with excessive IL-26 secretion. On the other hand, no significant differences were detected in the other downstream target of IL-26, STAT1, between tumor and adjacent normal tissues. STAT1 and STAT3 are thought to play opposite roles in tumorigenesis [18]. STAT1 has a complex array of functions in both tumor cells and the immune system and is usually considered as a tumor suppressor because of its role in growth inhibition and apoptosis promotion [32]. In summary, we showed that IL-26 promotes cell growth and prevents apoptosis of human gastric cancer cells by modulating the balance of STAT1/STAT3 signaling, indicating that IL-26 may be a valuable prognostic indicator and therapeutic target in gastric cancer patients. ==== Refs References 1 Ott K , Lordick F , Blank S , Buchler M (2011 ) Gastric cancer: surgery in 2011 . Langenbecks Arch Surg 396 : 743 –758 .21234760 2 Backert S , Clyne M , Tegtmeyer N (2011 ) Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori . Cell Commun Signal 9 : 28 .22044679 3 Asaoka Y , Ikenoue T , Koike K (2011 ) New targeted therapies for gastric cancer . Expert Opin Investig Drugs 20 : 595 –604 . 4 Watanabe M, Kato J, Inoue I, Yoshimura N, Yoshida T, et al.. (2012) Development of gastric cancer in nonatrophic stomach with highly active inflammation identified by serum levels of pepsinogen and Helicobacter pylori antibody together with endoscopic rugal hyperplastic gastritis. Int J Cancer. 5 Weyand CM , Younge BR , Goronzy JJ (2011 ) IFN-gamma and IL-17: the two faces of T-cell pathology in giant cell arteritis . Curr Opin Rheumatol 23 : 43 –49 .20827207 6 Ghoreschi K , Laurence A , Yang XP , Hirahara K , O'Shea JJ (2011 ) T helper 17 cell heterogeneity and pathogenicity in autoimmune disease . Trends Immunol 32 : 395 –401 .21782512 7 Hahn JN , Falck VG , Jirik FR (2011 ) Smad4 deficiency in T cells leads to the Th17-associated development of premalignant gastroduodenal lesions in mice . J Clin Invest 121 : 4030 –4042 .21881210 8 Arnold IC , Dehzad N , Reuter S , Martin H , Becher B , et al (2011 ) Helicobacter pylori infection prevents allergic asthma in mouse models through the induction of regulatory T cells . J Clin Invest 121 : 3088 –3093 .21737881 9 Zhang B , Rong G , Wei H , Zhang M , Bi J , et al (2008 ) The prevalence of Th17 cells in patients with gastric cancer . Biochem Biophys Res Commun 374 : 533 –537 .18655770 10 Caruso R , Pallone F , Monteleone G (2007 ) Emerging role of IL-23/IL-17 axis in H pylori-associated pathology . World J Gastroenterol 13 : 5547 –5551 .17948927 11 Knappe A , Hor S , Wittmann S , Fickenscher H (2000 ) Induction of a novel cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri . J Virol 74 : 3881 –3887 .10729163 12 Donnelly RP , Sheikh F , Dickensheets H , Savan R , Young HA , et al (2010 ) Interleukin-26: an IL-10-related cytokine produced by Th17 cells . Cytokine Growth Factor Rev 21 : 393 –401 .20947410 13 Commins S , Steinke JW , Borish L (2008 ) The extended IL-10 superfamily: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29 . J Allergy Clin Immunol 121 : 1108 –1111 .18405958 14 Fickenscher H , Pirzer H (2004 ) Interleukin-26 . Int Immunopharmacol 4 : 609 –613 .15120646 15 Conti P , Kempuraj D , Frydas S , Kandere K , Boucher W , et al (2003 ) IL-10 subfamily members: IL-19, IL-20, IL-22, IL-24 and IL-26 . Immunol Lett 88 : 171 –174 .12941475 16 Jiang R , Xia Y , Li J , Deng L , Zhao L , et al (2010 ) High expression levels of IKKalpha and IKKbeta are necessary for the malignant properties of liver cancer . Int J Cancer 126 : 1263 –1274 .19728335 17 Eyerich S , Eyerich K , Pennino D , Carbone T , Nasorri F , et al (2009 ) Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling . J Clin Invest 119 : 3573 –3585 .19920355 18 Yu H , Pardoll D , Jove R (2009 ) STATs in cancer inflammation and immunity: a leading role for STAT3 . Nat Rev Cancer 9 : 798 –809 .19851315 19 Aujla SJ , Dubin PJ , Kolls JK (2007 ) Th17 cells and mucosal host defense . Semin Immunol 19 : 377 –382 .18054248 20 Allam JP , Duan Y , Winter J , Stojanovski G , Fronhoffs F , et al (2011 ) Tolerogenic T cells, Th1/Th17 cytokines and TLR2/TLR4 expressing dendritic cells predominate the microenvironment within distinct oral mucosal sites . Allergy 66 : 532 –539 .21087216 21 Sabat R (2010 ) IL-10 family of cytokines . Cytokine Growth Factor Rev 21 : 315 –324 .21112807 22 Takeuchi H , Maehara Y , Tokunaga E , Koga T , Kakeji Y , et al (2001 ) Prognostic significance of natural killer cell activity in patients with gastric carcinoma: a multivariate analysis . Am J Gastroenterol 96 : 574 –578 .11232710 23 Ishigami S , Natsugoe S , Tokuda K , Nakajo A , Che X , et al (2000 ) Prognostic value of intratumoral natural killer cells in gastric carcinoma . Cancer 88 : 577 –583 .10649250 24 Cella M , Fuchs A , Vermi W , Facchetti F , Otero K , et al (2009 ) A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity . Nature 457 : 722 –725 .18978771 25 Hughes T , Becknell B , McClory S , Briercheck E , Freud AG , et al (2009 ) Stage 3 immature human natural killer cells found in secondary lymphoid tissue constitutively and selectively express the TH 17 cytokine interleukin-22 . Blood 113 : 4008 –4010 .19244159 26 Sheikh F , Baurin VV , Lewis-Antes A , Shah NK , Smirnov SV , et al (2004 ) Cutting edge: IL-26 signals through a novel receptor complex composed of IL-20 receptor 1 and IL-10 receptor 2 . J Immunol 172 : 2006 –2010 .14764663 27 Hor S , Pirzer H , Dumoutier L , Bauer F , Wittmann S , et al (2004 ) The T-cell lymphokine interleukin-26 targets epithelial cells through the interleukin-20 receptor 1 and interleukin-10 receptor 2 chains . J Biol Chem 279 : 33343 –33351 .15178681 28 Oshima H , Oshima M (2012 ) The inflammatory network in the gastrointestinal tumor microenvironment: lessons from mouse models . J Gastroenterol 47 : 97 –106 .22218775 29 Kohanbash G , Okada H (2012 ) MicroRNAs and STAT interplay . Semin Cancer Biol 22 : 70 –75 .22210182 30 Fitzpatrick LR (2012 ) Novel Pharmacological Approaches for Inflammatory Bowel Disease: Targeting Key Intracellular Pathways and the IL-23/IL-17 Axis . Int J Inflam 2012 : 389404 .22506136 31 He G , Karin M (2011 ) NF-kappaB and STAT3 - key players in liver inflammation and cancer . Cell Res 21 : 159 –168 .21187858 32 Stark GR , Darnell JE (2012 ) The JAK-STAT pathway at twenty . Immunity 36 : 503 –514 .22520844	23704922	PMC3660585	CC BY	2021-01-05 17:27:49	yes	PLoS One. 2013 May 21; 8(5):e63588
==== Front Mol CancerMol. CancerMolecular Cancer1476-4598BioMed Central 1476-4598-12-342363484310.1186/1476-4598-12-34ResearchExtranuclear ERα is associated with regression of T47D PKCα-overexpressing, tamoxifen-resistant breast cancer Perez White Bethany 1bethanyelena@gmail.comMolloy Mary Ellen 1mmollo2@uic.eduZhao Huiping 1hpzhao58@uic.eduZhang Yiyun 2yiyunzhang@yahoo.comTonetti Debra A 1dtonetti@uic.edu1 Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60611, USA2 Current address: Department of Medicine, Harvard Medical School, Boston, MA 02115, USA2013 1 5 2013 12 34 34 14 8 2012 26 4 2013 Copyright © 2013 Perez White et al.; licensee BioMed Central Ltd.2013Perez White et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCα)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCα preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17β-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCα tumor regression requires extranuclear ERα and interaction with the extracellular matrix. Methods T47D:A18/PKCα cells were grown in vitro using two-dimensional (2D) cell culture, three-dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERα) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERα with caveolin-1. Results We report that although T47D:A18/PKCα cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERα rapidly translocates to extranuclear sites during T47D:A18/PKCα tumor regression in response to both raloxifene and E2, whereas ERα is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERα to the nucleus. T47D:A18/neo tumors that do not overexpress PKCα maintain ERα in the nucleus during tamoxifen-mediated regression. An association between ERα and caveolin-1 increases in tumors regressing in response to E2. Conclusions Extranuclear ERα plays a role in the regression of PKCα-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERα in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCα-expressing tumors encountered in the clinic. Breast cancerPKCαExtranuclear ERαTamoxifenRaloxifene ==== Body Introduction Patients with estrogen receptor α (ERα)-positive breast cancer are candidates for treatment with endocrine therapies such as the selective estrogen receptor modulator (SERM) tamoxifen (TAM), aromatase inhibitors (AIs) letrozole, anastrozole, or exemestane or the selective estrogen receptor downregulator (SERD), fulvestrant. However, both de novo and acquired endocrine resistance represent a significant clinical problem. Mechanisms of endocrine resistance include activation of growth factor signaling and downstream pathway activation including phosphatidyl inositol 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) (reviewed in [1]). Numerous reports from our laboratory and others suggest that activation of protein kinase C (PKC) signaling, specifically PKCα, is associated with endocrine resistance in the clinic [2-4]. We developed and previously described a preclinical TAM-resistant model where PKCα is stably overexpressed in the T47D:A18 breast cancer cell line [5]. Under two-dimensional (2D) culture conditions, T47D:A18/PKCα cells exhibit both TAM-resistance and hormone-independence characterized by proliferation in the presence and absence of 17β-estradiol (E2). Paradoxically when T47D:A18/PKCα cells are grown in vivo as xenograft tumors, E2 administration inhibits tumor growth and induces complete tumor regression in established tumors [6,7]. Similarly, we previously reported that the MCF-7 TAM tumor model that exhibits the E2-inhibitory phenotype [8] also overexpresses PKCα [7]. Previous mechanistic studies in our laboratory determined that E2-induced T47D:A18/PKCα tumor regression is dependent upon ERα, increased Fas/FasL–mediated apoptosis and decreased AKT signaling [9]. Moreover, we showed that T47D:A18/PKCα cultured in three-dimensional (3D) Matrigel™ partially recapitulated the in vivo E2-inhibitory effects by inhibiting colony formation. Further, the membrane impermeable E2-BSA conjugate was shown to inhibit T47D:A18/PKCα colony formation in a manner similar to E2, suggesting the potential involvement of a plasma membrane localized ERα [9]. In addition to genomic signaling by nuclear ERα, examples of nongenomic rapid responses of extranuclear ERα in the presence of E2 are abundant in the literature [10-14]. Extranuclear ERα plays an important role in cell proliferation, cell cycle regulation and blockade of cell death by activating MAPK [15,16] and the AKT signaling pathways [17-19] in breast cancer cell lines. There is evidence that extranuclear ERα interacts with several growth factor receptors as a mechanism for endocrine-resistant breast cancer by promoting downstream proliferation and survival signals [20-22]. In the present study we determined that in 2D and 3D cell culture, TAM-resistant T47D:A18/PKCα cells exhibit cross-resistance to raloxifene (RAL). Similar to the paradoxical effects of E2 in this model, RAL induces T47D:A18/PKCα tumor regression. Based on our previous findings showing the dependence of ERα in tumor regression and the involvement of extranuclear ERα in colony inhibition, in this study we determined the subcellular localization of ERα in T47D:A18/PKCα tumors during regression (E2 and RAL) and during proliferation (absence or presence of TAM) using immunofluorescence (IF) confocal microscopy. Interestingly, ERα localizes to the nucleus in tumors proliferating in a hormone-independent manner or in mice treated with TAM, whereas ERα localizes to extranuclear sites in tumors undergoing regression with either E2 or RAL. Withdrawal of RAL treatment results in the resumption of T47D:A18/PKCα tumor growth accompanied by relocalization of ERα back into the nucleus. We further report an association of extranuclear ERα with caveolin-1 suggesting a mechanism whereby ERα may influence growth factor signaling. These findings are in agreement with our previous report that E2-induced tumor regression is accompanied by downregulation of AKT signaling in this model [9]. To our knowledge this is the first study to report an association of extranuclear ERα with tumor regression, as opposed to the activation of growth factor receptor signaling. With the renewed interest in the use of E2 for treatment of endocrine resistant breast cancer [23,24], our model offers a potential inhibitory mechanism involving extranuclear ERα. Results RAL exerts opposite proliferative effects on T47D:A18/PKCα in vitro and in vivo We previously reported that overexpression of PKCα in T47D:A18 cells (T47D:A18/PKCα) results in TAM-resistant and hormone-independent cell growth in 2D culture. When xenografts are established from these cells, tumors are growth-inhibited and completely regress in the presence of E2 [7]. To determine whether these cells also exhibit cross-resistance to RAL, a DNA assay in 2D culture was performed. Whereas the parental T47D:A18/neo cells are E2-dependent and growth inhibited by both 4-hydroxytamoxifen (4-OHT) and RAL (Figure 1A), the TAM-resistant T47D:A18/PKCα cells exhibit cross-resistance to RAL (Figure 1B). When cultured in 3D Matrigel™, T47D:A18/PKCα colony formation is inhibited by E2 as previously reported [9] but grew in the presence of both 4-OHT and RAL (Figure 1C,D). Therefore T47D:A18/PKCα cells display similar cross-resistance to 4-OHT and RAL in 2D and 3D culture. Figure 1 T47D:A18/PKCα cells are resistant to 4-OHT and RAL in 2D and 3D cell culture. DNA and Matrigel™ colony formation assays were performed as described in materials and methods. Cells were grown in the presence of vehicle (DMSO, 0.1%), E2 (10-9M), 4-OHT (10-7M) or RAL (10-7M) with media changes every three days. A. T47D:A18/neo cells. B. T47D:A18/PKCα cells. RFU, relative fluorescence units. C. Quantification of T47D:A18/PKCα colonies. Graphs are representative of at least three independent experiments and error bars represent SEM. P < 0.05 compared to vehicle; by one-way ANOVA followed by Bonferroni’s post-test. D. Photographic representation of T47D:A18/PKCα colonies. Total magnification: 6X. To examine whether T47D:A18/PKCα cells are similarly resistant to RAL in vivo, we bilaterally injected T47D:A18/PKCα cells into the mammary fat pads of ovariectomized athymic mice and began treatment with TAM (1.5 mg/day), low dose RAL (0.5 mg/day) or high dose RAL (1.5 mg/day) (Figure 2A). As expected, T47D:A18/PKCα tumors are TAM-resistant as previously described [7] compared to the TAM and RAL-sensitive T47D:A18/neo tumors (Figure 2C). However, mice receiving the lower dose of RAL (0.5 mg/day), experienced tumor growth until week 5, followed by tumor stabilization and partial regression. Mice receiving the higher dose of RAL (1.5 mg/day) exhibited minimal tumor growth and achieved tumor stabilization by week 3 followed by tumor regression after 10 weeks of treatment (Figure 2A). These results indicate that (1) RAL is capable of inhibiting the growth of T47D:A18/PKCα TAM-resistant tumors and (2) RAL exerts contradictory in vitro and in vivo growth effects on T47D:A18/PKCα cells in a manner similar to E2. The distinction between E2 and RAL activity is that E2 but not RAL inhibits colony formation in 3D culture (Figure 1C, D) [9]. Figure 2 RAL inhibits TAM-resistant T47D:A18/PKCα xenograft tumors. Xenograft tumors were formed as described in materials and methods. Treatments for TAM and RAL were given by oral gavage 5 days/week. A. T47D:A18/PKCα tumors. Mice (10/group) were given TAM (1.5 mg/day) or RAL (0.5 mg/day or 1.5 mg/day). B. LT-TAM-treated T47D:A18/PKCα tumors. Mice (10/group) received TAM (1.5 mg/day) or RAL (1.5 mg/day). C. T47D:A18/neo tumors. Mice (6/group) were given no treatment, E2 capsule (1.0 cm), TAM (1.5 mg/day) or RAL (1.5 mg/day). The dotted line indicates initiation of TAM or RAL treatment following 8 weeks of E2 treatment. Error bars represent SEM. To more closely parallel the clinical situation where TAM is given to patients for 5 years, we created the long-term TAM (LT-TAM) tumor model by serially passaging T47D:A18/PKCα tumors in mice treated with 1.5 mg TAM 5 days/week for 5 years. We then asked whether RAL was capable of causing tumor regression in this LT-TAM tumor model. LT-TAM tumors were established and groups were treated with either 1.5 mg TAM or 1.5 mg RAL per day. During the first 7 weeks of treatment, both the TAM and RAL groups exhibited similar tumor growth. However between weeks 8–10, tumors in the RAL treated group began to regress (Figure 2B). These results suggest that RAL is a potential lead compound as an alternative to E2 for second-line treatment following tumor progression on TAM in those tumors that overexpress PKCα. E2 and RAL induce ERα translocation from the nucleus to extranuclear sites in vivo We previously reported that ERα and the extracellular matrix (ECM) are required for T47D:A18/PKCα tumor regression and that plasma membrane-associated ERα is likely to mediate the inhibitory effects of E2 [9]. To test our hypothesis that extranuclear ERα participates in E2-induced T47D:A18/PKCα tumor regression, we asked whether ERα localization differs in E2 and RAL-induced T47D:A18/PKCα regressing tumors compared with TAM-stimulated T47D:A18/PKCα tumors or E2-stimulated T47D:A18/neo tumors. To address this question, we established T47D:A18/neo and T47D:A18/PKCα tumors in athymic mice (Figures 3A- 3D) and as previously reported, T47D:A18/neo tumors are stimulated by E2 (Figure 3A) and are TAM and RAL-sensitive (Figure 2C), whereas T47D:A18/PKCα tumors are TAM-resistant and hormone-independent (Figure 3B) and regress following E2 treatment (Figures 3C and 3D) [7]. As we report here for the first time, RAL induces T47D:A18/PKCα tumor regression, although the degree of regression with RAL is not as complete as is seen with E2 (Figure 3C). Upon withdrawal of RAL, we observed re-growth of T47D:A18/PKCα tumors. In contrast, no resumption of tumor growth is seen upon discontinuation of E2 treatment for up to 31 weeks (Figure 3D). Since the E2 capsules maintain constant serum E2 levels for only 8–10 weeks, we are confident that the E2 capsule is depleted by week 20 and have confirmed no detectable serum E2 by mass spectrometry at 31 weeks (data not shown). Figure 3 Growth of T47D:A18/neo and T47D:A18/PKCα xenograft tumors. Xenograft tumors were formed as described in materials and methods. A. T47D:A18/neo tumors (NT, 15 mice/group and E2, 3 mice/group). B. T47D:A18/PKCα tumors (10 mice/group). C. T47D:A18/PKCα tumors. Tumors were grown to an average size of 0.5 cm2. Mice were then randomized into NT, RAL or E2 groups (large arrow, 9 mice/group). Two weeks later RAL treatment was stopped (small arrow). D. T47D:A18/PKCα tumors (5 mice/group). Tumors were grown to an average size of 0.3 cm2. Mice were then randomized into NT or E2 groups (arrow). IF confocal microscopy of T47D:A18/neo E2-stimulated tumors and TAM- and RAL-regressing tumors illustrates that ERα is mainly localized in the nucleus (Figure 4A). The T47D:A18/neo no treatment (NT) group is not available for comparison since T47D:A18/neo cells required E2 for tumor growth. Similarly, ERα is located within the nucleus in T47D:A18/PKCα NT and TAM treatment groups. However, ERα is almost completely localized to extranuclear sites in E2- and RAL-induced regressing T47D:A18/PKCα tumors. Interestingly, following withdrawal of RAL (RAL W/D) tumors resume growth and ERα re-localizes to the nucleus. Semi-quantitative analysis of ERα signals from tumor sections showed a significant re-localization from the nucleus to the cytoplasm in E2- and RAL-treated T47D:A18/PKCα tumors compared to NT, TAM or RAL W/D (Figure 4B). ERα translocation to extranuclear sites by E2 was verified with the 1D5 ERα antibody directed towards a different epitope of ERα (Additional file 1). ERα protein levels from each tumor group were also assessed by western blot (Figure 4C). As previously reported, ERα protein expression is elevated in T47D:A18/PKCα tumors even though ER function as determined by ERE-luciferase activity is decreased [5]. The abundance of ERα protein as assessed by western blot is in agreement with the IF image ERα signal intensity (Figures 4A,C). The observed downregulation of ERα protein by E2 and ERα stabilization by antiestrogens is considered classic ERα regulation as previously established [25-28]. Figure 4 ERα localizes to extranuclear sites in E2- and RAL-induced T47D:A18/PKCα regressing tumors. A. Tissue sections were immunostained as described in materials and methods. Images are representative photographs of immunostained tumor sections. Sections were costained for ERα (green) and nuclei (blue). Scale bar = 20 μm. All images were acquired and processed using parameters described in materials and methods. PKCα, T47D:A18/PKCα; neo, T47D:A18/neo. B. Quantification of ERα localization in tumor sections. At least three fields from each tumor were counted. T47D:A18/neo is represented by two individual tumors. Bars representing T47D:A18/PKCα tumors show the mean (± SEM) of three individual tumors. *, P < 0.001 compared to PKCα NT, TAM and RAL W/D by two-way ANOVA. C. Expression of ERα in whole cell tumor lysates. Molecular weights of ERα and β-actin are 67 kDa and 42 kDa, respectively. Values represent β-actin-normalized ERα expression relative to T47D:A18/neo E2-treated tumors. Therefore TAM and RAL which oppositely regulate T47D:A18/PKCα tumor growth, induces differential ERα subcellular localization. Furthermore, T47D:A18/PKCα tumor regression induced by either E2 or RAL is associated with extranuclear ERα. The finding that ERα is localized to the nucleus during RAL and TAM-induced T47D:A18/neo tumor regression suggests that it is not simply regression that triggers ERα to exit from the nucleus, but localization may be influenced by PKCα overexpression. Association of ERα with caveolin-1 ERα does not have a membrane localization sequence thus it does not behave like a transmembrane receptor [29]. Membrane ERα normally exists as a cytoplasmic pool and can be tied to the inner face of the plasma membrane bilayer through binding to the lipid raft protein caveolin-1 [30,31]. To determine whether there is a direct physical interaction between ERα and caveolin-1, we prepared total protein extract from tumors and performed co-immunoprecipitation (co-IP) using an ERα antibody followed by western blot analysis (Figure 5A). As expected, the level of total ERα was lower in tumors from the E2 treatment group. However, immunodetection with a caveolin-1 antibody showed a significant increase in complex formation between ERα and caveolin-1 in T47D:A18/PKCα tumors from the E2 treatment group compared with the T47D:A18/PKCα NT group and the T47D:A18/neo E2 group (Figure 5B). These results indicate that the abundance of the ERα/caveolin-1 complex is increased in response to E2, but not from treatment with TAM or RAL. We conclude that ERα/caveolin-1 complex formation correlates with durable tumor regression produced with E2, but not with transient tumor regression as observed with RAL, nor with proliferating T47D:A18/PKCα tumors (NT, TAM, RAL W/D). This result is consistent with the hypothesis that E2-induced tumor regression is accompanied by ERα exit from the nucleus and association at the plasma membrane, perhaps via caveolin-1. Figure 5 ERα/caveolin-1 complex formation in response to E2, TAM and RAL treatment in T47D:A18/PKCα tumors. A. Representative western blot of co-IP experiments in T47D:A18/PKCα and T47D:A18/neo tumor extracts as detailed in materials and methods. B. Densitometric quantification of three co-IP experiments from three independent tumors for each group. Error bars represent SEM. , P < 0.05 compared to all groups determined by one-way ANOVA followed by Bonferroni’s post-test. ERα localization in the 2D and 3D microenvironment As previously described [9], the ECM is required for the growth inhibitory effect of E2 on T47D:A18/PKCα cells; E2 stimulates T47D:A18/PKCα cells proliferation on 2D cell culture, yet E2 inhibits colony formation in 3D Matrigel™. However we report here that T47D:A18/PKCα cells are resistant to RAL both on 2D and 3D (Figures 1B, C), yet RAL inhibits tumor growth (Figure 2). Therefore we wanted to determine whether extranuclear ERα correlates with inhibition of growth (on 2D and 3D) and/or colony regression. Inhibition of colony formation by E2 in 3D culture is analogous to the in vivo phenotype whereby E2 prevents tumor establishment [7]. However, unlike the in vivo phenotype, E2 is incapable of initiating regression of an established T47D:A18/PKCα colony in Matrigel™. To determine whether extranuclear ERα is a response to E2 and RAL treatment in 3D culture or whether ERα translocation occurs only during regression in tumors, we compared ERα subcellular localization in T47D:A18/neo and T47D:A18/PKCα cells grown in 2D and 3D culture. In 2D culture ERα is both nuclear and cytoplasmic in T47D:A18/neo cells, whereas ERα is mainly nuclear in T47D:A18/PKCα cells following 1 h exposure to E2, 4-OHT or RAL (Additional file 2). These results indicate that ERα localization does not change in T47D:A18/neo and T47D:A18/PKCα following 1 h treatment in 2D culture. To address ERα localization in 3D culture, T47D:A18/neo and T47D:A18/PKCα cells were plated in Matrigel™ under two treatment paradigms. The first paradigm is known to inhibit colony formation in the presence of E2 where cells are plated (as shown in Figure 1C, D) and given continuous treatment for 6 days with media changes every third day. Under these conditions, T47D:A18/neo cells in colonies showed nuclear ERα expression in the E2 treatment group and no expression in vehicle control, 4-OHT or RAL groups and T47D:A18/PKCα colonies had cells with nuclear ERα expression in all groups (Additional file 3). These results indicate that ERα subcellular localization does not change as a result of continuous treatments in 3D culture (Additional file 3). The second paradigm was designed to mimic tumor regression. Colonies were allowed to establish for 10 days when treatments were initiated and continued for either 24 h or 10 days with E2, 4-OHT or RAL. In contrast to E2-induced tumor regression seen in vivo, treating colonies does not cause a decrease in colony number or size (data not shown). Following 24 h treatment of established T47D:A18/neo colonies, there was no ERα expression in the vehicle and E2 treatment groups and sparse staining in the 4-OHT and RAL groups (Additional file 4). Examination of T47D:A18/PKCα colonies under the same conditions, shows strong ERα nuclear staining in the vehicle, 4-OHT and RAL treated groups. However, in the 24 h E2 treatment group, some colonies showed nuclear staining while other colonies showed membrane and/or cytoplasmic staining (Additional file 4). To determine if treating established colonies for a longer period would lead to the complete translocation of ERα from the nucleus to the cytoplasm, we extended treatment for 10 days with media changes every three days before IF staining. Under these conditions, ERα is localized to the nucleus in all groups of T47D:A18/neo colonies as well as T47D:A18/PKCα vehicle control, 4-OHT and RAL groups (Figure 6). However, ERα is completely extranuclear in all cells growing in response to E2. Taken together these findings suggest that ERα localization does not correlate with proliferative response in 2D cell culture nor with inhibition of colony formation in 3D Matrigel™. However, under conditions that mimic tumor regression, T47D:A18/PKCα colonies exhibit complete ERα translocation out of the nucleus in response to E2 after 10 days and this effect is seen as early as 24 h. While E2 administration to established colonies in Matrigel™ induces ERα translocation to extranuclear sites, ERα translocation alone is not sufficient to induce regression likely due to the requirement of additional factors found in the tumor microenvironment, but not in Matrigel™. We also find E2 and RAL exert opposite effects on ERα localization in T47D:A18/PKCα cells plated in 3D Matrigel™, but similar localization in vivo. Figure 6 E2 induces complete relocalization of ERα in established T47D:A18/PKCα colonies after 10 days. A. T47D:A18/neo colonies (neo) and T47D:A18/PKCα colonies (PKCα) colonies were immunostained for ERα (green) and nuclei (blue). All images were acquired and processed using parameters described in materials and methods. Colonies were grown for 10 days then treated for 10 days with vehicle (EtOH, 0.1%), E2 (10-9 M), 4-OHT (10-7 M) or RAL (10-7M). Scale bar = 20 μm. B. Expression of ERα in whole cell colony lysates. Molecular weights of ERα and β-actin are 67 kDa and 42 kDa, respectively. Values represent β-actin-normalized ERα expression relative to T47D:A18/neo E2-treated colonies. Discussion In this paper we have shown by IF confocal microscopy that ERα translocates from the nucleus to the extranuclear space upon E2 and RAL-induced tumor regression in our T47D:A18/PKCα preclinical TAM-resistant model. This model is clinically relevant as evidenced by the reported success of E2 in the clinic [23,24]. We initially associated PKCα expression with TAM resistance [2], and others further identified PKCα as a marker of endocrine resistance and breast cancer aggressiveness [3,4]. Extranuclear ERα was previously reported to play a role in endocrine-resistant breast cancers specifically by interacting with growth factor receptors to activate proliferative and pro-survival signals [20-22]. However we demonstrate here that ERα translocation is associated with tumor regression only in PKCα overexpressing tumors in response to E2 and RAL. Our findings imply that a specific subset of endocrine-resistant breast cancers that express PKCα may be uniquely susceptible to E2 therapy. Although the literature is conflicting regarding the level of PKCα expression in breast cancers compared to the normal breast [32-36], variability in PKCα expression amongst breast cancers and the link to endocrine resistance and tumor aggressiveness is clear. Based on three reports in the literature, the prevalence of PKCα expression in all breast cancers ranges between 28% to as high as 70% [3,4,37]. Even if the lowest estimate of 28% prevalence is the most accurate, this still represents a significant number of patients that may benefit from E2 treatment. There are numerous reports of nongenomic signaling by estrogen in breast cancer cell lines [38,39] and there is evidence that this pathway is upregulated in endocrine resistant breast cancers. Translocation of nuclear ERα to extranuclear sites is reported to be involved in cytoskeletal remodeling, migration and invasion [40] and recently shown to play an important role in breast cancer cell motility and metastasis [41]. High expression of the MTA1 protein is reported to sequester ERα in the cytoplasm and activate MAPK signaling [42], and the same group reported that overexpression of Her-2 causes ERα nuclear to cytoplasmic translocation [43]. Fan et al.[44] showed that long term exposure to TAM causes translocation of ERα from the nucleus to the cytoplasm and enhances the interaction between ERα and EGFR. All of these examples in the literature describe the activation of signaling pathways by extranuclear ERα leading to cancer cell proliferation and survival. However in our study, we present a novel finding that translocation of ERα from the nucleus to extranuclear sites occurs following E2- and RAL-induced T47D:A18/PKCα tumor regression. We previously reported that E2-induced regression is accompanied by apoptosis mediated in part by Fas/FasL and downregulation of the AKT pathway [9]. An additional novel finding is that TAM and RAL elicit opposite growth effects in our T47D:A18/PKCα tumor model. We hypothesize that PKCα, a cytoplasmic protein that translocates to the plasma membrane when activated [45], may physically interact with other growth factor receptors and signaling pathways [46]. A recent publication by Guttierez et al. shows that translocation of ERα to the plasma membrane in response to E2 results in activation of PKCα/ERK 1/2 signaling in anterior pituitary cells, yet PKCα is not responsible for mediating the physical translocation of ERα to the plasma membrane [47]. Src kinase is one of the important molecules of the signalosome complex which plays a critical role in E2-mediated nongenomic signaling [48]. It has been reported in the literature that Her-2 upregulates and activates PKCα through src kinase in Her-2 mediated cancer cell invasion [49]. Longo et al. has shown that a PKCα-src kinase-ERα interaction is critical in the modulation of estrogen responsiveness and the differentiation process in osteoblasts [50]. However, we were unable to detect a physical interaction between PKCα and ERα, Her2 or src in our tumor model. We detected a physical interaction between ERα and caveolin-1 by co-IP (Figures 5A-B). These results suggest that caveolin-1 may be responsible for transporting ERα to the plasma membrane during E2-induced tumor regression. Palmitoylation of ERα is known to be necessary for the physical association with caveolin-1 and in particular palmitoylation of the E domain of ERα at C447 along with nine flanking amino acids are required for association with caveolin-1 [30,31,51,52]. The ERα-caveolin-1 complex in turn facilitates the translocation of the caveolae rafts to the plasma membrane. Caveolin-1 serves as a scaffold protein at the membrane in the recruitment of signaling molecules to form a signalosome complex that can include ERα. Taken together these results suggest that perhaps PKCα is capable of modifying the interaction of ERα and caveolin-1, potentially at the membrane via the proposed signalosome to effect tumor regression. It is interesting to note that ERα/caveolin-1 complex formation correlates with durable tumor regression produced with E2, but not with transient tumor regression as observed with RAL, nor with proliferating T47D:A18/PKCα tumors (NT, TAM, RAL W/D). Although ERα translocation to extranuclear sites does occur in Matrigel™ in response to E2 (Figure 6), colony regression is not initiated perhaps because a component in the tumor microenvironment is also required to initiate the regression signal. As shown in Figures 3C-D, E2-induced tumor regression occurs rapidly and tumors are gone within 2–3 weeks. Matrigel™ results reveal that the translocation of ERα may be an early event as ERα was seen in the membrane and cytoplasm in some colonies at 24 h further illustrating a rapid response to E2 treatment. Our results regarding ERα translocation in the Matrigel™ environment compared with in vivo tumors highlight the importance of the ECM in triggering tumor regression. Since we and others have reported that PKCα expression can be a predictive marker of TAM resistance [2-4] our T47D:A18/PKCα model suggests that detection of extranuclear ERα can be used to monitor therapeutic response in TAM-resistant, PKCα-expressing breast cancers. Unfortunately, extranuclear ERα is not currently measured clinically and although pathologists may observe such staining, it is not reported. A recent report by Welsh et al.[53] with the purpose of testing a panel of ERα-specific antibodies to detect non-nuclear ERα in clinical specimens found the average incidence to be only 1.5%. In an accompanying commentary, Levin points out that while it is possible that the number of breast tumors that express extranuclear ERα may indeed be small, it is also possible that more sensitive techniques are required to detect the very small ERα pools located outside of the nucleus [54]. We offer the possibility that extranuclear ERα may be detected more frequently in PKCα-expressing tumors that are regressing possibly indicating a response to treatment. It remains to be seen whether other techniques will be developed that may improve the detection of extranuclear ERα in clinical specimens. We have previously suggested that PKCα may be used as predictive biomarker for the use of E2 or an E2-like compound to effect tumor regression [9], and in fact the utility of using E2 was demonstrated [23]. We report here that not only E2, but RAL is capable of eliciting T47D:A18/PKCα tumor regression, despite the fact that these tumors are TAM-resistant. Further we have shown that following 5 years of TAM treatment, these tumors are still sensitive to RAL-induced tumor regression (Figure 2B). Although RAL may be considered as a potential treatment for patients with PKCα-expressing breast cancers, RAL is not as durable as E2 to elicit complete tumor regression (Figure 3D). Since RAL has poor bioavailability, we are currently testing a series of benzothiophene analogues in our T47D:A18/PKCα preclinical model for improved tumor inhibitory activity. Conclusions In summary, we report for the first time the involvement of extranuclear ERα in an endocrine resistant-tumor model to be associated with tumor regression and not growth stimulation. Key to this phenomenon may be expression of PKCα, frequently associated with endocrine resistance and a potential biomarker for the use of E2 or RAL-like compounds for the treatment of endocrine-resistant breast cancer. Methods Reagents For in vitro experiments dimethylsulfoxide (DMSO), ethanol, E2, 4-OHT and RAL were obtained from Sigma-Aldrich (St. Louis, MO USA). For in vivo experiments E2 and TAM were obtained from Sigma. RAL (Evista®, Eli Lilly and Company, Indianapolis, IN USA) was purchased from the University of Illinois at Chicago Hospital Pharmacy. Cell culture reagents were obtained from Life Technologies (Carlsbad, CA USA). Tissue cultureware was purchased from Becton-Dickinson (Franklin Lakes, NJ USA). The following antibodies were used: rabbit monoclonal ERα (for tissue and cells, SP1, Lab Vision, Thermo Scientific, Kalamazoo, MI USA), mouse monoclonal ERα (alternative epitope to confirm specificity for tissue, 1D5, N-terminal epitope, Abcam, Cambridge, MA USA), rabbit polyclonal ERα (for colonies, HC20, Santa Cruz Biotechnology, Santa Cruz, CA USA), and mouse monoclonal caveolin-1 (Clone2234, BD Transduction Laboratories, Franklin Lakes, NJ USA). Secondary antibodies included: anti-rabbit Alexa Fluor 488 (Life Technologies, Carlsbad, CA USA), anti-mouse Cy3 (Jackson Immunoresearch Laboratories, West Grove, PA USA) and HRP-cojungated anti-rabbit and anti-mouse (GE Healthcare UK Limited, Buckinghamshire, UK). Cell culture conditions T47D:A18/neo and T47D:A18/PKCα [5] cells were maintained in RPMI 1640 with phenol red supplemented with 10% fetal bovine serum (FBS) and G418 (500 μg/ml) at 37°C, 5% CO2. Prior to experiments cell lines were placed in phenol red-free RPMI 1640 supplemented with 10% stripped FBS (E2-depleted media) for 3 days and maintained in the same manner for the duration of experiments. Cell lines were tested for Mycoplasm contamination on a regular basis (MycoAlert™ Mycoplasm Detection Kit, Lonza Ltd., Rockland, ME, USA). Cell lines were not authenticated by the authors. DNA growth assay Cells were plated at a density of 15,000 cells/well in 24-well plates. Treatment media (vehicle, DMSO [0.1%], E2 [10-9M], 4-OHT [10-7M] or RAL [10-7M]) was added the following day (Day 1) and changed every three days. Growth was determined by incubating cells with Hoechst 33342 cell permeable dye (Life Technologies, Carlsbad, CA USA) for 1 h at 37°C and reading fluorescence at excitation 355 nm/emission 460 nm on a Perkin Elmer Victor3 V (Waltham, MA USA) plate reader. Matrigel™ colony formation assay Treatments (ethanol [0.1%], E2 [10-9M], 4-OHT [10-7M] or RAL [10-7M]) were added to liquefied phenol-red free Matrigel™ matrix (BD Biosciences, Franklin Lakes, NJ USA) and used to coat 6-well plates and solidified at 37°C for 30 min. Cells (5000) were seeded in E2-depleted media containing treatments on top of pre-gelled Matrigel™ and incubated at 37°C with 5% CO2. Treatment media were changed every three days. Colonies were stained with 0.25% crystal violet (Sigma-Aldrich, St. Louis, MO USA) solution for 30 min and then destained with 0.9% saline for 20 min at room temperature. Colony number was determined by counting five 1.0 cm2 areas. Xenograft tumor establishment All procedures involving animals were approved by the Animal Care and Use Committee of the University of Illinois at Chicago according to institutional and national guidelines. T47D:A18/neo and T47D:A18/PKCα tumors were established in 4–6 week old ovariectomized athymic nude mice (Harlan Laboratories) as previously described [7]. LT-TAM tumors were derived by in vivo serial transplantation in the presence of TAM for 5 years. Where indicated, mice were given the following treatments as previously described: E2 (1.0 cm silastic capsule, s.c.), TAM (1.5 mg/day, p.o.), RAL (0.5 mg/day, p.o.), or RAL (1.5 mg/day, p.o.) [55]. Tumor cross-sectional area was determined at least weekly and sometimes daily using digital calipers and calculated using the formula: length/2 × width/2 × π. Mice were euthanized by CO2 inhalation and cervical dislocation. Tumors were immediately excised and either fixed in 10% buffered formalin for paraffin block preparation or snap frozen in liquid nitrogen and stored at −80°C for co-immunoprecipitation and western blot analysis. Tumor IF confocal microscopy and co-localization analysis Tumors sections (4 μm) were prepared from paraffin blocks for IF staining by deparaffinization and rehydration. Antigen retrieval was performed by incubating slides in Tris-EDTA (pH = 9.0) buffer at 90°C and allowed to cool at room temperature for 45 min. Slides were blocked with antibody diluent (DAKO, Carpinteria, CA USA) for 20 min followed by primary antibody at 1:100 in antibody diluent for 1 h at room temperature. Slides were incubated with fluorescence-conjugated secondary antibodies at 1:100 in antibody diluent for 45 min at room temperature followed by 4’, 6-diamidino-2-phenylindole (DAPI) (1 μg/mL), DAKO, Carpinteria, CA USA) for 15 min and mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA USA). Confocal microscopy was performed with a Zeiss LSM 510 microscope (Carl Zeiss, Incorporated, North America, Thornwood, NY USA). The objective used was a C-Apochromat 63X with a numerical aperture of 1.2. Image acquisition scaling was X: 0.14 μm and Y: 0.14 μm and stack size was X: 142.86 and Y: 142.86, these two parameters were kept constant across samples. Pinholes and laser intensities were kept constant for each wavelength (green: λ = 488 nm, laser = 15%, pinhole = 228 μm and blue: λ = 405 nm, laser = 5%, pinhole 194 μm) across all samples. Images were modified following acquisition using the Zeiss LSM Image Browser by similarly enlarging images 2X and increasing the brightness and contrast by 10%. Co-IP and western blot Tumors were ground into a fine powder in liquid nitrogen and resuspended in cell lysis buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, with protease [Sigma, St. Louis, MO] and phosphatase [Calbiochem, Bilerica, MA] inhibitor cocktails) and homogenized using a Polytron hand-held homogenizer (Fisher Scientific, Pittsburgh, PA USA). Protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA USA). Equal amounts of total tumor extract (500 μg) were immunoprecipitated by rotating for 2 hr at 4°C with antibody followed by overnight rotation with protein-A Dynabeads (Life Technologies, Carlsbad, CA), at 4°C. Samples were washed and boiled for 10 min then eluted from beads with sample buffer containing 2-mercaptoethanol (Sigma, St. Louis, MO USA). Samples were subjected to 8% SDS-PAGE, followed by western blot with respective primary and secondary antibodies. Proteins were detected by chemiluminescence using a Chemi Doc Gel Documentation System (Bio-Rad Laboratories, Hercules, CA USA). Cell IF microscopy Cells were seeded in phenol red-containing media onto Lab-Tek II 4-well chamber slides (Millipore, Billerica, MA) at a density of 3 × 104 cells/well. The following day cells were placed in E2-depleted media for 3 days then given treatment media (DMSO [0.1%], E2 [10-9M], 4-OHT [10-7M] or RAL [10-7M]). For IF, cells were fixed in 100% methanol overnight at −20°C and stained as described above for tissue sections. Cells were imaged using Zeiss Axiovision Observer D1 microscope (Carl Zeiss, LLC, Thornwood, NY USA). Colony IF microscopy Colonies were formed by ding cells in Matrigel™ as described above and treated with DMSO (0.1%), E2 (10-9M), 4-OHT (10-7M) or RAL (10-7M). Colonies were extracted from the Matrigel™ by adding ice-cold PBS-EDTA to the rinsed and aspirated wells. Gel was lifted from the bottom of the well with a cell scraper and plates were shaken gently on ice. Colonies were then transferred to a conical tube and shaken on ice for an additional 30 min until Matrigel™ was completely dissolved, collected by centrifugation at 115g for 2 min and pipetted onto a slide. Slides were then fixed in ice cold methanol and stored at −80°C until staining (as described above). Confocal microscopy was performed with a Zeiss LSM 510 microscope. The objective used was a C-Apochromat 63X with a numerical aperture of 1.2. Image acquisition scaling was X: 0.14 μm and Y: 0.14 μm and stack size was X:142.86 and Y: 142.86, these two parameters were kept constant across samples. Pinholes and laser intensities were kept constant for each wavelength (green: λ = 488 nm, laser = 10%, pinhole = 200 μm and blue: λ = 405 nm, laser = 13%, pinhole 92 μm) across all samples. Images were modified following acquisition using the Zeiss LSM Image Browser by similarly enlarging images 2X and increasing the brightness and contrast by 10%. Statistical analysis The specific statistical test applied to the data is described in the figure legends. All of the statistics on the data were performed using GraphPad Prism 5.02 Software (La Jolla, CA USA). Abbreviations 4-OHT: 4-Hydroxytamoxifen; AI: Aromatase inhibitor; co-IP: Co-immunoprecipitation; DAPI: 4’, 6-Diamidino-2-phenylindole; DMSO: Dimethylsulfoxide; E2: 17β-Estradiol; ERα: Estrogen receptor alpha; ECM: Extracellular matrix; IF: Immunofluorescence; LT-TAM: Long-term TAM; MAPK: Mitogen activated protein kinase; PI3K: Phosphatidylinositol 3’kinase; PKCα: Protein kinase C alpha; RAL: Raloxifene; SERM: Selective estrogen receptor modulator; SERD: Selective estrogen receptor downregulator; TAM: Tamoxifen. Competing interests The authors declare that they have no competing interests. Authors’ contributions BPW and MEM contributed equally to this study and contributed to writing portions of the manuscript. BPW made figures and designed layout. All authors contributed to xenograft experiments, HZ and YZ developed the LTTAM tumor model, HZ performed IF staining and microscopy on cell lines, BPW and MEM performed IF and confocal microscopy on tumor sections and colonies. DAT conceived of the study and wrote the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1 ERα is localized to extranuclear sites in E2-regressing tumors with an antibody directed to an alternative epitope. Tissue sections were immunostained as described in materials and methods. Images are representative photographs of immunostained tumor sections. Sections were costained for ERα (green) and nuclei (blue). Scale bar = 20 μm. Click here for file Additional file 2 ERα localization does not change in cells grown in 2D culture. T47D:A18/neo (neo) and T47D:A18/PKCα (PKCα) cells were immunostained for ERα (green) and nuclei (blue) as detailed in materials and methods. Cells were treated (Vehicle [EtOH 0.1%], E2 [10-9 M], 4-OHT [10-7 M] or RAL [10-7M]) for 1 h. Scale bar = 50 μm. Click here for file Additional file 3 Continuous E2 treatment inhibits colony formation but does not induce extranuclear ERα in T47D:A18/PKCα cells. T47D:A18/neo colonies (neo) and T47D:A18/PKCα colonies (PKCα) colonies were immunostained for ERα (green) and nuclei (blue) as detailed in materials and methods. Colonies were given treatment upon plating with vehicle (EtOH, 0.1%), E2 (10-9 M), 4-OHT (10-7 M) or RAL (10-7M) and were treated continuously for 6 days. Scale bar = 20 μm. Click here for file Additional file 4 E2 treatment in established T47D:A18/PKCα colonies induces partial extranuclear ERα following 24 h treatment. T47D:A18/neo colonies (neo) and T47D:A18/PKCα (PKCα) colonies were immunostained for ERα (green) and nuclei (blue) as detailed in materials and methods. Colonies were grown for 10 days then treated for 24 h with vehicle (EtOH, 0.1%), E2 (10-9 M), 4-OHT (10-7 M) or RAL (10-7M). N: nuclear, M/C: membrane/cytoplasmic. Scale bar = 20 μm. Click here for file Acknowledgements These studies were supported in part by NIH/NCI RO1 CA122914 (to DAT) and NIH/NIGMS T32 BM070388 (to BPW and MEM). The authors thank Jae Woo Choi for performing mass spectrometry on serum samples in the laboratory of Dr. Gregory Thatcher. We also would like to acknowledge the Histopathology Core and the Confocal Microscopy Facility of the Research Resources Center at the University of Illinois at Chicago for providing services and expertise. ==== Refs Osborne CK Schiff R Mechanisms of endocrine resistance in breast cancer Annu Rev Med 2011 62 233 247 10.1146/annurev-med-070909-182917 20887199 Tonetti DA Morrow M Kidwai N Gupta A Badve S Elevated protein kinase C alpha expression may be predictive of tamoxifen treatment failure Br J Cancer 2003 88 1400 1402 10.1038/sj.bjc.6600923 12778068 Assender JW Gee JM Lewis I Ellis IO Robertson JF Nicholson RI Protein kinase C isoform expression as a predictor of disease outcome on endocrine therapy in breast cancer J Clin Pathol 2007 60 1216 1221 10.1136/jcp.2006.041616 17965220 Lonne GK Cornmark L Zahirovic IO Landberg G Jirstrom K Larsson C PKCalpha expression is a marker for breast cancer aggressiveness Mol Cancer 2010 9 76 10.1186/1476-4598-9-76 20398285 Tonetti DA Chisamore MJ Grdina W Schurz H Jordan VC Stable transfection of protein kinase C alpha cDNA in hormone-dependent breast cancer cell lines Br J Cancer 2000 83 782 791 10.1054/bjoc.2000.1326 10952784 Lin X Yu Y Zhao H Zhang Y Manela J Tonetti D Overexpression of PKCα is required to impart estradiol inhibition and tamoxifen-resistance in a T47D human breast cancer tumor model Carcinogenesis 2006 27 1538 1546 16513679 Chisamore MJ Ahmed Y Bentrem DJ Jordan VC Tonetti DA Novel antitumor effect of estradiol in athymic mice injected with a T47D breast cancer cell line overexpressing protein kinase Calpha Clin Cancer Res 2001 7 3156 3165 11595710 Yao K Lee ES Bentrem DJ England G Schafer JI O’Regan RM Jordan VC Antitumor action of physiological estradiol on tamoxifen-stimulated breast tumors grown in athymic mice Clin Cancer Res 2000 6 2028 2036 10815929 Zhang Y Zhao H Asztalos S Chisamore M Sitabkhan Y Tonetti DA Estradiol-induced regression in T47D:A18/PKCalpha tumors requires the estrogen receptor and interaction with the extracellular matrix Mol Cancer Res 2009 7 498 510 10.1158/1541-7786.MCR-08-0415 19372579 Nemere I Farach-Carson MC Membrane receptors for steroid hormones: a case for specific cell surface binding sites for vitamin D metabolites and estrogens Biochem Biophys Res Commun 1998 248 443 449 10.1006/bbrc.1998.8492 9703943 Watson CS Gametchu B Membrane-initiated steroid actions and the proteins that mediate them Proc Soc Exp Biol Med 1999 220 9 19 10.1046/j.1525-1373.1999.d01-2.x 9893163 Molloy CA May FE Westley BR Insulin receptor substrate-1 expression is regulated by estrogen in the MCF-7 human breast cancer cell line J Biol Chem 2000 275 12565 12571 10.1074/jbc.275.17.12565 10777546 Simoncini T Hafezi-Moghadam A Brazil DP Ley K Chin WW Liao JK Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase Nature 2000 407 538 541 10.1038/35035131 11029009 Stoica GE Franke TF Wellstein A Czubayko F List HJ Reiter R Morgan E Martin MB Stoica A Estradiol rapidly activates Akt via the ErbB2 signaling pathway Mol Endocrinol 2003 17 818 830 10.1210/me.2002-0330 12554767 Kelly MJ Levin ER Rapid actions of plasma membrane estrogen receptors Trends Endocrinol Metab 2001 12 152 156 10.1016/S1043-2760(01)00377-0 11295570 Song RX McPherson RA Adam L Bao Y Shupnik M Kumar R Santen RJ Linkage of rapid estrogen action to MAPK activation by ERalpha-Shc association and Shc pathway activation Mol Endocrinol 2002 16 116 127 10.1210/me.16.1.116 11773443 Castoria G Migliaccio A Bilancio A Di Domenico M de Falco A Lombardi M Fiorentino R Varricchio L Barone MV Auricchio F PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells EMBO J 2001 20 6050 6059 10.1093/emboj/20.21.6050 11689445 Marquez DC Pietras RJ Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells Oncogene 2001 20 5420 5430 10.1038/sj.onc.1204729 11571639 Stoica GE Franke TF Moroni M Mueller S Morgan E Iann MC Winder AD Reiter R Wellstein A Martin MB Stoica A Effect of estradiol on estrogen receptor-alpha gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway Oncogene 2003 22 7998 8011 10.1038/sj.onc.1206769 12970748 Nicholson RI Johnston SR Endocrine therapy–current benefits and limitations Breast Cancer Res Treat 2005 93 Suppl 1 S3 S10 16247594 Schiff R Massarweh SA Shou J Bharwani L Arpino G Rimawi M Osborne CK Advanced concepts in estrogen receptor biology and breast cancer endocrine resistance: implicated role of growth factor signaling and estrogen receptor coregulators Cancer Chemother Pharmacol 2005 56 Suppl 1 10 20 16273359 Song RX Chen Y Zhang Z Bao Y Yue W Wang JP Fan P Santen RJ Estrogen utilization of IGF-1-R and EGF-R to signal in breast cancer cells J Steroid Biochem Mol Biol 2010 118 219 230 10.1016/j.jsbmb.2009.09.018 19815064 Ellis MJ Gao F Dehdashti F Jeffe DB Marcom PK Carey LA Dickler MN Silverman P Fleming GF Kommareddy A Lower-dose vs high-dose oral estradiol therapy of hormone receptor-positive, aromatase inhibitor-resistant advanced breast cancer: a phase 2 randomized study JAMA 2009 302 774 780 10.1001/jama.2009.1204 19690310 Lonning PE Taylor PD Anker G Iddon J Wie L Jorgensen LM Mella O Howell A High-dose estrogen treatment in postmenopausal breast cancer patients heavily exposed to endocrine therapy Breast Cancer Res Treat 2001 67 111 116 10.1023/A:1010619225209 11519859 Nardulli AM Katzenellenbogen BS Dynamics of estrogen receptor turnover in uterine cells in vitro and in uteri in vivo Endocrinology 1986 119 2038 2046 10.1210/endo-119-5-2038 3533520 Reid G Hubner MR Metivier R Brand H Denger S Manu D Beaudouin J Ellenberg J Gannon F Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling Mol Cell 2003 11 695 707 10.1016/S1097-2765(03)00090-X 12667452 Seo HS Larsimont D Querton G El Khissiin A Laios I Legros N Leclercq G Estrogenic and anti-estrogenic regulation of estrogen receptor in MCF-7 breast-cancer cells: comparison of immunocytochemical data with biochemical measurements Int J Cancer 1998 78 760 765 9833770 Wijayaratne AL McDonnell DP The human estrogen receptor-alpha is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators J Biol Chem 2001 276 35684 35692 10.1074/jbc.M101097200 11473106 Song RX Barnes CJ Zhang Z Bao Y Kumar R Santen RJ The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane Proc Natl Acad Sci U S A 2004 101 2076 2081 10.1073/pnas.0308334100 14764897 Razandi M Alton G Pedram A Ghonshani S Webb P Levin ER Identification of a structural determinant necessary for the localization and function of estrogen receptor alpha at the plasma membrane Mol Cell Biol 2003 23 1633 1646 10.1128/MCB.23.5.1633-1646.2003 12588983 Acconcia F Ascenzi P Bocedi A Spisni E Tomasi V Trentalance A Visca P Marino M Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol Mol Biol Cell 2005 16 231 237 15496458 O’Brian C Elevated protein kinase C expression in human breast tumor biopsies relative to normal tissue Cancer Res 1989 49 3215 3217 2720675 Lahn M Köhler G Sundell K Su C Li S Paterson BM Bumol TF Protein kinase C alpha expression in breast and ovarian cancer Oncology 2004 67 1 10 15459489 Ali S Al-Sukhun S El-Rayes BF Sarkar FH Heilbrun LK Philip PA Protein kinases C isozymes are differentially expressed in human breast carcinomas Life Sci 2009 84 766 771 10.1016/j.lfs.2009.03.007 19324060 Ainsworth PD Winstanley JH Pearson JM Bishop HM Garrod DR Protein kinase C alpha expression in normal breast, ductal carcinoma in situ and invasive ductal carcinoma Eur J Cancer 2004 40 2269 2273 10.1016/j.ejca.2004.06.027 15454252 Kerfoot C Huang W Rotenberg SA Immunohistochemical analysis of advanced human breast carcinomas reveals downregulation of protein kinase C alpha J Histochem Cytochem 2004 52 419 422 10.1177/002215540405200314 14966210 Tonetti DA Gao W Escarzaga D Walters K Szafran A Coon JS PKC alpha; and ER beta; are associated with triple-negative breast cancers in African American and Caucasian patients Int J Breast Cancer 2012 2012. Migliaccio A Di Domenico M Castoria G de Falco A Bontempo P Nola E Auricchio F Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells EMBO J 1996 15 1292 1300 8635462 Kousteni S Bellido T Plotkin LI O’Brien CA Bodenner DL Han L Han K DiGregorio GB Katzenellenbogen JA Katzenellenbogen BS Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity Cell 2001 104 719 730 11257226 Giretti MS Fu XD De Rosa G Sarotto I Baldacci C Garibaldi S Mannella P Biglia N Sismondi P Genazzani AR Simoncini T Extra-nuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion PLoS One 2008 3 e2238 10.1371/journal.pone.0002238 18493596 Chakravarty D Nair SS Santhamma B Nair BC Wang L Bandyopadhyay A Agyin JK Brann D Sun LZ Yeh IT Extranuclear functions of ER impact invasive migration and metastasis by breast cancer cells Cancer Res 2010 70 4092 4101 10.1158/0008-5472.CAN-09-3834 20460518 Kumar R Wang RA Mazumdar A Talukder AH Mandal M Yang Z Bagheri-Yarmand R Sahin A Hortobagyi G Adam L A naturally occurring MTA1 variant sequesters oestrogen receptor-alpha in the cytoplasm Nature 2002 418 654 657 10.1038/nature00889 12167865 Yang Z Barnes CJ Kumar R Human epidermal growth factor receptor 2 status modulates subcellular localization of and interaction with estrogen receptor alpha in breast cancer cells Clin Cancer Res 2004 10 3621 3628 10.1158/1078-0432.CCR-0740-3 15173068 Fan P Wang J Santen RJ Yue W Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells Cancer Res 2007 67 1352 1360 10.1158/0008-5472.CAN-06-1020 17283173 Newton AC Protein kinase C: poised to signal Am J Physiol Endocrinol Metab 2009 298 E395 E402 19934406 Levin ER Pietras RJ Estrogen receptors outside the nucleus in breast cancer Breast Cancer Res Treat 2008 108 351 361 10.1007/s10549-007-9618-4 17592774 Gutierrez S Sosa L Petiti JP Mukdsi JH Mascanfroni ID Pellizas CG De Paul AL Cambiasso MJ Torres AI 17beta-Estradiol stimulates the translocation of endogenous estrogen receptor alpha at the plasma membrane of normal anterior pituitary cells Mol Cell Endocrinol 2012 355 169 179 10.1016/j.mce.2012.02.008 22366173 Yudt MR Vorojeikina D Zhong L Skafar DF Sasson S Gasiewicz TA Notides AC Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation Biochemistry 1999 38 14146 14156 10.1021/bi9911132 10571988 Tan M Li P Sun M Yin G Yu D Upregulation and activation of PKC alpha by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKC alpha and Src inhibitors Oncogene 2006 25 3286 3295 10.1038/sj.onc.1209361 16407820 Longo M Brama M Marino M Bernardini S Korach KS Wetsel WC Scandurra R Faraggiana T Spera G Baron R Interaction of estrogen receptor alpha with protein kinase C alpha and c-Src in osteoblasts during differentiation Bone 2004 34 100 111 10.1016/j.bone.2003.09.007 14751567 Pedram A Razandi M Sainson RC Kim JK Hughes CC Levin ER A conserved mechanism for steroid receptor translocation to the plasma membrane J Biol Chem 2007 282 22278 22288 10.1074/jbc.M611877200 17535799 Acconcia F Ascenzi P Fabozzi G Visca P Marino M S-palmitoylation modulates human estrogen receptor-alpha functions Biochem Biophys Res Commun 2004 316 878 883 10.1016/j.bbrc.2004.02.129 15033483 Welsh AW Lannin DR Young GS Sherman ME Figueroa JD Henry NL Ryden L Kim C Love RR Schiff R Rimm DL Cytoplasmic estrogen receptor in breast cancer Clin Cancer Res 2012 18 118 126 10.1158/1078-0432.CCR-11-1236 21980134 Levin ER Elusive extranuclear estrogen receptors in breast cancer Clin Cancer Res 2012 18 6 8 10.1158/1078-0432.CCR-11-2547 22215901 O’Regan RM Gajdos C Dardes RC De Los Reyes A Park W Rademaker AW Jordan VC Effects of raloxifene after tamoxifen on breast and endometrial tumor growth in athymic mice J Natl Cancer Inst 2002 94 274 283 10.1093/jnci/94.4.274 11854389	23634843	PMC3661391	CC BY	2021-01-05 01:57:57	yes	Mol Cancer. 2013 May 1; 12:34
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23717633PONE-D-12-4059710.1371/journal.pone.0064576Research ArticleBiologyDevelopmental BiologyCell Fate DeterminationEmbryologyModel OrganismsAnimal ModelsZebrafishMolecular Cell BiologySignal TransductionSignaling CascadesWNT Signaling CascadeSignaling in Selected DisciplinesDevelopmental Signaling Eafs Control Erythroid Cell Fate by Regulating c-myb Expression through Wnt Signaling Eafs Control Erythroid Cell FateMa Xufa 1 Liu Jing-Xia 2 * 1 College of Fisheries, Huazhong Agricultural University, Wuhan, P. R. China 2 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, P. R. China Eisenberg Leonard Editor New York Medical College, United States of America * E-mail: ichliu@ihb.ac.cnCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: XM JXL. Performed the experiments: XM JXL. Analyzed the data: XM JXL. Wrote the paper: XM JXL. 2013 22 5 2013 8 5 e6457619 12 2012 16 4 2013 © 2013 Ma and Liu2013Ma and LiuThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.ELL associated factor 1 and ELL associated factor 2 (EAF1/2 factors) are reported to play important roles in tumor suppression and embryogenesis. Our previous studies showed that eaf factors mediated effective convergence and extension (C&E) movements and modulated mesoderm and neural patterning by regulating both non-canonical and canonical Wnt signaling in the early embryonic process. In this study, through knockdown of both eaf1 and eaf2 in embryos, we found that differentiation of primary erythroid cells was blocked, but hematopoietic precursor cells maintained in eafs morphants. Co-injection of c-myb-MO rescued the erythroid differentiation in eafs morphants, as indicated by the restored expression of the erythroid-specific gene, βe3 globin. In addition, low dosage of c-myb effectively blocked the βe3 globin expression in embryos, and did not affect the expression of markers of hematopoietic progenitor cells and other mesoderm, which was similar to the phenotypes we observed in eafs morphants. We also revealed that knockdown Wnt signaling by transiently inducing dn-Tcf in embryos at the bud stage down-regulated the increased c-myb to normal level and also restored βe3 globin expression in eafs morphants. Our evidence points to a novel role for eaf factors in controlling erythroid cell fate by regulating c-Myb expression through canonic Wnt signaling. The work is supported by innovation project of Chinese Academy of Sciences, grant number is KSCX2-EW-Q-12 (website: http://www.bio.cas.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Human hemoglobinopathies are a group of genetic disorders caused by abnormal expression of globin genes, that result in anemia in patients. The molecular mechanism underlying the globin genes induction is evolutionarily conserved among vertebrates, and the process appears to depend on the extensive sharing of lineage-restricted transcriptional factors [1]. Gata1 is the erythroid factor, which is highly expressed in megakaryoytic/erythroid progenitors [2], [3], [4], [5]. It is essential for erythroid specification and differentiation [3], [5] and cooperates with runx1 and fli1, which are involved in the divergence of megakaryoytic from the erythroid lineage [6], [7]. On the contrary, Pu.1, a myeloid factor, is lineage-restrictedly required for granulocyte/macrophage induction [2], [5]. Lmo2, gata2 and scl are required earlier in the hematopoiesis process and restrict hemoto-vascular development of lateral mesoderm [8], [9]; in addition, they are also in a complex required for full erythroid cell maturation [10]. The c-Myb transcriptional factor is a key regulator of the hematopoietic stem and progenitor cells, one that is essential for the establishment of hematopoiesis as evidenced by the defects in the development of multiple blood lineages in the Myb null mice [11], [12], [13]. Conditional c-Myb knockout in adult hematopoietic stem cells leads to loss of self-renewal due to impaired proliferation and accelerated differentiation [14]. The c-Myb level must be down-regulated in order for hematopoietic cells terminal differentiation and maturation [15]. Constitutive expression of c-Myb blocks Friend murine erythroleukemia cell differentiation [16]. Expression of c-Myb was repressed by GATA-1 to allow further differentiation of erythrocyte at the onset of terminal differentiation in erythroblast [17], [18]. On the contrary, by using microRNAs to knockdown c-Myb expression in primary human erythroid progenitor cells, fetal and embryonic hemoglobin genes displayed elevated expression. This suggests that c-Myb plays an important role in silencing the fetal and embryonic hemoglobin genes [19]. In addition, those microRNAs elevate fetal hemoglobin expression via c-Myb in human trisomy13 [19]. During zebrafish embryogenesis, eaf factors have been revealed to mediate effective C&E movements through maintaining expression of non-canonical Wnt signaling ligands, Wnt5 and Wnt11 [20], and to modulate mesoderm and neural patterning by inhibiting canonical Wnt signaling [21]. In addition, eaf factors form a negative loop with another non-canonical Wnt signaling ligand, Wnt4, in embryos and mammalian cell lines [22]. In the embryos with morpholina-mediated eaf1 and eaf2 knockdown, we observed the high percentage of erythroid differentiation defects, but we still know little about the underlying mechanisms. In this study, by screening potential repressors of erythroid differentiation in eafs morphants using morpholino-mediated knockdown, we selectively and respectively co-injected morpholinos of genes that had been reported to inhibit erythroid differentiation and also displayed increased expression in eafs morphants. By co-injecting with c-myb-MO [23], we found that knockdown c-myb in eafs morphants could effectively rescue the erythroid differentiation defects, indicated by restored βe3 globin expression. Consistently, an over-expressed c-myb in embryos could nearly phenocopy the erythroid differentiation defects shown in eafs morphants. In addition, in eafs morphants, by transiently inducing dn-Tcf, a dominant negative form of Wnt signaling, we found that knockdown of canonical Wnt signaling can not only restore the increased expression of c-myb to normal levels, but also restore the reduced βe3 globin expression. In our study, we reveal a novel genetic cascade in the process of erythroid differentiation, in which Wnt signaling up-regulates expression of hematopoietic progenitor-restricted transcriptional factors, including c-myb, and the increased c-myb mediates the suppression of erythroid differentiation. Materials and Methods Fish Stocks As previously discussed, wild-type zebrafish (Danio rerio) (AB) maintenance, breeding, and staging were performed [20] and the hs:dnTCF-GFP transgenic line was performed as reported previously [24], [25]. Heat-shock Modulation Embryonic heat-shock experiments were conducted at 38°C for 20 minutes. Genotype was determined by the presence of GFP fluorescence at 3 hours post heat shock, then the non-fluorescent (wild-type) siblings were sorted and used as controls [24], [25]. Morpholino and mRNA Synthesis The translation blocking morpholinos (ATG targeted) eaf1-MO1 and eaf2-MO1, and splicing-blocking morpholinos, eaf1-MO3 and eaf2-MO3, have been described previously [20], [21], as the c-myb antisense morpholino have been described previously [23]. All morpholinos were purchased from Gene Tools, LLC (Philomath, Oregon, USA) and their sequences are listed in Table S1. For mRNA preparation, capped mRNA of c-myb was synthesized using the Ampticap SP6 High Yield message maker kit (Epicenter Biotechnologies). The synthesized mRNAs were diluted into different concentrations and injected into one-cell stage embryos, and the plasmid for c-myb mRNA was described previously [26]. Whole Mount in situ Hybridization and O-Dianisidine Staining Probes for gata2, fli1, scl, lmo2, runx1, gata1, pu.1, c-myb and flk1 were reported previously [3], [26], [27]. Probes for pax2a and myoD were kindly provided by Dr. Schier (Harvard University, Molecular and Cellular Biology), probe for cdh5 was kindly provided by Dr. Wang (Institute of Hydrobiology, Chinese Academy of Science), and probe for ntl was reported previously [20]. The procedures for in situ hybridization and o-dianisidine staining were performed as described previously [20], [28]. Western Blot Western blot was performed as described previously [29]. The embryos at the 24 hpf were washed with ice-cold PBS buffer and then lysed in RIPA (radioimmune precipitation) buffer containing 50 mM Tris, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA, pH 8.0, 150 mM NaCl, 1 mM NaF, 1 mM PMSF (phenylmethylsulphonyl fluoride), 1 mM Na3VO4 (sodium orthovanadate) and 1∶100 dilution of protease inhibitor cocktail (Sigma). After homogenizing, lysates were centrifuged for 15 minutes at 12,000 g at 4°C, and the supernatants were boiled with 1× SDS sample buffer, separated on SDS-PAGE and transferred to PVDF membrane (Millipore). The Western blot analysis was performed as described previously using the indicated anti-βe3 globin antibody (Z-Fish™ (Zebrafish)) and anti-β-actin antibody (Abcam). Results Eafs are Required for Erythroid Differentiation In our previous studies, we observed C&E movement defects and forebrain defects in eaf1 and eaf2 morphants, revealing that both eaf1 and eaf2 mediate C&E movements by non-canonical Wnt signaling and modulate mesoderm and neural patterning by inhibiting canonical Wnt signaling [20], [21]. In the late stage, in eafs morphants at 2 dhf, we observed significant defects including heart edema, reduced circular blood cells, and a lack of blood flow or clumps of blood cells located in front of the beating heart in embryos knockdown both of eaf1 and eaf2 (data not shown), suggesting that the eafs morphants displayed hematopoietic defects, as observed in mll morphants [30]. Here, in order to further test the molecular characters underlying the phenotypes, we applied blood markers to detect the hematopoietic defects in eafs morphants. Knockdown of both eaf1 and eaf2 in embryos by injection of either translation-blocking ATG-MO (indicated by eafs-MO1, and shown in Figure 1) or “splicing-blocking” morpholinos (data not shown) caused a striking hematopoietic phenotype. Morpholinos reduced functional eafs message, yielded obviously erythroid defects (Figure 1). Hemoglobin, indicated by o-dianisidine staining, reduced significantly in eafs morphants (Figure 1, A2, A3 and A4) compared to its control group (Figure 1, A1, A3 and A4), and nearly 100% of morphants displayed dramatically reduced differentiated mature erythrocytes (Figure 1, A3 and A4). By whole mount in situ hybridization (WISH), transcripts of βe3 globin, which marked mature erythroid, displayed obviously reduced expression in over half of the detected eafs morphants (59 of 95 morphants, Figure 1, B2); its protein level also reduced significantly in eafs morphants compared to its expression in control embryos (Figure 1, C). 10.1371/journal.pone.0064576.g001Figure 1 Eafs are required for specification of erythroid cells. (A) Erythroid defects in eafs morphants were detected by o-dianisidine staining for hemoglobin. (B) Eafs morphants displayed erythroid defects, indicated by reduced mRNA of βe3 globin. (C) Eafs morphants displayed reduced protein of βe3 globin. Erythroid Defects in Eafs Morphants are Specific To determine the specificity of eafs-MO induced erythroid defects, we examined the integrity of non-hematopoietic tissues and other markers which labeling vasculature and hematopoietic progenitors (Figure 2). Eafs morphants displayed obviously anterior neuroectoderm truncation at 24 hpf (Figure 2, A2 and A4, indicated by black arrowhead). These observations were consistent with our previous report that eafs were required for forebrain formation [21]. But the posterior mesoderm, including muscle and notochord, at 24 hpf, were intact but displayed subtle patterning defects in eafs morphants. MyoD displayed more compact expression and striped muscles was irregular (Figure 2, A2), it might be a phenotype of C&E movement defects as we observed previously [20]. Notochord (ntl: Figure 2, A3 and A4) was also normal in a representative morphant which displayed severe anterior truncation (Figure 2, A4, anterior truncation is indicated by black arrowhead). Functional vasculature and primitive hematopoietic progenitors were normal and maintained in eafs morphants. By WISH, eafs-MO injected embryos had a few increased progenitors (c-myb: A5, A6; scl: A7, A8, indicated by black arrow), and had increased vasculature (chd5; A9, A10; flk1; A11, A12). 10.1371/journal.pone.0064576.g002Figure 2 Erythroid defects in eafs morphants are specific. (A) At 24 hpf to 26 hpf, the expression detection of control or eafs-MO injected embryos (8 ng per embryo) processed by WISH for tissue specific genes: somites (A1, A2), notochord (A3, A4), vasculature (cdh5: A9, A10; flk1: A11, A12), and primitive progenitors (c-myb: A5, A6; scl: A7, A8, indicated by black arrow). (B) At the 8 somites stage, primitive hematoposis in eafs morphants, indicated by primitive progenitor genes (scl: B1, B2; lmo2: B3, B4). (C) At the 10 somites stage, progenitors for blood (gata2: C1, C2; lmo2: C3, C4; runx1: C7, C8; c-myb: C15, C16), progenitors for vasculature (fli: C11, C12), progenitors for both blood and vasculature (scl: C5, C6), more mature erythroid progenitors (gata1: C9, C10), and more mature myeloid progenitors (Pu.1: C13, C14). A1–A12, C13–C16, lateral view, anterior to the left. B1–B4, C1–C12, dorsal view, anterior to the up. In order to test whether the initiation and progress of primitive hematopoiesis were normal in eafs morphants, we detected blood progenitor-restricted transcriptional factors during early somitogenesis. At the 8 somites stage, blood progenitors indicated by scl (Figure 2, B1 and B2), which is required earlier in the hematopoiesis and is important for both blood and vessel development, showed normal in eafs morphants. Similarly, lmo2, another important transcriptional factor required earlier in the hematopoiesis process and restricting hemoto-vascular development of lateral mesoderm, also displayed normal in eafs morphants (Figure 2, B3 and B4). When the morphants developed to the 10 somites stage (Figure 2, C), however, there was a little overexpression in scl (Figure 2, C5 and C6), and lmo2 (Figure 2, C3 and C4), compared to their respective control; Gata2 (Figure 2, C1 and C2), runx1 (Figure 2, C7 and C8), and pu.1 (Figure 2, C13 and C14) displayed increased expression in eafs morphants from the 10 somites stage; A slight overexpression was also observed in case of fli (Figure 2, C3 and C4), gata1 (Figure 2, C9 and C10), and c-myb (Figure 2, C15 and C16). Knockdown C-myb in Eafs Morphants Restored βe3 globin Expression The above observations indicated that blood precursor-restricted transcriptional factors, including gata2, fli, scl, runx1, gata1, lmo2, pu.1, and c-myb increased in eafs morphants from the 10 somites stage (Figure 2), but βe3 globin reduced dramatically (Figure 1). Gata1 is essential for erythroid cell fate determination [3], [5], and gata2, scl, and lmo2 are factors in the transcriptional complex for erythroid maturation [8], [9], [10]. However, pu.1, runx1, fli, and c-myb are negative factors that antagonize erythroid differentiation and maturation [6], [7]. Thus, we analyzed whether the reduced βe3 globin expression might result from the up-regulation of pu.1, runx1, fli, or c-myb in eafs morphants. In order to investigate this hypothesis further, we performed morphorlino-mediated knockdown of those transcriptional factors in eafs morphants respectively. We found that knockdown of c-myb could restore βe3 globin expression in eafs morphants to some extent (Figure 3, A3), far fewer embryos (22 of 86 specimen) displayed reduced βe3 globin expression after co-injected with c-myb-MO (Figure 3, A3) compared to eafs signal morphants (51 of 95 specimen) (Figure 3, A2). In addition, βe3 globin expression also increased in rescued embryos compared to its level in eafs morphants (Figure 2, A3). 10.1371/journal.pone.0064576.g003Figure 3 Eafs act upstream of c-myb in primitive hamatopoiesis. (A) In situ hybridization of βe3 globin showing rescue of erythroid differentiation defects in eafs morphants by knockdown of c-myb, the numbers of embryos displayed reduced βe3 globin in total detected embryos was shown in (A2, A3). (B) βe3 globin expression in eafs morphants (B2) and in,c-myb morphants (4 ng per embryo) (B3) and in morphants injected with combined eafs-MO and c-myb-MO (8 ng eafs-MO per embryo and 2 ng c-myb-MO) (B4). (C) Morphology of representative embryos injected with c-myb-MO (C2), and mesoderm pattern indicated by expression of pax2a and myoD in c-myb morphants at the bud stage (C4). (D) C-myb function downstream of eafs in primitive hematopoiesis. Early hematopoietic progenitors, lmo2, displayed obviously reduced expression from the 6 somites stage both in c-myb morphants (4 ng per embryo) (D2) and in morphants injected with combined c-myb-MO and eafs-MO (8 ng eafs-MO/per embryo and 2 ng c-myb-MO) (D3). Gata1 and scl also displayed obviously reduced expression in embryos injected with combined eafs-MO and c-myb-MO (D6, D9) as in embryos injected with c-myb-MO single (D5, D8) at the 10 somites stage. A1–A3, D1–D9, dorsal view, anterior to the up; C3, C4, dorsal view, anterior to the left. B1–B4, C1, C2, lateral view, anterior to the left. Contrary to eafs morphants (Figure 3, B2), c-myb morphants displayed little elevation of expression of βe3 globin (Figure 3, B3); the morphants injected with both eafs-MO and c-myb-MO displayed in-between expressions of βe3 globin (Figure 3, B4). We also analyzed the morphogenesis of c-myb morphants and defects of non-blood mesoderm in c-myb morphants. As shown in Figure 3C, the c-myb morphants displayed similar phenotypes to those reported previously such as small eyes and small brain (Figure 3, C2) [23], but no obvious defects displayed in the posterior body. In c-myb morphants, the induction of non-blood mesoderm, indicated by pax2a and myoD, also displayed normal expression at the bud stage, although there appear to be subtle patterning defects such as short myoD domain in c-myb morphants (Figure 3, C4). C-myb was Required for Maintaining of Hematopoietic Precursors Although we found that knockdown c-myb in eafs morphants could rescue βe3 globin expression effectively, we still knew little about the genetic cascade between c-myb and eaf factors in this process. In order to further understand the genetic pathway between c-myb and eaf factors in hematopoiesis, we detected more blood markers in c-myb morphants and in morphants co-injected with c-myb-MO and eafs-MO together. In PLM (posterior lateral mesoderm), the initiation of c-myb was detected around the 4 somites stage [31], [32], so we detected the expression of blood markers in c-myb morphants from the 6 somites stage. As shown in Figure 3D, the expression of lmo2 reduced significantly in c-myb morphants from the 6 somites stage (27 of 27 specimen, Figure 3, D2). In embryos co-injected with both c-myb-MO and eafs-MO, lmo2 displayed obviously reduced expression (10 of 13 specimen, Figure 3 D3). In addition, gata1 (14 of 15 specimen, Figure 3, D5) and scl (19 of 21 specimen, Figure 3, D8) also displayed dramatically reduced expression in c-myb morphants. Similarly, in embryos injected with both c-myb-MO and eafs-MO, gata1 (8 of 10 specimen, Figure 3, D6) and scl (13 of 15 specimen, Figure 3, D9) also displayed reduced expression, as we observed in c-myb morphants. All the observations suggested that c-myb might be required to maintain the hematopoietic progenitor cells and could also act downstream of eafs in specification blood cells or regulating expression of blood transcriptional factors. C-myb Suppressed βe3 globin Expression, but Had no Influence on Hematopoietic Precursors With all the above observations taken together, we assumed that c-myb might be required for hematopoietic precursor cells, but would block erythroid differentiation in zebrafish, thus functioning similarly with its mammalian orthologs [14], [17], [19]. To further detect its roles in hematopoiesis in vivo, we over-expressed c-myb in embryos by mRNA injection. We injected the embryos with different dosages of c-myb mRNA, from 50 pg per embryo to 200 pg per embryo, and the embryos did not display defects of morphology (data not shown). We analyzed the development of hematopoietic cells in embryos injected with different dosages of c-myb mRNA respectively. Most embryos injected with c-myb mRNA displayed obviously reduced expression of βe3 globin, either the dosage was 50 pg per embryo (Figure 4, A2) or was 200 pg per embryo (Figure 4, A4), however, gata1, displayed normal expression in all the detected embryos injected with different dosages of c-myb mRNA (Figure 4, A6 and A8). The observations here suggested that, c-myb could effectively suppress βe3 globin expression in vivo even at a very low dosage (50 pg per embryo). In order to further detect the roles of c-myb in hematopoiesis in zebrafish embryos, we detected more molecular markers labeling non-hematopoietic tissues or hematopoietic cells, with all embryos injected with the same dosage of c-myb mRNA (50 pg per embryo). At this low dosage (50 pg per embryo), c-myb could effectively suppress βe3 globin expression in vivo (37 of 70 specimen, Figure 4, B2), and the corresponding injected c-myb mRNA displayed ubiquitously distribution (Figure 4, B4). We wondered whether reduced βe3 globin expression resulted from mesoderm pattern defects, then we detected the expression of other molecular markers, including markers labeling hematopoietic precursors and axis mesoderm, in embryos with ectopic c-myb expression (50 pg per embryo). At the 10 somites stage, the erythroid precursor marker, gata1, displayed similar expression levels in embryos with ectopic expression of c-myb (Figure 4, C2) as in control embryos (Figure 4, C1). Similarly, scl, another erythroid precursor marker, also displayed normal expression in embryos injected with c-myb mRNA (Figure 4, C3 and C4). In addition, non-blood mesoderm markers, pax2a and myoD, showed normal expression in embryos with ectopic c-myb expression (Figure 4, C6) when compared to control embryos (Figure 4, C5). All the data suggested that c-myb specifically inhibited βe3 globin expression, but had no influence on expression of axis mesoderm and hematopoietic precursor cells in embryos. 10.1371/journal.pone.0064576.g004Figure 4 C-myb suppressed specification of mature erythroid cells, and the phenotype of c-myb gain of function is specific. (A) Different dosage of c-myb on specification of mature erythroid cells (βe3 globin: A2, 50 pg per embryo; A4, 200 pg per embryo) and on erythroid progenitors (gata1: A6, A8). (B) In situ hybridization of βe3 globin showed that erythroid differentiation was blocked in embryos with ectopic c-myb expression (50 pg per embryo) (B2), the number of embryos displayed reduced βe3 globin was shown in (B2), and the in situ hybridization of c-myb shown its ectopic expression in corresponding embryos (B4). (B) Hematopoietic progenitor cells including gata1 (C1, C2) and scl (C3, C4), and other mesoderm cells including pax2a and myoD (C5, C6) specified and maintained normally in embryos with ectopic c-myb expression. A1–A8, B1, B2, C1–C4, dorsal view, anterior to the up; B3, B4, C5, C6, lateral view, anterior to the left. Eaf Regulate Erythroid Cell Differentiation by Modulating C-myb Expression through Wnt Signaling The above observations suggested that the increased expression of c-myb might mediate the suppression of erythroid differentiation in eafs morphants. High Wnt signaling was revealed in eafs morphants [21], and Wnt signaling was reported as being required for stem cell renewal and for inhibiting terminal multi-linage cells differentiation [33], [34], [35]. In addition, down-regulating Wnt activities resulted in significantly reduced expression of gata1 in intermediate cell mass (ICM) [36], and c-myb, in aorta-gonad- mesonephros (AGM) [25], in treated embryos. In this study, by applying hs:dnTCF-GFP transgenic embryos [24], [25], [36], which is a stable line that express a dominant negative of TCF/LEF, we down-regulated Wnt activities by transiently induceing dn-Tcf expression in embryos, and detected some hematopoietic cell markers including gata1, c-myb and erythroid specific markers. The scheme of knockdown Wnt signaling in embryos using hs:dnTCF-GFP fish was shown in Figure 5A. We heat-shocked the embryos at the bud stage, there was no obvious morphology defects observed in the GFP positive embryos at the 16 somites stage or later (data not shown), which differs from the previous report that the embryos showed malformation after heat-shocking at 75% epiboly stage [36]. We detected the expression of hematopoietic precursors and erythroid markers in dn-Tcf transgene induced embryos. Compared to their control offsprings (Figure 5, B1 and B3), embryos with expression of transiently induced dn-Tcf, both c-myb (Figure 5, B2) and gata1 (Figure 5, B4) displayed obviously reduced expression, the observations here were consistent with previous reports [25], [36]. 10.1371/journal.pone.0064576.g005Figure 5 Knockdown Wnt signaling in hs:dnTCF-GFP embryos by heat shock at the bud stage resulted in reduced progenitor cells and accelerated differentiation of erythroid cells. (A) Scheme of using hs:dnTCF-GFP fish to knockdown Wnt signaling in embryos. (B) Reduced progenitor blood cells, labeled by c-myb (B1, B2) and gata1 (B3, B4), but accelerated erythroid cells differentiation, labeled by βe3 globin (B5, B6, B9, B10) and band3 (B7, B8) displayed in hs:dnTCF-GFP positive embryos, and black arrow indicate the increased expression of βe3 globin expression in hs:dnTCF-GFP positive embryos (B10) compared to its control siblings (B9). Other mesoderm, labeled by pax2a and myoD (B9, B10, B11, B12), was normal in hs:dnTCF-GFP positive embryos. B1, B2, B7–B12, lateral view, anterior to the left; B3–B6 dorsal view, anterior to the up. The differentiated erythroid markers, βe3 globin (Figure 5, B6 and B10) and band3 (Figure 5, B8),however, showed small increase in the embryos with induced dn-Tcf expression. We could not detect the expression change of non-blood axis mesoderm, such as pax2a and myoD, in the embryos with transiently induced dn-Tcf expression (Figure 5, B11 and B12), consistent with the observations that no obvious morphology defects displayed in the heat-shocked GFP positive embryos (data not shown). We then contemplated whether down-regulating Wnt signaling in eafs morphants could rescue the erythroid defects and down-regulate the expression of precursor markers. Figure 6A shows the scheme of using hs:dnTCF-GFP fish to do rescue experiments. As expected, transiently inducing expression of dn-Tcf in eafs morphants could restore the expression of βe3 globin significantly (Figure 6, B and C). In a total of 27 eafs morphants, 48.2% of morphants displayed strongly reduced βe3 globin expression, 22.2% of morphants displayed mildly reduced βe3 globin expression, and 29.6% of morphants shown normal (Figure 6, C); but after heat-shocking to induce dn-Tcf expression in eafs morphants, we found that in a total of 29 detected embryos, only 6.9% of morphants showed strongly reduced expression of βe3 globin. Another 34.5% of morphants even shown increased expression of βe3 globin (Figure 6, B and.C), suggesting that dn-Tcf might act downstream of eafs and be very effective to rescue differentiation defects of erythroid cells in eafs morphants. In addition, the increased expression of c-myb was also restored to a normal level by transiently inducing dn-Tcf expression in eafs morphants (Figure 6, B and C). 10.1371/journal.pone.0064576.g006Figure 6 Knockdown Wnt signaling in eafs morphants by transiently inducing dn-Tcf expression rescued defects of c-myb expression and erythroid cells specification. (A) Scheme of rescuing experiments in eafs morphants by using hs:dnTCF-GFP embryos. (B) Increased c-myb expression and reduced βe3 globin expression were restored in eafs morphants by transiently inducing dn-Tcf in embryos at the bud stage. B1–B8, dorsal view, anterior to the up. Discussion Erythroid Differentiation is Blocked in Eafs Morphants, and the Phenotype is Specific Eaf factors play important roles in tumor suppression and embryogenesis. In zebrafish, by morpholino-mediated knockdown, we revealed that eaf patterned the embryonic axis by regulating both non-canonical and canonical Wnt signaling at early developmental stages [20], [21]. At later stages, we observed significant defects including reduced circular blood cells and a lack of blood flow in eafs morphants. By further detecting the molecular markers of hematopoietic cells in eafs morphants, obvious defects of erythroid differentiation were revealed, indicated by reduced mRNA and protein level of βe3 globin and significant reduced o-dianisidine staining hemoglobin in eafs morphants (Figure 1). Normal vasculature (Figure 2, A9–A12), the integrity of posterior non-hematopoietic tissues indicated by myoD and ntl (Figure 2, A1–A4), and increased expression of precursors of blood and vessel, indicated by increased expression of gata1, scl, lmo2, gata2, pu.1, runx1, fli1, and c-myb in eafs morphants from the 10 somites stage (Figure 2, A5–A8 and C), both suggested that defects of erythroid specification in eafs morphants were highly specific and not due to the expense of nearby tissues. Morphants at 24 hpf displayed severe anterior truncation (Figure 2, A2 and A4, indicated by black arrowhead), the observations here were consistent with our previous reports that eafs are required for forebrain formation in embryos [20], [21], [22]. In eafs morphants, the specification and formation of the erythroid progenitors were normal, but erythroid differentiation was blocked, suggested that the cells in the primary hematopoietic process might keep and accumulate on the precursor stage. The following observations help to support this point. Firstly, in our study, all precursor markers, including gata1, scl, lmo2, gata2, c-myb and other genes, displayed increased expression from the 10 somites stage (Figure 2, A and C), but their expressions were still normal, even when detected by the 8 somites stage (Figure 2, B), and it was impossible that some factors could generally up-regulate all the precursor markers in such a short time. Second, it is reported that the initiation of erythroid began around the 8–10 somites stage in zebrafish embryos [37] (personal communication with Jared J. Ganis). In our study, the increased expression of precursor markers began shortly after the initiation of erythroid differentiation (Figure 2, B and C), this might only happen after the blocking of erythroid differentiation. If we could count the whole number of blood cells and the ratio of precursor cells and differentiated erythroid cells in both control embryos and in eafs morphants, we might perceive more clear mechanism clues underlying the phenotypes in eafs morphants. Of course, we still could not remove the possibility that the increased expression of precursor markers might come from the accelerated proliferation of progenitor cells or from their increased expression in a single cell. In eaf2 knockout mice, Xiao et al also found hematopoiesis defects [38], [39]. Very small number of eaf2 knockout mice developed extramedullary hematopoiesis, they suggested that eaf2 inactivation may disrupt the normal hematopoiesis in bone marrow, causing a compensatory response from spleen and liver in the eaf2 knockout mice [38], [39]. In addition, eaf2 deletion enhanced B-cell lymphoma development in eaf2 knockout mice [39], and eaf1 and eaf2 have been implicated in human hematopoietic cancers [40], [41]. Our data here showed more a detail character of the hematopoiesis defects cause by eaf1 and eaf2 knockdown in vivo, and the data here also suggested that the roles of eaf factors in regulating hematopoiesis might be conserved from zebrafish to mice. C-myb was Downstream of Eafs in Hematopoietic Developmental Process We observed increased expression of precursor markers but dramatically suppressed erythroid differentiation in eafs morphants (Figure 1, Figure 2). Through a morphorlino-mediated knockdown of c-myb in eafs morphants, we found that knockdown c-myb could significantly restore βe3 globin expression in eafs morphants (Figure 3, A and B). C-myb morphants displayed a morphogenesis phenotype as reported previously [23], characterized by a small brain but normal body axis formation (Figure 3, C2). Consistent with its normal formation of posterior body axis by morphology, we also observed normal pattern of mesoderm markers in c-myb morphants, indicated by pax2a and myoD expression (Figure 3, C4). But the primary hematopoietic wave failed in c-myb morphants, indicated by reduced expression of progenitor markers and accelerated erythroid differentiation before 24 hpf (Figure 3, B and D). On the contrary, embryos with ectopic expression of c-myb displayed reduced expression of erythroid markers but normal expression of hematopoietic precursors in fish (Figure 4). It is reported that c-Myb is required for the development of hematopoietic precursors [12], [13], and conditional c-Myb knockout in adult hematopoietic stem cells leads to impaired proliferation and accelerated differentiation in mouse[14]. In addition, c-Myb suppresses erythroid differentiation [16] and plays an important role in silencing the fetal and embryonic hemoglobin genes [19]. Our data here supported that c-myb is required for maintaining hematopoietic precursor markers, but blocks erythroid differentiation, and suggests that the roles of c-myb in hematopoiesis were conserved from zebrafish to mammalian. C-myb is a transcriptional factor which is important in hematopoiesis; it can bind with a vast number of different proteins in specifying different lineage of blood cells. The conditional c-myb knockout mice displayed hematopoietic defects in bone marrow [14], totally opposite to hematopoietic defects in a c-myb M303 mutant [42]. Similarly with the observations in mice, in this study, the hematopoietic defects occurred in c-myb morphants, which are different from what Thompson et al observed in c-myb b316 mutants; they found that gata2, lmo2, and gata1 displayed normal expression in the mutants around 18 hpf [26]. We speculate, c-myb b316 mutants, similar to mice c-Myb M303 mutant, maybe only lost binding ability with some specific protein or lost some specific roles important for embryogenesis development, but its ability in regulating primary hematopoiesis still exists. The limitation of this mutant model can not truly or really tell us about the hematopoietic specification as the c-myb gene is disrupted by morpholino mediating knockdown in embryos. C-Myb specifically inhibited erythroid specification was convinced in this study (Figure 4), although we still know little about the underlying mechanisms. Countless literatures report that c-Myb is a myeloid lineage regulator, cooperates with C/EBPa, to activate transcription of myeloid genes [43], [44]. Recently, hematopoietic genes, especially myeloid other than erythroid genes, have been identified as direct targets of c-Myb by ChIP-Seq (chromatin immunoprecipitation followed by massively parallel sequencing) [45], [46]. Myeloerythroid lineage cells initiate expression of both myeloid and erythroid lineage regulator [2], and myeloid lineage regulator C/EBPa could compete with erythroid lineage regulator gata1, to shift binding of SMAD1 to sites of nonerythroid [47]. Since our observations showed that c-myb inhibited erythriod specification (Figure 3 and Figure 4), taken the above mentioned reports together, we suppose that, c-myb might act as a nonerythroid lineage regulator, compete with erythroid lineage regulator gata1 or others, to bind with general master regulators, such as SMAD1, function indirectly in erythroid specification, However, we need more evidences to prove this speculation. All the hematopoietic markers in c-myb morphants displayed the opposite expression pattern to their expression in eafs single morphants (Figure 3, B and D). In morphants injected with c-myb-MO and eafs-MO together, the hematopoietic precursors also reduced dramatically (Figure 3, D), similar to what we observed in c-myb morphants (Figure 3, D) but opposite to their expression in eafs morphants (Figure 2, A and C). Combining the similarity of βe3 globin expression in c-myb morphants and in morphants knockdown with both eafs-MO and c-myb-MO, but opposite to its expression in eafs morphants together, we speculated that c-myb might act downstream of eaf factors in hematopoietic cells specification and differentiation. Eaf Control Erythroid Cell Fate by Regulating C-myb Expression through Wnt Signaling Eaf factors have been revealed to modulate mesoderm and neural patterning by inhibiting canonical Wnt signaling, and high activity of canonical Wnt/β-catenin is revealed in eafs morphants [21]. Wnt signaling was reported to be required for renewal of stem cells and progenitor cells and for inhibiting terminal multi-linage cells differentiation [33], [34], [35]. In addition, the expression of a hematopoietic precursor marker, gata1, reduced significantly in ICM in embryos with down-regulating Wnt activities [36]. In this study, we down-regulated Wnt activities in embryos by transiently induceing expression of dn-Tcf transgene, and revealed that including the reported gene gata1 (Figure 5, B3 and B4) [36], other precursor markers, specially c-myb (Figure 5, B1 and B2) [25] and pu.1 (data not shown), all displayed reduced expression. Our data was not only consistent with the previous study [25], [36], but also suggested that down-regulating Wnt activities in embryos could result in general down-regulating expression of most precursor markers. But how Wnt signaling acts on those cells in primitive hematopoiesis? Is this specific or direct? Since Goessling et al found that Wnt acts directly in AGM to promote hematopoietic stem cells proliferation [25], we need to evaluate how Wnt signaling act in blood forming regions in primitive hematopoiesis by applying one or more Wnt reporter lines in future days. However, we could not detect any morphology defects under macroscopic detection (data not shown) or by staining of axis mesoderm markers (Figure 4, B11 and B12) in the treated embryos with transiently induced dn-Tcf expression. The reason for this might be that we treated embryos at the bud stage (Figure 4, A), and it is well-known that Wnt signaling has no influence on the axis pattern after 90% epiboly. By transiently inducing dn-Tcf transgene expression, we rescued βe3 globin expression in eafs morphants, and the increased c-myb expression was restored to normal level (Figure 6, B and C). Some morphants even showed increased expression of βe3 globin after heat-shocking (Figure 6, C), suggesting that dn-Tcf might act downstream of eafs and be more efficient than eafs in the determination and specification of erythroid cells. As such, we elicit a model of hematopoietic process in eafs morphants in which knockdown eafs in embryos resulted in constitutive Wnt activities in the initiation and progress of hematopoietic cells specification and differentiation. The high Wnt activities induced increased expression hematopoietic precursor-restricted transcriptional factors, including c-myb, then the increased c-myb suppressed erythroid differentiation. Our data here also provided a novel mechanism that c-myb might mediate Wnt signaling in erythroid differentiation. Supporting Information Table S1 Morpholinos used in this study. (DOC) Click here for additional data file. We are grateful to Dr. Leonard Zon for helping to conceive the experiments, and appreciate Leonard Zon, Dr. Eirini Trompouki and Dr. Teresa V. Bowman for the generous gifts of reagents and resources, and we are also grateful to Leonard Zon for his kind support in performing some experiments in his lab for this manuscript. We are grateful to Xiaoying Bai, and Christian Mosimann, Owen J. Tamplin for their comments. ==== Refs References 1 Stamatoyannopoulos G (2005 ) Control of globin gene expression during development and erythroid differentiation . Exp Hematol 33 : 259 –271 .15730849 2 Arinobu Y , Mizuno S , Chong Y , Shigematsu H , Iino T , et al (2007 ) Reciprocal activation of GATA-1 and PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and myelolymphoid lineages . Cell Stem Cell 1 : 416 –427 .18371378 3 Galloway JL , Wingert RA , Thisse C , Thisse B , Zon LI (2005 ) Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos . Dev Cell 8 : 109 –116 .15621534 4 Fujiwara Y , Browne CP , Cunniff K , Goff SC , Orkin SH (1996 ) Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1 . Proc Natl Acad Sci U S A 93 : 12355 –12358 .8901585 5 Rhodes J , Hagen A , Hsu K , Deng M , Liu TX , et al (2005 ) Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish . Dev Cell 8 : 97 –108 .15621533 6 Elagib KE , Racke FK , Mogass M , Khetawat R , Delehanty LL , et al (2003 ) RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation . Blood 101 : 4333 –4341 .12576332 7 Starck J , Cohet N , Gonnet C , Sarrazin S , Doubeikovskaia Z , et al (2003 ) Functional cross-antagonism between transcription factors FLI-1 and EKLF . Mol Cell Biol 23 : 1390 –1402 .12556498 8 Burns CE , Traver D , Mayhall E , Shepard JL , Zon LI (2005 ) Hematopoietic stem cell fate is established by the Notch-Runx pathway . Genes Dev 19 : 2331 –2342 .16166372 9 Gering M , Yamada Y , Rabbitts TH , Patient RK (2003 ) Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1 . Development 130 : 6187 –6199 .14602685 10 Porcher C , Liao EC , Fujiwara Y , Zon LI , Orkin SH (1999 ) Specification of hematopoietic and vascular development by the bHLH transcription factor SCL without direct DNA binding . Development 126 : 4603 –4615 .10498694 11 Emambokus N , Vegiopoulos A , Harman B , Jenkinson E , Anderson G , et al (2003 ) Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb . EMBO J 22 : 4478 –4488 .12941699 12 Greig KT , Carotta S , Nutt SL (2008 ) Critical roles for c-Myb in hematopoietic progenitor cells . Semin Immunol 20 : 247 –256 .18585056 13 Mucenski ML , McLain K , Kier AB , Swerdlow SH , Schreiner CM , et al (1991 ) A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis . Cell 65 : 677 –689 .1709592 14 Lieu YK , Reddy EP (2009 ) Conditional c-myb knockout in adult hematopoietic stem cells leads to loss of self-renewal due to impaired proliferation and accelerated differentiation . Proc Natl Acad Sci U S A 106 : 21689 –21694 .19955420 15 Gonda TJ , Metcalf D (1984 ) Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukaemia . Nature 310 : 249 –251 .6205276 16 Clarke MF , Kukowska-Latallo JF , Westin E , Smith M , Prochownik EV (1988 ) Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation . Mol Cell Biol 8 : 884 –892 .2832742 17 Vegiopoulos A , Garcia P , Emambokus N , Frampton J (2006 ) Coordination of erythropoiesis by the transcription factor c-Myb . Blood 107 : 4703 –4710 .16484593 18 Bartunek P , Kralova J , Blendinger G , Dvorak M , Zenke M (2003 ) GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter . Oncogene 22 : 1927 –1935 .12673198 19 Sankaran VG , Menne TF , Scepanovic D , Vergilio JA , Ji P , et al (2011 ) MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13 . Proc Natl Acad Sci U S A 108 : 1519 –1524 .21205891 20 Liu JX , Hu B , Wang Y , Gui JF , Xiao W (2009 ) Zebrafish eaf1 and eaf2/u19 mediate effective convergence and extension movements through the maintenance of wnt11 and wnt5 expression . J Biol Chem 284 : 16679 –16692 .19380582 21 Liu JX , Zhang D , Xie X , Ouyang G , Liu X , et al (2013 ) Eaf1 and Eaf2 negatively regulate canonical Wnt/beta-catenin signaling . Development 140 : 1067 –1078 .23364330 22 Wan X , Ji W , Mei X , Zhou J , Liu JX , et al (2010 ) Negative feedback regulation of Wnt4 signaling by EAF1 and EAF2/U19 . PLoS One 5 : e9118 .20161747 23 Lin YC , Kuo MW , Yu J , Kuo HH , Lin RJ , et al (2008 ) c-Myb is an evolutionary conserved miR-150 target and miR-150/c-Myb interaction is important for embryonic development . Mol Biol Evol 25 : 2189 –2198 .18667440 24 Weidinger G , Thorpe CJ , Wuennenberg-Stapleton K , Ngai J , Moon RT (2005 ) The Sp1-related transcription factors sp5 and sp5-like act downstream of Wnt/beta-catenin signaling in mesoderm and neuroectoderm patterning . Curr Biol 15 : 489 –500 .15797017 25 Goessling W , North TE , Loewer S , Lord AM , Lee S , et al (2009 ) Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration . Cell 136 : 1136 –1147 .19303855 26 Thompson MA , Ransom DG , Pratt SJ , MacLennan H , Kieran MW , et al (1998 ) The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis . Dev Biol 197 : 248 –269 .9630750 27 Ransom DG , Bahary N , Niss K , Traver D , Burns C , et al (2004 ) The zebrafish moonshine gene encodes transcriptional intermediary factor 1gamma, an essential regulator of hematopoiesis . PLoS Biol 2 : E237 .15314655 28 Paffett-Lugassy N , Hsia N , Fraenkel PG , Paw B , Leshinsky I , et al (2007 ) Functional conservation of erythropoietin signaling in zebrafish . Blood 110 : 2718 –2726 .17579187 29 Zhou J , Feng X , Ban B , Liu J , Wang Z , et al (2009 ) Elongation factor ELL (Eleven-Nineteen Lysine-rich Leukemia) acts as a transcription factor for direct thrombospondin-1 regulation . J Biol Chem 284 : 19142 –19152 .19447890 30 Wan XY , Hu B , Liu JX , Feng X , Xiao WH (2011 ) Zebrafish mll Gene Is Essential for Hematopoiesis . Journal of Biological Chemistry 286 : 33345 –33357 .21784840 31 Patterson LJ , Gering M , Patient R (2005 ) Scl is required for dorsal aorta as well as blood formation in zebrafish embryos . Blood 105 : 3502 –3511 .15644413 32 Patterson LJ , Gering M , Eckfeldt CE , Green AR , Verfaillie CM , et al (2007 ) The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish . Blood 109 : 2389 –2398 .17090656 33 Reya T , Duncan AW , Ailles L , Domen J , Scherer DC , et al (2003 ) A role for Wnt signalling in self-renewal of haematopoietic stem cells . Nature 423 : 409 –414 .12717450 34 Fleming HE , Janzen V , Lo Celso C , Guo J , Leahy KM , et al (2008 ) Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo . Cell Stem Cell 2 : 274 –283 .18371452 35 Sato N , Meijer L , Skaltsounis L , Greengard P , Brivanlou AH (2004 ) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor . Nat Med 10 : 55 –63 .14702635 36 Lengerke C , Schmitt S , Bowman TV , Jang IH , Maouche-Chretien L , et al (2008 ) BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway . Cell Stem Cell 2 : 72 –82 .18371423 37 Ganis JJ , Hsia N , Trompouki E , de Jong JL , DiBiase A , et al (2012 ) Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR . Dev Biol 366 : 185 –194 .22537494 38 Xiao W , Ai J , Habermacher G , Volpert O , Yang X , et al (2009 ) U19/Eaf2 binds to and stabilizes von hippel-lindau protein . Cancer Res 69 : 2599 –2606 .19258512 39 Xiao W , Zhang Q , Habermacher G , Yang X , Zhang AY , et al (2008 ) U19/Eaf2 knockout causes lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostatic intraepithelial neoplasia . Oncogene 27 : 1536 –1544 .17873910 40 Simone F , Luo RT , Polak PE , Kaberlein JJ , Thirman MJ (2003 ) ELL-associated factor 2 (EAF2), a functional homolog of EAF1 with alternative ELL binding properties . Blood 101 : 2355 –2362 .12446457 41 Simone F , Polak PE , Kaberlein JJ , Luo RT , Levitan DA , et al (2001 ) EAF1, a novel ELL-associated factor that is delocalized by expression of the MLL-ELL fusion protein . Blood 98 : 201 –209 .11418481 42 Sandberg ML , Sutton SE , Pletcher MT , Wiltshire T , Tarantino LM , et al (2005 ) c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation . Dev Cell 8 : 153 –166 .15691758 43 Tahirov TH , Sato K , Ichikawa-Iwata E , Sasaki M , Inoue-Bungo T , et al (2002 ) Mechanism of c-Myb-C/EBP beta cooperation from separated sites on a promoter . Cell 108 : 57 –70 .11792321 44 Verbeek W , Gombart AF , Chumakov AM , Muller C , Friedman AD , et al (1999 ) C/EBPepsilon directly interacts with the DNA binding domain of c-myb and cooperatively activates transcription of myeloid promoters . Blood 93 : 3327 –3337 .10233885 45 Zhao L , Glazov EA , Pattabiraman DR , Al-Owaidi F , Zhang P , et al (2011 ) Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb . Nucleic Acids Res 39 : 4664 –4679 .21317192 46 Lorenzo PI , Brendeford EM , Gilfillan S , Gavrilov AA , Leedsak M , et al (2011 ) Identification of c-Myb Target Genes in K562 Cells Reveals a Role for c-Myb as a Master Regulator . Genes Cancer 2 : 805 –817 .22393465 47 Trompouki E , Bowman TV , Lawton LN , Fan ZP , Wu DC , et al (2011 ) Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration . Cell 147 : 577 –589 .22036566	23717633	PMC3661582	CC BY	2021-01-05 17:47:15	yes	PLoS One. 2013 May 22; 8(5):e64576
==== Front Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 10.1155/2013/467245Research ArticleDecursin Isolated from Angelica gigas Nakai Rescues PC12 Cells from Amyloid β-Protein-Induced Neurotoxicity through Nrf2-Mediated Upregulation of Heme Oxygenase-1: Potential Roles of MAPK Li Li 1 2 Du Ji-kun 3 Zou Li-yi 1 Wu Tie 1 Lee Yong-woo 2 Kim Yong-ho 2 1Department of Pharmacology, Guangdong Medical College, Dongguan 523-808, China2Department of Smart Food and Drugs, Graduate School, Inje University, Gimhae 621-749, Republic of Korea3Department of Clinical Laboratory, Shenzhen Shajing Affiliated Hospital of Guangzhou Medical University, Shenzhen 518-104, ChinaYong-woo Lee: mlsywlee@inje.ac.kr and Yong-ho Kim: mlskimyh@inje.ac.krAcademic Editor: Youn Chul Kim 2013 9 5 2013 2013 46724529 1 2013 27 3 2013 7 4 2013 Copyright © 2013 Li Li et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Decursin (D), purified from Angelica gigas Nakai, has been proven to exert neuroprotective property. Previous study revealed that D reduced Aβ 25‒35-induced cytotoxicity in PC12 cells. Our study explored the underlying mechanisms by which D mediates its therapeutic effects in vitro. Pretreatment of cells with D diminished intracellular generation of ROS in response to Aβ 25‒35. Western blot revealed that D significantly increased the expression and activity of HO-1, which was correlated with its protection against Aβ 25‒35-induced injury. Addition of ZnPP, an HO-1 competitive inhibitor, significantly attenuated its protective effect in Aβ 25‒35-treated cells, indicating the vital role of HO-1 resistance to oxidative injury. Moreover, D induced Nrf2 nuclear translocation, the upstream of HO-1 expression. While investigating the signaling pathways responsible for HO-1 induction, D activated ERK and dephosphorylated p38 in PC12 cells. Addition of U0126, a selective inhibitor of ERK, blocked D-induced Nrf2 activation and HO-1 induction and meanwhile reversed the protection of D against Aβ 25‒35-induced cell death. These findings suggest D augments cellular antioxidant defense capacity through both intrinsic free radical scavenging activity and activation of MAPK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction, thereby protecting the PC12 cells from Aβ 25‒35-induced oxidative cytotoxicity. ==== Body 1. Introduction Alzheimer's disease (AD) is the most common form of senile dementia, affecting millions of people worldwide. It is an age-related neurodegenerative disease pathologically characterized by deposition of senile plaques, intracellular neurofibrillary tangles (NFT), and loss of neurons in the brain. Amyloid β-peptide (Aβ), a 39- to 43-amino acid peptide fragment derived from an amyloid precursor protein via a sequential cleavage by β- and γ-secretases, is the major component of senile plaques and is considered to be tightly related to the development and progress of AD. Extensive evidence indicates that the brains of individuals with AD are characterized by exaggerated oxidative stress [1–8], and the overproduction of Aβ leads to Aβ-associated free radical production and cell death [9–13]. Not only does Aβ increase oxidative stress, but its generation is also increased as a result of oxidative stress, which in turn causes more oxidative damage. Given the important role of oxidative stress in AD, therapeutic strategies which are directed at early interventions targeted at oxidative stress may be effective in delaying AD development and slowing its progression. Indeed, increased antioxidant activity confers protection and has been reported to lower the risk of AD [14]. Thus, an approach which simultaneously enhances various intracellular oxidative defense capacities may be more effective in combating neurodegeneration. Among the various cytoprotective enzymes, heme oxygenase (HO) has received considerable attention, which consists of three isoforms: HO-1, HO-2, and HO-3. Although HO-2 and HO-3 are constitutively expressed, HO-1 is inducible in many cell types, such as neuronal cells [15, 16]. HO-1 is one of the major antioxidant/cytoprotective enzymes that are readily induced in response to oxidative stress. HO-1 catalyzes the rate-limiting step in the heme degradation process, releasing iron, carbon monoxide (CO), and biliverdin. The antioxidant potential of HO-1-generated metabolic products highlights the HO-1 pathway as a therapeutic target for pharmacological intervention of various diseases including neurological disorders [17–19]. The induction of HO-1 resulted in a relatively higher resistance to glutamate- and H2O2-mediated oxidative damage and MPTP- or Aβ-induced neurotoxicity [20–23]. Transcriptional regulation of the ho-1 gene is linked to the transcription factor NF-E2-related factor (Nrf2), which plays a crucial role in cellular defense. Nrf2 is a basic leucine zipper transcription factor that resides in the cytoplasm bound to its inhibitor protein, Keap1, and translocated to the nucleus after stimulation. It then binds to the antioxidant response element (ARE) sequences in the promoter regions of cluster of antioxidant/detoxifying genes, such as ho-1 [24–26]. Activation of Nrf2 pathway has been demonstrated to be involved in the protection of the nerve cells against oxidative damage in vivo and in vitro [27–29]. Neurons lacking Nrf2 are highly sensitive to oxidative stress but can be rescued by transfection with a functional Nrf2 construct [30]. In addition, activation of the Nrf2/ARE pathway in astrocytes by tert-butylhydroquinone (tBHQ), an Nrf2 activity inducer, is able to protect neurons from subsequent oxidative stress [31]. To date, multiple signaling kinases related to cell survival/proliferation have been reported to regulate the nuclear translocation of Nrf2, including mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC) [32–34]. MAPK is one of the most common signaling pathways that serve to coordinate the cellular response to a variety of extracellular stimuli. These are well characterized in mammals and include c-Jun N-terminal kinase (JNK), p38 MAP kinase (p38), and mitogenic signaling, extracellular signal-regulated kinase 1/2 (ERK). Several members of MAPK family are implicated in neurodegenerative processes [35] and its activation also modulates several gene and protein expressions, such as that of HO-1 [36, 37]. Thus, in light of the cytoprotective role of HO-1, the specific activation of Nrf2 and upregulation of HO-1 gene expression by pharmacological modulation may represent a novel target form therapeutic intervention of AD. Angelica gigas Nakai (Umbelliferae) root is used in traditional oriental herbal medicine to treat female afflictions and is regarded by herbalists as female ginseng for its hemopoietic and health-promoting activities [38]. Decursin (D) is a pyranocoumarin which is the major active ingredient present in Angelica gigas Nakai. Reported in vivo and in vitro studies indicated that D possesses anticancer [39–43], antibacterial [44], antiplatelet aggregation [45], antinematodal activities [46], and antioxidant activities [47] properties. Growing bodies of evidence have supported the fact that D exhibits potent neuroprotective activity against glutamate-induced neurotoxicity in primary cultures of rat cortical cells [48] and greatly improves scopolamine-induced amnesia in mice [49, 50]. Our previously study indicated that D increases cellular resistance to Aβ-induced oxidative injury in the rat pheochromocytoma (PC12) cells, presumably through not only the induction of Nrf2 and related antioxidant enzymes, but also the antiaggregation of Aβ [51]. However, the upstream signaling and the detailed molecular mechanisms by which D exerts its neuroprotective effects in vivo remain largely unresolved. To gain a further insight into the biological roles of D, we attempt, in this study, to elucidate the correlation between its neuroprotection effect and HO-1 production. We designed an experiment to investigate whether the D-induced HO-1 expression is associated with the activation of MAPKs/Nrf2 in PC12 cells following treatment with Aβ as an in vitro model. 2. Materials and Methods 2.1. Materials Amyloid beta-protein (25–35) trifluoroacetate salt (Aβ 25–35) was provided by Bachem California (Torrance, CA). RPMI+GlutaMAX-l, penicillin-streptomycin, fetal bovine serum (FBS), and horse serum (HS) were purchased from Invitrogen (Grand Island, NY). BCA protein assay kit was purchased from Thermo Fisher Scientific (Barrington, IL). The assay kit for cytotoxicity (WST-8) was supplied by Cayman Chemical Company (Ann Arbor, MI). Lipid peroxidation colorimetric assay kit was obtained from Oxford Biochemical Research (Rochester Hills, MI). SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and Protoporphyrin IX zinc (II) (Znpp, HO-1 Inhibitor) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO). Antibodies to Nrf2 (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA); phosphor-SAPK/JNK rabbit mAb, phospho-p44/42 MAPK (ERK1/2) rabbit mAb, phosphor-p38 MAP kinase rabbit mAb, HO-1 (P249) rabbit mAb, glyceraldehydes-3-phosphate dehydrogenase (GAPDH) rabbit mAb, anti-rabbit IgG alkaline-phosphatase- (AP-) linked antibodies, and U0126 (ERK inhibitor) were obtained from Cell Signaling Technology (Danvers, MA). All the other reagents were of the highest grade and were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. 2.2. Preparation of Decursin D was prepared by Dr. Kim's lab in the Department of Smart Foods and Drugs, Inje University, as described previously [52]. Briefly, dried and powdered root of A. gigas Nakai (1 kg) was extracted with 5 L of 95% ethanol for 24 h at room temperature. Extracts were filtered through Whatman No. 1 filter paper and were concentrated using a rotary evaporator (R-200, Büchi Labortechnik AG, Flawil, Switzerland) under reduced pressure, and 50 g A. gigas Nakai ethanol extract (AGNEX) was obtained. D was purified from AGNEX using recycling preparative HPLC (LC-9104, JAI, Tokyo, Japan). The AGNEX (20 g) was dissolved in 30 mL of 70% acetonitrile/water and filtered with a 0.45 μm membrane filter. 3 mL of sample was injected to the JAIGEL ODS-AP column (20 × 500 mm, JAI) at a flow rate of 4 mL/min. Isocratic elution was applied using 70% acetonitrile/water as the mobile phase, and the peaks were detected using an RI and UV/Vis detector at 328 nm. Finally, 5.3 g of D was obtained. 2.3. Preparation of Aβ 25–35 Stock Solution Aβ 25–35, the most toxic peptide fragment derived from the amyloid precursor protein (APP), was dissolved in deionized distilled water at a concentration of 1 mM and was incubated in 37°C for 3 d to induce maximal aggregation according to the previous report [53]. In order to create stable conditions for the aged stock solution, the solution was stored at −80°C and diluted in serum-free medium to desired concentrations immediately before use. 2.4. Cell Culture The rat pheochromocytoma (PC12) cell line kindly provided by Dr. Kam (Inje University) was maintained in RPMI+GlutaMAX-l supplemented with 5% FBS, 10% HS, and 1% penicillin-streptomycin and cultured at 37°C in a humidified atmosphere of 5% CO2. All cells were plated in poly-L-lysine coated culture dishes. The medium was changed every other day, and the cells were plated at an appropriate density according to the scale of each experiment. After the 24 h subculture, cells were switched to serum-free medium for treatment. 2.5. Assay for Cell Viability The cell viability was assessed by the WST-8 cell proliferation assay kit according to the manufacturer's instructions (Cayman Chemical Company, Ann Arbor, MI). Briefly, PC12 cells were seeded in 96-well culture plates. After incubation, the media were supplemented with 10 μL/well WST for 2 h prior to spectrophotometric evaluation. Conversion of WST to formazan was measured at 450 nm by fluorescence multidetection reader (Synergy HT, Biotek, Highland Park, IL). This reaction reflects the reductive capacity of the cells, representing the viability of the cells, and the results were expressed as the percentage of control (untreated) cells. Decreased WST reduction was taken as an indication of neuronal cell injury. 2.6. Assay of Intracellular Reactive Oxygen Species ROS production in PC12 cells was measured using the redox-sensitive fluorescent dye H2DCF-DA. Briefly, PC12 cells were seeded in 96-well plates, following treatment, the cells were loaded with 10 μM H2DCF-DA at 37°C for 30 min in the dark and then washed twice with PBS, and finally, the fluorescence intensity was measured at the excitation wavelength of 485 nm and the emission wavelength of 530 nm using a fluorescence microplate reader (Synergy HT, Biotek, Highland Park, IL). Data were analyzed and expressed as a percentage of the control. 2.7. Assay of HO Activity The HO activity was measured by spectrophotometric determination of bilirubin formation according to the previously described procedures. Briefly, microsomes obtained from harvested cells were added to a reaction mixture (1 mL final volume, pH 7.4) containing NADPH, bilirubin reductase from rat liver cytosol, and the substrate hemin. The reaction was conducted at 37°C in the dark for 1 h, terminated by the addition of 1 mL of chloroform, and the extracted bilirubin was measured by the difference in absorbance between 464 and 530 nm. 2.8. Nuclear and Cytosolic Lysate Preparation Cells were treated with various chemicals, as detailed in the figure legends. Nuclear extracts were prepared with a commercial kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA). All steps were carried out on ice or at 4°C unless stated otherwise. Protease inhibitors (10 μg/mL aprotinin, 10 μg/mL leupeptin) and a reducing agent (1 mM dithiothreitol, 1 mM phenylmethyl sulfonyl fluoride) were added to each buffer just prior to use. Briefly, cells were incubated in 5 vol of hypotonic buffer A (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, and 10 mM KCl) on ice for 15 min and homogenized. Nuclei were recovered by centrifugation at 3000 ×g for 15 min, and the supernatant was kept as the cytoplasmic extract. The nuclei were washed once using nuclei wash buffer (10 mM HEPES (pH 7.9), 0.2 mM MgCl2, and 10 mM KCl) and extracted for 30 min on ice in buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, and 1.5 mM MgCl2). Insoluble material was removed by centrifugation at 21,000 ×g for 10 min. The supernatant was used as the nuclear extract. All the protein fractions were stored at −70°C until use, and the protein concentrations were measured with a BCA protein assay kit (ThermoFisher Scientific, Barrington, IL). 2.9. Western Blot Analysis Western blot was performed by the standard method. Equal amounts of proteins were fractionated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to an Immun-Blot PVDF membrane (0.2 μM pore size, Bio-Rad). Membranes were blocked overnight at 4°C in Tris-buffered saline (TBS), 0.05% (v/v) Tween-20, 150 mM NaCl, and 5% (w/v) bovine serum Albumin (BSA, Santa Cruz Biotechnology, Santa Cruz, CA), followed by 2 h incubation with primary antibody diluted in the same buffer (Nrf2 1 : 250, HO-1 1 : 1000, phosphor-p42/44 MAPK (ERK1/2) 1 : 1000, phosphor-SAPK/JNK 1 : 1000, phosphor-p38 MAPK 1 : 1000, and GAPDH 1 : 1000). After washing with 0.1% (v/v) Tween-20 in TBS, the membrane was incubated with anti-rabbit IgG AP-linked secondary antibody for 1 h at room temperature and then washed with the same buffer. The immunoblotted membrane was developed with 5-bromo-4-chloro-3-indoyl phosphate (BCIP)/nitro blue tetrazolium (NBT) color-developing solution. The blots in the samples were quantified by densitometry analysis using PDQuest software (version 7.0, Bio-Rad, Hercules, CA). All data from three independent experiments were expressed as the relative intensity compared to the control group for the statistical analyses. 2.10. DPPH-Scavenging Capacities D and α-tocopherol were evaluated for their activities to scavenge the stable DPPH radical according to a previously described method. The affinity of the test material to quench the DPPH free radical was evaluated according to the equation scavenging % = (Ac − As)/Ac × 100%. As and Ac are the absorbance at 517 nm of the reaction mixture with sample and control, respectively. 2.11. Trolox Equivalent Antioxidant Capacity (TEAC) Analysis The ABTS radical cation was prepared by mixing an ABTS stock solution (7 mM in water) with 2.45 mM potassium persulfate. This mixture has to remain for 12–24 h until the reaction is complete and the absorbance is stable. For measurement, the ABTS•+ solution was diluted to an absorbance of 0.700 ± 0.020 at 734 nm. 1 mL ABTS•+ solution and 100 μL of the antioxidant solution were mixed for 45 s and the absorbance at 734 nm was recorded after 1 min of incubation. TEAC is defined as the concentration (mM) of Trolox having the antioxidant activity equivalent to a 1.0 mM concentration of D. 2.12. Statistical Analysis Data bars represent the means ± SD (standard deviation) for at least three independent experiments in all cases. Two group comparisons were evaluated by student's t-tests as appropriate. Differences were considered statistically significant when P value was <0.05. 3. Results 3.1. Effect of D on Cell Viability of PC12 Cells Initially, the cytotoxic potential of D on PC12 cells was measured. No cytotoxic effects of D were reported up to a concentration of 10 μM, using the WST assay. However, higher amount D reduced the viability of the PC12 cells (Figure 1(a)). Thus, for further experiments, the cells were treated with D in the concentration range of 0.01–10 μM. Furthermore, 10 μM D treatment for 24, 48, and 72 h did not show any toxic effect on cultured PC12 cells (Figure 1(b)). 3.2. Effect of D on HO-1 Expression and HO Activity of PC12 Cells As HO-1 is an important component of the cellular defense against oxidative stress, we assessed whether noncytotoxic concentrations (0.01–10 μM) of D affected HO-1 protein expression and HO activity. PC12 cells exposed to D for 24 h caused a dose-dependent increase in HO-1 expression (Figure 2(a)) and HO activity (Figure 2(c)). At a concentration of 10 μM D, HO-1 induction was evident at 3 h, peaked at around 6 h, and decreased after 12 h in PC12 cells (Figure 2(b)). Consistently, D-induced HO activity directly correlated with enhanced HO-1 protein level (Figure 2(d)). 3.3. Effect of D on Aβ 25–35-Induced Cytotoxicity and Intracellular ROS Generation in PC12 Cells To evaluate the in vitro neuroprotective effect of D, we tested its protective effect on Aβ 25–35-induced cytotoxicity in PC12 cells. PC12 cells were treated with various sub-lethal concentrations of D for 3 h, followed by further incubation for 24 h in the presence or the absence of Aβ 25–35. Treatment with Aβ 25–35 (25 μM) for 24 h induced approximately 40% cell death, whereas D, at non-cytotoxic concentrations (0.01–10 μM), resulted in marked enhancement of survival of the PC12 cells as compared to the Aβ 25–35-treated group (Figure 3(a)). Maximal rescue occurred at a concentration of 10 μM of D. These results showed that Aβ 25–35 treatment significantly reduced the viability of PC12 cells and that D blocked the injury caused by Aβ 25–35. It has been reported that Aβ impairs mitochondrial redox activity and increases the generation of ROS [54–56]. The degree of intracellular ROS generation in cells was measured using fluorescence assay with H2DCF-DA probe. H2DCF-DA can be deacetylated in cells, where it can react quantitatively with intracellular radicals, mainly H2O2, and convert into its fluorescent products DCF, which are retained within the cell. Therefore, this assay provides an index of cell cytosolic oxidation. As shown in Figure 3(b), treatment with D significantly reduced the Aβ 25–35-induced ROS generation, indicating that D attenuates ROS production. Moreover, as shown in Figure 3(c), this effect was dependent on the duration of D pretreatment. Thus, attenuation of ROS released by D required the presence of D at least 3 h prior to the addition of D. These results suggest that D induced the expression of a gene(s) essential to ROS antagonism. 3.4. Effect of HO-1 Expression on Aβ 25–35-Induced Oxidative Neurotoxicity Mediated by D in PC12 Cells Recent reports have described the expression of HO-1 as an adaptive and protective response against oxidative insult in a wide variety of cells, such as PC12 cells. To test whether the protective effect of D is related to its inductive effect on HO-1 expression, we tried to block the activity of HO-1 using Znpp, the inhibitor of HO activity. As shown in Figure 4, D-induced HO-1 expression was required for suppressing Aβ 25–35-induced cell death and ROS generation. The HO-1 inducer CoPP showed comparable protection to D. ZnPP, abrogated the protective effect of D on Aβ 25–35-induced cytotoxicity, and partially reversed the inhibitory effects of D on ROS production. These results suggest that the cytoprotective effect of D is mediated through HO-1 induction. 3.5. Effects of D on Nrf2 Nuclear Translocation in PC12 Cells Several studies have reported that nuclear translocation of activated Nrf2 is an important upstream contributor to the mechanism of HO-1 expression. Therefore, we examined whether D could induce the translocation of Nrf2 to the nucleus in PC12 cells. Using Western blot analysis, we tested the presence of Nrf2 proteins in nuclear compartments of PC12 cells. The cells were incubated with 10 μM D for 0, 1, 3, and 6 h. As shown in Figure 5(a), the nuclear fractions of D-treated PC12 cells showed a gradual increase in Nrf2 levels which was strongly correlated with the increase in HO-1 expression and HO-1 activity (Figure 2), whereas they were decreased concomitantly in the cytoplasmic fractions (Figure 5(b)). 3.6. Involvement of MAPK Pathway in D-Induced HO-1 Expression and Nrf2 Nuclear Translocation in PC12 Cells MAPK is activated in response to oxidative stress and other various stressors. Several studies have demonstrated that the activation of the MAPK pathways is involved in regulating the translocation of Nrf2 and ARE-mediated HO-1 gene expression [57, 58]. To further elucidate the upstream signaling pathway involved in D-mediated Nrf2 activation and HO-1 induction, we examined the effect of D on activation of MAPKs in PC12 cells. Cells were exposed to D, total protein was harvested, and then Western blots were performed using anti-phospho-JNK, ERK, and p38 antibodies. At a concentration of 10 μM, which strongly induced the levels of HO-1, D caused prolonged ERK activation. As illustrated in Figure 6, phosphorylation of ERK was observed 0.5 h after D treatment and was sustained for up to 1 h after D treatment. In contrast, phosphorylation of p38 kinases was decreased. No changes in the expression of phospho-JNK protein were detected, verifying that similar amounts of proteins were loaded in each lane. Next, to address the role of MAPK in D-induced HO-1 expression against Aβ 25–35, we examined the effects of specific inhibitors of ERK (U0126), JNK (SP600125), and p38 (SB203580) on the levels of HO-1 and cell viability, by Western blot and WST assay. As shown in Figures 7(a) and 7(b), The U0126 significantly reduced D-induced HO-1 expression and activity. Likewise, D against Aβ 25–35-induced cell death was effectively abolished by U0126 (Figure 7(c)), whereas p38 inhibitor increased these items. The inhibitor of JNK did not show any changes at any of the tested time periods. Therefore, we suggested that D-induced expression of HO-1 was mediated through ERK pathway phosphorylation and decreased p38 activation in the PC12 cells. U0126, SP600125, and SB203580 alone did not alter cell viability in control or Aβ 25–35-treated cells (data not shown). Furthermore, we examined whether the MAPK pathway was involved in D-induced Nrf2 nuclear translocation. As shown in Figure 7(d), inhibitor of the ERK MAPK pathway blocked D-induced Nrf2 nuclear translocation; on the contrary, p38 inhibitor facilitated the translocation of Nrf2. These results indicate a role for MAPK signaling in D-mediated HO-1 induction through nuclear translocation of Nrf2. 3.7. Effect of D on Free Radical Scavenging Activities To evaluate the antioxidant activity of D, we started by investigating its DPPH-scavenging actions. The DPPH stable free radical method is an easy, rapid, and sensitive way to survey the antioxidant activity of compounds or extracts. Figure 8(a) demonstrates that DPPH-scavenging potentials increased as the concentrations of D and α-tocopherol increased. This result indicates that the antioxidant effect of D is similar to α-tocopherol for trapping DPPH. The TEAC of D was further measured from the decolorization of ABTS•+, which was measured spectrophotometrically at 734 nm. Figure 8(b) shows that D has compatible antioxidant potential with positive control Trolox. 4. Discussion There is considerable evidence supporting oxidative stress is implicated in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD), cerebral ischemia, and Parkinson's disease (PD) [59, 60]. Several lines of evidence indicate that beta amyloid peptide (Aβ) has a causal role in the development and progress of AD. In AD, oxidative stress is suspected to be generated by Aβ [56]. Recent studies showed that there is a vicious circle among Aβ production/accumulation and oxidative stress [61, 62]. Given the important role of oxidative stress in AD, therapeutic strategies which are directed at early interventions targeted at oxidative stress may be effective in delaying Aβ development and slowing its progression. A feasible ways to prevent ROS-mediated cellular damage is to augment the intracellular oxidative defense capacity through dietary or pharmacological intake of antioxidants. Moreover, the induction of endogenous phase II detoxifying enzymes or antioxidative proteins seems to be a reasonable strategy for delaying disease progression and the toxic effects associated with Aβ-mediated cytotoxicity [63–66]. Indeed, increased cellular antioxidant activity confers protection and has been reported to lower the risk of AD [14]. Previous studies have revealed that D could increase cellular resistance to Aβ 25–35-induced oxidative injury in PC12 cells. However, the mechanisms by which D mediates its therapeutic effects against Aβ 25–35-induced neuronal death in vitro remain an interesting speculation that awaits further investigation. Since previous findings support the importance of HO-1 in protection of neurons against Aβ-induced oxidative stress-dependent injury [67], as a part of our continuing research, in this study, we attempted to explain the possible molecular mechanisms underlying the antioxidant effects of D against Aβ-induced oxidative cell death, with focus on upregulation of HO-1 and the underlying regulatory signaling pathways. The extent and the rapidity of quenching the DPPH free radical and ABTS radical cation are the criteria commonly used to assess relative antioxidant capacity of flavonoid compared with standard antioxidants, α-tocopherol, and Trolox [68, 69]. Current research demonstrates that D has compatible antioxidant potential with positive control, α-tocopherol, and Trolox (Figure 8). These results showed that D possesses powerful antioxidant potency, which are consistent with the previous reports showing that D is an oxygen-free radical scavenger. This was also confirmed with our cell-based antioxidant assay in which D significantly reduced the basal intracellular level of ROS (Figure 3). Aβ 25–35 caused a significant decrease in PC12 cell viability, and pretreatment cells with D concentration-dependently increased cell viability (Figure 4(a)). These results showed a clear and strong correlation among free radical-quenching activities, ROS scavenging activity, and the enhanced resistance to Aβ 25–35-induced oxidative damage. As a consequence, the intrinsic antioxidant capacity may play a role for D, in contributing towards the partial or total alleviation of cellular oxidative stress. HO-1, also known as heat-shock protein 32 (HSP 32) or inducible HO, is a 32-kDa protein transiently activated by a wide variety of noxious stimuli including oxidative stress. Upregulation of HO-1 leading to elevation of HO activity has been shown to provide neuroprotective effects by converting the prooxidant heme to biologically active antioxidant by-products such as biliverdin/bilirubin and also to heme inactivating carbon monoxide. The transgenic mice overexpression HO-1 in the brain attenuated neuronal cell injury caused by ischemic stroke [70]. HO-1 overexpressing cells derived from transgenic mice or the cells treated with a HO-1 inducer are relatively resistant to oxidative stress [20, 22]. Induction of HO-1 is highly recognized as an important therapeutic target for pharmacological intervention of oxidative disorders. It has been suggested that an elevation of HO-1 by various stimuli may be protective cellular response to delay the cell death. Therefore, we are interested in determining the potential role of HO-1 in the Aβ 25–35-induced PC12 cells damage and the D-mediated neuroprotection. We have provided evidence for the induction of HO-1 by D in PC12 cells and showed that D-induced HO-1 protein expression and HO activity occurred in a concentration- and time-dependent manner (Figure 2). Furthermore, the increase of HO-1 expression by D conferred cytoprotection against Aβ 25–35-induced oxidative stress (Figure 3). In addition, we showed that ZnPP, a potent inhibitor of HO activity, can partially reverse the protective effects of D, thus providing further evidence for HO-1 as a possible cytoprotective pathway for D (Figure 4(a)). The induction of HO-1 expression was also required to suppress Aβ 25–35-induced ROS generation (Figure 4(b)). These results strongly indicate that in our experimental setting, D may have multiple mechanisms of action that affect cytoprotection both by reducing ROS generation and boosting HO-1 induction for ROS detoxification. The central sensor of intracellular oxidative stress is the cytosolic Keap1-Nrf2 complex. In response to oxidative stress, Nrf2 is released from Keap1 and transmits the stress signal to the nucleus for activation of distinct set of genes encoding phase II detoxifying enzymes as well as several stress responsive proteins including HO-1 [71, 72]. Recently, Nrf2-ARE signaling pathways have emerged as key therapeutic targets for treatment of a variety of oxidative stress-related neurodegenerative insults [73]. Nrf2-null mice resulted in a decrease in the basal expression level of detoxifying or antioxidant genes including HO-1 [74]. In contrast, Nrf2+/+ mice protect the brain from cerebral ischemia in vivo [75], whereas primary neuronal cultures treated with chemical activators of the Nrf2-ARE pathway displayed significantly greater resistance to oxidative stress-induced neurotoxicity [73]. Phytochemicals including sulforaphane, caffeic acid phenethyl ester (CAPE), and curcumin activate the Nrf2-ARE system [76, 77]. Using Western blot, we examined whether D activated Nrf2 in PC12 cells and found that Nrf2 was promoted translocation into the nucleus in PC12 cells exposed to a nontoxic concentration of D (Figure 5). The translocation of Nrf2 into the nucleus following D treatment was associated with a marked increase in HO-1 induction (Figure 2). Therefore, our results suggest that the transcriptional activation of Nrf2 is involved in the increased expression of HO-1 and the cytoprotection against Aβ 25–35 induced in PC12 cells (Figure 7(c)). The upstream signaling pathways regulating Nrf2 transactivation remain poorly defined. Recent studies have implicated a major role for the MAPK in Nrf2-dependent translocation and HO-1 activation, although other kinases including tyrosine kinases, PI3K, and PKC have also emerged as potential contributing mechanisms coordinately or separately [78, 79]. In vertebrates, the three major kinase cascades are represented by ERK, JNK, and p38 MAPK [80]. The current experiments were designed to verify a possible role of MAPK pathway in D-induced Nrf2 activation and HO-1 expression, and D was found to facilitate the phosphorylation of ERK and dephosphorylation of p38 (Figure 6). In addition, upregulation of HO-1 as well as Nrf2 nuclear translocation by D was remarkably inhibited by U0126, a highly selective inhibitor of ERK pathway. These investigations suggested that the activation of ERK pathway by D contributed to HO-1 expression (Figures 7(a) and 7(b)), Nrf2 nuclear translocation (Figure 7(d)), and cytoprotection (Figure 7(c)) in PC12 cells. It is generally acknowledged that MAPK can be differentially regulated by the same stimuli in diverse cell types. ERK pathway is thought to mediate cellular responses to growth and differentiation factors, whereas JNK and p38 pathway is activated by distinct and overlapping sets of stress-related stimuli [81]. For instance, quercetin induces Nrf2 nuclear translocation and HO-1 upregulation via the ERK and p38 pathway but not the JNK pathway in human hepatocytes [82]. 3H-1, 2-dithiole-3-thione increases nuclear Nrf2 accumulation via the ERK pathway but not the p38 and JNK pathway in murine keratinocytes [83]. In PC12 cells, 15-Deoxy-D12, 14-prostaglandin J2 induces HO-1expression via the ERK and Akt/PI3K pathway but not the p38 and JNK pathway [84]. Shen et al. [79] have shown that the transcriptional activity of Nrf2 transactivation domain was stimulated by ERK and JNK signaling pathways while the p38 MAPK plays a negative role. This may be due to the diverse assortment and intensity of the signaling pathways activated by different inducers in different cell types. However, further studies are extensively ongoing to define the exact role of the MAPK pathways in HO-1 expression. In summary, the intrinsic free radical scavenging activity and inducing the upregulation of HO-1 expression through activation of Nrf2 exposed to D confer protection against the Aβ 25–35-induced oxidative damage in PC12 cells. One of the most salient features of our present study is that ERK is involved in HO-1 induction via Nrf2 activation in the D-stimulated cells. Thus, pharmacological inhibition of ERK suppressed Nrf2 activation and subsequent HO-1 expression. Results of our study imply the potential involvement of these upstream kinases in Nrf2 activation and HO-1 upregulation by D. However, the complete molecular milieu that links all these events needs to be elucidated. Continued attempts to identify novel target molecules responsible for the HO-1 regulation and to clarify their cross-talk with upstream and downstream signaling molecules will pave the way to exploiting preventive and/or therapeutic strategies for the management of oxidative stress-mediated disorders. Moreover, considering that the D has a property that can cross the blood-brain barrier, further in vivo study with D should substantiate this therapeutic potential of this compound. These findings indicate that D might prove to be a promising therapeutic approach to combat neural demise in AD and other oxidative stress-related diseases. Acknowledgment The authors gratefully acknowledge the financial support of this work by Guangdong Medical College (B2011011). Figure 1 Effect of D on cell viability of PC12 cells. (a) PC12 cells were incubated for 24 h with various concentrations of D (0.01–10 μM). (b) PC12 cells were incubated with 10 μM D for 24, 48, and 72 h. Cell viability was estimated by the WST-8 assay. Data are expressed as percent of values in untreated control cultures and represent the means ± SD for three experiments with triplicates. P < 0.05 compared with control. Figure 2 Effects of D on HO-1 expression and HO activity in PC12 cells. (a) Cells were incubated with various concentrations of D for 24 h. (b) Cells were incubated for indicated periods with 10 μM of D. Expression of HO-1 was determined by Western blot analysis, and representative blots of three independent experiments are shown. (c) HO activity was determined via bilirubin formation at 24 h after treatment with various concentrations of D. (d) PC12 cells were treated with 10 μM of D, and HO activity was measured at the indicated time points. Each bar represents the means ± SD of three independent experiments with triplicates. P < 0.05 compared with control. P < 0.01 compared with control. P < 0.001 compared with control. Figure 3 Effect of D on Aβ 25–35-induced oxidative neurotoxicity and intracellular ROS generation in PC12 cells. Effect of D on Aβ 25–35-induced oxidative neurotoxicity in PC12 cells. (a) PC12 cells were pretreated with various concentrations of D for 3 h and then incubated with and without 25 μM Aβ 25–35 for 24 h. Cell viability was estimated by the WST-8 assay. Effect of D on Aβ 25–35-induced intracellular ROS generation in PC12 cells. (b) PC12 cells were pretreated with various concentrations of D for 3 h and then incubated with and without 25 μM Aβ 25–35 for 24 h. After that the cells were washed with PBS and then treated with 10 μM H2DCF-DA for 30 min. (c) PC12 cells were incubated with D (10μM) for the indicated time. They were then treated with 25 μM Aβ 25–35 for 24 h and with 10 μM H2DCF-DA for 30 min. Intracellular ROS production was measured at the excitation wavelength of 485 nm and the emission wavelength of 530 nm using the fluorescence microplate reader. Each bar represents mean ± SD from three experiments with triplicates. P < 0.05 compared with control. P < 0.01 compared with control. P < 0.001 compared with control. # P < 0.05 compared with the group treated by Aβ 25–35 alone. ### P < 0.001 compared with the group treated by Aβ 25–35 alone. Figure 4 Effect of HO-1 expression on Aβ 25–35-induced oxidative neurotoxicity mediated by D in PC12 cells. (a) Cells were treated with 10 μM of D or 20 μM CoPP in the presence or absence of 50 μM ZnPP and then exposed to Aβ 25–35 (25 μM) for 24 h. (b) Exposure of PC12 cells to 25 μM Aβ 25–35 for 24 h increased ROS production. D-induced HO-1 effectively inhibited ROS production. Each bar represents mean ± SD from three experiments with triplicates. P < 0.01 compared with control. *P < 0.001 compared with control. ## P < 0.01 compared with the group treated by Aβ 25–35 alone. ### P < 0.001 compared with the group treated by Aβ 25–35 alone. Figure 5 Effects of D on Nrf2 nuclear translocation in PC12 cells. Cells were treated with 10 μM D for 0, 1, 3, and 6 h, after which the nuclear (a) and cytosolic (b) Nrf2 proteins were determined by Western blot analyses. Data shown represent the means ± SD expressed as fold of 0 h group values obtained from three separated experiments with triplicates. P < 0.05 compared with 0 h group. *P < 0.01 compared with 0 h group. Figure 6 Effects of D on ERK (a), JNK (b), and p38 MAPK (c) in PC12 cells. Cells were treated with 10 μM D for the indicated times. Cell extracts were analyzed by Western blot with antibodies specific for phosphorylated ERK (p-ERK), phosphorylated JNK (p-JNK), or phosphorylated p38 (p-p38). Data shown represent the means ± SD expressed as fold of 0 h group values obtained from three separated experiments with triplicates. P < 0.05 compared with 0 h group. *P < 0.01 compared with 0 h group. Figure 7 Effects of D-induced MAPK activation on expression (a) and activity (b) of HO-1, neurotoxicity (c), and Nrf2 translocation (d) in Aβ 25–35-induced PC12 cells. Cells were treated with 10 μM D with and without the inhibitors of MAPK. Cell extracts were analyzed by Western blot with specific antibodies. Each bar represents means ± SD from three experiments with triplicates. P < 0.05 compared with control. **P < 0.01 compared with control. # P < 0.05 compared with the group treated by Aβ 25–35 alone. ## P < 0.01 compared with the group treated by Aβ 25–35 alone. ### P < 0.001 compared with the group treated by Aβ 25–35 alone. Figure 8 Effects of D on DPPH (a) and ABTS•+ (b) free radical scavenging. Data represent the means ± SD of three independent experiments. ==== Refs 1 Bonda DJ Wang X Perry G Oxidative stress in Alzheimer disease: a possibility for prevention Neuropharmacology 2010 59 4-5 290 294 20394761 2 Chauhan V Chauhan A Oxidative stress in Alzheimer’s disease Pathophysiology 2006 13 3 195 208 16781128 3 Gibson GE Huang HM Oxidative processes in the brain and non-neuronal tissues as biomarkers of Alzheimer’s disease Frontiers Bioscience 2002 1 7 1007 1015 4 Montine TJ Neely MD Quinn JF Lipid peroxidation in aging brain and Alzheimer’s disease Free Radical Biology Medcine 2002 33 5 620 626 5 Arlt S Beisiegel U Kontush A Lipid peroxidation in neurodegeneration: new insights into Alzheimer’s disease Current Opinion in Lipidology 2002 13 3 289 294 2-s2.0-0035985698 12045399 6 Lyras L Caims NJ Jenner A Jenner P Halliwell B An assessment of oxidative damage to proteins, lipids, and DNA in brain from patients with Alzheimer’s disease Journal of Neurochemstry 1997 68 5 2061 2069 7 Gabbita SP Lovell MA Markesbery WR Increased nuclear DNA oxidation in the brain in Alzheimer’s disease Journal of Neurochemstry 1999 71 5 2034 2040 8 Lovell MA Gabbita SP Markesbery WR Increased DNA oxidation and decreased levels of repair products in Alzheimer’s disease ventricular CSF Journal of Neurochemistry 1999 72 2 771 776 2-s2.0-0032933750 9930752 9 Harris ME Hensley K Butterfield DA Leedle RA Carney JM Direct evidence of oxidative injury produced by the Alzheimer’s β -amyloid peptide (1–40) in cultured hippocampal neurons Experimental Neurology 1995 131 2 193 202 2-s2.0-0028908516 7895820 10 Hensley K Butterfield DA Mattson M A model for beta-amyloid aggregation and neurotoxicity based on the free radical generating capacity of the peptide: implications of ‘molecular shrapnel’for Alzheimer’s disease Proceeding of Western Pharmacology Society 1995 38 113 120 11 Kadowaki H Nishitoh H Urano F Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation Cell Death Differentiation 2005 12 1 19 24 15592360 12 Monji A Utsumi H Ueda T The relationship between the aggregational state of the amyloid-beta peptides and free radical generation by the peptides Journal of Neurochemstry 2001 77 6 1425 1432 13 Sponne I Fifre A Drouet B Apoptotic neuronal cell death induced by the non-fibrillar amyloid-beta peptide proceeds through an early reactive oxygen species-dependent cytoskeleton perturbation The Journal of Biological Chemistry 2003 278 5 3437 3445 12435748 14 Engelhart MJ Geerlings MI Ruitenberg A Diet and risk of dementia: does fat matter? The Rotterdam study Neurology 2002 59 12 1915 1921 2-s2.0-0037168780 12499483 15 Schipper HM Heme oxygenase expression in human central nervous system disorders Free Radical Biology and Medicine 2004 37 12 1995 2011 2-s2.0-8544257019 15544918 16 Satoh T Baba M Nakatsuka D Role of heme oxygenase-1 protein in the neuroprotective effects of cyclopentenone prostaglandin derivatives under oxidative stress The European Journal of Neuroscience 2003 17 11 2249 2255 12814358 17 Maines MD The heme oxygenase system: a regulator of second messenger gases Annual Review of Pharmacology and Toxicology 1997 37 517 554 2-s2.0-0030923630 18 Prawan A Kundu JK Surh YJ Molecular basis of heme oxygenase-1 induction: implications for chemoprevention and chemoprotection Antioxidants and Redox Signaling 2005 7 11-12 1688 1703 2-s2.0-28844446987 16356130 19 Ryter SW Choi AM Heme oxygenase-1: redox regulation of a stress protein in lung and cell culture models Antioxidants Redox Signaling 2005 7 1-2 80 91 15650398 20 Chen K Gunter K Maines MD Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death Journal of Neurochemistry 2000 75 1 304 313 10854275 21 Hung SY Liou HC Kang KH Overexpression of heme oxygenase-1 protects dopaminergic neurons against 1-methyl-4-phenylpyridinium-induced neurotoxicity Molecular Pharmacology 2008 74 6 1564 1575 18799798 22 Le WD Xie WJ Appel SH Protective role of hemeoxygenase-1 in oxidative stress-induced neuronal injury Journal of Neuroscience Research 1999 56 6 652 658 10374820 23 Schipper HM Gupta A Szarek WA Suppression of glial HO-1 activity as a potential neurotherapeutic intervention in AD Current Alzheimer Research 2009 6 5 424 430 19874266 24 Lee MS Lee J Kwon DY Kim MS Ondamtanggamibang protects neurons from oxidative stress with induction of heme oxygenase-1 Journal of Ethnopharmacology 2006 108 2 294 298 16806762 25 Qiang W Cahill JM Liu J Activation of transcription factor Nrf-2 and its downstream targets in response to moloney murine leukemia virusts-induced thiol depletion and oxidative stress in astrocytes Journal of Virology 2004 78 21 111926 111938 26 Kim KM Pae HO Zheng M Park R Kim YM Chung HT Carbon monoxide induces heme oxygenase-1 via activation of protein kinase R-like endoplasmic reticulum kinase and inhibits endothelial cell apoptosis triggered by endoplasmic reticulum stress Circulation Research 2007 101 9 919 927 2-s2.0-37349006900 17823375 27 Kensler TW Wakabayashi N Biswal S Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway Annual Review of Pharmacology Toxicology 2007 47 89 116 28 Lee JM Shih AY Murphy TH NF-E2-related factor-2 mediates neuroprotection against mitochondrial complex I inhibitors and increased concentrations of intracellular calcium in primary cortical neurons The Journal of Biological Chemistry 2003 278 39 37948 37956 12842875 29 Yang C Zhang X Fan H Liu Y Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia Brain Research 2009 1282 133 141 2-s2.0-67649670106 19445907 30 Radjendirane V Joseph P Lee YH Disruption of the DT diaphorase (NQO1) gene in mice leads to increased menadione toxicity Journal of Biological Chemistry 1998 273 13 7382 7389 2-s2.0-0032571361 9516435 31 Shih AY Johnson DA Wong G Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress The Journal of Neuroscience 2003 23 8 3394 3406 12716947 32 Kong ANT Owuor E Yu R Induction of xenobiotic enzymes by the map kinase pathway and the antioxidant or electrophile response element (ARE/EpRE) Drug Metabolism Reviews 2001 33 3-4 255 271 2-s2.0-0035212419 11768769 33 Nakaso K Yano H Fukuhara Y PI3K is a key molecule in the Nrf2-mediated regulation of antioxidative proteins by hemin in human neuroblastoma cells FEBS Letters 2003 546 2-3 181 184 12832036 34 Numazawa S Ishikawa M Yoshida A Tanaka S Yoshida T A typical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress American Journal of Physiology Cell Physiology 2003 285 2 334 342 35 Mielke K Herdegen T JNK and p38 stress kinases-degenerative effectors of signal-transduction-cascades in the nervous system Progress Neurobiology 2000 61 1 45 60 36 Stanciu M Wang Y Kentor R Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a neuronal cell line and primary cortical neuron cultures The Journal of Biological Chemistry 2000 275 16 12200 12206 10766856 37 Iles KE Dickinson DA Wigley AF HNE increases HO-1 through activation of the ERK pathway in pulmonary epithelial cells Free Radical Biology Medcine 2005 39 3 355 354 38 Sarker SD Nahar L Natural medicine: the genus Angelica , Current Medcinal Chemistry 2004 11 3 1479 1500 39 Lee S Yeon SL Sang HJ Kuk HS Kim BK Sam SK Anti-tumor activities of decursinol angelate and decursin from Angelica gigas Archives of Pharmacal Research 2003 26 9 727 730 2-s2.0-12944306438 14560921 40 Kim HH Ahn KS Han H Choung SY Choi SY Kim IH Decursin and PDBu: two PKC activators distinctively acting in the megakaryocytic differentiation of K562 human erythroleukemia cells Leukemia Research 2005 29 12 1407 1413 2-s2.0-27644490806 15992925 41 Hyeon HK Sung SB Jin SC Han H Kim IH Involvement of PKC and ROS in the cytotoxic mechanism of anti-leukemic decursin and its derivatives and their structure-activity relationship in human K562 erythroleukemia and U937 myeloleukemia cells Cancer Letters 2005 223 2 191 201 2-s2.0-19344368158 15896453 42 Jiang C Lee HJ Li GX Potent antiandrogen and androgen receptor activities of an Angelica gigas -containing herbal formulation: identification of decursin as a novel and active compound with implications for prevention and treatment of prostate cancer Cancer Research 2006 66 1 453 463 2-s2.0-31544472904 16397261 43 Song GY Lee JH Cho M Decursin suppresses human androgen-independent PC3 prostate cancer cell proliferation by promoting the degradation of beta-catenin Molecular Pharmacology 2007 72 6 1599 1606 17855653 44 Lee S Shin DS Ju SK Oh KB Sam SK Antibacterial coumarins from Angelica gigas roots Archives of Pharmacal Research 2003 26 6 449 452 2-s2.0-2142854619 12877552 45 Lee YY Lee S Jin JL Yun-Choi HS Platelet anti-aggregatory effects of coumarins from the roots of Angelica genuflexa and A. gigas Archives Pharmacal Research 2003 26 9 723 726 46 Shiomi K Hatano H Morimoto H Decursin and decursinol angelate selectively inhibit NADH-Fumarate reductase of ascarissuum Planta Medica 2007 73 14 1478 1481 17948169 47 Lee S Lee YS Jung SH Shin KH Kim BK Kang SS Antioxidant activities of decursinol angelate and decursin from Angelica gigas roots Natural Product Sciences 2003 9 3 170 173 2-s2.0-0141886286 48 So YK Kim YC Decursinol and decursin protect primary cultured rat cortical cells from glutamate-induced neurotoxicity Journal of Pharmacy and Pharmacology 2007 59 6 863 870 2-s2.0-34447295443 17637179 49 Kim DH Kim DY Kim YC Nodakenin, a coumarin compound, ameliorates scopolamine-induced memory disruption in mice Life Sciences 2007 80 21 1944 1950 17382968 50 Kang SY Lee KY Sung SH Four new neuroprotective dihydropyrano coumarins from Angelica gigas Journal of Natural Products 2005 68 1 56 59 15679317 51 Li L Li W Jung SW Lee YW Kim YH Protective effects of decursin and decursinol angelate against amyloid β -protein-induced oxidative stress in the PC12 cell line: the role of Nrf2 and antioxidant enzymes Bioscience, Biotechnology and Biochemistry 2011 75 3 434 442 2-s2.0-79953771772 52 Kim KM Jung JY Hwang SW Kim MJ Kang JS Isolation and purification of decursin and decursinol angelate in Angelica gigas Nakai Journal of the Korean Society of Food Science and Nutrition 2009 38 5 653 656 2-s2.0-68949161768 53 Xu H Want H Zhuang LX Demonstration of an anti-oxidative stress mechanism of quetiapine: implications for the treatment of Alzheimer's disease The FEBS Journal 2009 275 14 3718 3728 54 Hensley K Carney JM Mattson MP A model for β -amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease Proceedings of the National Academy of Sciences of the United States of America 1994 91 8 3270 3274 2-s2.0-0028180518 8159737 55 Shearman MS Ragan CI Iversen LL Inhibition of PC12 cell redox activity is a specific, early indicator of the mechanism of beta-amyloid-mediated cell death Proceeding of the National Academy of Sciencesof the United States of America 1994 91 4 1470 1474 56 Behl C Davis JB Lesley R Hydrogen peroxide mediates amyloid beta protein toxicity Cell 1994 77 6 817 827 8004671 57 Kietzmann T Samoylenko A Immenschuh S Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes Journal of Biological Chemistry 2003 278 20 17927 17936 2-s2.0-0037705359 12637567 58 Choi BM Kim YM Jeong YR Induction of heme oxygenase-1 is involved in anti-proliferative effects of paclitaxel on rat vascular smooth muscle cells Biochemicaland Biophysical Research Communications 2004 321 1 132 137 59 Markesbery WR Neuropathological criteria for the diagnosis of Alzheimer's disease Neurobiology Aging 1997 18 4 13 19 60 Reddy PH Mitochondrial oxidative damage in aging and Alzheimer’s disease: implications for mitochondrially targeted antioxidant therapeutics Journal of Biomedicine and Biotechnology 2006 2006 13 pages 2-s2.0-33646349444 31372 61 Shen C Chen Y Liu H Hydrogen peroxide promotes Abeta production through JNK-dependent activation of gamma-secretase The Journal of Biology Chemistry 2008 283 25 17721 17730 62 Tamagno E Guglielmotto M Aragno M Oxidative stress activates a positive feedback between the gamma- and beta-secretase cleavages of the beta-amyloid precursor protein Journal of Neurochemstry 2008 104 3 683 695 63 Jen LS Hart AI Jen A Alzheimer’s peptide kills cells of retina in vivo Nature 1998 392 6672 140 141 2-s2.0-0032510580 9515959 64 Jang JH Surh YJ Protective effect of resveratrol on beta-amyloid-induced oxidative PC12 cell death Free Radical Biology Medicine 2003 34 8 1100 1110 12684095 65 Pappolla MA Sos M Omar RA Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide The Journal of Neuroscience 1997 17 5 1683 1690 9030627 66 Qin L Liu Y Cooper C Microglia enhance beta-amyloid peptide-induced toxicity in cortical and mesencephalic neurons by producing reactive oxygen species Journal of Neurochemitry 2002 83 4 973 983 67 Wruck CJ Ötz MEG Herdegen T Kavalactones protect neural cells against amyloid beta peptide-induced neurotoxicity via extracellular signal-regulated kinase 1/2-dependent nuclear factor erythroid 2-related factor 2 activation Molecular Pharmacology 2008 73 6 1785 1795 18334601 68 Tang YZ Liu ZQ Free-radical-scavenging effect of carbazole derivatives on DPPH and ABTS radicals Journal of American Oil Chemistry Society 2007 84 1095 1100 69 Musialik M Litwinienko G Scavenging of dpph• radicals by vitamin E is accelerated by its partial ionization: the role of sequential proton loss electron transfer Organic Letters 2005 7 22 4951 4954 2-s2.0-27644539327 16235930 70 Panahian N Yoshiura M Maines MD Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice Journal of Neurochemistry 1999 72 3 1187 1203 2-s2.0-0032972686 10037492 71 Itoh K Ishii T Wakabayashi N Yamamoto M Regulatory mechanisms of cellular response to oxidative stress Free Radical Research 1999 31 4 319 324 2-s2.0-0032827002 10517536 72 Itoh K Wakabayashi N Katoh Y Keap1 regulates both cytoplasmic-nuclearshuttling and degradation of Nrf2 in response to electrophiles Genes Cells 2003 8 4 379 391 12653965 73 Johnson JA Johnson DA Kraft AD The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration Annals of the New York Academy of Sciences 2008 1147 61 69 19076431 74 Lee JS Surh YJ Nrf2 as a novel molecular target for chemoprevention Cancer Letter 2005 224 2 171 184 75 Shih AY Imbeault S Barakauskas V Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo The Journal of Biology Chemistry 2005 280 24 22925 22936 76 Chen C Kong ANT Dietary chemopreventive compounds and ARE/EpRE signaling Free Radical Biology and Medicine 2004 36 12 1505 1516 2-s2.0-2942568014 15182853 77 Morimitsu Y Nakagawa Y Hayashi K A sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway The Journal of Biology Chemistry 2002 277 5 3456 3463 78 Ryter SW Tyrrell RM Singlet molecular oxygen: a possible effector of eukaryotic gene expression Free Radical Biology Medcine 1998 24 9 1520 1534 79 Shen CP Tsimberg Y Salvadore C Meller E Activation of Erk and JNK MAPK pathways by acute swim stress in rat brain regions BMC Neuroscience 2004 5 article 36 2-s2.0-12944268757 80 Johnson GL Lapadat R Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases Science 2002 298 5600 1911 1912 2-s2.0-0037032817 12471242 81 Alam J Wicks C Stewart D Mechanism of hemeoxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells The Journal of Biology Chemistry 2000 275 36 27694 27702 82 Yao P Nussler A Liu L Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways Journal of Hepatology 2007 47 2 253 261 2-s2.0-34347392509 17433488 83 Manandhar S Cho JM Kim JA Kensler TW Kwak MK Induction of Nrf2-regulated genes by 3H-1, 2-dithiole-3-thione through the ERK signaling pathway in murine keratinocytes European Journal of Pharmacology 2007 577 1–3 17 27 17854798 84 Kim JW Li MH Jang JH 15-Deoxy-D12, 14-prostaglandin J2 rescues PC12 cells from H2 O2 -induced apoptosis through Nrf2-mediated upregulation of heme oxygenase-1 potential roles of Akt and ERK1-2 Biochemical Pharmacology 2008 76 11 1577 1589 18775681	23762139	PMC3665219	CC BY	2021-01-05 10:53:12	yes	Evid Based Complement Alternat Med. 2013 May 9; 2013:467245
==== Front J Anal Methods ChemJ Anal Methods ChemJAMCJournal of Analytical Methods in Chemistry2090-88652090-8873Hindawi Publishing Corporation 10.1155/2013/617243Research ArticleThe Studies of Chlorogenic Acid Antitumor Mechanism by Gene Chip Detection: The Immune Pathway Gene Expression 0000-0002-8444-9889Kang Tian Yi 1 Yang Hua Rong 2 Zhang Jie 2 Li Dan 1 Lin Jie 1 Wang Li 1 Xu XiaoPing 1 1West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan Province, China2Jiuzhang Biochemical Engineering Science and Technology Development Co., Ltd, Chengdu 610041, Sichuan Province, ChinaXiaoPing Xu: xu106@sina.comAcademic Editor: Yu-Ming Fan 2013 9 5 2013 2013 61724331 1 2013 4 4 2013 Copyright © 2013 Tian Yi Kang et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Persistently increasing incident of cancer in human beings has served to emphasize the importance of studies on mechanism of antitumor substances. Chlorogenic acid (CA), extracted from folium cortex eucommiae, has been confirmed to have lots of biological activities encompassing inhibition of tumor. However, the anticancer mechanism of CA remains unclear. Here, we have utilized a whole mouse genome oligo microarray (4∗44K) to analyze gene expression level of female BALB/c mice (implanted with EMT-6 sarcoma cells) after treatment with low, medium, and high-dose CA (5 mg/kg, 10 mg/kg, and 20 mg/kg), docetaxel, interferon, and normal saline separately at 6 time points (3rd, 6th, 9th, 12th, 15th, and 18th days after administration). Differentially expressed genes screened out by time-series analysis, GO analysis, and pathway analysis, and four immune-related genes were selected for further confirmation using RT-qPCR. The results demonstrated that CA is able to change gene expression and that the responsive genes (CaN, NFATC2, NFATC2ip, and NFATC3) involved in immune pathways had been significantly upregulated by CA. Expression of immune factors such as IL-2R and IFN-γ can be improved by CA to promote activation and proliferation of T cells, macrophages, and NK cells, thus enhancing their surveillance and killing abilities, further suppressing the growth rate of tumor cells. ==== Body 1. Introduction Cancer dormant cell theory takes the view that activation of resting cells is the key factor causing cancer metastasis. Recent studies indicate that resting cells can be activated through escaping immune surveillance when immune function is weakened and that energy used for cell revivals can be provided by the newborn vessels [1–3]. Therefore, the study on improving body immune surveillance and restraining the energy of tumor growth will be an emergent mission as well as a breakthrough in the field of clinical treatment of cancer. Chlorogenic acid (CA), extracted from folium cortex eucommiae and the flower bud of Lonicera confusa, is a kind of depside formed by caffeic acid and quinic acid. A large number of studies on CA have demonstrated that CA has a wide range of biological activities including inhibition of tumor cells [4]. According to preliminary studies, we recognize that CA is able to inhibit tumor in mouse except for these with T-cells defect, which suggests that immune system can be one of the targets of tumor suppression. Meanwhile, in vivo studies of our group indicate that CA also changes the advantage state of Th2 drift of BALB/c EMT-6 mice. It significantly enhance the activities of BALB/c EMT-6 mice cytotoxic T lymphocyte and natural killer cells as well as strengthening macrophage phagocytosis activity and lymphocyte transcription activity, thus boosting specific and nonspecific cellular immune function to tumor cells. Recent studies show that antitumor property of CA may have a connection with its ability of enhancing the activities of aryl hydrocarbon hydroxylase, suppressing formation of 8-OH-dG, carcinogen-DNA adduct, and oxygen radical [5, 6]. Meanwhile, CA can guard against gastric cancer and colon cancer and even suppress related carcinogenic factors [7, 8]. In vitro studies revealed that CA can enhance T-cell proliferation caused by influenza virus antigen and can induce the generation of IFN-γ and IFN-α by human lymphocytes and peripheral blood leukocytes [9, 10]. Additionally, we find that CA can also activate neurocalcin to strengthen the activity of macrophagocyte [11]. Although much evidence has proved the anticancer property of CA, little is known about its exact targets on molecular level. Base on the principle of complementary base pairing, microarray technology can distinguish particular genes from the mixture of genes by taking advantage of gene probes. Different from normal PCR, RT-qPCR can take quantitative analysis of unknown system. Meanwhile, its sensitivity, accuracy, and specificity are better than those of normal PCR. To explore the expression level of genes in tumor cells, we utilized a microarray technique to detect BALB/c EMT-6 mice after treatment with CA, docetaxel, interferon, normal saline separately, and the differences in expression level were confirmed by RT-qPCR. Time-series analysis, GO analysis, and pathway analysis were used to screen out common genes and analyze the relationship between putative genes and anticancer process of CA. Our data has suggested that CA is able to inhibit the growth of tumor through regulating immune system. 2. Materials and Methods 2.1. Animal Model Construction and CA Treatment Female SPF mice (BALB/c) used in this experiment weigh 17-18 g on average, provided by the animal center of Sichuan University. EMT-6 sarcoma cells were provided by West China Hospital of Sichuan University Department of Health Engineering Key Laboratory of Transplantation and Transplantation Immunity. We took EMT-6 cell line out of the −152°C ultra low temperature refrigerator. After thawing, centrifuging, and primary culturing, we used 0.25% trypsin for digestion twice and then subcultured it to a required number. All the collected cells were diluted with phosphate-buffered saline (PBS) in the end. Each BALB/c mouse was injected with 0.2 mL cell solution. The tumor would not transfer until it grew to a certain size. We homogenized the tumor taken from the body of BALB/c tumor-bearing mice to cell suspension and then inoculated the cell suspension to other BALB/c mice. Mice used for microarray analysis were injected with high-dose CA (experimental group), docetaxel (control group), and normal saline (negative control group). Gene expression of experimental group was analyzed at six time points (3rd, 6th, 9th, 12th, 15th, and 18th days, resp., after administration), while the control group and negative control group were both analyzed at the 12th days after administration. RT-qPCR took mice injected with low, medium, and high-dose CA (5 mg/kg, 10 mg/kg, 20 mg/kg) as experimental group, docetaxel and interferon as control group, and normal saline as negative control group. Each of these groups was tested 12 days after administration. 2.2. RNA Extraction and Labeling Total RNA was extracted using mirVana RNA Isolation Kit (Applied Biosystem p/n AM1556) in 15 min after tissue collection. Quality and concentration of RNA were checked by spectrophotometer analysis and gel electrophoresis. All extracted samples had an A260/280 ratio of between 2.0 and 2.1 and a 28S/18S ratio of 2 [12]. Agilent 2100 Bioanalyzer was used for further verification and qualified RNA had an RIN greater than 7.0. Total RNA was transcribed to double-stranded cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas), with T7 Promoter Primer. The aaUTP-labeled cRNA was produced from cDNA by in vitro transcription. cRNA was dyed with Cy3 and purified using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the protocol of the RNeasy Plus Mini Kit. 2.3. Hybridization and Scanning Cy3 cRNA must be fragmented in a buffer of 11 μL 10× blocking agent, 2.2 μL 25× fragmentation buffer and a certain amount of nuclease-free water at 60°C for half an hour before hybridization. We added 55 μL 2× GEx hybridization buffer to the denatured and fragmented cRNA and then transfered them to microarrays at 65°C for 17 h with constant rotation. After hybridization, the microarrays were washed twice with buffer 1 for 1 minute followed by buffer 2 at 37°C for 1 minute too. In the end, the microarrays were autoscanned twice in 100% and 10% PMT, respectively, at 5 μm resolution. The data was analyzed by Agilent Feature Extraction software and the quantile normalization was finished by GeneSpring 10.0. 2.4. Microarray Data Preprocessing After acquisition of raw data, Agilent Feature Extraction software kit was used to eliminate the effect of background signals through automatic gridding. We took the log 2 of the normalized background adjusted values to narrow the threshold of fluorescence signal strength before quantile normalization by GeneSpring 10.0 and then used a linear model to estimate expression values on the log scale. Both programs were available in the limma package. We chose a standard among the microarray samples thus the whole microarray data varied on it to obtain the same baseline of average density. Unqualified probes should be filtered out. Differentially expressed genes are submitted to Student's t-test and P ≤ 0.05. 2.5. Interaction Network Analysis We inputted the data of probes used for gene expressing analysis into SBC Analysis System (V2010.05). 2.6. RT-qPCR RNA extracted from experimental group of CA, positive control group of docetaxel, and interferon, negative control group were used to measure expression of selected genes of interest by RT-qPCR. Genes CaN, Nfatc2, Nfatc3, and Nfatc2ip showed a different expression level following treatment with low, medium, and high-dose CA, respectively. Primers used for RT-qPCR validation were designed according to each gene sequence and finished by SBC. Primer names, accession numbers, and sequences are listed in Table 1. Total RNA was isolated using TRIzol reagents. The RNA integrity was verified with RNA formaldehyde electrophoresis and the quality was detected by spectrophotometry. The RNA samples had an A260/280 ratio of greater than 2 and a 20sRNA/18sRNA ratio of greater than 1.1. Single strand cDNA was synthesized using RevertAid First strand cDNA Synthesis Kit. Reverse transcription reactions were conducted at 42°C for 30 min and at 85°C for 5 min at a 20 μL total reaction volume containing the following reagents: 2 μL RNA, 1 μL oligo, 2 μL (10 mM) dNTP mixture, 1 μL RiboLock RNase Inhibitor, 4 μL 5× reaction buffer, 9 μL water, and 1 μL RevertAid M-MuLV. PCR was performed using a Sino Bio Taq 2× Master Mix. The RT-qPCR products were analyzed by 1.0% agarose gel electrophoresis. PCR reaction should be taken at a temperature gradient in the range of annealing temperature of CaN, Nfatc2, Nfatc3, and Nfatc2ip. RT-qPCR analysis was conducted to obtain crossing point (Ct) values of each gene and standard curve was established through the linearity between Ct value and log 2 of expression values. Before carrying out RT-qPCR analysis, cDNA and primers should be diluted in 20 μL reagents containing the following: 9 μL MIX, 8 μL H2O, 1 μL sense primer (F), 1 μL antisense primer (R), and 1 μL cDNA. The same cycling parameters were repeated for 40 times. Relative content of mRNA extracted from experimental group of CA, positive control group of docetaxel, and interferon negative control group was calculated by RT-qPCR detection system BIORAD IQ5. 3. Results and Discussion 3.1. Gene Expression Profiles of Tumor Cell after Being Treated by CA 34275 probes without repeat were retained after combination and filtration. We carried out a Student's t-test of these probes to select differentially expressed genes (P ≤ 0.05) and then drew curves reflecting different expression tendency of them at 6 time points. Eighty different curves were obtained after curve fitting. The smaller the P value is, the more similar each gene trend fits the curve. NO.21 fitting curve had a P value close to 0 and the expression profiles of genes covered in this fitting curve were analyzed systematically here. As shown in Figure 1, each inflection point corresponds to gene expression at different time points after administration. Genes covered in this curve were upregulated as seen from the whole time cycle; in other word, CA is capable of upregulating certain genes of tumor-bearing mice. Data of genes covered in NO.21 was put into SBC Analysis System (V2010.05) and compared with KEGG (Kyoto Encyclopedia of Genes and Genomes) database. As shown in Table 2, 96 pathways are found to be related to upregulated genes and 29 of them are found to be statistically significant including T-cell receptor signaling pathway, B-cell receptor signaling pathway, and natural killer cell mediated cytotoxicity. Immune-related genes expression is speculated-changed after treatment with CA and probably have a trend of upregulation. Previous studies on CA immune function verified that CA could increase the carbon clearance index and the content of serum hemolysin and enhance the phagocytic function. Expression level of IFN-γ and IL-2 increased whereas that for IL-4, IL-10 decreased. The release of tumor necrosis factor (TFN) was found reduced while the activation of cytotoxic T lymphocyte (CTL) which aimed at Lewis lung cancer was boosted. NK cells activation and lymphocyte transformation rate of Lewis lung cancer bearing mice were both significantly improved through the determination of immune function indexes. To sum up, CA has been proved capable of strengthening cellular immune functions to tumor. When 3 immune related pathways were found including upregulated genes, we inferred that the immunity of mouse had been improved after treatment of CA. GSK3B, NFATC2, NFATC3, CaN, VAV2, and NCK1 were found upregulated in the 3 immune related pathways as listed in Table 3. According to the functions, genes covered in NO.21 can be classified into 312 different GO (Gene Ontology) and 6 of them has a P value less than 0.05 as listed in Table 4. GO of statistical significance includes the following genes: Cyp2a4, Cyp2c37, Cyp2c38, Cyp27b1, Cyp51, E4f1, Fdps, Gata4, Eif2ak1, Myt1l, Rarg, Sod1, Sp1, Taf3, Nr2c2, Hnf4g, Zdhhc3, Phf6, Mynn, Zfp386, Zfp617, Zfp114, Zfp238, Zfp174, Zfp113, Zfp109, Zfp263, Pcdhb6, Pcdhb15, Pcdhb20, Pcdhb21, Cdyl, Nfatc3, Pbx2, Rest, Smarca5, and Nfatc2. After doing some queries on the function of genes covered in statistically significant pathways and GO, we found out immune related genes and common genes of pathway and GO. Nfatc2, Nfatc3, Nfatc2ip, and CaN were found through combing raw analysis data of gene expression profile, as shown in Table 5. 3.2. Validation of Differentially Expressed Genes by Fluorescence Quantitative PCR Relative expression level of the target genes Nfatc2, Nfatc3, Nfatc2ip, and CaN on average is shown in Table 6. Relative expression level of Nfatc2, Nfatc3, Nfatc2ip, and CaN in high-dose CA group, Nfatc2ip and CaN in medium-dose CA group, and CaN in low-dose CA group significantly improved compared with negative control group. The exact expression quantity is shown in Figure 2. As shown in Figure 2, relative expression level of Nfatc2ip, Nfatc3, Nfatc2, and CaN in high-dose CA group improved compared with negative control group, which corresponds to the result of microarray analysis. 3.3. Discussion of Genes Nfatc2ip, Nfatc3, Nfatc2, and CaN Here, a whole mouse genome oligo microarray (4∗44K) was used to analyze gene expression level of female BALB/c mice and to compare the expression of corresponding target genes of each group on the time series. Systematic error or accidental error can affect the accuracy of microarray analysis to some extent and the errors may come from the processes of RNA extraction, RNA reverse transcription, and hybridization as well as the quality of gene chips and RNA. While the results of microarray analysis can reflect expressing pattern at body's internal gene level quickly, false positive results possibly caused by systematic error or accidental error must be taken into consideration. Verifying experiments like ELISA test, western blot test, and PCR test should be taken for further confirmation. Here, the results of PCR test help completing the study as well as making it more convincing. Through the analysis of genes included in NO.21, expression of critical genes in T-cell receptor signaling pathway, B-cell receptor signaling pathway, and natural killer cell mediated cytotoxicity had changed and immune related genes Nfatc2ip, Nfatc3, Nfatc2, and CaN were found. NFAT (nuclear factor of activated T cells) which consists of 4 components NFATc1, NFATc2, NFATc3, and NFATc4 has been proved important in lymphocyte activation and development. Nfatc2 existing in cytoplasm translocates to the nucleus upon T-cell receptor stimulation and then becomes a member of the nuclear factor of activating T-cells transcription [13]. When the body lacks NFATc2, lymphocyte apoptosis will be significantly reduced which suggests lymphangiectasia and Th2-type response. Meanwhile, the Th1/Th2 balance will be destroyed which suggests a Th2 polarization. Immune regulatory function will disorder when Th1/Th2 balance is destroyed and tumor occurrence shows the preponderance state tendency of Th2. This state will weaken antitumor immune function and will induce tumor cells free from immune surveillance and immune attack. This may be one of the immune mechanisms of tumor development and provides a new idea of tumor treating. The reverse of Th1/Th2 abnormal drift is in favor of recovering antitumor immunocompetence and reducing tumor recurrence and metastasis to improve long-term survival rate finally. NFATc2 and NFATc3 have been proved to synergistically regulate the reaction of T cell receptor, cell division, and Th2 differentiation. Th2-type reaction which suggests the secretion of IL-4, IL-5, and IL-6 is increased whereas the decrease IL-2, IFN-γ, TNF-α, and IL-10 happens when the body lacks NFATc2 and NFATc3 [14–16]. When B cell and T-cell lack NFATc2 and NFATc3 simultaneously, the function of T cell will be weakened but the ability of T cell receptor mediating cell proliferation still exists, while B cells over-activate and show excessive differentiation [17]. The fact that expression of NFATc2 and NFATc3 is improved after treatment of CA indirectly indicates that CA could reverse Th1/Th2 drift. Meanwhile, secretion of IL-2, IFN-γ, TNF-α, and IL-10 is connected to CA too. High expression of NFATc2 and NFATc3 is in favor of the secretion of these cytokines which have already been used as nonspecific immunity treatment to cancer. NFATc2ip can induce the expression of T-cell cytokines, especially enhancing IL production. Three splice variants existing in NFATc2ip are able to methylate NFATc2ip after its translation to produce NFATc2ip regulatory factor. Expression of Th1-type and Th2-type cell factors will be suppressed when the methylation process is inhibited. Therefore, methylation process of NFATc2ip is an important controlling point of manipulating expression of NFAT-dependent cell factors but it will not have any influence on general transcription factor of Th1 and Th2 nor NFAT activation. CaN (Calcineurin) is known as the only serine/threonine protein phosphatase regulated by Ca(2+)-calmodulin so far and mainly aims at catalyzing dephosphorylation of phosphatidylserine and phosphatidyl threonine. It is a multifunctional signaling enzyme involved in function regulation of many cells and distributes in a wide range of tissues especially nerve tissue, T lymphocytes, heart, and skeletal muscle [18, 19]. NFAT family is the main substrate of CaN. NFAT regulates many genes expression as well as influencing many cells differentiation through CaN/NFAT signaling pathway. CaN is able to make NFAT existing in cytoplasm move into cell nucleus after dephosphorylation to finish following transcription and translation, and then Nfatc can combine with AP-1 family members alone or in groups to stimulate secretion of cell factors in certain areas such as IL-2. Transcription induction of IL-2 is a sign of T-cells activation. As mentioned above, NFAT is very important in regulating immunoreaction; meanwhile, NFATc3 and NFATc2 play a particularly key role in correcting body's immune function. Thus, high expression of CaN is a benefit to dephosphorylation of NFAT and indirectly has an effect upon immune system. 4. Conclusion The result of microarray analysis of differentially expressed genes of female BALB/c EMT-6 mice has indicated that the antitumor mechanism of CA is closely related to body's immune system. Through upregulating, the expression of CaN, NFATC2, NFATC2ip, and NFATC3, CA is able to improve the transcription of immune factors like IL-2R and IFN-γ, stimulate proliferation and activation of T cells, NK cells, and macrophage, strengthen monitoring and killing abilities of cancer cells, and inhibit growth of tumor finally. The understanding of the anticancer mechanism of CA has provided a reliable evidence of taking advantage of CA to fight against cancer. Disclosure Hua Rong Yang and Jie Zhang, employed by Jiuzhang Biochemical Engineering Science and Technology Development Co., Ltd, are cooperators of this project. All of the other authors are researchers of West China School of Pharmacy, Sichuan University, and have no conflict of interests. Reagents were supported by the fund (The Sichuan Province Science and Technology Support Project Fund 2011SZ0131) of this project. Some parts of the experiment were entrusted to the third party but not sponsored by it. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interests. Acknowledgments The Sichuan Province Science and Technology Support Project Fund 2011SZ0131 supported this work. The authors would like to thank Hongwei Liu (Shanghai Biochip Co., Ltd.) for his great help in microarray analysis. Figure 1 Expressing change of genes covered in NO.21 at 6 time points. Figure 2 Relative expressing quantity of Nfatc2ip, Nfatc3, Nfatc2, and CaN in each groups. (a) Comparsion of Nfatc2ip expressing quantity among every group. (b) Comparsion of Nfatc3 expressing quantity among every group. (c) Comparsion of Nfatc2 expressing quantity among every group. (d) Comparsion of CaN expressing quantity among every groups. Table 1 Primers used for RT-qPCR validation and additional expression profiling. Gene GenBank accession Forward Reverse Nfatc3 NM_010901 CTCCCTATCATAACCCA CTgAAggCAAATCTgTG Nfatc2ip NM_010900 AAAAgCAgAAAgCATACg gACAggCCCTTCCACTA Nfatc2 NM_010899 TgAgAAgATCgTAggCAAC gCTCgATgTCAgCgTTT CaN NM_001004025 TgggAAATgAggCgATT CCACgCTCAAAgAACCAg Table 2 Pathways of upregulated genes. Pathway name Hits Total Percent (%) Natural killer cell mediated cytotoxicity—Mus musculus (mouse) 4 161 2.48 B-cell receptor signaling pathway—Mus musculus (mouse) 5 85 5.88 T-cell receptor signaling pathway—Mus musculus (mouse) 6 132 4.55 Table 3 Upregulated genes in T-cell receptor signaling pathway, B-cell receptor signaling pathway, and natural killer cell mediated cytotoxicity. Pathway name GeneID ProbeID Symbol Natural killer cell mediated cytotoxicity—Mus musculus (mouse) 18019 Nfatc2 NFATC2 18021 Nfatc3 NFATC3 19059 CaN CAN 22325 Vav2 VAV2 T-cell receptor signaling pathway—Mus musculus (mouse) 17973 Nck1 NCK1 56637 Gsk3b GSK3B 18019 Nfatc2 NFATC2 18021 Nfatc3 NFATC3 19059 CaN CAN 22325 Vav2 VAV2 B-cell receptor signaling pathway—Mus musculus (mouse) 56637 Gsk3b GSK3B 18019 Nfatc2 NFATC2 18021 Nfatc3 NFATC3 19059 CaN CAN 22325 Vav2 VAV2 Table 4 GO of statistical significance. GO ID Name Hits Total Percent (%) GO:0009055 Electron carrier activity 7 107 6.54 GO:0003682 Chromatin binding 8 138 5.8 GO:0046906 Tetrapyrrole binding 6 107 5.61 GO:0030528 Transcription regulator activity 28 1053 2.66 GO:0009058 Biosynthetic process 73 3388 2.15 GO:0043167 Ion binding 78 3709 2.1 Table 5 Immune related genes covered in GO and pathway. GenBank accession GO ID Gene symbol NM_010899 GO:0001816 Nfatc2 NM_010900 GO:0001816 Nfatc2ip NM_010901 GO:0003677 Nfatc3 NM_001004025 GO:0004723 CaN Table 6 Relative expression level of target genes on average (x-±s, n = 10). Group Gene Nfatc2 Nfatc3 Nfatc2ip CaN Docetaxel 1.270 ± 1.216 1.311 ± 0.573 0.294 ± 0.160 1.136 ± 0.544 Interferon 3.831 ± 4.960 2.152 ± 1.089 0.562 ± 0.464 1.566 ± 0.914 High-dose CA 2.920 ± 2.047 4.341 ± 3.961 1.373 ± 1.096 1.961 ± 0.927 Mid-dose CA 1.086 ± 0.367 1.803 ± 1.255 0.937 ± 0.950 1.868 ± 1.160 Low-dose CA 1.185 ± 1.156 1.220 ± 1.303 0.507 ± 0.168 1.238 ± 0.310 Negative 1.085 ± 0.462 0.979 ± 0.839 0.769 ± 1.599 0.950 ± 0.418 ==== Refs 1 McKallip R Li R Ladisch S Tumor gangliosides inhibit the tumor-specific immune response Journal of Immunology 1999 163 7 3718 3726 2-s2.0-0033213878 2 Rosenberg SA Progress in human tumour immunology and immunotherapy Nature 2001 411 6835 380 384 2-s2.0-0035902080 11357146 3 Ostrand-Rosenberg S Animal models of tumor immunity immunotherapy and cancer vaccines Current Opinion in Immunology 2004 16 2 1430 1450 4 Zhang AL Ma Q Gao JM Studies on bioactivities of chlorogenic acid and its analogues Chinese Traditional and Herbal Drugs 2001 32 2 173 176 5 Kasai H Fukada S Yamaizumi Z Action of chlorogenic acid in vegetables and fruits as an inhibitor of 8-hydroxy-deoxyguanseine formation in vitro and in a rat carcinogenesis mode1 Food and Chemical Toxicology 2000 38 5 467 471 10762733 6 Ignatowicz E Balana B Vulimiri SV The effect of plant Phenolics on the formaiton of 7, 12-dimethylbenz[a] an-thracene-DNA adducts and TPA- stimulated polymorphonuclear neutrophifls chemiluminescence in vitro Toxicology 2003 189 3 199 209 12832153 7 Shimizu M Yoshimi N Yamach Y Suppressive effects of chlorogenic acid on N-methyl-N-nitrosourea-indueed glandular stomach carcinogenesis in male F344 rats Toxicological Sciences 1999 24 5 433 439 8 Matsunaga K Katayama M Sakata K Inhibitory effects of chlorogenic acid on azoxymethane-induced colon carcinogenesis in male F344 rats Asian Pacific Organization for Cancer Prevention 2002 3 2 163 166 9 Chiang LC Ng LT Chiang W Immunomodulatory activities of flavonoids, monoterponoids, triterpenoids, iridoid glycosides and phenolic compounds of Plantago species Planta Medica 2003 69 7 600 604 12898413 10 Hu KJ Qu FJ Wang ZL Experimental study on the induction of a-tinterferon of human leucocyte in vitro by chlorogenic acid Journal of Harbin Medical University 2004 38 120 122 11 Wu HZ Luo J Yin YX Effects of chlorogenic acid, an active compound activating calcineurin, purified from Flos Lonicerce on macrophage Acta Pharmacologica Sinica 2004 25 12 1685 1689 15569416 12 Rogers PD Barker KS Evaluation of differential gene expression in fluconazole-susceptible and -resistant isolates of Candida albicans by cDNA microarray analysis Antimicrobial Agents and Chemotherapy 2002 46 11 3412 3417 2-s2.0-0036839691 12384344 13 Baksh S Widlund HR Frazer-Abel AA NFATc2-mediated repression of cyclin-dependent kinase 4 expression Molecular Cell 2002 10 5 1071 1081 2-s2.0-0036864625 12453415 14 Hodge MR Ranger AM de la Brousse FC Hoey T Grusby MJ Glimcher LH Hyperproliferation and dysregulation of IL-4 expression in NF-ATp-deficient mice Immunity 1996 4 4 397 405 2-s2.0-0030002617 8612134 15 Kiani A Viola JPB Lichtman AH Rao A Down-regulation of IL-4 gene transcription and control of Th2 cell differentiation by a mechanism involving NFAT1 Immunity 1997 7 6 849 860 2-s2.0-0031415048 9430230 16 Ranger AM Hodge MR Gravallese EM Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc Immunity 1998 8 1 125 134 2-s2.0-0031933037 9462518 17 Schuh B Kneitz J Heyer K Retarded thymic involution and massive germinal center formation in NF-ATp-deficient mice The European Journal of Immunology 1998 28 8 2456 2466 9710223 18 Mukai H Chang CD Tanaka H Ito A Kuno T Tanaka C cDNA cloning of a novel testis-specific calcineurin B-like protein Biochemical and Biophysical Research Communications 1991 179 3 1325 1330 2-s2.0-0025940074 1718268 19 Dodge KL Scott JD Calcineurin anchoring and cell signaling Biochemical and Biophysical Research Communications 2003 311 4 1111 1115 14623297	23762780	PMC3665237	CC BY	2021-01-05 11:20:04	yes	J Anal Methods Chem. 2013 May 9; 2013:617243
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23724014PONE-D-13-0221110.1371/journal.pone.0064000Research ArticleBiologyDevelopmental BiologyStem CellsMesenchymal Stem CellsCell DifferentiationGeneticsMolecular GeneticsModel OrganismsAnimal ModelsMolecular Cell BiologyCellular TypesStem CellsMesenchymal Stem CellsNeuronsNeuroscienceDevelopmental NeuroscienceNeurogenesisNeurobiology of Disease and RegenerationMedicineNeurologyParkinson DiseaseConversion of Human Umbilical Cord Mesenchymal Stem Cells in Wharton’s Jelly to Dopamine Neurons Mediated by the Lmx1a and Neurturin In Vitro: Potential Therapeutic Application for Parkinson’s Disease in a Rhesus Monkey Model Stem Cell and Gene Therapy for Parkinson’s DiseaseYan Min 1 Sun Maosheng 1 Zhou Yan 1 Wang Wanpu 2 He Zhanlong 1 Tang Donghong 1 Lu Shuaiyao 1 Wang Xiaonan 1 Li Song 1 Wang Wenju 1 Li Hongjun 1 * 1 Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Engineering Research Center of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China 2 The First People's Hospital in Yunnan Province, Kunming, China Pant Aditya Bhushan Editor Indian Institute of Toxicology Research, India * E-mail: lihj6912@hotmail.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: HL. Performed the experiments: MY YZ Wanpu Wang XW S. Li. Analyzed the data: MY MS. Contributed reagents/materials/analysis tools: Wenju Wang ZH S. Lu DT. Wrote the paper: MY. 2013 28 5 2013 8 5 e6400014 1 2013 8 4 2013 © 2013 Yan et al2013Yan et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.hUC-MSCs hold great promise in vitro neuronal differentiation and therapy for neurodegenerative disorders including Parkinson’s disease. Recent studies provided that Lmx1α play an important role in the midbrain dopamine cells differentiation. Neurturin is desired candidate gene for providing a neuroprotective to DA neurons. In this study, we investigated a novel neuronal differentiation strategy in vitro with Lmx1α and NTN. We transferred these two genes to hUC-MSCs by recombinant adenovirus combined with Lmx1α regulatory factor and other inductor to improve the efficiency of inducing. Then those induced cells were implanted into the striatum and substantia nigra of MPTP lesioned hemi-parkinsonian rhesus monkeys. Monkeys were monitored by using behavioral test for six months after implantation. The result showed that cells isolated from the umbilical cord were negative for CD45, CD34 and HLA-DR, but were positive for CD44, CD49d, CD29. After those cells were infected with recombinant adenovirus, RT-PCR result shows that both Lmx1α and NTN genes were transcribed in hUC-MSCs. We also observed that the exogenous were highly expressed in hUC-MSCs from immunofluorescence and western blot. Experiments in vitro have proved that secretion NTN could maintain the survival of rat fetal midbrain dopaminergic neurons. After hUC-MSCs were induced with endogenous and exogenous factors, the mature neurons specific gene TH, Pitx3 was transcripted and the neurons specific protein TH, β-tubulinIII, NSE, Nestin, MAP-2 was expressed in those differentiated cells. In addition, the PD monkeys, transplanted with the induced cells demonstrated the animals’ symptoms amelioration by the behavioral measures. Further more, pathological and immunohistochemistry data showed that there were neuronal-like cells survived in the right brain of those PD monkeys, which may play a role as dopaminergic neurons. The findings from this study may help us to better understand the inside mechanisms of PD pathogenesis and may also help developing effective therapy for Parkinson’s disease. This research was financially supported by the Research Fund for Doctor Innovation of Beijing Union Medical College (2011-1001-32); Science and Technology Project of Yunnan Province (2012AE001, 2007C0012Z); National Natural Science Foundation of China (30971003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Parkinson disease (PD) is a neurodegenerative disorder in the elderly whose symptoms of tremor, rigidity, bradykinesia and postural instability are caused primarily by the degeneration of dopamine (DA) neurons in the substantia nigra [1], [2], [3]. The current existence therapies mostly confined to relieve symptoms but not fundamentally restore the lesion side function and loss of efficacy with disease progression [4]. Therefore, a therapy, which is safe and the functional recovery of the nigrostriatal system, is critical in clinical treatment of PD. Since only HSCs are widely used, but all other celluar therapy with SCs are experimental. In the search for a renewable source of dopamine-producing, human fetal brain tissue [5], embryonic stem cells [6], and neural SCs/progenitors [7] have been investigated. However, technical and ethical difficulties limited the application of this therapy [8], [9]. During the last few years isolations of adult mesenchymal stem cell from different sources have been reported [10], [11], [12]. The MCSs derived from umbilical cord tissue have low immunogenicity and contain few immune cells. Previously studys demonstrated that human umbilical cord mesenchymal stem cells (hUC-MSCs) could be induced to differentiate into neuron-like cells. Sarugaser et al. [13] and Karahuseyinoglu et al. [14] showed the isolation, culturing and differentiation behavior of human perivascular umbilical cells and obtained osteogenic nodules. Datta et al. [15], Fu et al. [16] and weiss et al. [17] demonstrated the differentiation capacity of hUC-MSCs into dopaminergic neurons and better than bone marrow derived MSCs. The LIM homeobox transcription factors 1 alpha (Lmx1α) is sufficient and required to trigger midbrain dopamine (mDA) neurons differentiation. It is accepted by most research that fully compatible dopaminergic differentiation requires extrinsic cues provided by signaling molecules such as sonic hedgeho (SHH) and the fibroblast growth factor (FGF-8) [18], [19], [20]. Ran Brazilay et al. [21] reported that Lmx1α forced expression, together with extrinsic signaling molecule is sufficient to produce cells that expressed high level of TH, the rate-limiting enzyme in dopamine synthesis, and secreted significantly higher levels of dopamine. Neurturin (NTN) is a potent trophic factor for dopaminergic neurons. NTN enhances dopaminergic neurons survival, prevents the loss of damaged nigral dopamine neurons in an animal model of PD and restores the neuronal micro-environment in vivo [22], [23]. We hypothesized that hUC-MSCs infected with adenoviral vectors expressing Lmx1α and NTN and cultured in chemical micro-environment could be transdifferentiated to undertake neuronal differentiation pathways. In this study, we examed the effects of promoting the differentiation potential and trophic effects on endogenous neural stem cells by continuous NTN and nutritional factors secretion in the transplanted neural-like cells. Materials and Methods Collection and use of human umbilical cord was approved by the Ethics Committee of the Institute of Medical Biology (YISHENGLUNZI [2011] 16), and was provided following caesarian section with written informed consent. The recombinant adenoviruses Ad-NTN and Ad-Lmx1α were provided by the Laboratory of Molecular Biology at the Institute of Medical Biology of the Chinese Academy of Medical Sciences. Ethics Statement All animal experimental procedures were carried out in strict accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals. The animal experiments were approved by the Ethics Committee of Animal Care & Welfare, Institute of Medical Biology, CAMS (Permit Number: SYXK (dian)2010-0007), in accordance with the animal ethics guidelines of Chinese National Health and Medical Research Council (NHMRC) and the Office of Laboratory Animal Management of Yunnan Province, China. Non-human primates were kept in a single cage and bred according to the guidelines of the Experimental Animals Ethics Committee of Institute of Medical Biology Chinese Academy of Medical Sciences (Permit Number SCXK-2005-0005). We injected penicillin to monkeys for diminishing inflammation after surgery. Rhesus monkeys were euthanized by an overdose of injection anaesthetic (sodium pentobarbital, 10 mg/kg). All efforts were made to minimize the number of animals used and their suffering. [24]. The rats we were obtained from Institute of Medical Biology of Chinese Academy of Medical Sciences. After the experiments rats were euthanized by dislocation of cervical vertebra; fetuses were euthanized by decapitation. Isolation and Expansion of hUC-MSCs Human umbilical cords were obtained from healthy full-term caesarian section births and aseptically store at 4°C PBS containing 1% antibiotics of penicillin and streptomycin. The cord was rinsed several times to drained blood from vessels and cords. Then cut it into 2–3 cm length and rinsed again. Umbilical arteries and veins were removed, and the remaining tissues were transferred to a sterile container and were dicied into small fragments in the PBS. The explants were transferred to flask contain minimum essential medium (α-MEM) along with 15% fetal bovine serum (FBS) and incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium was replaced every five days. The cells were reached 60%–70% confluence then transferred it to 6-well plates supplemented with 20 µM BrdU. After 48 h incubation, the cells were rinsed with PBS and fixed with 2% paraformaldehyde/0.2% Trinton-X 100/PBS for 30 min at 4°C and then treated with pre-cooled methanol for 10 min at 4°C. The cells were rinsed with PBS, subsequently treated with 2 M hydrogen chloride (HCl) for 20 min at 37°C and rinsed with PBS. Then the sample blocked with 2% bovine serum albumin (BSA) in PBS for 30 min at 37°C. Cells were incubated overnight at 4°C with the primary antibody (anti-BrdU mouse monoclonal antibodiy, ZSGB-BIO ORIGENE, diluted in 1∶50). The next day, cells were rinsed with PBS, then incubated with secondary antibody diluted in 2% BSA/0.2 Tween-20/PBS for 30 min at 37°C and counterstained DAPI (4′, 6-Diamidino-2-Phenylindole Dihydrochloride, Sigma-Aldrich) for 2 min at room temperature. The testing cells were observed under fluorescence microscope. Flow Cytometry To stain the hUC-MSCs, the cells were lifted with 0.125% trypsin. The digested cells were washed with PBS and resuspended into a single cell suspension at a concentration of 106/mL. The suspension cells were stained with the following labeled antibody: CD45-PerCP, CD34-PE, CD49d-PE, CD29-PE, CD44-PE and HLA-DR-FITC (BD Pharmingen), incubated in the dark for 30 min at room temperature and then analyzed by means of a FACSCalibur (BD Biosciences). Transmission Electron Microscopy The hUC-MSCs were detached with 0.125% trypsin and fixed in 2.5% glutaraldehyde in PBS for 2 h. Cells were rinsed with 0.1 M PBS and fixed in 1% osmic acid fixative for 3 h at 4°C. Cells underwent sequential dehydration for 15 min in 50%, 70% and 90% ethanol, a 1∶1 mixture of 90% ethanol and 90% acetone and then 90% acetone. The above steps were all operated at 4°C. Cells were washed with 100% acetone at room temperature for three times. Cells were embedded in epoxy resin, and cut it into 50–60 nm slices. The slices were double stained with 3% uranyl acetate-lead citrate and observed under TEM at 80 kV. Adipogenic Differentiation Potential hUC-MSCs were transferred to 6-well plates(1×105 cells per well) with cover slips cultured with MEM-α containing 15% FBS. When the cells reached 90% confluence, the medium was replaced with high-glucose Dulbecco’s Modified Eagle’s Medium(H-DMEM) containing 10% FBS, 1 µM dexamethasone (ENZO), 10 µg/mL insulin and 0.5 mM 3-isobuty-1-methylxanthine (Sigma). Four days later, the medium was replaced with H-DMEM containing 10% FBS and 200 µM indomethacin (Sigma-Aldrich). After four days inducing, the medium was replaced with H-DMEM containing 10% FBS, 1 µM dexamethasone, 10 µg/mL insulin, 0.5 mM 3-isobuty-1-methylxanthine and 200 µM indomethacin. The medium was replaced every four days. After 21 days of adipogenic stimulation, cells were rinsed with PBS, fixed in 4% paraformaldehyde at room temperature and incubated for 30 min with oil red O to stain lipid vacuoles and treated with hematoxylin (Beytime) to stain nuclei. Osteogenic Differentiation Potential hUC-MSCs were transferred to 6-well plates(1×105 cells per well) with cover slips cultured with MEM-α containing 15% FBS. When the cells reached 60%–70% confluence, the medium was replaced with H-DMEM containing 10% FBS, 10 nM dexamethasone, 10 mM β-glycerophosphate disodium salt hydrate and 150 µM L-ascorbic acid-2-phosphate (Sigma-Aldrich). Medium was changed every four days. After 21 days of osteogenic stimulation, cells were rinsed with PBS, fixed in 4% paraformaldehyde at room temperature and incubated for 30 min with alizarin red to stain calcium nodules. Alkaline phosphatase (ALP) activity was assayed by means of the GENMED alkaline phosphatase activity staining kit (GENMED Scientifics, Inc.). RT-PCR Analysis of the Transcription of the NTN and Lmx1α in hUC-MSCs hUC-MSCs were transferred to a flask (1×106 cells per well) and adenovirus was added in culture medium at multiplicity of infection (MOI, the appropriate dose for the virus between the number of infected cells and the virus titer) of 5 for 48 h. Total RNA was extracted from control cells and treated cells (Ad-NGFpreNTN and Ad-Lmx1α infected) with the use of the RNA extraction kit (OMEGA) as described by the supplier. RNA was reverse-transcribed with the use of M-MuLV reverse transcriptase in a 20 µL volume containing 0.2 µg Oligo (dT), 400 µmol/L dNTP and buffers supplied by the manufacture. With the above cDNA (1 µg) as a template, the sequences were used in the Polymerase chain reaction (PCR) was showed in Table 1. 10.1371/journal.pone.0064000.t001Table 1 Primers sequences used in reverse transcription-polymerase chain reaction and Real-time PCR. Gene Sequence(5′-3′) β-actin Forward: GGCATCCTCACCCTGAAGTA Reverse: GGGGTGTTGAAGGTCTCAAA NGFpreproNTN Forward: TGTCCATGTTGTTCTAC Reverse: TTACACGCAGGCGCACTC Lmx1α Forward: CTAAGATGTGAAGTAAG Reverse: CTAAGATGTGAAGTAAG TH Forward: GCACCTTCGCGCAGTTC Reverse: CCCGAACTCCACCGTGAA β-tubulinIII Forward: CGTGCCTCGAGCCATTCT Reverse: GCCCCCTCCGTGTAGTGA MAP-2 Forward: AGACCACCATTGACGACTCC Reverse: TCTCCGAGCTTCCTTTTCAG DAT Forward: CCCACTACGGAGCCTACATCTT Reverse: CAATGGCGTAGGCCAGTTTC Lmx1β Forward: TGTGCAAGGGTGACTACGAGAA Reverse: TTCATGTCCCCATCTTCATCCT Msx1 Forward: CTCCCTGAGTTCACTCTCCG Reverse: CAGGAGACATGGCCTCTAGC Pitx3 Forward: GGACTAGGCCCTACACACAGA Reverse: TCCGCGCACGTTTATTTC Foxα2 Forward: TTCAGGCCCGGCTAACTCT Reverse: AGTCTCGACCCCCACTTGCT Nurrl Forward:CGAAACCGAAGAGCCCACAGGA Reverse: GGTCATAGCCGGGTTGGAGTCG NES Forward: TTCACAGCCAATGTAGGGAT Reverse: ATGGGACAAGAGCAAAGCAC Nestin Forward: AGACTCTTCCCGACTCCACT Reverse: CATCCGCAAACCCATCAG EN1 Forward:TGGGTGTACTGCACACGTTATTC Reverse:GGAACTCCGCCTTGAGTCTCT Immunofluorescence Detected the Expression of NTN and Lmx1α hUC-MSCs hUC-MSCs were transferred to 6-well plates (1×105 cells per well) with cover slips and adenovirus (Ad-NGFpreproNTN, Ad-Lmx1α) was added in culture medium at MOI of 5 for 48 h. The cells were rinsed with PBS and fixed with 2% paraformaldehyde containing 0.2% Triton X-100 at 4°C for 30 min, Subsequently fixed with precooled methanol for 10 min at 4°C. After washing with PBS, samples were incubated with antibodies (NTN: Mouse Anti-Human Neurturin Monoclonal Antibody,R&D,1∶100; Lmx1α: Rabbit Anti-Human Lmx1α Monoclonal Antibody,Santa Cruz,1∶50) for 90 min at 37°C. Then rinsed the cells and incubated with the secondary antibody(NTN: FITC Labeled Rabbit Anti-Mouse IgG Secondary Antibody,KPL,1∶250; Lmx1α: anti-rabbit IgG-DylightTM549, RockLand immunochemicals, 1∶3000) for 30 min at 37°C. The cells were observed under a fluorescence microscope. hUC-MSCs without any treated were as control group. Western Blotting Detection of Lmx1α Expression and Mature NTN Secretion Adenovirus (Ad-NGFpreproNTN, Ad-Lmx1α) was added at MOI of 5 for 48 h. The condition medium and cells were harvested after adenovirus infection. The normal cells were as control group. These samples were separated by SDS-PAGE (12% gel) and transferred to PVDF membranes. The membranes were blocked at 37°C for 2 h with nonfat milk in PBS. Membranes were incubated with primary antibodies (NTN:Goat Anti-Human Neurturin Polyclonal Antibody,R&D,1∶250; Lmx1α: Rabbit Anti-Human Lmx1α Monoclonal Antibody,Santa Cruz,1∶50) for 2 h at 37°C, after washed three times the membranes were incubated for 2 h at 37°C with approximate horseradish peroxidase (HRP) conjugated secondary antibodies (NTN: Rabbit anti-Goat IgG Secondary antibody,KPL,1∶2000; Lmx1α: Goat anti-Rabbit IgG Secondary antibody,KPL,1∶2000). After washing, blots were visualized using DAB chromogenic reagent. The molecular weight of the analyzed proteins was estimated using PageRuler Prest Protein Ladder (Therom). ELISA Detection of the Extracellular Secretion of NTN by hUC-MSCs The medium was collected after hUC-MSCs infected with Ad-NGFpreproNTN at 24 h, 48 h, 72 h and 96 h. Control group was normal hUC-MSCs culture medium. The 96-well palte was incubated with coating buffer overnight at 4°C. The samples were washed three times with PBS-T (containing 0.5% Tween-20) and blocked with 3% BSA in PBS-T for 2 h at 37°C. After washing, each sample was incubated with primary antibody (Goat Anti-Human Neurturin Polyclonal Antibody, R&D, 1∶2000) for 1 h at 37°C. Then the plate was rinsed three times and incubated again with secondary antibody (Rabbit anti-Goat IgG Secondary antibody, KPL, 1∶2500) for 1 h at 37°C. The samples were visualized using TMB (3, 3′, 5, 5′-Tetramethylbenzidine) for 15 min at room temperature. The results of densitometry measurement were statically analyzed by student’s t test. The Bioassay of the Neurons Survival Promoting Activity of NTN Sprague Dawley (SD) rats that had been pregnant for 14 days were sacrificed by dislocation of cervical vertebra. The abdominal cavity was opened under aseptic conditions and put the uterus in a sterile dish containing precooled Hank’s Balanced Salt Solution (HBSS). Then removed the fetal rats carefully and opened the cranial cavity with forceps and the entire brain was dissected after clearing the meninges and blood vessels. The ventral midbrain area was removed and cut into pieces in precooled HBSS solution. The pieces were digested with 0.125% trypsin for 20 min before adding culture medium to terminate the digestion. Cells were dispersed by repeated pipetting. Digested cells were added to 6-well plates coated with poly-L-lysine with coverglasses, followed by incubation at 37°C with 5% CO for 24 h. The culture medium was together with the condition mediun and Neural Basal plus B27 seurm free supplements. The control group was supplemented with the culture supernatant from control hUC-MSCs. The DA neurons were incubated and the medium was replaced by half ervery three days. After 21day’s observation, immunohistochemisty was performanced to assay the DA neuros. Differentiation of hUC-MSC into Dopaminergic Neurons Like Cells To differentiated hUC-MSCs into a dopaminergic neurons like cells in vitro, hUC-MSCs were transferred to 6-well plates with cover slips (1×105 cells per well). hUC-MSCs were infected with Ad-NGFpreproNTN at MOI of 6 for 24 h. Subsequently the cells were infected with Ad-Lmx1α at MOI of 5 for 24 h. Then, the medium was replaced with neuronal pre-induction medium, consisting of MEM-α (20% FBS) medium with a cocktail of bFGF (Millpore) and β-mercaptoethanol (Sigma-Aldrich). Cells were induced for 24 h and replaced with neuronal induction medium, containing MEM-α (15% FBS) medium with a cocktail of bFGF, β-mercaptoethanol, SHH (R&D), FGF-8(R&D), RA (Sigma-Aldrich). Seven days later, the medium was replaced with the final induction medium, consisting of MEM-α (5% FBS) medium with a cocktail of bFGF, β-mercaptoethanol, SHH, FGF-8, RA. The neuronal indction medium was replaced every seven days. As a control, hUC-MSCs were also cultured with growth medium alone, without added adenovirus or induction medium. Real-time PCR analysis Total RNA was isolated with the use of the RNA extraction kits (OMEGA) as described by the supplier. RNA was reverse-transcribed with the use of M-MuLV reverse transcriptase in a 20 µL volume containing 0.2 µg Oligo (dT), 400 µmol/L dNTP and buffers supplied by the manufacture. With the above cDNA (1 µg) as a template, real-time PCR was performed with the use of SsoFast™ EvaGreen Supermix (Bio-Rad) and run on a CFX96™ real-time system instrument and software (Bio-Rad). Sequences of the primers are show in Table 1. The mRNA level relative to that of β-actin was calculated. Immunofluorescence staining For immunofluorescence staining, the induced cells were fixed with 2% paraformaldehyde containing 0.2% Triton X-100 for 30 min at 4°C. They were then fixed with pre-cooled methanol for 10 min at 4°C and subsequently blocked with 2% BSA in PBS for 30 min at 37°C. Cells were incubated with the following specific primary antibodies diluted in PBS: TH (Rabbit anti-Human TH,Millipore,1∶1000), NSE (Rabbit anti-Human/Monkey neuron-specific enolase,Thermo Fisher Scientific,60 µL/mL), Nestin (Anti-Nestin,miliipore,1∶200), β-tubulinIII (Anti-Tubulin, beta III isoform, C-terminus,millipore, 1∶100)and MAP-2 (Rabbit anti-human microtubule-associated protein 2,Cell Signaling Technology, 1∶50). The next day, the cells were washed and appropriate secondary antibodies including anti-mouse IgG, FITC (KPL) and anti rabbit IgG, FITC (Chemicon) were incubated for 30 min at 37°C. The negative controls were processed identically to the test sample. Animal Model Study Establishment of a semi-parkinsonian animal model Monkeys were anesthetized with hydrochloric acidulated ketamine (10 mg/kg) for inducement and sodium pentobarbital (20 mg/kg) for maintenance. The right carotid artery was exposed by surgery, and the common carotid artery was injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP-HCL,GBICO, 0.6 mg/kg), while the external carotid artery was being blocked. Stereotaxic surgery Nine hemiparkinsonism monkeys were selected and randomly divided into three groups: the test group, which was injected with induced cells, the control group, which was injected with HBSS and the normal group. Before the stereotaxic injection, magnetic resonance imaging (MRI) was conducted to locate the target sites (caudate nucleus, putamen and substantia nigra). All test monkeys were injected by the same parameters (CA: AP = 27 mm, ML = −5.5 mm and DV = 21 mm; PU: AP = 22 mm, ML = −11.5 mm and DV = 25 mm; and SN: AP = 9 mm, ML = −5 mm and DV = 33.5 mm) [25]. After anesthesia with hydrochloric acidulated ketamine and sodium pentobarbital was given, monkeys were fixed in a surgical frame. The cells culture medium was added BrdU to label the hUC-MSCs. The injection dosage was 107 per pore. For the control group, the HBSS injection dosage was 50 µL. Behavioral analysis by the parkinson’s disease rating scale Parkinsonian behavior was evaluated by Kordower et al. [23], with modifications (Table 2) including tremor, grait, hypokinesia, balance and so forth. The evaluation was conducted 3 times per week. An average score was calculated and ayalyzed by means of the student’s t test. 10.1371/journal.pone.0064000.t002Table 2 Unified Parkinson’s Disease Rating Scale. NO: Date: Recorder: Designed by: Comment: Items Detail Instruction For Rating Scale Week Week Week Week Week Week Week Week Facial Expression(0–3) 0 = Normal 1 = Slight but definitely abnormal diminution of facial expression 2 = Moderate hypomania; lips parted some of time 3 = Masked of fixed facial with severe of complete loss of facial expression Tremor(0–3) 0 = Absent 1 = Slight-low amplitude 2 = Moderate amplitude, present most of the time 3 = Severe-high amplitude, virtually continuous, interferes with function Posture(0–2) 0 = Normal Erect 1 = Stopped 2 = Face down Gait(0–5) 0 = Normal, use all 4 limbs smoothly 1 = One side circling 2 = Walk slowly 3 = Markedly impaired, able to ambulate but very slowly and with effort 4 = Severe decrease in ability to ambulate 5 = Unable to ambulate Gross Motor Skills (Left and right upper limbs) (0–4) 0 = Normal use limbs through a wide range of motion and activities 1 = Noticeable decrease in capability to use limb, but used consistently 2 = Severe decrease in capacity to use limb, rarely used 3 = Unable or refuse to use limb(will use for walking) 4 = Unable to use limb including walking Defense Reaction(0–2) 0 = Normal; Response to an attack actively 1 = Response to an attack with only a simple action or passively 2 = Without any response to an attack Balance(0–2) 0 = Normal balance 1 = Mild loss of balance on arising or with movement, holds onto cage for support 2 = Major lapses in balance Freezing(0–2) 0 = Absent 1 = Present for up to 10 s 2 = Present for over 10 s Hypokinesia(0–2) 0 = Normal amount of movements 1 = Moderate decrease in the amount of movements 2 = Essentially no movement (akinetic) Total Rating Scale Sum all the scores in each items Average/week R-(−) apomorphine-induced rotation A characteristic of the hemiparkinsonian state is rotation induced by R-(−) apomorphine (2 mg/kg, GBICO), a dopamine agonist. The induced rotations were examined after administration of the cell therapies to evaluate the recovery of DA neurons on the lesioned sides. The number of rotations of each animal was recorded and analyzed by means of the student’s t test. Histological evaluation of recovery About six months after treatment, the monkeys were sacrificed with the method of euthanasia. Brains were removed and fixed with paraformaldehyde for seven days. The tissues were sectioned in paraffin (7 µm) by a sliding microtome.The sections were incubated with the primary antibody mouse anti-monkey TH (1∶2000) and mouse anti-BrdU (1∶50), followed by treatment with HRP-labeled secondary antibodies. Grafted cells, which were labeled with BrdU, were detected in the right side substantia nigra of cell therapy monkeys (Fig. 20 C) and the left side had no significantly staining (Fig. 20 D). In the HBSS group there were not significantly staining in the left side (Fig. 20 A) and the lesion side (Fig. 20 B). Results Characteriztion of Cultured hUC-MSCs About 3×106 hUC-MSCs were collected from 20 cm of umbilical cord. Seven days after isolation, hUC-MSCs adhered and displayed a fibroblast-like appearance. Ten days later, they started to proliferate rapidly and reached 80% confluence within 2–3 weeks (Fig. 1 A). The cells isolation in vitro was stabled through 7 passages (Fig. 1 B). An amplification index assay indicated that 90% of DAPI-positive cells were BrdU positive (Fig. 2 A, B), confirming the capacity of hUC-MSCs for self-proliferation. 10.1371/journal.pone.0064000.g001Figure 1 Expansion of hUC-MSCs cultured in vitro. (A) Morphology of hUC-MSCs primary cultured for 20 days. Scale bar: 40 µm. (B) Morphology of hUC-MSCs cultured at the 7th generation. Scale bar: 100 µm. 10.1371/journal.pone.0064000.g002Figure 2 Characterizaton and proliferation index of hUC-MSCs cultured in growth medium. (A) hUC-MSCs counterstain with BrdU. (B) hUC-MSCs counterstain with DAPI. Scale bar: 100 µm. Flow Cytometry FACS analysis demonstrated that hUC-MSCs were stained positive for CD29, CD44 and CD49d but negative for CD45, CD34 and HLA-DR. The CD45 negative cells (Fig. 3 A), 82.76% were CD29+HLA-DR (Fig. 3 C), 81.25% were CD49d+HLA-DR (Fig. 3 D), 66.75% were CD44+HLA-DR (Fig. 3 B) and 99.38% were CD34+HLA-DR (Fig. 3 E). 10.1371/journal.pone.0064000.g003Figure 3 Flow cytometry analysis for immunophenotypic characterization of hUC-MSCs. The cells were stained with antibodies against CD29, CD49d, CD44, CD45, CD34 and HLA-DR. Ultrastructural of hUC-MSCs The hUC-MSCs were sphere-like (Fig. 4 A), and the surface of the cells membrane had many microvilli-like protrusions (Fig. 4 C). Cells had a large nuclear-cytoplasmic ratio, and the nuclei were oval or irregular with nuclear fussion (Fig. 4 D). The nuclear outer membrane was visibled and contained ribosomes (Fig. 4 E). The heterochromatin was distributed around the nucleus, which contained greater amount of enchromatin (Fig. 4 B), rough endoplasmic (Fig. 4 C) and concentration mitochondria (Fig. 4 F). The result indicated that hUC-MSCs were primitive, metabolically active and poorly differentiated. 10.1371/journal.pone.0064000.g004Figure 4 Ultrastructural characteristics of hUC-MSCs were detected by TME. Differentiation Capacity and Plasticity of hUC-MSCs In vitro differentiation into adipogenic cells Adipocytic phenotypes in induced hUC-MSCs were first signaled by the appearance multisized, most cells became round or cuboid and retracted their cellular extensions during induction, although a few retained their fusiform shape. After 5 days of adipogenic differentiation, tiny intracytoplasmic droplets could be observed. After 21 days adipogenic differentiation, most cultivation cells were became larger and the number of lipid droplets in cytoplasm increased (Fig. 5 A). The effectiveness of differentiation was assessed by histochemical staining. In the adipogenic differentiated cells, red stained intracellular vacuoles (Fig. 5 C). Control hUC-MSCs were only visibly stained with hematoxylin (Fig. 5 B). 10.1371/journal.pone.0064000.g005Figure 5 Differentiation of hUC-MSCs into adipocytes in vitro. (A) The lipid droplet of cells cultured in adipogenic medium for 21 days. (B) Staining was not observed in control hUC-MSCs cultured in growth medium. (C) Oil red O staining of the lipid droplet of cells cultured in adipogenic medium. Scale bar: 100 µm. In vitro differentiation into osteogenic cells After osteogenic induction, most cells became proximity to each other (Fig. 6 A, D). After 21days induction, alizarin red stained obvious calcium deposits (Fig. 6 B), whereas noninduced cells did not exhibit any calcium deposits (Fig. 6 C). ALP activity revealed blue-black stained in induced cells (Fig. 6 E), whereas non induced cells did not exibit any ALP activity (Fig. 6 F). 10.1371/journal.pone.0064000.g006Figure 6 Differentiation of hUC-MSCs into osteoblasts in vitro. (A, D) Morphology of hUC-MSCs cultured in osteogenic medium for 21 days. (B, E) In vitro differentiation of hUC-MSCs into osteoblasts, were shown by positive alizarin red (B) and ALP staining (E) of calcified extracellular matrix. (C, F) Staining was not observed in control hUC-MSCs cultured in growth medium. Scale bar: 100 µm. Transcription of NTN and Lmx1α in hUC-MSCs after Recombinant Adenoviruses After infected with Ad-NGFpreproNTN and Ad-Lmx1α for 48 h, the RT-PCR results shown that NTN and Lmx1α were transcripted in the cells infected with adenoviruses, but they were not transcripted in the control hUC-MSCs (Fig. 7). 10.1371/journal.pone.0064000.g007Figure 7 The transcription of NTN and Lmx1α in hUC-MSCs was analyzed by RT-PCR. Lane 1, the PCR product of Lmx1α in Ad-Lmx1α infected hUC-MSCs. Lane 2, the PCR product of β-actin in Ad-Lmx1α infected hUC-MSCs. Lane 3, the product of NTN in Ad-NGFpreproNTN infected hUC-MSCs. Lane 4, the product of β-actin in Ad-NGFpreproNTN infected hUC-MSCs. Lane 5, the product of Lmx1α in control hUC-MSCs. Lane 6, the product of NTN in control hUC-MSCs. Lane 7, the product of β-actin in control hUC-MSCs, DNA marker DL,2000. Identification of NTN and Lmx1α Protein Expression in hUC-MSCs and the Secretion of NTN Immunofluorescence results showed that NTN and Lmx1α proteins were expressed at high level in infected hUC-MSCs: NTN was expressed in the cytoplasm (Fig. 8 C), whereas Lmx1α was expressed in the nucleus (Fig. 8 A). These two proteins were not detected in control hUC-MSCs (Fig. 8 B, D). 10.1371/journal.pone.0064000.g008Figure 8 The expression of NTN and Lmx1α in hUC-MSCs was analyzed by immunofluorescence. (A) The expression of Lmx1α in hUC-MSCs infected with Ad-Lmx1α. (B) The expression of Lmx1α in control hUC-MSCs. (C) The expression of NTN in hUC-MSC infected with Ad-NGFpreproNTN. (D) The expression of NTN in control hUC-MSCs. Scale bar: 100 µm. From WB result we found that the band with a strong signal appearing in the membrane at approximately 12 kDa which characterized as NTN was observed in cytoplasm and culture medium (24 kDa was dimmer of mature NTN) (Fig. 9). However, the expression of Lmx1α was only be detected in cytoplasm (36 kDa) (Fig. 10). The expression of these two proteins was not detected in control group. 10.1371/journal.pone.0064000.g009Figure 9 Detection of the expression of NTN and secretion of NTN in conditioned medium by western blot. Lane 1, was loaded with the cell lysis infected with Ad-NGFpreproNTN. Lane 2, was loaded with the medium harvested from Ad-NGFpreproNTN infection. Lane 3, was loaded with the cell lysis from control hUC-MSCs. Lane 4, was loaded with the medium from control hUC-MSCs. 10.1371/journal.pone.0064000.g010Figure 10 Detction of the expression of Lmx1α by western blot. Lane 1, was loaded with the cell lysis infected with Ad-Lmx1α. Lane 2, was loaded with the medium infected with Ad-Lmx1α. Lane 3, was loaded with the cell lysis from control hUC-MSCs. Lane 4, was loaded with the medium from control hUC-MSCs. ELISA Detection of NTN Secretion The student’s t test compared the means of OD450 between the conditional medium and the control medium, there were significant difference between these two groups (Fig. 11).The expression of NTN at 48 h was higher than 24 h, 72 h and 96 h. These results indicated that NTN be secreted into the culture medium. 10.1371/journal.pone.0064000.g011Figure 11 Detection of NTN secretion in 24 h, 48 h, 72 h and 96 h of hUC-MSCs infected with Ad-NGFpreproNTN and control hUC-MSCs by ELISA (* p<0.05). Identification the Bioactivity of NTN Isolated neuronal cells were strong light refraction and slender axons. These cells were co-cultured with B27 and conditional medium. Cells in the experimental group were treated with NTN-conditioned medium were alived after 21 days cultured, according to observations, these cells exhibited neuronal morphology and the survival rate was 80% (Fig. 12 B). In contrast, a large number of cells in the control group were shrunk, and there were few surviving cells with intact morphology after 21 days cultured (Fig. 12 A). Immunohistochemistry showed TH expressed in the treated group, suggesting that the surviving cells were dopaminergic neurons (Fig. 12 C). 10.1371/journal.pone.0064000.g012Figure 12 The culture of neurons derived from embryonic 14.5 rat ventral mesencephalic progenitors test. (A) The neurons cultured in neural basal (2% B27) alone. Neurons are most dead. (B, C) the neurons cultured in neural basal (2% B27) and NTN condition medium (1∶1). The result of TH immunohistochemistry reveals some neurons are alive and grow well. Scale bar: 100 µm. Identification of Inducing hUC-MSCs Real-time PCR detected the neuron-specific genes After 21 days induction, the transcriptional levels of TH, Msx-1, Nestin, MAP-2, Lmx1β, Foxα2, Pitx3, DAT, β-tubulin III, Nurrl and EN1 were significantly upregulated. The upregulation of TH suggested the cells had been induced to dopaminergic neurons. Nestin is a marker that is specifically expressed in neuronal precursor cells, and these results showed that the transcription of nestin was significantly reduced on the 21st day induction, suggested the induced cells were going to differentiate into mature neurons. It is also worth mentioned that the NSE transcription level was similar between cells before and after induction, suggesting that genes were expressed highly in uninduced cells. The statistical analysis (student’s t test) revealed significant difference in the transcriptional levels of 11 genes (TH, Msx-1, nestin, MAP-2, Lmx1β, Foxα2, Pitx3, DAT, β-tubulin III, Nurrl and EN1) between cells induced for 21 days and normal cells, and between cells induced for 21 days and cells induced for 7 days (Fig. 13). 10.1371/journal.pone.0064000.g013Figure 13 The specific mRNA of induced hUC-MSCcs. Transcription of neural-specific genes like TH, Msx-1, Nestin, MAP-2, Lmx1β, Foxα2, Pitx3, DAT, β-tubulin III, Nurrl, EN1 and NES was analyzed by real-time PCR. All samples were normalized with β-actin (* p<0.05 compared with non-induecd cells; # p<0.05 compared with cells induced for 7 days). Immunofluorescence detection of neuron-specific protein expression After seven days induction, immunofluorescence was able to detect expression of the neuronal precursor cell-specific protein nestin (Fig. 14 F) but not the expression of proteins specific for mature neurons (Fig. 14 G, H, I, J). After 21 days of induction, the immunofluorescence results revealed the expression of proteins specific for mature neurons, including NSE (Fig. 15 F), MAP-2 (Fig. 15 H), β-tubulinIII (Fig. 15 G) and the dopaminergic neuron-specific antigen TH (Fig. 15 J), whereas the nestin expression level was significantly decreased (Fig. 15 I). The expression of these mature proteins was not detected in the control group (Fig. 15 A, B, C, D; Fig. 14 A, B, C, D, E). 10.1371/journal.pone.0064000.g014Figure 14 The expression of neural-specific marker containing TH, β-tubulinIII, MAP-2, NSE, Nestin and Lmx1α in hUC-MSCs induced 7 days was analyzed by immunofluorescence. Scale bar: 100 µm. 10.1371/journal.pone.0064000.g015Figure 15 The expression of neural-specific marker containing TH, β-tubulinIII, MAP-2, NSE, Nestin and Lmx1α in hUC-MSCs induced 21 days was analyzed by immunofluorescence. Scale bar: 100 µm. Effect of Induced hUC-MSCs in Parkinsonian Rhesus Monkeys Behavioral evaluation We found that all monkeys who were lesioned by MPTP displayed acute symptoms with reduced motor movements and facial expressions. After the administration of therapeutic cells, instant behavior restorations were observed in cell therapy group. One weeks after therapy, the monkeys injected with cells not only made more vigorous movements, but also show good balance ability and had no handicap in the motor movements in this entire experiment cours. However, the monkeys in the control group displayed no significant behaviors recovery expressed as reduced movement, loss of facial expression and holding the cage for balance. One week after surgery, behavior changes were evaluated by the UPDRS. The evaluation was conducted three times a week and last for six months (Fig. 16 A). Significant difference was observed in behavior rating scores between cell therapy group and HBSS control group. (Fig. 16 B). 10.1371/journal.pone.0064000.g016Figure 16 Establishiment and behavioral assessment of experimental animals. (A) Evaluation of the behavior restoration of the normal group, HBSS group and cell transplanted therapy group (n = 3). (B) Comparison of the behavior recovery rating score between the HBSS group and cell therapy group. (n = 3, * p<0.05 compared with cell therapy group; # p<0.05 compared with cell therapy group). Effect of Apomorphine-induced rotation Apomorphine was injected from the fourth week after the model was established, and the results were shown in Fig. 17 A. The rotation rate of the experimental animals in the cell transplantation group was reduced to 16 turns/min in the fourth week, whereas that of the HBSS group was increased to 34 turns/min. Following five months therapy, the cell therapy group animals could control their rotation behavior and stop rotation in 10 min. The final rotation rate in cell therapy group was 5 turns/min. In HBSS group, the animals could not control their rotaion behavior and the final rating was 40 turns/min. There was significant difference in rotation rating scores between cell therapy group and HBSS group (Fig. 17 B). 10.1371/journal.pone.0064000.g017Figure 17 Establishiment and behavioral assessment of experimental animals. (A) The apomorphine-induced revolution of the experimental animals in the therapy group and HBSS group. (B) Statistical illustration of the cycle per minute the monkeys revolved, induced by apomporphine (n = 3, * p<0.05 compared with cell therapy group; # p<0.05 compared with cell therapy group). Histological assessment In HBSS group, TH positive neurons in the lesioned substantia nigra side were more or less lost (Fig. 18 A). However, there were many TH positive cells in the left side (Fig. 18 B). Moreover, we have examined histopathological section of substantia nigra by HE staining (Fig. 19 A, B), the results were the same as TH immunohistochemical. However, the cell therapy group has a greater color reactive area and intensity compared to HBSS group, which indicated that the neuroprotection effect from NTN delivered by Ad-NTN has protected the DA neurons which were lesioned by MPTP (Fig. 18 C, D). The HE staining results were also showed in the right substantia nigra have a small number of neurons (Fig. 19 C) but were fewer than the left side (Fig. 19 D). The surviving neurons on the right side show normal morphology and only a few showed pyknosis. 10.1371/journal.pone.0064000.g018Figure 18 Immunohistochemistry of the TH-positive cells in the substantia nigra of the monkeys in two groups. Scale bar: 100 µm. 10.1371/journal.pone.0064000.g019Figure 19 Hematoxylin and eosin (HE) staining of the substantia nigra of the monkeys in two groups. Scale bar: 100 µm. 10.1371/journal.pone.0064000.g020Figure 20 Immunohistochemistry of the BrdU-ir cells in the substantia nigra of the monkeys in two groups. Scale bar: 100 µm. Discussion It has been reported that the human umbilical cord contained abundant of MSCs [13], [26], [27], [28]. Different culture methods were used to guide cells to express neuronal phenotypes [16], [17], endothelial phenotypes of myocardial cells markers [29], [30].The facts explained that these cells have multi-differentiation potential. The current methods used for the isolation of hUC-MSCs mainly include the enzyme digestion and the tissue explant adherent. The enzyme digestion has the advantage of rapid isolation, but the enzyme system may degrade the outer leaflet of the cell membrane, and damage cells if improper control of time. So we utilized the tissue explant adherent method to isolate hUC-MSCs. The hUC-MSCs obtained in this study showed similar to previous reports: including cell morphology, immunophenotype, cell proliferative and differentiation potential. Studies have suggested that stem cells could be induced and played a role as engineered cells to release neurotrophic factors, providing neuronal protection and promote self-healing [31], [32]. Cells therapy is undoubtedly the most promsing therapeutic approach for PD. The aim of this study was to replace lost neurons and restore the net work of striatal dopaminergic nerve terminals in PD. So we focused on inducing the hUC-MSCs into dopaminergic neurons. After inducing, Real-time PCR results showed that the transcriptional level of the Msx1 gene was upregulated after inducing. The Msx1 and Lmx1α genes were played important roles in the midbrain dopaminergic neurons during embryonic development, the upregulation of Msx1 expression resulted in a transcriptional cascade, leading to the induced cells into mature dopaminergic neurons expressing Pitx3 and TH. In the present studies, NTN secreted by hUC-MSCs was abled to enhance dopaminergic neurons survival and play a nutritive role in sensory neurons. In other words, NTN was abled to prevent the deterioration of induced neurons derived from hUC-MSCs. Lmx1α is presumed to be the first expressed intrinsic dopaminergic determinant. We hypothesized the expression of endogenous transcription factors (upregulation of Lmx1α and NTN genes) could activate the SHH/Lmx1α pathways involved in neuronal development. It has also been reported that transplated cells to substantia nigra and striatum together can enhance recovery in PD model [33]. So we choose to transplant induced cells into the substantia nigra, the caudate nucleus and putamen to alleviate the symptoms of PD. The animal experiment results indicated that the obvious behavioral recovery was not observed in HBSS control group. The symptoms of control monkeys get worsen. The symptoms in cell therapy animals were gradually alleviated. TH immunohistochemistry results also demonstrated that there were surviving neurons in the lesioned side substantia nigra of the cell therapy group and there were no significant difference between the caudate nucleus and the putamen. These results were the same as PD clinically pathological changes. In this study, the grafted cells which were labeled with BrdU were detected in the right side of substantia nigra. These results were also indicated that the transplanted cells were survival; on the one hand, transplanted neural-like cells are capable of replacing lost neurons and restoring the lost net work of striatal dopaminergic nerve terminals in PD. On the other hand, transplanted neural-like cells are capable of promoting the differentiation potential and providing trophic effects on endogenous neural stem cells probably through their continuous NTN and nutritional factors secretion. The furtuer secretion of NTN by induced DA neurons could provide neurons nutritional protection on the cells differentiated by induced cells and neural stem cells and form a virtuous circle. Conclusions In this study, we induced hUC-MSCs into neuron-like cells in vitro that are able to secret dopamine. In addition, we transplanted those cells into the brains of PD monkeys. These neuron-like cells could perform the physiological functions of dopaminergic neurons and may play a therapeutic role to ameliorate the symptoms of PD. We believe the results from this study would provide the basis for developing novel cell therapy for PD in future. We are grateful to Huaisheng Shi and Jinyuan Wu for assistance with image capture during the expriments. ==== Refs References 1 Vernier P , Moret F , Callier S , Snapyan M , Wersinger C , et al (2004 ) The degeneration of dopamine neurons in Parkinson’s disease: insights from embryology and evolution of the mesostriatocortical system . Ann N Y Acad Sci 1035 : 231 –249 .15681811 2 Prakash N , Wurst W (2006 ) Development of dopaminergic neurons in the mammalian brain . Cell Mol Life Sci 63 : 187 –206 .16389456 3 Prakash N , Wurst W (2006 ) Genetic networks controlling the development of midbrain dopaminergic neurons . J Physiol 575 : 403 –410 .16825303 4 Morris JG (1978 ) A review of some aspects of the pharmacology of levodopa . Clin Exp Neurol 15 : 24 –50 .386308 5 Stromberg I , Bygdeman M , Goldstein M , Seiger A , Olson L (1986 ) Human fetal substantia nigra grafted to the dopamine-denervated striatum of immunosuppressed rats: evidence for functional reinnervation . Neurosci Lett 71 : 271 –276 .2879264 6 Schulz TC , Noggle SA , Palmarini GM , Weiler DA , Lyons IG , et al (2004 ) Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture . Stem Cells 22 : 1218 –1238 .15579641 7 Ostenfeld T , Caldwell MA , Prowse KR , Linskens MH , Jauniaux E , et al (2000 ) Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation . Exp Neurol 164 : 215 –226 .10877932 8 Doi D , Morizane A , Kikuchi T , Onoe H , Hayashi T , et al (2012 ) Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson’s disease . Stem Cells 30 : 935 –945 .22328536 9 Hall VJ (2008 ) Embryonic stem cells and Parkinson’s disease: cell transplantation to cell therapy . Ann Acad Med Singapore 37 : 163 –162 .18392292 10 da Silva Meirelles L , Chagastelles PC , Nardi NB (2006 ) Mesenchymal stem cells reside in virtually all post-natal organs and tissues . J Cell Sci 119 : 2204 –2213 .16684817 11 Shih DT , Lee DC , Chen SC , Tsai RY , Huang CT , et al (2005 ) Isolation and characterization of neurogenic mesenchymal stem cells in human scalp tissue . Stem Cells 23 : 1012 –1020 .15941858 12 Zuk PA , Zhu M , Ashjian P , De Ugarte DA , Huang JI , et al (2002 ) Human adipose tissue is a source of multipotent stem cells . Mol Biol Cell 13 : 4279 –4295 .12475952 13 Sarugaser R , Lickorish D , Baksh D , Hosseini MM , Davies JE (2005 ) Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors . Stem Cells 23 : 220 –229 .15671145 14 Karahuseyinoglu S , Cinar O , Kilic E , Kara F , Akay GG , et al (2007 ) Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys . Stem Cells 25 : 319 –331 .17053211 15 Datta I , Mishra S , Mohanty L , Pulikkot S , Joshi PG (2011 ) Neuronal plasticity of human Wharton’s jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells . Cytotherapy 13 : 918 –932 .21696238 16 Fu YS , Cheng YC , Lin MY , Cheng H , Chu PM , et al (2006 ) Conversion of human umbilical cord mesenchymal stem cells in Wharton’s jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism . Stem Cells 24 : 115 –124 .16099997 17 Weiss ML , Medicetty S , Bledsoe AR , Rachakatla RS , Choi M , et al (2006 ) Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson’s disease . Stem Cells 24 : 781 –792 .16223852 18 Cai J , Donaldson A , Yang M , German MS , Enikolopov G , et al (2009 ) The role of Lmx1a in the differentiation of human embryonic stem cells into midbrain dopamine neurons in culture and after transplantation into a Parkinson’s disease model . Stem Cells 27 : 220 –229 .18832589 19 Friling S , Andersson E , Thompson LH , Jonsson ME , Hebsgaard JB , et al (2009 ) Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells . Proc Natl Acad Sci U S A 106 : 7613 –7618 .19383789 20 Andersson E , Tryggvason U , Deng Q , Friling S , Alekseenko Z , et al (2006 ) Identification of intrinsic determinants of midbrain dopamine neurons . Cell 124 : 393 –405 .16439212 21 Barzilay R , Ben-Zur T , Bulvik S , Melamed E , Offen D (2009 ) Lentiviral delivery of LMX1a enhances dopaminergic phenotype in differentiated human bone marrow mesenchymal stem cells . Stem Cells Dev 18 : 591 –601 .19298173 22 Kotzbauer PT , Lampe PA , Heuckeroth RO , Golden JP , Creedon DJ , et al (1996 ) Neurturin, a relative of glial-cell-line-derived neurotrophic factor . Nature 384 : 467 –470 .8945474 23 Kordower JH, Herzog CD, Dass B, Bakay RA, Stansell J 3rd, et al (2006 ) Delivery of neurturin by AAV2 (CERE-120)-mediated gene transfer provides structural and functional neuroprotection and neurorestoration in MPTP-treated monkeys . Ann Neurol 60 : 706 –715 .17192932 24 Gorska P (2000 ) Principles in laboratory animal research for experimental purposes . Med Sci Monit 6 : 171 –180 .11208307 25 Wang W , Sun M , Li H , Yan M (2012 ) The delivery of tyrosine hydroxylase accelerates the neurorestoration of Macaca Rhesus model of Parkinson’s disease provided by Neurturin . Neurosci Lett 524 : 10 –15 .22766137 26 Kestendjieva S , Kyurkchiev D , Tsvetkova G , Mehandjiev T , Dimitrov A , et al (2008 ) Characterization of mesenchymal stem cells isolated from the human umbilical cord . Cell Biol Int 32 : 724 –732 .18396423 27 Wang HS , Hung SC , Peng ST , Huang CC , Wei HM , et al (2004 ) Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord . Stem Cells 22 : 1330 –1337 .15579650 28 Covas DT , Siufi JL , Silva AR , Orellana MD (2003 ) Isolation and culture of umbilical vein mesenchymal stem cells . Braz J Med Biol Res 36 : 1179 –1183 .12937783 29 Rao MS , Mattson MP (2001 ) Stem cells and aging: expanding the possibilities . Mech Ageing Dev 122 : 713 –734 .11322994 30 Kadivar M , Khatami S , Mortazavi Y , Shokrgozar MA , Taghikhani M , et al (2006 ) In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells . Biochem Biophys Res Commun 340 : 639 –647 .16378596 31 Kitayama T , Onitsuka Y , Song L , Morioka N , Morita K , et al (2007 ) Assessing an eating disorder induced by 6-OHDA and the possibility of nerve regeneration therapy by transplantation of neural progenitor cells in rats . Nihon Shinkei Seishin Yakurigaku Zasshi 27 : 109 –116 .17633522 32 Shan X , Chi L , Bishop M , Luo C , Lien L , et al (2006 ) Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease-like mice . Stem Cells 24 : 1280 –1287 .16424396 33 Bjorklund A , Lindvall O (2000 ) Cell replacement therapies for central nervous system disorders . Nat Neurosci 3 : 537 –544 .10816308	23724014	PMC3665802	CC BY	2021-01-05 17:28:21	yes	PLoS One. 2013 May 28; 8(5):e64000
==== Front PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco, USA 23724090 PONE-D-13-02991 10.1371/journal.pone.0064751 Research Article Biology Model Organisms Animal Models Rat Medicine Anatomy and Physiology Clinical Immunology Clinical Research Design Drugs and Devices Pharmacokinetics Gastroenterology and Hepatology Stomach and Duodenum Gastritis Peptic Ulcer Disease Gastrointestinal Infections Gastrointestinal Motility Disorders In Vivo Antioxidant and Antiulcer Activity of Parkia speciosa Ethanolic Leaf Extract against Ethanol-Induced Gastric Ulcer in Rats Antiulcer Activity of Parkia speciosa Al Batran Rami 1 Al-Bayaty Fouad 1 Jamil Al-Obaidi Mazen M. 1 Abdualkader Abdualrahman Mohammed 2 Hadi Hamid A. 3 Ali Hapipah Mohd 3 Abdulla Mahmood Ameen 4 * 1 Center of Studies for Periodontology, Faculty of Dentistry, University Technology MARA (UiTM), Shah Alam, Selangor, Malaysia 2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang, Malaysia 3 Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia 4 Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Tanowitz Herbert B. Editor Albert Einstein College of Medicine, United States of America * E-mail: ammeen@um.edu.my Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: MAA. Performed the experiments: RAB. Analyzed the data: AMA RAB. Contributed reagents/materials/analysis tools: MMJ. Wrote the paper: FAB. Literature search: HMA HAH. 2013 28 5 2013 8 5 e6475120 1 2013 17 4 2013 © 2013 Al Batran et al 2013 Al Batran et al This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Background The current study was carried out to examine the gastroprotective effects of Parkia speciosa against ethanol-induced gastric mucosa injury in rats. Methodology/Principal Findings Sprague Dawley rats were separated into 7 groups. Groups 1–2 were orally challenged with carboxymethylcellulose (CMC); group 3 received 20 mg/kg omeprazole and groups 4–7 received 50, 100, 200 and 400 mg/kg of ethanolic leaf extract, respectively. After 1 h, CMC or absolute ethanol was given orally to groups 2–7. The rats were sacrificed after 1 h. Then, the injuries to the gastric mucosa were estimated through assessment of the gastric wall mucus, the gross appearance of ulcer areas, histology, immunohistochemistry and enzymatic assays. Group 2 exhibited significant mucosal injuries, with reduced gastric wall mucus and severe damage to the gastric mucosa, whereas reductions in mucosal injury were observed for groups 4–7. Groups 3–7 demonstrated a reversal in the decrease in Periodic acid-Schiff (PAS) staining induced by ethanol. No symptoms of toxicity or death were observed during the acute toxicity tests. Conclusion Treatment with the extract led to the upregulation of heat-shock protein 70 (HSP70) and the downregulation of the pro-apoptotic protein BAX. Significant increases in the levels of the antioxidant defense enzymes glutathione (GSH) and superoxide dismutase (SOD) in the gastric mucosal homogenate were observed, whereas that of a lipid peroxidation marker (MDA) was significantly decreased. Significance was defined as p<0.05 compared to the ulcer control group (Group 2). The authors express gratitude to the University of Malaya for the financial support of UM/MOHE High Impact Research Grant (HIR Grant No. F000009-21001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Peptic ulcers, which are characterized by the presence of mucosal damage, are predominantly caused by infection with Helicobacter pylori, antiplatelet agents such as acetylsalicylic acid [1], non-steroidal anti-inflammatory drugs (NSAIDs) such as oral bisphosphonates, potassium chloride, immunosuppressive medications [2], serotonin reuptake inhibitors [3], alcohol consumption and cigarette smoking [4]. These factors can cause submucosal erosion and inhibit cyclooxygenase, thus disturbing the protection of the gastric mucosal layer [5]. Anatomically, peptic ulcers occur mostly in the stomach and proximal duodenum. Peptic ulcers are caused by an imbalance between the defensive (mucus secretion, mucosal barrier, blood flow, cellular regeneration and endogenous protective agents) and destructive (acid and pepsin secretion) functions of the gastric system [6]. Alcohol-induced gastric lesions impair gastric defense factors such as mucus secretion and mucosal circulation [7]. Ethanol causes necrotic lesions in the gastric mucosa through multiple pathways, directly producing necrotic lesions, which in turn reduces defensive factors, bicarbonate secretion and mucus production [8]. The gastric wall mucus is thought to play an important role as a defensive barrier against gastrointestinal damage [9]. Mucus secretion is considered to be a crucial defensive factor that protects the gastric mucosa from lesions [10]. The level of gastric wall mucus has been evaluated previously and is used as an indicator of gastric mucus secretion [11]. Researchers have reported a large number of medicinal plants with antiulcer properties [12]–[14]. Plant-based medicines represent a vast untapped resource that has shown enormous therapeutic potential. Parkia speciosa is also known as stink bean or “petai.” It bears long, flat bean pods with green seeds. These beans are popular in Southeastern Asia, including Malaysia and Northeastern India. The beans are sold in the pods or as seeds that are already separated from the pods. The beans may are be jarred in brine and exported. In addition, P. speciosa is believed by the local residents to possess medicinal properties [15] and has been reported to exhibit hypoglycemic, antibacterial, anticancer and antioxidant activity [16]. The present study was performed to establish the antioxidant and antiulcer activity of Parkia speciosa ethanol leaf extract against ethanol-induced gastric ulcers in rats. Materials and Methods Materials and Reagents All materials and reagents were obtained from Sigma (Sigma Aldrich, Germany), and the MDA, SOD and GSH activity measurement kits were purchased from Cayman Chemical Company (Cayman, USA). Omeprazole was used as a reference antiulcer drug and was obtained from the University of Malaya Medical Centre (UMMC). The drug was dissolved in 0.5% (w/v) carboxymethylcellulose (CMC) and orally administered to the rats at a dosage of 20 mg/kg body weight (5 ml/kg) according to Mahmood et al [17]. Plant Material and Preparation of the Crude Extract Fresh Parkia speciosa leaves were obtained from Ethno Resources (Selangor, Malaysia). The identity of the plant was confirmed at the Rimba Ilmu Herbarium, Institute of Biological Sciences, University of Malaya through a comparison with the voucher specimen. Fresh leaves were hung upside down in a warm, dry place (away from direct sunlight) with good air circulation for two weeks to dry. Then, using an electrical blender, the dried leaves were converted to a fine powder. A quantity of 100 g of the powder was soaked for 3 days in a flask that contained 500 ml of 95% ethanol. The mixture was filtered using a fine muslin cloth and filter paper (Whatman No. 1). The filtered mixture was distilled using a rotary evaporator (Eyela, USA) and yielded nearly 17.4% dried mass. The dried extract was diluted in CMC and orally administered to the rats at a dose of 50, 100, 200 or 400 mg/kg body weight (in 0.5% CMC, 5 ml/kg body weight), in accord with earlier reports [18]. Acute Toxicity Healthy male and female Sprague Dawley rats (6–8 weeks old) were obtained from the animal house (University of Malaya, Ethics No. PM/07/05/2010/1111/MAA/R). The rats weighed between 180–200 g. The animals were given standard rat pellets and tap water ad libitum and were individually placed in separate cages with wide-mesh wire bottoms to prevent coprophagy during the experiment. An acute toxicity study was performed to determine a safe dose for the P. speciosa extract. A total of 48 rats (24 males and 24 females) were equally divided into 4 groups that received the vehicle (0.5% CMC, 5 ml/kg) or 1, 3 or 5 g/kg of leaf extract (5 ml/kg). The animals were fasted overnight (but allowed water) prior to dosing. Food was withheld for a further 3 to 4 h after dosing. The animals were observed for 48 hours after the administration of the powder for the onset of clinical or toxicological symptoms. Mortality, if any, was observed over a period of 2 weeks. The animals were sacrificed by an overdose of xylazine and ketamine anesthesia on the 15th day. Histological, hematological and serum biochemical parameters were determined according to standard methods [19]. The ethics committee for animal experimentation of the Faculty of Medicine, University of Malaya, approved the experiment. Throughout the experiments, all animals were treated humanely according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” [20]. Animal Stock Healthy adult Sprague Dawley rats (6–8 weeks old), each weighing between 200–220 g, were obtained from the animal house at the University of Malaya (Ethic No. PM/12/05/2010/MAA/R). The rats were randomly divided into 7 groups of 6 rats each and received a standard pellet diet and tap water ad libitum. The rats were individually placed in separate cages with wide-mesh wire bottoms to prevent coprophagy during the experiment. Gastric Ulcer Induction by Ethanol The animals were fasted for 24 h prior to the experiment [17]. Groups 1 and 2 received vehicle (0.5% CMC) orally. Group 3 received an oral dose of 20 mg/kg omeprazole in 0.5% CMC (5 ml/kg), and groups 4–7 received ethanolic extract of Parkia speciosa at doses of 50, 100, 200 or 400 mg/kg as a pretreatment. At 1 h after pretreatment, the vehicle or absolute ethanol was orally administered to groups 2–7 [12]. One hour later, the rats were euthanized, and their stomachs were dissected. Measurement of Gastric Juice Acidity The animals were sacrificed, and their stomachs were removed. The stomach contents were collected, measured, centrifuged, and subjected to analysis for titratable acidity against 0.01 N NaOH to pH 7 [21]. Determination of Gastric Wall Mucus (GWM) The modified procedure of Piper et al. was used to analyze the gastric wall mucus. The glandular segments from the stomachs of the control and treated rats were removed and weighed. Each segment was transferred immediately to 1% Alcian blue (in sucrose solution, buffered with sodium acetate, pH 5), and the excess dye was removed by rinsing with sucrose solution. The dye in complex with the gastric wall mucus was extracted with magnesium chloride. A 4 ml sample of the blue extract was then shaken with an equal volume of diethyl ether. The resulting emulsion was centrifuged, and the absorbance of the aqueous layer was measured at 580 nm. The quantity of Alcian blue extracted per gram (net) of glandular tissue was then calculated [22]. Macroscopic Gastric Lesion Evaluation The rat stomachs were examined under a stereomicroscope. The length and width (mm) of each individual hemorrhagic lesion was measured by a planimeter (10 × 10 mm2 = ulcer area) under a dissecting microscope (1.8×). The ulcer area (UA) was calculated using the sum of the areas of all lesions for each stomach according to a previously published protocol [23]. The UA was calculated using the following formula: Inhibition percentage (I%) was calculated as follows: Antioxidant Activity Preparation of homogenate Gastric tissue homogenate 10% (w/v) was prepared in ice-cold 50 mM phosphate buffer (pH 7.4) containing a mammalian protease inhibitor cocktail and then centrifuged at 4,000 rpm for 10 minutes (4°C). Measurement of superoxide dismutase (SOD) activity The SOD activity was measured according to the protocol of Sun et al [24]. The enzymatic activity was evaluated by measuring the enzyme’s capacity to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT). In this assay, the photochemical reduction of riboflavin generates O2−; this reduces the NBT to produce a formazan salt that absorbs light at a wavelength of 560 nm. In the presence of SOD, NBT reduction is inhibited because the enzyme converts the superoxide radical to peroxide. The results are expressed as the quantity of SOD necessary to inhibit the rate of NBT reduction by 50% in units of enzyme per gram of protein. The supernatants of the homogenates were centrifuged a second time (20 min, 12,000 rpm, 4°C), and the resulting supernatant was assayed. In a dark chamber, 1 ml of the reactant (50 mM phosphate buffer, 100 nM EDTA and 13 mM l-methionine, pH 7.8) was mixed with 30 µL of the sample, 150 µL of 75 µM NBT and 300 µL of 2 µM riboflavin. The solution was then exposed to fluorescent light (15 W) for 15 min and read using a spectrophotometer at 560 nm. Measurement of lipid peroxidation (MDA) Tissue malondialdehyde (MDA) (mmol/L) was determined using the double heating method of Draper and Hadley [25]. A reaction mixture containing 8.1% sodium dodecyl sulfate, 20% acetate buffer (pH 3.5) and 0.8% thiobarbituric acid (TBA) was mixed well with 0.2 ml of stomach tissue homogenate for 3 min and then incubated at 95°C for 60 min. After chilling, the TBA-reactive substance (MDA) was extracted with 1 ml of H2O and 2.5 ml of an n-butanol:pyridine mixture (15∶1, v/v). The upper organic layer containing the MDA, which was produced by lipid peroxidation, was measured at 532 nm. Determination of total glutathione (GSH) The gastric mucosa was weighed, minced with scissors and homogenized at 48°C in phosphate-buffered saline (PBS). The homogenate was immediately precipitated with 0.1 ml 25% trichloroacetic acid, and the precipitate was removed by centrifugation at 4200 rpm for 40 min at 4°C. The supernatant was used in a 5,5′-dithiobis(2-nitrobenzoic acid) assay to determine GSH. Absorbance was measured at 412 nm using a spectrophotometer [26]. Histological Examination of the Gastric Mucosa The gastric wall specimens were fixed in 10% buffered formalin for 24 h prior to paraffin tissue processing (Leica, Germany). The stomach tissues were sectioned at a thickness of 5 µm and stained with hematoxylin and eosin to evaluate histological degeneration [27]. Study of Mucosal Glycoproteins The sections of the glandular portion of the rat stomach were stained with Periodic acid-Schiff (PAS) as described by McManus et al [28]. Immunohistochemical Staining Immunohistochemical staining for HSP70 and BAX proteins was conducted according to the manufacturer’s protocol (DakoCytomation, USA). Statistical Analysis All values are reported as the mean ± S.E.M. and were analyzed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons using the Statistical Package for the Social Sciences software (SPSS 18). The differences between means were considered statistically significant when the p value was less than 0.05. Results Acute Toxicity Study No death or significant toxicity was observed in the control or treatment groups (extract doses of 1, 3 or 5 g/kg for 14 days), as evaluated based on clinical and histopathological observations. Gross Evaluation of Gastric Lesions The antiulcer activity of the Parkia speciosa leaf extract in the ethanol-induced gastric lesion model is shown in Table 1. The results demonstrated that rats pretreated with omeprazole or Parkia speciosa extracts prior to treatment with absolute ethanol (groups 3–7) exhibited significantly smaller areas of gastric ulceration than did group 2 (Figure 1). Absolute ethanol produced extensive and visible hemorrhagic lesions in the gastric mucosa. Parkia speciosa extract significantly inhibited the ulcer formation induced by absolute ethanol and obviously decreased the gastric mucosal damage in a dose-dependent manner, i.e., the Parkia speciosa extract significantly suppressed the formation of ulcers. Intriguingly, a flattening of the gastric mucosal folds was observed in group 7. The protection of the gastric mucosa was also the most prominent in group 7 (Table 1 and Figure 1). The inhibition of gastric ulcer formation in group 6 was significant and comparable to that observed in the omeprazole treatment group (group 3) (Table 1 and Figure 1). 10.1371/journal.pone.0064751.g001 Figure 1 The effect of P. speciosa on the macroscopic appearance of the gastric mucosa in ethanol-induced gastric mucosal lesions in rats. (G1) (Normal control group); (G2) (Ulcer control group); (G3) (Omeprazole); (G4) (50 mg/kg), (G5) (100 mg/kg), (G6) (200 mg/kg), (G7) (400 mg/kg) Parkia speciosa extract. 10.1371/journal.pone.0064751.t001 Table 1 The effect of Parkia speciosa ethanolic extract on gastric content pH, gastric ulcer area and the inhibition of ethanol-induced gastric mucosal lesions in rats. Experimental Group pH Ulcer Area Inhibition(%) CMC (vehicle) 6.57±0.21 0 0 CMC (control) 3.43±0.26 877.45±29.99 0 Omeprazole 5.43±0.21 209.46±12.78 76 P. speciosa (50 mg/kg) 4.24±0.20 373.04±22.23** 57 P. speciosa (100 mg/kg) 4.75±0.34* 215.09±23.43 75 P. speciosa (200 mg/kg) 5.36±0.20 102.37±7.59 88 P. speciosa (400 mg/kg) 6.04±0.27 39.18±3.55** 95 Rats pretreated with Parkia speciosa (100, 200, and 400 mg/kg) extracts had significantly reduced gastric ulcer areas and gastric content pH. All values are expressed as the mean ± standard error of the mean; differences between means were considered significant at p0.05 or p0.001 levels. The data were analyzed by one-way ANOVA using the Statistical Package for the Social Sciences software (SPSS 18). The Effect of Parkia Speciosa on GWM, SOD, MDA, and GSH The effects of P. speciosa on the gastric wall mucus in ethanol-induced gastric mucosal lesions in rats were examined. Group 2 exhibited a significant decrease in the Alcian blue binding capacity of the gastric wall mucus, whereas groups 4–7 demonstrated a significant enhancement in the Alcian blue binding capacity of the gastric mucosa. Similarly, in group 2, ethanol reduced the SOD activity, while groups 4–7 exhibited a significant increase in SOD enzyme activity compared with group 2. The MDA activity was significantly higher in group 2 than in group 1, while groups 4–7 demonstrated significantly decreased MDA activity. In addition, the effect of the Parkia speciosa extract on the total GSH in gastric mucosal homogenates was assessed. Ethanol treatment caused a significant depletion of GSH in group 2 compared to group 1. In contrast, groups 47 exhibited significantly augmented GSH content (Figure 2). 10.1371/journal.pone.0064751.g002 Figure 2 The effects of P. speciosa on SOD, MDA, GSH and GWM in the gastric mucosa homogenate of ethanol-induced gastric mucosal lesions in rats. All values are expressed as the mean ± standard error of the mean. All P. speciosa-treated groups were significantly different from the control groups at p0.05. The data were analyzed by one-way ANOVA using the Statistical Package for the Social Sciences software (SPSS 18). Histological Evaluation of Gastric Lesions Histological observations of group 1 indicated that there was no disruption of the surface epithelium, while the histological examination showed extensive damage to the gastric mucosa in group 2, with necrotic lesions penetrating deeply into the mucosa accompanied by extensive edema and leucocyte infiltration of the submucosal layer (Figure 3). Group 4 exhibited moderate disruption of the surface epithelium, with edema and leucocyte infiltration of the submucosal layer, and group 5 showed a mild disruption of the surface epithelium with edema and leucocyte infiltration into the submucosal layer. Group 6 showed mild disruption of the surface epithelium with edema and leucocyte infiltration of the submucosal layer. Group 7 showed mild edema and leucocyte infiltration of the submucosal layer, but no disruption of the surface epithelium. Group 3 exhibited a mild disruption of the surface epithelium, with submucosal edema and leucocyte infiltration. These results demonstrated that the plant extracts exerted cytoprotective effects in a dose-dependent manner (Figure 3). 10.1371/journal.pone.0064751.g003 Figure 3 The effect of P. speciosa on the histology of ethanol-induced gastric mucosal damage in rats. (G1) (Normal control group); (G2) (Ulcer control group); (G3) (Omeprazole); (G4) (50 mg/kg), (G5) (100 mg/kg), (G6) (200 mg/kg), (G7) (400 mg/kg) Parkia speciosa extract. The rats in groups 4–7 demonstrated comparatively better protection of the gastric mucosa, as shown by a reduction in or absence of the ulcer area, submucosal edema and leucocyte infiltration (H&E staining, 20×). Periodic Acid-Schiff (PAS) The gastric mucosa in animals pretreated with Parkia speciosa extract or omeprazole (group 3–7) displayed increased PAS staining intensity compared to the rats in group 2, indicating an increase in the glycoprotein content of gastric mucosa in pretreated rats (Figure 4). 10.1371/journal.pone.0064751.g004 Figure 4 The effect of Parkia speciosa on gastric tissue glycoprotein-PAS staining in ethanol-induced gastric ulcers in rats. (G1) (Normal control group); (G2) (Ulcer control group); (G3) (Omeprazole); (G4) (50 mg/kg), (G5) (100 mg/kg), (G6) (200 mg/kg), (G7) (400 mg/kg) Parkia speciosa extract (PAS stain 20×). Immunohistochemistry The expression of the HSP70 protein in the gastric mucosa was downregulated in group 2 but upregulated in group 3–7 (Figure 5). 10.1371/journal.pone.0064751.g005 Figure 5 Immunohistochemical analysis of HSP70 expression in the gastric mucosa of rats. (G1) (Normal control group); (G2) (Ulcer control group); (G3) (Omeprazole); (G4) (50 mg/kg), (G5) (100 mg/kg), (G6) (200 mg/kg), (G7) (400 mg/kg) Parkia speciosa extract. HSP70 protein expression was upregulated in rats pretreated with Parkia speciosa (magnification 20×). Immunohistochemical staining of the gastric mucosa of rats pretreated with Parkia speciosa extract or omeprazole demonstrated a downregulation of the BAX protein (Figure 6). In addition, the levels of BAX protein in group 2 rats were higher than those of groups 3–7 (Figure 6). 10.1371/journal.pone.0064751.g006 Figure 6 Immunohistochemical analysis of the expression of the BAX protein in the gastric mucosa of rats. (G1) (Normal control group); (G2) (Ulcer control group); (G3) (Omeprazole); (G4) (50 mg/kg), (G5) (100 mg/kg), (G6) (200 mg/kg), (G7) (400 mg/kg) Parkia speciosa extract. BAX protein expression was downregulated in rats pretreated with Parkia speciosa (magnification 20×). Discussion The acute toxicity test did not suggest any toxicity or mortality in the P. speciosa-treated rats. This test revealed that the plant is safe and has no toxicity when administered orally up to 5 g/kg. Certain antiulcer drugs have been reported to increase the amount of gastric mucus secretion in the gastric mucosa [29]. Pretreatment with Parkia speciosa extract significantly increased the gastric mucus content in rats with ethanol-induced ulcers, indicating that the gastroprotective effect of Parkia speciosa is mediated partly by preservation of the gastric wall mucus. This mucus consists of mucin-type glycoproteins, which can be detected by Alcian blue [30]; the increase in Alcian blue staining demonstrates the protective effect of orally administered Parkia speciosa, which may be mediated by the formation of protective complexes between Parkia speciosa and the mucus that acts as a barrier against necrotizing agents introduced to the stomach [31]. Here, the Parkia speciosa extract prevented the decrease in the concentration of gastric wall mucus upon treatment with ethanol. Thus, one possible mechanism by which the gastric mucosa is protected by Parkia speciosa involves the reinforcement of the mucosal barrier resistance, generated by a protective coating. It is likely that the protective effect of Parkia speciosa is due at least in part to the preservation of the mucus layer in the gastric mucosa; Parkia speciosa was observed to prevent ethanol-induced gastric wall mucus depletion. The results of the present study demonstrated that Parkia speciosa extract has an effective antiulcer activity against ethanol-induced gastric mucosal injury. The plant extract increased the mucus content of the gastric wall, which is consistent with results reported by Thirunavukkarasu et al. [32]. Our results revealed the protection of the gastric mucosa and inhibition of leucocyte infiltration into the gastric wall in rats pretreated with Parkia speciosa extract. The activation and infiltration of neutrophils appear to be involved in the initial processes that form these lesions. Similarly, Abdulla et al. [12] demonstrated that the reduction of neutrophil infiltration into ulcerated gastric tissues helped to prevent gastric ulcers in rats. Wasman et al. [33] showed that the oral administration of plant extract prior to ethanol administration significantly decreased neutrophil infiltration into the gastric mucosa. Ethanol causes extensive damage to the gastric mucosa and leads to increased neutrophil infiltration into this tissue. The present study established that pretreatment with Parkia speciosa reduced neutrophil infiltration into ulcerated tissue. We also observed a flattening of the mucosal folds, which suggests that the gastroprotective effect of the Parkia speciosa leaf extract might be attributed to a decrease in gastric motility. Changes in gastric motility have been implicated in the development and prevention of experimental gastric lesions. The relaxation of the circular muscles may protect the gastric mucosa through a flattening of the folds. This flattening increases the mucosal area exposed to necrotizing agents and reduces the volume of the gastric irritants that come into contact with the rugal crest [12], [33]. Gastric tissue homogenate prepared from the groups that were pretreated with plant extract exhibited significant antioxidant activity, with decreased levels of MDA and elevated levels of GSH and SOD, in response to oxidative stress due to ethanol treatment. SOD converts superoxide to hydrogen peroxide (H2O2), which is transformed into water by catalase in the lysosomes or by glutathione peroxidase in the mitochondria [34]. MDA is the final product of lipid peroxidation and is used to determine lipid peroxidation levels [35]. Lipid peroxidation causes a loss of membrane fluidity, impaired ion transport and membrane integrity and ultimately a loss of cellular function. Our experimental results indicate that Parkia speciosa ethanolic extract significantly inhibited the negative effects of ethanol on gastric GSH levels at all doses used. The gastric GSH level was highest for the 400 mg/kg dose and lowest for the 50 mg/kg dose, while the GSH level was decreased in the ulcer control group. The current results and previously published articles indicate that there is an important relationship between gastric GSH levels and ulcer severity. GSH and GSH-related enzymes are known as important tissue protective agents due to their antioxidant properties [36], [37]. The periodic acid-Schiff (PAS) histochemical method produces a characteristic carmine staining in stomach regions that secrete mucopolysaccharides. Tissue sections from the group of rats that was treated with 400 mg/kg plant extract showed intense staining reflecting mucus secretion in the gastric glands. Mucus production is one of the major mechanisms of local gastric mucosal defense [38]. BAX promotes apoptosis [39], while BCL-2 inhibits this process. Apoptosis may be caused by an imbalance in the expression of BCL-2 family antiapoptotic proteins and apoptotic BAX proteins in stress ulcers [40]. HSP70 is a 70 kDa protein from the HSP family that is expressed in mammalian cells. These proteins are responsible for protecting cellular homeostatic processes from environmental and physiologic injuries by preserving the structure of normal proteins and repairing or removing damaged proteins [41], and the study of this protein can thus provide interesting data for the elucidation of possible mechanisms of action. HSP70 defends cells from oxidative stress or heat shock. Ethanol-generated reactive oxygen species (ROS) normally act to inhibit the expression of HSP70 and increase the expression of BAX. HSP70 prevents these partially denatured proteins from aggregating and allows them to refold. The upregulation of HSP70 that was observed in this study suggests that the Parkia speciosa extract protected the gastric tissues through the upregulation of HSP70. Additionally, HSP70 has been suggested to exert its cytoprotective activity by protecting mitochondria and interfering with the stress-induced apoptotic program. Immunohistochemical staining showed that BAX protein expression was downregulated in rats pretreated with Parkia speciosa extract. Conclusion In conclusion, P. speciosa extract was clearly demonstrated to function as an antiulcer agent. The results suggest that the P. speciosa extract may act by enhancing the gastric mucosal defense and/or by inhibiting leukotriene synthesis. The plant extract protected the gastric tissues through the upregulation of the HSP70 and downregulation of the BAX protein. Parkia speciosa reversed the decrease in PAS staining induced by ethanol, significantly increased the GSH and SOD activities, and decreased the level of lipid peroxidation (MDA) in the P. speciosa-pretreated groups. The authors are thankful to the staff of Faculty of Medicine, and Animal House for the care and supply of rats and to others who participated in this work. ==== Refs References 1 Yeomans ND , Hawkey CJ , Brailsford W , Næsdal J (2009) Gastroduodenal toxicity of low-dose acetylsalicylic acid: a comparison with non-steroidal anti-inflammatory drugs. Current Medical Research & Opinion 25 : 2785–2793.19788350 2 Yuan L , Wang JH , Sun TM (2010) Total synthesis and anticancer activity studies of the stereoisomers of asperphenamate and patriscabratine. Chinese Chemical Letters 21 : 155–158. 3 Itatsu T , Nagahara A , Hojo M , Miyazaki A , Murai T , et al (2011) Use of selective serotonin reuptake inhibitors and upper gastrointestinal disease. Internal Medicine 50 : 713–717.21467703 4 Søberg T , Hofstad B , Sandvik L , Johansen M , Lygren I (2010) Risk factors for peptic ulcer bleeding. Tidsskrift for den Norske Laegeforening: Tidsskrift for Praktisk Medicin, ny Raekke 130 : 1135–1139. 5 Lanas A (2009) Nonsteroidal antiinflammatory drugs and cyclooxygenase inhibition in the gastrointestinal tract: a trip from peptic ulcer to colon cancer. The American Journal of the Medical Sciences 338 : 96.19680014 6 Maity P , Biswas K , Roy S , Banerjee RK , Bandyopadhyay U (2003) Smoking and the pathogenesis of gastroduodenal ulcer–recent mechanistic update. Molecular and Cellular Biochemistry 253 : 329–338.14619984 7 Kushima H , Hiruma-Lima CA , Santos MA , Viana E , Coelho-Ferreira M , et al (2005) Gastroprotective activity of Pradosia huberi on experimentally induced gastric lesions in rodents: Role of endogenous sulphydryls and nitric oxide. Journal of Ethnopharmacology 101 : 61–67.15908153 8 Marhuenda E , Martin M , Alarcon Lastra C (1993) Antiulcerogenic activity of aescine in different experimental models. Phytotherapy Research 7 : 13–16. 9 Davenport H (1968) Destruction of the gastric mucosal barrier by detergents and urea. Gastroenterology 54 : 175.5711899 10 Jainu M , Mohan KV , Devi CSS (2006) Gastroprotective effect of Cissus quadrangularis extract in rats with experimentally induced ulcer. Indian Journal of Medical Research 123 : 799.16885602 11 Lukie BE , Forstner GG (1972) Synthesis of intestinal clycoproteins inhibition of [1–14C]glucosamine incorporation by sodium salicylate in vitro. Biochimica et Biophysica Acta (BBA) - General Subjects 273 : 380–388.5080325 12 Abdulla MA , Ahmed KAA , Al-Bayaty FH , Masood Y (2010) Gastroprotective effect of Phyllanthus niruri leaf extract against ethanol-induced gastric mucosal injury in rats. African Journal of Pharmacy and Pharmacology 4 : 226–230. 13 Abdelwahab SI, Mohan S, Mohamed Elhassan M, Al-Mekhlafi N, Mariod AA, et al .. (2010) Antiapoptotic and antioxidant properties of Orthosiphon stamineus Benth (cat’s whiskers): intervention in the Bcl-2-mediated apoptotic pathway. Evidence-Based Complementary and Alternative Medicine 2011. 14 Wasman S , Mahmood A , Suan Chua L , Alshawsh MA , Hamdan S (2011) Antioxidant and gastroprotective activities of Andrographis paniculata (Hempedu Bumi) in Sprague Dawley rats. Indian Journal of Experimental Biology 49 : 767.22013743 15 Gan CY , Latiff AA (2011) Optimisation of the solvent extraction of bioactive compounds from Parkia speciosa pod using response surface methodology. Food Chemistry 124 : 1277–1283. 16 Ching LS , Mohamed S (2001) Alpha-tocopherol content in 62 edible tropical plants. Journal of Agricultural and food Chemistry 49 : 3101–3105.11410015 17 Mahmood A , Mariod AA , Al-Bayaty F , Abdel-Wahab SI (2010) Anti-ulcerogenic activity of Gynura procumbens leaf extract against experimentally-induced gastric lesions in rats. J Med Plants Res 4 : 685–691. 18 Mahmood A , Al-Bayaty F , Salmah I , Syuhada ABN , Harita H , et al (2011) Enhancement of gastric ulcer by Areca catechu nut in ethanol-induced gastric mucosal injuries in rats. Journal of Medicinal Plant Research 5 : 2562–2569. 19 CHEMICALS D (2005) OECD Guideline for testing of chemicals. 20 Resources IoLA (1996) Guide for the care and use of laboratory animals: National Academies Press. 21 Shay H (1945) A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology 4 : 43–61. 22 Piper D , Whitecross D , Leonard P , Clarke A (1970) Alcian blue binding properties of gastric juice. Gastroenterology 59 : 534.4920566 23 Bardi D , Khan MAS , Sabri S , Kadir F , Mahmood A , et al (2011) Anti-ulcerogenic activity of Typhonium flagelliforme aqueous leaf extract against ethanol-induced gastric mucosal injury in rats. Scientific Research and Essays 6 : 3232–3239. 24 Sun Y , Oberley LW , Li Y (1988) A simple method for clinical assay of superoxide dismutase. Clinical Chemistry 34 : 497–500.3349599 25 Draper H , Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods in enzymology 186 : 421.2233309 26 Sedlak J , Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Analytical biochemistry 25 : 192.4973948 27 Behmer OA (1976) Manual de técnicas para histologia normal e patológica: EDART/USP. 28 McManus J (1948) Histological and histochemical uses of periodic acid. Biotechnic & Histochemistry 23 : 99–108. 29 Robert A , Böttcher W , Golanska E , Kauffman Jr G (1984) Lack of correlation between mucus gel thickness and gastric cytoprotection in rats. Gastroenterology 86 : 670.6421648 30 Bolton JP , Palmer D , Cohen MM (1978) Stimulation of mucus and nonparietal cell secretion by the E 2 prostaglandins. Digestive Diseases and Sciences 23 : 359–364. 31 Clamp J , Allen A , Gibbons R , Roberts G (1978) Chemical aspects of mucus. British medical bulletin 34 : 25–41.626832 32 Thirunavukkarasu P , Ramanathan T , Ramkumar L , Shanmugapriya R (2010) Anti ulcer effect of Avicennia officinalis leaves in albino rats. World Applied Sci J 9 : 55–58. 33 Wasman S , Mahmood A , Salehhuddin H , Zahra A , Salmah I (2010) Cytoprotective activities of Polygonum minus aqueous leaf extract on ethanol-induced gastric ulcer in rats. J Med Plants Res 4 : 2658–2665. 34 Johansen JS , Harris AK , Rychly DJ , Ergul A (2005) Oxidative stress and the use of antioxidants in diabetes: linking basic science to clinical practice. Cardiovascular Diabetology 4 : 5.15862133 35 Dursun H , Bilici M , Albayrak F , Ozturk C , Saglam MB , et al (2009) Antiulcer activity of fluvoxamine in rats and its effect on oxidant and antioxidant parameters in stomach tissue. BMC gastroenterology 9 : 36.19457229 36 Repetto M , Llesuy S (2002) Antioxidant properties of natural compounds used in popular medicine for gastric ulcers. Brazilian Journal of Medical and Biological Research 35 : 523–534.12011936 37 La Casa C , Villegas I , Alarcon de La Lastra C , Motilva V , Martın Calero M (2000) Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions. Journal of Ethnopharmacology 71 : 45–53.10904145 38 Laine L , Takeuchi K , Tarnawski A (2008) Gastric mucosal defense and cytoprotection: bench to bedside. Gastroenterology 135 : 41–60.18549814 39 Emily HYAC , Wei MC , Weiler S , Flavell RA , Mak TW , et al (2001) BCL-2, BCL-XL sequester BH3 domain-only molecules preventing BAX-and BAK-mediated mitochondrial apoptosis. Molecular Cell 8 : 705–711.11583631 40 Konturek PC , Brzozowski T , Konturek S , Pajdo R , Konturek J , et al (1999) Apoptosis in gastric mucosa with stress-induced gastric ulcers. Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society 50 : 211.10424718 41 Tytell1 M, Hooper PL (2001) Heat shock proteins: new keys to the development of cytoprotective therapies. Emerging Therapeutic Targets 5 : 267–287.	23724090	PMC3665813	CC BY	2024-01-03 23:16:09	yes	PLoS One. 2013 May 28; 8(5):e64751
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23734258PONE-D-12-2992510.1371/journal.pone.0065456Research ArticleBiologyGenomicsChromosome BiologyMolecular Cell BiologyChromosome BiologyMedicineGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis BHepatitis CInfectious diseasesViral diseasesHepatitisHepatitis BHepatitis CAn Occult Hepatitis B-Derived Hepatoma Cell Line Carrying Persistent Nuclear Viral DNA and Permissive for Exogenous Hepatitis B Virus Infection New Hepatoma Cell Lines for HBV InfectionLin Chih-Lang 1 2 Chien Rong-Nan 1 Lin Shi-Ming 3 Ke Po-Yuan 4 Lin Chen-Chun 3 Yeh Chau-Ting 3 * 1 Liver Research Unit, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Keelung, Taiwan 2 Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan 3 Liver Research Center, Chang Gung Memorial Hospital, Taipei, Taiwan 4 Department of Biochemistry and Molecular Biology, Chang Gung University, Taoyuan, Taiwan Fung James Editor The University of Hong Kong, Hong Kong * E-mail: chautingy@gmail.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: RNC CTY. Performed the experiments: CLL SML CCL. Analyzed the data: PYK SML. Contributed reagents/materials/analysis tools: CTY. Wrote the paper: CLL CTY. 2013 29 5 2013 8 5 e6545629 9 2012 26 4 2013 © 2013 Lin et al2013Lin et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection. The study is supported by grants from Chang Gung Medical Research Council (CMRPG 371693; CMRPG 3A0522). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Chronic hepatitis B virus (HBV) infection is one of the major infectious diseases worldwide and may lead to severe liver diseases, including liver cirrhosis and hepatocellular carcinoma (HCC) [1], [2]. HBV is a small, enveloped partially double-stranded DNA virus of the family Hepadnaviridae, which replicates via reverse transcription. The 3.2-kb relaxed circular DNA (RC-DNA) genome encapsidated by the viral particles is transported into the host cell nuclei, where it is repaired to become covalently closed circular DNA (cccDNA)—the template for transcription of several subgenomic and genomic RNAs [3]. Much has been learned about HBV host–pathogen interactions since its discovery, but many unsolved issues remain, such as the mechanism of viral entry, the exact role of some viral components, the kinetics of HBV cccDNA production, and the precise mechanism that leads to the development of HCC [4]. A prime characteristic of HBV is that it exhibits a very narrow host range and a strong tropism for liver parenchymal cells [5], [6]. To investigate the complete life cycle of HBV infection—despite the usefulness of available human liver cell lines—there is clearly a strong need for appropriate in vitro infection systems, allowing for exogenous HBV infection [7]. HBV replication can be initiated by genomic DNA transfection into HepG2, Huh7, HepAD38, or primary hepatocytes [8]. However, until recent years, exogenous HBV infection has only been successfully achieved using primary human hepatocytes [9], [10]. However, the system is limited by a low susceptibility to infection and the cultured hepatocytes become non-permissive for HBV soon after plating. Until now, highly efficient HBV infection is accomplished in only one well differentiated hepatoma cell line (Hepa RG) established from a female patient suffering from HCC and hepatitis C virus (HCV) infection [5]. As such, establishment of new HBV permissive human hepatoma cell lines for the study of HBV biology and the development of new anti-HBV strategy is needed. The persistence of HBV genomes in the blood or liver of individuals negative for HBV surface antigen (HBsAg) is termed occult HBV infection [11]. It is not known why occult HBV carriers are HBsAg-negative. Some of these individuals are infected by viral variants that either produce an antigenically modified HBV surface protein—undetectable by even the most sensitive available HBsAg assays—or carry mutations capable of inhibiting surface protein gene expression and/or viral replication [12]. However, such mutations cannot be detected in a large proportion of occult hepatitis B patients. Several other hypotheses have been raised for the molecular mechanisms of occult hepatitis B infection, including HBV infection in host mononuclear cells and formation of immune complex interfering HBsAg detection. Experimental evidences are still in need to determine whether one or some of them stands. Substantial clues indicate that occult HBV infection is associated with the progression of liver fibrosis and cirrhosis development [11], [13], [14], and that it is a risk factor for HCC development [15], [16] in patients with cryptogenic liver disease. Several reports performed in the 1990s suggested that occult HBV may negatively influence chronic HCV infection by reducing the response to IFN therapy [17], [18], [19]. Thus, there is a strong need to establish appropriate cell-based systems to study the molecular mechanism of occult HBV infection. To our knowledge, cell culture systems derived from patients with occult HBV infection, capable of maintaining sustained HBV replication, have not yet been generated. In this study, we established 2 hepatoma cell lines which expressed a representative panel of liver-specific proteins and were susceptible to exogenous HBV infection. Notably, we produced the first stable hepatoma cell line derived from occult hepatitis B patient, wherein a nuclear form of HBV DNA persisted during multiple cell line passages without secretion of HBsAg. This cell line provides a novel tool suitable for studying several important aspects of occult HBV infection. Materials and Methods Cell line establishment and culture conditions This study was approved by the Institutional Review Board of Chang Gung Medical Center. Hep-Y1 cells were grown from the fine-needle-aspirated hepatoma cells of a male patient suffering from HCC without HBV and HCV infection. Hep-Y2 cells were isolated from the fine-needle-aspirated hepatoma cells of a female patient suffering from HCC with occult HBV and chronic HCV infection. Occult HBV infection is defined as persistence of viral genomes in the blood or liver of individuals negative for HBsAg. Chronic HCV infection is defined as seropositive for anti-HCV antibodies and HCV RNA for more than 6 months. HCC cells were aspirated under echo-guided procedure using a fine needle at the same time when aspiration cytology was needed for diagnosis. Human methothelial cells were isolated from ascites of cirrhotic patients and plated as a confluent monolayer in culture dishes. HCC cells were seeded on to the methothelial cells and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cells were grown in a 1∶1 mixture of RPMI-1640 medium (Gibco, Invitrogen Corporation, Carlsbad, CA) and PromoCell Hepatocyte Medium (PromoCell GmbH, Heidelberg, Germany) supplemented with 2 mM l-glutamine, 10 mM HEPES buffer, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. The cells were trypsinized and re-plated every 7 days, allowing for senescence and death of methothelial cells. After 5 passages, the cells were grown in only RPMI-1640 medium with supplements. After >20 passages, the cultured cells, if still growing, were collected and stored in liquid nitrogen [20]. Serological assays, HBV DNA assay, and HCV RNA assay HBsAg was measured by an enzyme immunoassay (Enzygnost HBsAg 5.0; Dade Behring Marburg GmbH, Marburg, Germany) and a radioimmunoassay (Ausria-II; Abbott, Abbott Park, IL). Serum antibody to HCV was assayed using commercially available second- or third-generation enzyme immunoassay kits (HCV EIA II or III; Abbott Laboratories). Serum HBV DNA levels were quantified using a COBAS TaqMan HBV test (Roche Molecular Systems, Inc., Pleasanton, CA) with a detection limit of 68 copies/mL. In this test, 5.82 copies/mL was equivalent to 1 IU/mL. HCV RNA in the serum sample and in the culture medium was assessed by use of a COBAS TaqMan HCV test (Roche Molecular Systems, Inc., Pleasanton, CA). Quantification of HBsAg was performed using Elecsys HBsAg II assay (Roche Diagnostic). Two HCC cell lines were described in this report. Hep-Y1 cells were derived from a HCC patient with negative anti-HCV antibody and negative HBsAg. HCV RNA was negative in both the serum sample of this patient and the culture medium of Hep-Y1 cells. Hep-Y2 was derived from a HCC patient with positive anti-HCV antibody. In this patient, the serum HCV RNA level was 12444 IU/mL. However, in the culture medium of Hep-Y2 cells, HCV RNA was negative. In the Hep-Y2-derived patient, the serum HBV DNA level was negative immediately and 6 months after the fine needle aspiration procedure. However, HBV DNA was positive in the liver tissue by PCR assay, which is in concordance with the definition of occult HBV infection [12]. HBV infection in cell lines Hep-Y1cells and Hep-Y2 cells were grown on coverslips with RPMI-1640 medium at 37°C in 5% CO2 air. At 24 h after plating, Hep-Y1cells and Hep-Y2 cells were inoculated with 500 µL of HBV-positive serum (4.2×10 9 copies/mL and 3.2 × 10 9 copies/mL, respectively). In control group, HBV-negative serum was used. During the inoculation, cells were maintained in the presence of 2% dimethyl sulfonxide (DMSO) and harvested at the indicated time-points. Extraction of HBV DNA and polymerase chain reaction (PCR) To isolate HBV DNA, viral replicative DNA intermediates were isolated from whole cell lysates. Cells were trypsinized and separated into nuclear and cytoplasmic fractions using cell lysis buffer (100 mM Tris-HCl [pH 7.4], 0.5% NP40, and 150 mM NaCl). DNA in the nucleus was further partitioned by the Hirt (1% SDS, 10 mM EDTA, 5 mM EGTA [pH 7.5], and 1 M NaCl) procedure into supernatant (extrachromosome) and pellet (chromosome) fractions. Cytoplasmic and nuclear extrachromosomal fractions were mixed with TEN buffer (1 M Tris-HCl [pH 7.5], 0.5 M EDTA [pH 8.0], 5 M NaCl, and 10% SDS) supplemented with proteinase K (1 mg/ml) and incubated at 55°C for 3 hr. For HBV DNA extraction from the serum and culture medium, 100 µL sample was mixed with 300 µL of buffer (13.3 mM Tris-HCl, pH 8.0, 6.7 mM EDTA, 0.67% SDS and 133 mg/mL proteinase K) and incubated at 55°C for 4 h. Two phenol/chloroform extractions were followed by one chloroform extraction, and DNA was precipitated with cold ethanol. The precipitate was dissolved in TE buffer (10 mM Tis-HCl [pH 8.0] and 1 mM EDTA). PCR was performed with 10 µL of DNA, 2 units of Super Tag (HT Biotechnology, Cambridge, UK), 200 µmol/L deoxynucleotide triphosphate, 100 pmol of each primer, and the reaction buffer that was provided with the enzyme. The PCR primers used were as follows: 5′-CTTATTGGTTCTTCTGGATTATC-3′ (P1, nt. 433–455, sense) and 5′-GTTTAAATGTATACCCAGAGAC-3′ (P2, nt. 837–816, antisense) [21]. The program cycle consisted of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Amplification proceeded for 30 cycles in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). A serum sample from a normal subject and an aliquot of water were included as negative controls. Nucleic acids were analyzed on a 2% agarose gel. The sensitivity and specificity of the aforementioned assays were previously tested according to the methods of Liaw et al [2]. Southern and western blot analyses The sequence flanked by primers P1 and P2 was amplified, labeled, and used as the probe for Southern blot analysis. The detailed methods for probe labeling and Southern blotting have previously been described [22]. The medium (100 µL) from the cell culture plates was loaded directly onto the nitrocellulose membrane. The following antibodies (1∶1000 dilution) were tested: albumin polyclonal antibody (lot A80–129A; Bethyl Laboratories, Montgomery, TX), aldolase B monoclonal antibody (lot GTX62246; GeneTex, Irvine, CA), and transferrin polyclonal antibody (lot GTX112729; GeneTex). To perform sodium dodecyl sulfate–polyacrylamide gel electrophoresis, cells were lysed in Tris-buffered saline (TBS) (10 mmol/L Tris-HCl [pH 7.2], 150 mmol/L NaCl) containing 0.5% Nonidet P-40 (Sigma Chemical Co., St. Louis, MO) and were centrifuged at 1500 g. Both the soluble (cytoplasmic) fraction and the culture medium were subjected to SDS–PAGE, followed by western blot analysis. As a control, GAPDH was detected by the use of anti-GAPDH antibody (6C5; Novus Biologicals, Littleton, CO). Immunofluorescence analysis Hep-Y1cells and Hep-Y2 cells were grown on coverslips and infected using 500 ul of HBV-positive serum (4.2 × 10 9 copies/mL and 3.2 × 10 9 copies/mL). Forty-eight hours post-transfection, the cells were fixed in acetone at −20°C for 2 min. Rabbit polyclonal anti-HBs and anti-HBc antibody (ViroStat; 1∶100 dilution) and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Leinco Technologies, Inc., St. Louis, MO; 1∶150 dilution) were used as the primary and secondary antibodies, respectively. To visualize the nuclei, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; 200 ng/mL). Chromosome preparation and cytogenetic analysis Standard Giemsa-banded karyotype analysis was performed according to the manufacturer's instructions, with modifications as previously described [20]. Briefly, after standard culturing of cell lines, chromosome spreads were prepared for performing karyotype analysis. The cells were then treated with hypotonic solution (0.1 M MgCl2), fixed with Carnoy's acetic solution, and stained with 0.8% Giemsa solution. The clonality criteria and the karyotype description followed the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN) [23]. Results Morphology of the cell lines Morphological assessment of both Hep-Y1 and Hep-Y2 (Figure 1A) cells by phase-contrast microscopy revealed a typical monolayer of bright and spherically shaped cells with characteristic well-contrasted borders. The majority of cells adhered to each other, presenting with a fascicular pattern of growth and granular hepatocyte-like appearance. Binuclei were observed in the majority of cells, the hepatic plate-like structures were noted, and the boundaries between hepatocytes were perfectly clear and bright. The presumably bile canaliculi and sinusoidal complexes were vague, and short microvilli were scattered over the plasma membrane surface. 10.1371/journal.pone.0065456.g001Figure 1 Phase-contrast and transmission electron micrographs under proliferating conditions. (A) Morphology of Hep-Y1 and Hep-Y2 cells in monolayer culture by phase-contrast microscopy. Cells were maintained in RPMI-1640 medium. Arrows indicated granular hepatocyte-like appearance. Arrowheads indicated hepatic plate-like structure. (B) Low- magnification views of a single Hep-Y2 cell by transmission electron microscopy. Arrows indicated prominent nucleoli. Arrowheads indicated mitochondria. (C) High-magnification views of a single Hep-Y2 cell by transmission electron microscopy. Arrowheads indicated mitochondria. (D) Low- magnification views of a single Hep-Y1 cell by transmission electron microscopy. Arrows indicated prominent nucleoli. Arrowheads indicated mitochondria. (E) High-magnification views of a single Hep-Y1 cell by transmission electron microscopy. Arrowheads indicated mitochondria. Transmission electron microscopy of Hep-Y2 cells (Figure 1A, B, and C) and Hep-Y1 cells (Figure 1A, D and E) demonstrated normal hepatocyte subcellular architecture with plentiful mitochondria. In addition, endoplasmic reticulum and Golgi; nuclei with prominent nucleoli; and well-defined nuclear pores, lipid vacuoles, and pools of glycogen were observed. Structures identical to bile canaliculi were also delineated by typical junctional complexes and presented with microvilli (Figure 1C and 1E). Cytogenetic studies Cytogenetic studies by standard Giemsa-banded karyotype analysis showed both structural and chromosomal abnormalities (Figure 2). Consistent with the findings of DNA content, the karyotype results of Hep-Y1 and Hep-Y2 cells revealed aneuploidy, presenting with 75, XX <3N> and 57, X <2N>, respectively. Complicated genetic abnormalities of chromosomal structure in Hep-Y1 cells, including +del(1)(p13), +der(1)t(1;?)(q10;?), add(2)(p23), +i(8)(q10), add(9)(p24), der(15;17)(q10;q10), +del(16)(q22), and +add(17)(p11.2) were observed. Among analyzed cells, chromosome gains, involving chromosomes 3, 7, 20, and 22, and chromosome losses, involving chromosomes 4, 5, 6, 9, 15, 18, and 21, and additional 5 marker chromosomes were noted in Hep-Y1 cells. The genetic abnormalities seen in Hep-Y2 cells were del(1)(q10), der(2)t(2;?)(p23;?), der(3)t(3;?)(p11;?), +i(7)(q10), +der(7)t(7;?)(q11.2;?), der(11)t(1;11)(q23;q23), and +ass(19)(q13.4). Among analyzed cells, chromosome number alterations of chromosomes in Hep-Y2 cells comprised gains in chromosomes 6, 14, 15, and 20, and losses on chromosome 17, and 5 marker chromosomes. 10.1371/journal.pone.0065456.g002Figure 2 Standard Giemsa-banded karyotype analysis. Representative chromosomal structures of Hep-Y1 cell (A) and Hep-Y2 cell (B) displayed aneuploidy and complicated genetic abnormalities. Arrows indicated chromosome gains. Arrowheads indicated marker chromosomes. Expression of liver-specific functions To further determine the liver-specific functional competencies of Hep-Y1 and Hep-Y2 cells, we subsequently tested for the secretion of alpha-fetoprotein (AFP) and cellular expression of albumin, transferrin, and glycolytic enzyme aldolase B. The AFP levels of Hep-Y1 and Hep-Y2 cells—detected using a commercial enzyme-linked immunosorbent assay—varied from days 1–3 dependent on growth conditions, with a peak level of 11.5 and 6.3 ng/mL, respectively. Western blot analysis of albumin, transferrin, and aldolase B were performed (Figure 3A). Albumin was strongly expressed in Hep-Y1, Hep-Y2, and HepG2 cells but not in 293-EBNA kidney cells. Transferrin, an iron-binding blood plasma glycoprotein that is mainly produced in the liver, was expressed by Hep-Y1, Hep-Y2, and HepG2 cells with increasing levels but not in 293-EBNA kidney cells. Aldolase B, a liver-type aldolase that played a key role in both glycolysis and gluconeogenesis, was strongly expressed in Hep-Y1 and HepG2 cells but weakly expressed in Hep-Y2 and 293-EBNA kidney cells. 10.1371/journal.pone.0065456.g003Figure 3 Liver-specific protein expression in culture cells and HBV DNA detection in culture medium. (A) Expression of liver-specific proteins in Hep-Y1 and Hep-Y2 cells. Western blot analysis of albumin, transferrin, and aldolase B in Hep-Y1, Hep-Y2, HepG2, and 293-EBNA kidney cells. (B) PCR products in the medium of cultured Hep-Y1 cells and Hep-Y2 cells on a 2% agarose gel. M, molecular weight marker; P, PCR product from HBV DNA using the same PCR primers as positive control. Lanes 1 to 3, and lanes 4 to 6 were results of triplicate experiments. Detection of sustained HBV DNA in Hep-Y2 cells The culture medium from both Hep-Y1 and Hep-Y2 cells were tested negative for HBsAg by either the enzyme immunoassay (Enzygnost HBsAg 5.0) or the radioimmunoassay (Ausria-II). To determine whether HBV replication was sustained in Hep-Y2 cells, a cell line derived from an occult HBV-infected patient, we assayed HBV DNA in the culture medium by PCR analysis. Figure 3B showed the presence of viral DNA in the medium of cultured Hep-Y2 cells. However, no viral DNA was detected in Hep-Y1 cells. HBsAg was negative in the culture medium of both cell lines. The intracellular HBV DNA level in Hep-Y1 cells was undetectable and in Hep-Y2 cells was 3.1719 × 106 copies per 106 cells, respectively. Permissiveness of HBV infection for Hep-Y1 and Hep-Y2 cells Hep-Y1 and Hep-Y2 cells were found to share many morphological characteristics with normal human hepatocytes. Thus, we examined their susceptibility to HBV infection. To this end, we employed the same conditions that have previously been validated for in vitro infection of primary human hepatocytes [5]. Figure 4A showed the increasing levels of HBV DNA in culture medium from day 1 to 5 after HBV infection in Hep-Y1 cells by Southern blot analysis of the PCR amplicons. In contrast, HBV DNA was not detectable in normal serum (HBV-negative) infection. 10.1371/journal.pone.0065456.g004Figure 4 HBV DNA replication in Hep-Y1 cells after HBV infection. (A) Southern blot kinetic analysis of PCR products after infection of Hep-Y1 cells with control normal HBV-negative serum and HBV-containing serum. P1 and P2, 1 pg and 50 pg of PCR products derived from HBV DNA were loaded. (B) Quantification of HBV DNA. The HBV-DNA levels in 106 cells after HBV infection were measured by Cobas TaqMan HBV assay. D1-3, Day 1–3 after infection. 1 IU = 5.82 copies. After HBV infection, the intracellular levels (cytoplasmic + extrachromosomal nuclear forms) of HBV DNA were also assessed by Cobas TaqMan HBV assays (Figure 4B). A progressively increasing HBV DNA level was observed in both Hep-Y1 and Y2 cells and the levels were higher when compared to those in HepG2 cells. Therefore, Hep-Y2 cells were also likely susceptible to HBV infection. To verify this point, we employed Southern blot analysis to compare the levels of HBV DNA produced in the cytosol after HBV infection. Cells were fractionated into cytoplasmic and nuclear fractions followed by Hirt's extraction. In Hep-Y1 cells, replicate intermediates of HBV DNA were detectable in the cytosol on day 5 after HBV infection by southern blot, confirming the permissiveness of HBV infection (Figure 5A). 10.1371/journal.pone.0065456.g005Figure 5 Permissiveness of Hep-Y2 and Hep-Y1 cells for HBV infection. (A) Southern blot kinetic analysis of cytosolic and nuclear HBV DNA after infection of Hep-Y2 cells with control normal serum and HBV-containing serum. (B) Southern blot kinetic analysis of cytosolic and nuclear HBV DNA after infection of Hep-Y1 cells with control normal serum and HBV-containing serum. C, un-infected HepG2 cells as negative control. P, 20 pg of PCR product from HBV DNA as positive hybridization control. In Hep-Y2 cells, southern blot analysis showed that extrachromosomal HBV DNA was detected in the nuclear but not cytoplasmic fractions even when control (HBV-negative) serum was used for infection (Figure 5B). This free form of HBV DNA was maintained in the nuclear fractions before and after day 9 of HBV infection, consistent with the status of occult HBV infection. Interestingly, replication intermediates of HBV DNA were detected in the cytosol on day 5 after HBV infection, which decreased gradually from day 5 to 9, suggesting permissiveness of HBV infection in Hep-Y2 cells (Figure 5B). Two species of HBV replication intermediates were discernable in Figure 5. The upper band corresponded to the relaxed circular form and the lower band corresponded to the double stranded linear form. The single stranded form and cccDNA were not seen in this study. HBV DNA levels in the culture medium of infected Hep-Y2 cells were 26.4, 68.1, 388.2, and 32.8 ×104 copies/mL at D3, D5, D7, and D9, respectively. HBV DNA level in the culture medium of infected Hep-Y1 cells was 280 copies/mL on D5 but was undetectable in D3, D7, and D9. HBeAg in the medium was negative in both cell lines after infection (D3 to 9). HBsAg in the medium was 1.46, 2.67, 3.24, and 2.89 IU/mL (D3, 5, 7, 9 after infection) in Hep-Y1 cells and 2.10, 3.54, 3.71 and 0.98 IU/mL (D3, 5, 7, 9 after infection) in Hep-Y2 cells using a quantification assay. Immunofluorescence analysis was performed for Hep-Y1 and Y2 cells after HBV infection using a polyclonal anti-HBc (hepatitis B core) antibody (Figure 6) and anti-HBs antibody (Figure 7). Prominent cytoplasmic HBsAg and nuclear HBcAg expressions were detected in both Hep-Y1 and Y2 cells after infection. The infected cells ranged from 75 to 95% among independent experiments. These results indicated that Hep-Y1 and Y2 cells were efficiently infected by HBV. 10.1371/journal.pone.0065456.g006Figure 6 Immunofluorescence analysis of core protein expression after HBV infection. (A) HBV-infected HepG2 and Hep-Y1 cells (left two panels) and HBV-negative serum inoculated Hep-Y1 and Hep-Y2 cells (right two panels, as negative controls) (B) HBV infected Hep-Y2 cells. Data of three independent experiments were shown (top to bottom). A polyclonal anti-HBc antibody was used to detect HBcAg. Nuclei were visualized by DAPI staining. 10.1371/journal.pone.0065456.g007Figure 7 Immunofluorescence analysis of surface protein expression after HBV infection. (A) HBV-infected HepG2, Hep-Y1, and Hep-Y2 cells. (B) HBV-negative serum inoculated Hep-Y1 and Hep-Y2 cells. A polyclonal anti-HBs antibody was used to detect HBsAg. Nuclei were visualized by DAPI staining. Discussion In this study, we established 2 HCC-derived cell lines, Hep-Y1 and Hep-Y2 cells. They maintained hepatocyte-like morphology as revealed by phase-contrast and transmission electron microscopy. Furthermore, optimal liver-specific functions such as the production of AFP, albumin, transferrin, and aldolase B were also observed. Similar to HepG2 cells, Hep-Y1 and Hep-Y2 cells were susceptible to HBV infection after treated with 2% DMSO. After infection with HBV-containing serum, HBcAg and HBsAg in Hep-Y1 and Hep-Y2 cells were detectable by immunofluorescence analysis. Compared with Hep-Y1 and Hep-Y2 cells, HepG2 cells infected with HBV produced much weaker immunofluorescence signals and lower HBV DNA levels. Hence, Hep-Y1 and Hep-Y2 cells seemed to be more susceptible to HBV infection than HepG2 cells. However, comparison between Hep-Y1, Hep-Y2, and another HBV-susceptible cell line—Hepa RG cells—has not been done in this study. Judging from the published data for Hepa RG cells, the HBV replication level in this cell line was even higher. Occult HBV infection is defined as the persistence of viral genomes in the liver tissue—and, in some cases, also in the serum—of HBsAg-negative individuals [12]. The efficient eradication as well as monitoring of occult HBV infection is still a challenge in modern medicine. The Hep-Y2 cells, which were derived from an occult HBV infected patient, showed a phenotype consistent with occult HBV infection. To our knowledge, this is the first report of an occult hepatitis B-derived cell line carrying persistent extrachromosomal HBV DNA. In addition, this cell line is permissive for further exogenous HBV infection, a property never been observed before. Several issues could be explored by use of this cell line in the future. For example, how the nuclear HBV DNA in Hep-Y2 cells is maintained? Is there HBV DNA integration in the chromosome? Is there a complete HBV replication cycle in Hep-Y2 cells? Is there complete virion secretion? Is there S gene mutation? Several important experiments in the future are required to solve these puzzles. Previous reports have indicated that in growing cells, replication intermediates of HBV mostly located in the cytosol. However, in quiescent cells (G0/G1 phase), HBV DNA could accumulate in the nuclei [24], [25]. Presumably, this is caused by cell cycle regulation of nuclear transport for HBV core proteins [26]. In Hep-Y2 cells, however, a nuclear form of HBV DNA was maintained in growing cells. Conceivably, some unknown host and viral factors are involved. The serum sample from the occult HBV infected patient (from whom Hep-Y2 cells were derived) was negative for HBV DNA, whereas the aspirated liver cells were positive for HBV DNA. As such, we were unable to obtain the “initial” strain of HBV for comparison because of insufficient amount of tissue sample. In Hep-Y2 cells, partial HBV genome derived from the extrachromosomal nuclear form was cloned and sequenced. However, there remained a gap of around 1000 bp between the core and pre-S coding regions (data not shown). Presumably, this is caused by either deletion or recombination. Another important question to be asked was whether an integrated form of HBV genome existed in Hep-Y2 cells. These tissues should be clarified in future studies. Occult HBV may have an important impact in several different clinical contexts, including the transmission of occult HBV infection by blood transfusion or organ transplantation and acute reactivation under immunosuppressive status. Moreover, evidence has suggested that it can enhance the progression of liver fibrosis and, above all, the development of HCC [12]. Accordingly, how to adequately detect and treat occult HBV infection is an important issue, which is still unclear at this time. By use of Hep-Y2 cells, we may be able to identify more effective and accurate therapeutic strategies with the presently available antiviral agents, which include nucleot(s)ide analogs and interferon. In summary, we established Hep-Y1 and Hep-Y2 hepatoma cell lines which were permissive for HBV infection after DMSO treatment. Furthermore, Hep-Y2 cells retained the phenotype of occult HBV infection and maintained an extrachromosomal form of persistent HBV DNA in the nuclei. Therefore, these cell lines serve as important tools for further studies on HBV host–pathogen interaction. Additionally, Hep-Y2 cells provide a powerful tool for investigating the molecular virology of occult HBV infection. The authors appreciated the help from members of Liver Research Center and Jin-Hou Wu from the Division of Hematology-Oncology, Chang Gung Memorial Hospital for providing technical help. ==== Refs References 1 Yeh CT , Chien RN , Chu CM , Liaw YF (2000 ) Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy . Hepatology 31 : 1318 –1326 .10827158 2 Liaw YF , Chien RN , Yeh CT , Tsai SL , Chu CM (1999 ) Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy . Hepatology 30 : 567 –572 .10421670 3 Sun D , Nassal M (2006 ) Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus . J Hepatol 45 : 636 –645 .16935386 4 Fellig Y , Almogy G , Galun E , Ketzinel-Gilad M (2004 ) A hepatocellular carcinoma cell line producing mature hepatitis B viral particles . Biochem Biophys Res Commun 321 : 269 –274 .15358171 5 Gripon P , Rumin S , Urban S , Le Seyec J , Glaise D , et al (2002 ) Infection of a human hepatoma cell line by hepatitis B virus . Proc Natl Acad Sci U S A 99 : 15655 –15660 .12432097 6 Dandri M , Lutgehetmann M , Volz T , Petersen J (2006 ) Small animal model systems for studying hepatitis B virus replication and pathogenesis . Semin Liver Dis 26 : 181 –191 .16673296 7 Weiss L , Kekule AS , Jakubowski U , Burgelt E , Hofschneider PH (1996 ) The HBV-producing cell line HepG2–4A5: a new in vitro system for studying the regulation of HBV replication and for screening anti-hepatitis B virus drugs . Virology 216 : 214 –218 .8614990 8 Narayan R , Gangadharan B , Hantz O , Antrobus R , Garcia A , et al (2009 ) Proteomic analysis of HepaRG cells: a novel cell line that supports hepatitis B virus infection . J Proteome Res 8 : 118 –122 .19053806 9 Gripon P , Diot C , Guguen-Guillouzo C (1993 ) Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration . Virology 192 : 534 –540 .8421898 10 Galle PR , Hagelstein J , Kommerell B , Volkmann M , Schranz P , et al (1994 ) In vitro experimental infection of primary human hepatocytes with hepatitis B virus . Gastroenterology 106 : 664 –673 .8119538 11 Torbenson M , Thomas DL (2002 ) Occult hepatitis B. Lancet Infect Dis . 2 : 479 –486 . 12 Raimondo G , Allain JP , Brunetto MR , Buendia MA , Chen DS , et al (2008 ) Statements from the Taormina expert meeting on occult hepatitis B virus infection . J Hepatol 49 : 652 –657 .18715666 13 Chen YC , Sheen IS , Chu CM , Liaw YF (2002 ) Prognosis following spontaneous HBsAg seroclearance in chronic hepatitis B patients with or without concurrent infection . Gastroenterology 123 : 1084 –1089 .12360470 14 Chemin I , Trepo C (2005 ) Clinical impact of occult HBV infections . J Clin Virol 34 Suppl 1S15 –21 .16461218 15 Brechot C (2004 ) Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms . Gastroenterology 127 : S56 –61 .15508104 16 Donato F , Gelatti U , Limina RM , Fattovich G (2006 ) Southern Europe as an example of interaction between various environmental factors: a systematic review of the epidemiologic evidence . Oncogene 25 : 3756 –3770 .16799617 17 Cacciola I , Pollicino T , Squadrito G , Cerenzia G , Orlando ME , et al (1999 ) Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease . N Engl J Med 341 : 22 –26 .10387938 18 Sagnelli E , Coppola N , Scolastico C , Mogavero AR , Stanzione M , et al (2001 ) Isolated anti-HBc in chronic hepatitis C predicts a poor response to interferon treatment . J Med Virol 65 : 681 –687 .11745931 19 Zignego AL , Fontana R , Puliti S , Barbagli S , Monti M , et al (1997 ) Relevance of inapparent coinfection by hepatitis B virus in alpha interferon-treated patients with hepatitis C virus chronic hepatitis . J Med Virol 51 : 313 –318 .9093946 20 Tai DI , Shen YJ , Weng WH , Chen HW , Kang CC , et al (2011 ) New sarcomatoid cancer cell line SAR-HCV established from a hepatitis C virus-related liver tumour lesion . Anticancer Res 31 : 129 –137 .21273590 21 Lin CL , Chien RN , Hu CC , Lai MW , Yeh CT (2012 ) Identification of hepatitis B virus rtS117F substitution as a compensatory mutation for rtM204I during lamivudine therapy . J Antimicrob Chemother 67 : 39 –48 .22001270 22 Hsu CW , Yeh CT , Chang ML , Liaw YF (2007 ) Identification of a hepatitis B virus S gene mutant in lamivudine-treated patients experiencing HBsAg seroclearance . Gastroenterology 132 : 543 –550 .17258721 23 Gonzalez Garcia JR , Meza-Espinoza JP (2006 ) Use of the International System for Human Cytogenetic Nomenclature (ISCN) . Blood 108 : 3952 –3953 author reply 3953.17114573 24 Kann M , Lu X , Gerlich WH (1995 ) Recent studies on replication of hepatitis B virus . J Hepatol 22(1 suppl) : 9 –13 .7602085 25 Yeh CT , Chiu HT , Chu CM , Liaw YF (1998 ) G1 phase dependent nuclear localization of relaxed-cricular hepatitis B virus DNA and aphidicolin-induced accumulation of covalently closed circular DNA . J Med Virol 55 : 42 –50 .9580885 26 Yeh CT , Wong SW , Fung YK , Ou JH (1993 ) Cell cycle regulation of nuclear localization of hepatitis B virus core protein . Proc Natl Acad Sci USA 90 : 6459 –6463 .8341655	23734258	PMC3667124	CC BY	2021-01-05 17:28:31	yes	PLoS One. 2013 May 29; 8(5):e65456
==== Front Mol VisMol. VisMVMolecular Vision1090-0535Molecular Vision 1242012MOLVIS0025Research ArticleSuprachoroidal delivery in a rabbit ex vivo eye model: influence of drug properties, regional differences in delivery, and comparison with intravitreal and intracameral routes Kadam Rajendra S. 1Williams Jason 1Tyagi Puneet 1Edelhauser Henry F. 2Kompella Uday B. 11 Nanomedicine and Drug Delivery Laboratory, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO2 Emory Eye Center, Emory University, Atlanta, GACorrespondence to: Uday B. Kompella, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus 12850 E. Montview Blvd., Aurora, CO 80045 Phone: (303) 724 4028, Fax: (303) 724-4666, email: uday.kompella@ucdenver.edu2013 30 5 2013 19 1198 1210 11 1 2012 28 5 2013 Copyright © 2013 Molecular Vision.2013Molecular VisionThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Purpose First, to determine the influence of drug lipophilicity (using eight beta-blockers) and molecular weight (using 4 kDa and 40 kDa fluoroscein isothiocyanate [FITC]-dextrans) on suprachoroidal delivery to the posterior segment of the eye by using a rabbit ex vivo eye model. Second, to determine whether drug distribution differs between the dosed and undosed side of the eye following suprachoroidal delivery. Third, to compare the suprachoroidal delivery of sodium fluorescein (NaF) with the intracameral and intravitreal routes by using noninvasive fluorophotometry. Methods Using a small hypodermic 26G needle (3/8”) with a short bevel (250 µm), location of the suprachoroidal injection in an ex vivo New Zealand white rabbit eye model was confirmed with India ink. Ocular tissue distribution of NaF (25 µl of 1.5 µg/ml) at 37 °C was monitored noninvasively using the Fluorotron MasterTM at 0, 1, and 3 h following suprachoroidal, intravitreal, or intracameral injections in ex vivo rabbit eyes. For assessing the influence of lipophilicity and molecular size, 25 µl of a mixture of eight beta-blockers (250 µg/ml each) or FITC-dextran (4 kDa and 40 kDa, 30 mg/ml) was injected into the suprachoroidal space of excised rabbit eyes and incubated at 37 °C. Eyes were incubated for 1 and 3 h, and frozen at the end of incubation. Ocular tissues were isolated in frozen condition. Beta-blocker and FITC-dextran levels in excised ocular tissue were measured by liquid chromatography–tandem mass spectrometry and spectrofluorometry, respectively. Results Histological sections of India ink-injected albino rabbit eye showed the localization of dye as a black line in the suprachoroidal space. Suprachoroidal injection of NaF showed signal localization to the choroid and retina at 1 and 3 h post injection when compared with intravitreal and intracameral injections. Drug delivery to the vitreous after suprachoroidal injection decreased with an increase in solute lipophilicity and molecular weight. With an increase in drug lipophilicity, drug levels in the choroid–retinal pigment epithelium (RPE) and retina generally increased with some exceptions. Beta-blockers and FITC-dextrans were localized more to the dosed side when compared to the opposite side of the sclera, choroid–RPE, retina, and vitreous. These differences were greater for FITC-dextrans as compared to the beta-blockers. Conclusions The suprachoroidal route of injection allows localized delivery to the choroid–RPE and retina for small as well as large molecules. Suprachoroidal drug delivery to the vitreous declines with an increase in drug lipophilicity and molecular weight. Drug delivery differs between the dosed and opposite sides following suprachoroidal injection, at least up to 3 h. GalleyStatusExport to XML ==== Body Introduction Age- and life style-related ocular disease, including age-related macular degeneration and diabetic retinopathy, are the major cause of visual impairment and blindness in the industrialized developed world [1]. Recent changes in demographics indicate an increase in the aged population [2]. According to the latest release by the United Nations, there were 759 million people aged above 60 in 2010, and this number is expected to increase to 2 billion by 2050 [3]. Rapid progress in the biologic sciences is leading to the development of various small and large therapeutic molecules, including tyrosine kinase inhibitors, monoclonal antibodies, small interfering RNAs, and aptamers, to combat these back of the eye diseases. However, convenient and safe delivery of therapeutic agents to the target posterior ocular tissues remains a major pharmacotherapeutic challenge. Drug delivery to the target tissues, such as the choroid and retina, is hindered by unique anatomic and physiologic barriers, including the outer and inner blood–retinal barriers, and blood circulation that prevent the entry of foreign molecules [4]. Development of drug delivery systems that are safe, minimally invasive, and effective in local delivery of drug to the target tissues in a therapeutically effective concentration is an unmet need for back of the eye drugs. Conventional topical ophthalmic dosing is effective in delivery of drugs to the anterior segment ocular tissues such as the cornea, iris–ciliary body, and aqueous humor, but typically fails to deliver adequate quantities of therapeutic agents to the back of the eye. Topical eye drops result in less than 5% bioavailability in the anterior segment eye tissues and substantially lower delivery to the posterior segment eye tissues, including the retina [5,6]. Systemic delivery to the eye is restricted by the requirement of large doses of a drug, nonspecific distribution, increased systemic side effects, and limited delivery to the choroid–retina due to outer and inner blood–retinal barriers. Current clinical methods for local drug delivery to the posterior segments are periocular (off-label) and intravitreal (approved for some products) injections [7]. Although both of these methods are effective in delivery of drugs to the choroid and retina, they are associated with their own limitations. With periocular injection, when drug is placed adjacent to the sclera, the drug has to diffuse across the sclera and the choroid–RPE barriers before it reaches the retina. Although periocular routes are less invasive than intravitreal or suprachoroidal injections, significant amount of drug gets eliminated by the conjunctival and episcleral circulations [8]. Permeability of drug across the sclera is anticipated to be passive, while that across the choroid–RPE may be passive or carrier/receptor mediated. Although the sclera is permeable for high molecular compounds [9], it offers about 40%–50% resistance for permeability of beta-blockers across the sclera–choroid–RPE in rabbits [10]. The intravitreal route is an effective mode to localize drug in the vicinity of retinal tissue. The intravitreal route allows sustained drug delivery using implants, drug suspensions, and macromolecule solutions [5]. However, intravitreal injection is invasive and carries the risk of retinal detachment and endophthalmitis [11]. Our long-term goal is to explore suprachoroidal injection as an alternative route for localized drug delivery to the posterior ocular tissues. Following suprachoroidal delivery or placement of drug molecules in the potential space between the sclera and the choroid–RPE, drug is expected to be exposed at high concentrations to the choroid followed by the retina. For the first time in 2002, Einhmal et al. showed the feasibility of suprachoroidal delivery in sustained ocular drug delivery in a rabbit model [12]. Olsen et al. showed the safety and delivery ability of suprachoroidal injection for triamcinolone acetonide and bevacizumab by using microcannulation in pigs [13,14]. Recently Patel et al. showed the applicability of glass microneedles for suprachoroidal delivery of micro- and nanoparticles in rabbit, pig, and human eyes [15]. Although these previous studies have shown the application of suprachoroidal injection for intraocular drug delivery, there is no report available evaluating the effect of solute physicochemical properties, such as lipophilicity and molecular weight, on suprachoroidal drug delivery. Thus, the first objective of this study was to determine the effect of drug physicochemical properties, such as lipophilicity (beta-blockers) and molecular weight (fluoroscein isothiocyanate [FITC]-dextran), on suprachoroidal delivery in ex vivo rabbit eyes. Since other local routes of delivery, such as the transscleral route, are known to distribute drug more on the dosed side and less on the opposite side [16], the second objective of this study was to evaluate the differences in regional distribution of drug molecules after suprachoroidal injection. Since intravitreal injections are commonly used and because intracamerally injected materials drain via the uveoscleral pathway [17], which includes the choroid region, the third objective of this study was to compare suprachoroidal injection with intracameral and intravitreal injections for drug delivery to the retina and choroid region. Methods Materials Atenolol (99.0%), sotalol hydrochloride (~98%), nadolol (~98%), pindolol (98%), timolol maleate (98%), metoprolol tartrate (99%), betaxolol (~98%), labetalol hydrochloride (99%), propranolol hydrochloride (99%), triethyl amine (99.5%), sodium hydroxide, formic acid (88%), sodium fluorescein (NaF), ammonium formate (99.9%), and 4 kDa and 40 kDa FITC-dextrans were purchased from Sigma-Aldrich (St. Louis, MO). High performance liquid chromatography-grade acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). All chemicals and reagents used in this study were of analytic reagent grade. Suprachoroidal injection in ex vivo rabbit eyes New Zealand white rabbit eyes were obtained from Pel Freez Biologicals (Rogers, AR) within 24 h of harvesting and shipped to the laboratory in Hank’s balanced salt solution (Catalogue # H1641, Sigma Aldrich, St Louis, MO) on wet ice. Eyes were used immediately after arrival. For the suprachoroidal injection, excised rabbit eyes were placed on a ceramic tile and held with forceps to avoid movement of the eye during injection. Suprachoroidal injections were performed using a 50 μl Hamilton syringe (Hamilton Company, Reno, NV) and a small hypodermic needle (26G, 3/8” in length, 250-µm bevel) commonly used for intradermal injections (BD Biosciences, Franklin Lake, NJ; Product no. 305110). The needle was inserted at a 45° angle through the conjunctiva and sclera, 5 to 7 mm posterior from the limbus, in the posterior direction with the bevel facing downward. A small bevel oriented inward allows delivery of a solution to the suprachoroidal space with minimal leakage in the sclera. When the needle was in the suprachoroidal space, there was little resistance to the injection, whereas there was significant resistance to solution injection when the needle was in the sclera. Once the needle was in the suprachoroidal space, 25 µl of drug solution was injected. The accuracy of the injection technique was validated using an injection of India ink in the suprachoroidal space and dissection of the eyes immediately after injection. Prior pilot studies indicated that increasing the volume of injection to 50 µl resulted in an obvious leakage of the drug solution back through the needle track. Immediately after injection, each eye was placed in 1 ml of assay buffer (122 mM NaCl, 25 mM NaHCO3, 1.2 mM MgSO4, 0.4M K2HPO4, 1.4 mM CaCl2, 10 mM HEPES, and 10 mM glucose) in a 12-well tissue culture plate maintained at 37 °C in a shaking water bath. Eyes were incubated vertically for 1 or 3 h with the cornea facing upward. During incubation, the eye surface was kept moist by applying 30 microliters of assay buffer every 30 min. Although this study used bevel facing downward to reduce egress of drug solution during suprachoroidal injection, for in vivo studies, bevel facing upward towards sclera might be safer in preventing any choroid blood vessel rupture. Histological examination of the suprachoroidal injection To visualize the spread of the injected solution and the accuracy of injection techniques, histological examination of the suprachoroidal injection of India ink was performed in albino rabbit eyes. We used the hypodermic needle to inject 25 µl of 5% India ink in the suprachoroidal space. Eyes were transferred to Davidson’s fixative [18] immediately after suprachoroidal injection. After 48 h in Davidson’s fixative, eyes were embedded in paraffin and 5-µm sections were obtained. Sections were further stained with hematoxylin and eosin (H & E). Pigmented rabbit and albino rabbit eye sections without India ink injections were used as controls. Cryosections (5 µm thick) were obtained and stained with hematoxylin and eosin (H&E). The stained sections were examined with a light microscope (Zeiss Axioscope 2; Carl Zeiss, Inc. Jena, Germany), and images were captured with an AxioCamMrC5 camera (Carl Zeiss, Inc.) using 10X and 20X objectives. Fluorophotometric monitoring of suprachoroidal, intravitreal, and intracameral injections Ocular tissue distribution of NaF in ex vivo rabbit eyes after the suprachoroidal, intravitreal, and intracameral injection was monitored noninvasively with the Fluorotron MasterTM (OcuMetrics, Mountain View, CA). Ex vivo rabbit eyes were injected with 25 µl of NaF (1.5 µg/ml) either by suprachoroidal, intravitreal, or intracameral injections. Fluorotron scans were acquired as described previously [19]. Fluorotron Master reports sodium fluorescein or its equivalent concentrations (ng/ml) at 149 data points representing various distances along the visual axis in the posterior to anterior direction of the eye. Rabbit eyes were held on a specially designed adjustable stand in such a way that the cornea faced the fluorophotometer lens. Fluorotron scans were acquired immediately after injection and at 1 and 3 h post injection to study the distribution of NaF in ex vivo rabbit eyes. Blank albino rabbit eye Fluorotron scans were acquired as baseline spectra. Fluorotron scans were acquired for the whole eye and assigned to four different regions: choroid–retina, vitreous, aqueous humor, and cornea as described previously [20]. In this study, the location of fluorescence peak immediately after intracameral injection was assigned to aqueous humor (between data points 105 and 115). The peak corresponding to intravitreal injection between data points 55 and 80 was assigned to vitreous humor. The fluorescence peak between data points 20 and 30 was assigned to choroid-retina. Suprachoroidal delivery of fluoroscein isothiocyanate-dextrans and beta-blockers To evaluate the effect of solute molecular weight on suprachoroidal delivery, ex vivo rabbit eyes (n=4) were injected with 25 µl of a 30-mg/ml solution of FITC-dextrans (4 or 40 kDa) in PBS (8.5 gm/l NaCl, 0.95 gm/l Na2HPO4, and 0.455 gm/l NaH2PO4) in the suprachoroidal space of rabbit eyes (0.75 mg of FITC-dextran per eye). To evaluate the effect of solute lipophilicity on suprachoroidal delivery, ex vivo rabbit eyes (n=4) were injected with 25 µl of a beta-blocker cocktail (250-µg/ml solution of each beta-blocker, i.e., 6.25 µg of each beta-blocker per eye) in PBS in the suprachoroidal space. Immediately after injection, each eye was placed in 1 ml of assay buffer in a 12-well tissue culture plate maintained at 37 °C in a shaking water bath. Eyes were incubated vertically for 1 or 3 h with the cornea facing upward. At the end of incubation, the eyes were snap frozen in a dry ice–isopentane bath and stored at −80 °C until further processing. Eye dissection and collection of various ocular tissues Ex vivo rabbit eyes injected with FITC-dextran and beta-blockers were dissected in frozen condition to isolate various ocular tissues. All dissection procedures were performed on a cooled ceramic tile placed on a dry ice–isopentane bath (<0 °C) to avoid thawing of the eye during dissection. After separation of the anterior part of the eye, the remaining posterior globe was cut longitudinally along the visual axis into two parts: the injected side and the opposite side. The retina, choroid, vitreous, and sclera were collected from the injected as well as opposite side. A new surgical blade was used for each eye. To prevent the transfer of drug between the tissues of each eye, surgical accessories were thoroughly rinsed with saline followed by methanol followed by saline and blotted dry. All samples were weighed and stored at −80 °C until further processing. Spectrofluorometric analysis of fluoroscein isothiocyanate-dextran Concentrations of FITC-dextran (4 and 40 kDa) in rabbit ocular tissues were measured using a spectrofluorometer after aqueous extraction of drugs from the tissues. Briefly, weighed amounts of ocular tissues were mixed with 1 ml of PBS (pH 7.4) in a 2-ml microcentrifuge tube, vortexed for 10 min, and then homogenized using a hand homogenizer in an ice bath. The aqueous layer was separated from the tissue matrix by centrifugation at 10,000 × g for 10 min, and the separated aqueous layer (100 µl) was transferred into clear 96-well plates. Fluorescence was measured using a spectrofluorometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA) at excitation and emission wavelengths of 485 and 525 nm, respectively. Blank rabbit ocular tissues were processed similarly to actual tissue samples in order to measure tissue background. Liquid chromatography–tandem mass spectrometry analysis of beta-blockers Concentrations of beta-blockers in rabbit ocular tissue samples were measured by means of liquid chromatography–tandem mass spectrometry as described previously [21]. An API-3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) coupled with a PerkinElmer series-200 liquid chromatography (PerkinElmer, Inc., Waltham, MA) system was used for analysis. The concentration of beta-blockers in rabbit ocular tissue samples was measured after liquid–liquid extraction using a dichloromethane and ethyl acetate (1:1 volume [v]/v) mixture. The calibration curve for analysis was prepared using blank rabbit ocular tissues samples as the matrix. Blank rabbit ocular tissue samples without beta-blocker injections were processed and analyzed to measure the tissue background. The mobile phase was a mixture of 5 mM ammonium formate adjusted to pH 3.5 (A) and acetonitrile–methanol (75:25) containing 0.02% triethylamine, adjusted to pH 3.5 (B). The linear gradient elution at a flow rate of 0.4 ml/min with a total run time of 13 min was as follows: 90% A (0–1.0 min), 10% A (6–9 min), and 90% A (10–13 min). The following transitions were monitored: 276/145 (atenolol); 273/255 (sotalol); 310/254 (nadolol); 249/116 (pindolol); 317/261 (timolol); 268/133 (metoprolol); 308/116 (betaxolol); 260/116 (propranolol); and 327/162 (labetalol). Statistical analysis The data were presented as mean ± standard deviation (SD). For statistical comparisons between injected versus opposite side tissues, the dependent samples Student t test was used. Comparison between 4 kDa and 40 kDa FITC-dextran was performed using the independent sample Student t test. Comparison of the mean between multiple beta-blockers was performed using one-way analysis of variance followed by Tukey’s post hoc analysis (SPSS, ver. 11.5; IBM, Armonk, NY). The results were considered statistically significant at p<0.05. Results Histological examination of suprachoroidal injection of India ink Histological sectioning of India ink-injected albino rabbit eye after the suprachoroidal injections was performed to confirm the accuracy of the suprachoroidal injection technique. India ink was used as a dye of choice because of its black color, which is easy to visualize in the albino rabbit eye that is devoid of melanin pigment. A histological cross-section of the India ink-injected albino rabbit eye showed a black line of India ink (Figure 1C,D) between the sclera and choroid–RPE, and a collaged light microscopic image showed the insertion of the needle through the sclera into the suprachoroidal space without any damage or penetration into the choroid–retina, thus confirming the accuracy of the injection. The injection site in the sclera was filled with India ink (Figure 1), and no black stain was observed in the sclera (except the injection site) or choroid–RPE, confirming that the injection specifically occurred in the suprachoroidal space. An albino rabbit eye without any injection of India ink was used as a negative control, and no black color was observed in any part of this eye. A histological cross-section of the albino rabbit eye showed both sclera–choroid and retina with a pink color and the choroid was attached to the sclera without any gap between these tissues. (Figure 1A). Figure 1 Suprachoroidal injection of India ink showed a thin black line between sclera and choroid-RPE in albino rabbit eye. Eyes were injected with 25 µl of 5% India ink dispersion in the suprachoroidal space. Eyes were fixed in Davidson’s fixative for 48 h immediately after injection and embedded in paraffin blocks, and 5-µm sections were obtained. Hematoxylin and eosin (H&E) stained sections were examined. A: Representative histological images of albino rabbit eye are shown. B: Representative histological images of pigmented rabbit eye are shown. C: Representative histological images of albino rabbit eye after suprachoroidal injection of India ink are shown. D: Collaged light microscopy image of H&E stained section of albino rabbit eye after suprachoroidal injection of India ink is shown. Readers may misinterpret the black color from India ink as melanin pigment in the choroid–RPE. Therefore, for comparative purposes pigmented rabbit eyes were included as a positive control to show the appearance of pigment in the choroid–RPE. Histological cross-section of a pigmented rabbit eye showed the distribution of melanin pigment throughout the choroid–RPE (Figure 1, middle panel), which is distinct in its pattern from the India ink-injection. Noninvasive fluorophotometry monitoring of suprachoroidal, intravitreal, and intracameral injections Ocular tissue distribution of NaF after suprachoroidal injection was compared with intravitreal and intracameral injections in ex vivo rabbit eyes using ocular fluorophotometry. A baseline spectrum for the albino rabbit eye without NaF injection was included as a control. As shown in Figure 2, tissue background showed negligible readings, with fluorescence intensity below 8 ng/ml. Immediately after intracameral and intravitreal injections of NaF, sharp peaks were observed in the anterior segment and vitreous, respectively. After suprachoroidal injection, a broad dispersed peak of NaF was observed posterior to the vitreous in the sclera–choroid–retina region of the eye. Due to low spatial resolution (980 µm) of the Fluorotron Master fluorophotometer, it was difficult to distinguish the thin ocular tissues, i.e., choroid from retina and sclera. A time course of Fluorotron scans of NaF in ex vivo rabbit eyes showed that after intracameral and intravitreal injections, the peak intensity rapidly diminished over 3 h due to dilution or distribution of the drug to adjacent tissues. With intravitreal injection, the NaF peak broadened during the 3-h study due to the spread of the signal within the vitreous followed by distribution of NaF to the anterior and posterior segment ocular tissues. With the suprachoroidal injection, the peak intensity remained unchanged for 3 h, possibly due to adsorption or binding of NaF to the sclera–choroid–retina and lack of vascular clearance mechanisms in the ex vivo model. Figure 2 Fluorophotometry scans show sodium fluorescein levels at various depths of the eye along the visual axis. Representative fluorophotometry scans attained using Fluorotron Master™ in albino rabbit eyes after intracameral, intravitreal, and suprachoroidal injections of sodium fluorescein are shown. Scans were collected using Fluorotron Master at 0, 1, and 3 h post injection. Peaks for different regions were assigned from left to right at 20-30, 55-80, and 105-115 data points corresponding to choroid–retina, vitreous, and aqueous humor regions, respectively. Ocular tissue distribution of fluoroscein isothiocyanate-dextrans after suprachoroidal injection Ocular tissue distribution of FITC-dextrans after suprachoroidal injection in ex vivo rabbit eyes was assessed to evaluate the effect of solute molecular weight on tissue distribution. Blank rabbit ocular tissues showed no interference at the excitation and emission wavelengths employed. As shown in Figure 3, retinal and vitreal (total tissue) delivery was significantly higher for 4 kDa FITC-dextran than 40 kDa FITC-dextran at both 1 and 3 h. Sclera and choroid–RPE levels were higher for 40 kDa FITC-dextran than 4 kDa FITC-dextran because of the low diffusivity of 40 kDa FITC-dextran (Figure 3). Figure 3 Total retinal and total vitreal delivery after suprachoroidal injection decreases with an increase in solute molecular weight. Ocular tissue distribution of fluorescein isothiocyanate (FITC)-dextran in ex vivo albino rabbit eyes at (A) 1 h and (B) 3 h after suprachoroidal is shown. Data is represented as mean ± standard deviation for n=4. * means the value is significantly different from FITC-dextran 40 kDa at p≤0.05. Both 4 and 40 kDa FITC-dextran showed regional differences in ocular tissue distribution. Drug levels in the injected side sclera, choroid–RPE, retina, and vitreous were several fold higher than in the opposite side (Figure 4 and Table 1). These differences in the regional distribution of FITC-dextran decreased significantly at 3 h as compared to 1 h, possibly due to convection/diffusion of the drug to the opposite side as well as adjacent tissues over a given incubation time. Drug levels were higher in the sclera and choroid–RPE and lower in the retina and vitreous. Further, drug levels were higher in tissues adjacent to the site of injection than in those in the anterior segment. Figure 4 Suprachoroidal injection of fluoroscein isothiocyanate-dextrans (FITC-dextrans) showed regional differences in distribution, with drug levels being several fold higher on the injected side than the opposite side. Differences in regional distribution decreased with an increase in incubation time. Ocular tissue distribution of (A) FITC-dextran 4 kDa at 1 h, (B) FITC-dextran 4 kDa at 3 h, (C) FITC-dextran 40 kDa at 1 h, and (D) FITC-dextran 40 kDa at 3 h in ex vivo albino rabbit eyes after suprachoroidal injection is shown. Data is represented as mean ± standard deviation for n=4. means the value is significantly different from opposite side tissues at p≤0.05. Table 1 Comparison of regional distribution of FITC-dextran in sclera, choroid-RPE, retina, and vitreous of ex-vivo albino rabbit eyes after suprachoroidal injection. Data are expressed as mean ± SD (n=4) Tissue Time (h) FITC-Dextran 4 kDa (µg/g of tissue) FITC-Dextran 40 kDa (µg/g of tissue) Injected side Opposite side Injected side: Opposite side Ratio Injected side Opposite side Injected side: Opposite side Ratio Sclera 1 h 847.36±181.72 17.74±3.14 49.73±17.48 1111.34±593.69 23.60±4.32 44.94±19.12 3 h 903.80±277.97 127.04±25.65 7.45±3.02 1558.15±656.68 216.03±81.61 8.01±3.84 Choroid-RPE 1 h 1329.31±369.76 20.39±14.86 187.74±256.07 2869.71±495.43 74.77±84.96 85.32±62.03 3 h 796.84±368.76 137.08±49.71 5.77±1.05 1293.17±742.66 250.83±175.55 5.95±2.84 Retina 1 h 687.40±137.67 37.14±22.74 28.71±25.90 494.71±76.21 63.48±66.10 15.85±6.28 3 h 344.51±275.03 56.06±15.50 7.39±7.72 57.77±24.11 30.22±10.26 2.18±1.30 Vitreous 1 h 6.10±2.31 2.22±3.46 17.09±14.03 4.30±3.56 0.92±0.46 4.02±2.55 3 h 49.17±18.49 20.63±4.52 2.50±1.17 26.93±17.85 10.24±1.62 2.80±2.13 As the suprachoroidal space is closer to the exterior surface of the eye, diffusion of FITC-dextran from the suprachoroidal space into the incubation medium was measured. As shown in Figure 5, both 4 and 40 kDa FITC-dextran showed continuous entry into the incubation medium. The 3 h cumulative percentage release of FITC-dextran into the medium was 4.0% of the administered dose for 4 kDa FITC-dextran and 2.9% for 40 kDa FITC-dextran. Figure 5 Fluoroscein isothiocyanate-dextran entered the incubation medium after suprachoroidal injection. Figure shows the cumulative percentage entry of fluoroscein isothiocyanate (FITC)-dextran from the suprachoroidal space of ex vivo rabbit eyes into the incubation medium. Data represent mean±standard deviation for n=4. Ocular tissue distribution of beta-blockers after suprachoroidal injection The effect of solute lipophilicity on ocular tissue distribution after suprachoroidal injection was evaluated in ex vivo rabbit eyes using a cocktail of beta-blockers. As shown in Figure 6, choroidal and retinal delivery of beta-blockers increased with an increase in solute lipophilicity, with tissue levels being the highest for lipophilic beta-blocker propranolol and the least for hydrophilic beta-blocker atenolol. For vitreous, solute lipophilicity showed an inverse relation with the vitreal concentration, with an increase in solute lipophilicity resulting in a decrease in vitreal concentration (Figure 6E). Correlation of beta-blocker lipophilicity (Log D, pH 7.4) showed a linear positive correlation with choroidal and retinal concentrations, with the correlation coefficient (R2) being 0.753 or greater for both the choroid–RPE and retina (Figure 6B,D). In the vitreous, solute lipophilicity showed an inverse correlation with vitreal concentrations with R2 ≥ 0.923 (Figure 6F). These differences were more evident at 3 h compared to 1 h. Figure 6 Total vitreal delivery decreases, whereas choroid–RPE and retinal delivery increases with increase in solute lipophilicity after suprachoroidal injection. Effect of beta-blocker lipophilicity on (A) choroid–RPE, (C) retina, and (E) vitreous delivery in ex vivo albino rabbit eyes after suprachoroidal injection is shown. Beta-blocker lipophilicity showed a direct linear correlation with (B) choroid–RPE and (D) retinal levels and an inverse correlation with (F) vitreal levels. Data represent mean±standard deviation for n=4. means the value is significantly different from atenolol, sotalol, nadolol, and pindolol at p≤0.05 All beta-blockers showed regional differences in distribution after suprachoroidal injection in posterior ocular tissues. Drug levels on the injected side of posterior ocular tissues were higher than the opposite side tissues (Figure 7). For beta-blockers, tissue concentrations were 1.5- to 7.9-fold higher on the injected side choroid–RPE, retina, and vitreous than the opposite side (Figure 7). These differences in the regional distribution of beta-blockers decreased at 3 h compared to 1 h. Beta-blocker levels were lower in the anterior segment tissues than in the posterior segment tissues. Figure 7 Suprachoroidal injection of beta-blockers showed regional differences in distribution, with drug levels being higher on the injected side tissues than the opposite side. Differences in regional distribution decrease with an increase in incubation time. Data represent mean±standard deviation for n=4. * means the value is significantly different from injected side tissues at p≤0.05. Discussion In the current study we successfully showed the feasibility of suprachoroidal injection in ex vivo rabbit eyes by using a simple short-bevel hypodermic needle and syringe. We observed that suprachoroidal injection results in enhanced exposure of NaF to the choroid and retina compared to intracameral and intravitreal injections. Drug physiochemical properties, including molecular weight and lipophilicity, influenced suprachoroidal drug delivery. Delivery to the vitreous after suprachoroidal injection decreased with an increase in molecular weight and lipophilicity. Suprachoroidal injection of beta-blockers and FITC-dextran showed regional differences in distribution in a given tissue, with drug levels being higher on the dosed side than the opposite side for the choroid, retina, and the vitreous. Differences in regional distribution decreased with an increase in exposure time. The first objective of this study was to show the feasibility of suprachoroidal injection with a short bevel hypodermic needle and syringe. Previous studies showed the use of the potential space between the sclera and choroid for sustained drug delivery to the posterior ocular tissues [12,13]. However, these earlier studies employed a surgical procedure to insert a cannula or a specially designed microneedle for suprachoroidal injections [12-15]. Insertion of a cannula in the suprachoroidal space is too complicated a procedure to perform routinely in the laboratory setting and requires surgical work. Although suprachoroidal injection using microneedles is more user friendly, this approach needs special tailoring of needles for different species, and no commercial source is available for microneedles intended for the rabbit. In this study we showed the feasibility of suprachoroidal injection using a simple needle and syringe assembly. We used a small tapering head 26G hypodermic needle (3/8”) with an ultra short bevel (250 µm); this allows injection of material specifically in the suprachoroidal space with little leakage. Accuracy of the injection technique was confirmed by histological sectioning of the albino rabbit eyes immediately after suprachoroidal injection of India ink. These histological sections showed a black line of India ink in the space between the sclera and choroid–RPE, without any black stain in the sclera and choroid–RPE (Figure 1). The mean particle size of India ink particles was 150.73±0.76 nm, and these were retained at the site of injection without further movement across the tissue barriers. India ink particles cannot readily move across intact tissue barriers, and hence they are used as a marker to test the corneal barrier integrity after cataract surgery [22]. Injection of drug material in the suprachoroidal space results in expansion of the suprachoroidal space between the sclera and choroid–RPE (Figure 1). Seiler et al. [23] and Kim et al. [24] also reported the expansion of the suprachoroidal space to accommodate the injection volume. A similar expansion of the suprachoroidal space was seen after suprachoroidal injection of particles ex vivo in pig eyes [15]. The second objective of our study was to compare ocular tissue distribution of NaF in ex vivo albino rabbit eyes after suprachoroidal, intravitreal, and intracameral injections. As shown in Figure 2, suprachoroidal injection of NaF sustained signal intensity for 3 h. However, intravitreal and intracameral injections resulted in a steep decline in signal intensity within 3 h. We injected 37.5 ng of NaF (25 µl of 1.5 µg/ml) in the suprachoroidal space. Such a small amount of NaF might largely remain bound to the tissue at the site of injection, accounting for the sustained signal intensity over the duration of the study. Previous reports indicated that NaF binds to sclera with a maximum binding capacity of 80±5 mM [25]. In the case of intravitreal and intracameral injections, NaF was injected in the ocular fluid matrix, which is composed of 99% water. This allows rapid diffusion of NaF to adjacent tissues, resulting in a rapid decrease in signal intensity within 3 h. In ex vivo rabbit eyes, suprachoroidal injection of NaF showed sustained signal intensity in the choroid–retina region for 3 h. However, NaF signal intensity is expected to be influenced by choroidal and retinal blood clearance in the live animal. Kim et al. showed rapid clearance of gadolinium diethylene triamine pentacetate (Gd-DTPA) from the suprachoroidal space within an hour, with an elimination half-life of 11.8 min [24]. The suprachoroidal injection of NaF showed a broad peak in the posterior ocular tissues; this can be explained by the focal diamond theory of fluorophotometry [26]. As noted by McCarey, the suprachoroidal injection of NaF showed a broad peak in posterior ocular tissues possibly due to the halation of the choroid–retina response [26]. Halation or secondary fluorescence occurs when light passing through the choroid–retina is reflected back by the choroid and scattered beyond its boundaries. This causes the fluorescence to bleed through, resulting in tailing of the choroid–retina response [27]. The third objective of our study was to study the effect of drug physicochemical properties, such as solute molecular weight and lipophilicity (log D), on ocular tissue distribution after suprachoroidal injection. To evaluate the effect of molecular weight, we used 4 and 40 kDa FITC-dextran. Different molecular weights of FITC-dextran were previously used to evaluate the effect of solute molecular weight on permeability across the sclera as well as the choroid–RPE [9,28]. Solute molecular weight showed a significant influence on retinal and vitreal delivery of FITC-dextran after suprachoroidal injection. The 40 kDa FITC-dextran showed significantly lower retinal and vitreal levels than the 4 kDa FITC-dextran (Figure 3). Pitkanen et al. reported that the permeability of 40 kDa FITC-dextran was fivefold lower than 4 kDa FITC-dextran across bovine choroid–RPE. Possibly due to lower diffusivity and permeability, 40 kDa FITC-dextran was retained to a greater extent at the site of injection, resulting in significantly higher levels in the sclera and choroid–RPE compared to 4 kDa FITC-dextran. Because of its lower diffusivity, 40 kDa FITC-dextran showed 1.38-fold lower egress into the incubation medium than 4 kDa FITC-dextran (Figure 5). We have not observed any significant effect of molecular weight on tissue distribution in the anterior segment tissues, such as cornea, aqueous humor, and conjunctiva. This might be due to the low solute levels observed in anterior segment tissues (15- to1500-fold lower than choroid–RPE levels). At such low levels, assay variability is high, making it difficult to discern any molecular weight-related differences. Solute levels in the anterior segment ocular tissues were most likely due to diffusion in conjunction with convection along the suprachoroidal space or needle track. Despite the difference in molecular weight and the extent of delivery, FITC-dextran (both 4 and 40 kDa) showed similar trends in tissue distribution after suprachoroidal injection. Drug distribution in posterior ocular tissues was in the following order: choroid–RPE~sclera>retina>vitreous. For anterior segment tissues, the general trend was conjunctiva>iris–ciliary body~cornea>aqueous humor. The effect of solute lipophilicity on suprachoroidal delivery was evaluated using a cassette of beta-blockers with a broad range of lipophilicity [29]. Beta-blockers are the most commonly used series of molecules to evaluate the effect of solute lipophilicity because of their narrow range of molecular weight and pKa and wide range of lipophilicity [29]. As shown in Figure 6, solute lipophilicity influenced drug distribution to the choroid–RPE, retina, and vitreous after suprachoroidal delivery. Total drug delivery to the choroid–RPE and retina increased with an increase in solute lipophilicity, whereas vitreal delivery decreased with an increase in lipophilicity (Figure 6). Our previous results with in vitro tissue partitioning of beta-blockers in the bovine choroid–RPE and retina also showed that tissue partitioning of beta-blockers increases with an increase in lipophilicity [29]. As shown in Figure 6E,F, solute lipophilicity showed an inverse correlation with vitreal delivery of beta-blockers after suprachoroidal injection. Reduced vitreal delivery for lipophilic beta-blockers is possibly due to retention of lipophilic molecules in the choroid–RPE and retina and low partitioning into the vitreous. We previously observed a decrease in sclera–choroid–RPE transport with an increase in solute lipophilicity due to accumulation of lipophilic drugs in the sclera–choroid–RPE [10]. The last objective of our study was to evaluate the differences in the regional distribution of drug molecules after suprachoroidal injection. We observed that drug levels on the injected side of the choroid, retina, and vitreous were significantly higher than the opposite side for both high and low molecular weight compounds (Figure 4 and Figure 7). Higher drug levels on the injected side tissue were due to a rapid uptake of the injected drug by tissues near to the site of injection and then slow diffusion to the adjacent tissues. The upward orientation of the eye during incubation was also a possible contributor to regional differences in solute distribution. The orientation of eyes in this study favors convection of the drug solution toward the posterior pole of the eye. Subconjunctival injection of FITC-dextran (70 kDa) in mice showed regional differences in distribution, with the injected-side sclera and choroid levels being significantly higher than the opposite side at initial time points [16]. Differences in regional tissue distribution decrease over time due to the diffusion of molecules to tissues on the other side. Previous reports of the suprachoroidal injection of a contrast agent in ex vivo porcine eyes also indicated regional differences in distribution, with mean pixel intensity of the contrast agent being significantly higher on the injected side than the opposite side [23]. In our study, these differences were higher for FITC-dextran than beta-blockers. Lower diffusivity of macromolecules might contribute to greater differences between dosed and opposite sides (Figure 4 and Figure 7). These differences decrease with an increase in exposure duration (Table 1). One of the limitations of the current study is that we used ex vivo rabbit eyes, which were devoid of blood circulation and other clearance mechanisms. The main objective of this study, however, was to show the effect of drug physicochemical properties on ocular tissue distribution after suprachoroidal injection in the absence of vascular clearance mechanisms. In our previous study we observed a good correlation between in vitro tissue partitioning and in vivo delivery for beta-blockers. Further, in vitro transport across isolated tissues, which was indirectly measured in the present study, is commonly used to predict in vivo delivery. Another limitation of the current study is that the eyes were used 18–24 h post harvesting. The time gap between enucleation of the eyes and the actual experiment may have altered ocular barrier properties. Majumdar et al. showed that the preservation of eyes for 24 h in Hank’s balanced salt solution results in no significant alteration in the transport of diazepam and L-arginine across cornea [30]. However, the permeability of passive permeability marker mannitol was increased threefold in corneas preserved for 24 h compared to freshly isolated corneas, indicating an increase in permeability of the corneal epithelial barrier after death [30]. The half-life of zona occludens-1 (ZO-1) in Madin Darby Canine Kidney (MDCK) cells is about 5 h based on metabolic radiolabeling studies [31]. In our study we cannot rule out the possibility of an increase in permeability of ocular barriers at 18–24 h after harvesting. However, the trends observed for delivery may hold true in vivo with some differences in the extent of delivery. In summary, this study showed the feasibility of the suprachoroidal space as a targeted delivery location for drug delivery to the choroid and retina. Suprachoroidal injection of NaF resulted in better signal localization to the choroid–retina when compared to intravitreal and intracameral injections. Suprachoroidal delivery to the vitreous decreases with an increase in molecular weight and lipophilicity of drug molecules. Suprachoroidal injection results in region-selective distribution of drug molecules, with drug levels being higher on the injected side than the opposite side. However, these differences are likely to be less prominent than those reported for periocular injections. Acknowledgments This work was supported by NIH grants R24EY017045 (through Emory University), R01EY018940, and R01EY017533, and an unrestricted grant from RPB. ==== Refs References 1 Del Amo EM Urtti A Current and future ophthalmic drug delivery systems. A shift to the posterior segment. Drug Discov Today 2008 13 135 43 18275911 2 Wiener JM Tilly J Population ageing in the United States of America: implications for public programmes. Int J Epidemiol 2002 31 776 81 12177018 3 United Nations. Current status of the social situation, wellbeing, participation in development and rights of older persons worldwide;August 2010 4 Sunkara G, Kompella UB. Membrane transport processes in the eye. In: Ophthalmic drug Delivery Systems. New York: Marcel Dekker Inc.; 2003. 5 Kompella UB Kadam RS Lee VH Recent advances in ophthalmic drug delivery. Ther Deliv 2010 1 435 56 21399724 6 Maurice DM Drug delivery to the posterior segment from drops. Surv Ophthalmol 2002 47 Suppl 1 S41 52 12204700 7 Raghava S Hammond M Kompella UB Periocular routes for retinal drug delivery. Expert Opin Drug Deliv 2004 1 99 114 16296723 8 Robinson MR Lee SS Kim H Kim S Lutz RJ Galban C Bungay PM Yuan P Wang NS Kim J Csaky KG A rabbit model for assessing the ocular barriers to the transscleral delivery of triamcinolone acetonide. Exp Eye Res 2006 82 479 87 16168412 9 Ambati J Canakis CS Miller JW Gragoudas ES Edwards A Weissgold DJ Kim I Delori FC Adamis AP Diffusion of high molecular weight compounds through sclera. Invest Ophthalmol Vis Sci 2000 41 1181 5 10752958 10 Kadam RS Cheruvu NP Edelhauser HF Kompella UB Sclera-choroid-RPE transport of eight beta-blockers in human, bovine, porcine, rabbit, and rat models. Invest Ophthalmol Vis Sci 2011 52 5387 99 21282583 11 Shah CP Garg SJ Vander JF Brown GC Kaiser RS Haller JA Outcomes and risk factors associated with endophthalmitis after intravitreal injection of anti-vascular endothelial growth factor agents. Ophthalmology 2011 118 2028 34 21705087 12 Einmahl S Savoldelli M D'Hermies F Tabatabay C Gurny R Behar-Cohen F Evaluation of a novel biomaterial in the suprachoroidal space of the rabbit eye. Invest Ophthalmol Vis Sci 2002 43 1533 9 11980871 13 Olsen TW Feng X Wabner K Conston SR Sierra DH Folden DV Smith ME Cameron JD Cannulation of the suprachoroidal space: a novel drug delivery methodology to the posterior segment. Am J Ophthalmol 2006 142 777 87 16989764 14 Olsen TW Feng X Wabner K Csaky K Pambuccian S Cameron JD Pharmacokinetics of pars plana intravitreal injections versus microcannula suprachoroidal injections of bevacizumab in a porcine model. Invest Ophthalmol Vis Sci 2011 52 4749 56 21447680 15 Patel SR Lin AS Edelhauser HF Prausnitz MR Suprachoroidal drug delivery to the back of the eye using hollow microneedles. Pharm Res 2011 28 166 76 20857178 16 Kim TW Lindsey JD Aihara M Anthony TL Weinreb RN Intraocular distribution of 70-kDa dextran after subconjunctival injection in mice. Invest Ophthalmol Vis Sci 2002 43 1809 16 12036983 17 Bernd AS Aihara M Lindsey JD Weinreb RN Influence of molecular weight on intracameral dextran movement to the posterior segment of the mouse eye. Invest Ophthalmol Vis Sci 2004 45 480 4 14744888 18 Latendresse JR Warbrittion AR Jonassen H Creasy DM Fixation of Testes and Eyes Using a Modified Davidson's Fluid: Comparison with Bouin's Fluid and Conventional Davidson's Fluid. Toxicol Pathol 2002 30 524 33 12187944 19 Berezovsky DE Patel SR McCarey BE Edelhauser HF In vivo ocular fluorophotometry: delivery of fluoresceinated dextrans via transscleral diffusion in rabbits. Invest Ophthalmol Vis Sci 2011 52 7038 45 21791594 20 Ghate D Brooks W McCarey BE Edelhauser HF Pharmacokinetics of intraocular drug delivery by periocular injections using ocular fluorophotometry. Invest Ophthalmol Vis Sci 2007 48 2230 7 17460284 21 Kadam RS Kompella UB Cassette analysis of eight beta-blockers in bovine eye sclera, choroid-RPE, retina, and vitreous by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009 877 253 60 19117816 22 Taban M Sarayba MA Ignacio TS Behrens A McDonnell PJ Ingress of India ink into the anterior chamber through sutureless clear corneal cataract wounds. Arch Ophthalmol 2005 123 643 8 15883283 23 Seiler GS Salmon JH Mantuo R Feingold S Dayton PA Gilger BC Effect and distribution of contrast medium after injection into the anterior suprachoroidal space in ex vivo eyes. Invest Ophthalmol Vis Sci 2011 52 5730 6 21685338 24 Kim SH Galban CJ Lutz RJ Dedrick RL Csaky KG Lizak MJ Wang NS Tansey G Robinson MR Assessment of subconjunctival and intrascleral drug delivery to the posterior segment using dynamic contrast-enhanced magnetic resonance imaging. Invest Ophthalmol Vis Sci 2007 48 808 14 17251481 25 Lin CW Wang Y Challa P Epstein DL Yuan F Transscleral diffusion of ethacrynic acid and sodium fluorescein. Mol Vis 2007 13 243 51 17356511 26 McCarey BE. Fluorophotometry for Pharmacokinetic Assessment In: Drug Product Development for the Back of the EyeNew York: Springer; 2011. 27 Gray JR Mosier MA Ishimoto BM Optimized protocol for Fluorotron Master. Graefes Arch Clin Exp Ophthalmol 1985 222 225 9 3979849 28 Pitkänen L Ranta VP Moilanen H Urtti A Permeability of retinal pigment epithelium: effects of permeant molecular weight and lipophilicity. Invest Ophthalmol Vis Sci 2005 46 641 6 15671294 29 Kadam RS Kompella UB Influence of lipophilicity on drug partitioning into sclera, choroid-retinal pigment epithelium, retina, trabecular meshwork, and optic nerve. J Pharmacol Exp Ther 2010 332 1107 20 19926800 30 Majumdar S Hingorani T Srirangam R Evaluation of active and passive transport processes in corneas extracted from preserved rabbit eyes. J Pharm Sci 2010 99 1921 30 19890936 31 Chen Y Lu Q Schneeberger EE Goodenough DA Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell 2000 11 849 62 10712504	23734089	PMC3669536	CC BY	2021-01-04 22:47:45	yes	Mol Vis. 2013 May 30; 19:1198-1210
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23750283PONE-D-12-3711110.1371/journal.pone.0066275Research ArticleBiologyBiochemistryProteinsModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCellular TypesEndothelial CellsMedicineOncologyCancer TreatmentAntiangiogenesis TherapyCancers and NeoplasmsBreast TumorsBasic Cancer ResearchRole of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer Rac1/Cdc42 in Breast CancerMa Ji 1 2 Xue Yan 1 * Liu Wenchao 1 Yue Caixia 3 Bi Feng 3 Xu Junqing 4 Zhang Jian 5 Li Yan 5 Zhong Cuiping 6 Chen Yan 1 1 Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shannxi, China 2 Department of Breast Surgery, Lanzhou General Hospital of People's Liberation Army, Lanzhou, Gansu, China 3 Laboratory of Signal Transduction and Molecular Targeted Therapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China 4 Department of Radiology, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shannxi, China 5 Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi’an, Shannxi, China 6 Department of Ear Nose Throat Surgery, Lanzhou General Hospital of People's Liberation Army, Lanzhou, Gansu, China Ushio-Fukai Masuko Editor University of Illinois at Chicago, United States of America * E-mail: xueyan2011@yahoo.com.cnCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: JM YX. Performed the experiments: WL JZ. Analyzed the data: CY JX YL. Contributed reagents/materials/analysis tools: FB. Wrote the paper: CZ YC. 2013 4 6 2013 8 6 e6627527 11 2012 3 5 2013 © 2013 Ma et al2013Ma et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin. This work was supported by grants from the National Natural Science Foundation of China (nos. 81202085, 81201209 and 30770823). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Tumor angiogenesis represents one of the most important pathological mechanisms in tumor growth, metastasis and recurrence [1], [2]. The treatment of tumor angiogenesis is impaired by its complicated and poorly understood pathogenesis. Until now, the main targeted inhibitor of tumor angiogenesis is bevacizumab, an inhibitor of vascular endothelial growth factor (VEGF). While the early success of anti-VEGF therapy in cancer patients is certainly encouraging, the effects of long-term VEGF inhibition will require close monitoring [3]. Tumor angiogenesis is initiated through the disruption of the balance of angiogenesis promoters and inhibitors [4]. As the most powerful angiogenesis promoter, VEGF plays a critical role in the development and maintenance of tumor vasculature and is regulated by classic signaling pathways, including the PI3K/AKT, Ras/MAPK and FAK/paxillin pathways [5], [6]. Moreover, accumulating evidence confirms that there are other key signaling molecules involved in tumor angiogenesis that regulate VEGF expression. A number of studies have indicated that some Rho guanosine triphosphatase (GTPase) family members are involved in tumor angiogenesis [7], [8]. Our previous findings demonstrated that in human breast cancer specimens, not only was high RhoA expression correlated with high VEGF expression, but RhoA can also increase VEGF expression to promote angiogenesis through an interaction with Murine Double Minute2 (MDM2) protein [9], [10]. In addition, other members of the Rho GTPase family Rac1 and Cdc42 also have significant effects on angiogenesis. Active Rac1 has been demonstrated to improve pathologic VEGF neovessel architecture and reduce vascular leak [11]. In addition, Rac1 signaling has been implicated in VEGF-mediated angiogenesis [12], [13]. Cdc42 reportedly participates in the processes of endothelial cell proliferation, migration and invasion [14], [15]. Although Rac1/Cdc42 signaling has been implicated in angiogenesis regulation, a comprehensive analysis of clinical data and the specific mechanisms by which Rac1/Cdc42 mediates VEGF-induced angiogenesis remain to be elucidated. In our previous study, we observed that the disruption of Rac1 or Cdc42 expression results in up-regulation of p53 and down-regulation of VEGF [16]. In addition, p53 inhibited VEGF expression and thus affected angiogenesis development by regulating specificity protein-1(Sp1) and v-src sarcoma viral oncogene homolog (src) kinase activity [17]–[19]. Notably, some indications have emerged that activated Cdc42 can protect the epidermal growth factor receptor (EGFR) from ubiquitin degradation [20]. Furthermore, Rac1 regulated participation of the ubiquitin-ligase Skp2 in the cell proliferation process [21]. These findings suggest that activated Rac1/Cdc42 regulates some target molecules by affecting protein stability. Given this evidence, we hypothesized that activated Rac1/Cdc42 participates in VEGF-dependent tumor angiogenesis by increasing the degradation of angiogenesis inhibitors, including the p53 protein. In the present study, we used MCF-7 human breast cancer cells, which express wild-type p53, a nude mouse model of breast cancer, and human breast cancer specimens to verify this hypothesis. We observed the effects of inhibiting the ubiquitin-proteasome pathway on HUVEC proliferation, tube formation, and the specific molecular mechanism induced by activated Rac1/Cdc42. Furthermore, in human tissue specimens, we analyzed the correlation of Rac1/Cdc42 with clinical features and angiogenesis factors. The results indicate that Rac1/Cdc42 is a vascular regulator involved in tumor angiogenesis, and that it may reduce the stability of p53 protein to increase VEGF levels by enhancing p53 protein ubiquitination. Our results indicate a novel molecular mechanism for tumor angiogenesis. Results Effect of MG132 on HUVEC proliferation and tube formation induced by activated Rac1/Cdc42 To observe whether Rac1/Cdc42 participates in tumor angiogenesis, the effects of activated Rac1/Cdc42 in MCF-7 cells on HUVEC proliferation and tube formation were examined. The constitutively activated Rac1 plasmid (V12Rac1) and Cdc42 plasmid (L61Cdc42) were used to produce activated Rac1 or Cdc42; pCEFL-GST served as the control plasmid. HUVECs were incubated with conditioned medium from MCF-7 cells stably transfected with one of these plasmids. After 24 h, the changes in HUVEC proliferation were not very obvious. However, after 48 h, the proliferation rates differed among the different groups. Compared to the control group, the medium derived from V12Rac1 or L61Cdc42–MCF-7 cells promoted HUVEC proliferation. Moreover, after treatment with the ubiquitin-proteasome inhibitor MG132, this increased effect was significantly inhibited (Fig. 1A). In the tube formation assay, HUVECs induced by V12Rac1– or L61Cdc42–MCF-7 cells associated with one another and formed more microtubes than did the control group. After MG132 treatment 48 h, however, the number of tubes formed by HUVECs induced by media from V12Rac1– or L61Cdc42–MCF-7 cells was reduced significantly (Fig. 1B and 1C). These results indicated that activated Rac1/Cdc42 in MCF-7 cells can accelerate HUVEC proliferation and tube formation to promote angiogenesis, and that these effects were partly inhibited by MG-132. However, the specific mechanism of angiogenesis induced by activated Rac1/Cdc42 needs to be further investigated. 10.1371/journal.pone.0066275.g001Figure 1 Effect of MG132 on HUVEC proliferation and tube formation induced by active Rac1/Cdc42. The MTT and tube formation assays were performed as described in Material and Methods. (A) HUVECs were grown to confluence and were then cultured in preconditioned media (derived from GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells pretreated or not with MG132 for 12 h) for 24 h or 48 h; the GST–MCF-7 group was used as a control. (B) HUVECs were plated on Matrigel and incubated with the different preconditioned media for 6 h. Photographs were taken in five random power fields (200 ×). (C) Tube lengths were measured with Image-Pro Plus software. Histograms represent quantification of the HUVEC. The GST–MCF-7 group was used as a control. All data are expressed as mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed using one-way ANOVA and Student's t-test. P<0.05, P<0.01, for the GST–MCF-7 group vs the V12Rac1– or L61Cdc42– MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132 group. Activated Rac1/Cdc42 regulates p53 expression to affect VEGF expression in MCF-7 cells To investigate whether p53 and VEGF are regulated by Rac1/Cdc42, the protein expressions of p53 and VEGF were measured in MCF-7 cells transfected with siRac1, V12Rac1, siCdc42 or L61Cdc42. Western blot results showed p53 expression to be increased in siRac1/Cdc42 groups, but decreased in V12Rac1/L61Cdc42 groups. At the same time, VEGF expression was reduced in the siRac1/Cdc42 groups, but augmented in the V12Rac1/L61Cdc42 groups (Fig. 2A–D). To further confirm whether p53 was regulated by Rac1/Cdc42 to affect VEGF expression, we used small interfering RNA of Rac1/Cdc42 and p53 (sip53) or V12Rac1/L61Cdc42 and wild type p53 (wt-p53) to transfect MCF-7 cells at the same time. Western blot results showed that although siRac1/Cdc42 could increase p53 and inhibit VEGF expression, reduced expression of VEGF caused by siRac1/Cdc42 disappeared after p53 knockdown (Fig. 2E and 2G). Moreover, V12Rac1/L61Cdc42 could decrease p53 and augment VEGF expression, but up-regulated VEGF caused by V12Rac1/L61Cdc42 was attenuated after p53 over-expression (Fig. 2F, H). These assays indicate that the effect of Rac1/Cdc42 on VEGF was at least partly mediated by p53. 10.1371/journal.pone.0066275.g002Figure 2 Rac1/Cdc42 regulates p53 expression to affect VEGF expression in MCF-7 cells. (A–D) MCF-7 cells were transfected with Rac1/Cdc42 small-interfering RNA (siRac1/Cdc42) or with negative control siRNA and with the plasmid pCEFL-GST-V12Rac1 (V12Rac1), pCEFL-GST-L61Cdc42 (L61Cdc42) or pCEFL-GST-neo using lipofectamine 2000 for 48 h. Protein and RNA were then extracted and subjected to western blot analyses. Total protein was subjected to 15% SDS-PAGE; membranes were incubated with anti-Rac1, anti-Cdc42, anti-p53, anti-VEGF, or anti-β-actin antibody. (E–H) MCF-7 cells were transfected with siRac1/Cdc42 or with p53 siRNA (sip53) and either activated Rac1/Cdc42 plasmid (V12Rac1/L61Cdc42) or wild-type p53 expression plasmid (wt-p53) with lipofectamine 2000 for 48 h. Protein was then extracted and subjected to 15% SDS-PAGE; membranes were incubated with anti-p53, anti-VEGF, or anti-β-actin antibody. The data represent three independent experiments. Activated Rac1/Cdc42 promotes ubiquitin-mediated degradation of p53 to increase VEGF production in MCF-7 cells Given that the amount of p53 protein could be modified by changes in the rate of synthesis or degradation [22], we hypothesized that Rac1/Cdc42 affected degradation of p53. To test this hypothesis, the ubiquitin-proteasome inhibitor MG-132 was utilized to analyze p53 protein levels. Western blot results revealed that p53 protein was inhibited by V12Rac1/Cdc42, but a large accumulation of p53 protein was observed in the presence of V12Rac1/Cdc42, together with MG-132 (Fig. 3A). Additionally, immunoprecipitation data showed that V12Rac1/Cdc42 could reduce p53 protein levels and increase the amount of ubiquitinated p53 (Fig. 3B). The VEGF ELISA assay also showed that in MCF-7 cells, V12Rac1/LCdc42 increased VEGF secretion after 24 h and 48 h, compared to the control group. However, VEGF expression induced by V12Rac1 or L61Cdc42 decreased after treatment with MG132 (Fig. 3C). These results confirmed that active Rac1/Cdc42 affected and promoted ubiquitin-mediated degradation of p53 to increase VEGF production. 10.1371/journal.pone.0066275.g003Figure 3 Effect of MG132 on VEGF and p53 expression induced by active Rac1/Cdc42. (A) GST–MCF-7, V12Rac1–MCF-7, and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 12 and 24 h, respectively. After incubation, the media were analyzed for VEGF levels using ELISA assay; cell numbers were counted. Data are expressed as the mean ± standard deviation (SD) for three independent experiments. Statistical significance was assessed with one-way ANOVA and Student's t-test. P<0.05, *P<0.01, for the GST–MCF-7 group vs V12Rac1– or L61Cdc42–MCF-7 group. #P<0.05, ##P<0.01, for the MG132-added group vs the no-MG132-added group. (B, C) MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells were incubated, and MG132 was added to select samples for 24 h. After incubation, the cells were analyzed by western blot assay. p53, p21 and β-actin expression was examined. β-actin protein levels were used as a loading control. (D) The immunoprecipitation assay was performed as described in Material and Methods. Total protein was extracted from MCF-7, GST–MCF-7, V12Rac1–MCF-7 and L61Cdc42–MCF-7 cells and subjected to an immunoprecipitation assay. p53 protein expression was examined. β-actin protein levels were used as a loading control. Effect of intratumoral injections of MG132 on vascularization induced by activated Rac1/Cdc42 in MCF-7 cell xenografts To further evaluate the effects of MG132 on tumor angiogenesis induced by V12Rac1/LCdc42, we injected 10 mg/kg MG132 or PBS every 2 days into pre-established, MCF-7 breast tumors (approximately 200 mm3) grown in nude mice. Immunolabeling for VEGF was much higher in tumors excised from mice in the V12Rac1 and L61Cdc42 groups than in those from the control group (Fig. 4). Moreover, p53 expression was lower in the V12Rac1 and L61Cdc42 groups than in the blank or control groups. However, MG312 injections dramatically inhibited the increase in VEGF expression and enhanced the decrease in p53 expression in the V12Rac1 and L61Cdc42 groups compared with the control group. There was no significant difference between the blank and control groups. These data revealed that in vivo, MG132 could reverse the effects of activated Rac1/Cdc42 on p53 and VEGF expression to suppress tumor angiogenesis. 10.1371/journal.pone.0066275.g004Figure 4 Effect of intratumoral injections of MG132 on vascularization induced by active Rac1/Cdc42 in MCF-7 cell xenografts. These experiments were described in the Materials and Methods section. Stably transfected cells (MCF-7, GST–MCF-7, V12Rac1–MCF-7, L61Cdc42–MCF-7) were used for xenografts in nude mice. When the tumors reached a volume of 200 mm3, the mice were treated with intratumoral injections of a specific dose of MG132 (10 mg/kg) or PBS. (A, B) Intratumoral vascularization was assessed by VEGF and p53 immunolabeling (400 × power) on paraffin-embedded MCF-7 cell tumor sections. Representative images are shown. Integrated optical density (IOD) values of VEGF and p53 protein expression were evaluated. ImagePro Plus software was used to analyze the IOD values of the positive areas of immunohistochemical staining. The resulting histograms are presented here. A statistical analysis was performed using a one-way ANOVA. The results are presented as mean ± SD for six mice. P<0.05; **P<0.01. Relationship between Rac1/Cdc42 expression and clinical histopathologic characteristics in breast cancer specimens To assess the significance of Rac1/Cdc42 protein expression in the development and progression of breast cancer, we compared histopathologic characteristics of tumors from 339 patients with available Rac1/Cdc42 protein status in breast cancer samples. The correlation of Rac1/Cdc42 protein expression with different clinical histopathologic factors is presented in Table 1. Statistically significant correlations were found between high Rac1/Cdc42 expression and advanced TNM staging (P<.001), proliferation index (Ki67 status; P<.001), ER status (P<.001), and positive invasive features, including lymph node metastasis (P<.001) and tumor invasion (P<.001) (Table 1). Correlation coefficients are presented in Table 2. However, Rac1/Cdc42 expression did not correlate with patient age, tumor size, histology differentiation or Her-2 status. 10.1371/journal.pone.0066275.t001Table 1 Statistical results of Rac1/Cdc42 expression in 339 breast cancer specimens. Variable No. Rac1 expression p Cdc42 expression p Positive (%) Negative (%) Positive (%) Negative (%) Age(years) 185a .989a ≤ 50 141 105 (74.5) 36 87 (61.7) 54 >50 198 (25.5) 65 (38.3) 76 133 (67.2) 122 (61.6) (32.8) (38.4) Tumor size .280a .311a ≤ 2 cm 142 95 (66.9) 47 83 (58.5) 59 >2 cm 197 (33.1) (41.5) 143 (72.6) 54 126 (64.0) 71 (27.4) (36.0) TNM stage <.0001a <.0001a I∼II 116 22 (19.0) 94 35 (30.2) 81 III∼IV 223 (81.0) 79 (69.8) 144 (64.6) 128 (57.4) 95 (35.4) (42.6) Lymph node <.0001a <.0001a Metastasis 228 195 (85.5) 33 184 (80.7) 44 Positive 111 (14.5) (19.3) 86 Negative 43 (38.7) 68 25 (22.5) (61.3) (77.5) Histology .464b .621b Poorly 105 75 (71.4) 30 68 (64.8) 37 differentiated (28.6) (35.2) Moderately 106 differentiated 78 (73.6) 28 66 (62.3) 40 Well 128 (26.4) (37.7) differentiated 85 (66.4) 43 75 (58.6) 53 (33.6) (41.4) Tumor invasion <.0001a <.0001a Yes 254 206 (81.1) 488 188 (74.0) 66 No 85 (18.9) ER status 32(37.6) 53 <.0001a 21 (24.7) 64 Positive 185 (62.4) 75.3 Negative 154 Her-2 status 90(48.6) .237a 86(46.5) .268a Positive 68 95(51.4) 99(53.5) Negative 271 148(96.1) 6(3.9) 123(79.9) Ki67 status <.0001a 31(20.1) <.0001a Positive 227 52(76.5) Negative 112 16(23.5) 46(67.6) 186(68.6) 22(32.4) 85(31.4) 163(60.1) 108(39.9) 184(81.1) 43(18.9) 169(74.4) 54(48.2) 58(25.6) 58(51.8) 40(35.7) 72(64.3) a The Fisher's exact test was used for statistical analyses. P values <0.05 were considered statistically significant. b The Pearson's chi square test was used for statistical analyses. 10.1371/journal.pone.0066275.t002Table 2 Correlation of Rac1/Cdc42 expression with clinical histopathologic characteristics in 339 breast cancer specimens. Variable Rac1 expression p a Cdc42 expression p a Correlation coefficient (rs) Correlation coefficient (rs) Age(years) .079 .149 .001 .987 Tumor size −.061 .260 −.056 .305 TNM stage −.433 .0001 −.295 .0001 Lymph node metastasis .480 .0001 0.562 .0001 Differentiated status .049 .365 .053 .332 Tumor invasion .412 .0001 .440 .0001 ER status −.517 .0001 −.342 .0001 Her-2 status .069 .208 .062 .257 Ki67 status .338 .0001 .375 .0001 a The Spearman correlation test was used for statistical analyses. P values <0.05 were considered statistically significant. Correlation of Rac1/Cdc42 expression with wild-type p53 and VEGF expression in breast cancer specimens Because the wild-type p53 is the functional form of the p53 protein, examining the level of this form is critical. To estimate the degree of correlation between Rac1/Cdc42 expression and wt-p53 and VEGF in the tumor specimens, we identified 145 specimens that expressed wt-p53 protein among the 339 breast cancer specimens by p53 gene mutation analysis. The statistical analysis indicated that both Rac1 and Cdc42 protein expression were inversely correlated with wt-p53 expression (correlation coefficient [rs] = –.406, P<.0001; and rs = –.263, P = .001; respectively). In contrast, statistically significant positive correlations were observed between expressions of Rac1 and VEGF (rs = .268; P = .001) and between Cdc42 and VEGF (rs = .224; P = .007) (Table 3). These data indicate that high Rac1/Cdc42 expression is correlated with low wt-p53 expression and high VEGF expression. 10.1371/journal.pone.0066275.t003Table 3 Correlation of Rac1/Cdc42 expression with wild-type p53 and VEGF protein in 145 breast cancer specimens containing the wild-type p53 protein. Variable No. Rac1 Expression p a Cdc42 Expression p a Positive (%) Negative (%) Positive (%) Negative (%) wt-p53 <.0001b 30 (44.1) 38 .001d Positive 68 29 (42.6) 39 (55.9) Negative 77 (57.4) 54 (70.1) 23 (29.9) 63 (81.8) 14 .007e (18.2) VEGF .001c Positive 105 75 (71.4) 30 68 (64.8) 37 Negative 40 (28.6) (35.2) 17 (42.5) 23 16 (40.0) 24 (57.5) (60.0) a The Spearman correlation test was used for statistical analyses. P values <0.05 were considered statistically significant. b Correlation coefficient (rs) = −.406. c rs = .268. d rs = −.263. e rs = .224. Discussion Although the Rac1 and Cdc42 oncogenes have been showed to promote carcinogenesis progression and angiogenesis, and to play critical roles in VEGF-dependent tumor angiogenesis [11]–[15], comprehensive clinical data and the specific mechanisms of Rac1/Cdc42-mediated tumor angiogenesis require further investigation. In the present study, we investigated the regulatory role of active Rac1/Cdc42 on p53 and VEGF in vitro and in vivo and investigated the correlation of Rac1/Cdc42 expression with tumor angiogenesis in breast cancer specimens. Our data indicate that Rac1/Cdc42 is the vascular regulator involved in tumor angiogenesis and that it may reduce the stability of p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin. Rac1/Cdc42 plays a key role in HUVEC proliferation and tube formation. Some studies indicate that silencing Rac1/Cdc42 in HUVECs inhibits VEGF-mediated tube formation, cell migration, invasion and proliferation [28], [29]. Furthermore, endothelial-specific excision of Rac1/Cdc42 leads to defective development of vessels and embryonic lethality, supporting an essential role for Eac1/Cdc42 in vascular development [13], [30]. Coincident with these findings, our data demonstrate that activated Rac1/Cdc42 increased HUVEC proliferation and tube formation. In present study of angiogenesis, we focused on the effects of activated Rac1/Cdc42 in MCF-7 cells on HUVECs. Whatever the context of the study, VEGF remains the important factor affecting vascular endothelial cells. We believe that activating of Rac1/Cdc42 in cancer cells is a critical step in tumor angiogenesis to increase VEGF secretion and promote formation of primary vessels from vascular endothelial cells. Recent studies suggest that Rac1/Cdc42 regulates its target molecule to participate in tumorigenesis through both gene transcription regulation and post-translational modification. Activated Cdc42 influences the interactions of the ubiquitin- ligase c-Cbl with the EGFR to protect the EGFR from ubiquitin degradation [20]. Furthermore, activated Rac1 increases the activity of the ubiquitin- ligase Skp2 protein by down-regulating p27 to promote cell proliferation [21]. In our previous study, we observed that disruption of Rac1 or Cdc42 expression results in up-regulation of p53 and down-regulation of VEGF [16], [31]. However, whether Rac1/Cdc42 affects p53 expression by ubiquitination remains unclear. In this study, activated Rac1/Cdc42 inhibited p53 and increased VEGF expression. Importantly, the inhibitory effects of activated Rac1/Cdc42 on p53 expression were reversed after MG132 treatment. Moreover, activated Rac1/Cdc42 was able to reduce p53 protein levels and increase the amount of ubiquitinated p53. These results suggest that Rac1/Cdc42 can regulate p53 protein by enhancing its degradation, thereby promoting VEGF expression. This finding represents a new insight into the mechanism of Rac1/Cdc42-mediated tumor angiogenesis; and suggests a novel strategy for tumor angiogenesis treatment. However, ubiquitin-proteasome inhibitor MG132 which is a kind of unspecific inhibitor could reverse some tumor suppressor protein levels in cancer cells to play the anticancer effects. In this study, using MG132 just indicated that active Rac1/Cdc42 in cancer cells may promote the degradation of antiangiogenesis factors through ubiquitin-proteasome pathway. Therefore, the specific ubiquitin molecule mediates this ubiquitination progression remains unclear and requires further investigation. To date, studies examining the association of Rac1/Cdc42 and the clinical histopathologic characteristics of human breast cancer tissues are rare. Fritz et al. reported that in 50 breast cancer specimens, high Rac1 expression was significantly related to tumor histological grade and proliferation index, but not to Her-2 status [32]. Coincident with these findings, we examined Rac1/Cdc42 expression in 339 human breast cancer specimens and determined that high levels of Rac1/Cdc42 expression were correlated with advanced TNM staging and proliferation index (Ki-67 status) but not with Her2 status. However, the Fritz study did not indicate the relationship between Rac1/Cdc42 expression and other hormone status, or invasiveness. In our study, we found that high Rac1/Cdc42 expression was significantly correlated with lymph node metastasis, tumor invasion and low ER expression. Su et al. determined that as a modulator, active Rac1/Cdc42 decreased ER transcription, and that inhibition of Rac1/Cdc42 enhanced ER transcriptional activity in MCF-7 cells [33]. These findings are consistent with our data in human breast cancer specimens. Importantly, we observed that high Rac1/Cdc42 expression was correlated with low wt-p53 expression and high VEGF expression in 145 specimens that expressed wt-p53. Although expression of Rac1/Cdc42 in breast cancer specimens has been investigated previously, to the best of our knowledge, the current report is the first comprehensive analysis of the regulatory relationship between these angiogenesis molecules in breast cancer tissues. The results of this study fully support our previous data collected in vitro. Taken together, our data demonstrate that Rac1/Cdc42 signaling is critical in tumor angiogenesis; activated Rac1/Cdc42 reduces the stability of p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin. Moreover, Rac1/Cdc42 is significantly correlated with metastasis, invasion and tumor angiogenesis in breast cancer and is a potential prognosis marker in breast cancer. Although the contribution of Rac1/Cdc42 to tumor angiogenesis is complicated and not yet fully understood, our findings provide further understanding and imply a novel target for tumor angiogenesis treatment. Materials and Methods Cell lines and reagents The breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (Rockville, Md) and was cultured according to the supplier's directions and guidelines. HUVECs were isolated, cultured, and characterized as previously described [22]. Cells were maintained at 37°C in a humidified incubator with 5% CO2. Mouse monoclonal anti-Rac1 (1∶1000 dilution; Abcam, Hong Kong, China), mouse monoclonal anti-Cdc42 (1∶1000 dilution; Abcam), mouse monoclonal anti-p53 (1∶500 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, California), rabbit monoclonal anti-p21 (1∶300 dilution; Santa Cruz Biotechnology), mouse monoclonal anti-ubiquitin (1∶300, Santa Cruz Biotech), rabbit polyclonal anti-VEGF (1∶1000 dilution; Cell Signaling, Danvers, Mass), rabbit polyclonal anti-β-actin (1∶3000 dilution; BIOS Biotechnology, Beijing, China), VEGF ELISA assay kit (R&D Corporation), Geneticin (G418, Invitrogen), protein G-sepharose (Sigma-Aldrich, St. Louis, Mo), and MG132 (Sigma-Aldrich, St. Louis, Mo) were used according to the manufactures' instructions. Gene transfection and stable transfected cells The eukaryotic expression plasmids pCEFL-GST-neo control, pCEFL-GST-V12Rac1 and pCEFL-GST-L61Cdc42 (constitutively activated Rac1 and Cdc42) were generously provided by Professor Zheng Yi (University of Cincinnati, Cincinnati, USA). According to the manufacturer's instructions, MCF-7 cells were plated in a 6-well plate and grown overnight to 70–80% confluence without antibiotics. Cells were then transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, California) and different plasmids. Five hours later, the transfection medium was replaced with DMEM supplemented with serum. The transfected cells were selected with 450 µg/ml G418 2 days after transfection. After 2 weeks, G418 was reduced to the maintenance dose of 300 µg/ml. After 4 weeks, these polyclonal, stable transfected cells were trypsinized and transferred to 10 cm tissue culture dishes. Monoclones were picked in a 6-well plate and expanded for an additional 2 months. MTT assay HUVECs were plated at a density of 4 × 104 cells/cm2 in 24-well plates in complete medium and allowed to adhere overnight. Stably transfected MCF-7 cells were cultured, and MG132 was added at a concentration of 2.5 µmol/L for 12 h. Conditioned culture medium from the stably transfected MCF-7 cells was subsequently placed in the HUVEC-containing wells; the whole plate was incubated in a culture chamber for 24 h or 48 h. HUVECs were then incubated for 4 h with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Formazan crystals thus formed were dissolved in 750 µl of dimethyl sulfoxide after the medium was aspirated. The solution was transferred to a 96-well plate, and optical density value was recorded at 570 nm on a microplate reader (Model 680, Bio-Rad, USA). The results are expressed relative to the OD value of HUVEC monocultures on day 1 of the assay. Tube formation assay Sterile 24-well plates were coated with 200 µl Matrigel and incubated at 37°C for 1 h to form gels. After polymerization of the gels, 1.0 × 105 HUVECs were seeded into each well and incubated with 1.0 ml DMEM containing 1% FBS. Stably transfected MCF-7 cells were then cultured, and MG132 was added at a concentration of 2.5 µmol/L for 48 h. Conditioned culture medium from the stably transfected MCF-7 cells was placed in the HUVEC-containing wells for 6 h in a culture chamber. Five different fields were chosen randomly in each well, and photographed. Lengths of the tubes were measured using Image-Pro Plus software (Media Cybernetics, L.P., Silver Spring, MD, USA) and were each expressed as total length (mm) per microscopic field for each well. ELISA assay To assess VEGF secretion in the supernatants of the stably transfected cells, the cells were incubated in serum-free medium with 2.5 µmol/L MG132. After 24 or 48 h, the media were collected, centrifuged to remove cellular debris, and stored at −70°C until being assayed for VEGF. VEGF secreted in the culture medium was measured by ELISA according to the manufacturer's instructions. The data are expressed in pg of VEGF/105 cells/ml. Western blot MCF-7 cells were homogenized in RIPA lysis buffer, and insoluble material was removed by centrifugation at 4°C. From each sample, 80–100 µg of total protein extract was resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). The membranes were blocked in 5% milk and probed with the antibodies overnight at 4°C. The membranes were washed and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotech) for 1 h at 37°C. Chemiluminescent HRP substrate solution (Millipore, USA) was used to develop the images. Immunoprecipitation assay The immunoprecipitation assay was performed as described previously [23]. Cell lysates were incubated with an anti-p53 antibody. The immune- complexes were precipitated with protein A- or protein G-Sepharose 4B (Amersham Biosciences). After washing with lysis buffer, the precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). The blots were probed with anti-ubiquitin overnight at 4°C. A western blot analysis was then performed. Xenograft study in nude mice For inoculation into nude mice, the stably transfected MCF-7 cells were washed with PBS, digested with trypsin, and resuspended in serum-free DMEM medium. After centrifugation (800 rpm), the cell pellets were resuspended in DMEM. The cell suspension (5 × 106 cells in 100 µl of PBS) was injected subcutaneously into the hind legs of 4-week-old female BALB/C athymic (nu/nu) mice (SLAC Laboratory Animal Company, Shanghai, China)[10]. When tumors reached volumes of 200 mm3, the mice were arbitrarily assigned to different groups ((n = 6 each) to receive intratumoral injections of a specific dose of MG132 (10 mg/kg) or PBS. Intratumoral injections were repeated every 3 days for a total of 20 days. On day 20, the mice were sacrificed and their tumors were removed for analysis. All of the experimental procedures were conducted in accordance with the Detailed Rules for the Administration of Animal Experiments for Medical Research Purposes issued by the Ministry of Health of China and received ethical approval by the Animal Experiment Administration Committee of the Fourth Military Medical University (Xi'an, P. R. China). All efforts were made to minimize the animals' suffering and to reduce the number of animals used. Immunohistochemistry Immunohistochemical staining was performed to assess expression of VEGF and p53 proteins, as described previously [9]. For immunohistochemistry, formalin-fixed tumor tissues were embedded in paraffin, and serial 4- µm sections were obtained using a Leica microtome. For staining, tumor sections were dewaxed in toluene, rehydrated in an alcohol gradient, and permeabilized in citrate buffer (pH 6.0). To eliminate endogenous peroxidase activity, the tumor sections were incubated with 3% H2O2 for 5 min, washed in PBS, incubated overnight with different antibodies, and subsequently incubated with biotinylated goat anti-rat or anti-rabbit IgG antibody for 15 min. After washing, the sections were incubated with streptavidin-peroxidase, lightly counterstained with hematoxylin, and observed under a photomicroscope. Staining evaluation This study was approved by the Clinical Trials Administration Committee and the Ethics Committee of the Fourth Military Medical University (Xi'an, P. R. China). All samples were obtained from the Tissue Bank of the Fourth Military Medical University Cancer Center and coded anonymously in accordance with local ethical guidelines. All patients agreed to participate in this study. Written informed consent was obtained from patients, and protocol was approved by the Review Board of Fourth Military Medical University Cancer Center. Fresh breast carcinoma specimens were collected from 339 female patients at the Xijing Hospital of the Fourth Military Medical University (Xi’an, China) and Lanzhou General Hospital of CPLA (Lanzhou, China) from 2006 to 2011. Genomic DNA from 339 tissue specimens was extracted for p53 gene mutation analysis [24]. Rac1/Cdc42 expression was detected in all of the specimens, and wt-p53 and VEGF expression was detected in 145 specimens containing wt-p53 protein. Tissue specimens were examined separately by two pathologists under double-blinded conditions without prior knowledge of the clinical status of the specimens. Molecule expression was scored as positive if >10% of the cells exhibited moderate to strong staining. Expression was scored as negative if either cytoplasmic or membranous staining was noted in <10% of the cells or if neither cytoplasmic nor membranous staining was observed [25]. The Ki67 immunohistochemistry results were reported as the percent positivity of neoplastic cells. For this analysis, we classified the Ki67 levels as negative if <16% or positive if ≥16% [26]. HER2 positivity was defined as 3+ staining on immunohistochemistry or an amplification ratio for fluorescent in situ hybridization if >2.2 [27]. Statistical analysis Experiments in vitro were performed in triplicate. Data from all quantitative assays are expressed as mean ± standard error and were analyzed statistically using a one-way ANOVA followed by Student's t-test. P<0.05 was considered statistically significant. In the in vivo study, associations between Rac1/Cdc42 expression and categorical variables were analyzed by the χ2 test or Fisher's exact test, as appropriate. Correlations between Rac1/Cdc42 expression and categorical variables were analyzed by the Spearman correlation test. ==== Refs References 1 Bergers G , Benjamin LE (2003 ) Tumorigenesis and the angiogenic switch . Nat Rev Cancer 3 : 401 –410 .12778130 2 Jain RK , Carmeliet P (2012 ) SnapShot: Tumor angiogenesis . Cell 149 : 1408 –1408.e1 .22682256 3 Jubb AM , Harris AL (2010 ) Biomarkers to predict the clinical efficacy of bevacizumab in cancer . Lancet Oncol 11 : 1172 –1183 .21126687 4 Meng D , Mei A , Liu J , Kang X , Shi X , et al (2012 ) NADPH Oxidase 4 Mediates Insulin-Stimulated HIF-1α and VEGF Expression, and Angiogenesis In Vitro . PLoS One 10 : e48393 . 5 Lichtenberger BM , Tan PK , Niederleithner H , Ferrara N , Petzelbauer P , et al (2010 ) Autocrine VEGF signaling synergizes with EGFR in tumor cells to promote epithelial cancer development . Cell 140 : 268 –279 .20141840 6 Grothey A , Galanis E (2009 ) Targeting angiogenesis: progress with anti-VEGF treatment with large molecules . Nat Rev Clin Oncol 6 : 507 –518 .19636328 7 Hoang MV , Whelan MC , Senger DR (2004 ) Rho activity critically and selectively regulates endothelial cell organization during angiogenesis . Proc Natl Acad Sci U S A 101 : 1874 –1879 .14769914 8 Sawada N , Li Y , Liao JK (2010 ) Novel aspects of the roles of Rac1 GTPase in the cardiovascular system . Curr Opin Pharmacol 10 : 116 –121 .20060361 9 Ma J , Xue Y , Cui W , Li Y , Zhao Q , et al (2012 ) Ras homolog gene family, member A promotes p53 degradation and vascular endothelial growth factor-dependent angiogenesis through an interaction with murine double minute 2 under hypoxic conditions . Cancer 118 : 4105 –4116 .22907703 10 Ma J , Zhang J , Ma Y , Zheng J , Cheng Y , et al (2012 ) Adenovirus-mediated RhoA shRNA suppresses growth of esophageal squamous cell carcinoma cells in vitro and in vivo . Med Oncol 29 : 119 –126 .21161732 11 Hoang MV , Nagy JA , Senger DR (2011 ) Active Rac1 improves pathologic VEGF neovessel architecture and reduces vascular leak: mechanistic similarities with angiopoietin-1 . Blood 117 : 1751 –1760 .21030561 12 Garrett TA , Van Buul JD , Burridge K (2007 ) VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2 . Exp Cell Res 313 : 3285 –3297 .17686471 13 Bijman MN , van NAGP , Laurens N , van HVW , Boven E (2006 ) Microtubule-targeting agents inhibit angiogenesis at subtoxic concentrations, a process associated with inhibition of Rac1 and Cdc42 activity and changes in the endothelial cytoskeleton . Mol Cancer Ther 5 : 2348 –2357 .16985069 14 de Toledo M , Anguille C , Roger L , Roux P , Gadea G (2012 ) Cooperative Anti-Invasive Effect of Cdc42/Rac1 Activation and ROCK Inhibition in SW620 Colorectal Cancer Cells with Elevated Blebbing Activity. PLoS One . 11 : e48344 . 15 Cascone I , Giraudo E , Caccavari F , Napione L , Bertotti E , et al (2003 ) Temporal and spatial modulation of Rho GTPases during in vitro formation of capillary vascular network. Adherens junctions and myosin light chain as targets of Rac1 and RhoA . J Biol Chem 278 : 50702 –50713 .12972426 16 Xue Y , Bi F , Zhang X , Zhang S , Pan Y , et al (2006 ) Role of Rac1 and Cdc42 in hypoxia induced p53 and von Hippel-Lindau suppression and HIF1alpha activation . Int J Cancer 118 : 2965 –72 .16395716 17 Zietz C , Rossle M , Haas C , Sendelhofert A , Hirschmann A , et al (1998 ) MDM-2 oncoprotein overexpression, p53 gene mutation, and VEGF up-regulation in angiosarcomas . Am J Pathol 153 : 1425 –1433 .9811333 18 Pal S , Datta K , Mukhopadhyay D (2001 ) Central role of p53 on regulation of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) expression in mammary carcinoma . Cancer Res 61 : 6952 –6957 .11559575 19 Mukhopadhyay D , Knebelmann B , Cohen HT , Ananth S , Sukhatme VP (1997 ) The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity . Mol Cell Biol 17 : 5629 –5639 .9271438 20 Wu WJ , Tu S , Cerione RA (2003 ) Activated Cdc42 sequesters c-Cbl and prevents EGF receptor degradation . Cell 114 : 715 –725 .14505571 21 Bond M , Wu YJ , Sala-Newby GB , Newby AC (2008 ) Rho GTPase, Rac1, regulates Skp2 levels, vascular smooth muscle cell proliferation, and intima formation in vitro and in vivo . Cardiovasc Res 80 : 290 –298 .18599477 22 Zhao W , Wang YS , Hui YN , Zhu J , Zhang P , et al (2008 ) Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1alpha with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system . Graefes Archive for Clinical and Experimental Ophthalmology 246 : 1413 –1422 . 23 Takata K , Morishige K , Takahashi T , Hashimoto K , Tsutsumi S , et al (2008 ) Fasudil-induced hypoxia-inducible factor-1alpha degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells . Mol Cancer Ther 7 : 1551 –1561 .18566226 24 Ang C , Guiot MC , Ramanakumar AV , Roberge D , Kavan P (2010 ) Clinical significance of molecular biomarkers in glioblastoma . Can J Neurol Sci 37 : 625 –630 .21059509 25 Lee DC , Kang YK , Kim WH , Jang YJ , Kim DJ , et al (2008 ) Functional and clinical evidence for NDRG2 as a candidate suppressor of liver cancer metastasis . Cancer Res 68 : 4210 –4220 .18519680 26 Viale G , Regan MM , Mastropasqua MG , Maffini F , Maiorano E , et al (2008 ) Predictive value of tumor Ki-67 expression in two randomized trials of adjuvant chemoendocrine therapy for node-negative breast cancer . J Natl Cancer Inst 100 : 207 –212 .18230798 27 Yaziji H , Goldstein LC , Barry TS , Werling R , Hwang H , et al (2004 ) HER-2 testing in breast cancer using parallel tissue-based methods . JAMA 291 : 1972 –1977 .15113815 28 Koh W , Mahan RD , Davis GE (2008 ) Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling . J Cell Sci 121 : 989 –1001 .18319301 29 Fiedler LR (2009 ) Rac1 regulates cardiovascular development and postnatal function of endothelium . Cell Adh Migr 3 : 143 –145 .19287203 30 Spindler V , Schlegel N , Waschke J (2010 ) Role of GTPases in control of microvascular permeability . Cardiovasc Res 87 : 243 –253 .20299335 31 Xue Y , Bi F , Zhang X , Pan Y , Liu N , et al (2004 ) Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells . Biochem Biophys Res Commun 320 : 1309 –1315 .15303276 32 Fritz G , Brachetti C , Bahlmann F , Schmidt M , Kaina B (2002 ) Rho GTPases in human breast tumours: expression and mutation analyses and correlation with clinical parameters . Br J Cancer 87 : 635 –644 .12237774 33 Su LF , Knoblauch R , Garabedian MJ (2001 ) Rho GTPases as modulators of the estrogen receptor transcriptional response . J Biol Chem 276 : 3231 –7 .11060289	23750283	PMC3672132	CC BY	2021-01-05 17:29:04	yes	PLoS One. 2013 Jun 4; 8(6):e66275
==== Front Front Hum NeurosciFront Hum NeurosciFront. Hum. Neurosci.Frontiers in Human Neuroscience1662-5161Frontiers Media S.A. 10.3389/fnhum.2013.00238NeuroscienceOriginal Research ArticleSpontaneous pre-stimulus fluctuations in the activity of right fronto-parietal areas influence inhibitory control performance Chavan Camille F. 1Manuel Aurelie L. 2Mouthon Michael 1Spierer Lucas 11Neurology Unit, Department of Medicine, Faculty of Sciences, University of FribourgFribourg, Switzerland2Neuropsychology and Neurorehabilitation Service, Vaudois University Hospital Center and University of LausanneLausanne, SwitzerlandEdited by: John J. Foxe, Albert Einstein College of Medicine, USA Reviewed by: Redmond O'Connell, Trinity College Dublin, Ireland; Kevin Whittingstall, Université de Sherbrooke, Canada Correspondence: Camille F. Chavan, Laboratory for Cognitive and Neurological Sciences, Neurology Unit, Department of Medicine, Faculty of Sciences, University of Fribourg, PER 09, Chemin du Musée 5, CH-1700 Fribourg, Switzerland e-mail: camille.chavan@unifr.ch06 6 2013 2013 7 23812 3 2013 15 5 2013 Copyright © 2013 Chavan, Manuel, Mouthon and Spierer.2013This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Inhibitory control refers to the ability to suppress planned or ongoing cognitive or motor processes. Electrophysiological indices of inhibitory control failure have been found to manifest even before the presentation of the stimuli triggering the inhibition, suggesting that pre-stimulus brain-states modulate inhibition performance. However, previous electrophysiological investigations on the state-dependency of inhibitory control were based on averaged event-related potentials (ERPs), a method eliminating the variability in the ongoing brain activity not time-locked to the event of interest. These studies thus left unresolved whether spontaneous variations in the brain-state immediately preceding unpredictable inhibition-triggering stimuli also influence inhibitory control performance. To address this question, we applied single-trial EEG topographic analyses on the time interval immediately preceding NoGo stimuli in conditions where the responses to NoGo trials were correctly inhibited [correct rejection (CR)] vs. committed [false alarms (FAs)] during an auditory spatial Go/NoGo task. We found a specific configuration of the EEG voltage field manifesting more frequently before correctly inhibited responses to NoGo stimuli than before FAs. There was no evidence for an EEG topography occurring more frequently before FAs than before CR. The visualization of distributed electrical source estimations of the EEG topography preceding successful response inhibition suggested that it resulted from the activity of a right fronto-parietal brain network. Our results suggest that the fluctuations in the ongoing brain activity immediately preceding stimulus presentation contribute to the behavioral outcomes during an inhibitory control task. Our results further suggest that the state-dependency of sensory-cognitive processing might not only concern perceptual processes, but also high-order, top-down inhibitory control mechanisms. inhibitory controlGo/NoGopre-stimulus periodinferior frontalEEGtopographyelectrical source estimation ==== Body Introduction Inhibitory control, the ability to suppress planned or ongoing cognitive or motor processes is necessary to ensure flexible and adapted goal-directed behavior in ever-changing environments (Aron, 2007; Dillon and Pizzagalli, 2007). Converging functional neuroimaging, transcranial magnetic stimulation and lesion data indicate that inhibitory control relies on a cortico-subcortical network involving the right inferior frontal gyrus (rIFG), the pre-supplementary motor area (SMA) and the basal ganglia (Garavan et al., 1999; Aron et al., 2003; Chambers et al., 2009; Majid et al., 2012), and manifesting at latencies of 150–400 ms after the onset of the stimuli associated with the inhibition goals (Kaiser et al., 2006; Smith and Douglas, 2011). Although inhibitory control performance mostly depends on how the stimuli triggering the inhibition are processed, mounting evidence indicates that the cognitive- and brain-states preceding the presentation of these stimuli also contribute to the success of the inhibition. In Eriksen flanker tasks, contrasts between event-related potentials (ERPs) time-locked to the motor responses to trial preceding error vs. accurate trials revealed a specific ERP component peaking 50–100 ms post-response onset over frontal electrode sites [The “error related positivity” (EPP) Ridderinkhof et al., 2003; Allain et al., 2004; Hajcak et al., 2005]. Britz and Michel (2010) extended these results by showing that dorsolateral prefrontal cortices were engaged differentially during the 100 ms preceding the stimulus onset in error vs. correct trials during a classical color stroop task. These collective results suggest that errors are foreshadowed by a disruption of prefrontal task monitoring systems before the actual need for inhibitory control (see also Eichele et al., 2010; Masaki et al., 2012; Steinhauser et al., 2012). Additional support for the critical role of pre-stimulus brain states in inhibitory control performance comes from studies in which the occurrence of the inhibition stimuli was predictable. In such cases, proactive inhibitory mechanisms are engaged before the stimulus presentation and interact with stimulus-driven reactive inhibitory mechanisms to eventually enhance inhibitory control (Claffey et al., 2010; Jahfari et al., 2010; Aron, 2011; Cai et al., 2012; Duque et al., 2012; Majid et al., 2012). By manipulating the degree of predictability of inhibition trials, these studies showed that when response inhibition can be prepared, effector-selective proactive inhibition mechanisms, mediated by the dorsolateral prefrontal cortex, are engaged to support fronto-basal reactive inhibition mechanisms (Aron, 2011 for review). Criaud et al. (2012) further demonstrated that proactive mechanisms persist once established at the beginning of each trial, suggesting that proactive inhibition has not only a transient effect but also modifies the general response mode of the participants. Taken together, these findings suggest that inhibitory control performance does not solely depend on how participants manage the conflict induced by the stimulus but also on the state of the task-monitoring and control systems before the trial. However, the studies conducted so far on the state-dependency of inhibitory control were based on response- or stimulus-evoked ERPs and thus could not reveal whether spontaneous, not-time-locked fluctuations of ongoing brain activity preceding unpredictable inhibition-stimuli impact on inhibitory control performance. The averaging of the EEG signal in ERP studies indeed canceled out the activity not time-locked to the event of interest and thus dismissed this substantial fraction of the variability of the raw electrical brain activity (e.g., Arieli et al., 1996). Based on the current evidence for the state-dependency of behavioral and brain responses during various types of perceptual tasks (e.g., Lehmann et al., 1994; Ress et al., 2000; Fox et al., 2006; Fox and Raichle, 2007; Britz et al., 2009, 2011), we hypothesize that spontaneous brain states immediately preceding the presentation of inhibition-triggering stimuli might also influence the success or failure to inhibit responses during inhibitory control tasks. To address this question, we used well-established methods of single-trial topographic analyses of EEG to determine whether specific voltage topographies present at the moment of the onset of unpredictable were associated with correct rejection (CR) vs. false alarms (FAs) to NoGo stimuli during a Go/NoGo task (Lehmann et al., 1994; Kondakor et al., 1995, 1997; Koenig et al., 1999; Muller et al., 2005; Britz et al., 2009; Brodbeck et al., 2012). The current investigation was based on a reanalysis of the data from Manuel et al. (2010) in which EEG was recorded in eleven healthy participants during an auditory spatial Go/NoGo task. Material and methods Participants Eleven healthy volunteers participated in the study, all male and right-handed (Oldfield, 1971), aged 22–39 years (mean ± SD, 29.4 ± 1.6 years). Each participant provided written, informed consent to participate in the study. No participant had a history of neurological or psychiatric illness, and all reported normal hearing. All procedures were approved by the Ethics Committee of the Faculty of Biology and Medicine of the Vaudois University Hospital Center and University of Lausanne. Stimuli Auditory stimuli were 150-ms noise bursts (200–500 Hz bandpass filtered; 5 ms rise/fall), lateralized by means of a right- or left-ear leading interaural time difference of 770 μ s resulting in a perceived lateralization of ~80° from the central midline (Blauert, 1997). The sounds were presented via ER-4P Etymotic earphones. Procedure and task The current study is based on a reanalysis of the data from Manuel et al. (2010), in which the procedure and task are already detailed; we thus present only the main task parameters here. Participants were seated in an electrically shielded and sound-attenuated booth in front of a 19-in screen. Stimulus delivery and response recording were controlled using E-prime 2.0. The paradigm comprised an auditory spatial Go/NoGo task in which participants had to respond as quickly as possible via a button to left-lateralized sounds (Go stimuli, hereafter termed LG) and to withhold responses to right-lateralized sounds (NoGo stimuli, termed RNG). Each trial started with the presentation of a centrally presented gray cross on a black background for a randomly determined duration ranging from 1000 to 1900 ms. At the same time that the cross disappeared, the LG and RNG sounds were presented and response collection window was opened. In the Go conditions, a feedback on accuracy and response speed was provided immediately after the response. LG and RNG trials were presented with an equal probability of 0.5. The experiment was divided into three sessions. Each session started with a calibration block of 16 randomly presented trials (eight LG and eight RNG), followed by two test blocks of 80 randomly presented trials each (40 LG and 40 RNG). The calibration blocks were used to individually adjust the task difficulty and to maintain time pressure across the whole experiment. During each calibration phase, the mean response time (RT) to LG trials was calculated online and used to determine the individual participant's RT threshold (RTt), which was set at 80% of the mean RT from the calibration block. During the test block, a Go response RT was considered as correct if it was below the 80% RTt of the immediately preceding calibration phase. Otherwise, a feedback screen indicating “too late!” was displayed immediately after the Go response (slow hit). On each trial, the mean percentage of correct trials, including fast hit and CR, was displayed. Participants were not informed about this thresholding procedure. Except for the global accuracy, no visual feedback was displayed after fast hits or FAs (see Vocat et al., 2008 for a similar procedure). The whole Go/NoGo training session included a total of 528 stimuli (160 stimuli in the test block + 16 stimuli in the calibration block × 3 sessions = 528) and lasted for a total of ~ 35 min. After the completion of each session, a rest period of 10 min was provided to participants. EEG acquisition and preprocessing Continuous EEG was acquired at 1024 Hz through a 128-channel Biosemi ActiveTwo system referenced to the common mode sense/driven right leg ground. All the EEG analyses were conducted with the Cartool software (Brunet et al., 2011). Before the single trial analyses, data at artifact electrodes from each participant were interpolated (Perrin et al., 1987). EEG epochs of the 50 ms preceding the stimulus onset were extracted from the raw EEG data, for each participant, for RNG CR and for the RNG FAs conditions. Because there was less FA than CR (see the behavioral results), we balanced the number of epochs included in each condition before the analyses to ensure that any potential differences between the two conditions did not follow from difference in statistical power. First, the same number of CR as FA epochs was randomly extracted for each participant separately. On the resulting epochs, a ±80 μV artifact rejection criterion was applied to exclude trials with eye blinks or other artifacts. When necessary, the number of epochs was then again balanced across conditions to eventually result in the inclusion of 25.4 ± 16.8 (mean ± SD) epochs in each condition for the single-trial analyses. Behavioral data analyses Behavioral data were analyzed to determine whether FA commission occurred randomly within the sequence of NoGo stimuli (see the Discussion section). We analyzed the pattern of FA occurrence by calculating the temporal auto-correlation function of the FA response type. If there was a relationship between FA occurrence at one trial with FA occurrence at previous or subsequent trials, as could for instance occur during periods of decrease in attention inducing series of FA, it should manifest as an increase in the autocorrelation coefficient (Britz et al., 2009, 2011; Bernasconi et al., 2011). We first performed a binary classification on the FAs and the Hits and Misses (respectively responded and missed Go trials), as well as CR of NoGo trials. To determine if the patterns of FA of the participants were different from a random distribution, we permuted 1000 times the sequence of each participant and then compared the autocorrelation coefficients of the sequence of the participant to the distribution of the autocorrelation coefficients of the randomized sequence of the participant. The autocorrelation coefficients of the FA occurrence were computed for each trial n with that in trial n + m for m = 1 − 20. Single-trial topographic analyses The time-locked averaging of the EEG signal across multiple repetitions of an event (e.g., the presentation of a stimulus or a behavioral response) to build ERPs cancels out all the fluctuations of brain activity not time-locked to the event of interest, because by definition, the phase of these fluctuations varies across trials. To circumvent this problem and to investigate whether non-stimulus-locked variations in the prestimulus activity impact on inhibitory control proficiency, we utilized previously published methods of single-trial EEG analysis (Lehmann et al., 1994; Kondakor et al., 1995, 1997; Koenig et al., 2002; Mohr et al., 2005; Britz et al., 2009; see for review Britz and Michel, 2010; Eichele et al., 2010; Steinhauser et al., 2012). This approach is based on evidence that evoked and induced ongoing EEG signal is not random, but rather organized in a succession of quasi-stable topographies of the electric potentials. Since the configuration of the electric potentials at the scalp reflects the sum of all active sources in the brain at each moment in time (Lehmann et al., 1987), the observation of stable topographies or “maps” suggest that during these time intervals, the functional state of the brain is stable (e.g., Michel et al., 2009). The short periods of functional stability have been referred to as “functional microstate” and typically comprise 80–120 long segments of stable configuration of the scalp-recorded voltage topography of the electric potential (Lehmann and Skrandies, 1980; Koenig et al., 2002). While EEG topography remains stable during a microstate, the strength of the electric potentials, as indexed, for example, by the Global Field Power (GFP), increases and then decreases. The GFP represents a single-number index of the strength of electric potentials; it is calculated as the spatial standard deviation of the electric potentials: the square root of the sum of all squared potentials divided by the number of electrodes (Lehmann and Skrandies, 1980; Murray et al., 2008). Compelling relationships have been found between spontaneously occurring microstates preceding the presentation of a stimulus and the behavioral and brain response to this stimulus (Lehmann et al., 1994; Kondakor et al., 1995, 1997; Mohr et al., 2005; Muller et al., 2005; Britz et al., 2009), suggesting that microstate-based single-trial EEG topographic analyses enable reliable investigations of the effects of variations in the raw ongoing EEG signal. The single-trial topographic analysis comprises the following processing steps, which mainly consist in reducing the raw EEG data into a limited number of common stable microstates within and then across participants (for similar procedures see also Koenig et al., 2002; Mohr et al., 2005; Britz et al., 2009, 2011). The first step involved determining the topographies differentiating the FAs and CR conditions for each participant: (1) We extracted from each EEG epoch, for each participant and each condition the topography manifesting at the single time frame when the GFP was maximal (i.e., when the signal-to noise ratio of the microstate was the highest) within the 50 ms pre-stimulus onset. We selected this pre-stimulus time frame because it corresponds to the half of the duration of an average microstate and thus this procedure enables to extract topography at the time frame best representing the microstate present at the moment when the stimulus is presented (the total length of a microstate ranges between 80 to 120 ms; Britz et al., 2011); (2) For each participant separately, we applied a spatial k-means cluster analysis (e.g., Pascual-Marqui et al., 1995) on these topographies to identify the most dominant topographies among all topographies extracted in step (1). (3) The optimal number of clusters, or “template topographies,” was determined based on a modified Krzanowski-Lai criterion (Krzanowski and Lai, 1988); (4) A fitting procedure, in which each original topography of step (1) was relabeled with the template map of step (2) with which it best correlated (Pegna et al., 1997), enabled us to identify the two template topographies that best differentiated the FA and CR conditions: the template map with the highest frequency of occurrence in the FA and in the CR conditions were retained. The second step comprised determining the topographies differentiating the FA and CR across participants: (5) We applied a second k-mean cluster analysis on the groups of FA and CR map identified during the first step for each participant and back-fitted the resulting template topographies on the original data using the same procedure as steps (1), (2), and (3). The statistical comparison between the frequency of occurrence of the maps in the FA and CR conditions revealed the topographies differentiating the two conditions at the group level. Electrical source estimations Electrical sources underlying the template map(s) of interest were estimated using a distributed linear inverse solution based on a local autoregressive average (LAURA) regularization approach (Grave De Peralta Menendez et al., 2001; Grave-De Peralta et al., 2004; see also Michel et al., 2004 for a comparison of inverse solution methods) implemented in the Cartool software (Brunet et al., 2011). LAURA selects the source configuration that mimics the biophysical behavior of electric potential (i.e., activity at one point depends on the activity at neighboring points). The solution space is based on a realistic head model and included 3005 solution points homogeneously distributed within the gray matter of the average brain of the Montreal Neurological Institute (courtesy of R. Grave de Peralta Menendez and S. Gonzalez Andino, University Hospital of Geneva, Geneva, Switzerland). Results Behavior Reaction time to Go stimuli was 251 ± 21 ms (mean ± SEM). The percentage of FAs was 10.9 ± 0.95% (Manuel et al., 2010). The autocorrelation coefficients of the sequence of FA of the participants were all between z = −0.37 and 0.64 SD from the mean of the coefficients from the randomized sequences, indicating no significant (−1.96 < z < 1.96; p > 0.05) differences between the distributions of FA in the random vs. actual sequences for lags 1–20 (Figure 1). Figure 1 Analysis of the sequence of false alarms. For lags 1–20, the maximal (blue), mean (green), and minimal (red) autocorrelation coefficients are represented, for the sequences of the participants (plain lines) and for randomized sequences (dotted lines). The sequences of FA from the participant did not differ statistically from the random sequences (see the Results section), supporting that random fluctuations in the pre-stimulus period may account for FA occurrence. Single-trial topographic analyses Consistent with previous applications of the current single-trial topographic analysis (Britz et al., 2009, 2011), the k-means cluster analysis applied during the first step of the analysis (see the Method section) identified on average 5.9 ± 1.6 maps for each participant as optimally explaining the data. These template maps accounted for 72.7 ± 7.3% of the global explained variance. The maps were then grouped for each conditions across participants and a second cluster analysis was applied. The best clustering during the second step explained 93% of the variance with 11 template maps. The resulting template maps were then back-fitted to the original individual subject data for each conditions to determine their frequency of occurrence. Two-tailed pairwise t-tests were conducted to compare the frequency of occurrence of each template map between the CR and FA conditions. A single topographic map was found to differ in its frequency of occurrence between the FA and CR conditions (Figure 2, violet square): This topography occurred significantly more often in the CR than FA condition [t(10) = 2.83; p = 0.018; dz = 0.9; Figure 2]. To test for the probability of type 1 errors, we generated 1000 randomized permutations of the raw data maps and computed, for each of the permutation, the same t-tests as those we applied to the actual data. The results revealed a probability of 1.7% to have the same pattern as in the real data (i.e., one p-values < 0.019 among the 11 tests) in the 1000 permutations, i.e., when there was no structure in the data. Figure 2 Topographies of the prestimulus microstates as revealed by the cluster analysis. The map that significantly differentiated the correct rejections is framed in violet. The red lines indicate the t-test p < 0.05 significance threshold and an asterisk is placed where significance has been reached. Electrical source estimations The estimation of the electric sources at the origin of the template topography differentiating the CR from the FA condition showed that it resulted from the activation of a right hemispheric parieto-frontal network extending from the inferior parietal lobule to the frontal gyri (Figure 3). The largest hub of activation was centered around the supramarginal gyrus/primary auditory cortex. Importantly, this result is only a visualization of the sources underlying the EEG topography and not the result of a statistical contrast; it should thus be interpreted with caution. Figure 3 LAURA distributed source estimations corresponding to the topography identified as best characterizing the pre-stimulus period before correct rejections identified a right hemispheric fronto-temporal prestimulus activation. Discussion The present study suggests that performance in a classical Go/NoGo task is influenced by the momentary state of the ongoing brain activity immediately preceding the onset of unpredictable NoGo stimuli. A specific EEG voltage topography manifested more frequently before successful response-inhibition to NoGo stimuli than before FAs. There was no evidence for topographies specifically preceding FA trials. Electrical source estimations localized the source of the topography preceding successful inhibition within a right fronto-temporal network. Our results contribute to current knowledge on the influence of pre-stimulus brain state on inhibitory control by indicating that inhibition performance is modulated by the momentary brain state present when the stimuli are presented. Fluctuations in the ongoing brain activity have been shown to represent almost 90% of the variability in the EEG signal (Eichele et al., 2010). This source of variability was mostly dismissed in previous ERPs studies on the effect of pre-stimulus activity because time-locked averaging of the EEG signal were applied to extract ERPs (e.g., Hajcak et al., 2005; Masaki et al., 2012); in such procedures, the signal not time-locked to the event is cancelled out and usually considered as background physiological noise. In line with our findings, mounting evidence indicate that ongoing spontaneous EEG fluctuations are actually functionally relevant and account for a substantial fraction of the variability in the behavioral and brain responses to stimuli in various experimental contexts (Picton et al., 2000; O'Connell et al., 2009 for the role of pre-stimulus endogenous modulation in oscillatory activity in task performance; Britz and Michel, 2010 for discussion). However, the dependency of sensory-cognitive processes to single trial variability in brain activity was so far demonstrated in perceptual but not executive tasks. For instance, Mohr et al. (2005) showed that a specific topographic map with a left anterior-right posterior dipole orientation spontaneously manifesting before word presentation predicted an enhanced discrimination of emotional word when the word was presented in the left visual field but only for men during a bilateral lexical decision task. Lehmann et al. (1994) and Kondakor et al. (1995, 1997) further demonstrated that the brain responses to identical tones depended on the spontaneously occurring topography preceding their presentation. Corroborating and extending these findings, our results suggest that spontaneous fluctuations of ongoing brain activity not only modulate perceptual processes but also high-order, top-down executive mechanisms as those supporting inhibitory control in conflict tasks. The sources of the scalp topography preceding more frequently CR than FAs were maximal within a right fronto-parietal network. The right rIFG has been repeatedly involved in inhibitory control by functional neuroimaging (Garavan et al., 1999; Rubia et al., 2003; Aron and Poldrack, 2006; Aron et al., 2007) and lesion studies (Decary and Richer, 1995; Aron et al., 2003; Rieger et al., 2003; Floden and Stuss, 2006; Picton et al., 2007). The rIFG is thought to trigger motor inhibition via its connections to the subthalamic nucleus (STN; Inase et al., 1999; Aron et al., 2007; Aron, 2011). Most of these studies, however, involved the rIFG in inhibitory control by contrasting the post-stimulus brain responses to stimuli associated with vs. without response inhibition. In the current study, we showed that random fluctuations in the activity of rIFG before the demand for inhibition also play a critical role in inhibitory control proficiency. In conditions when the onset of the NoGo stimuli cannot be predicted, if the NoGo stimuli are presented when the activity of the rIFG is high, the probability of a correct inhibition increases. Although speculative, an account for this effect could be that a pre-activation of the rIFG increases the speed of inhibition process because less time is needed to reach the elicitation threshold of the inhibitory command from frontal to subcortical structures. Right inferior parietal areas have been previously involved in response inhibition (Garavan et al., 1999; Liddle et al., 2001; Menon et al., 2001; Rubia et al., 2003) or response conflict resolution (Braver et al., 2001; Van Veen et al., 2001). These studies suggest that inferior parietal cortices mediate attention to the task and might thus, in turn, modulate performance (see also Hampshire et al., 2010). Increase in the activity of the precentral gyrus in condition of successful inhibition has also been reported in stop-signal task (though more superior as in the current study; Li et al., 2006), and have been interpreted as “negative motor areas” (Ikeda et al., 2000; Yazawa et al., 2000), whose direct stimulation elicits response inhibition (Luders et al., 1995). Alternatively, since participants had to respond with their right hand, an activation of ipsilateral (right) motor areas might have inhibited contralateral, homotopic motor areas via interhemispheric inhibition mechanisms and in turn facilitated the rejection of NoGo stimuli (e.g., Ferbert et al., 1992). Hand and arm motor representations are, however, higher along the central sulcus as the area found in the current study. The precise role of the right temporal structures in inhibitory control is more difficult to delineate. These areas have been shown to interact with higher level prefrontal regions during inhibitory control tasks (Egner and Hirsch, 2005) and to be modulated in inhibition-related disorders (Tamm et al., 2004; Solanto et al., 2009), but their precise role remains unclear. An alternative account for the role of right temporal areas in FA commission would be their involvement in the processing of the auditory spatial features distinguishing Go from NoGo stimuli in the current task. Go and NoGo goals activation indeed first require discriminating whether the stimulus is presented on the left or right auditory hemifield. Right temporal areas have been repeatedly involved in the early stage of auditory spatial processing (Spierer et al., 2007, 2011) and their pre-activation could have facilitated the detection of NoGo stimuli and in turn response inhibition. In line with this hypothesis, Manuel et al. (2010) suggested an important role of early auditory analyses of the lateralization of the stimuli in inhibitory control proficiency during the current auditory spatial Go/NoGo task. Importantly, however, we would like to emphasize that our source estimations were only visualizations, rather than a statistical analysis, of the network likely underlying the topography preceding FA commission. These results should thus be interpreted with caution. Although our pattern of result is highly consistent with current evidence for a crucial role of the rIGF in stimulus-induced inhibitory control processes, it contrasts with previous data on the brain regions whose pre-stimulus activity modulates inhibition proficiency. Electrophysiological studies on trials preceding error during an Eriksen flankers task identified a specific response-locked ERP component at 100 ms (EPP; Ridderinkhof et al., 2003; Allain et al., 2004; Hajcak et al., 2005). The EPP was specific to trials preceding an error and its amplitude was larger on frontal, central and parietal midline electrodes, compatible with a generator located within the anterior cingulate cortex (ACC). Using a stroop task, Britz and Michel (2010) showed a specific pattern of stimulus-locked fronto-parietal activity before erroneous vs. correct trials. The modulation of prefrontal activity before error commission found in the studies reviewed above was interpreted as reflecting a transient disruption of action monitoring and executive control systems. Our results complement these finding by demonstrating that in addition to the impact of maintaining a minimal level of action monitoring during the task found by response- or stimulus-locked ERP approaches, spontaneous ongoing activity within the networks mediating stimulus-induced response inhibition also influences inhibitory control performance. In line with this assumption, growing evidence indicate that as soon as the need for response inhibition can be predicted, proactive inhibitory control mechanisms are engaged to facilitate the inhibitory control of task-irrelevant responses (Claffey et al., 2010; Jahfari et al., 2010; see Aron, 2011 for review; Cai et al., 2012; Criaud et al., 2012). “Active breaking” mechanisms would be engaged before the stimulus onset to decrease the activity of motor area and in turn facilitate response inhibition (Cai et al., 2012; Duque et al., 2012). Anticipatory mechanisms have been shown to rely on a fronto-striatal network partly overlapping with the estimated sources of the topography prominently associated with successful inhibition in the current study. We cannot rule out that the frequency of occurrence of the topography preceding successful inhibition was influenced by proactive preparatory mechanisms in the current study. According to this hypothesis, the anticipation of NoGo stimuli would have increased the frequency of occurrence of the “facilitatory” topography or, in other word, pre-activated inhibitory control mechanisms. Speaking against this hypothesis, there was no strategic advantage of intentionally maintaining inhibitory control networks active in our task because such decrease in the response elicitation threshold would have resulted in a general decrease in response speed to Go stimuli. In our paradigm, emphasis was put on response speed: slow Hits (correctly responded Go stimuli but above the RTt determined in calibration blocks) were considered as error and reported explicitly as such by the visual feedback provided after each trial. Although there was a probability of 0.5 for NoGo stimuli occurrence, response prepotency was maintained very high by the recurrent feedback on responses speed. The threshold for considering a response to Go trials to slow was indeed individually adjusted to 80% of the mean response speed calculated during the calibration blocks intervening between experimental blocks (see the Method section). Hence, we interpret our results as indicating that when the fronto-temporal brain network was already (spontaneously) activated before a NoGo stimulus was presented, inhibition performance was improved. Compatible with this hypothesis, the analyses of the distribution of FA occurrence across the experimental session showed that participants committed FA following the same pattern as when FA were randomly distributed within the sequence. The adoption of any strategy by the participant would have most likely resulted in non-random sequences consisting of series of FA or CR because a specific task set could unlikely be maintained constantly during the whole experiment (Palva and Palva, 2012). Our results cannot disentangle the relationship between proactive inhibition and the occurrence of specific microstates. To our knowledge, no study has so far investigated whether top-down, strategic implementation of task sets can impact on the frequency of occurrence-specific microstate and thus whether proactive mechanism could have influenced our pattern of result. Further studies focusing on longer epochs and manipulating the engagement of proactive inhibitory mechanisms are necessary to elucidate this question. Of note, we found a specific EEG topography preceding successful trials, but no specific topography preceding errors. This pattern of result suggests that while the engagement of specific cognitive set may enhance motor responses inhibition in conditions of high response prepotency, many different deviations from this optimal brain-state could result in inhibition failure, in turn explaining that we did not identify a specific topographic map preceding errors. In this regard, a limitation of the current study is that by defining our FA and CR conditions only based on RT (and lack thereof), we possibly included in the CR condition some trials in which erroneous motor activation was actually engaged but inhibited before reaching activation threshold. Referred to as “covert” errors, these partial-error trials can only be detected with the recording of electromyography activations and have been suggested to depend on distinct mechanisms as overt errors (Burle et al., 2002; Allain et al., 2004; Boulinguez et al., 2008). In any case, the inclusion of covert error trials in the CR condition would have increased noise in the data and thus most likely increased the probability of type 2 but not type 1 errors. Second, because FAs were quite rare in our data, our single trial topographic analyses included only a limited number of trials. While the capacity to deal with small numbers of EEG epochs is an advantage of this approach and we reach reliable statistical results, how the number of epochs impacts the statistical outcome of our approach remains to be determined. Collectively, our results point out that spontaneous fluctuations within inhibitory control networks at the moment when NoGo stimuli are presented influence inhibition performance. The question remains open, however, as to whether and how prestimulus modulations impact on the processing of the inhibition-related stimuli. It would be notably interesting to link directly the current results with previous literature on pre-target predictor of inhibition performance and to examine potential differences in the early sensory processing of the NoGo stimuli between FA and CR trials. Further studies comparing the pre-stimulus with the post-stimulus activity would be necessary to elucidate this question. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by a grant from the Swiss National Science Foundation to Lucas Spierer (#320030_143348). The Cartool software has been programmed by Denis Brunet (Functional Brain Mapping Laboratory, Geneva, Switzerland) and supported by the Center for Biomedical Imaging of Geneva and Lausanne. We thank David Magezi, David Souto and the reviewers for their valuable comments. ==== Refs References Allain S. Carbonnell L. Falkenstein M. Burle B. Vidal F. (2004 ). The modulation of the Ne-like wave on correct responses foreshadows errors . Neurosci. Lett . 372 , 161 –166 10.1016/j.neulet.2004.09.036 15531109 Arieli A. Sterkin A. Grinvald A. Aertsen A. (1996 ). Dynamics of ongoing activity: explanation of the large variability in evoked cortical responses . Science 273 , 1868 –1871 10.1126/science.273.5283.1868 8791593 Aron A. R. (2007 ). The neural basis of inhibition in cognitive control . Neuroscientist 13 , 214 –228 10.1177/1073858407299288 17519365 Aron A. R. (2011 ). From reactive to proactive and selective control: developing a richer model for stopping inappropriate responses . Biol. Psychiatry 69 , e55 –e68 10.1016/j.biopsych.2010.07.024 20932513 Aron A. R. Durston S. Eagle D. M. Logan G. D. Stinear C. M. Stuphorn V. (2007 ). Converging evidence for a fronto-basal-ganglia network for inhibitory control of action and cognition . J. Neurosci . 27 , 11860 –11864 10.1523/JNEUROSCI.3644-07.2007 17978025 Aron A. R. Fletcher P. C. Bullmore E. T. Sahakian B. J. Robbins T. W. (2003 ). Stop-signal inhibition disrupted by damage to right inferior frontal gyrus in humans . Nat. Neurosci . 6 , 115 –116 10.1038/nn1003 12536210 Aron A. R. Poldrack R. A. (2006 ). Cortical and subcortical contributions to Stop signal response inhibition: role of the subthalamic nucleus . J. Neurosci . 26 , 2424 –2433 10.1523/JNEUROSCI.4682-05.2006 16510720 Bernasconi F. Manuel A. L. Murray M. M. Spierer L. (2011 ). Pre-stimulus beta oscillations within left posterior sylvian regions impact auditory temporal order judgment accuracy . Int. J. Psychophysiol . 79 , 244 –248 10.1016/j.ijpsycho.2010.10.017 21056064 Blauert J. (1997 ). Spatial Hearing (revised edition) . Cambridge, MA : Massachusetts Institute of Technology Boulinguez P. Jaffard M. Granjon L. Benraiss A. (2008 ). Warning signals induce automatic EMG activations and proactive volitional inhibition: evidence from analysis of error distribution in simple RT . J. Neurophysiol . 99 , 1572 –1578 10.1152/jn.01198.2007 18171709 Braver T. S. Barch D. M. Gray J. R. Molfese D. L. Snyder A. (2001 ). Anterior cingulate cortex and response conflict: effects of frequency, inhibition and errors . Cereb. Cortex 11 , 825 –836 10.1093/cercor/11.9.825 11532888 Britz J. Landis T. Michel C. M. (2009 ). Right parietal brain activity precedes perceptual alternation of bistable stimuli . Cereb. Cortex 19 , 55 –65 10.1093/cercor/bhn056 18424780 Britz J. Michel C. M. (2010 ). Errors can be related to pre-stimulus differences in ERP topography and their concomitant sources . Neuroimage 49 , 2774 –2782 10.1016/j.neuroimage.2009.10.033 19850140 Britz J. Pitts M. A. Michel C. M. (2011 ). Right parietal brain activity precedes perceptual alternation during binocular rivalry . Hum. Brain Mapp . 32 , 1432 –1442 10.1002/hbm.21117 20690124 Brodbeck V. Kuhn A. Von Wegner F. Morzelewski A. Tagliazucchi E. Borisov S. (2012 ). EEG microstates of wakefulness and NREM sleep . Neuroimage 62 , 2129 –2139 10.1016/j.neuroimage.2012.05.060 22658975 Brunet D. Murray M. M. Michel C. M. (2011 ). Spatiotemporal analysis of multichannel EEG: CARTOOL . Comput. Intell. Neurosci . 2011 :813870 10.1155/2011/813870 21253358 Burle B. Possamai C. A. Vidal F. Bonnet M. Hasbroucq T. (2002 ). Executive control in the Simon effect: an electromyographic and distributional analysis . Psychol. Res . 66 , 324 –336 10.1007/s00426-002-0105-6 12466929 Cai W. George J. S. Verbruggen F. Chambers C. D. Aron A. R. (2012 ). The role of the pre-supplementary motor area in stopping action: two studies with event-related transcranial magnetic stimulation . J. Neurophysiol . 108 , 380 –389 10.1152/jn.00132.2012 22514296 Chambers C. D. Garavan H. Bellgrove M. A. (2009 ). Insights into the neural basis of response inhibition from cognitive and clinical neuroscience . Neurosci. Biobehav. Rev . 33 , 631 –646 10.1016/j.neubiorev.2008.08.016 18835296 Claffey M. P. Sheldon S. Stinear C. M. Verbruggen F. Aron A. R. (2010 ). Having a goal to stop action is associated with advance control of specific motor representations . Neuropsychologia 48 , 541 –548 10.1016/j.neuropsychologia.2009.10.015 19879283 Criaud M. Wardak C. Ben Hamed S. Ballanger B. Boulinguez P. (2012 ). Proactive inhibitory control of response as the default state of executive control . Front. Psychol . 3 :59 10.3389/fpsyg.2012.00059 22403563 Decary A. Richer F. (1995 ). Response selection deficits in frontal excisions . Neuropsychologia 33 , 1243 –1253 10.1016/0028-3932(95)00040-A 8552227 Dillon D. G. Pizzagalli D. A. (2007 ). Inhibition of action, thought, and emotion: a selective neurobiological review . Appl. Prev. Psychol . 12 , 99 –114 10.1016/j.appsy.2007.09.004 19050749 Duque J. Labruna L. Verset S. Olivier E. Ivry R. B. (2012 ). Dissociating the role of prefrontal and premotor cortices in controlling inhibitory mechanisms during motor preparation . J. Neurosci . 32 , 806 –816 10.1523/JNEUROSCI.4299-12.2012 22262879 Egner T. Hirsch J. (2005 ). The neural correlates and functional integration of cognitive control in a Stroop task . Neuroimage 24 , 539 –547 10.1016/j.neuroimage.2004.09.007 15627596 Eichele H. Juvodden H. T. Ullsperger M. Eichele T. (2010 ). Mal-adaptation of event-related EEG responses preceding performance errors . Front. Hum. Neurosci . 4 :65 10.3389/fnhum.2010.00065 20740080 Ferbert A. Priori A. Rothwell J. C. Day B. L. Colebatch J. G. Marsden C. D. (1992 ). Interhemispheric inhibition of the human motor cortex . J. Physiol . 453 , 525 –546 1464843 Floden D. Stuss D. T. (2006 ). Inhibitory control is slowed in patients with right superior medial frontal damage . J. Cogn. Neurosci . 18 , 1843 –1849 10.1162/jocn.2006.18.11.1843 17069475 Fox M. D. Raichle M. E. (2007 ). Spontaneous fluctuations in brain activity observed with functional magnetic resonance imaging . Nat. Rev. Neurosci . 8 , 700 –711 10.1038/nrn2201 17704812 Fox M. D. Snyder A. Z. Zacks J. M. Raichle M. E. (2006 ). Coherent spontaneous activity accounts for trial-to-trial variability in human evoked brain responses . Nat. Neurosci . 9 , 23 –25 10.1038/nn1616 16341210 Garavan H. Ross T. J. Stein E. A. (1999 ). Right hemispheric dominance of inhibitory control: an event-related functional MRI study . Proc. Natl. Acad. Sci. U.S.A . 96 , 8301 –8306 10.1073/pnas.96.14.8301 10393989 Grave-De Peralta R. Gonzalez-Andino S. Gomez-Gonzalez C. M. (2004 ). The biophysical foundations of the localisation of encephalogram generators in the brain. The application of a distribution-type model to the localisation of epileptic foci . Rev. Neurol . 39 , 748 –756 15514904 Grave De Peralta Menendez R. Gonzalez Andino S. Lantz G. Michel C. M. Landis T. (2001 ). Noninvasive localization of electromagnetic epileptic activity. I. Method descriptions and simulations . Brain Topogr . 14 , 131 –137 10.1023/A:1012944913650 11797811 Hajcak G. Nieuwenhuis S. Ridderinkhof K. R. Simons R. F. (2005 ). Error-preceding brain activity: robustness, temporal dynamics, and boundary conditions . Biol. Psychol . 70 , 67 –78 10.1016/j.biopsycho.2004.12.001 16168251 Hampshire A. Chamberlain S. R. Monti M. M. Duncan J. Owen A. M. (2010 ). The role of the right inferior frontal gyrus: inhibition and attentional control . Neuroimage 50 , 1313 –1319 10.1016/j.neuroimage.2009.12.109 20056157 Ikeda A. Ohara S. Matsumoto R. Kunieda T. Nagamine T. Miyamoto S. (2000 ). Role of primary sensorimotor cortices in generating inhibitory motor response in humans . Brain 123(Pt 8) , 1710 –1721 10.1093/brain/123.8.1710 10908200 Inase M. Tokuno H. Nambu A. Akazawa T. Takada M. (1999 ). Corticostriatal and corticosubthalamic input zones from the presupplementary motor area in the macaque monkey: comparison with the input zones from the supplementary motor area . Brain Res . 833 , 191 –201 10.1016/S0006-8993(99)01531-0 10375694 Jahfari S. Stinear C. M. Claffey M. Verbruggen F. Aron A. R. (2010 ). Responding with restraint: what are the neurocognitive mechanisms? J. Cogn. Neurosci . 22 , 1479 –1492 10.1162/jocn.2009.21307 19583473 Kaiser S. Weiss O. Hill H. Markela-Lerenc J. Kiefer M. Weisbrod M. (2006 ). N2 event-related potential correlates of response inhibition in an auditory Go/Nogo task . Int. J. Psychophysiol . 61 , 279 –282 10.1016/j.ijpsycho.2005.09.006 16298004 Koenig T. Lehmann D. Merlo M. C. Kochi K. Hell D. Koukkou M. (1999 ). A deviant EEG brain microstate in acute, neuroleptic-naive schizophrenics at rest . Eur. Arch. Psychiatry Clin. Neurosci . 249 , 205 –211 10.1007/s004060050088 10449596 Koenig T. Prichep L. Lehmann D. Sosa P. V. Braeker E. Kleinlogel H. (2002 ). Millisecond by millisecond, year by year: normative EEG microstates and developmental stages . Neuroimage 16 , 41 –48 10.1006/nimg.2002.1070 11969316 Kondakor I. Lehmann D. Michel C. M. Brandeis D. Kochi K. Koenig T. (1997 ). Prestimulus EEG microstates influence visual event-related potential microstates in field maps with 47 channels . J. Neural Transm . 104 , 161 –173 10.1007/BF01273178 9203079 Kondakor I. Pascual-Marqui R. D. Michel C. M. Lehmann D. (1995 ). Event-related potential map differences depend on the prestimulus microstates . J. Med. Eng. Technol . 19 , 66 –69 7494212 Krzanowski W. J. Lai Y. T. (1988 ). A criterion for determining the number of groups in a data set using sum-of-squares clustering . Biometrics 44 , 23 –34 Lehmann D. Michel C. M. Pal I. Pascual-Marqui R. D. (1994 ). Event-related potential maps depend on prestimulus brain electric microstate map . Int. J. Neurosci . 74 , 239 –248 7928108 Lehmann D. Ozaki H. Pal I. (1987 ). EEG alpha map series: brain micro-states by space-oriented adaptive segmentation . Electroencephalogr. Clin. Neurophysiol . 67 , 271 –288 2441961 Lehmann D. Skrandies W. (1980 ). Reference-free identification of components of checkerboard-evoked multichannel potential fields . Electroencephalogr. Clin. Neurophysiol . 48 , 609 –621 10.1016/0013-4694(80)90419-8 6155251 Li C. S. Huang C. Constable R. T. Sinha R. (2006 ). Imaging response inhibition in a stop-signal task: neural correlates independent of signal monitoring and post-response processing . J. Neurosci . 26 , 186 –192 10.1523/JNEUROSCI.3741-05.2006 16399686 Liddle P. F. Kiehl K. A. Smith A. M. (2001 ). Event-related fMRI study of response inhibition . Hum. Brain Mapp . 12 , 100 –109 10.1002/1097-0193(200102)12:2<100::AID-HBM1007>3.0.CO;2-6 11169874 Luders H. O. Dinner D. S. Morris H. H. Wyllie E. Comair Y. G. (1995 ). Cortical electrical stimulation in humans. The negative motor areas . Adv. Neurol . 67 , 115 –129 8848964 Majid D. S. Cai W. George J. S. Verbruggen F. Aron A. R. (2012 ). Transcranial magnetic stimulation reveals dissociable mechanisms for global versus selective corticomotor suppression underlying the stopping of action . Cereb. Cortex 22 , 363 –371 10.1093/cercor/bhr112 21666129 Manuel A. L. Grivel J. Bernasconi F. Murray M. M. Spierer L. (2010 ). Brain dynamics underlying training-induced improvement in suppressing inappropriate action . J. Neurosci . 30 , 13670 –13678 10.1523/JNEUROSCI.2064-10.2010 20943907 Masaki H. Murphy T. I. Kamijo K. Yamazaki K. Sommer W. (2012 ). Foreshadowing of performance accuracy by event-related potentials: evidence from a minimal-conflict task . PLoS ONE 7 :e38006 10.1371/journal.pone.0038006 22701541 Menon V. Adleman N. E. White C. D. Glover G. H. Reiss A. L. (2001 ). Error-related brain activation during a Go/NoGo response inhibition task . Hum. Brain Mapp . 12 , 131 –143 10.1002/1097-0193(200103)12:3<131::AID-HBM1010>3.0.CO;2-C 11170305 Michel C. M. Koenig T. Brandeis D. Gianotti L. Wackermann J. (2009 ). Electrical Neuroimaging . Cambridge, MA : Cambridge University Press Michel C. M. Murray M. M. Lantz G. Gonzalez S. Spinelli L. Grave De Peralta R. (2004 ). EEG source imaging . Clin. Neurophysiol . 115 , 2195 –2222 10.1016/j.clinph.2004.06.001 15351361 Mohr C. Michel C. M. Lantz G. Ortigue S. Viaud-Delmon I. Landis T. (2005 ). Brain state-dependent functional hemispheric specialization in men but not in women . Cereb. Cortex 15 , 1451 –1458 10.1093/cercor/bhi025 15689523 Muller T. J. Koenig T. Wackermann J. Kalus P. Fallgatter A. Strik W. (2005 ). Subsecond changes of global brain state in illusory multistable motion perception . J. Neural Transm . 112 , 565 –576 10.1007/s00702-004-0194-z 15340871 Murray M. M. Brunet D. Michel C. M. (2008 ). Topographic ERP analyses: a step-by-step tutorial review . Brain Topogr . 20 , 249 –264 10.1007/s10548-008-0054-5 18347966 O'Connell R. G. Dockree P. M. Robertson I. H. Bellgrove M. A. Foxe J. J. Kelly S. P. (2009 ). Uncovering the neural signature of lapsing attention: electrophysiological signals predict errors up to 20 s before they occur . J. Neurosci . 29 , 8604 –8611 10.1523/JNEUROSCI.5967-08.2009 19571151 Oldfield R. C. (1971 ). The assessment and analysis of handedness: the Edinburgh inventory . Neuropsychologia 9 , 97 –113 10.1016/0028-3932(71)90067-4 5146491 Palva J. M. Palva S. (2012 ). Infra-slow fluctuations in electrophysiological recordings, blood-oxygenation-level-dependent signals, and psychophysical time series . Neuroimage 62 , 2201 –2211 10.1016/j.neuroimage.2012.02.060 22401756 Pascual-Marqui R. D. Michel C. M. Lehmann D. (1995 ). Segmentation of brain electrical activity into microstates: model estimation and validation . IEEE Trans. Biomed. Eng . 42 , 658 –665 10.1109/10.391164 7622149 Pegna A. J. Khateb A. Spinelli L. Seeck M. Landis T. Michel C. M. (1997 ). Unraveling the cerebral dynamics of mental imagery . Hum. Brain Mapp . 5 , 410 –421 10.1002/(SICI)1097-0193(1997)5:6<410::AID-HBM2>3.0.CO;2-6 20408244 Perrin F. Pernier J. Bertrand O. Giard M. H. Echallier J. F. (1987 ). Mapping of scalp potentials by surface spline interpolation . Electroencephalogr. Clin. Neurophysiol . 66 , 75 –81 10.1016/0013-4694(87)90141-6 2431869 Picton T. W. Bentin S. Berg P. Donchin E. Hillyard S. A. Johnson R. Jr. (2000 ). Guidelines for using human event-related potentials to study cognition: recording standards and publication criteria . Psychophysiology 37 , 127 –152 10.1017/S0048577200000305 10731765 Picton T. W. Stuss D. T. Alexander M. P. Shallice T. Binns M. A. Gillingham S. (2007 ). Effects of focal frontal lesions on response inhibition . Cereb. Cortex 17 , 826 –838 10.1093/cercor/bhk031 16699079 Ress D. Backus B. T. Heeger D. J. (2000 ). Activity in primary visual cortex predicts performance in a visual detection task . Nat. Neurosci . 3 , 940 –945 10.1038/78856 10966626 Ridderinkhof K. R. Nieuwenhuis S. Bashore T. R. (2003 ). Errors are foreshadowed in brain potentials associated with action monitoring in cingulate cortex in humans . Neurosci. Lett . 348 , 1 –4 10.1016/S0304-3940(03)00566-4 12893411 Rieger M. Gauggel S. Burmeister K. (2003 ). Inhibition of ongoing responses following frontal, nonfrontal, and basal ganglia lesions . Neuropsychology 17 , 272 –282 10.1037/0894-4105.17.2.272 12803433 Rubia K. Smith A. B. Brammer M. J. Taylor E. (2003 ). Right inferior prefrontal cortex mediates response inhibition while mesial prefrontal cortex is responsible for error detection . Neuroimage 20 , 351 –358 10.1016/S1053-8119(03)00275-1 14527595 Smith J. L. Douglas K. M. (2011 ). On the use of event-related potentials to auditory stimuli in the Go/NoGo task . Psychiatry Res . 193 , 177 –181 10.1016/j.pscychresns.2011.03.002 21764566 Solanto M. V. Schulz K. P. Fan J. Tang C. Y. Newcorn J. H. (2009 ). Event-related FMRI of inhibitory control in the predominantly inattentive and combined subtypes of ADHD . J. Neuroimaging 19 , 205 –212 10.1111/j.1552-6569.2008.00289.x 19594667 Spierer L. De Lucia M. Bernasconi F. Grivel J. Bourquin N. M. Clarke S. (2011 ). Learning-induced plasticity in human audition: objects, time, and space . Hear. Res . 271 , 88 –102 10.1016/j.heares.2010.03.086 20430070 Spierer L. Tardif E. Sperdin H. Murray M. M. Clarke S. (2007 ). Learning-induced plasticity in auditory spatial representations revealed by electrical neuroimaging . J. Neurosci . 27 , 5474 –5483 10.1523/JNEUROSCI.0764-07.2007 17507569 Steinhauser M. Eichele H. Juvodden H. T. Huster R. J. Ullsperger M. Eichele T. (2012 ). Error-preceding brain activity reflects (mal-)adaptive adjustments of cognitive control: a modeling study . Front. Hum. Neurosci . 6 :97 10.3389/fnhum.2012.00097 22536179 Tamm L. Menon V. Ringel J. Reiss A. L. (2004 ). Event-related FMRI evidence of frontotemporal involvement in aberrant response inhibition and task switching in attention-deficit/hyperactivity disorder . J. Am. Acad. Child Adolesc. Psychiatry 43 , 1430 –1440 10.1097/01.chi.0000140452.51205.8d 15502603 Van Veen V. Cohen J. D. Botvinick M. M. Stenger V. A. Carter C. S. (2001 ). Anterior cingulate cortex, conflict monitoring, and levels of processing . Neuroimage 14 , 1302 –1308 10.1006/nimg.2001.0923 11707086 Vocat R. Pourtois G. Vuilleumier P. (2008 ). Unavoidable errors: a spatio-temporal analysis of time-course and neural sources of evoked potentials associated with error processing in a speeded task . Neuropsychologia 46 , 2545 –2555 10.1016/j.neuropsychologia.2008.04.006 18533202 Yazawa S. Ikeda A. Kunieda T. Ohara S. Mima T. Nagamine T. (2000 ). Human presupplementary motor area is active before voluntary movement: subdural recording of Bereitschaftspotential from medial frontal cortex . Exp. Brain Res . 131 , 165 –177 10766269	23761747	PMC3674319	CC BY	2021-01-04 22:48:44	yes	Front Hum Neurosci. 2013 Jun 6; 7:238
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23762395PONE-D-13-0287010.1371/journal.pone.0065556Research ArticleBiologyComputational BiologyMolecular GeneticsGene ExpressionGeneticsGene ExpressionMolecular Cell BiologyCell AdhesionCadherinsSignal TransductionNuclear Receptor SignalingSignaling PathwaysGene ExpressionMedicineObstetrics and GynecologyBreast CancerOncologyBasic Cancer ResearchMetastasisCancers and NeoplasmsBreast TumorsAIB1 Cooperates with ERα to Promote Epithelial Mesenchymal Transition in Breast Cancer through SNAI1 Activation AIB1 with ERα Promotes EMT via SNAI1Wang Miao Zhao Feng Li Shujing Chang Alan K. Jia Zhaojun Chen Yixuan Xu Feihong Pan Hongming Wu Huijian * School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China Müller Rolf Editor Philipps University, Germany * E-mail: wuhj@dlut.edu.cnCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: HW. Performed the experiments: MW. Analyzed the data: MW. Contributed reagents/materials/analysis tools: FZ SL AKC ZJ YC FX HP. Wrote the paper: HW. 2013 7 6 2013 8 6 e6555615 1 2013 25 4 2013 © 2013 Wang et al2013Wang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Epithelial Mesenchymal Transition (EMT) plays a major role in cancer metastasis. Several genes have been shown to play a role in EMT, and one of these is Amplified-in-breast cancer 1 (AIB1), which has oncogenic function and is known to be amplified in breast cancer. However, the role of AIB1 in EMT remains largely undefined at the molecular level. In this study, the effect of AIB1 overexpression on the EMT of the breast cancer cell line T47D was investigated. Overexpression of AIB1 disrupted the epithelial morphology of the cells. At the same time, the cells displayed a strong metastasis and reduced level of the epithelial marker E-cadherin. In contrast, knockdown of AIB1 in T47D cells increased cell-cell adhesion and produced weak metastasis, as well as a higher level of E-cadherin expression. We proposed that the regulation of EMT by AIB1 occurred through the action of the transcription factor SNAI1, and demonstrated that such interaction required the participation of ERα and the presence of ERα-binding site on SNAI1 promoter. The expression level of E-cadherin and the extent of cell migration and invasion in SNAI1-knocked down T47D cells that overexpressed AIB1 were similar to those of T47D cells that did not overexpress AIB1 and had no SNAI1 knockdown. Taken together, these results suggested that AIB1 exerted its effect on EMT through its interaction with ERα, which could directly bind to the ERα-binding site on the SNAI1 promoter, allowing the AIB1-ERα complex to promote the transcription of SNAI1 and eventually led to repression of E-cadherin expression, consistent with the loss of E-cadherin being a hallmark of EMT. This research was supported by grants (31171353, 31271500 to H.W.) from National Natural Science Foundation of China and grants (973 Program 2011CB504201 to H.W.) from the Ministry of Science and Technology of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Breast cancer is the most common malignancy among women, and it is the second leading cause of death among women having cancer [1]. Breast cancer mortality is largely attributed to metastasis. Despite its clinical relevance, research on the molecular mechanisms of epithelial mesenchymal transition (EMT) has not been extensively pursued, partly due to a lack of appropriate experimental models and difficulties in identifying metastasis-specific regulators and mediators [2]. EMT is considered as an important step in metastasis, during which non-motile, polarized epithelial cells dissolve their cell-cell junctions and convert into individual and motile mesenchymal cells [3], [4]. Cells undergoing EMT have several prominent features, including a change in cell morphology from round compact epithelial shape to spindle-scattered mesenchymal phenotype accompanied by the loss of E-cadherin [5], [6]. However, some cells undergoing partial EMT instead of complete EMT, and these cells retain some of the characteristics of epithelium and also display features of mesenchymal cells, a phenomenon which is recognized as partial EMT [7]. Several transcription factors are known to play a central role in the activation of EMT by acting as EMT inducers. These transcription factors include SNAI1 (Snail), SNAI2 (Slug) and ZEB1, all of which interact with the proximal E-boxes of E-cadherin promoter [8]. Therefore, understanding the regulation of these transcriptional factors would provide important insights into the molecular mechanisms implicated in breast tumor metastasis. Amplified-in-breast cancer 1 (AIB1 also known as SRC-3, ACTR, p/CIP, RAC3, TRAM1 and NCOA3) is a member of the p160 family, which also contains SRC-1 and SRC-2 [9], [10], [11], [12], [13], [14], [15], [16]. AIB1 is a transcriptional coactivator that promotes the transcriptional activity of multiple nuclear receptors such as estrogen receptors α (ERα) and other transcription factors such as Sp1, AP-1 and E2F1 [17], [18], [19]. ERα is a well-known estrogen (E2)-dependent receptor that plays a critical role in breast cancer development, and its functions are primarily mediated by AIB1 [9], [10], [13], [14]. Although the role of AIB1 in the proliferation of primary tumor in the mammary gland is well established, a role for this oncogenic coregulator in tumor cell motility and metastasis has been elucidated only recently. In the nucleus, AIB1 is essential for proteolytic breakdown of the extracellular matrix by matrix-metalloproteinases, a process which enables primary tumor cells to invade the surrounding stroma [20], [21]. At the plasma membrane, an exon-4-truncated isoform of AIB1 (AIB1Δ4) lacking the N-terminal bHLH domain (which contains a nuclear localization signal) serves as a signaling adaptor for the epidermal growth factor, focal adhesion kinase and c-Src signal transduction pathway, all of which are implicated in metastasis and invasion [22]. Together, these studies underscore a pivotal role of AIB1 not only as a proto-oncogene, but also as a prometastatic factor during the early stages of metastasis [23]. However, the contribution of AIB1 in the regulation of tumor metastasis and the underlying mechanism of this process is largely unknown. We have previously shown that the AIB1 is required for breast cancer cell proliferation and demonstrated that its transcriptional activity is up-regulated by phosphorylation and down-regulated by sumoylation, and identified PIAS1 as the SUMO E3 ligase that can enhance the sumoylation of AIB1, thereby down-regulating AIB1 transcriptional activity [24], [25]. In this study, we used a combined molecular and cellular approach to characterize the role of AIB1 in EMT. We showed that cooperation between AIB1 and ERα raised SNAI1 expression and repressed E-cadherin transcriptional activation, resulting in the promotion of EMT in breast cancer cells. Results AIB1 is Associated with Cell-cell Adhesion in Breast Cancer Cells Elevated level of AIB1 is frequently associated with distant metastasis, high tumor grade and poor prognosis, especially for breast tumor. The morphologies of three estrogen receptor alpha-positive (ERα+) human breast cancer cell lines (ZR-75-1, MCF-7 and T47D) with different invasive capabilities were analyzed and compared. The order of increasing invasiveness for the three cell lines was ZR-75-1<MCF-7<T47D. Thus T47D cells showed the least cell-cell contact compared to the other two cell lines, whereas ZR-75-1 cells exhibited the most compact colonies (Fig. 1A). Analysis of the protein levels of AIB1 and ERα in these cell lines showed an increased level of AIB1 in T47D cells, but no obvious change in the level of ERα was detected among the three cell lines (Fig. 1B). This implied that AIB1, which is often highly expressed in breast cancer cells, might play a role in controlling the morphological characteristics of these cells well as the extent of their cell-cell contact. 10.1371/journal.pone.0065556.g001Figure 1 AIB1 regulates the morphologies of breast cancer cells. (A) Light microscopic images showing the morphology of different breast cancer cells. (B) Expression levels of AIB1, ERα and E-cadherin in different breast cancer cells as detected by western blot analysis. Cell extracts were prepared from the different cell lines and probed with specific antibody against AIB1, ERα and E-cadherin or β-actin. (C) Light microscopic images showing EMT morphological changes induced in T47D cells after treatment with EGF (50 ng/ml) or E2 (20 nM) for 24 h. (D) Western blot analysis showing the levels of AIB1, ERα, E-cadherin and β-actin expressions in T47D cells after treatment with EGF or E2. (E) Coimmunoprecipitation showing AIB1 and ERα complex increased after treatment with EGF or E2. T47D cells treated without or with EGF (50 ng/ml) or E2 (20 nM) for 24 h were subjected to immunoprecipitation with anti-AIB1 or control IgG antibodies, followed by western blot analysis with anti-ERα and anti-AIB1 antibodies. (F) Light microscopic images showing T47D cells without or with AIB1 knockdown (shAIB1#1 and shAIB1#2), ERα knockdown (shERα) or both AIB1 and ERα knockdown. Cells were treated with the corresponding iRNA and then plated out in 50-mm dishes and incubated for 3 days before observing. (G) The cells from (F) were counted and plotted as percentage of clustered or scattered cells relative to total number of cells (400–500). (H) Western blot analysis showing the levels of AIB1, ERα, E-cadherin and β-actin proteins in T47D cells from (F). Previous study has shown that EGF signal pathways can reduce cell-cell contact and induce metastasis [26]. Treatment of T47D cells with EGF leads to activation of MAPK pathway, and the resulting phosphorylation of MAPK enhances the transcriptional activity of AIB1 [27]. We also observed changes in the morphology of T47D cells after treatment with EGF. The cells became scattered, with loss of close cell-cell junctions and formation of spindle-shaped appearance, which are morphological changes that are reminiscent of the cells belonging to the mesenchymal lineage (Fig. 1C). In addition, these cells also showed reduced expression level of the critical epithelial protein E-cadherin (Fig. 1D). Treatment of T47D cells with E2 also induced mesenchymal morphology and reduced cell-cell contact. Most importantly, no significant upregulation of AIB1 and ERα expressions was observed when these cells were treated with EGF or E2 (Fig. 1D). Our previously study has shown that treatment of MCF-7 cells with E2 resulted in increased levels of phosphorylated AIB1 and enhancement of AIB1 transcriptional activity [24]. The interaction between AIB1 and ERα in T47D cells was demonstrated by coimmunoprecipitation experiments, and the result showed that formation of AIB1 and ERα complex increased almost two folds in EGF- or E2-treated cells compared to non-treated cells (Fig. 1E). This implied that AIB1 and ERα both might play a role in the loss of cell-cell contact and in the process that would ultimately cause the cells to become more scattered. The effect of AIB1 or ERα on cell-cell contact was investigated by knocking down AIB1 or ERα or both AIB1 and ERα in T47D cells, and comparing the extent of cell-cell contact in these cells. Two shAIB1 sequences, shAIB1#1 and shAIB#2, were used to knock down AIB1 in the cells. Knocking down AIB1 with shAIB1#1 increased the overall proportion of clustered cells by 15%, whereas knocking down AIB1 with shAIB#2 increased it by 10%, relative to control cells. Knocking down ERα resulted in just 5% increase in the overall proportion of clustered cells (Figs. 1F&1G). Knocking down both AIB1 and ERα yielded the highest level of cell-cell contact, and increased the overall proportion of clustered cells by about 35% relative to control cells (Figs. 1F&1G). Knockdown of AIB1 increased the level of E-cadherin, a protein that is important for cell adhesion. Knockdown of AIB1 by shAIB1#1 increased the level of E-cadherin to more than two fold the level of control cells, whereas knockdown of AIB1 by shAIB1#2 increased the level of E-cadherin to about 1.5 fold the level of control cells. Since shAIB1#1 was more effective than shAIB1#2 at knocking down AIB1, all subsequent AIB1-knockdown experiments were conducted with shAIB1#1. Knockdown of ERα also caused some increase in the level of E-cadherin, but knockdown of both AIB1 and ERα caused the highest increase, almost three fold the level of control (Fig. 1H). These data implied that the role of AIB1 in cell-cell adhesion could in part be due to increases in the level of AIB1-ERα complex. Since AIB1 knockdown caused more disruption to cell-cell contact and higher increase in E-cadherin expression compared to ERα knockdown, part of the effect exerted by AIB1 on cell-cell contact in breast cancer cells might not be associated with ERα, the nature of which requires further investigation. AIB1 is Important in Breast Cancer Cells Motility and Invasion Given that AIB1 could reduce cell-cell adhesion (Fig. 1 data), it then became important to know the relationship between AIB1 and cell motility and invasion in these tumor cells. AIB1 was therefore overexpressed in T47D cells and the effect its overexpression had on cell motility and invasion was determined by wound healing and transwell assays. Cells that overexpressed AIB1 showed faster wound healing with 50% more cells migrating through the uncoated membrane in the chamber compared to control cells that did not overexpress AIB1 (Fig. 2A). In addition, the migration of these cells through the Matrigel coated membrane (invasion assay) increased to 2.5 fold the number of control cells (Fig. 2B). Knockdown of AIB1 in T47D cells weakened the motility and invasiveness of the cells, as shown by wound healing and transwell migration and invasion assays (Fig. 2B). In order to confirm that the higher numbers of migrating and invading cells observed for cells that over expressed AIB1 were not a result of increase in cell proliferation caused by AIB1, we carried out MTT assay to compare differences in cell proliferation among control cells, T47D cells that over expressed AIB1 and those that had AIB1 knockdown. No significant differences in cell proliferation were observed among these different groups and no significant increases were detected at 24 h (Fig. 2C). This showed that the effect of AIB1 on cell proliferation within a 24-h period was insignificant, and therefore faster recovery of the wound achieved by cells that over expressed AIB1 and the stronger migration displayed by these cells compared to AIB1 knockdown cells were not due to cell proliferation caused by AIB1. This suggested that AIB1 might play an important role in tumor cell motility and invasion, and that this role is not contributed by its effect on cell proliferation. 10.1371/journal.pone.0065556.g002Figure 2 AIB1 promotes cell motility and invasion in T47D cells. (A) Scratch wound-healing assay showing the effect of AIB1 on cell motility in T47D cells. Top panel: Images of T47D cells that were transfected with empty vector (pcDNA) or AIB1, and cells without (NC) or with AIB1 knockdown (shAIB1#1) before and after wound-healing assay. The cell layers were carefully wounded using a sterile 200-µl tip and then cultured for 24 h before evaluation. Bottom panel: Western blot analysis showing the levels of AIB1, ERα and β-actin expressions in pcDNA- or AIB1-transfected T47D cells, and of T47D cells without or with AIB1 knockdown. (B) Transwell migration and invasion assays showing the effect of AIB1 on cell motility and invasion ability in T47D cells. Images showing the migration and invasion of T47D cells that were transfected with empty vector (pcDNA) or AIB1, and of T47D cells without (NC) or with AIB1 knockdown (shAIB1#1). For NC group, the cells were transfected with a negative control scrambled shRNA synthesis DNA cloned into siRNA expression vector pRNAT carries GFP marker. Cell migration and invasion assays were performed in 24-well chambers without and with Matrigel, respectively. Cells (1000 per well) were transfected with GFP-AIB1 or just GFP (pEGFPC) and then transferred to the upper chamber. After 48 h of incubation, the numbers of migrating and invasive cells on the lower surface of the filter were counted under a fluorescent microscope. The bar graphs on the right of the images show the number of migrating and invading cells for each category of cells. (C) MTT assays showing the effect of AIB1 on cell proliferation in T47D cells. Cells were transfected as in (A) and then subjected to MTT assay within 24 hours. AIB1 Promotes EMT via Reduced Expression of E-cadherin Increased motility and invasiveness shown by tumor cells are reminiscent of the events that occur during epithelial mesenchymal transition (EMT), and loss of E-cadherin expression is an essential event in EMT [26]. AIB1 might regulate cell motility and invasion through targeting the expression of E-cadherin, either at the protein or mRNA level or both. T47D cells that overexpressed AIB1 showed 50% reduction in the level of E-cadherin protein and 30% increase in the level of N-cadherin protein (a mesenchymal protein which is also important for EMT) compared to control cells (as determined by western blot), and similar results were also observed for MCF-7 cells that overexpressed AIB1 (Fig. 3A ). Knocking down AIB1 in these cells resulted in the opposite effect. In the case of T47D cells, AIB1-knockdown resulted in about 50% increase in the level of E-cadherin and about 60% decrease in the level of N-cadherin compared to control cells (Fig. 3A). In the case of MCF-7 cells, knockdown of AIB1 produced very similar result as in T47D cells. A decreased level of E-cadherin and increased level of N-cadherin in both T47D and MCF-7 cells caused by AIB1 overexpression led to induction of mesenchymal cells morphology, as shown by immunofluorescence staining (Fig. 3C). Change in the level of E-cadherin mRNA paralleled with the change in level of E-cadherin protein, with about 40% less in cells that overexpressed AIB1 compared to control cells, as shown by real time PCR (Fig. 3D). However, reporter gene activity assay showed a much higher decrease in E-cadherin reporter activity in cells that over expressed AIB1 relative to control cells (Fig. 3E). At the same time, the level of E-cadherin mRNA in these cells also increased by about 30% (Fig. 3D), while the level of E-cadherin reporter activity increased almost by 100% over control cells (Fig. 3E). These data confirmed that the main effect AIB1 had on E-cadherin was on its gene transcription. 10.1371/journal.pone.0065556.g003Figure 3 AIB1 regulates E-cadherin expression through E-box-dependent transcription. (A) Western blot analysis of E-cadherin and N-cadherin levels in T47D and MCF-7 cells that overexpressed AIB1 or had AIB1 knockdown. Cells were transfected with either empty vector (pcDNA) or AIB1, or with the negative control scrambled shRNA (NC) or shAIB1#1 (in the case of AIB1 knockdown). Western blot analysis was carried out using cell extract and antibody against AIB1, E-cadherin, N-cadherin or β-actin. (B) Immunofluorescence study showing the regulation of AIB1 on the expression of E-cadherin and N-cadherin. Cells overexpressed AIB1 and empty vector or AIB1 knockdown and negative control scrambled shRNA (NC) were cultured in the chamber slide and fixed, immunostained with anti-E-cadherin and anti-N-cadherin antibodies followed by the secondary antibody, Texas-red Fluor 589 anti-Rabbit. Nuclear protein was stained with DAPI. (C) Fluorescence microscopic images showing the morphology of T47D and MCF-7 cells that overexpressed AIB1 and the empty vector pEGFPC1. (D) RT-PCR analysis showing the regulation of AIB1 on the transcription of E-cadherin. The mRNA level of E-cadherin was expressed relative to GAPDH transcript level. (E) AIB1-regulated E-cadherin luciferase reporter activity. T47D cells were co-transfected with the E-cadherin-Luc reporter vector (pGL3-Ecad) and AIB1 or empty vector. Cells were harvested 48 h after transfection and subjected to luciferase activity assay. (F) Dependence of AIB1-suppressed E-cadherin transcription on E-box as shown by luciferase reporter activity. T47D cells were cotransfected with empty vector (pcDNA) or vector plus AIB1 insert (pcDNA-AIB1) together with the E-box wild-type (pGL3-Ecad) or E-box mutant (pGL3-Ecad-mut) E-cadherin-Luc reporter. Cells were harvested 48 h after transfection and subjected to luciferase activity. Relative luciferase activity was normalized to β-galactosidase activity, used as control to monitor the transfection efficiency. Each experiment was performed in triplicates and repeated at least three times. Data are the means ± SDs. Statistically significant differences (P<0.05) in paired Student’s t-test are marked with an asterisk. Repression of E-cadherin transcription by AIB1 might be dependent on the presence of E-box in the promoter of E-cadherin. This was investigated by comparing the luciferase activity of T47D cells that over expressed AIB1 and E-cadherin-promoter-driven luciferase, in which the E-cadherin promoter contained wild-type E-box, and comparing it to the luciferase activity of T47D cells that overexpressed AIB1 but luciferase driven by E-cadherin promoter containing mutant box. The result showed that the level of luciferase activity obtained from wild-type E-cadherin promoter was only 25% the level obtained from the mutant E-cadherin promoter (Fig. 3F). In the case of wild-type E-cadherin promoter, overexpression of AIB1 also led to some but significant increase in luciferase activity compared to control cells (not overexpressing AIB1), whereas in the case of mutant E-cadherin promoter, no difference in luciferase activity was detected between cells that overexpressed AIB1 and control cells. Thus repression of E-cadherin expression by AIB1 required a functional E-box in the E-cadherin promoter. AIB1 Potentiates ERα-mediated SNAI1 Expression EMT repressors such as SNAI1, SNAI2 and ZEB1 are implicated in E-box-dependent transcriptional repression of E-cadherin [28], and since AIB1 could repress E-cadherin expression it may achieve this through interacting with these transcription factors. Analysis of the levels of SNA1 and ZEB1 mRNAs in T47D cells that overexpressed AIB1 showed that only the level of SNAI1 mRNA was increased, reaching 2.5 fold the level of control cells, but the level of SNAI1 mRNA in AIB1-knockdown cells was reduced by 40% compared to control cells (Fig. 4A). Increases in SNAI1-promoter-driven luciferase activity in T47D cells that overexpressed AIB1 also occurred, and in a dose-dependent manner (Fig. 4B). These results indicated that AIB1 regulated SNAI1 expression at the transcriptional level. 10.1371/journal.pone.0065556.g004Figure 4 AIB1 induces SNAI1 expression. (A) RT-PCR analysis showing the regulation of the expression of EMT-inducing transcription factor by AIB1. The mRNA levels of three EMT-inducing transcription factors, SNAI1, SNAI2 and ZEB1, in T47D cells without (pcDNA) or with AIB1 overexpression (AIB1) or without (NC) and with AIB1 knockdown (shAIB1#1) were measured by RT-PCR. The mRNA levels of SNAI1, SNAI2 and ZEB1 are expressed relative to GAPDH transcripts. (B) AIB1-regulated SNAI1 luciferase reporter activity. T47D cells were cotransfected with SNAI1-Luc and different amounts (0.1–0.5 ng) of pcDNA-AIB1 construct. Luciferase activity level in cells transfected with the empty vector pcDNA was set to 1. (C) Regulation of the activity of SNAI1 promoter by different transcription factors without and with AIB1 overexpression. T47D cells were transfected with SNAI1-Luc reporter construct and the transcription factor Sp1, ERα, E2F or Jun-D construct with or without AIB1 construct. (D) Regulation of the activity of SNAI1 promoter by nuclear receptors without and with AIB1 overexpression. T47D and HEK293T cells were transfected with SNAI1-Luc reporter vector with ERα or AR construct with and without AIB1 construct. (E) Effect of hormone and nuclear receptor inhibitor on AIB1-regulated SNAI1 activity. T47D cells were transfected with SNAI1-Luc reporter construct with or without AIB1 knockdown, and cells were then treated with 20 nM E2 or 10 nM ICI (inhibitor of estrogen receptor) or without treatment (control) for 12 h. The levels of luciferase were normalized to β-galactosidase activity, used to evaluate transfection efficiency. Each experiment was performed in triplicates and repeated at least three times. Data are the means ± SDs. Statistically significant differences (P<0.05) in paired Student’s t-test are marked with an asterisk. The 2-kb SNAI1 promoter region used to drive the luciferase activity (SNAI-luc construct) contains binding sites for ERα, Sp1, Jun-D, E2F as predicted by TESS: Transcription Element Search System. Some of these transcription factors have been shown to regulate SNAI1 promoter activity [7]. In order to identify which of these transcription factors might interact with AIB1, T47D cells were transfected with SNAI1-luc reporter, AIB1 and either Sp1, ERα, Jun-D or E2F. The levels of SNAI1-driven luciferase activity in cells that overexpressed Sp1, Jun-D or E2F along with AIB1 did not increase compared to cells that did not overexpress AIB1 (Fig. 4C), suggesting that AIB1 did not play a role in the regulation of SNAI1 expression by these transcription factors. In contrast, the level of luciferase activity in cells that overexpressed both ERα and AIB1 increased to more than 2-fold the level of cells that overexpressed ERα only (Fig. 4C), indicating that the combined action of ERα and AIB1 could promote the activity of SNAI1 promoter further. In addition, T47D cells that overexpressed AR (androgen receptor, which is another NR family member) together with AIB1 had higher level (40% more) of SNAI1-drive luciferase activity compared to cells that overexpressed AR only (Fig. 4D). Comparable increases in SNAI1-drivern luciferase activity were observed for HEK293T cells that overexpressed AIB1 and AR versus those that overexpressed AR only, indicating that increase in the level of SNAI1 promoter activity was not affected by the high level of endogeneous ERα as in the case of T47D cells, since HEK293T cells do not have a high level of endogenous ERα compared to T47D cells. T47D cells in which AIB1 was knocked down showed reduced SNAI1-driven luciferase activity compared to control cells (no AIB1 knockdown). However, compared to untreated cells, cells treated with E2 exhibited no change in the level of luciferase activity, but cells treated with the ERα inhibitor, ICI, exhibited almost 50% reduction in luciferase activity (Fig. 4E). This tends to suggest that ERα might regulate SNAI1 activity through coorperation with AIB1 as well as independent of AIB1. When the endogenous AIB1 of the cells was retained, treatment of the cells with E2 caused some increase in SNAI1-driven luciferase activity when the cells were treated with E2, while treatment of the cells with ICI caused some decrease in luciferase activity, but both were not significant (Fig. 4E). Thus much of the activity of SNAI1 induced by AIB1 did not appear to be contributed by the co-action of ERα, and hence E2 responsive, since the inhibition of ERα by ICI only caused slight reduction in SNAI1 activity. AIB1 Cooperates with ERα to Activate SNAI1 Transcription The relevant section of the SNAI1 promoter showing the locations of ERα-binding sites and E-boxes is schematically shown in Figure 5A. To obtain further information regarding the regulation of SNAI1 promoter activity by AIB1 and ERα we used ChIP assay to analyze the region of the SNAI1 promoter that interacted with AIB1-ERα. The results revealed that AIB1 and ERα specifically associated with regions A but not with region B or C (Fig. 5B). The 2-kb SNAI1 promoter region contained multiple ERα-binding sites and E-Boxes, and three primer pairs were designed to amplify regions represented by A, B and C along the promoter as depicted in Fig. 5A. To further examine the effect that each of the regions (A–C) has on SNAI1 activity, three different truncated forms of the promoter (Fig. 5A) were constructed and each was fused to a luciferase gene to yield a reporter construct. From the ChIP assay data, it could be inferred that among the three truncated SNAI1 promoters, AIB1 specifically associated with SNAI1-a(−1061/+108), which contained regions A, B and C, and therefore all the ERα-binding sites and E-Boxes. AIB1 did not associated with SNAI1-b(−497/+108), which contained only regions B and C, or with SNAI1-c(−227/+108), which contained only region C. This suggested that AIB1 was recruited to the ERα-binding sites and E-Boxes within region A of the SNAI1 promoter (Fig. 5A), and this could be the region where ERα would actually bind to and activate SNAI1 transcription. This was subsequently confirmed by reporter gene assay, in which T47D cells transfected with the luc gene fused to SNAI1-a exhibited significant increase in luciferase activity when the cells over expressed either ERα, AIB1 or ERα and AIB1 compared to control cells (transfected with SNAI1-a-luc and pcDNA only) (Fig. 5C). Increases in luciferase activity over control cells were about three fold, five fold and seven folds, respectively, for cells that overexpressed ERα, AIB1, and both ERα and AIB1. No significant increase in luciferase activity was observed for cells that overexpressed AIB1 or ERα alone or together compared to control cells when the luc gene was fused to SNAI1-b or SNAI1-c. However, within the control cells, the level of luciferase activity was highest when the luc gene was fused to SNAI1-c, being almost three fold the level exhibited by SNAI1-a, and four fold the level of SNAI1-b. This suggested that SNAI1-b might contain only elements that are associated with the suppression of its activity whereas SNAI1-c probably contained no regulatory element, and the reporter activity observed was a result of unregulated threshold expression. Taken together, these results indicated that AIB1 promoted ERα-medicated SNAI1 transcription mainly via the region A of the SNAI1 promoter, which contained the first groups of ERα-binding sites. 10.1371/journal.pone.0065556.g005Figure 5 AIB1 regulates ERα-mediated SNAI1 expression. (A) Schematic illustration of ERα-binding elements in SNAI1 promoter. Fragments A to C were chosen for PCR amplification in ChIP assays. Three truncated versions of the SNAI1 promoter were made, and the length of each is shown in the illustration. (B) ChIP assays showing AIB1- and ERα-SNAI1 promoter interaction in T47D cells. Cross-linked chromatin was extracted from T47D cells and subjected to immunoprecipitation with anti-AIB1, anti-ERα or control IgG, and the resulting precipitated DNA was used as template for PCR-ampαlification of SNAI1 promoter using specific primer covering region A, B or C of the promoter region. (C) Reporter gene assays of different truncated versions of SNAI1 promoter in the presence of AIB1 or ERαoverexpression. Each of the truncated SNAI1 promoters was fused to luciferase gene in pGL3 and the resulting construct was introduced into T47D cells along with AIB1 or ERα construct or both. The levels of luciferase activity in these cells were determined 48 h after transfection. Luciferase activity was normalized to β-galactosidase activity, used to evaluate transfection efficiency. Each experiment was performed in triplicates and repeated at least of three times. Data are the means ± SDs. Statistically significant differences (P<0.05) in paired Student’s t-test are marked with an asterisk. SNAI1 Mediates the Role of AIB1 in Promoting Breast Cancer Cell EMT The data obtained from the preceding experiments suggested that in breast cancer cells AIB1 may suppress E-cadherin expression and promote EMT through upregulation of SNAI1. For T47D cells overexpressing AIB1, the level of SNAI1 expression was markedly reduced, at least by more than 50% (at both mRNA and protein levels) in cells with SNAI1 knockdown compared to control cells (Fig. 6A). The higher level of SNAI1 expression caused by overexpression of AIB1 was also supported by the lower SNAI1 expression in T47D cells that did not overexpress AIB1, but without SNAI1 knockdown. As for E-cadherin, the transcript and protein levels were both reduced by more than 50% in SNAI1-knocked down T47D cells that overexpressed AIB1, compared to cells that did not overexpress AIB1, with or without SNAI1 knockdown, and this reaffirmed that the difference in E-cadherin expression was caused by AIB1 (Figs. 6A&6B). These data again demonstrated that AIB1-induced EMT was dependent on SNAI1 activation, which also affected E-cadherin expression. 10.1371/journal.pone.0065556.g006Figure 6 SNAI1 mediates the role of AIB1 in breast cancer cell EMT. (A) Western blot analysis showing the reversion of repressed E-cadherin expression in SNAI1-knockdown cells by overexpression of AIB1. T47D cells without or with SNAI1 knockdown were transfected with AIB1 and the levels of SNAI1, E-cadherin and β-actin in the cells were analyzed by western blot using the corresponding antibodies. For comparison, the levels of these proteins in T47D cells without SNAI1 knockdown and AIB1 overexpression were also analyzed. (B) RT-PCR analysis showing the level of SNAI1 or E-cadherin transcript in the different groups of cells in A. The mRNA levels of SNAI1 and E-cadherin are expressed relative to GAPDH transcripts. (C) Effect of SNAI1 knockdown on cell motility in T47D cells. The cell motility of T47D cells without or with SNAI1 knockdown that over expressed AIB1 were evaluated by scratch wound healing assay. The motility of cells without SNAI1 knockdown and AIB1 overexpression was also evaluated for comparison purpose. (D) Effect of SNAI1 knockdown on cell migration and invasion abilities of T47D cells. T47D cells without or with SNAI1 knockdown that over expressed AIB1 were subjected to transwell migration and invasion assays. Cells that migrated through the uncoated filter or invaded the Matrigel-coated filters of the chamber were detected by fluorescence imaging. The graph shows the actual number of migrated and invaded cells for each group. In addition, scratch wound healing and transwell assays demonstrated that T47D cells with SNAI1 knockdown that overexpressed AIB1 showed reduced cell motility and invasion compared to cells without SNAI1 knockdown, but did not overexpress of AIB1 (Figs. 6C& 6D). The levels of cell motility and invasion exhibited by these cells were similar to cells without SNAI1 knockdown and AIB1 overexpression, suggesting that as in AIB1-induced EMT, which depended on SNAI1 activation, AIB1-induced cell motility and invasion also depended on SNAI1 activation. The relevance of our findings to human breast cancer was validated by analyzing the levels of AIB1, SNAI1 and E-cadherin proteins in the invasive front of human ERα-positive breast tumor tissues. AIB1 protein level was aberrantly upregulated in invasive tumor cells, whereas SNAI1 protein level was moderately upregulated and E-cadherin protein level was downregulated in these cells (Fig. 7A). Significant correlation was observed between AIB1 and SNAI1 as well as between AIB1 and E-cadherin when the levels of these proteins in 31 ERα-positive-primary invasive breast tumor samples were compared. Although only 58% of the samples displayed high level of AIB1, 72% of these also displayed high level of SNAI1 with no detectable E-cadherin expression (Fig. 7B), which is in agreement with our speculation that AIB1 synergistically induced SNAI1 expression and E-cadherin repression, resulting in induction of EMT in the progression of breast cancer. 10.1371/journal.pone.0065556.g007Figure 7 Correlation between AIB1/SNAI1 expressions and E-cadherin expression in human breast tissue samples. (A) Representative results of immunohistochemistry of AIB1, SNAI1 and E-cadherin in serial sections of primary tumor and invasive tumor tissues (n = 31) respectively. Each sample was incubated with antibody against ERα, AIB1, SNAI1 or E-cadherin. Positive staining and negative staining are indicated by brown and blue staining, respectively (×400 Magnification). (B) Association between AIB1/SNAI1 expressions and E-cadherin proteins in ERα+ breast cancer tissue samples. Fisher exact test, P<0.0001. Discussion AIB1 belongs to the p160 family of transcriptional coregulators, and it interacts with nuclear receptors ERα and other specific transcription factors, forming complexes that will recruit chromatin remodeling and other transcriptional proteins to facilitate the assembly of general transcription factors, eventually leading to the transcriptional activation of many genes [15]. Moreover, high levels of AIB1 are also associated with poor prognosis in breast cancer. Although AIB1 has multiple functions, a role of AIB1 in the onset of distant metastasis is still unclear. In this study, we found that AIB1 could control the morphological characteristics of a cell and cell-cell contact. Our results showed that knockdown of AIB1 in T47D cells increased cell-cell adhesion and induced epithelial-type morphology. Furthermore, cells treated with EGF or E2 showed a scattered distribution, and although the expression of AIB1 and ERα did not show any significant upregulation, more AIB1 existed in the form of complex with ERα (Figs. 1D&E), and AIB1 deficiency attenuated E2 signaling by reducing the ERα-activated SNAI1 promoter activity (Fig. 4E). Overexpression of AIB1 in T47D cells promoted significant cell motility and invasion, but had only a slight effect on cell proliferation within 24 h (Fig. 2). Although we have shown here the importance of AIB1 overexpression and its subsequent effect on SNAI11/E-cadherin expressions, which ultimately impacted on cell motility and migration, we cannot rule out other factors that may also contribute to these two aspects and at the same time, may also be connected to AIB1 overexpression. For example, AIB1 overexpression has been shown to increase the level of IGF (insulin-like growth factor) and production of other cytokines that promote migration and metastasis [20], [29]. AIB1 amplification is known to be associated with breast tumors, especially for ERα-positive breast cancers [30], and ERα-regulated gene expression is in part dependent on the recruitment of coregulators such as AIB1 [27], [31]. Thus aberrant expression of AIB1 in a cell is likely to play a major role in controlling the morphological characteristics of the cell. Tumor cell EMT is associated with increased cell migration, invasion, and metastasis. The well-established hallmark of EMT is the loss of E-cadherin, which disrupts the stable cell-cell adhesion between epithelial cells. Apart from E-cadherin, there is so far no other EMT inducer that has been reported. Our data showed that AIB1 expression inversely correlated with E-cadherin expression, suggesting that AIB1 may promote EMT through inhibition of E-cadherin expression, probably through interacting with the whole E-box in the proximal promoter. If transcription of E-cadherin is blocked, adhesion molecules such as N-cadherin, which has E-box in the intron, are induced during metastatic progression. In our study, the expression of N-cadherin was also increased in cells (MCF-7 and T47D) that overexpressed AIB1 (Figs. 3A&B). Expression of N-cadherin has been shown to be induced by TWIST in prostate cancer cell [32], although TWIST mRNA level in T47D cells was not affected by overexpression of AIB1 and ERα (data not shown). This may imply that AIB1 might regulate N-cadherin expression through other proteins or transcription factors, and this will be a subject of future investigation. The expression of E-cadherin is subject to regulation by Snail, which functions as a transcriptional repressor, and is the most widely studied effector of EMT and E-cadherin expression. AIB1 could regulate the expression of SNAI1, but not of SNAI2 and ZEB1, probably through the ERα-binding sites located at −1061 to −790 bp of the SNAI1 promoter (Figs. 5B&C), where the ERα-AIB1 complex could bind, leading to subsequent activation of gene expression. Interestingly, the loss of these ERα-binding sites and presumably the E-box at −594 bp (SNAI1b, Fig. 5B) resulted in inhibition of SNAI1-reporter activity, whereas additional truncation that resulted in the loss of ERα-binding sites at −594 to −388 bp (SNAI1c, Fig. 5B) resulted in increased SNAI1-reporter activity. This seemed to suggest that the sequence within the −594 bp region of SNAI1 promoter may contain binding sites for repressors, and loss of this region would result in deregulation of SNAI1 expression. Conceivably, AIB1 may also enhance SNAI1 expression through other transcription factors and future investigation is required to identify these unknown transcription factors. As for the lack of change in reporter activity observed when AIB1 was coexpressed with SNAI2-luciferase or ZEB1-luciferase in T47D cells, we could only speculate that AIB1 either did not interact with these genes in the regulation of E-cadherin expression or that additional accessory proteins or transcriptional factors are needed, which also need to be overexpressed to produce noticeable changes in the corresponding luciferase activity. Besides inhibiting the expression of E-cadherin (Figs. 6A&B), SNAI1 also been shown to inhibit the expression of epithelium-specific genes such as PTEN, Muc1, and some nuclear factor receptors [33], and in addition to its association with tumor metastasis, SNAI1 is also associated with other cancer hallmarks such as p53. Finally, this study addressed what effect SNAI1 may have on the mammary tumor cells by acting as a target gene of AIB1. Knockdown of SNAI1 in AIB1-overexpressing T47D cells increased E-cadherin expression and decreased cell invasion capability. This appeared to suggest that upregulated SNAI1 expression was the major effect exerted by AIB1 on SNAI1 in controlling cell morphology and invasiveness. Aberrant AIB1 expression followed by SNAI1 activation and repression of E-cadherin were clearly detected at the tumor invasive front in invasive breast cancer tissue (Fig. 7A). A positive connection between AIB1 and SNAI1 expressions in breast cancer was detected since 72% of the samples with high level of AIB1 also had high level of SNAI1, although 9.6% of the samples with low AIB1 expression also had high level of SNAI1 (Fig. 7B). Furthermore, samples that showed high levels of AIB1 and SNAI1 expression also had no detectable level of E-cadherin. From a clinical perspective, since AIB1 can regulate SNAI1/E-cadherin expression, which is an important factor in tumor invasiveness, AIB1 could therefore be considered as potential marker for detecting the malignancy likelihood of breast tumor. Short-term expression of either AIB1 or SNAI1 at moderate levels was not sufficient to induce complete EMT of human breast tumor cell line T47D cultured in vitro. This could be due to the lack of in vivo tumor progression environment or insufficient time for completing EMT. Furthermore, in addition to AIB1, several other coactivators have been implicated in breast cancer metastasis. For example, AIB1 can increase the function of Ets (E-twenty six) family transcription factors PEA3, AP-1 and E2F1, the activity of the IGF1 signaling pathway, epidermal growth factor (EGFR) and ERBB2, and the expression of MMPs to promote breast-tumor cell proliferation, migration, invasion and metastasis [20], [23]. Targeting AIB1 may help to reduce the extent of tumor metastasis, but since AIB1 is merely one of the genes that is associated with tumor development and progression, further study into the mechanism of AIB1-SNAI1 interaction and the identification of genes whose products are directly involved in cell motility and invasiveness will shed more light on tumor proliferation and metastasis. Materials and Methods Cell Culture and Experimental Reagents Human breast cancer cell line ZR-75-1, MCF7 and T47D have been used in our previous study [24], [34], [35]. ZR-75-1 and MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Hyclone, Logan, UT), whereas T47D cell cells were cultured in RPMI-1640 Medium containing 0.2 Units/ml bovine insulin, 10% fetal bovine serum, 100 µg/ml penicillin and 100 µg/ml streptomycin at 37°C in the presence of 5% CO2. Anti-AIB1, anti-SNAI1, anti-N-cadherin, anti-β-actin and anti-ERα antibodies were obtained from Santa Cruz (Santa Cruz, CA). Anti-E-cadherin antibody was obtained from Cell Signaling (Boston, MA). EGF was obtained from Peprotech (Rehovot, Israel), and 17β-estradiol was obtained from Abcam (Cambridge, UK). Plasmid Construction Human SNAI1 (Gene ID 6615) was amplified from a human cDNA library by RT-PCR and then cloned into the expression vector pEGFPC1. Human AIB1 was obtained from Dr. Anna T. Riegel (Georgetown University Medical Center, USA). The promoter regions of human SNAI1 and E-cadherin were amplified from human genomic DNA by PCR and each was separately cloned into the plasmid pGL3. Construct naming was based on the region of the promoter that was covered. Four truncated versions of SNAI1 promoter were constructed, and each was fused to a luciferase reporter gene. These were SNAI1-Luc (amplified by sense primer, 5′-GGGGTACCGGAAAGGTCTGGGATGT-3′ and antisense primer, 5′- CCGCTCGAGCCTGACGAGGAAAGAGC-3′); SNAI1-1061/+108 (amplified by sense primer, 5′-GGGGTACCTAACCAGGTCCCTCCTCA-3′, and antisense primer, 5′-CCGCTCGAGCCTGACGAGGAAAGAGC-3′); SNAI1-497/+108 (amplified by sense primer, 5′-GGGGTACCCCAGTGATGTGCGTTTC-3′, and antisense 5′-CCGCTCGAGCCTGACGAGGAAAGAGC-3′); and SNAI1-227/+108 (amplified by sense primer, 5′-GGGGGTACCGCGCTGCGCCAGCG-3′ and antisense primer, 5′-CCGCTCGAGCCTGACGAGGAAAGAGC-3′). E-cadherin-luc was amplified by sense primer, 5′-GGGGTACCCGAGGCAGAGTGCAGTGGCTC-3′, and antisense primer, 5′-CCGCTCGAGTGAAC TGACTTCCGCAAGCTC-3′. Luciferase Reporter Assay Promoter activity was determined by a luciferase assay system, 24 h after transfection. For some experiments, T47D cells were transfected with SNAI1-promoter or E-cadherin promoter-luciferase construct and without or with AIB1, and luciferase activity of the cells was assayed 48 h after transfection. Luciferase activity was measured using Centro LB 960 Microplate Luminometer (BERTHOLD TECHNOLOGIES GmbH & Co KG, Germany).To evaluate the efficiency of transfectin, cells were co-transfected with the β-galactosidase plasmid followed by chemiluminescent assay [36]. RNA Extract and RT-PCR Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions [37]. RNA was quantified by optical density (A260) and stored at −80°C until used. One microgram of total RNA was reverse transcribed by oligo (dT) primer using Reverse Transcription System (TAKARA, Dalian, China). The single-stranded cDNA was amplified by PCR using specific primers: 5′-GGGGTACCATGCCGCGCTCTTTCCT-3′ (sense) and 5′-CGAGATCTTCAGCGGG GACATCCT-3′ (antisense) for SNAI1; 5′-CCCAAGCTTGCGGCCGCGATG-3′ (sense) and 5′-CTTCTAGATCACCGGTGCTTC-3′ (antisense) for AIB1; 5′-TCCATTTCTTGGTCTACGCC-3′(sense) and 5′-CACCTTCAGCCATCCTGTTT-3′ (antisense) for E-cadherin; 5′-GTTTCCCCCCACTCAACAGCG-3′ (sense) and 5′-TCCCTTGTCATTGGTACTGGC-3′ (antisense) for ERα; 5′-TCGTGCGTGACAT TAAGGAG-3′ (sense) and 5′-ATGCCAGGGTACATGGTGGT-3′ (antisense) β-actin. PCR was carried out for 30 cycles, with each cycle consisted of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s). The PCR products were analyzed by electrophoresis using a 1% agarose gel. Western Blot Analysis Cells were washed three times with PBS and then lysed in HEPES containing 0.5% NP-40 and a mixture of protease inhibitors. The lysate was centrifuged to obtain the clear extract. An aliquot of the clear cell extract was used to determine the protein concentration using Bradford reagent. The cell lysates were resolved by SDS-PAGE using 10% gel. After electrophoresis, protein bands in the gel were transferred onto polyvinylidene difluoride (PVDF) membrane and were probed with the appropriate primary antibodies followed by the appropriate secondary antibodies. Positive bands of the blot were detected by ChemiLuminescence (ECL) regents (Pierce, Rockford, IL) [35]. Immunoprecipitation Cells were lysed in hypotonic buffer (0.5% NP-40, 20 mM HEPES (pH 7.9), 1 mM EDTA, 20 mM NaF, 1 mM dithiothreitol (DTT), 0.4 mM PMSF). After centrifugation at 13000× g for 2 min, the pellet (which contained the nuclear fraction) was extracted with a buffer containing 450 mM NaCl, 20% glycerol, 20 mM HEPES (pH 7.9), 1 mM EDTA, 20 mM NaF, 1 mM dithiothreitol (DTT), and 0.4 mM PMSF to yield the nuclear extract. Aliquot (500 µl) of the nuclear extract (containing 200–400 µg total protein) was incubated with specific antibody or with control IgG at 4°C for overnighte followed by addition of 25 µl of protein A and a further 2-h incubation at the same temperature (Amresco, Solon, OH) [38]. RNA Interference To create the AIB1 shRNA expression vector, the pRNAT-U6.1 vector (GenScript, Piscataway, NJ) was used for DNA vector-based shRNA synthesis. The sequences of AIB1 used for AIB1 knockdown study were 5′-TCCTGCAGTGTATAGTATG-3′ (shAIB1#1) [39], and 5′-GGTCTTACCTGCAGTGGTGAA-3′ (shAIB1#2) [40] and the sequence used for SNAI1 knockdown study was 5′-GGACAAAGGCTGACAGACT-3′ [41], and the sequence used for ERα knockdown experiment was 5′-CCGCTACTGTTTGCTCCTAAC-3′ [42]. The sequence of the negative control scrambled shRNA was 5′-GACGCTTACCGATTCAGAA-3′, which had no significant homology to human gene sequences [43]. Immunofluorescence Cells were cultured on coverslips and fixed in 4% paraformaldehyde for 15 min. After washing, the cells were permeabilized in PBS containing 0.5% Triton-X100 for 5 min. The cover slips were blocked with 0.8% BSA for 1 h and incubated with antibody against E-cadherin or N-cadherin at 4°C for overnight, followed by washing with PBS and further incubation with secondary antibody (Alexa Fluor 568) for 1 h. The cover slips were then washed with PBS and mounted on glass slides with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI), and examined and photographed using a Nikon TE2000-U microscope. Chromatin Immunoprecipitation Assays Cells were crosslinked with 1% formaldehyde in PBS for 10 min at room temperature. Crude cell lysates were sonicated (typically, six 15-sec pulses followed by 45-sec rest periods at output 6.0) to generate 300 to 1500-bp DNA fragments. The sheared chromatin (25 mg) was diluted 1∶10 in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.0) containing protein G and AIB1 antibody or rabbit IgG (as a negative control) and placed on a rolling shaker at 4°C for overnight. The immunoprecipitated chromatin was purified from the chromatin-antibody mixture and the chromatin immunoprecipitated DNA was eluted in elution buffer (50 mM Tris-Cl pH8, 01% SDS, and 10 mM EDTA). The purified DNA was subjected to PCR to amplify the promoter region using specific primers: 5′-TAACCAGGTCCCTCCTCA-3′ (sense) and 5′-ACGCGGCGACCGTTAAGA-3′ (antisense) for SNAII; 5′-GTGCTCTTGGCTAGATG-3′ (sense) and 5′-GACACCTGACCTTCCG-A-3′ (antisense) for; and 5′-GCACCTGCTCGGGGAGT-3′ (sense) and 5′-CCGCTCGAGCCTGACGAGGAAAGAGC-3′ (antisense) for; and 5′-CCGCTCGAGAGAGGCTGCTCCAAG-3′ (sense) and 5′-ACTCCAGGCTAGAGGGTCACC -3′ (antisense) for E-cadherin. Wound Healing Assay Cells were seeded at a density of 3.0×105 cells in 35-mm culture dish, and after 24 h, wounds were incised by scratching the cell monolayers with a 200-µl pipette tip. Photographs were taken under phase-contrast microscopy immediately and 24 h after incision. Transwell Migration and Invasion Assays Transwell migration and invasion assay were performed in 24-well modified chambers precoated with (invasion) Matrigel (BD Transduction, Franklin Lake, NJ) or without precoating (migration). Cells in serum-free medium were transferred into the upper chamber. Following 48 h of incubation, the migrated cells in the lower chamber with 10% fetal bovine serum were counted in five random fields. Each assay was performed in triplicate. Immunohistochemical Assay Sections of the tumors were first fixed in 10% buffered formalin. After fixing they were embedded in paraffin, and then deparaffinized and rehydrated using standard procedures. For antibody staining, antigen retrieval was performed in citrate buffer (pH 6.0, 30 min) and stained for the presence of ERα, AIB1, SNAI1 or E-cadherin using antibodies against these proteins. An immunohistochemistry kit (Maixin Bio, China) and DAB (diaminobenzidine) were used as chromagen for the antibody. Statistical Analysis Data were analyzed by analysis of variance using the Student’s t-test. Values of p<0.05 were considered to indicate statistical significance. The authors thank Shanshan Lan 1st Affiliated Hospital of Jilin University for providing the samples from breast cancer tissues. ==== Refs References 1 DeSantis C , Siegel R , Bandi P , Jemal A (2011 ) Breast cancer statistics, 2011 . CA Cancer J Clin 61 : 409 –418 .21969133 2 Welch DR , Steeg PS , Rinker-Schaeffer CW (2000 ) Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis . Breast Cancer Res 2 : 408 –416 .11250734 3 Voulgari A , Pintzas A (2009 ) Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers and strategies to overcome drug resistance in the clinic . Biochim Biophys Acta 1796 : 75 –90 .19306912 4 Thiery JP , Sleeman JP (2006 ) Complex networks orchestrate epithelial-mesenchymal transitions . Nat Rev Mol Cell Biol 7 : 131 –142 .16493418 5 Ross JS , Figge HL , Bui HX , del Rosario AD , Fisher HA , et al (1994 ) E-cadherin expression in prostatic carcinoma biopsies: correlation with tumor grade, DNA content, pathologic stage, and clinical outcome . Mod Pathol 7 : 835 –841 .7530850 6 Richmond PJ , Karayiannakis AJ , Nagafuchi A , Kaisary AV , Pignatelli M (1997 ) Aberrant E-cadherin and alpha-catenin expression in prostate cancer: correlation with patient survival . Cancer Res 57 : 3189 –3193 .9242448 7 de Herreros AG , Peiro S , Nassour M , Savagner P (2010 ) Snail family regulation and epithelial mesenchymal transitions in breast cancer progression . J Mammary Gland Biol Neoplasia 15 : 135 –147 .20455012 8 van Roy F , Berx G (2008 ) The cell-cell adhesion molecule E-cadherin . Cell Mol Life Sci 65 : 3756 –3788 .18726070 9 Anzick SL , Kononen J , Walker RL , Azorsa DO , Tanner MM , et al (1997 ) AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer . Science 277 : 965 –968 .9252329 10 Chen H , Lin RJ , Schiltz RL , Chakravarti D , Nash A , et al (1997 ) Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300 . Cell 90 : 569 –580 .9267036 11 Li H , Gomes PJ , Chen JD (1997 ) RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2 . Proc Natl Acad Sci U S A 94 : 8479 –8484 .9238002 12 Onate SA , Tsai SY , Tsai MJ , O'Malley BW (1995 ) Sequence and characterization of a coactivator for the steroid hormone receptor superfamily . Science 270 : 1354 –1357 .7481822 13 Takeshita A , Cardona GR , Koibuchi N , Suen CS , Chin WW (1997 ) TRAM-1, A novel 160-kDa thyroid hormone receptor activator molecule, exhibits distinct properties from steroid receptor coactivator-1 . J Biol Chem 272 : 27629 –27634 .9346901 14 Torchia J , Rose DW , Inostroza J , Kamei Y , Westin S , et al (1997 ) The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function . Nature 387 : 677 –684 .9192892 15 Xu J , Wu RC , O'Malley BW (2009 ) Normal and cancer-related functions of the p160 steroid receptor co-activator (SRC) family . Nat Rev Cancer 9 : 615 –630 .19701241 16 Alan KC , Huijian Wu (2012 ) The role of AIB1 in breast cancer (Review) . Oncology Letter 4 : 588 –594 . 17 Mussi P , Yu C , O'Malley BW , Xu J (2006 ) Stimulation of steroid receptor coactivator-3 (SRC-3) gene overexpression by a positive regulatory loop of E2F1 and SRC-3 . Mol Endocrinol 20 : 3105 –3119 .16916939 18 Louie MC , Zou JX , Rabinovich A , Chen HW (2004 ) ACTR/AIB1 functions as an E2F1 coactivator to promote breast cancer cell proliferation and antiestrogen resistance . Mol Cell Biol 24 : 5157 –5171 .15169882 19 Yan J , Yu CT , Ozen M , Ittmann M , Tsai SY , et al (2006 ) Steroid receptor coactivator-3 and activator protein-1 coordinately regulate the transcription of components of the insulin-like growth factor/AKT signaling pathway . Cancer Res 66 : 11039 –11046 .17108143 20 Qin L , Liao L , Redmond A , Young L , Yuan Y , et al (2008 ) The AIB1 oncogene promotes breast cancer metastasis by activation of PEA3-mediated matrix metalloproteinase 2 (MMP2) and MMP9 expression . Mol Cell Biol 28 : 5937 –5950 .18644862 21 Li LB , Louie MC , Chen HW , Zou JX (2008 ) Proto-oncogene ACTR/AIB1 promotes cancer cell invasion by up-regulating specific matrix metalloproteinase expression . Cancer Lett 261 : 64 –73 .18162290 22 Long W , Yi P , Amazit L , LaMarca HL , Ashcroft F , et al (2010 ) SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration . Mol Cell 37 : 321 –332 .20159552 23 Lydon JP , O'Malley BW (2011 ) Minireview: steroid receptor coactivator-3: a multifarious coregulator in mammary gland metastasis . Endocrinology 152 : 19 –25 .21047941 24 Wu H , Sun L , Zhang Y , Chen Y , Shi B , et al (2006 ) Coordinated regulation of AIB1 transcriptional activity by sumoylation and phosphorylation . J Biol Chem 281 : 21848 –21856 .16760465 25 Li S , Yang C , Hong Y , Bi H , Zhao F , et al (2012 ) The transcriptional activity of co-activator AIB1 is regulated by the SUMO E3 Ligase PIAS1 . Biol Cell 104 : 287 –296 .22283414 26 Perl AK , Wilgenbus P , Dahl U , Semb H , Christofori G (1998 ) A causal role for E-cadherin in the transition from adenoma to carcinoma . Nature 392 : 190 –193 .9515965 27 Font de Mora J , Brown M (2000 ) AIB1 is a conduit for kinase-mediated growth factor signaling to the estrogen receptor . Mol Cell Biol 20 : 5041 –5047 .10866661 28 Nieto MA (2002 ) The snail superfamily of zinc-finger transcription factors . Nat Rev Mol Cell Biol 3 : 155 –166 .11994736 29 Torres-Arzayus MI , Font de Mora J , Yuan J , Vazquez F , Bronson R , et al (2004 ) High tumor incidence and activation of the PI3K/AKT pathway in transgenic mice define AIB1 as an oncogene . Cancer Cell 6 : 263 –274 .15380517 30 Bautista S , Valles H , Walker RL , Anzick S , Zeillinger R , et al (1998 ) In breast cancer, amplification of the steroid receptor coactivator gene AIB1 is correlated with estrogen and progesterone receptor positivity . Clin Cancer Res 4 : 2925 –2929 .9865902 31 Wu H , Chen Y , Liang J , Shi B , Wu G , et al (2005 ) Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis . Nature 438 : 981 –987 .16355216 32 Alexander NR , Tran NL , Rekapally H , Summers CE , Glackin C , et al (2006 ) N-cadherin gene expression in prostate carcinoma is modulated by integrin-dependent nuclear translocation of Twist1 . Cancer Res 66 : 3365 –3369 .16585154 33 Peinado H , Olmeda D , Cano A (2007 ) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7 : 415 –428 .17508028 34 Zhang H , Yi X , Sun X , Yin N , Shi B , et al (2004 ) Differential gene regulation by the SRC family of coactivators . Genes Dev 18 : 1753 –1765 .15256502 35 Li S, Wang M, Ao X, Chang AK, Yang C, et al.. (2012) CLOCK is a substrate of SUMO and sumoylation of CLOCK upregulates the transcriptional activity of estrogen receptor-alpha. Oncogene: doi:10.1038/onc.2012.1518. 36 Wang M , Bao YL , Wu Y , Yu CL , Meng X , et al (2008 ) Identification and characterization of the human testes-specific protease 50 gene promoter . DNA Cell Biol 27 : 307 –314 .18462069 37 Wang M , Bao YL , Wu Y , Yu CL , Meng XY , et al (2010 ) Basic FGF downregulates TSP50 expression via the ERK/Sp1 pathway . J Cell Biochem 111 : 75 –81 .20506264 38 Hong Y , Xing X , Li S , Bi H , Yang C , et al (2011 ) SUMOylation of DEC1 protein regulates its transcriptional activity and enhances its stability . PLoS One 6 : e23046 .21829689 39 Yang C , Li S , Wang M , Chang AK , Liu Y , et al (2013 ) PTEN suppresses the oncogenic function of AIB1 through decreasing its protein stability via mechanism involving Fbw7 alpha . Mol Cancer 12 : 21 .23514585 40 Louie MC , Revenko AS , Zou JX , Yao J , Chen HW (2006 ) Direct control of cell cycle gene expression by proto-oncogene product ACTR, and its autoregulation underlies its transforming activity . Mol Cell Biol 26 : 3810 –3823 .16648476 41 Mikami S , Katsube K , Oya M , Ishida M , Kosaka T , et al (2011 ) Expression of Snail and Slug in renal cell carcinoma: E-cadherin repressor Snail is associated with cancer invasion and prognosis . Lab Invest 91 : 1443 –1458 .21808237 42 Shi B , Liang J , Yang X , Wang Y , Zhao Y , et al (2007 ) Integration of estrogen and Wnt signaling circuits by the polycomb group protein EZH2 in breast cancer cells . Mol Cell Biol 27 : 5105 –5119 .17502350 43 Zhou L , Bao YL , Zhang Y , Wu Y , Yu CL , et al (2010 ) Knockdown of TSP50 inhibits cell proliferation and induces apoptosis in P19 cells . IUBMB Life 62 : 825 –832 .21086474	23762395	PMC3676316	CC BY	2021-01-05 17:29:15	yes	PLoS One. 2013 Jun 7; 8(6):e65556
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23762387PONE-D-13-0535610.1371/journal.pone.0065539Research ArticleBiologyBiochemistryNucleic acidsRNARNA interferenceGeneticsCancer GeneticsImmunologyImmunologic TechniquesImmunohistochemical AnalysisMolecular Cell BiologyCell GrowthGene ExpressionMathematicsStatisticsBiostatisticsMedicineGastroenterology and HepatologyGastrointestinal CancersOncologyBasic Cancer ResearchMetastasisCancer Detection and DiagnosisEarly DetectionCancer Risk FactorsGenetic Causes of CancerCancers and NeoplasmsGastrointestinal TumorsOverexpression of YAP and TAZ Is an Independent Predictor of Prognosis in Colorectal Cancer and Related to the Proliferation and Metastasis of Colon Cancer Cells Synergy of YAP and TAZ in Colorectal CancerWang Lijuan 1 Shi Shengjia 2 Guo Zhangyan 2 Zhang Xiang 2 Han Suxia 1 Yang Angang 2 Wen Weihong 2 * Zhu Qing 1 * 1 Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi'an, P. R. China 2 State Key Laboratory of Cancer Biology, Department of Immunology, Fourth Military Medical University, Xi’an, P. R. China Hong Wanjin Editor Institute of Molecular and Cell Biology, Singapore * E-mail: wenweih@fmmu.edu.cn (WW); newzhuqing1972@yohoo.com (QZ)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: QZ WW. Performed the experiments: LW SS. Analyzed the data: SS LW. Contributed reagents/materials/analysis tools: AY ZG XZ SH. Wrote the paper: SS LW. 2013 10 6 2013 8 6 e655394 2 2013 25 4 2013 © 2013 Wang et al2013Wang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background and Objective Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are nuclear effectors of the Hippo pathway. Although they are abundantly expressed in the cytoplasm and nuclei of human colorectal cancer (CRC), and related to tumor proliferation status, there have been few studies on the predictive role of YAP and TAZ expression on the overall survival of patients with CRC. This study investigated YAP and TAZ expression in both CRC patients and colon cancer cell lines, and assessed their prognostic value. Methods Paraffin-embedded specimens from 168 eligible patients were used to investigate YAP and TAZ expression by immunohistochemistry, and compared with experimental results in colon cancer HCT116 cell line to explore their clinical significance in CRC. Results Statistically significant positive correlations were found between protein expression of YAP and TAZ in CRC tissues. Patients with higher YAP or TAZ expression showed a trend of shorter survival times; more importantly, our cohort study indicated that patients with both YAP and TAZ overexpression presented the worst outcomes. This was supported by multivariate analysis. In HCT116 colon cancer cells, the capacity for proliferation, metastasis, and invasion was dramatically reduced by knockdown of YAP and TAZ expressions by siRNA. Conclusions Co-overexpression of YAP and TAZ is an independent predictor of prognosis for patients with CRC, and may account for the higher proliferation, metastasis, and poor survival outcome of these patients. This work was supported by the National Natural Science Foundation of China (No. 81072117). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The Hippo pathway is an important regulator of cell growth, proliferation, and apoptosis. It was first discovered by genetic mosaic screens in Drosophila melanogaster [1], [2]; however, there is an increasing body of evidence demonstrating that the Hippo pathway also limits organ size in mammalian systems [3], [4] by inhibiting cell proliferation and promoting apoptosis. Components of this pathway are highly conserved in mammals, and include Mst1/2; WW45; Lats1/2; Mob1; YAP; TAZ; NF; FRMD6; and Fat4 [2]. Mst1/2 and Last1/2 are kinases, and WW45 and Mob1 act as adaptors/activators in mammals, together these make up the core kinase cassette of the Hippo pathway. The Yki homologs: Yes-associated protein (YAP) and its paralog, transcriptional co-activator with PDZ-binding motif (TAZ), are the principle targets of the core Hippo kinase cascade, and are considered to be the nuclear effectors of Hippo pathway [5]. Previous studies have reported that aberrant alteration of the key components of the Hippo pathway leads to uncontrolled cell growth, and is associated with cancer development [6], [7]. This implies that the Hippo pathway plays a critical role in suppressing tumor growth [8]. Aberrant activation of YAP has been associated with poor prognosis in multiple of human cancers [5], [9]–[11], including hepatocellular, ovarian, and malignant mesothelioma, and may act as an oncogene in breast cancer [15]. As such, YAP has been proposed as a candidate oncogene. Furthermore, overexpression of YAP was found to enhance liver size and eventually lead to tumor development in conditional transgenic mice models [5], [12], [13]. Additional studies have shown that TAZ, and TAZ-dependent secretion of amphiregulin (AREG), also plays a significant role in breast tumorigenesis and metastasis: when overexpressed TAZ is knocked down in non-small-cell lung carcinoma (NSCLC), its proliferation and oncogenic properties are suppressed [16]. Although both YAP and TAZ have been shown to be involved in the progression of cancers originating from various tissues, it is necessary to investigate the tissue-specific role of YAP and TAZ expression in order to investigate the role of YAP and TAZ in human colorectal cancer (CRC) and further understand the function of Hippo pathway. Recently published data suggests that overexpressed YAP may interplay with β-catenin to drive proliferation of colon cancer cells, implying that YAP could play a role in cancer therapy [17]; however, to our knowledge, research into the clinical significance of YAP and TAZ, and the prognostic value of YAP and TAZ, has been limited, and the significance of YAP and TAZ co-overexpression in CRC, remains elusive. In this study, we investigated the prognostic value of both YAP and TAZ in a retrospective cohort study with a five years follow-up, to determine the independent predictive role of YAP and TAZ in patients with CRC. We identified a synergy between these two proteins and a potential mechanism by which the Hippo pathway modulates human CRC progression. Materials and Methods Patients and Follow-up This study was approved by the Ethics Committee of the Fourth Military Medical University (FMMU; Xi'an, China), and all participating patients gave their written informed consent. The retrospective cohort included 168 patients diagnosed with potentially resectable CRC between February 2006 and December 2007 at the Department of Gastrointestinal surgery of Xijing Hospital, FMMU. Patients with the following criteria were excluded from participation: had received adjuvant chemotherapy prior to surgery; had been diagnosed with gastrointestinal stromal tumor or lymphoma; had additional cancers diagnoses; or refused consent. All the clinical specimens were retrieved from the tissue archive of Department of Pathology, Xijing Hospital, FMMU. Follow-up information was updated every three months, from all participants, by telephone. Overall survival was defined as the time elapsed from surgery to the time of patient death. Patient death was established from their family. Immunohistochemistry and Scoring Paraffin-embedded sections of normal and tumor tissues were stained for YAP (H-125: sc-15407; Santa Cruz Biotechnology, USA) and TAZ (NP_056287: ab118373; Abcam, UK) expression. As the antibody against YAP used in this study is a polyclonal antibody, we used Western blotting to identify the antibody specificity of YAP (Figure S1). Immunohistochemistry (IHC) for YAP and TAZ were performed as previously described [7] with minor modifications. Briefly, slides were deparaffinized in xylene, and rehydrated through a graded alcohol series, before endogenous peroxidase activity was blocked with 3% H2O2 in methanol. After blocking against nonspecific protein binding, samples were incubated overnight with YAP or TAZ primary antibodies, diluted to the recommended concentrations (1∶500), in a humidity chamber at 4°C. Samples were washed three times with phosphate-buffered saline (PBS), before biotinylated secondary antibody was applied for 30 min at room temperature. Visualization was performed using 3,3′ Diaminobenzidine (DAB) chromogen for 2–3 min. Negative controls were prepared by replacing the primary antibody with preimmune rabbit serum. YAP and TAZ staining were scored by two independent pathologists, blinded to the clinical characteristics of the patients. The scoring system used to grade the expression of YAP and TAZ is described in Table 1. 10.1371/journal.pone.0065539.t001Table 1 Immunohistochemcal scoring system. Extensional standards score (i) number of positive stained cell <5% 0 number of positive stained cell 6%–25% 1 number of positive stained cell 26%–50% 2 number of positive stained cell 51%–75% 3 number of positive stained cell >75% 4 (ii) Colorless 0 Pallideflavens 1 Yellow 2 Brown 3 The extensional standards (i) and (ii) were multipled. Score 0–4 and 5–12 was considered as negative and positive, respectively. Cell Culture The following human colon cancer cell lines: HCT116, LS174T, LOVO, SW480, and SW480 were used in this study. All the cell lines were obtained from the American Type Culture Collection (ATCC). HCT116 and LOVO cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone); SW480 and LS174T cells were cultured in RPMI1640 (Invitrogen, CA, USA) medium supplemented with 10% FBS; SW620 cells were cultured in Leibovitz's L-15 (Invitrogen, CA, USA) medium with 10% FBS. All the cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Transfection and Gene Silencing For small interfering RNA (siRNA) transfection, the following siRNA duplexes were synthesized (Genepharma, Shanghai, China): (5′-GGUGAUACUAUCAACCAAATT-3′), targeting the YAP gene; (5′-GGAUACAGGAGAAAACGCATT-3′), targeting the TAZ gene; and the negative control duplex, (5′-CCUACGCCACCAAUUUCGU-3′). These siRNA duplexes (100 nmol/L) were transfected into HCT116 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. HCT116 cells were harvested 48 h post-transfection for gene or protein analysis. Quantitative Real-time Reverse-transcriptase Polymerase Chain Reaction Total RNA was extracted from cell lines using TRIzol reagent (Invitrogen), and subsequent synthesis of cyclic DNA (cDNA; TaKaRa, Japan), were carried out according to the manufacturers’ protocols. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed using the CFX96TM Real-Time PCR system (BioRad, CA, USA) with the SYBR Green II kit (#DRR041A; TaKaRa, Japan) according to the manufacturers’ instructions. QRT-PCR analysis was carried out in a total volume of 20 µl with the following amplification steps: an initial denaturation step at 95°C for 10 min; followed by 40 cycles of denaturation at 95°C for 15 s; and then elongation at 55°C for 30 s. The expressions were normalized to the human β-actin gene. The following primer sequences were used: 5′-ACCCACAGCTCAGCATCTTCG-3′ (sense) and 5′-TGGCTTGTTCCCATCCATCAG-3′ (antisense) for YAP; 5′-GTCACCAACAGTAGCTCAGATC-3′ (sense) and 5′-AGTGATTACAGCCAGGTTAGAAAG-3′ (antisense) for TAZ; 5′-CGTCTTCCCCTCCATCGT-3′ (sense) and 5′-GAAGGTGTGGTGCCAGATTT-3′ (antisense) for β-actin. Western Blot Analysis Cells were harvested in radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology). Proteins were separated by SDS-PAGE, and transferred onto nitrocellulose membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk in PBS buffer for 2 h at room temperature, before being targeted ith the following antibodies according the manufacturers’ instructions: anti-Yap (1∶500); anti-TAZ (1∶500); and anti-actin (1∶5,000; AC40: A4700; Sigma-Aldrich, USA). Membranes were incubated with their associated horseradish peroxidase-conjugated (HPC) secondary antibodies, and the antibody-bound proteins were visualized by chemiluminescence (New England Nuclear, MA, USA). Cell Growth Assay (MTT) Cell proliferation in vitro was analyzed using tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT); because yellow MTT dye is reduced to a blue formazan product by respiratory enzymes that are only active in viable cells, the degree of color change is indicative of cell proliferation. HCT116 cells were transfected for 48h with no siRNA (parental); specific siRNAs (si-Con, si-YAP, or si-TAZ); or co-transfected with si-YAP and si-TAZ (si-YAP-TAZ), and suspended in DMEM with 10% FBS. Briefly, 2000 cells of each clone (parental, si-Con, si-YAP, si-TAZ, and si-YAP-TAZ) were plated in five 96-well plates in 200 µl of DMEM medium. For analysis: 20 µl of MTT substrate (from a 2.5 mg/ml stock solution in PBS) was added to each well; the plates were returned to the incubator for an additional 4 h at 37°C in a humidified atmosphere of 5% CO2; the medium was removed; the cells were solubilized in 150 µl dimethylsulfoxide; and colorimetric analysis was performed (wavelength, 490 nm). One plate was analyzed immediately after the cells adhered (approximately 4 h after plating), and the remaining plates were assayed over the next four consecutive days. Flow Cytometric Analysis of Apoptotic Cells HCT116 cells were transfected for 48 h with no siRNA (parental); specific siRNA (si-Con, si-YAP, or si-TAZ ); or co-transfected with si-YAP and si-TAZ (si-YAP-TAZ), before being suspended in PBS at a density of 1 × 106 cells/ml. Apoptotic cells were analyzed by flow cytometry using a CYTOMICS FC 500 flow cytometer (Beckman Coulter), after incubating the cells with a reagent containing Annexin V-FITC and Propidium Iodide (BD Bioscience, CA, USA) for 15 min in darkness at room temperature. Analysis of Invasiveness and Mobility (Migration and Invasion Assays) Cell invasion and migration potentials were measured in vitro by Transwell assays (Millipore, Billerica, MA) as follows: HCT116 cells were transfected for 48 h with no siRNA (parental); specific siRNA (si-Con, si-YAP, or si-TAZ ); or co-transfected with si-YAP and si-TAZ (si-YAP-TAZ); the cells were suspended in DMEM with 10 g/l BSA at a density of 50 cells/µl; 200 µl cell suspensions were seeded into the upper chambers of the Transwells, in which the porous membrane was either coated with Matrigel (BD Bioscience) for the invasion assays, or left uncoated for the migration assays. DMEM with 10% serum (500 µl) was added to the bottom chamber as a chemoattractant. After migration for 24 h, or invasion for 48 h, the cells that had penetrated the filters were fixed in methanol, and stained in 4g/l crystal violet. The numbers of migrated and invasive cells were determined from five random fields under an Olympus microscope (Olympus) at ×10 magnification. Statistical Analysis Statistical analysis was undertaken using IBM SPSS Statistical software (version 20.0). Spearman’s rank test was used to assess the correlation between YAP and TAZ expressions; survival curves were estimated using the Kalplan-Meier method; and distributions were evaluated by the long-rank test. Cox’s proportional hazards modeling of factors potentially related to survival were conducted to calculate hazard ratios (HR), and identify which factors might have a significant influence on survival. Differences in characteristics between the two groups were examined by the Pearson’s chi-square (χ2) test and Fisher’s exact test. All P values were determined from 2-tailed tests and differences with a P-value <0.05 were considered to be statistically significant. Results Clinical Significance of YAP and TAZ Overexpression in Colorectal Cancer Tissue To determine the prevalence and clinical significance of YAP and TAZ in CRC, we assessed the expression of YAP and TAZ protein by IHC in tumor tissue samples from our retrospective cohort of 168 from CRC patients after tumor resection. Among the 168 patients, 122 (72.6%) were positive for YAP expression, either nuclear or cytoplasmic (Figure 1A); whereas 46 (27.4%) were negative for YAP expression (Figure 1B); 97 (57.8%) samples were positive for TAZ expression; and 71 (42.2%) were negative for TAZ expression (Figures 1E and F, respectively). In contrast, only 3 (18.75%) and 2 (12.5%) out of the 16 adjacent normal tissue samples were found positive for YAP and TAZ expression, respectively (Figures 1C, D, G, and H). In addition, there was a significant positive correlation between YAP and TAZ (r = 0.630, P<0.001; Table S1), indicating a potential correlation between these proteins in CRC. 10.1371/journal.pone.0065539.g001Figure 1 Immunohistochemically stained tissues from CRC patients and adjacent normal control tissues. (A) YAP-positive tumor; (B) YAP-negative tumor; (C) YAP-positive normal tissue; (D) YAP-negative normal tissue; (E) TAZ-positive tumor; (F) TAZ-negative tumor; (G) TAZ-positive normal tissue; (H) TAZ-negative normal tissue. Representative images were taken under a microscope (x10). These results indicate the clinical significance of YAP and TAZ overexpression in CRC tissue. As a result of these observations, we evaluated the relationship between YAP and TAZ expression and the clinical features of CRC patients by Pearson’s chi-square test or Fisher’s exact test. Our results showed that expression of YAP and TAZ were significantly associated with the lymph node status in colorectal tumors (P = 0.001 and P = 0.013, respectively), but were not significantly correlated to gender, tumor location, tumor size, cell differentiation, or TNM stage (Table 2). The high prognostic impact of lymph node metastases, and the total number of lymph nodes to be resected, is well established. A recent study demonstrated that the cut-off values of lymph node ratio (the ratio of tumor-infiltrated nodes to resected lymph nodes) are strong independent prognostic factors for CRC patients, based on the analyses of clinical and histopathological data from 3026 patients at a single surgical center over a 25-year period [18]. These findings led us to investigate the role of YAP and TAZ in the prognosis of CRC patients. 10.1371/journal.pone.0065539.t002Table 2 Clinical correlation of YAP and/or TAZ expression in colorectal cancer. ClinicopathologicFeatures Total No. of patients, N = Yap POSITIVE p TAZ POSITIVE p Age (mean ± SD),years 168(59.8±12.5) 122 0.304 71 0.553 ≤40 19 14 10 40–60 66 52 28 ≥60 83 56 33 Sex 0.082 0.156 Men 95 64 45 Women 73 58 26 Tumor location 0.832 0.352 Right 73 54 27 Left 77 56 34 Rectum 18 12 10 Tumor size, cm 0.545 0.469 >3 127 92 56 ≤3 41 32 15 T status 0.223 0.814 T1 1 0 0 T2 14 9 6 T3 134 105 60 T4 10 8 5 N status 0.001* 0.013* N0 74 43 25 N1 79 66 37 N2 15 13 11 M status 0.380 0.899 M0 152 112 64 M1 16 10 7 TNM stage 0.057 0.893 I 12 8 6 II 60 37 26 III 83 68 33 IV 13 9 6 Differentiation 0.113 0.930 well 76 56 33 moderately 67 52 29 poorly 25 14 9 * Statistically significant. YAP and TAZ Co-overexpression were Associated with Poor Overall Survival To determine the prognostic significance of YAP and TAZ expression in CRC patients, we attempted to relate YAP and TAZ expression to the clinical outcomes. The overall median survival time among the patients in our retrospective cohort was 43 months (range: 1–56 months), and at the end of the follow-up period (60 months), 70 of the 168 patients were alive, and 98 patients were dead. The association between YAP and TAZ protein expression and overall survival of CRC patients was investigated using Kaplan-Meier analysis and log-rank test for significance estimates. Patients were divided into two groups: those with positive expression of YAP or TAZ, and those with negative expression of YAP or TAZ. A statistically significant difference was found between the overall survival of the two groups (long-rank test: P<0.02 and P<0.001, respectively). Patients with higher expressions of YAP or TAZ tended to have a higher risk of death compared to patients with lower expressions of YAP or TAZ, with the unadjusted HR being 1.617 and 1.643, respectively. In addition, cell differentiation, lymph node metastasis, and TNM stage were found to be associated with prognosis for CRC patients (Table 3). Multivariate analysis showed that higher expression of YAP or TAZ was associated with a reduction of overall survival, with the adjusted HR being 2.168 (95% CI: 1.125–4.179; P<0.001) and 1.544 (95% CI: 0.999–2.451; P = 0.05), respectively, indicating that the expression of YAP or TAZ could be a prognostic factor independent of these adjusted clinicopathologic characteristics (Table 3). 10.1371/journal.pone.0065539.t003Table 3 Cox regression analysis of overall survival (n = 168). Univariate Multivariate N HR 95% CI P Value HR 95%CI P Value YAP No expression 46 1.000 1.000 Expression 122 1.617 (1.151–2.273) 0.006* 2.168 (1.125–4.179) 0.021* TAZ No expression 71 1.000 1.000 Expression 97 1.643 (1.210–2.230) <0.001* 1.544 (0.999–2.451) 0.050* Age ≤40 19 1.000 1.000 40–60 66 0.964 (0.554–1.679) 0.898 1.161 (0.648–2.080) 0.606 ≥60 83 0.971 (0.707–1.333) 0.856 1.004 (0.711–1.419) 0.982 Sex Men 95 1.000 1.000 Women 73 1.258 (0.789–1.447) 0.856 1.050 (0.765–1.440) 0.764 Tumor location Right 73 1.000 1.000 Left 77 1.015 (0.606–1.701) 0.955 1.506 (1.251–2.023) 0.058 Rectum 78 0.981 (0.587–1.639) 0.943 1.528 (1.258–2.069) 0.076 Tumor size, cm ≤3.0 41 1.000 1.000 >3.0 127 1.045 (0.731–1.496) 0.808 1.601 (0.964–2.661) 0.069 TNM stage I 12 1.000 1.000 II 60 1.501 (1.228–2.099) 0.085 1.537 (1.258–2.119) 0.097 III 83 2.597 (1.382–3.038) 0.023* 1.416 (1.179–1.971) 0.043* IV 13 2.569 (1.482–4.932) <0.001* 1.499 (1.255–1.977) 0.042* Differentiation Well 76 1.000 1.000 Moderately 67 1.721 (1.341–2.132) 0.087 1.663 (1.406–2.083) 0.242 Poorly 25 1.605 (1.393–2.266) 0.014* 1.549 (1.340–1.887) 0.120 YAP-TAZ- 11 1.000 1.000 1.000 YAP+TAZ- 60 1.252 (0.659–2.380) 0.008* 2.072 (0.751–5.716) 0.047* YAP-TAZ+ 35 1.226 (0.624–2.410) 0.492 1.127 (0.380–3.344) 0.160 YAP+TAZ+ 62 4.388 (1.249–4.566) <0.001* 3.118 (1.017–9.559) <0.001* 95%CI indicates 95% confidence interval. * Statistically significant. We next investigated whether co-overexpression of YAP and TAZ could have a prognostic role in CRC. Our cohort patients were divided into four groups according to their combined expression levels of YAP and TAZ. A statistically significant difference was observed in overall survival outcome between these four subgroups of patients; those with positive co-overexpression of YAP and TAZ had the worst overall survival (Figure 2). Cox’s proportional hazards model, adjusted for gender, age, tumor size, tumor location, differentiation status, and TNM stage, compared with YAP or TAZ expression, showed that negative expression of both YAP and TAZ were associated with significantly improved overall survival; whereas, expression of both YAP and TAZ were associated with significantly poor overall survival (Table 3), with the adjusted HR being 3.118 (95% CI: 1.017–9.559; P<0.001). These data demonstrated that YAP and TAZ co-overexpression may have a specific and independent prognostic impact among CRC patients. 10.1371/journal.pone.0065539.g002Figure 2 Kaplan-Meier analysis of overall survival (cumulative survival) of CRC patients relative to YAP and TAZ expression. (A) Correlation of YAP expression with overall survival; (B) correlation of TAZ expression with overall survival; (C) correlation of combined YAP and TAZ expression with overall survival. A statistically significant difference is shown in overall survival outcome between the different groups of patients, with those having positive co-overexpression of YAP and TAZ having the worst overall survival. Overexpression of YAP and TAZ in HCT116 Colon Cancer Cell Line In order to further investigate the effect of YAP and TAZ on the progression of CRC, we tested our preliminary cohort study conclusions in cell line experiments. We first examined the gene and protein expression levels of YAP and TAZ in HCT116, LS174T, LOVO, SW620, and SW480 colon cancer cell lines. HCT116 cells showed the highest expression levels of both YAP and TAZ (Figures 3A and B); therefore, the HCT116 cell line was chosen for subsequent experiments to investigate YAP and TAZ in CRC progression. 10.1371/journal.pone.0065539.g003Figure 3 Assessment of YAP and TAZ expression in six human colon adenocarcinoma cell lines. (A) QRT-PCR analysis of YAP and TAZ expressions; (B) Western blot analysis of YAP and TAZ expressions. β-actin is used as an internal loading control. HCT116 cells show the highest expression levels of both YAP and TAZ. Effect of YAP or TAZ siRNA on YAP or TAZ Levels in Human HCT116 Colon Cancer Cell Lines We next examined the effect of YAP and TAZ in the HCT116 cell line through knockdown of YAP or TAZ expression using targeted siRNAs. The specificity of siRNA targeting for YAP and TAZ was confirmed by Western blot analysis. Compared to cells that had been transfected with control siRNA, the expression of YAP and TAZ was strongly suppressed in HCT116 cells transfected with 10 µg of targeted siRNAs (Figure 4A and Figure S2). 10.1371/journal.pone.0065539.g004Figure 4 Effect of YAP and TAZ expression on proliferation and apoptosis of HCT116 cells. (A) Western blot analysis of cell lines with reduced YAP and TAZ levels after transfection with siRNAs: parental HCT116 cells carrying no siRNA (parental); control siRNA (si-Con); siRNA to YAP (si-YAP); siRNA to TAZ (si-TAZ); and co-transfected with siRNA to YAP and siRNA to TAZ (si-YAP-TAZ). (B) MTT cell growth assays of control cells (parental, si-Con); cells with reduced YAP or TAZ expression (si-YAP, si-TAZ); or cells with reduced YAP and TAZ expression (si-YAP-TAZ). Data are presented as mean ± SD, N = 3, P<0.05. (C) Flow cytometric analyses of cells stained with Annexin-V-FITC and PI: cells carrying no siRNA (parental); control siRNA (si-Con); siRNA to YAP (si-YAP); siRNA to TAZ (si-TAZ); and cells with reduced YAP and TAZ expression (si-YAP-TAZ). These results indicate that YAP expression has a greater effect on cell proliferation and suppression of apoptosis compared to TAZ expression; however, a synergistic role between YAP and TAZ increases their combined individual effects. Effect of YAP and TAZ Expression on Cell Proliferation MTT assays were used to determine the effects of reduced YAP and TAZ expression on HCT116 colon cancer cell growth rates. The group transfected with YAP siRNA showed a significant reduction in proliferation rate relative to the group transfected with parental or control siRNAs. A similar trend was observed with TAZ siRNA; however, the effect was not as strong. The group that was co-transfected with YAP and TAZ siRNAs showed the most dramatic, and highly significant (P<0.05), decrease in proliferation rate compared to the parental or control siRNA groups (Figure 4B); therefore, these results demonstrated that YAP and TAZ possessed a synergistic role of on the proliferation of colon cancer cells. Effect of YAP and TAZ Expression on HCT116 Cells Apoptosis Ratio We then investigated the difference in apoptosis ratio between the YAP and/or TAZ knockdown groups and the control group to confirm our retrospective cohort study results. Flow cytometry analysis showed that the apoptosis ratio was decreased in HCT116 cells transfected with YAP siRNA compared to HCT116 cells transfected with parental or si-Con siRNA (17.48% vs. 1.62%, P<0.05; 17.48% vs. 2.28%, P<0.05, respectively; Figure 4C); the effect was slightly weaker in HCT116 cells transfected with TAZ siRNA relative to those transfected with parental or si-Con siRNA (6.48% vs.1.62%, P<0.05; 6.48% vs. 2.28%, P<0.05, respectively; Figure 4C); however, the apoptosis ratio was most dramatically decreased in HCT116 cells co-transfected with YAP and TAZ siRNAs compared to those transfected with parental and si-Con (33.60% vs. 1.62%, P<0.01; 33.60% vs. 2.28%, P<0.05, respectively; Figure 4C). Collectively, these results indicated that YAP expression had a major role in promoting cell proliferation and suppressing cell apoptosis; TAZ played an important, though less significant role, in this progress; and a synergistic effect between YAP and TAZ increased their impact on cell proliferation and apoptosis further. Effect of YAP and TAZ on Migration and Invasion YAP and TAZ activity has previously been shown to mediate tumor cell migration and invasion. Our observation that a reduction of YAP and TAZ expression played a synergistic role in the inhibition of cell proliferation and increase in apoptosis of HCT116 cells led us to assess the effect of YAP and TAZ siRNA co-transfection on the migration and invasion capabilities of colon cancer cells. Standard, and Matrigel-coated transwell assays were used to assess migration and invasion, respectively. Both YAP and TAZ siRNA transfected cells displayed statistically significantly reduced migration compared to control cells; the reduction was more significant in the group transfected with TAZ siRNA; however, the group transfected with both YAP and TAZ showed the most significant effect on the reduction of migration (Figures 5A and B). The trends were similar in the Matrigel invasion assays: clones with reduced YAP or TAZ levels showed a statistically significant reduction of invasiveness compared to control cells; the effect was more significant in the TAZ knockdown group; however, the most significant reduction of invasiveness was observed when both YAP and TAZ were knocked down (Figure 5C and D). 10.1371/journal.pone.0065539.g005Figure 5 Effect of YAP and TAZ expression on the migration and invasion of HCT116 colon cancer cells. (A) Representative images (×10) of migration assays of HCT116 cells with normal levels of YAP and TAZ expression (parental and si-Con); cells with suppressed levels of YAP or TAZ expression (si-YAP or si-TAZ); and cells with suppressed YAP and TAZ expression (si-YAP-TAZ). (B) Mean number of cells from the five independent migration assays described in previously. (C) Invasion assay of parental HCT116 cells; si-Con cells; si-YAP; si-TAZ; and si-YAP-TAZ cells in modified Boyden chambers with Matrigel-coated membranes. After 24 h, invasive cells that had moved through the Matrigel membrane were stained and counted under a microscope at ×10 magnification. (D) Graphical representation of invasive cells calculated as mean value ± SD from five fields. These show statistically significantly reduced migration in both YAP and TAZ siRNA transfected cells compared to control cells; the effect is greatest in the group transfected with both YAP and TAZ. [Asterisks indicate statistically significant differences in YAP or TAZ siRNA transfected cells vs. si-Con or parental cells, (, P<0.05; **, P<0.01).]. Discussion YAP and TAZ are major downstream targets of the Hippo signaling pathway. To date, there has been a considerable body of evidence that links the YAP/TAZ oncogene to tumorigenicity in several solid types of cancers, including ovary, breast, prostate, liver, and lung [7], [9], [12], [15]. Overexpression of YAP has been reported to aberrantly activate an array of target genes responsible for cell proliferation, survival, anti-apoptosis, and migration [19], [20]. Furthermore, previous studies have identified YAP as an independent prognostic marker for overall survival and disease-free times of hepatocellular carcinoma (HCC) patients; and clinicopathologically associated with tumor differentiation [21]. Based on the established views that YAP may increase organ size, and function as an oncogene [5], [9]; and that TAZ may promote cell proliferation, induce epithelial-mesenchymal transition (EMT), and increase cell migration and invasion [22]; combined with the potential similarity of tissue-specific functions of YAP and TAZ, we examined and characterized the clinical significance of YAP and TAZ as an independent prognostic factor for determining the outcomes of CRC patients. Our results were consistent with a previous study [7], in that the expression levels of both YAP and TAZ were significantly elevated in the majority of CRC tissues compared to adjacent normal tissues; however, in contrast to reports on breast cancer and lung squamous cell carcinoma, we found no discrepancy between cytoplasmic and nuclear expression of YAP or TAZ in CRC tissues [15]. Variations in the expression of YAP between different cancer tissues are probably due to their complicated tumor microenvironment. Using multivariate Cox regression analyses, we found that high co-expression of YAP and TAZ was a prognostic predictor for overall survival of CRC patients, independent of gender, age, tumor size, differentiation status, vascular invasion, and TNM stage. Furthermore, the lowest overall survival rates were identified in patients who co-expressed YAP and TAZ, and were associated with tumors having the greatest capacities of migration and invasion. YAP is acknowledged as a candidate oncogene, and became the focus of research, after it was identified in human chromosome 11q22 amplicon which is evident in several human cancers [14]. TAZ is a YAP paralog, initially identified as a 14-3-3 binding protein [23]; TAZ has approximately 50% sequence identity with YAP, and a similar topology; TAZ acts as a transcriptional co-activator in similar manner to YAP [23]; of note, was the discovery that YAP and TAZ share the downstream transcription factor, TEAD, to promote cell proliferation, migration, anchorage-independent growth, and EMT, which are all involved in cancer initiation and progression [24]. In addition, the mechanisms of TAZ and YAP regulation by Hippo are similar. These findings suggest shared regulation and function between TAZ and YAP; however, there are some apparent differences between them [23]: Although YAP knockout animals are embryonic lethal [25], TAZ null mice are characterized by renal cysts that lead to end stage kidney disease [26], [27]; however, mice lacking both YAP and TAZ die exceptionally early, suggesting there is a potential synergy between YAP and TAZ that exacerbates the individual effects of YAP and TAZ knockout during the embryonic period of mice [28]. As a result of all these evidences, we postulated that YAP and TAZ, which are both downstream targets of the Hippo signaling pathway, cooperate in CRC progression as opposed to acting as two distinct oncogenic proteins. Our results, discussed above, had revealed that patients with high expressions of both YAP and TAZ showed the worst survival outcome in our retrospective cohort study; therefore, we wished to further explore YAP and TAZ cooperation in the initiation and progression of CRC. The purpose of this study was to contribute to accurate prediction of prognosis for patients following surgery, and enable treatments to be tailored to each patient. In addition, because the Hippo signaling pathway has been identified as a vital growth regulator of cell proliferation and apoptosis, establishing a synergistic effect of YAP and TAZ, which are known effectors of the Hippo pathway in other common solid tumors, could enhance understanding of the underlying mechanisms regulating the initiation and progression of cancer. We confirmed the synergy of YAP and TAZ in the progression of CRC by investigating the effect of YAP and/or TAZ on the proliferation, invasion, migration, and apoptosis of HCT116 colon cancer cell lines. Consistent with a previous report, we found a shared and distinct relationship between YAP and TAZ function [29]: Compared to TAZ, YAP had a more significant effect on the proliferation and apoptosis of CRC cells; in contrast, the converse situation was observed for migration and invasion; as expected, all of these effects were most suppressed when both YAP and TAZ were knocked down. In combination, the colon cancer cell line results confirmed the tissue results of patients with CRC by showing that YAP and TAZ could cooperate to enhance their effects during CRC progression. Considering the multiple functions of the Hippo signaling pathway in the development of cell proliferation and apoptosis [6], these results may partly explain why mice with both YAP and TAZ knockdown died significantly earlier than mice with either YAP or TAZ knockdown [25]–[28]. Several possible reasons may account for the synergy in function between YAP and TAZ: firstly, both YAP and TAZ are major downstream targets of the Hippo pathway, and share many upstream and downstream proteins, such as TEAD [30]–[33], Angiomotin (AMOT) family proteins [34], and Wbp2 [35], [36]. This suggests that YAP and TAZ inevitably share similar functions, as opposed to competitive inhibition, and these may be enhanced through cooperation; secondly, in the same way that two signals are needed to activate T cells, co-expression of YAP and TAZ may be required for optimal functioning of the Hippo signaling pathway. Two recent studies have been published concerning the functions of YAP and TAZ in CRC [37] [38]. These have similarities with our work, but also show some differences. The first report, by Yuen et al. (2013), reported that mRNA expression of TAZ, but not YAP, was a prognostic indicator for colon cancer progression by virtue of being associated with increased expression of genes involved in colon cancer progression [37]. The second report, by Barry et al. (2013), demonstrated that a decrease of YAP expression could predict worse patient survival outcomes, and was associated with high-grade, stage IV disease, compared to YAP-positive groups [38]. The discrepancies between these two studies and our work may reflect the different pathological features selected by each study, and the different methods employed: the study by Yuen et al. choose to detect mRNA levels of YAP and TAZ; in contrast, our work focused on the protein levels of YAP and TAZ; the study by Barry et al. was a prospective study, compared to our retrospective study; furthermore, the clinicopathologic features of the two study cohorts were different. For example, patients receiving chemotherapy were excluded from our cohort study, but included in their study; patients in their cohort study group were predominantly white; whereas, our cohort group was confined to Chinese yellow patients, which was a common limitation in these type studies. These limitations indicate that more detailed work is required to clarify the relationship between YAP and TAZ, to further understand the Hippo signaling pathway. To our knowledge, this study presents the first clinical evidence identifying the relationship between YAP and TAZ as well as the predictive role of co-expression of them in colorectal cancer. Our results indicated that co-overexpression of YAP and TAZ may play an important role in the regulation of tumor progression. Although our sample size was modest, and our analyses will need to be confirmed in a larger patient population, our retrospective study provided convincing evidence that co-overexpression of YAP and TAZ could be an independent predictor of prognosis of patients with CRC. In conclusion, targeting YAP and TAZ could prove to be a promising therapeutic strategy for the treatment of CRC. Supporting Information Figure S1 Western blot analysis of YAP expression in HCT116 cells. The molecular weight of YAP is approximately 65 kDa. (TIF) Click here for additional data file. Figure S2 Densitometric quantitation of the relative intensities of β-actin, YAP, and TAZ given in the Western blot analysis of Figure 4A. (TIF) Click here for additional data file. Table S1 Correlation between the positive staining of YAP and TAZ (DOC) Click here for additional data file. ==== Refs References 1 Edgar BA (2006 ) From cell structure to transcription: Hippo forges a new path . Cell 124 : 267 –273 .16439203 2 Pan D (2007 ) Hippo signaling in organ size control . Genes Dev 21 : 886 –897 .17437995 3 Lai ZC , Wei X , Shimizu T , Ramos E , Rohrbaugh M , et al (2005 ) Control of cell proliferation and apoptosis by mob as tumor suppressor, mats . Cell 120 : 675 –685 .15766530 4 Wu S , Huang J , Dong J , Pan D (2003 ) hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts . Cell 114 : 445 –456 .12941273 5 Dong J , Feldmann G , Huang J , Wu S , Zhang N , et al (2007 ) Elucidation of a universal size-control mechanism in Drosophila and mammals . Cell 130 : 1120 –1133 .17889654 6 Hong W , Guan KL (2012 ) The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway . Semin Cell Dev Biol 23 : 785 –793 .22659496 7 Steinhardt AA , Gayyed MF , Klein AP , Dong J , Maitra A , et al (2008 ) Expression of Yes-associated protein in common solid tumors . Hum Pathol 39 : 1582 –1589 .18703216 8 Olson MF (2005 ) Modeling human cancer: report on the Eighth Beatson International Cancer Conference . Cancer Res 65 : 11247 –11250 .16357126 9 Zhao B , Wei X , Li W , Udan RS , Yang Q , et al (2007 ) Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control . Genes Dev 21 : 2747 –2761 .17974916 10 Zender L , Spector MS , Xue W , Flemming P , Cordon-Cardo C , et al (2006 ) Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach . Cell 125 : 1253 –1267 .16814713 11 Goulev Y , Fauny JD , Gonzalez-Marti B , Flagiello D , Silber J , et al (2008 ) SCALLOPED interacts with YORKIE, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila . Curr Biol 18 : 435 –441 .18313299 12 Camargo FD , Gokhale S , Johnnidis JB , Fu D , Bell GW , et al (2007 ) YAP1 increases organ size and expands undifferentiated progenitor cells . Curr Biol 17 : 2054 –2060 .17980593 13 Zhang N , Bai H , David KK , Dong J , Zheng Y , et al (2010 ) The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals . Dev Cell 19 : 27 –38 .20643348 14 Overholtzer M , Zhang J , Smolen GA , Muir B , Li W , et al (2006 ) Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon . Proc Natl Acad Sci U S A 103 : 12405 –12410 .16894141 15 Wang X , Su L , Ou Q (2012 ) Yes-associated protein promotes tumour development in luminal epithelial derived breast cancer . Eur J Cancer 48 : 1227 –1234 .22056638 16 Zhou Z , Hao Y , Liu N , Raptis L , Tsao MS , et al (2011 ) TAZ is a novel oncogene in non-small cell lung cancer . Oncogene 30 : 2181 –2186 .21258416 17 Rosenberg R , Friederichs J , Schuster T , Gertler R , Maak M , et al (2008 ) Prognosis of patients with colorectal cancer is associated with lymph node ratio: a single-center analysis of 3,026 patients over a 25-year time period . Ann Surg 248 : 968 –978 .19092341 18 Avruch J , Zhou D , Bardeesy N (2012 ) YAP oncogene overexpression supercharges colon cancer proliferation . Cell Cycle 11 : 1090 –1096 .22356765 19 Zhao B , Ye X , Yu J , Li L , Li W , et al (2008 ) TEAD mediates YAP-dependent gene induction and growth control . Genes Dev 22 : 1962 –1971 .18579750 20 Hao Y , Chun A , Cheung K , Rashidi B , Yang X (2008 ) Tumor suppressor LATS1 is a negative regulator of oncogene YAP . J Biol Chem 283 : 5496 –5509 .18158288 21 Xu MZ , Yao TJ , Lee NP , Ng IO , Chan YT , et al (2009 ) Yes-associated protein is an independent prognostic marker in hepatocellular carcinoma . Cancer 115 : 4576 –4585 .19551889 22 Chan SW , Lim CJ , Guo K , Ng CP , Lee I , et al (2008 ) A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells . Cancer Res 68 : 2592 –2598 .18413727 23 Kanai F , Marignani PA , Sarbassova D , Yagi R , Hall RA , et al (2000 ) TAZ: a novel transcriptional co-activator regulated by interactions with 14–3-3 and PDZ domain proteins . EMBO J 19 : 6778 –6791 .11118213 24 Mahoney WM Jr, Hong JH , Yaffe MB , Farrance IK (2005 ) The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members . Biochem J 388 : 217 –225 .15628970 25 Morin-Kensicki EM , Boone BN , Howell M , Stonebraker JR , Teed J , et al (2006 ) Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65 . Mol Cell Biol 26 : 77 –87 .16354681 26 Makita R , Uchijima Y , Nishiyama K , Amano T , Chen Q , et al (2008 ) Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ . Am J Physiol Renal Physiol 294 : F542 –553 .18172001 27 Hossain Z , Ali SM , Ko HL , Xu J , Ng CP , et al (2007 ) Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1 . Proc Natl Acad Sci U S A 104 : 1631 –1636 .17251353 28 Nishioka N , Inoue K , Adachi K , Kiyonari H , Ota M , et al (2009 ) The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass . Dev Cell 16 : 398 –410 .19289085 29 Zhao B , Lei QY , Guan KL (2008 ) The Hippo-YAP pathway: new connections between regulation of organ size and cancer . Curr Opin Cell Biol 20 : 638 –646 .18955139 30 Vassilev A , Kaneko KJ , Shu H , Zhao Y , DePamphilis ML (2001 ) TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm . Genes Dev 15 : 1229 –1241 .11358867 31 Wu S , Liu Y , Zheng Y , Dong J , Pan D (2008 ) The TEAD/TEF family protein Scalloped mediates transcriptional output of the Hippo growth-regulatory pathway . Dev Cell 14 : 388 –398 .18258486 32 Zhang H , Liu CY , Zha ZY , Zhao B , Yao J , et al (2009 ) TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition . J Biol Chem 284 : 13355 –13362 .19324877 33 Chan SW , Lim CJ , Loo LS , Chong YF , Huang C , et al (2009 ) TEADs mediate nuclear retention of TAZ to promote oncogenic transformation . J Biol Chem 284 : 14347 –14358 .19324876 34 Harvey KF , Zhang X , Thomas DM (2013 ) The Hippo pathway and human cancer . Nat Rev Cancer 13 : 246 –257 .23467301 35 Chan SW , Lim CJ , Huang C , Chong YF , Gunaratne HJ , et al (2011 ) WW domain-mediated interaction with Wbp2 is important for the oncogenic property of TAZ . Oncogene 30 : 600 –610 .20972459 36 Zhang X , Milton CC , Poon CL , Hong W , Harvey KF (2011 ) Wbp2 cooperates with Yorkie to drive tissue growth downstream of the Salvador-Warts-Hippo pathway . Cell Death Differ 18 : 1346 –1355 .21311569 37 Yuen HF , McCrudden CM , Huang YH , Tham JM , Zhang X , et al (2013 ) TAZ expression as a prognostic indicator in colorectal cancer . PLoS One 8 : e54211 .23372686 38 Barry ER , Morikawa T , Butler BL , Shrestha K , de la Rosa R , et al (2013 ) Restriction of intestinal stem cell expansion and the regenerative response by YAP . Nature 493 : 106 –110 .23178811	23762387	PMC3677905	CC BY	2021-01-05 17:29:19	yes	PLoS One. 2013 Jun 10; 8(6):e65539
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23785404PONE-D-12-3764310.1371/journal.pone.0064707Research ArticleBiologyMolecular Cell BiologySignal TransductionSignaling CascadesApoptotic Signaling CascadeMedicineOncologyBasic Cancer ResearchTumor PhysiologyCancer Risk FactorsGenetic Causes of CancerPredisposing Conditions and SyndromesViral and Bacterial Causes of CancerCancers and NeoplasmsGastrointestinal TumorsGastric CancermiR-23a Targets Interferon Regulatory Factor 1 and Modulates Cellular Proliferation and Paclitaxel-Induced Apoptosis in Gastric Adenocarcinoma Cells Regulation of IRF1 by miR-23aLiu Xue 1 Ru Jing 1 Zhang Jian 1 Zhu Li-hua 1 2 Liu Min 1 Li Xin 1 Tang Hua 1 * 1 Tianjin Life Science Research Center and Department of Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China 2 Department of Pathogen Biology and Immunology, College of Basic Medicine, Hebei United University, Tangshan, Hebei Province, China Cheng Jin Q. Editor H.Lee Moffitt Cancer Center & Research Institute, United States of America * E-mail: htang2002@yahoo.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: HT X. Li. Performed the experiments: X. Liu JR JZ LHZ. Analyzed the data: X. Liu JR JZ LHZ. Contributed reagents/materials/analysis tools: ML. Wrote the paper: HT X. Liu. 2013 10 6 2013 8 6 e6470722 11 2012 17 4 2013 © 2013 Liu et al2013Liu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.MicroRNAs are a class of non-coding RNAs that function as key regulators of gene expression at the post-transcriptional level. In our previous research, we found that miR-23a was significantly up-regulated in human gastric adenocarcinoma cells. In the current study, we demonstrate that miR-23a suppresses paclitaxel-induced apoptosis and promotes the cell proliferation and colony formation ability of gastric adenocarcinoma cells. We have identified tumor suppressor interferon regulator factor 1 (IRF1) as a direct target gene of miR-23a. We performed a fluorescent reporter assay to confirm that miR-23a bound to the IRF1 mRNA 3′UTR directly and specifically. The ectopic expression of IRF1 markedly promoted paclitaxel-induced apoptosis and inhibited cell viability and colony formation ability, whereas the knockdown of IRF1 had the opposite effects. The restoration of IRF1 expression counteracted the effects of miR-23a on the paclitaxel-induced apoptosis and cell proliferation of gastric adenocarcinoma cells. Quantitative real-time PCR showed that miR-23a is frequently up-regulated in gastric adenocarcinoma tissues, whereas IRF1 is down-regulated in cancer tissues. Altogether, these results indicate that miR-23a suppresses paclitaxel-induced apoptosis and promotes cell viability and the colony formation ability of gastric adenocarcinoma cells by targeting IRF1 at the post-transcriptional level. This work was supported by the National Natural Science Foundation of China (numbers 31270818; 91029714; 31071191) and the Natural Science Foundation of Tianjin (12JCZDJC25100; 09JCZDJC17500). The websites of the funders are http://www.tstc.gov.cn/ and http://www.nsfc.gov.cn/ respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Gastric cancer is a disease that is associated with a poor prognosis and a high mortality rate [1], [2]. Gastric cancer is the second leading cause of cancer death worldwide after lung cancer [3]. Approximately 90% of gastric cancers are adenocarcinomas, which originate from the glandular epithelium of the gastric mucosa [4]. Previous studies have suggested that gastric adenocarcinoma is a multifactorial disease [5]. Numerous studies have also revealed that oncogenes or tumor suppressors may play important roles in the tumorigenesis and progression of gastric cancer [6], [7]. However, the molecular mechanisms of gastric cancer development and progression remain unresolved. miRNAs are a class of small, non-coding RNAs that can regulate gene expression by either inducing the degradation of target mRNAs or by impairing the translation of their target mRNAs. miRNAs can also up-regulate gene expression by targeting the 5′ untranslated region (UTR) of their target genes. Many studies have revealed that aberrantly expressed miRNAs participate in tumorigenesis in temporal and spatial manners [8]. Some miRNAs become over-expressed in tumor cells and function as oncogenes. miR-223 has been shown to stimulate gastric cancer cell migration and invasion in vitro and in vivo [9]. miR-27a is highly expressed in gastric adenocarcinoma tissue and promotes cell growth [10], [11]. However, other miRNAs are deleted or reduced in tumor cells and act as tumor suppressor genes. The miR-200bc/429 cluster, miR-497 and miR-181b, have been shown to be down-regulated in gastric cancer cell lines [12], [13], [14], and these miRNAs have been suggested to play a role in the development of multidrug resistance by modulating apoptosis through the regulation of BCL2 [15]. Recently, several reports have demonstrated that miR-23a has diverse functions in tumor biology. miR-23a, is located in the miR-23a/24/27a cluster and regulates the TGF-β-induced epithelial–mesenchymal transition (EMT) by targeting E-cadherin in lung cancer cells [16]. The miR-23a cluster is a downstream target of PU.1 and is involved in antagonizing lymphoid cell fate [17]. miR-23a promotes colon carcinoma cell growth, invasion and metastasis through the inhibition of the MTSS gene [18]. miR-23a also targets glutaminase (GLS) mRNA and inhibits the expression of the GLS protein [19]. The miR-23a/24/27a cluster appears to function as an antiapoptotic and proliferation-promoting factor in liver cancer cells [20], and miR-23a has been shown to be significantly up-regulated in bladder cancers compared to normal bladder mucosa [21]. miR-23a was also found to act as an oncogene in gastric cancer. In a previous study that was conducted in our lab [22], we analyzed gastric adenocarcinoma-related miRNAs using miRNA microarrays, and we identified miR-23a as an oncogenic miRNA. In the current study, we show that human miR-23a can promote cell proliferation and suppress paclitaxel-induced apoptosis in gastric adenocarcinoma cell lines. We further validate interferon regulatory factor 1 (IRF1) as a target gene of miR-23a. The miR-23a-induced malignant phenotypes of gastric adenocarcinoma appear to occur through the down-regulation of IRF1 expression. An increase in miR-23a expression and a concomitant decrease in IRF1 expression in gastric adenocarcinoma cells appear to contribute to the tumorigenesis of gastric adenocarcinomas. Materials and Methods Human cancer tissue samples and RNA isolation Fresh frozen human gastric adenocarcinoma tissue samples and matched normal gastric tissue samples were obtained from the Tumor Bank Facility of Tianjin Medical University Cancer Institute and Hospital and from the National Foundation for Cancer Research. The detailed information of gastric samples is shown in Table S1. The subtype of each tumor was confirmed by histological analysis. All human materials were used in accordance with the policies of our Institutional Review Board. RNA extraction from cells or tissue samples was performed by using the mirVana miRNA Isolation Kit (Ambion), according to the manufacturer's instructions. Large RNAs (larger than 200 nt) and small RNAs (smaller than 200 nt) were separated and purified by this procedure. The integrity of the large RNAs was confirmed by 1% denatured agarose gel electrophoresis. Plasmid construction The construction of the pcDNA3/pri-miR-23a (pri-miR-23a) plasmid was described in our previous published study [22]. We also commercially synthesized a 2′-O-methyl-modified antisense oligonucleotide for miR-23a (ASO-23a) (GenePharm, Shanghai, China). The sequence of the ASO-23a construct used in this study was listed in Table 1. The enhanced green fluorescence protein (EGFP) expression vector (pcDNA3/EGFP) was provided by our lab. The 3′UTR fragment of the IRF1 gene containing the predicted miR-23a binding site was amplified by PCR using the primers listed in Table 1. PCR products were cloned into the pcDNA3/EGFP plasmid between the BamHI and EcoRI restriction sites. The resulting vector was named pcDNA3/EGFP - IRF1 3′UTR. Moreover, a mutant fragment of the IRF1 3′UTR, containing a mutated miR-23a binding site, was amplified by PCR site-directed mutagenesis and was cloned into the pcDNA3/EGFP plasmid between the BamHI and EcoRI restriction sites (pcDNA3/EGFP - IRF1 3′UTR-mut). All insertions were confirmed by sequencing. To construct the pSilencer/shRNA-IRF1 (sh-IRF1) vector, a 70-bp double-stranded fragment, which was designed to contain BamHI and HindIII restriction sites at the ends, was obtained via an annealing step using the two single-stranded sequences listed in Table 1. The fragment was then cloned into the pSilencer2.1/neo vector (Ambion) between the BamHI and HindIII sites. The pcDNA3 vector was used to generate an IRF1 over-expression plasmid. The full-length human IRF1 cDNA sequence (GenBank TM, NM_017423.2) was amplified by PCR using cDNA isolated from HL60 cells in which IRF1 expression is rich,as the template and the primers listed in Table 1, and then the IRF1 cDNA gene was cloned into the EcoRI and XhoI restriction sites. 10.1371/journal.pone.0064707.t001Table 1 Oligonucleotides used in this study. name Sequence (5′---- 3′) pri-miR-23a forward 5′ – GCGAGATCTGGCTCCTGCATATGAG – 3′ pri-miR-23a reverse 5′ – GATGAATTCCAGGCACAGGCTTCGG – 3′ ASO-23a 5′ – GGAAATCCCTGGCAATGTGAT – 3′ ASO-NC 5′ – GTGGATATTGTTGCCATCA – 3′ IRF1-3′UTR-S 5′ – CGCGGATCCAGAAAAGCATAACACCAATCC – 3′ IRF1-3′UTR-A 5′ – CGGAATTCGTGGCAAGATCCACACCGA – 3′ IRF1-3′UTR-MS 5′ – CCAAAGCCAGTGATAAGAGTGAAAGTGGG – 3′ IRF1-3′UTR-MA 5′ – CCCACTTTCCTACTCTTATCACTGGCTTTGG – 3′ IRF1-S-EcoRI 5′ – CGGAATTCGCCAACATGCCCATCACTCGG – 3′ IRF1-AS-XhoI 5′ – CCAGGCTCGAGGCTACGGTGCACAGGGAATG – 3′ IRF1-qPCR-S: 5′ ACATTCCTGTCATAGGAAC 3′ IRF1-qPCR-AS: 5′ GCCTCAAAACTTAACACTC 3′ IRF1-siR-Top 5′ – GATCCGCTGAGGACATCATGAAGCTTTCAAGAGAAGCTTCATGATGTCCTCAGTTTTTTGGAAA – 3′ IRF1-siR-Bot 5′ – AGCTTTTCCAAAAAACTGAGGACATCATGAAGCTTCTCTTGAAAGCTTCATGATGTCCTCAGCG – 3′ β-Actin-S 5′ – CGTGACATTAAGGAGAAGCTG – 3′ β-Actin-A 5′ – CTAGAAGCATTTGCGGTGGAC – 3′ miR-23a-RT 5′ – GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACGGAAATCC – 3′ miR-23a forward 5′ – TGCGGATCACATTGCCAGG – 3′ U6 RT 5′ – GTCGTATCCAGTGCAGGGTCCGAGGTGCACTGGATACGACAAAATATGG – 3′ U6 forward 5′ – TGCGGGTGCTCGCTTCGGCAGC – 3′ U6 Reverse 5′ – CCAGTGCAGGGTCCGAGGT – 3′ GAPDH-S 5′ – GCGAATTCCGTGTCCCCACTGCCAACGTGTC – 3′ GAPDH-AS 5′ – GCTACTCGAGTTACTCCTTGGAGGCCATGTGG – 3′ Real-time PCR Real-time PCR was performed to detect the level of miR-23a in the tissue samples. Two micrograms of small RNA extracted from the tissue samples were reverse transcribed to cDNA using M-MLV reverse transcriptase (Promega, Madison, WI), and the primers (miR-23a RT and U6 RT), which can fold into a stem-loop structure, are shown in Table 1. The cDNA was used for the amplification of mature miR-23a and an endogenous control U6 snRNA through PCR. The PCR cycling conditions used were as follows: 94°C for 3 min followed by 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s. To quantify IRF1 gene expression, 5 µg of RNA extracted from cells or tissue samples was reverse transcribed to cDNA using the M-MLV reverse transcriptase. The cDNA was used for the PCR amplification of IRF1 and the endogenous control gene β-actin. The PCR cycling conditions used were as follows: 94°C for 3 min followed by 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. The SYBR Premix Ex Taq™ kit (TaKaRa, Otsu, Shiga, Japan) was used to measure the amplified DNA, and real-time PCR was performed using an iQ5 real-time PCR detection system (Bio-Rad). The relative gene expression levels were calculated using the 2−ΔΔct method [23]. All primers were purchased from AuGCT, Inc. (Beijing, China), and the sequences of the primers used are shown in Table 1. Cell culture and transfection The human gastric adenocarcinoma cell lines MGC803 and BGC823 were obtained commercially from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. They were maintained in RPMI1640 (GIBCO BRL, Grand Island, NY, USA) and were supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin. The cell lines were incubated at 37°C in a humidified chamber supplemented with 5% CO2. Transient transfections were performed in this study using the Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's suggested protocol. Oligonucleotides were used at a final concentration of 100 nM, and plasmids were used at a final concentration of 5 ng/ µL. Approximately 2×104 cells were seeded in a 48-well plate one day before transfection. Cells were approximately 65% confluent at the time of transfection. Both oligonucleotides and plasmids were incubated in antibiotic-free Opti-MEM medium (Invitrogen, Carlsbad, CA, USA). To monitor the transfection efficiency, we transfected pcDNA3-EGFP as well as the control pcDNA3 into the two cell lines and observed the cells for EGFP expression 48 h after transfection by fluorescence microscopy. When we used more than one construct, we used the total amount of DNA as a reference to determine the transfection efficiency. As expected, the transfection efficiency was approximately 60–70%. Fluorescent report assay MGC803 cells (approximately 2×104 cells) were transfected in 48-well plates with 0.2 µg per well of the IRF1 EGFP reporter vector with wild-type or mutated 3′UTR. The cells were also cotransfected with 20 pmol of miR-23a ASO or 0.2 µg of pcDNA3/pri-23a per well. The assay was normalized to cells transfected with 0.05 µg per well of the red fluorescent protein expression vector pDsRed2-N1 (Clontech, Mountain View, CA, USA). Forty-eight hours after transfection, cells that were approximately 80–90% confluence were lysed in lysis buffer (0.15 M NaCl, 0.05 M Tris-HCl pH 7.2, 1% Triton X-100, and 0.1% SDS). Each experimental group was conducted in triplicate. The fluorescence intensities of EGFP and the red fluorescent protein were detected using a Fluorescence Spectrophotometer F-4500 (Hitachi, Tokyo, Japan). MTT and colony formation assays Cells were transfected with 100 nM ASO or 5 ng/µL of each plasmid as described above. Cells were then trypsinized, counted, and seeded 24 h after transfection in a 96-well plate at a concentration of 4000 cells per well in 100 µL of cell culture medium. The cells were incubated at 37°C in the presence of 5% CO2. After incubation for 48 h and 72 h, the cells reached approximately 40–50% confluence and were incubated with 10 µL of MTT (at a final concentration of 0.5 mg/mL) at 37°C for 4 h. The medium was removed and the precipitated formazan was dissolved in 100 µL of DMSO. After shaking for 10 min, the absorbance at 570 nm was detected using an lQuant Universal Microplate Spectrophotometer (Bio-tek Instruments). Each group was repeated in triplicate. After transfection with 100 nM of ASO or 5 ng/µL of each plasmid, the cells were trypsinized, counted, and seeded for a colony formation assay in 12-well plates at concentrations of 300 BGC823 cells and 200 MGC803 cells per well. During colony growth, the culture medium was replaced every three days. A colony was counted if it was composed of more than 50 cells, and the number of colonies was determined on the 10th day for MGC803 and the 14th day for BGC823 after seeding. Before counting, 300 µL of crystal violet staining solution was added to each well to stain the colonies purple. Each group was repeated in triplicate. TUNEL assay The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was carried out using the In-Situ Cell Death Detection Kit (Roche Diagnostics Corporation, Indianapolis, IN, USA). Cells were transfected with 100 nM ASO or 5 ng/µL of each plasmid, as described previously. Cells were then trypsinized, counted, and seeded at 5000 cells per well 24 h after transfection in 14-well plates. Each group was conducted in triplicate. Then, the cells (approximately 70–80% confluence) were treated with 0.5 ppc paclitaxel for 1 h and were fixed with paraformaldehyde (4% in PBS, pH 7.4). The cells were then incubated in permeabilization solution (0.1% TritonX-100 in 0.1% sodium citrate) for 2 minutes on ice. After washing the cells, 4 µL of the TUNEL reaction mixture (3.6 µL of the labeling solution and 0.4 µL of the enzyme solution) was added to each well. The slide was incubated for 60 minutes at 37°C in the dark, and then TUNEL reactions were then performed according to the manufacturer's suggested protocol. DAPI (4, 6- diamidino-2- phenylindole, Dojindo Molecular Technologies, Inc., Japan) was used to stain the nuclei. Fluorescence images were observed using a Nikon Digital sight DS-U1 scanning microscope (Nikon, Tokyo, Japan). The images were superimposed using NIS Elements F 2.20 imaging software (Nikon, Tokyo, Japan). Western blot analysis Cells were transfected with 100 nM ASO or 5 ng/µL of each plasmid, and 72 h later, the cells (approximately 80%–90% confluence) were lysed with RIPA lysis buffer and the proteins were harvested. The protein concentration of cell lysates was determined using BCA regents from Promega. Approximately 25 ug/lane of proteins was resolved by SDS-denatured polyacrylamide gel electrophoresis and was then transferred onto a nitrocellulose membrane. Membranes were incubated overnight at 4°C with anti-IRF1 (1∶200) and anti-tubulin (1∶1000). Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1∶1000). Protein expression was observed by incubating the membranes with enhanced chemiluminescence and exposing the membranes to autoradiographic film. LabWorks™ Image Acquisition and Analysis Software (UVP) were used to quantify the band intensities. The rabbit anti-IRF1 and anti-Tubulin antibodies were purchased from Saier Inc. (Tianjin, China). Bioinformatics methods and statistical analysis The miRNA targets predicted by computer-aided algorithms were obtained from PicTar (http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi) and TargetScan Release 4.0 (http://www.targetscan.org). The two-tailed Student's t test was used to assess statistical significance where appropriate. The threshold for statistical significance was set at a p-value less than 0.05. Results miR-23a promotes cell proliferation and suppresses paclitaxel-induced apoptosis of gastric adenocarcinoma cells Human gastric adenocarcinoma cells were transfected with pcDNA3, pri-miR-23a, ASO-23a or ASO-NC. In BGC823 cells quantitative real-time PCR was performed to validate the efficiency of pri-miR-23a or ASO-23a compared to the controls, pcDNA3 and ASO-NC (Fig. 1A). The MTT assay was performed to determine the effects of miR-23a on cell viability. We observed that pri-miR-23a could increase cell viability by 24%±0.6% (p = 0.0003) and 20%±2.1% (p = 0.0014) in MGC803 and BGC823 cells, respectively, compared to the control pcDNA3 group. In contrast, ASO-23a reduced the cell viability of MGC803 cells to 71%±3.2% (p = 0.0415) and reduced that of BGC823 cells to 83%±7.3% (p = 0.0456) (Fig. 1B). To determine the effects of miR-23a on the long-term and independent growth activity, we performed colony formation assays. Compared with the control group, the colony formation ability of MGC803 cells showed a 1.33-fold (p = 0.0037) increase in the pri-miR-23a group and a 77%±3.8% (p = 0.0004) decrease in the ASO-23a group (Fig. 1C). In consonance with these findings, we observed the same effect in BGC823 cells. The colony formation ability of BGC823 cells was increased 1.81-fold (p = 0.0312) in the pri-miR-23a group and that of BGC823 cells was reduced to 52%±4.7% (p = 0.0065) in the ASO-23a group (Fig. 1C). Given that miR-23a can promote cell growth viability and colony formation ability, we also performed TUNEL assays to determine the effect of miR-23a expression on paclitaxel-induced apoptosis. 10.1371/journal.pone.0064707.g001Figure 1 Biological effects of the over-expression or knockdown of miR-23a in human gastric adenocarcinoma cells. Cells were transfected with pcDNA3, pri-miR-23a, ASO-NC or ASO-23a. (A) RNA was extracted from the transfected BGC823 cells, and the expression of miR-23a was measured by real-time PCR (n = 3, * p<0.05). (B) The cell viability of MGC803 and BGC823 cells was determined by MTT assay 72 h after transfection (n = 3, * p<0.05; *** p<0.001). (C) Cell clonogenicity was measured by the colony formation assay. MGC803 cells were cultivated for 10 days, and BGC823 cells were cultivated for 14 days. The photos depict the stained colonies (n = 3,* p<0.05; p<0.01; * p<0.001). Cells were transfected with pcDNA3, pri-miR-23a, ASO-23a or ASO-NC. The over-expression of miR-23a suppresses paclitaxel-induced apoptosis of MGC803 and BGC823 cells (Fig. 2A and 2B). Conversely, blocking miR-23a promotes paclitaxel-induced apoptosis in MGC803 and BCG823 cells (Fig. 2A and 2B). Compared with the control group, the induced-apoptosis index of MGC803 cells was reduced to approximately 62%±0.8% (p<0.05) in the pri-miR-23a group, and the induced-apoptosis index was increased 1.69-fold (p<0.01) in the ASO-23a group. As expected, we obtained consistent results in BGC823 cells. The induced-apoptosis index of BGC823 cells was reduced to 54%±1% (p<0.05) in the pri-miR-23a group and was increased to 1.78-fold (p<0.01) in the ASO-23a group (Fig. 2C). These results indicate that miR-23a can repress paclitaxel-induced apoptosis and promote cell viability, which are the long-term growth ability and the independent growth ability of MGC803 and BGC823 cells. 10.1371/journal.pone.0064707.g002Figure 2 The effects of over-expression or knockdown of miR-23a in MGC803 and BGC823 cells on paclitaxel-induced apoptosis. MGC803 (A) and BGC823 (B) cells were transfected with pcDNA3, pri-miR-23a, ASO-NC or ASO-23a. Paclitaxel (0.5 ppc) was added to the cells to induce cell apoptosis, and apoptotic cells were detected by the TUNEL assay 48 h after transfection. (C) The induced-apoptosis indexes were expressed quantitatively (n = 3, p<0.05, p<0.01). IRF1 is directly targeted by miR-23a The up-regulated miRNAs, such as miR-23a, may function as oncogenes and may promote tumor growth by suppressing their target genes. Therefore, we used bioinformatic analysis methods to predict potential target genes that could mediate the cell growth functions of miR-23a. In our previous study, we demonstrated that IL6R is a target gene of miR-23a [22]. The tumor suppressor IRF1 appears to be another possible target gene of miR-23a, which is consistent with a model whereby miR-23a is an oncogenic miRNA that promotes tumor development by targeting and negatively regulating tumor suppressors. The 3′UTR of IRF1 mRNA contains miR-23a complementary binding sites, and these binding sites are conserved among several species (Fig. 3A). To determine whether miR-23a represses IRF1 expression by binding directly to its 3′UTR, we first used various algorithms to predict potential miR-23a binding sites in the 3′UTR of IRF1 (Fig. 3A). To confirm whether miR-23a can bind to the target site in IRF1 3′UTR directly, we constructed an enhanced green fluorescence protein reporter vector (EGFP-IRF1 3′UTR), in which the predicted target regions were inserted downstream of the EGFP coding region. MGC803 cells were co-transfected with the reporter vector and either pcDNA3, pri-miR-23a, ASO-NC or ASO-23a. As shown in Figure 3B, the relative intensity of EGFP was increased in the ASO-23a-transfected cells and was decreased in the pri-miR-23a group relative to the negative control group. Similarly, we constructed another EGFP reporter vector (EGFP-IRF1 3′UTR mutant) containing mutations in the miR-23a binding sites (Fig. 3A). Our results show that neither ASO-23a nor pri-miR-23a affects the intensity of the EGFP-IRF1 3′UTR mutant (Fig. 3C). These observations suggest that miR-23a binds directly to the 3′UTR of IRF1 and represses IRF1 expression. 10.1371/journal.pone.0064707.g003Figure 3 IRF1 is directly repressed by miR-23a. (A) As predicted by the TargrtScan and PicTar database, the IRF1 3′UTR contained a miR-23a binding site. The mutated IRF1 3′UTR containing several mutated nucleotides within the miR-23a binding site is shown. (B) The direct interaction of miR-23a and IRF1 3′UTR was confirmed by using a fluorescent reporter assay. MGC803 cells were transfected with the EGFP-IRF1 3′UTR reporter gene together with pcDNA3, pri-miR-23a, ASO-NC or ASO-23a. The cells were lysed 72 h after transfection, and the EGFP intensity was measured by spectrophotometry (n = 3, p<0.05). (C) MGC803 cells were transfected with the EGFP vector, the IRF1 3′UTR reporter or the mutant EGFP-3′UTR reporter in addition to pcDNA3, pcDNA3/pri-miR-23a, ASO-NC or ASO-23a. The fluorescence intensity was detected through the method described previously (n = 3, * p<0.05). (D) MGC803 and BGC823 cells were transfected with pcDNA3, pcDNA3/pri-miR-23a, ASO-NC or ASO-23a. RNA was extracted from the transfected cells, and the expression of IRF1 mRNA was measured by real-time PCR (n = 3, * p<0.05). (E) We determined the protein expression level of IRF1 in MGC803 and BGC823 cells by western blot. The numerals above the western blot image show the ratios of the densitometry of IRF1 and GAPDH when compared with the control group. To examine whether miR-23a antagonizes endogenous IRF1 expression, quantitative real-time PCR and western blot analyses were performed to detect IRF1 mRNA and protein expression. We transfected MGC803 and BGC823 cells with pri-miR-23a, ASO-23a, pcDNA3 or ASO-NC. When miR-23a expression was inhibited, IRF1 mRNA levels increased, whereas when miR-23a was over-expressed, IRF1 mRNA levels were decreased relative to the control group (Fig. 3D). To confirm the results obtained above, we also analyzed the protein expression level of IRF1 in the above four groups. As expected, the results of the protein expression analysis were consistent with the quantitative real-time PCR results. The IRF1 protein level of pri-miR-23a-transfected cells decreased 0.56 fold in MGC803 cells and 0.66 fold in BGC823 cells relative to the control-transfected cells (Fig. 3E). Conversely, IRF1 expression in ASO-23a-transfected cells showed a 1.55-fold increase in MGC803 cells and a 1.44-fold increase in BGC823 cells relative to the ASO-NC groups (Fig. 3E). Collectively, these results suggest that miR-23a regulates endogenous IRF1 expression at the post-transcriptional level. Inverse expression of miR-23a and IRF1 in gastric adenocarcinoma tissues In a previous study from our lab, we showed that miR-23a is up-regulated in gastric adenocarcinomas based on oligonucleotide microarrays. To further confirm the up-regulation of miR-23a in gastric adenocarcinomas, quantitative real-time PCR was applied to detect the expression level of miR-23a in 9 pairs of gastric adenocarcinoma tissue samples and matched normal gastric tissue samples. The results showed that miR-23a was remarkably up-regulated in gastric adenocarcinoma tissue samples (Fig. 4A). The relative expression levels of miR-23a in 9 pairs of gastric adenocarcinoma tissue samples were 7.89, 2.22, 2.06, 37.44, 7.09, 5.49, 7.09, 0.64 and 0.76 (p<0.05), respectively, compared to matched normal gastric tissue samples (Fig. 4A). We also detected the expression level of IRF1 mRNA in these samples. Fig. 4B shows that IRF1 mRNA is consistently down-regulated in gastric adenocarcinoma tissue samples when compared with the matched normal gastric tissue samples. The relative expression levels of IRF1 mRNA in 9 pairs of gastric adenocarcinoma tissue samples were 0.17, 1.14, 0.27, 0.19, 0.20, 0.84, 0.38, 1.00 and 0.03 (p<0.05), respectively, compared with the matched normal gastric tissue samples (Fig. 4B). To further confirm the IRF1 level in tissues, we utilized an immunohistochemistry assay to detect the IRF1 expression level in these tissues. As shown in Figure 4C, the expression levels of IRF1 in cancer tissues were significantly lower than those in matched normal tissues. Thus, these results provide strong evidence that miR-23a is prominently over-expressed in gastric adenocarcinomas, and the expression of IRF1 in gastric adenocarcinomas is much lower than normal, which supports the hypothesis that miR-23a negatively regulates IRF1 in gastric adenocarcinoma tissues. 10.1371/journal.pone.0064707.g004Figure 4 Differential expressions of miR-23a and IRF1 in gastric adenocarcinoma tissues and matched normal tissues. (A) The expression of miR-23a was detected by real-time PCR in 9 pairs of gastric adenocarcinoma tissue and the corresponding adjacent normal tissue. U6 RNA was included as an endogenous housekeeping gene, and the relative miR-23a expression is shown (n = 9, p<0.05). (B) The expression of IRF1 mRNA was detected by real-time PCR, and β-actin was used as an endogenous control (n = 9, p<0.05). (C) The expression of IRF1 in gastric adenocarcinoma tissues and matched normal tissues by immunohistochemistry (n = 9, p<0.05). IRF1 represses cell proliferation and promotes paclitaxel-induced apoptosis in gastric adenocarcinoma cells Previous studies have shown that IRF1 plays an important role in suppressing tumor cell proliferation and functions as a tumor suppressor. Accordingly, to further confirm that miR-23a promotes the growth of gastric adenocarcinoma cells by down-regulating IRF1, we constructed pSilencer/sh-IRF1 plasmids to knockdown the expression of IRF1. We also constructed the expression vector pCD3/IRF1, which lacks a 3′UTR and is thus not subjected to miR-23a regulation. Quantitative real-time PCR and western blot analysis were used to confirm IRF1 expression in transfected MGC803 and BGC823 cells. Our results show that pSilencer/sh-IRF1 effectively suppresses both the mRNA and protein expression of IRF1. The IRF1 mRNA level of sh-IRF1-transfected cells decreased to 62%±2% (p<0.05) in MGC803 cells and 52%±1.5% (p<0.05) in BGC823 cells relative to the control cells (Fig. 5A). The knockdown of IRF1 caused a 45%±1.2% (p<0.01) or 55%±2.1% (p<0.01) reduction in the protein expression of IRF1, respectively, in MGC803 cells and BGC823 cells (Fig. 5B). Furthermore, pcDNA3/IRF1 significantly increased the protein expression level of IRF1. The over-expression of IRF1 caused a 1.88-fold (p<0.01) increase in IRF1 protein expression in MGC803 cells and a 1.44-fold (p<0.01) increase in IRF1 protein expression in BGC823 cells (Fig. 5B). 10.1371/journal.pone.0064707.g005Figure 5 Knockdown or over-expression of IRF1 alters the growth and colony formation ability of gastric adenocarcinoma cells. The two cell lines MGC803 and BGC823 were transfected with pSilencer/sh-IRF1, pcDNA3/IRF1 or the appropriate control vectors. (A) Real-time PCR was performed to detect the efficiency of pSilencer/sh-IRF1 (n = 3, * p<0.05). RNA was extracted 48 h after transfection. (B) The IRF1 protein expression level of the transfected MGC803 and BGC823 cells was detected by western blot analysis. The numerals above the western blot image show the ratio of the densitometry of IRF1 and GAPDH when compared with the control. (C) The cell growth viability of the two cell lines was determined by the MTT assay 72 h after transfection (n = 3, *** p<0.001). (D) Cell clonogenicity was measured by colony formation assay. MGC803 cells were grown for 10 days, and BGC823 cells were grown for 14 days. The images represent the stained colonies (n = 3,* p<0.05). The MTT assay was used to determine the effect of IRF1 expression on cell viability. The knockdown of IRF1 showed an increase in cell viability, whereas the over-expression of IRF1 decreased the cell viability (Fig. 5C). The cell viability of sh-IRF1-transfected cells increased 1.39-fold (p = 0.0004) in MGC803 cells and 1.66-fold (p<0.0001) in BGC823 cells relative to the control cells. Conversely, the cell viability of IRF1-transfected cells showed a 0.86-fold (p = 0.0003) decrease in MGC803 cells and a 0.80-fold (p = 0.0008) decrease in BGC823 cells relative to the pcDNA3 groups (Fig. 5C). The colony formation assay was performed to detect the long-term and independent cell growth ability of these cells upon modulating IRF1 expression. The knockdown of IRF1 expression showed a 1.39-fold (p = 0.0238) increase in MGC803 cells and a 1.66-fold (p = 0.0002) increase in BGC823 cells, whereas the over-expression of IRF1 caused a 51%±5.7% (p = 0.0028) decrease in the clonogenicity of MGC803 cells and a 59%±3.4% (p = 0.0001) decrease in that of BGC823 cells (Fig. 5D). Next, the TUNEL assay was used to detect paclitaxel-induced apoptosis in gastric adenocarcinoma cell lines. Compared to the control group, the over-expression of miR-23a suppressed paclitaxel-induced apoptosis in MGC803 and BGC823 cells, whereas the knockdown of miR-23a caused the opposite results (Fig. 6A and 6B). The induced-apoptosis index of MGC803 cells was reduced 37%±5.1% (p<0.01) in the sh-IRF1 group and increased 2.32-fold (p<0.01) in the IRF1 group. Similar results were observed in BGC823 cells. Compared with the control group, the induced-apoptosis index of BGC823 cells was reduced 59%±1.2% (p<0.05) in the sh-IRF1 group and was increased 1.84-fold (p<0.05) in the IRF1 group (Fig. 6C). These data indicate that IRF1 represses cell proliferation and promotes paclitaxel-induced apoptosis in gastric adenocarcinoma cell lines. Ultimately, IRF1 appears to play the role of a tumor suppressor in the tumorigenesis of gastric adenocarcinomas. 10.1371/journal.pone.0064707.g006Figure 6 The effects of the over-expression or knockdown of IRF1 in gastric adenocarcinoma cells on paclitaxel-induced apoptosis. Gastric adenocarcinoma cells were transfected with pSilencer, pSilencer/sh-IRF1, pcDNA3 or pcDNA3/IRF1. Paclitaxel (0.5 ppc) was added to the cells to induce apoptosis, and the TUNEL assay was performed to detect apoptosis in the transfected cells. The images in (A) show the paclitaxel-induced apoptosis of MGC803 cells, and the images in (B) show the paclitaxel-induced apoptosis of BGC823 cells. (C) The induced-apoptosis results are expressed quantitatively (n = 3, p<0.05, p<0.01). Restoration of IRF1 expression counteracts the effects of miR-23a To validate whether the effects of miR-23a expression on cell growth and paclitaxel-induced apoptosis in MGC803 and BGC823 cells are mediated by IRF1, we transfected pri-miR-23a into MGC803 and BGC823 cells along with either the control pcDNA3 or pcDNA3/IRF1. Relative to the control, expression of pcDNA3/IRF1 reversed the negative effects of miR-23a on IRF1 protein expression in MGC803 cells and BGC823 cells (Fig. 7A). The ectopic expression of IRF1 reversed increased in cell viability caused by miR-23a from approximately 1.34-fold (p = 0.0048) to 0.74-fold (p = 0.0005) in MGC803 cells and from 1.41-fold (p<0.0001) to 1.01-fold (p<0.0001) in BGC823 cells compared to control group (Fig. 7B). Similar results were observed in the colony formation assay (Fig. 7C). The ectopic expression of IRF1 also counteracted the inhibition of paclitaxel-induced apoptosis caused by miR-23a expression in the TUNEL assay (Fig. 8 A and 8B). Relative to the control vector, the expression of the pcDNA3/IRF1 construct reversed the negative effects of miR-23a on paclitaxel-induced apoptosis from approximately 42%±8% (p<0.05) to 89%±9% (p<0.05) in MGC803 cells and approximately from 39%±7% (p<0.05) to 92%±8% (p<0.05) in BGC823 cells (Fig. 8C). These results suggest that IRF1 is a tumor suppressor, functions as a target of miR-23a and is involved in the miR-23a–mediated malignant phenotype of gastric adenocarcinoma cells. 10.1371/journal.pone.0064707.g007Figure 7 Restoration of IRF1 counteracts the miR-23a-induced cellular phenotypes in gastric adenocarcinoma cells. MGC803 and BGC823 cells were co-transfected with pri-23a and either pcDNA3-IRF1or pcDNA3; the pcDNA3 group was used as the control. (A) Proteins were extracted 48 h after transfection, and western blot analysis was used to detect IRF1 protein expression. The numerals above the western blot image show ratios of the densitometry of IRF1 and GAPDH when compared with the control. (B) The cell growth viability of BGC823 cells was determined by MTT assay at 72 h after transfection. (C) Cell clonogenicity was measured by colony formation assay. Pictures under the graph show the stained colonies (n = 3, p<0.05). 10.1371/journal.pone.0064707.g008Figure 8 Restoration of IRF1 counteracts the effect of miR-23a on paclitaxel-induced apoptosis in gastric adenocarcinoma cells. MGC803 and BGC823 cells were co-transfected with pcDNA3-IRF1 and either pri-23a or pcDNA3. Paclitaxel (0.5 ppc) was added to the cells after transfection to induce apoptosis. The TUNEL assay was then performed to detect paclitaxel-induced apoptosis in the transfected cells. The photos in Figure 8A show apoptotic MGC803 cells. The photos in Figure 8B show apoptotic BGC823 cells. (C) The induced-apoptosis results were expressed quantitatively (n = 3, p<0.05, *p<0.01). Discussion The prior study by Nozawa showed that the loss of functional IRF-1 is critical for the development of human gastric cancers [24]. Consistent to our study, IRF-1 was shown to be a transcription factor which acts as a tumor suppressor in gastric cancer. It also demonstrated the loss of functional IRF1 is critical for the development of human gastric cancers. The loss of heterozygosity (LOH) observed in human gastric cancer strongly suggests the existence of tumor suppressor genes at the concerned locus. The IRF1 locus on chromosome 5q31.1 is one of the common minimal regions of LOH in gastric cancer. A prior study reported a case of gastric adenocarcinoma with a point mutation in the second exon of the IRF1 gene of the residual allele, leading to the production of functionally impaired IRF1 [25]. These alternations are an important mechanism of the IRF1-mediated regulation of carcinogenesis at the gene expression level. However, epigenetic mechanisms have been reported to contribute to the decreased expression of IRF1 in various cancers. Yamashita M et al. proposed that the epigenetic inactivation of IRF1 plays a key role in the tumorigenesis of gastric cancer and that the inhibition of DNA methylation may restore the antitumor activity of interferons through the up-regulation of IRFs [26], [27]. Kondo T et al. reported that a nuclear factor, nucleophosmin (NPM), inhibited the DNA-binding and transcriptional activity of IRF-1 in human cancer development. This mechanism represents an alternative pathway by which IRF1 may be inactivated [28]. Here, we have reported a post-transcriptional mechanism in which miR-23a directly targets IRF1 and down-regulates its expression level in gastric adenocarcinoma cell lines (Fig. 9). This conclusion was arrived at from our findings, which can be summarized in six major points. (a) We combined bioinformatic prediction software including TargetScan, PicTar, miRBase and mirnaviewer, with human gene associations for cell proliferation and apoptosis and compiled the resulting data using the AmiGO website. Interferon regulatory factor 1 was predicted to be a candidate target for further study. (b) We found that miR-23a is up-regulated, whereas IRF1 is down-regulated in gastric adenocarcinoma tissues compared with matched normal tissues. (c) miR-23a negatively regulated IRF1 at both the mRNA and protein level. (d) The expression of the EGFP reporter containing the 3′-UTR of IRF1 was inhibited when miR-23a was over-expressed. (e) The over-expression of IRF1 suppressed cellular proliferation and promoted apoptosis [29], [30]. (f) The expression of IRF1 was sufficient to counteract the miR-23a-mediated promotion of cellular proliferation and the repression of cell apoptosis. The results of this study suggest that miR-23a promotes the tumorigenesis of gastric adenocarcinoma by negatively regulating IRF1. As expected, IRF1 can also be regulated by other miRNAs [31]. IRF1 has been shown to be a functional target of hCMV-miR-UL 112 by fluorescent reporter assays [32]. Another study revealed that the down-regulation of miR-383 is associated with male infertility and testicular germ cell tumors through its targeting of IRF1 [33]. In acute promyelocytic leukemia cells, IRF1 expression is associated with miR-342 [34]. We also cannot exclude the possibility that other miRNAs may regulate the expression of IRF1 in gastric cancer cells. In addition to the finding that IRF1 behaves as a tumor suppressor gene in various cancers, it is also involved in the regulation of apoptosis by many pathways. Our study is consistent with previous studies that showed that IRF-1 induced apoptosis in gastric cancer cells [35]. In this study, we performed the TUNEL assay to show that IRF1 can inhibit cellular apoptosis in gastric adenocarcinoma cells. IRF1 has been shown to activate the caspase cascade and induce apoptosis in breast cancer, which can occur in a p53-dependent or p53-independent manner [36]. IRF1 is also known to activate caspase1, caspase3, caspase7, and caspase8 [37], [38]. 10.1371/journal.pone.0064707.g009Figure 9 Distinct Mechanisms Used by IRF1 in gastric tumorigenesis In post-transcription level, IRF1 expression level was suppressed and directly targeted by miR-23a, which plays an important role in tumorigenesis of gastric cancer. IRF1 gene deletion and methylation repressed its expression in gene expression and epigenetic level, respectively. IRF1 was originally identified as a transcriptional activator in the interferon (IFN) system and has been shown to bind to the promoter regions of IFN α/β. Based on previous studies, a novel anti-cancer mechanism involving IFN-c/IRF1 signaling which down-regulates hTERT expression has been suggested [39]. It is well known that the type I and type II IFNs play important roles in regulating immune responses during bacterial and viral infections. Helicobacter pylori infection is believed to be the cause of most stomach cancer. In more detail, Helicobacter pylori are the main risk factor in 65–80% of gastric cancers [40]. The mucosal inflammatory response to H. pylori infection is complex and IRF1 make a role in this procedure [41]. IRF1 is an activator of IFNs that also has antiviral properties [42]. Epstein-Barr virus (EBV) DNA is found within the malignant cells of 10% of gastric cancers [43]. Schaefer et al. reported that IRF1 and IRF2 can constitutively activate the promoter of the EBV BamHI Q fragment [43], but whether miR-23a-mediated IRF1 suppression is involved in EBV infection in gastric cancer remains unknown. Various evidence has shown a correlation between IRF1 expression and EBV [44], HPV [45], HBV [46], [47], HIV [48] and West Nile virus infections. Whether IRF1 is related to EBV infection in gastric cancer needs further research. In summary, we have demonstrated that miR-23a down-regulates IRF1 expression by targeting the 3′UTR of IRF1 to regulate pro-proliferative and anti-apoptotic activity in gastric cancer cells. This reported role for miR-23a may provide new insight into the tumorigenesis of gastric cancer and suggests that miR-23a may have potential value as a diagnostic and treatment marker in cancers. Supporting Information Table S1 The information of gastric adenocarcinoma tissues used in this study. (DOC) Click here for additional data file. We thank Prof. Da-lin Ren for technical assistance with fluorescence detection. ==== Refs References 1 Abbasi SY , Taani HE , Saad A , Badheeb A , Addasi A (2011 ) Advanced gastric cancer in jordan from 2004 to 2008: a study of epidemiology and outcomes . Gastrointest Cancer Res 4 : 122 –127 .22368735 2 Fock KM , Ang TL (2010 ) Epidemiology of Helicobacter pylori infection and gastric cancer in Asia . J Gastroenterol Hepatol 25 : 479 –486 .20370726 3 Kim YH , Liang H , Liu X , Lee JS , Cho JY , et al (2012 ) AMPKalpha modulation in cancer progression: multilayer integrative analysis of the whole transcriptome in Asian gastric cancer . Cancer Res 72 : 2512 –2521 .22434430 4 Bornschein J , Rokkas T , Selgrad M , Malfertheiner P (2011 ) Gastric cancer: clinical aspects, epidemiology and molecular background . Helicobacter 16 Suppl 145 –52 .21896085 5 Mandong BM , Manasseh AN , Tanko MN , Echejoh GO , Madaki AJ (2010 ) Epidemiology of gastric cancer in Jos University Teaching Hospital Jos a 20 year review of cases . Niger J Med 19 : 451 –454 .21526638 6 Shin A , Kim J , Park S (2011 ) Gastric cancer epidemiology in Korea . J Gastric Cancer 11 : 135 –140 .22076217 7 Shridhar R , Dombi GW , Finkelstein SE , Meredith KL , Hoffe SE (2011 ) Improved survival in patients with lymph node-positive gastric cancer who received preoperative radiation: an analysis of the Surveillance, Epidemiology, and End Results database . Cancer 117 : 3908 –3916 .21365627 8 Katada T , Ishiguro H , Kuwabara Y , Kimura M , Mitui A , et al (2009 ) microRNA expression profile in undifferentiated gastric cancer . Int J Oncol 34 : 537 –542 .19148490 9 Li X , Zhang Y , Zhang H , Liu X , Gong T , et al (2011 ) miRNA-223 promotes gastric cancer invasion and metastasis by targeting tumor suppressor EPB41L3 . Mol Cancer Res 9 : 824 –833 .21628394 10 Liu T , Tang H , Lang Y , Liu M , Li X (2009 ) MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin . Cancer Lett 273 : 233 –242 .18789835 11 Sun Q , Gu H , Zeng Y , Xia Y , Wang Y , et al (2010 ) Hsa-mir-27a genetic variant contributes to gastric cancer susceptibility through affecting miR-27a and target gene expression . Cancer Sci 101 : 2241 –2247 .20666778 12 Zhu W , Shan X , Wang T , Shu Y , Liu P (2010 ) miR-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines . Int J Cancer 127 : 2520 –2529 .20162574 13 Zhu W , Xu H , Zhu D , Zhi H , Wang T , et al (2012 ) miR-200bc/429 cluster modulates multidrug resistance of human cancer cell lines by targeting BCL2 and XIAP . Cancer Chemother Pharmacol 69 : 723 –731 .21993663 14 Zhu W , Zhu D , Lu S , Wang T , Wang J , et al (2012 ) miR-497 modulates multidrug resistance of human cancer cell lines by targeting BCL2 . Med Oncol 29 : 384 –391 .21258880 15 Nakajima A , Nishimura K , Nakaima Y , Oh T , Noguchi S , et al (2009 ) Cell type-dependent proapoptotic role of Bcl2L12 revealed by a mutation concomitant with the disruption of the juxtaposed Irf3 gene . Proc Natl Acad Sci U S A 106 : 12448 –12452 .19617565 16 Cao M , Seike M , Soeno C , Mizutani H , Kitamura K , et al (2012 ) MiR-23a regulates TGF-beta-induced epithelial-mesenchymal transition by targeting E-cadherin in lung cancer cells . Int J Oncol 41 : 869 –875 .22752005 17 Kong KY, Owens KS, Rogers JH, Mullenix J, Velu CS, et al. (2010) MIR-23A microRNA cluster inhibits B-cell development. Exp Hematol.38 : : 629–640e621. 18 Wang Z , Wei W , Sarkar FH (2012 ) miR-23a, a critical regulator of “migR”ation and metastasis in colorectal cancer . Cancer Discov 2 : 489 –491 .22684455 19 Rathore MG , Saumet A , Rossi JF , de Bettignies C , Tempe D , et al (2012 ) The NF-kappaB member p65 controls glutamine metabolism through miR-23a . Int J Biochem Cell Biol 44 : 1448 –1456 .22634383 20 Huang S , He X , Ding J , Liang L , Zhao Y , et al (2008 ) Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells . Int J Cancer 123 : 972 –978 .18508316 21 Gottardo F , Liu CG , Ferracin M , Calin GA , Fassan M , et al (2007 ) Micro-RNA profiling in kidney and bladder cancers . Urol Oncol 25 : 387 –392 .17826655 22 Zhu LH , Liu T , Tang H , Tian RQ , Su C , et al (2010 ) MicroRNA-23a promotes the growth of gastric adenocarcinoma cell line MGC803 and downregulates interleukin-6 receptor . FEBS J 277 : 3726 –3734 .20698883 23 Schmittgen TD , Livak KJ (2008 ) Analyzing real-time PCR data by the comparative C(T) method . Nat Protoc 3 : 1101 –1108 .18546601 24 Nozawa H , Oda E , Ueda S , Tamura G , Maesawa C , et al (1998 ) Functionally inactivating point mutation in the tumor-suppressor IRF-1 gene identified in human gastric cancer . Int J Cancer 77 : 522 –527 .9679752 25 Cavalli LR , Riggins RB , Wang A , Clarke R , Haddad BR (2010 ) Frequent loss of heterozygosity at the interferon regulatory factor-1 gene locus in breast cancer . Breast Cancer Res Treat 121 : 227 –231 .19697121 26 Yamashita M , Toyota M , Suzuki H , Nojima M , Yamamoto E , et al (2010 ) DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae . Cancer Sci 101 : 1708 –1716 .20507321 27 Dhayalan A , Kudithipudi S , Rathert P , Jeltsch A (2011 ) Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase . Chem Biol 18 : 111 –120 .21276944 28 Kondo T , Minamino N , Nagamura-Inoue T , Matsumoto M , Taniguchi T , et al (1997 ) Identification and characterization of nucleophosmin/B23/numatrin which binds the anti-oncogenic transcription factor IRF-1 and manifests oncogenic activity . Oncogene 15 : 1275 –1281 .9315094 29 Hong S , Kim HY , Kim J , Ha HT , Kim YM , et al (2013 ) Smad7 Protein Induces Interferon Regulatory Factor 1-dependent Transcriptional Activation of Caspase 8 to Restore Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Apoptosis . J Biol Chem 288 : 3560 –3570 .23255602 30 Li P , Du Q , Cao Z , Guo Z , Evankovich J , et al (2012 ) Interferon-gamma induces autophagy with growth inhibition and cell death in human hepatocellular carcinoma (HCC) cells through interferon-regulatory factor-1 (IRF-1) . Cancer Lett 314 : 213 –222 .22056812 31 Muhie S, Hammamieh R, Cummings C, Yang D, Jett M (2012) Transcriptome characterization of immune suppression from battlefield-like stress. Genes Immun. 32 Stevens AM , Wang YF , Sieger KA , Lu HF , Yu-Lee LY (1995 ) Biphasic transcriptional regulation of the interferon regulatory factor-1 gene by prolactin: involvement of gamma-interferon-activated sequence and Stat-related proteins . Mol Endocrinol 9 : 513 –525 .7659094 33 Lian J , Tian H , Liu L , Zhang XS , Li WQ , et al (2010 ) Downregulation of microRNA-383 is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation by targeting IRF1 . Cell Death Dis 1 : e94 .21368870 34 De Marchis ML , Ballarino M , Salvatori B , Puzzolo MC , Bozzoni I , et al (2009 ) A new molecular network comprising PU.1, interferon regulatory factor proteins and miR-342 stimulates ATRA-mediated granulocytic differentiation of acute promyelocytic leukemia cells . Leukemia 23 : 856 –862 .19151778 35 Gao J , Senthil M , Ren B , Yan J , Xing Q , et al (2010 ) IRF-1 transcriptionally upregulates PUMA, which mediates the mitochondrial apoptotic pathway in IRF-1-induced apoptosis in cancer cells . Cell Death Differ 17 : 699 –709 .19851330 36 Ning Y , Riggins RB , Mulla JE , Chung H , Zwart A , et al (2010 ) IFNgamma restores breast cancer sensitivity to fulvestrant by regulating STAT1, IFN regulatory factor 1, NF-kappaB, BCL2 family members, and signaling to caspase-dependent apoptosis . Mol Cancer Ther 9 : 1274 –1285 .20457620 37 Stang MT , Armstrong MJ , Watson GA , Sung KY , Liu Y , et al (2007 ) Interferon regulatory factor-1-induced apoptosis mediated by a ligand-independent fas-associated death domain pathway in breast cancer cells . Oncogene 26 : 6420 –6430 .17452973 38 Liu L , Qiu W , Wang H , Li Y , Zhou J , et al (2012 ) Sublytic C5b-9 complexes induce apoptosis of glomerular mesangial cells in rats with Thy-1 nephritis through role of interferon regulatory factor-1-dependent caspase 8 activation . J Biol Chem 287 : 16410 –16423 .22427665 39 Lee SH , Kim JW , Lee HW , Cho YS , Oh SH , et al (2003 ) Interferon regulatory factor-1 (IRF-1) is a mediator for interferon-gamma induced attenuation of telomerase activity and human telomerase reverse transcriptase (hTERT) expression . Oncogene 22 : 381 –391 .12545159 40 Kikuchi S (2002 ) Epidemiology of Helicobacter pylori and gastric cancer . Gastric Cancer 5 : 6 –15 .12021854 41 Yamaoka Y , Kudo T , Lu H , Casola A , Brasier AR , et al (2004 ) Role of interferon-stimulated responsive element-like element in interleukin-8 promoter in Helicobacter pylori infection . Gastroenterology 126 : 1030 –1043 .15057743 42 Schoggins JW , Wilson SJ , Panis M , Murphy MY , Jones CT , et al (2011 ) A diverse range of gene products are effectors of the type I interferon antiviral response . Nature 472 : 481 –485 .21478870 43 Schaefer BC , Paulson E , Strominger JL , Speck SH (1997 ) Constitutive activation of Epstein-Barr virus (EBV) nuclear antigen 1 gene transcription by IRF1 and IRF2 during restricted EBV latency . Mol Cell Biol 17 : 873 –886 .9001242 44 Tang W , Morgan DR , Meyers MO , Dominguez RL , Martinez E , et al (2012 ) Epstein-barr virus infected gastric adenocarcinoma expresses latent and lytic viral transcripts and has a distinct human gene expression profile . Infect Agent Cancer 7 : 21 .22929309 45 Lace MJ , Anson JR , Klingelhutz AJ , Harada H , Taniguchi T , et al (2009 ) Interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (IRF)-1 . Carcinogenesis 30 : 1336 –1344 .19541854 46 Alcantara FF , Tang H , McLachlan A (2002 ) Functional characterization of the interferon regulatory element in the enhancer 1 region of the hepatitis B virus genome . Nucleic Acids Res 30 : 2068 –2075 .11972347 47 Guidotti LG , Morris A , Mendez H , Koch R , Silverman RH , et al (2002 ) Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice . J Virol 76 : 2617 –2621 .11861827 48 Su RC , Sivro A , Kimani J , Jaoko W , Plummer FA , et al (2011 ) Epigenetic control of IRF1 responses in HIV-exposed seronegative versus HIV-susceptible individuals . Blood 117 : 2649 –2657 .21200019	23785404	PMC3677940	CC BY	2021-01-05 17:29:20	yes	PLoS One. 2013 Jun 10; 8(6):e64707
==== Front Front Behav NeurosciFront Behav NeurosciFront. Behav. Neurosci.Frontiers in Behavioral Neuroscience1662-5153Frontiers Media S.A. 10.3389/fnbeh.2013.00063NeuroscienceReview ArticleEpisodic Memory: A Comparative Approach Martin-Ordas Gema 1Call Josep 21Center on Autobiographical Memory Research, Aarhus University, Aarhus, Denmark2Max Planck Institute for Evolutionary Anthropology, Leipzig, GermanyEdited by: Ekrem Dere, University Pierre and Marie Curie Paris, France Reviewed by: Thomas Zentall, University of Kentucky, USA; Jonathon D. Crystal, Indiana University, USA Correspondence: Gema Martin-Ordas, Department of Psychology, Center on Autobiographical Memory Research, Bartholins Allé 9, 8000 Århus C, Denmark e-mail: ordas@psy.au.dk11 6 2013 2013 7 6306 3 2013 22 5 2013 Copyright © 2013 Martin-Ordas and Call.2013This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Historically, episodic memory has been described as autonoetic, personally relevant, complex, context-rich, and allowing mental time travel. In contrast, semantic memory, which is theorized to be free of context and personal relevance, is noetic and consists of general knowledge of facts about the world. The field of comparative psychology has adopted this distinction in order to study episodic memory in non-human animals. Our aim in this article is not only to reflect on the concept of episodic memory and the experimental approaches used in comparative psychology to study this phenomenon, but also to provide a critical analysis of these paradigms. We conclude the article by providing new avenues for future research. episodic memoryepisodic-like memorysemantic memoryautobiographical memorynon-human animals ==== Body A crucial component of cognition is memory. Memory is made up of a number of different and inter-related systems that are defined, among other features, by how we access them or the type of information encoded (Squire, 1992; Miyashita, 2004). In the study of memory, one of the most influential distinctions is between semantic and episodic memory systems (Tulving, 1972). While semantic memory refers to relative permanent store of general world knowledge that persists over the years and is not related to specific events, episodic memory refers to specific personal past events (Tulving, 1983, 2005). The field of comparative psychology has adopted this distinction in order to study episodic memory in non-human animals (e.g., Clayton and Dickinson, 1998). Our aim in this article is not only to reflect on the concept of episodic memory and experimental approaches used in comparative psychology, but also to provide new avenues for future research. First, we comprehensively review Tulving’s definition of episodic memory. Next, we review the research done in the field of comparative psychology and we highlight some of its limitations. Drawing on this, we will provide a critical analysis of the consequences of basing the study of episodic memory exclusively on Tulving’s definition. Based on the autobiographical memory framework, we conclude the article by providing new avenues for future research. Episodic Memory Vs. Semantic Memory In the study of memory, one of the most influential distinctions is between semantic and episodic memory systems (Tulving, 1972). In his seminal work, Tulving (1972) defined semantic memory as our database of knowledge about the world, including words, objects, places, and people, and their inter-relationships. In contrast, episodic memory was defined as “an information processing system that (a) receives and stores information about temporally dated episodes or events, and about temporal-spatial relations among these events, (b) retains various aspects of this information, and (c) upon instructions transmits specific retained information to other systems, including those responsible for translating it into behavior and conscious awareness” (Tulving, 1972, p. 385). Thus, when we state that Leipzig is a city in Germany we are drawing on semantic memory; however when we remember biking to the zoo in Leipzig last summer, we are drawing on episodic memory. While Tulving’s first definition of episodic memory was instrumental in precipitating a new area of study, it lacked empirical support. On the matter of his early definition, Tulving (1983) wrote, “[I]t was impressionistic, incomplete, and somewhat muddled, whatever evidence existed to support it was all anecdotal” (Tulving, 1983, pp. 9). Thus, in order to examine his ideas experimentally, Tulving (1983) extensively cataloged the distinguishing features of semantic and episodic memory along the following three dimensions: kind of information processed, characteristics of operations, and applications (see Table 3.1 for complete summary, Tulving, 1983, Chap. 3). In addition, a new definition of episodic was born: it “is a recently evolved, late-developing, and early deteriorating past-oriented memory system, more vulnerable than other memory systems to neuronal dysfunction, and probably unique to humans. It makes possible mental time travel through subjective time, from the present to the past, thus allowing one to re-experience, through autonoetic awareness, one’s own previous experiences. Its operations require, but go beyond, the semantic memory system. Retrieving information from episodic memory (remembering or conscious recollection) is contingent on the establishment of a special mental set, dubbed episodic “retrieval mode” (p. 5, Tulving, 2005). Its operations depend on semantic memory, and it is sub-served by multiple brain regions including medial temporal lobes and prefrontal cortex. This definition of episodic memory was only superficially similar to the initial one. “Past events” become “past experiences” in an attempt to distinguish between semantic events and personal events. Tulving (1983) also added ideas about the evolution and development of episodic memory and he mentioned some neural substrates that could be involved in the retrieval of personal past events. Finally, a new operation was identified: “recollective experience.” Recollective experiences generate particular “feeling tones” (Tulving, 1983) that tell the rememberer that his memory relates to a personal experience he had in the past. This construct later evolved into “autonoetic consciousness,” or “the capacity that allows adult humans to mentally represent and to become aware of their protracted existence across time” (Wheeler et al., 1997, p. 335). It is for this reason that Tulving (2002) has argued that one of the cardinal features of episodic memory is that it operates in “subjective time,” and, therefore, it differs from semantic memory not only in being oriented to the past but also specifically in the past of the owner of that memory. So while some semantic knowledge does involve a datable occurrence (e.g., knowing when you were born), these memories are fundamentally different from episodic memories as they do not require any mental time travel (Tulving, 2002). Since then, Tulving (2002, 2005) has consistently emphasized that the critical distinction is not so much the type of information being processed, but instead the type of phenomenological experience that seems to play a crucial role in such a distinction. Of course, episodic memory still presupposes that the individual can retrieve the spatial-temporal context in which the to-be-remembered event occurred, and therefore, spatial-temporal context remains a critical component of episodic memory. In contrast, noetic (as opposed to autonoetic) consciousness is considered the defining property of semantic memory and is expressed without any such self-recollection but simply in awareness of familiarity or knowing. In a clear departure from previous usage, Tulving used the term “remembering” to refer to expressions of autonoetic consciousness and the term “knowing” to refer to expressions of noetic awareness. Comparative Approach For more than a decade, researchers have undertaken an extensive effort to identify processes in non-human animals that bear some relationship to the case of episodic memory in humans. However, while studies with humans can test the subjective and behavioral components of episodic memory, studies with non-human animals focus exclusively on the behavioral elements. This is so because we lack behavioral markers for subjective experiences. Next we review the main empirical approaches to the study of episodic memory in non-human animals. Episodic-like memory Clayton et al. (2003a) developed behavioral criteria for studying episodic memory that focus on Tulving (1972) classic definition of episodic memory: what occurred, where it took place, and when it transpired. This conceptualization is significant because it can be evaluated in non-human animals (henceforth animals). The focus is on the content of memory – knowledge of what, where, and when a unique event occurred. Clayton and Dickinson (1998) introduced the term episodic-like memory to emphasize that behavioral criteria do not assess subjective experiences. What-where-when Clayton and Dickinson (1998) used a food-caching and recovery paradigm to examine whether or not scrub-jays (Aphelocoma coerulescens) are capable of episodic-like memory, by testing their ability to remember what, where and when they have cached a particular food, based on a trial-unique experience of caching. Birds were allowed to cache perishable wax worms and non- perishable peanuts, and recover these items either after a short (4 h) or long (124 h) retention interval. Jays prefer wax worms to peanuts, so when given a choice between the two food types they would preferentially recover and eat worms. Of crucial importance was the fact that for one group of jays wax worms became inedible after 124 h but not 4 h, whereas for a second group of jays wax worms were edible after the short and long retention interval. For both groups, peanuts remained edible after both intervals. While the group of jays that experienced the perishability of the worms rapidly adopted the strategy of visiting worm locations before peanut locations after 4 h but visiting peanut locations before worm locations after 5 days, the other group recovered worms independently of the duration of the retention interval. In follow-up studies, Clayton and colleagues (Clayton and Dickinson, 1999a,b,c; Clayton et al., 2001, 2003b; de Kort et al., 2005) demonstrated that scrub-jays have detailed representations of what, where and when the food was cached. Other bird and mammal species have also been shown to possess such type of memory: rodents (Ergorul and Eichenbaum, 2004; Babb and Crystal, 2005, 2006; Ferkin et al., 2008; Roberts et al., 2008; Zhou and Crystal, 2009; see Bird et al., 2003 and McKenzie et al., 2005 for negative results), birds [Henderson et al., 2006 (when and where, but not what), Zinkivskay et al., 2009; see Skov-Rackette et al., 2006 for negative results], non-human primates (Martín-Ordás et al., 2010; see Hampton et al., 2005 and Dekleva et al., 2011 for negative results). Episodic-like memory met the behavioral criteria of what-where-when memory, originally suggested by Tulving (1972) but was called episodic-like because the introspective property of autonoetic or personal consciousness later introduced by Tulving (1983) could not be assessed in an animal. Thus, animals might be displaying a type of memory that has some but not all of the properties of human episodic memory. In fact, Suddendorf and Busby (2003) pointed out that this type of memory should be more properly called www-memory rather than episodic-like memory. They argued that one could know what happened, where and when (e.g., know when you were born) just by using semantic memory and without necessarily having to remember the event. In reply, Clayton et al. (2003a) indicated that in addition to the content of the memories, flexibility (i.e., flexible deployment of information) and structure (i.e., integrated “what-where-when” representation) of a memory are criteria that have to be met in order to define a memory as “episodic-like.” We now turn to these two aspects. Flexibility This criterion refers to the use of the encoded information in a variable way depending on the context. Clayton et al. (2003b) (see also Salwiczek et al., 2008) have argued that because episodic memories are embedded within the declarative system, which also encodes factual information (e.g., Tulving and Markowitsch, 1998), the information should not only be generalized across situations but also updated when new information is acquired after the encoding of the original information. Perhaps the most impressive demonstration of flexibility comes from a study in which the jays were allowed to cache perishable and non-perishable items, but then discovered in the interval between caching and recovery that the perishable food type degrades more quickly than originally learned (Clayton et al., 2003b; Babb and Crystal, 2006). Clayton and colleagues reasoned that if the birds do use a flexible declarative memory system, then they should update their knowledge about the rate of perishability of the food and change their search behavior at recovery accordingly. They should do so even if the episodic information about the caching event was encoded prior to the acquisition of the new knowledge about the decay rates. The jays behaved accordingly: if they cached perishable and non-perishable items in different locations in one tray and then subsequently discovered that the perishable items from another tray had degraded more quickly than they expected, then when given the original tray back the birds switched their search preference in favor of the nuts. Scrub-jays continued to search for the perishable food if it had been cached recently, thus showing that they had not simply developed a general aversion to searching for food that might perish (Clayton et al., 2003b). Thus, learning about the properties of the food items during training could be viewed as the acquisition of semantic information that is applicable to different events in a flexible way (Clayton et al., 2001, 2003a). Structure Clayton et al. (2003a) argued that the “what,” ”where,” and “when” should be bound together so they represent the same event, and therefore retrieving one of the components will imply the retrieval of the other components as well. This feature is crucial because it allows us to distinguish between episodes that share some of the components (e.g., going to have dinner with the same friend to the same restaurant on different occasions). In fact, remembering what-where-when is not sufficient to characterize a memory as episodic, unless it is also proven that these components are integrated in a representation. A demonstration of this feature comes from a study in which trained jays were allowed to cache peanuts and worms in one tray on 1 day, and then at a later time they cached the same food types in a second tray, after which the jays were allowed to recover from both trays (Clayton et al., 2001). The retention intervals are such that the worms will be decayed in the first tray while still being fresh in the second tray, and the critical question is whether the jays show the appropriate search pattern for each of the two trays. If the birds retrieved the “when” component separately, they could not have distinguished between the caching episodes because, by that account, the jays would simply associate caching the worms with a temporal tag, and the memory of caching worms at recovery would retrieve temporal tags for both the long and short retention intervals. In short, a linear mnemonic structure does not support the appropriate recovery pattern, namely searching for peanuts in the first tray and worms in the second tray. However, jays do in fact search appropriately, a result that suggests that they do form integrated memories, because they can distinguish in memory between the two caching episodes in terms of their time and location, even though they involved the same food items. Clayton et al. (2003a,b) interpreted this finding as evidence that the jays’ behavior met what they called the structural criterion for episodic-like memory [see Martín-Ordás et al. (2010) for evidence of integrative memories in great apes and Skov-Rackette et al. (2006) for lack of integrative memories in pigeons]. The know/remember paradigm The remember/know paradigm has been used in the study of recognition to distinguish between recollection and familiarity. Whereas recollection is associated with the retrieval of episodic memories, familiarity is associated with semantic memory (Yonelinas, 2001). Familiarity is determined by the strength of a perceptual match to prior exposure and, consequently, is susceptible to variations in superficial sensory qualities of the stimuli. In contrast, recollection allows the recovery of the previous episode in which the stimulus was experienced, and emphasizes conceptual properties (e.g., the meaning of the object to be recognized) as well as associations of the object, including the spatial and temporal context in which it was experienced. One way to empirically differentiate recollection and familiarity is the analysis of the receiver operating characteristics (ROC) functions of recognition-correct responses plotted against false positives as a function of response confidence across different decision criteria (Yonelinas, 2001). In order to obtain pairs of hit and false alarm rates at different decision criteria, participants are asked to provide confidence ratings for their yes/no recognition decisions. A pair of hit and false alarm rates is calculated for each level of confidence, and the paired values are plotted across the confidence levels to construct an ROC (Yonelinas, 2001). In tests of familiarity without recollection, the ROCs are curvilinear and symmetrical, whereas in recognition tests involving recollection, ROCs are linear and asymmetric in shape (Yonelinas, 1997, 1999a,b; Rotello et al., 2000; Slotnick et al., 2000; Kelley and Wixted, 2001; Arndt and Reder, 2002). Using the ROC procedure, Fortin et al. (2004) carried out an odor recognition judgment experiment. Rats first received a series of odors, each consisting of a series of different spices mixed in with playground sand in a plastic cup. After a time delay rats were presented with old and new odors. They were only rewarded for responding to the new odors. To produce the ROC function, authors compared hit and false alarm rates across different response criteria, which were obtained by using a combination of variations in the height of the test cup, making it more or less difficult to respond to that cup, and manipulations of the reward magnitudes associated with correct responses to the test and the unscented cup. Their results showed a very similar asymmetric curvilinear ROC to that of humans suggesting two component processes. Fortin et al. (2004) also explored the role of hippocampus in this task. Rats were divided into two groups (both matched on performance components): one group received lesions of the hippocampus and the other group received sham control operations. After recovery, authors tested recognition performance. Their results showed that the ROC of the control rats continued to reflect both recollection and familiarity components. In contrast, the ROC curves of the rats with lesions in the hippocampus were fully symmetrical and curvilinear, characteristic of familiarity based recognition in humans. Likewise, Sauvage et al. (2008) found that rats, who were required to recognize whether pairs of scents and substrates had been previously presented together or in a different pairing, produced curvilinear ROCs in rats with hippocampal lesions but linear ROCs in controls (see Wixted and Squire (2008) for critical interpretation of the results). In a similar vein, Basile and Hampton (2011) tested recollection and familiarity in five rhesus macaques. Authors trained the rhesus monkeys on a novel recall test in which they had to reproduce a simple figure on a touchscreen from memory. During the study phase, monkeys saw a simple shape composed of two or three colored boxes located on a grid on a computer touchscreen. During the test phase, one of the boxes appeared in a new location. Monkeys could reproduce the absent box or boxes by touching the appropriate grid locations. When successful, they earned food; errors were followed by a time out and no food. Critically, monkeys could not solve this memory test using familiarity, because the image to-be-remembered was not present during the test phase to experience as familiar. Performance was significantly above chance levels. In comparison with a recognition test matched (i.e., delayed match to sample test), they found that recognition accuracy was higher than recall accuracy at short delays but declined more rapidly. These results resemble those found in adult humans (Hockley, 1992; Yonelinas, 2002). However the extent to which Basile and Hampton’s task can be considered a pure recall task is debatable. This is because at test subjects were presented with cues (e.g., small white crosses), which indicated which response locations were available. Thus, subjects could identify the location in which they previously saw the (now) absent box by matching the representation in their memory to one or more choices (indicated by the white crosses). Unexpected question One of the characteristics of human episodic memories is that they are incidentally encoded and we usually encode features or details of an event without any conscious intent to do so (Morris and Frey, 1997; Zentall et al., 2001; Salwiczek et al., 2008). In fact, a recent study with adult humans on episodic memory (Holland and Smulders, 2011) has shown that when subjects were instructed to memorize the details of a what-where-when task, their performance was better than when subjects did not receive the instructions. Authors suggested that instructing people to memorize the details of the task could have increased their attention, which could have led to more accurate episodic memories for the event. Along the same line, Zentall et al. (2001) argued that in those experiments in which extensive training is required, animals might use semantic knowledge in the what-where-when discrimination because the contingencies are explicitly trained. Consequently, Zentall et al. (2001) suggested that using an unexpected question about a recent past event could be an advantageous method to test episodic-like memory in animals. They used a delayed-matching to sample task with pigeons in which subjects were required to remember whether or not they performed a particular action in the past. Their results clearly indicated that pigeons could reliably indicate whether or not they had pecked. Moreover, they were above chance on the very few first trials, when the element of surprise was still present. However, there is an important caveat in this experiment. As pointed out by Crystal (2009), episodic memory is defined as a long-term memory system and the unexpected question experiment carried out by Zentall et al. (2001) only dealt with short-time delays between the encoding event and the experimental question. Recently, Zhou et al. (2012) used the unexpected question after incidental encoding paradigm in a new way. Rats were trained on two tasks. In the first one, subjects were placed in a five-arm radial maze, three of which were open and had food placed at the end. Rats could visit these three arms and retrieve the food. After a delay period, rats were again given access to the five arms, although the food was only available in the two not previously visited arms. The second task consisted of learning to navigate a T-maze. At the beginning of a trial, a rat either received food or not: food delivery (or not) was the cue as to which way to turn at the end of the maze to retrieve additional food items (i.e., one direction if the rat had just eaten food, and the other if it had not). Once the rats were proficient in both tasks, they were presented with the crucial test: rats had access to three of the five arms of the radial maze, however they either received food or not. Next they were presented with the T-maze test, where they had to respond based on whether they had received or not food in the radial maze. Zhou et al. (2012) then temporarily inactivated the CA3 region of the hippocampus in some rats, and found very selective effects on performance in these tasks. They found that the inactivation of the CA3 region affected only performance to the unexpected questions. However, the more general responses in the T-maze when an expected question was asked (and the rats presumably could implement a planned action pattern) were not affected by the inactivation of the CA3 region. It seemed clear that hippocampus involvement was necessary for the rats to encode whether they ate (or not) so that they could later retrieve such information when the unexpected question was asked. Zhou et al.’s (2012) study offers new insights on episodic memory in non-human animals. However, would it be possible for the rat to simply know whether she received food (or not) in the radial maze and use the information to perform in the T-maze? That is, is the memory of the episode (e.g., contextual information) or the memory of having received food that is driving rats’ behavior? Unique trial learning Evidence for episodic-like memory using what-where-when, as reviewed above, is based on food-reward behavior, which usually requires extensive training procedures. We have already mentioned that one of the consequences of this training is that animals might encode the event information semantically. One way to address this criticism is by using the spontaneous unique trial paradigm. Episodic-like memory (what-where-when) This paradigm has been successfully implemented in rodents (rats: Kart-Teke et al., 2006; mice: Dere et al., 2005). Kart-Teke et al. (2006) (see also Dere et al., 2005) presented rats with a three-trial object exploration task in which memory for what (object recognition), where (location of the objects) and when (temporal order for the presentation of the objects) were combined. In the first sample trial, subjects explored four copies of a novel object. After a time delay, subjects were presented with a second sample trial, identical to the first, except that four novel objects were present, which were arranged in a different spatial configuration. After another delay, the subjects received a test trial identical to the second sample trial, except that two copies of the object from sample trial 1 (“old familiar” objects) and two copies of the object known from sample trial 2 (“recent familiar” objects) were present and one of the “old familiar” objects was shifted to a location in which it was not encountered during the sample trial 1. The results from these experiments showed that rats were sensitive not only to the location of the objects, but also to the temporal order in which they were presented. These results led the authors to conclude that rats integrated what, where and when an event happened. Overall, this paradigm seems to fulfill several features of human episodic memory. First of all, subjects are asked to remember a specific episode rather than learn over multiple trials to apply procedural rules (Zentall et al., 2001; Schwartz et al., 2005). Second, this task also shows the integration of information for what-where-when (Clayton and Dickinson, 1998). The length of the retention intervals (up to 50 min.) rules out the possibility that the animal’s performance during the test trial relies on short-term memory (Hampton and Schwartz, 2004). However, like in other attempts to model episodic memory, this paradigm does not allow the assessment of “conscious recollection.” Episodic-like memory (what-where-which) Humans are very poor at using information about the timing of events (Friedman, 1993, 2007). In those occasions in which temporal information is available, it often helps to dissociate between two similar memories. However, when no temporal information is available, we use contextual cues to help us differentiate events from one another. For example, we can differentiate between two events happening at the same restaurant because each time we went with a different friend. Accordingly, some authors have suggested that the concept of what-where-when should be broadened in order to include any contextual cue that defines a specific occasion in which an event occurred (Eacott and Norman, 2004; Eacott and Gaffan, 2005; Eacott et al., 2005). This what-where-which definition can include temporal contexts when the temporal cues define the exact occasion. However, when the temporal information is poor, other contextual markers can be used to help us recall the memory. That is, episodic memories contain not only the memory of what happened and where but also the complex visuo-spatial background in which the event took place (Gaffan, 1991, 1994). Eacott and Norman (2004) developed an experimental paradigm to test if rats remember what happened, where, and in which context. In their experiment, rats were allowed to explore an E-shaped maze containing three novel objects. Two different contexts were created by covering the maze with black cloth on one occasion and wire netting on another. For each context, the objects were placed in different positions and they were never visible from the entrance. After exposure to both contexts, subjects were habituated to two of the novel objects. Since rats have a natural preference for novel objects, the authors expected rats to go to the area containing the non-habituated object. This is what they found: when placed back in the maze (with the objects present but out-of-sight), rats often headed straight to the area containing the non-habituated item. Since rats remembered what was where in which context, authors suggested that this was evidence for episodic-like memory. They further argue that this experiment is powerful because subjects’ responses require no training and, therefore, no specific “rules” are acquired. Additionally, this paradigm reduces potential confounds caused by reinforced learning (Eacott and Norman, 2004). Furthermore, since exploring novelty is a natural response for many species, the recall of the more novel object/location/context appears to be unexpected, which meets the criteria for recollection. Thus, by using what-where-which rather than what-where-when, Eacott and colleagues seem to have demonstrated recollection of episodic (like) memory in rats (Eacott et al., 2005). Replacing “when” with “which” might get closer to the phenomenology of human episodic memory since the “when” element can be inaccurate or even absent but the rich context is central (Friedman, 1993, 2007). However, in terms of a behavioral criterion “which” can be interchange with “what” or “where” (e.g., rats would only need to remember the location of the objects within two different mazes). In consequence, Cheke and Clayton (2010) have argued that even though episodic-like memory of an event may not need to require the recollection of “when” the event occurred, this component is necessary to behaviorally confirm that memory is for a specific episode rather than for timeless facts about the spaces or objects involved in that event. Spontaneous recall (what-where-who) There is evidence that animals may engage in free recall. Schwartz et al. (2002) investigated whether a gorilla could remember who did what. In the training phase, the gorilla had to learn to associate five types of food and their English words with five wooden cards in which a picture of each food was represented. The gorilla also had to associate two trainers with their respective names. In the experimental condition, the two trainers were present, although only one of them gave him one of the food items. Sometime later (either 10 min or 24 h) the gorilla was provided with a set of seven cards, five for the different types of food and two for the two trainers. He was asked what he ate and who gave him the food in that particular episode. The gorilla was able to hand over the card that represented the type of food that was given to him and the card with the name of the trainer who had given him the food after the delay (Schwartz et al., 2002). However, it is still an open question whether the gorilla recalled the event or simply chose the cards that were more familiar to him (Schwartz, 2005; Schwartz et al., 2005). Menzel (2005) carried out a free recall experiment with a language-trained chimpanzee. In the study, the chimpanzee could see the caretaker hiding foods and assorting objects to an outdoor enclosure, but she was moved to indoor enclosure before she could get the food. At a retention interval as long as 16 h, the chimpanzee indicated which type of food was hidden and also where it was hidden based on unique events. Note that one possible alternative explanation for the chimpanzee’s performance is spatial semantic memory; that is, the chimpanzee may have updated her memory about spatial landmarks without recalling the food-hiding event. Episodic foresight It has been argued that the function of episodic memory lies not with the benefits of remembering per se, but that its function is to support future-planning, the ability to travel forwards in the mind’s eye to imagine future events and scenarios (Suddendorf and Corballis, 1997; Dudai and Carruthers, 2005; Schacter and Addis, 2007). This function of episodic memory and its role on episodic foresight has been one of the most explored areas in animal cognition. This comparative research has been based on the classic “spoon-test” (Tulving, 2005). Tulving describes an Estonian tale in which a girl dreamed that she went to a party and found that she could not eat a delicious chocolate pudding because she did not have a spoon with her. The next night, she falls asleep while holding a spoon in her hand because she wants to avoid making the same mistake again. Based on the “spoon-test,” Mulcahy and Call (2006) carried out a tool-use study with orangutans and bonobos. Subjects were presented with an out-of-reach reward and with a set of useful and useless tools, which they could take into a waiting room. To obtain the reward, subjects had to return to the room where the out-of-reach reward was placed, carrying the useful tool either an hour or 24 h after having seen the reward. Mulcahy and Call showed that great apes were capable of saving tools needed in a distant future (see Naqshbandi and Roberts, 2006; Dufour and Sterck, 2008; Osvath and Osvath, 2008 for similar results; however, see Suddendorf, 2006; Suddendorf and Corballis, 2007 for a critical view on these experiments). Likewise, scrub-jays have been demonstrated to have future-planning skills (Correia et al., 2007; Raby et al., 2007). One of the strongest evidence comes from Correia et al. (2007) study in which they provided evidence for scrub-jays being able to anticipate future specific hunger in the absence of a current immediate need. In this experiment, scrub-jays that were prefed one type of food (i.e., food A) preferentially cached a different type of food (i.e., food B) 3 h later. However, between caching and recovery, one group of scrub-jays was prefed with the alternative food (e.g., B) before being allowed to recover what they had cached. The next day and after they were prefed food A, instead of caching food B, jays preferentially cached food A, that they had been prefed, in anticipation of being prefed food B after caching and prior to recovery. Thus, Correia et al. (2007) concluded that in the absence of a specific hunger for a type of food (A), scrub-jays preferentially cached that type of food (A), in anticipation of being prefed food B prior to recovery (for a study with another corvid species also showing evidence for planning see Cheke and Clayton, 2011). Problems for and from the Comparative Approach We have reported that animals can, at least, remember information about past events. We have also described that animals are able to use this information to plan for future events. Whether they experience such events in the same way as humans and, in particular, whether they have any sense of personally having experienced those events is still unknown and perhaps unknowable. Next we critically examine some limitations of the episodic (like) memory approaches. Episodic vs. semantic memory The taxonomic distinction between episodic and semantic memory was a central feature of Tulving’s original conceptualization that has stood the test of time. Perhaps the most compelling evidence for the distinction between episodic and semantic memory comes from brain-based studies, particularly neuropsychological studies (Kapur, 1999; Conway and Fthenaki, 2000; Wheeler and McMillan, 2001). It has been shown that patients with medial temporal lobe lesions (e.g., those with Alzheimer’s disease-type temporal lobe degeneration) lose the ability to use episodic memory while retaining other classes of memory, including semantic memory (Vargha-Khadem et al., 1997; Hirano and Noguchi, 1998; Gadian et al., 2000). Conversely, patients with semantic dementia whose neural damage typically involves frontotemporal lobar degeneration (Neary et al., 1998; Hodges and Miller, 2001) are characterized by severe semantic memory loss, while their episodic memory is relatively spared (Snowden et al., 1994; Graham et al., 2003; McKinnon et al., 2006). However evidence for a clear-cut division between these two types of memories is somewhat contentious (Squire et al., 2004; Tulving, 2005). In fact, other lines of neuropsychological research have shown an interdependent relation between episodic and semantic memory (i.e., semantic memories are the basic material from which complex and detailed episodic memories are constructed; see Greenberg and Verfaellie, 2010 for a review). Autobiographical memory research has also adopted a more integrative approach in relation to this issue. Indeed, most of the memories reported in autobiographical memory studies have been described to include both semantic and episodic components (Rubin et al., 2003). The field of comparative psychology has adopted the distinction between episodic and semantic memory and, as described in the previous section, is still in widespread use. However, focusing on the study of episodic memory as an independent memory system has an important disadvantage: comparative psychologists have devoted less attention to the ways in which one form of memory might influence the other. What is the content of episodic memories: The role of the temporal component The temporal component is one of the main features of the episodic (like) memory definition. Remarkably there is a clear lack of agreement in the way that the temporal component has been operationalized. In some studies when is considered as “in which occasion” [e.g., order of events (Eichenbaum et al., 2005; Eacott and Easton, 2010)], in others defined when is defined as sensitivity to “how long ago” the caching/baiting event took place (Clayton and Dickinson, 1998; Clayton et al., 2003b) and in others “when” is defined as “in which moment” (Roberts et al., 2008; Zhou and Crystal, 2009). In fact, the extent to what remembering “order of events” require the same memory system as remembering “how long ago” is still an open question (Easton et al., 2012). Alternatively, some authors have suggested that the temporal component is not part of episodic memory (Menzel, 2005; Friedman, 2007; Suddendorf and Corballis, 2007; Zhou et al., 2012) and others have argued that an explicit temporal aspect is not always crucial (Easton et al., 2012). As we previously described, Eacott and colleagues’ definition of episodic-like memory does not specifically include temporal cues. Nonetheless, it is strikingly impaired by lesions within the hippocampal system, which has been described to be involved in episodic recall (Eacott and Norman, 2004; Easton et al., 2009; Langston and Wood, 2010). In contrast, some temporal cues, such as those relating to how long ago an event occurred, are vulnerable to a non-hippocampal lesion (Eacott and Easton, 2012). In a study designed to mirror the what-where-when and what-where-which tasks given to rats, human participants were sequentially presented with two complex scenes, each containing the same abstract objects but in different locations within the scene and each scene having a different background (Easton et al., 2012). Participants were then asked to make two-choice judgments about what they had seen, where and when (first or second scene) or in which location (based on the distinctive background of the scene). In addition, for each judgment participants made they were asked whether their memory for what they had seen came with a feeling of remembering (associated with episodic memory) or a feeling of knowing (associated with familiarity in the absence of episodic memory). Results showed that participants were able to correctly make the former judgments (first vs. second) even when they were not using episodic memory, as evidenced by their reports of the subjective feeling of knowing, rather than remembering. In contrast, judgments which asked participants on which occasion they had seen objects in particular locations (what-where-which occasion) were reliant on episodic memory as they could not be reliably answered when the participant did not have an experience of remembering which is associated with episodic memory (Easton et al., 2012). Therefore, episodic memory might specifically be about discriminating complex events from one another based on the arrangements of items on a particular occasion. The occasion may be defined by a number of cues, but crucially they do not have to be temporal in nature (Eacott and Easton, 2012). Autonoetic awareness Episodic memories involve re-experiencing situations. According to Tulving’s definition (1983), this feature implies conscious awareness of being engaged in the act of recollection. We mentioned already that it has proven exceptionally difficult to develop animal models of episodic memory processing. At the heart of this issue are the difficulties in precisely defining the terms episodic and semantic for animals without assuming that animals have a similar form of consciousness as is attributed to humans. Consider a chimpanzee that sees an experimenter hiding a tool in an enclosure and retrieves it the following day. How does the chimpanzee accomplish this task? Perhaps the chimpanzee mentally travels back in time and re-experiences the hiding event, as we might. Alternatively, the chimpanzee may simply know that the enclosure is some place in which tools are hidden and may be able to make use of salient cues to locate the object it desires. Likewise the chimpanzee may know exactly where the tool is without remembering the episode in which it was placed there. In all these cases, the behavioral outcome might be the same (i.e., the chimpanzee finds the tool); however what the chimpanzee has in mind when she is retrieving the tool differs drastically in each case. If we assume that subjective (autonoetic) awareness is the central and crucial component of episodic memory, then demonstrating this capacity in non-verbal animals is going to be a very difficult, if not impossible, task. However, two recent studies help to shed light on this issue. Recently, Lu et al. (2012) have demonstrated that rats’ brain has a default mode network. Such network has been identified as being involved in autonomous mental activity in humans. Since the hippocampus is a critical region in the default mode network and is also involved in recalling and planning, thus subjective experience might also be present in non-human animals (Corballis, 2013). The implication of this study is crucial for the field of comparative psychology, since the main missing component in the behavioral studies is the subjective one (Premack, 2007; Suddendorf and Corballis, 2007). On the other hand, Klein and Nichols (2012) have reported the case of an amnesic patient who was able to relive personal past events, although he was unable to experience them as his own past experiences; that is, his episodic memories were disconnected from autonoetic awareness. Klein (2013) argues that the difference between episodic and semantic memory is not at the content level but at the retrieval level. Although it still remains to be specified when autonoetic awareness comes at play at retrieval, it seems that the relation between episodic memory and autonoetic awareness might be more complicated than originally thought (Klein, 2012). The role of episodic memory in episodic foresight Martín-Ordás et al. (2012) critically analyzed the contribution of episodic and semantic memory to episodic foresight in humans and non-human animals. They suggest that despite the current claim that episodic memory is necessary for episodic foresight there is no clear evidence in the literature for such statement. In fact, only few studies in the human literature have empirically addressed this issue. D’Argembeau and Mathy (2011) examined the content of people’s thoughts when they were attempting to think about a possible personal future event. Their results showed that general personal knowledge plays a crucial role in the construction of episodic future thoughts. In fact, they reported that when participants attempted to construct specific future events in response to cue words, they most frequently activated personal semantic information and/or general events before producing the specific future event. There is also evidence that a substantial amount of people’s future-oriented thoughts consist of abstract representations that do not refer to specific events (Anderson and Dewhurst, 2009; D’Argembeau et al., 2011). Overall these studies seem to suggest that general knowledge or semantic memory plays a crucial role in constructing and thinking about future personal events. We have mentioned that some animals pass the spoon-test proposed by Tulving (2005). Nonetheless, this research has generally been criticized for two main reasons: first, solving these tasks does not necessarily reflect self-projection in the future event and second, semantic memory suffices to solve future-planning tasks. However, if humans can foresee future events based only on general events or semantic information (as we mentioned above), the same might be true for animals. A New Route: Autobiographical Memory Numerous researchers understand autobiographical memory as the kind of memory that allows one to remember personal past events (Tulving, 1983; Wheeler et al., 1997; Conway and Pleydell-Pearce, 2000; Pillemer, 2003; Rubin, 2006; Bauer, 2007; Berntsen, 2009). However, personal events can vary substantially regarding their temporal, spatial and social complexity. For example, remembering using a tool to crack-open a nut might be a simpler event compared to remembering using a tool to crack-open nut while sitting at the forest with other group mates in a warm spring day. In the latter example, the event (using a tool to crack-open a nut) embeds other personal events (other past experiences involving using tools to crack-open nuts in different locations) and is itself embedded in another event (searching for food). In addition, it also involves general knowledge about when and where to find nuts and how to open them. Thus, autobiographical memories include vivid contextual information, such as the image of the nut, who was also in the group, the location (i.e., episodic components); but also general knowledge about how to open a nut (i.e., semantic components). In addition, having an autobiographical memory requires a successful binding of contextual information, which will facilitate events to be distinguished one from each other. Thus, autobiographical memory research makes a clear distinction between the different components of an event (content and context) and, in addition, measures very lengthy intervals (Conway, 2009; Piolino et al., 2009). The assessment of autobiographical memory makes it possible to investigate not only the ability to recall specific and meaningful personal events, locating it in time and space, but also the ability to travel back into the past and relive specific details of that event, which distinguish it from any similar ones. Consequently, this has lead researchers to study autobiographical memory by using complex real life events (e.g., Rubin et al., 1986; Thompson et al., 1996; Rubin, 2006; Bauer, 2007). This is in contrast with episodic memory, which has been tested in the lab using word-list tasks (e.g., Tulving, 1983). Autobiographical memories are not always based on events that happened only once (e.g., memories from high-school). This is a crucial feature since this latter type of memory seems to not have a specific temporal component. Thus, one might be tempted to conclude that they are not necessarily drawn from episodic memory. Remarkably, these memories can also be rich in other contextual details (e.g., spatial location). In fact, if we were asked to remember one of our classrooms from high-school, we could probably provide precise details about the location, the color of the walls or what the arrangement of the classroom was like. Thus, the recollection of specific spatial contexts suggests that these memories have not been decontextualized and, therefore, they are not drawn from semantic memory. Likewise, when recollecting these types of memories, we might not seem to be conscious of a particular prior experience, but instead we seem to be conscious of a group of several previous experiences. Memories like these are not explicitly addressed in the classic episodic-semantic model, but they appear to fit Neisser’s (1981) idea of merging of memories for past events into one representative event, Barsalou’s (1988) concept of summarized or extended events, or Conway’s (2001) “general events” level of autobiographical knowledge. In terms of retrieval, autobiographical memories can be voluntarily retrieved (i.e., following a controlled and goal-directed retrieval process) but can also come to mind spontaneously and without any conscious or deliberate attempt to retrieve them, so-called involuntary autobiographical memories (Berntsen, 1996). Involuntary memories tend to be cued by some feature of the context at retrieval (most often something external to the person remembering), which matches distinctive features of the memory (Berntsen, 2009). Although involuntary autobiographical memories are generally recognized as important for our understanding of memory (see, e.g., Neisser, 1981; Mandler, 1994), were mentioned as one of three basic manifestations of memory by Ebbinghaus (1964), and have been observed in clinical settings in relation to a wide range of disturbances (e.g., Horowitz, 1975; Stevenson and Cook, 1995; Reynolds and Brewin, 1999), experimental psychologists have tended to neglect them (Tulving, 1983). Note, though, that research has shown that involuntary autobiographical memories are universal and they occur as often as voluntary autobiographical memories (Berntsen, 2009). Thus far, we have provided a brief review of research on autobiographical memory. We will draw on this research to suggest new research avenues. Future Directions Altogether the pieces of evidence reported here indicate that the distinction between episodic memory and semantic memory might not be as clear as it might seem. Also, the role that the temporal component plays in episodic memory is still under debate. In addition, no animal has unequivocally been shown to have episodic memory as described by Tulving (1983, 2005). Remarkably, if one were to apply all the behavioral and phenomenological criteria that have been put forward for animal studies to current human studies, these unfortunately fall short of measuring episodic memory (Dere et al., 2006; Eacott and Easton, 2012). Human studies do not demonstrate free recall of an integrated “what, where, and when” memory for unique experiences, whereby the memory test was unexpected, required conscious recollection from long-term memory and flexible use of such memory in novel situations. Thus, there is a need to define objective behavioral criteria by which memory for past events can be assessed in both humans and non-human animals. We believe that a way in which this can be achieved is by stepping out-of Tulving’s framework and broadening the field of comparative research to other theoretical frameworks. We suggest that the autobiographical memory research could play a pivotal role in such enterprise and will help to open new and promising lines of research. Since autobiographical memory research has shown that memories for personal past events seem to be integrated by episodic and semantic components, it is crucial to investigate what the contribution of each component could be. It is also clear that episodic memories are not only stored information about what happened or where the event took place. There is evidence that people store specific details but also general or external details about the past experience (Levine et al., 2002). Thus, the content of a past event can be integrated by episodic elements, such as the location where the event took place (e.g., my parents’ house) or what we were doing (e.g., eating a cake), and external or semantic elements (e.g., it was in summer because I was wearing summer clothes). Indeed, Eacott and Easton’s paradigm on what-where-which indirectly touches on this idea of internal (e.g., specific location of the objects in the maze) and external details (e.g., the maze). We believe that pursuing this line of research could help us better understand what animals remember about their past. Do animals remember in which context an event happened? Do they remember details about the context (e.g., spatial information, who was there)? Do they distinguish between events that have elements in common? Along the same line, future research should address in a more concise way how the relations between stimuli are encoded and bound together in relation to a context (Chalfonte and Johnson, 1996; Eichenbaum, 1997; Newcombe et al., 2007). Clayton et al. (2003a) proposed that a critical element of episodic-like memory is that the retrieved memory is about an integrated event; consequently, the representation of what-where-when should be integrated. However an important element that has not received enough attention is the context in which a past event took place. It still remains to be assessed whether the “where” component in the episodic-like memory tasks is equivalent to a context in a real-life complex event (e.g., meeting an old friend at a reunion party in Spain), as pointed out in the autobiographical memory literature (Cabeza and St Jacques, 2007). Thus, memories for past events might involve not only binding “what” (e.g., meeting a friend), “where” (e.g., in Spain) something happened, but also the context in which those elements took place (e.g., at a reunion party). In a similar vein, a recent study with rats investigated the issue of source memory (Crystal et al., 2013). Crystal et al. (2013) showed that rats remembered the source of encoded information by discriminating between events in which they found the food and events in which the experimenter placed the rat at the food. Their results also demonstrated that the inactivation of the CA3 region eliminated source memory. Even though these results have a crucial value for the field of episodic memory, we believe that it is still an open question whether non-human animals remember the source of encoded information after single trial exposures and after incidental encoding. We mentioned before that autobiographical memories can refer to specific unique episodes (e.g., your first talk at a conference) or general events, defined as summaries of repeated events or events extended in time (e.g., what you normally do and experience when you give talks at conferences). The recall of general and unique events has not been addressed in non-human animals. Most of the previous research on episodic-like memory investigated the recall a series of repeated unique events. We have already mentioned that an important limitation of using repeated trials is that at encoding subjects could anticipate that they would be tested later and, thus, only encode the information semantically. Thus, we suggest that future research should investigate whether animals can recall general and unique events. One way to address this issue would be to present animals with events that happened only once (e.g., food A is hidden in location A) and a series of similar events that happened more than once (e.g., subjects experience food B being hidden in location B1 and B2 more than once). Recall could be assessed by using the unexpected question paradigm. After a retention interval (e.g., 2 weeks), subjects are presented with a cue, either food A or food B. Would they remember where to search for food? Also if there are memories that are neither truly episodic nor semantic (i.e., general event), then a similar system oriented toward the future might also exist (Martín-Ordás et al., 2012). If so, this type of future thinking should differ from what Atance and O’Neill (2001) coined as “semantic” [i.e., knowing about a future situation (knowing that the next general elections in Spain are going to be in 2015)] and “episodic” future thinking [i.e., the capacity to self-projection in a future situation (imagining my next job interview)]. Similarly, lacking the self-knowing awareness of a past event might still allow us to have episodic foresight. Investigating when episodic memory is necessary for episodic foresight is crucial in order to understand the extent to which autonoetic awareness is necessary for episodic foresight. One possibility, as pointed out in the autobiographical memory research (Pillemer, 2001, 2003), is that the recollection of old episodic information could be used to solve problems in the present and to predict future events. This could occur through specific memories being related to, or representative of, important situations in life (e.g., remembering who is willing to help in a cooperative situation). Such events might be used as a touchstone to decide what action to take (e.g., choosing the helpful partner in order to solve a problem together). However, it is equally possible that one might also be able to do so without recollecting episodic information. Although we might have a less accurate image of the future scene, be less flexible in the way we imagine possible future scenarios, or plan less effectively than when we use episodic memory, we may still be able to project ourselves into the future scenario by using semantic knowledge (also Suddendorf and Corballis, 2007). Imagine a tool-use context in which animals are provided with three sources of experiences: a unique event, a general event and general/semantic knowledge. Are these three sources of experience equally useful to plan a future event (e.g., which tool I will need to solve the task)? Does semantic knowledge suffice for future-planning? This hypothesis should be tested. Alternatively, some authors (Nelson, 1992; Dessalles, 2007; Boyer, 2008) have attributed a social function to episodic memory (e.g., to tell stories, to share specific information or people’s reliability as coalition partners). Raby and Clayton (2009) further hypothesize that perhaps different evolutionary pressures drove the development of the two cognitive systems. They speculate that semantic memory could have evolved as a mechanism for learning from previous experience, and that episodic memory could have evolved as a social tool to promote a sense of self and understanding of others, in conjunction with theory of mind. We believe that empirically addressing these issues would provide us with a more comprehensive understanding of the evolution of episodic and semantic memory. Conclusion We have described various methodological approaches that have been used to study animals’ episodic memory. Findings suggest that the capacity to remember what-where-when-which is present in rodents, corvids, and non-human primates. We have pointed out some of the limitations of the classical approach to the study of episodic-like memory. We also suggest that it is necessary to approach the comparative study of episodic memory from a broader perspective. Turning our attention to the autobiographical memory framework might be helpful in order to use a more innovative and compelling approach to the study of this phenomenon in animals. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by the Danish National Research Foundation (DNRF93). ==== Refs References Anderson R. J. Dewhurst S. A. (2009 ). Remembering the past and imagining the future: differences in event specificity of spontaneously generated thought . Memory 17 , 367 –373 10.1080/09658210902751669 19235018 Arndt J. Reder L. M. (2002 ). Word frequency and receiver operating characteristic curves in recognition memory: evidence for a dual process interpretation . J. Exp. Psychol. Learn. Mem. Cogn. 28 , 830 –842 10.1037/0278-7393.28.5.830 12219793 Atance C. M. O’Neill D. K. (2001 ). Episodic future thinking . Trends Cogn. Sci. 5 , 533 –539 10.1016/S1364-6613(00)01804-0 11728911 Babb S. J. Crystal J. D. (2005 ). Discrimination of what, when and where: implications for episodic-like memory in the rat . Learn. Motiv. 36 , 177 –189 10.1016/j.lmot.2005.02.009 17118573 Babb S. J. Crystal J. D. (2006 ). Discrimination of what, when and where is not based on the time of the day . Learn. Behav. 34 , 124 –130 10.3758/BF03193188 16933798 Barsalou L. W. (1988 ). “The content and organization of autobiographical memories,” in Remembering Reconsidered: Ecological and Traditional Approaches to the Study of Memory , eds Neisser U. Winograd E. (New York : Cambridge University Press ), 193 –243 Basile B. Hampton R. R. (2011 ). Monkeys recall and reproduce simple shapes from memory . Curr. Biol. 21 , 774 –778 10.1016/j.cub.2011.03.044 21530257 Bauer P. J. (2007 ). Remembering the Times of Our Lives. Memory in Infancy and Beyond . Mahwah, NJ : Lawrence Erlbaum Berntsen D. (1996 ). Involuntary autobiographical memories . Appl. Cogn. Psychol. 10 , 435 –454 10.1002/(SICI)1099-0720(199610)10:5<435::AID-ACP408>3.0.CO;2-L Berntsen D. (2009 ). Involuntary Autobiographical Memories: An Introduction to the Unbidden Past . Cambridge : Cambridge University Press Bird L. R. Roberts W. A. Abroms B. Kit K. A. Crupi C. (2003 ). Spatial memory for food hidden by rats (Rattus norvegicus) on the radial maze: studies of memory for what, where and when . J. Comp. Psychol. 117 , 176 –187 10.1037/0735-7036.117.2.176 12856788 Boyer P. (2008 ). Evolutionary economics of mental time travel? Trends Cogn. Sci. 12 , 219 –224 10.1016/j.tics.2008.03.003 18468941 Cabeza R. St Jacques P. (2007 ). Functional neuroimaging of autobiographical memory . Trends Cogn. Sci. 11 , 219 –227 10.1016/j.tics.2007.02.005 17382578 Chalfonte B. L. Johnson M. K. (1996 ). Feature memory and binding in young and older adults . Mem. Cognit. 24 , 403 –416 10.3758/BF03200930 8757490 Cheke L. G. Clayton N. S. (2010 ). Mental time travel in animals . Cogn. Sci. 1 , 915 –930 Cheke L. G. Clayton N. S. (2011 ). Eurasian jays (Garrulus glandarius) overcome their current desires to anticipate two distinct future needs and plan for them accordingly . Biol. Lett. 8 , 171 –175 10.1098/rsbl.2011.0909 22048890 Clayton N. S. Bussey T. J. Dickinson A. (2003a ). Can animals recall the past and plan for the future? Nat. Rev. Neurosci. 4 , 685 –691 10.1038/nrn1180 12894243 Clayton N. S. Yu K. S. Dickinson A. (2003b ). Interacting cache memories: evidence for flexible memory use by western scrub-jays (Aphelocoma coerulescens) . J. Exp. Psychol. Anim. Behav. Process. 29 , 14 –22 10.1037/0097-7403.29.1.14 12561130 Clayton N. S. Dickinson A. (1998 ). Episodic-like memory during cache recovery by scrub jays . Nature 395 , 272 –274 10.1038/26216 9751053 Clayton N. S. Dickinson A. (1999a ). Memory for the contents of caches by scrub jays (Aphelocoma coerulescens) . J. Exp. Psychol. Anim. Behav. Process. 25 , 82 –91 10.1037/0097-7403.25.1.82 9987859 Clayton N. S. Dickinson A. (1999b ). Motivational control of caching behavior in the scrub jay. Aphelocoma coerulescens . Anim. Behav. 57 , 435 –444 10.1006/anbe.1998.0989 10049484 Clayton N. S. Dickinson A. (1999c ). Scrub jays (Aphelocoma coerulescens) remember the relative time of caching as well as the location and content of their caches . J. Comp. Psychol. 113 , 403 –416 10.1037/0735-7036.113.4.403 10608564 Clayton N. S. Yu K. S. Dickinson A. (2001 ). Scrub-jays (Aphelocoma coerulescens) form integrated memories of the multiple features of caching episodes . J. Exp. Psychol. Anim. Behav. Process. 27 , 17 –29 10.1037/0097-7403.27.1.17 11199511 Conway M. A. (2001 ). Sensory-perceptual episodic memory and its context: autobiographical memory . Philos. Trans. R. Soc. Lond. B Biol. Sci. 356 , 1375 –1384 10.1098/rstb.2001.0940 11571029 Conway M. A. (2009 ). Episodic memories . Neuropsychologia 47 , 2305 –2313 10.1016/j 19524094 Conway M. A. Fthenaki A. (2000 ). “Disruption and loss of autobiographical memory,” in Handbook of Neuropsychology: Memory and its Disorders , 2nd Edn , eds Boiler F. Grafman J. (Amsterdam : Elsevier Science ), 281 –312 Conway M. A. Pleydell-Pearce C. W. (2000 ). The construction of autobiographical memories in the self-memory system . Psychol. Rev. 107 , 261 –288 10.1037/0033-295X.107.2.261 10789197 Corballis M. C. (2013 ). Mental time travel: a case for evolutionary continuity . Trends Cogn. Sci. 17 , 5 –6 10.1016/j.tics.2012.10.009 23153675 Correia S. P. C. Dickinson A. Clayton N. S. (2007 ). Western scrub-jays anticipate future needs independently of their current motivational state . Curr. Biol. 17 , 856 –861 10.1016/j.cub.2007.03.063 17462894 Crystal J. D. (2009 ). Elements of episodic-like memory in animal models . Behav. Processes 80 , 269 10.1016/j.beproc.2008.09.009 18950694 Crystal J. D. Alford W. T. Zhou W. Hohmann A. G. (2013 ). Source memory in the rat . Curr. Biol. 23 , 387 –391 10.1016/j.cub.2013.01.023 23394830 D’Argembeau A. Mathy A. (2011 ). Tracking the construction of episodic future thoughts . J. Exp. Psychol. Gen. 140 , 258 –271 10.1037/a0022581 21401291 D’Argembeau A. Renaud O. Van der Linden M. (2011 ). Frequency, characteristics, and functions of future-oriented thoughts in daily life . Appl. Cogn. Psychol. 35 , 96 –103 10.1002/acp.1647 de Kort S. R. Dickinson A. Clayton N. S. (2005 ). Retrospective cognition by food-caching western scrub-jays . Learn. Motiv. 36 , 159 –176 10.1016/j.lmot.2005.02.008 Dekleva M. Dufour V. de Vries H. Spruijt B. M. Sterck E. H. M. (2011 ). Chimpanzees (Pan troglodytes) fail a what-where-when task but find rewards by using a location-based association strategy . PLoS ONE 6 :e16593 10.1371/journal.pone.0016593 21359204 Dere E. Huston J. P. De Souza Silva M. A. (2005 ). Integrated memory for objects, places and temporal order: evidence for episodic-like memory in mice . Neurobiol. Learn. Mem. 84 , 214 –221 10.1016/j.nlm.2005.07.002 16102980 Dere E. Kart-Teke E. Huston J. P. De Souza Silva M. A. (2006 ). The case for episodic memory in animals . Neurosci. Biobehav. Rev. 30 , 1206 –1224 10.1016/j.neubiorev.2006.09.005 17079013 Dessalles J. (2007 ). Storing events to retell them . Behav. Brain Sci. 30 , 321 –322 10.1017/S0140525X07002063 Dudai Y. Carruthers M. (2005 ). The Janus face of Mnemosyne . Nature 434 , 567 10.1038/434567a 15800602 Dufour V. Sterck E. H. M. (2008 ). Chimpanzees fail to plan in an exchange task but succeed in a tool-using procedure . Behav. Processes 79 , 19 –27 10.1016/j.beproc.2008.04.003 18502593 Eacott M. J. Easton A. (2010 ). Episodic memory in animals: remembering which occasion . Neuropsychologia 48 , 2273 –2280 10.1016/j.neuropsychologia.2009.11.002 19913043 Eacott M. J. Easton A. (2012 ). Remembering the past and thinking about the future: is it really about time? Learn. Motiv. 43 , 200 –208 10.1016/j.lmot.2012.05.012 Eacott M. J. Easton A. Zinkivsky A. (2005 ). Recollection in an episodic-like memory task in the rat . Learn. Mem. 12 , 221 –223 10.1101/lm.92505 15897259 Eacott M. J. Gaffan E. A. (2005 ). The roles of perirhinal cortex, postrhinal cortex, and the fornix in memory for objects, contexts, and events in the rat . Q. J. Exp. Psychol. B 58 , 202 –217 10.1080/02724990444000203 16194965 Eacott M. J. Norman G. (2004 ). Integrated memory for object, place and context in rats: a possible model of episodic-like memory in rats? J. Neurosci. 24 , 1948 –1953 10.1523/JNEUROSCI.2975-03.2004 14985436 Easton A. Webster L. A. D. Eacott M. J. (2012 ). The episodic nature of episodic-like memories . Learn. Mem. 19 , 146 –150 10.1101/lm.025676.112 22411421 Easton A. Zinkivskay A. Eacott M. J. (2009 ). Recollection is impaired, but familiarity remains intact in rats with lesions of the fornix . Hippocampus 19 , 837 –843 10.1002/hipo.20567 19235228 Ebbinghaus H. (1964 ). Memory: A Contribution to Experimental Psychology . New York : Dover. (Original work published 1885) Eichenbaum H. (1997 ). How does the brain organize memories? Science 277 , 330 –332 10.1126/science.277.5324.330 9518364 Eichenbaum H. Fortin N. J. Ergorul C. Wright S. P. Agster K. L. (2005 ). Episodic recollection in animals: "If it walks like a duck and quacks like a duck …." Learn . Motiv. 36 , 190 –207 10.1016/j.lmot.2005.02.006 Ergorul C. Eichenbaum H. (2004 ). The hippocampus and memory for "what," "where," and "when." Learn . Mem. 11 , 397 –405 10.1101/lm.73304 15254219 Ferkin M. H. Combs A. del Barco-Trillo J. Pierce A. A. Franklin S. (2008 ). Meadow voles, Microtus pennsylvanicus, have the capacity to recall the “what,” “where” and “when” of a single past event . Anim. Cogn. 11 , 147 –159 10.1007/s10071-007-0101-8 17653778 Fortin N. J. Wright S. P. Eichenbaum H. (2004 ). Recollection-like memory retrieval in rats is dependent on the hippocampus . Nature 431 , 188 –191 10.1038/nature02853 15356631 Friedman W. J. (1993 ). Memory for the time of past events . Psychol. Bull. 113 , 44 –66 10.1037/0033-2909.113.1.44 Friedman W. J. (2007 ). The meaning of “time” in episodic memory and mental time travel . Behav. Brain Sci. 30 , 323 10.1017/S0140525X07002087 Gadian D. G. Aicardi J. Watkins K. E. Porter D. A. Mishkin M. Vargha-Khadem F. (2000 ). Developmental amnesia associated with early hypoxic–ischaemic injury . Brain 123 , 499 –507 10.1093/brain/123.3.499 10686173 Gaffan D. (1991 ). Spatial organization of episodic memory . Hippocampus 1 , 262 –264 10.1002/hipo.450010311 1669303 Gaffan D. (1994 ). Scene-specific memory for objects: a model of episodic memory impairment in monkeys with fornix transection . J. Cogn. Neurosci. 6 , 305 –320 10.1162/jocn.1994.6.4.305 Graham K. S. Lee A. C. H. Brett M. Patterson K. (2003 ). The neural basis of autobiographical and semantic memory: new evidence from three PET studies . Cogn. Affect. Behav. Neurosci. 3 , 234 –254 10.3758/CABN.3.3.234 14672158 Greenberg D. L. Verfaellie M. (2010 ). Interdependence of episodic and semantic memory: evidence from neuropsychology . J. Int. Neuropsychol. Soc. 16 , 748 –753 10.1017/S1355617710000676 20561378 Hampton R. R. Hampstead B. M. Murray E. A. (2005 ). Rhesus monkeys (Macaca mulatta) demonstrate robust memory for what and where, but not for when, in an open-field test of memory . Learn. Motiv. 36 , 245 –259 10.1016/j.lmot.2005.02.004 Hampton R. R. Schwartz B. L. (2004 ). Episodic memory in nonhumans: what, and where, is when? Curr. Opin. Neurobiol. 14 , 192 –197 10.1016/j.conb.2004.03.006 15082324 Henderson J. Hurly T. A. Bateson M. Healy S. D. (2006 ). Timing in free living rufous humming birds Selasphorus rufus . Curr. Biol. 16 , 512 –515 10.1016/j.cub.2006.01.054 16527747 Hirano M. Noguchi K. (1998 ). Dissociation between specific personal episodes and other aspects of remote memory in a patient with hippocampal amnesia . Percept. Mot. Skills 87 , 99 –107 10.2466/pms.1998.87.1.99 9760633 Hockley W. E. (1992 ). Item versus associative information: further comparisons of forgetting rates . J. Exp. Psychol. Learn. Mem. Cogn. 18 , 1321 –1330 10.1037/0278-7393.18.6.1321 Hodges J. R. Miller B. (2001 ). The classification, genetics and neuropathology of frontotemporal dementia. Introduction to the special topic papers: part I . Neurocase 7 , 31 –35 10.1093/neucas/7.2.113 11239074 Holland S. M. Smulders T. V. (2011 ). Do humans use episodic memory to solve a What-Where-When memory task? Anim. Cogn. 14 , 95 –102 10.1007/s10071-010-0346-5 20661614 Horowitz M. J. (1975 ). Intrusive and repetitive thought after experimental stress: a summary . Arch. Gen. Psychiatry 32 , 1457 –1463 10.1001/archpsyc.1975.01760290125015 1200767 Kapur N. (1999 ). Syndromes of retrograde amnesia: a conceptual and empirical synthesis . Psychol. Bull. 125 , 800 –825 10.1037/0033-2909.125.6.800 10589303 Kart-Teke E. De Souza Silva M. A. Huston J. P. Dere E. (2006 ). Wistar rats show episodic-like memory for unique experiences . Neurobiol. Learn. Mem. 85 , 173 –182 10.1016/j.nlm.2005.10.002 16290193 Kelley R. Wixted J. T. (2001 ). On the nature of associative information in recognition memory . J. Exp. Psychol. Learn. Mem. Cogn. 27 , 701 –722 10.1037/0278-7393.27.3.701 11394675 Klein S. B. (2012 ). The self and its brain . Soc. Cogn. 30 , 474 –516 10.1521/soco.2012.30.4.474 Klein S. B. (2013 ). Making the case that episodic recollection is attributable to operations occurring at retrieval rather than to content stored in a dedicated subsystem of long-term memory . Front. Behav. Neurosci. 7 :3 10.3389/fnbeh.2013.00003 23378832 Klein S. B. Nichols S. (2012 ). Memory and the sense of personal identity . Mind 121 , 677 –702 10.1093/mind/fzs080 Langston R. F. Wood E. R. (2010 ). Associative recognition and the hippocampus: differential effects of hippocampal lesions on object-place, object-context and object-place-context memory . Hippocampus 20 , 1139 –1153 10.1002/hipo.20714 19847786 Levine B. Svoboda E. Hay J. F. Winocur G. Moscovitch M. (2002 ). Aging and autobiographical memory: dissociating episodic from semantic retrieval . Psychol. Aging 17 , 677 –689 10.1037/0882-7974.17.4.677 12507363 Lu H. Zou Q. Gu H. Raichle M. E. Stein E. A. Yang Y. (2012 ). Rat brains also have a default mode network . Proc. Natl. Acad. Sci. U.S.A. 109 , 3979 –3984 10.1073/pnas.1200506109 22355129 Mandler G. (1994 ). “Hypermnesia, incubation, and mind-popping: on remembering without really trying,” in Attention and Performance: Conscious and Unconscious Information Processing , eds Umiltà C. Moscovitch M. (Cambridge, MA : MIT Press ), 3 –33 Martín-Ordás G. Atance C. Louw A. (2012 ). The role of episodic and semantic memory in episodic foresight . Learn. Motiv. 43 , 209 –219 10.1016/j.lmot.2012.05.011 Martín-Ordás G. Haun D. Colmenares F. Call J. (2010 ). Keeping track of time: evidence of episodic-like memory in great apes . Anim. Cogn. 13 , 331 –340 10.1007/s10071-009-0282-4 19784852 McKenzie T. L. B. Bird L. R. Roberts W. A. (2005 ). The effects of cache modification on food caching and retrieval behavior by rats . Learn. Motiv. 36 , 260 –278 10.1016/j.lmot.2005.02.011 McKinnon M. C. Black S. E. Miller B. Moscovitch M. Levine B. (2006 ). Autobiographical memory in semantic dementia: implications for theories of limbic-neocortical interaction in remote memory . Neuropsychologia 44 , 2421 –2429 10.1016/j.neuropsychologia.2006.04.010 16765999 Menzel E. (2005 ). “Progress in the study of chimpanzee recall and episodic memory,” in The Missing Link in Cognition: Origins of Self-Reflective Consciousness , eds Terrace H. Metcalfe J. (New York : Oxford University Press ), 188 –224 Miyashita Y. (2004 ). Cognitive memory: cellular and network machineries and their top-down control . Science 306 , 435 –440 10.1126/science.1101864 15486288 Morris R. G. M. Frey U. (1997 ). Hippocampal synaptic plasticity: role in spatial learning or the automatic recording of attended experience? Philos. Trans. R. Soc. Lond. B Biol. Sci. 352 , 1489 –1503 10.1098/rstb.1997.0136 9368938 Mulcahy N. J. Call J. (2006 ). Apes save tools for future use . Science 312 , 1038 –1040 10.1126/science.1125456 16709782 Naqshbandi M. Roberts W. A. (2006 ). Anticipation of future events in squirrel monkeys (Saimiri sciureus) and rats (Rattus norvegicus): tests of the Bischof- Köhler hypothesis . J. Comp. Psychol. 120 , 345 –357 10.1037/0735-7036.120.4.345 17115855 Neary D. Snowden J. S. Gustafson L. Passant U. Stuss D. Black S. (1998 ). Frontotemporal lobar degeneration. A consensus on clinical diagnostic criteria . Neurology 51 , 1546 –1554 10.1212/WNL.51.6.1546 9855500 Neisser U. (1981 ). John Dean’s memory: a case study . Cognition 9 , 1 –22 10.1016/0010-0277(81)90011-1 7196816 Nelson K. (1992 ). Emergence of autobiographical memory at age 4 . Hum. Dev. 35 , 172 –177 10.1159/000277149 Newcombe N. S. Lloyd M. E. Ratliff K. R. (2007 ). “Development of episodic and autobiographical memory: a cognitive neuroscience perspective,” in Advances in Child Development and Behavior , Vol. 35 , ed. Kail R. V. (San Diego : Elsevier ), 37 –85 Osvath M. Osvath H. (2008 ). Chimpanzee (Pan troglodytes) and orangutan (Pongo abelii) forethought: self-control and pre-experience in the face of future tool use . Anim. Cogn. 11 , 661 –674 10.1007/s10071-008-0157-0 18553113 Pillemer D. (2001 ). Momentous events in the life story . Rev. Gen. Psychol. 5 , 123 –134 10.1037/1089-2680.5.2.123 Pillemer D. (2003 ). Directive functions of autobiographical memory: the guiding power of specific episodes . Memory 11 , 193 –202 10.1080/741938208 12820831 Piolino P. Desgranges B. Eustache F. (2009 ). Episodic autobiographical memories over the course of time: cognitive, neuropsychological and imaging findings . Neuropsychologia 47 , 2314 –2329 10.1016/j.neuropsychologia.2009.01.020 19524095 Premack D. (2007 ). Human and animal cognition: continuity and discontinuity . Proc. Natl. Acad. Sci. U.S.A. 104 , 13861 –13867 10.1073/pnas.0706147104 17717081 Raby C. R. Alexis D. M. Dickinson A. Clayton N. S. (2007 ). Planning for the future by western scrub-jays . Nature 445 , 919 –921 10.1038/nature05575 17314979 Raby C. R. Clayton N. S. (2009 ). Prospective cognition in animals . Behav. Processes 80 , 314 –324 10.1016/j.beproc.2008.12.005 20522320 Reynolds M. Brewin C. R. (1999 ). Intrusive memories in depression and posttraumatic stress disorder . Behav. Res.Ther. 37 , 201 –215 10.1016/S0005-7967(98)00132-6 10087639 Roberts W. A. Feeney M. C. MacPherson K. Petter M. McMillan N. Musolino E. (2008 ). Episodic-like memory in rats: is it based on when or how long ago? Science 320 , 113 –115 10.1126/science.1152709 18388296 Rotello C. M. Macmillan N. A. Van Tassel G. (2000 ). Recall-toreject in recognition: evidence from ROC curves . J. Mem. Lang. 43 , 67 –88 10.1006/jmla.1999.2701 Rubin D. C. (2006 ). The basic systems model of episodic memory . Perspect. Psychol. Sci. 1 , 277 –311 10.1111/j.1745-6916.2006.00017.x Rubin D. C. Schrauf R. W. Greenberg D. L. (2003 ). Belief and recollection of autobiographical memories . Mem. Cogn. 31 , 887 –901 10.3758/BF03196443 14651297 Rubin D. C. Wetzler S. E. Nebes R. D. (1986 ). “Autobiographical memory across the adult lifespan,” in Autobiographical Memory , ed. Rubin D. C. (New York : Cambridge University Press ), 202 –221 Salwiczek L. H. Dickinson A. Clayton N. S. (2008 ). “What do animals remember about their past?,” in Cognitive Psychology of Memory , ed. Menzel R. (Oxford : Elsevier ), 441 –459 Sauvage M. M. Fortin N. J. Owens C. B. Yonelinas A. P. Eichenbaum H. (2008 ). Recognition memory: opposite effects of hippocampal damage on recollection and familiarity . Nat. Neurosci. 11 , 16 –18 10.1038/nn2016 18037884 Schacter D. L. Addis D. R. (2007 ). The ghosts of past and future . Nature 445 , 27 10.1038/445027a 17203045 Schwartz B. L. (2005 ). “Do humans primates have episodic memory?,” in The Missing Link in Cognition , eds Terrace H. S. Metcalfe J. (Oxford : Oxford University Press ), 225 –241 Schwartz B. L. Colon M. R. Sanchez I. C. Rodriguez I. A. Evans S. (2002 ). Single-trial learning of “what” and “who” information in a gorilla (Gorilla gorilla gorilla): implications for episodic memory . Anim. Cogn. 5 , 85 –90 10.1007/s10071-002-0132-0 12150040 Schwartz B. L. Hoffman M. L. Evans S. (2005 ). Episodic-like memory in a gorilla: a review and new findings . Learn. Motiv. 36 , 226 –244 10.1016/j.lmot.2005.02.012 Skov-Rackette S. I. Miller N. Y. Shettleworth S. J. (2006 ). What-where-when memory in pigeons . J. Exp. Psychol. Anim. Behav. Process. 32 , 345 –358 10.1037/0097-7403.32.4.345 17044738 Slotnick S. D. Klein S. A. Dodson C. S. Shimamura A. P. (2000 ). An analysis of signal detection and threshold models of source memory . J. Exp. Psychol. Learn. Mem. Cogn. 26 , 1499 –1517 10.1037/0278-7393.26.6.1499 11185779 Snowden J. Griffiths H. Neary D. (1994 ). Semantic dementia: autobiographical contribution to preservation of meaning . Cogn. Neuropsychol. 11 , 265 –288 10.1080/02643299408251976 16934432 Squire L. R. (1992 ). Declarative and nondeclarative memory: multiple brain systems supporting learning and memory . J. Cogn. Neurosci. 4 , 232 –243 10.1162/jocn.1992.4.3.232 Squire L. R. Stark C. E. L. Clark R. E. (2004 ). The medial temporal lobe . Annu. Rev. Neurosci. 27 , 279 –306 10.1146/annurev.neuro.27.070203.144130 15217334 Stevenson I. Cook E. W. (1995 ). Involuntary memories during severe physical illness or injury . J. Nerv. Ment. Dis. 183 , 452 –458 10.1097/00005053-199507000-00005 7623017 Suddendorf T. (2006 ). Foresight and evolution of the human mind . Science 312 , 1006 –1007 10.1126/science.1129217 16709773 Suddendorf T. Busby J. (2003 ). Like it or not? The mental time travel debate . Trends Cogn. Sci. 7 , 437 –438 10.1016/j.tics.2003.08.010 Suddendorf T. Corballis M. C. (1997 ). Mental time travel and the evolution of the human mind . Genet. Soc. Gen. Psychol. Monogr. 123 , 133 –167 9204544 Suddendorf T. Corballis M. C. (2007 ). The evolution of foresight: what is mental time travel and is it unique to humans? Behav. Brain Sci. 30 , 299 –313 10.1017/S0140525X07001975 17963565 Thompson C. V. Skowronski J. S. Larsen S. F. Betz A. L. (1996 ). Autobiographical Memory: Remembering What and Remembering When . New York : Lawrence Erlbaum Tulving E. (1972 ). “Episodic and semantic memory,” in Organization of Memory , eds Tulving E. Donaldson W. (New York : Academic Press ), 381 –403 Tulving E. (1983 ). Elements of Episodic Memory . New York : Oxford Univ Press Tulving E. (2002 ). Episodic memory: from mind to brain . Annu. Rev. Psychol. 53 , 1 –25 10.1146/annurev.psych.53.100901.135114 11752477 Tulving E. (2005 ). “Episodic memory and autonoesis: uniquely human?” in The Missing Link in Cognition , eds Terrace H. S. Metcalfe J. (Oxford : Oxford University Press ), 3 –56 Tulving E. Markowitsch H. J. (1998 ). Episodic and declarative memory: role of the hippocampus . Hippocampus 8 , 198 –204 10.1002/(SICI)1098-1063(1998)8:3<198::AID-HIPO2>3.3.CO;2-J 9662134 Vargha-Khadem F. Gadian D. G. Watkins K. E. Connelly A. Van Paesschen W. Mishkin M. (1997 ). Differential effects of early hippocampal pathology on episodic and semantic memory . Science 277 , 376 –380 10.1126/science.277.5324.376 9219696 Wheeler M. A. McMillan C. T. (2001 ). Focal retrograde amnesia and the episodic-semantic distinction . Cogn. Affect. Behav. Neurosci. 1 , 22 –36 10.3758/CABN.1.1.22 12467101 Wheeler M. A. Stuss D. T. Tulving E. (1997 ). Toward a theory of episodic memory: the frontal lobes and autonoetic consciousness . Psychol. Bull. 121 , 331 –354 10.1037/0033-2909.121.3.331 9136640 Wixted J. T. Squire L. R. (2008 ). Constructing receiver operating characteristics (ROCs) with experimental animals: cautionary notes . Learn. Mem. 15 , 687 –690 10.1101/lm.1077708 18772256 Yonelinas A. P. (1997 ). Recognition memory ROCs for item and associative information: the contribution of recollection and familiarity . Mem. Cognit. 25 , 747 –763 10.3758/BF03211318 9421560 Yonelinas A. P. (1999a ). The contribution of recollection and familiarity to recognition and source-memory judgments: a formal dual-process model and an analysis of receiver operating characteristics . J. Exp. Psychol. Learn. Mem. Cogn. 25 , 1415 –1434 10.1037/0278-7393.25.6.1415 10605829 Yonelinas A. P. (1999b ). Recognition memory ROCs and the dual-process signal-detection model: comment on Glanzer, Kim, Hilford, and Adams (1999) . J. Exp. Psychol. Learn. Mem. Cogn. 25 , 514 –521 10.1037/0278-7393.25.2.514 Yonelinas A. P. (2001 ). Components of episodic memory: the contribution of recollection and familiarity . Philos. Trans. R. Soc. Lond. B Biol. Sci. 356 , 1363 –1374 10.1098/rstb.2001.0939 11571028 Yonelinas A. P. (2002 ). The nature of recollection and familiarity: a review of 30 years of research . J. Mem. Lang. 46 , 441 –517 10.1006/jmla.2002.2864 Zentall T. R. Clement T. S. Bhatt R. S. Allen J. (2001 ). Episodic-like memory in pigeons . Psychon. Bull. Rev. 8 , 685 –690 10.3758/BF03196204 11848586 Zhou W. Crystal J. D. (2009 ). Evidence for remembering when events occurred in a rodent model of episodic memory . Proc. Natl. Acad. Sci. U.S.A. 106 , 9525 –9529 10.1073/pnas.0904360106 19458264 Zhou W. Hohmann A. G. Crystal J. D. (2012 ). Rats answer an unexpected question after incidental encoding . Curr. Biol. 22 , 1149 –1153 10.1016/j.cub.2012.04.040 22633809 Zinkivskay A. Nazir F. Smulders T. V. (2009 ). What–where–when memory in magpies (Pica pica) . Anim. Cogn. 1 , 10.1007/s10071-008-0176-x 18670793	23781179	PMC3678104	CC BY	2021-01-04 22:49:30	yes	Front Behav Neurosci. 2013 Jun 11; 7:63
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23776453PONE-D-13-0156410.1371/journal.pone.0065217Research ArticleBiologyDevelopmental BiologyMorphogenesisCell MigrationMedicineDiagnostic MedicinePathologyGeneral PathologyBiomarkersObstetrics and GynecologyBreast CancerOncologyBasic Cancer ResearchMetastasisCancer TreatmentAntibody TherapyGene TherapyCancers and NeoplasmsBreast TumorsVascular Endothelial Growth Factor Receptor-1 Activation Promotes Migration and Invasion of Breast Cancer Cells through Epithelial-Mesenchymal Transition VEGFR-1 and EMT in Breast CancerNing Qian 1 Liu Caigang 2 Hou Lei 1 Meng Min 1 Zhang Xiaojin 1 Luo Minna 1 Shao Shan 1 Zuo Xiaoxiao 1 Zhao Xinhan 1 * 1 Department of Oncology, the First hospital Affiliated to School of Medicine of Xi'an Jiaotong University, Xi'an, Shaanxi Province, China 2 Department of Oncology, First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China Batra Surinder K. Editor University of Nebraska Medical Center, United States of America * E-mail: zhaoxinhanprof@163.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: QN CL X. Zhao. Performed the experiments: LH MM X. Zhang ML SS X. Zuo. Analyzed the data: QN. Contributed reagents/materials/analysis tools: X. Zhao. Wrote the paper: QN. Harvestd the breast cancer samples: QN ML. 2013 11 6 2013 8 6 e6521710 1 2013 23 4 2013 © 2013 Ning et al2013Ning et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Vascular endothelial growth factor receptor-1 (VEGFR-1 or Flt-1), a tyrosine kinase receptor, is highly expressed in breast cancer tissues, but near absent in normal breast tissue. While VEGFR-1 expression is associated with poor prognosis of women with breast cancer, it is not clear whether it is involved in the aggressiveness of breast cancer. Thus, the present study examined whether VEGFR-1 activation is associated with the invasiveness of breast cancer. We reported that VEGFR-1 was detected in 60.6% of invasive breast carcinoma tissue sections. In addition, VEGFR-1 expression positively correlated with lymph node-positive tumor status, low expression level of membranous E-cadherin, and high expression levels of N-cadherin and Snail. We found that PlGF-mediated VEGFR-1 activation promoted migration and invasion in MCF-7 (luminal) cells and led to morphologic and molecular changes of epithelial-mesenchymal transition (EMT). This was blocked by the down-regulation of VEGFR-1. Conversely, down-regulation of VEGFR-1 in MDA-MB-231 (post-EMT) cells resulted in morphologic and molecular changes similar to mesenchymal-epithelial transition (MET), and exogenous PlGF could not reverse these changes. Moreover, VEGFR-1 activation led to an increase in nuclear translocation of Snail. Finally, MDA-MB-231 cells expressing shRNA against VEGFR-1 significantly decreased the tumor growth and metastasis capacity in a xenograft model. Histological examination of VEGFR-1/shRNA-expressing tumor xenografts showed up-regulation of E-cadherin and down-regulation of N-cadherin and Snail. These findings suggest that VEGFR-1 may promote breast cancer progression and metastasis, and therapies that target VEGFR-1 may be beneficial in the treatment of breast cancer patients. This work was supported by National Nature Science Foundation of China (No: 30801119). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Breast cancer is one of the most common malignant tumors in Chinese women. It's estimated that there will be more than 100 new cases per 100,000 women aged 55–69 years by 2021 [1]. Understanding the molecular mechanisms underlying the progression of breast cancer may provide ways for the development of novel antineoplastic therapies. Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor that binds vascular endothelial growth factor (VEGF)-A, VEGF-B and placental growth factor (PlGF). VEGFR-1 is the sole tyrosine kinase receptor for the later two. While VEGFR-1 is highly expressed in breast cancer tissues and breast cancer cell lines, its expression is absent or near background in normal breast tissue [2], [3]. This suggests that VEGFR-1 may play a role in tumorigenesis of breast cancer or even tumor progression and metastasis. Indeed, it has been suggested that VEGFR-1 may be an independent predicator for poor prognosis in breast carcinoma patients [4]. Epithelial-mesenchymal transition (EMT) is an essential developmental process through which cells of epithelial origin lose cell-cell contacts and cell polarity and acquire mesenchymal phenotypes, including fibroblast-like morphology with cytoskeleton reorganization, increased potential for motility, invasiveness and metastasis [5], [6]. The concept of EMT, initially developed in the field of embryology, has recently been extended to tumor invasion and metastasis. As a feature of aggressive tumors, EMT is characterized by the down-regulation of E-cadherin expression and up-regulation of N-cadherin expression [7]–[9]. Consistent with this notion, invasive ductal carcinoma exhibits a decrease in E-cadherin expression and an increase in N-cadherin expression [10], [11]. Although the role of EMT in tumor invasion and metastasis becomes a topic of interest, the molecular mechanism by which EMT is regulated has not been fully understood. One of the key EMT regulators is Snail, which is a zinc-finger transcription factor, that represses expression of E-cadherin mRNA by binding to E-boxes in the promoter, leading to the disruption of adherin junctions (AJ) [12], [13]. Thus, Snail-deficient mouse embryos die during gastrulation due to a failure to undergo EMT [14]. The dissolution of the E-cadherin-mediated AJ is a key preliminary step in EMT. This is also the first step for tumor cells to invade surrounding tissues. Consistent with this notion, previous reports have shown that Snail mRNA is not detected in normal breast epithelium, but is expressed in 47% of infiltrating ductal breast carcinomas, and that Snail protein is over-expressed in 40.9% of invasive ductal breast carcinomas [15], [16]. It appears that the expression level of Snail is reversely correlated with E-cadherin in various carcinomas, including breast carcinoma [16]. A previous study showed that EMT resulted in an increased expression of VEGFR-1 in colonic organoids. In addition, blocking VEGFR-1 function caused massive apoptosis only in cells that underwent EMT [17]. Treatment with VEGF-A and VEGF-B, the VEGFR-1 ligands, led to morphologic and expression changes characteristic of EMT in pancreatic cells. Blocking VEGFR-1 function inhibited these changes [17], [18]. These studies demonstrated that VEGFR-1 expression or activation was associated with EMT. Since VEGFR-1 supports growth and survival of human breast carcinoma and EMT is associated with breast cancer metastasis [19]–[21], we sought to examine the association between VEGFR-1 and EMT in human breast carcinoma. In the present study, we systemically analyzed the association between clinicopathological variables with expression levels of VEGFR-1 and EMT-related proteins in 94 cases of primary invasive breast carcinoma. Finally, we demonstrated that VEGFR-1/PlGF regulated EMT in breast cancer cells in vitro and in vivo. Methods Cell lines, culture conditions and treatment Human umbilical vein endothelial cells (HUVECs) and breast carcinoma cell lines (MDA-MB-453, MDA-MB-231, SK-BR-3, T-47D, BT-474 and MCF-7) were obtained from the American Type Culture Collection (ATCC, VA, USA) and cultured as instructed by ATCC [19], [22], [23]. All cells used in our experiments were at passages 3 to 15 after obtaining them from the suppliers. Cells were cultured in the absence of serum overnight prior to the treatment with PlGF (2 nmol/L, PeproTech Inc.) for the indicated periods. Patients and samples We evaluated 94 invasive breast cancer samples and the matched adjacent non-cancerous samples harvested from surgical resections in the First Affiliated Hospital, College of Medicine at Jiaotong University from 2008 to 2011. All the patients (median age, 37 years; range, 21–53 years) did not receive chemotherapy, radiotherapy or hormone therapy before surgery. Their clinicopathological parameters are summarized in Table 1. Histologic types were classified according to the World Health Organization (2003). TNM staging was defined according to the American Joint Committee on Cancer (AJCC) (the 6th version, 2002). All the cases were individually categorized by independent pathologists. 10.1371/journal.pone.0065217.t001Table 1 Association analysis of clinicopathological features with expression levels of VEGFR-1, E-cadherin, N-cadherin and Snail in 94 invasive breast carcinoma samples. Definition VEGFR-1 E-cadherin N-cadherin Snail Low High P Low High P Low High P Low High P Age <50 years 14 24 0.680 20 18 0.136 19 19 0.168 16 22 0.452 ≥50 years 23 33 38 18 20 36 28 28 Histology Ductal 23 41 0.321 36 28 0.112 30 34 0.122 30 34 0.985 Lobular 14 16 22 8 9 21 14 16 Tumor size ≤2 cm 13 18 0.938 13 18 0.022 18 13 0.007 19 12 0.029 2–5 cm 16 26 30 12 18 24 20 22 >5 cm 8 13 15 6 3 18 5 16 Grading Ι 5 5 0.569 3 7 <0.001 7 3 <0.001 9 1 0.015 ΙΙ 20 28 22 26 28 20 20 28 ΙΙΙ 12 24 33 3 4 32 15 21 LN status Negative 22 21 0.032 28 15 0.532 25 18 0.003 36 7 <0.001 Positive 15 36 30 21 14 37 8 43 Staging Ι 13 16 0.072 12 17 0.001 7 22 <0.001 16 13 0.013 ΙΙ 11 30 24 17 28 13 23 18 ΙΙΙ 13 11 22 2 4 20 5 19 Menopausal status Pre- 18 26 0.773 28 16 0.717 16 28 0.344 17 27 0.136 Post- 19 31 30 20 23 27 27 23 ER Negative 17 33 0.257 40 10 <0.001 15 35 0.016 10 40 <0.001 Positive 20 24 18 26 24 20 34 10 PR Negative 25 27 0.054 37 15 0.036 24 28 0.307 25 27 0.784 Positive 12 30 21 21 15 27 19 23 c-erbB-2 0 3 17 0.09 7 13 0.088 6 14 0.418 11 9 0.546 1 13 16 19 10 14 15 16 13 2 7 11 13 5 8 10 6 12 3 5 7 9 3 3 9 5 7 Not specified 9 6 10 5 8 7 6 9 Total 37 57 58 36 39 55 44 50 LN: lymph node; ER: estrogen receptor; PR, progesterone receptor. P<0.05 was considered as significant. Ethics Statement The procedure was approved by the Committee for the Conduct of Human Ethics Committee of the First Affiliated Hospital, College of Medicine at Jiaotong University. Written informed consent was obtained from each patient enrolled in the study. Immunohistochemical staining Fresh tissue specimens were fixed in 4% paraformaldehyde, embedded in paraffin, cut at 2–3 µm, and placed on slides. The slides were deparaffinized with xylene, dehydrated in graduate decreasing concentrations of ethanol, and subsequently incubated with 0.3% hydrogen peroxide for 10 min in order to block endogenous peroxidase activity. For antigen retrieval, tissue sections were heated in citrate buffer in a microwave oven followed by incubation in phosphate-buffered saline (PBS). Slides were incubated with 10% normal goat serum (in PBS) for 15 min at room temperature to block unspecific labeling and then incubated with the following primary antibodies in a humidified chamber overnight at 4°C: a rabbit polyclonal VEGFR-1 antibody (1∶50; Beijing Biosynthesis Biotechnology); a rabbit polyclonal E-cadherin antibody (1∶100; Beijing Biosynthesis Biotechnology); a rabbit polyclonal N-cadherin antibody (1∶100; Beijing Biosynthesis Biotechnology); and a rabbit polyclonal Snail antibody (1∶50; Beijing Biosynthesis Biotechnology). Following washes in PBS, sections were incubated with an appropriate secondary antibody (polymerized horseradish peroxidase [HRP]-anti rabbit IgG; Maixin-Bio, Fuzhou) for 15 min at 37°C, followed by incubation with streptavidin-peroxidase (DAKO) for 15 min at 37°C. 3,3′ -diaminobenzidine (DAB; DAKO) was applied as the chromogenic agent. Then slides were counterstained with hematoxylin, dehydrated in graded ethanol, and coverslipped. The primary antibody was replaced with PBS or normal goat serum for the blank or negative control, respectively. Immunohistochemical evaluation The expression levels of VEGFR-1, E-cadherin, N-cadherin and Snail were independently evaluated by two investigators. Semi-quantitative analysis of staining distribution was scored as negative, +, ++, and +++ according to the percentage of cells showing immunoreactivity. Negative indicated the complete absence or weak staining in<1% of the tumor cells, + indicated focal staining in<1–10% of tumor cells, ++ indicated positive staining in 11–50% of tumor cells, and +++ indicated positive staining in>50% of tumor cells. Tumors were defined as immunopositive when>10% of tumor cells show immunoreactivity. Expression of Snail protein was observed in the cytoplasm or nucleus or both; however, expression in only the nuclear compartment was counted as immunopositive for Snail. Expression of VEGFR-1, E-cadherin and N-cadherin were distributed in the cytoplasm and/or membrane, and both cytoplasmic and membranous expressions were considered as positive events. Western blotting analysis Cells were lysed in RIPA buffer with proteinase inhibitors for 30 min on ice, and then cleared at 12,000 rpm for 20 min at 4°C. The supernatant was aliquoted for total cellular protein and its concentration was determined using the Bradford assay (Sigma Chemicals, Bangalore, India). Nuclear extraction was performed according to the manufacturer's instructions (Pioneer Biotechnology, Inc.). Equivalent amounts of total cellular protein (10–30 µg) were subjected to reducing SDS-PAGE (8–12%) followed by blotting on a polyvinylidene difluoride (PVDF) membrane. After blocking in 5% nonfat dry milk for 2 h, membranes were incubated with the following antibodies overnight at 4°C: a rabbit monoclonal VEGFR-1 antibody (1∶10,000; Abcam); a rabbit polyclonal N-cadherin antibody (1∶800; Proteintech Group Inc.); a rabbit polyclonal vimentin antibody (1∶2,000; Proteintech Group Inc.); a rabbit polyclonal E-cadherin antibody (1∶800; Proteintech Group Inc.); a rabbit polyclonal occludin antibody (1∶1,000; Proteintech Group Inc.); a rabbit polyclonal β-catenin antibody (1∶800; Proteintech Group Inc.); a rabbit polyclonal lamin B1 antibody (1∶1,500; Proteintech Group Inc.); and a mouse monoclonal β-actin antibody (1∶1,000; Santa Cruz Biotechnology, Inc.). The membranes were then washed and incubated with a HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature and visualized by enhanced chemiluminescence (Amersham Biosciences). Images were documented by a scanner and analyzed by Quantity One software. β-actin expression was used as a loading control for whole cell lysates, while lamin B1 expression was used as a loading control for nuclear lysates. Enzyme-linked immunoabsorbent assay (ELISA) Breast carcinoma cells (1×106) were cultured in 75 cm2 culture flasks containing complete medium with 10% FBS. Conditioned medium was collected on day 3 and PlGF levels were assessed using ELISA kits (R&D Systems) according to the manufacturer's instructions. Generation of MDA-MB-231 and MCF-7 cells stably transfected with VEGFR-1 shRNA VEGFR-1-shRNA and control shNC in eukaryotic pGPU6/GFP/Neo plasmid vectors were obtained from Genepharma Co., Ltd (China). The target sequence for VEGFR-1-shRNA was GGACGTAACTGAAGAGGATTT as previously reported [24]. 24 h before transfection, cells were seeded onto 6-well culture plates. When cells grew to 40–70% confluence, transfection was performed using TurboFect in vitro Transfection Reagent (Fermentas) according to the manufacturer's instructions. 48 h post-transfection, G418 (Gibco) was added to the cell culture media at a final concentration of 500 µg/ml (MDA-MB-231) or 800 µg/ml (MCF-7) for 3 weeks. Media was changed once every 3 days. After 3 weeks, G418-resistant colonies were isolated and cultured in 96-well culture plates for further experiments. Cell migration and invasion assays For in vitro migration assays, 24-well plates inserted with 8.0 µm pore transwells (Millipore) were utilized. 2×104 cells in 200 µl of complete medium without FBS were placed on the top chamber of the insert. In the lower chamber, 500 µl of complete medium containing 10% FBS with or without PlGF was added as a chemoattractant. After 6 h (MDA-MB-231, MDA-MB-231/VEGFR-1 shRNA) or 24 h (MCF-7, MCF-7/VEGFR-1 shRNA), the cells on the top surface of the membrane were removed with a cotton swab. Migrated cells adhering to the underside of the membrane were fixed with methanol, stained with crystal violet solution and counted in 10 random fields under a microscope. For in vitro invasion assays, the procedure was similar to the migration assay described above, except the membranes were coated with Matrigel and prehydrated in serum-free medium. Cells were incubated for 36 h prior to fixing and staining. Immunofluorescence Cells were seeded on poly-L-lysine-coated glass coverslips in 6-well plates and received different treatments. After being fixed with 4% paraformaldehyde for 20 min, cells were permeabilized in 0.2% Triton X-100 for 10 min and blocked with normal goat serum for 30 min. Subsequently, cells were incubated with the following antibodies overnight at 4°C: a rabbit polyclonal ZO-1 antibody (1∶50 in 1%BSA; Proteintech Group Inc.); a mouse monoclonal E-cadherin antibody (1∶100 in 1%BSA; Invitrogen); a rabbit polyclonal vimentin antibody (1∶50 in 1%BSA; Proteintech Group Inc.). Slides were then washed in PBS, followed by incubation with a TRITC-conjugated secondary antibody (Wuhan Boster Biological Engineering Co., Ltd) for 2 h at room temperature. After washing in PBS, cells were incubated with DAPI (1 µg/ml, diluted in PBS) for nuclear staining for 3 min and mounted onto slides. Cells were observed under a fluorescent microscope. Tumor formation in athymic nude mice Athymic nude mice (Silaike Laboratory Animal Co., Ltd, Shanghai, China) were used to assess the effect of VEGFR-1 shRNA on tumor growth and metastasis in vivo. The protocol was approved by the Animal Care and Use Committee of Xi'an Jiaotong University. Mice were divided into two groups, with six per group. Approximately 2×106 cells (MDA-MB-231/shNC, MDA-MB-231/VEGFR-1 shRNA), which were suspended in 0.2 ml serum-free medium, were inoculated into the fat pad of mice. Tumor formation was measured every day. The volume of tumors was calculated using the following formula: length x width2×0.5. The experiments were terminated after 30 days because more than half of the mice became cachectic. Tumor-bearing athymic nude mice were observed by IVIS imaging system (IVIS spectrum, Xenogen, CA, USA) before sacrifice. The tumor tissues were removed for immunohistochemical staining. Statistical analysis Statistical analysis was performed using SPSS 13.0 for Windows software (SPSS Inc., Chicago, IL). Chi-square tests and Fisher's exact tests (two-sided) were performed to assess the correlation between clinicopathological parameters and protein expression. A Spearman's rho test was used to determine the relationship among expression levels of each protein. One-way ANOVA or two-tailed Student's t-tests were used for comparisons between groups. P<0.05 was considered statistically significant. Results Immunohistochemical analysis of VEGFR-1, E-cadherin, N-cadherin and Snail in invasive breast carcinoma samples We attempted to examine the expression of VEGFR-1, E-cadherin, N-cadherin and Snail in 94 invasive breast carcinoma tissues by immunohistochemistry. Association analysis between expression levels of VEGFR-1, E-cadherin, N-cadherin and Snail and clinicopathological features are summarized in Table 1. Consistent with the previous studies, VEGFR-1 protein was not detectable in normal breast tissues (Figure 1A), but was predominantly expressed in the cytoplasm of the tumor cells (Figure 1B) in 60.6% of patients (Table 1). It should be noted that both vascular endothelial cells of newly formed blood vessels and stromal cells were positive for VEGFR-1 (Figure 1B); however, only the expression of VEGFR-1 in cancer cells will be discussed in this report. VEGFR-1 expression was detected in 48.8% of samples in the lymph node-negative group and 70.6% of samples in the lymph node-positive group (Table 1). A significant association was observed between the cytoplasmic expression of VEGFR-1 and the node-positive tumor status (P = 0.032). 10.1371/journal.pone.0065217.g001Figure 1 Immunohistochemical analysis of VEGFR-1, E-cadherin, N-cadherin and Snail in normal breast tissues and breast carcinoma tissues. VEGFR-1 (A), E-cadherin (C), N-cadherin (E), and Snail (G) expression in normal breast tissues, and VEGFR-1 (B), E-cadherin (D), N-cadherin (F) and Snail (H) expression in tumor tissues (magnification: A, E and G: 200×; B, C, D, F and H: 400×). E-cadherin was normally present in the cell membranes of normal breast tissues, but failed to express in tumor tissues (Figure 1C and D). Approximately 61.7% (58/94) of the tumor sections showed loss or reduction of E-cadherin expression (Table 1). The reduction of E-cadherin expression was observed in 91.7% (22/24) of the late-stage and 91.7% (33/36) of the high-grade tumors, significantly more than 41.4% (12/29) of the early-stage and 30% (3/10) of the low-grade tumors (P = 0.001 and P<0.001, respectively) (Table 1). In addition, low E-cadherin expression was associated with a large tumor size and the status of estrogen receptor (ER) and progesterone receptor (PR) negativity (P = 0.022, P<0.001, and P = 0.036, respectively) (Table 1). N-cadherin was absent in normal breast tissues, but highly expressed in tumor tissues (Figure 1E and F). Consistent with the association in the low-expressed E-cadherin subgroup, high N-cadherin expression was strongly associated with high-grade and late-stage tumors (P<0.001) (Table 1). Positive membranous N-cadherin expression also correlated with the lymph-node positive group and a negative ER status (P = 0.003 and P = 0.016, respectively) (Table 1). Snail was not detected in normal breast tissue, but was detected in 53.2% of the tumors (Figure 1G and H and Table 1). Positive nuclear expression of Snail was associated with all adverse clinicopathologic variables, including high tumor grade, late tumor stage, lymph node positivity, and a negative ER status (Table 1). Finally, no statistically significant correlation was found between the expression levels of these four proteins and age, histology (ductal or lobular), menopausal status or the expression of c-erbB-2 (Table 1). Association between expression levels of VEGFR-1, E-cadherin, N-cadherin and Snail in invasive breast carcinoma samples We next wanted to analyze the correlation between the expression levels of VEGFR-1 and EMT-related proteins in breast carcinoma tissues. In concordance with protein changes during EMT, high expression levels of N-cadherin and Snail were associated with weak expression of membranous E-cadherin (P = 0.009, r = −0.269; P = 0.002, r = −0.314, respectively) (Table 2). In addition, high N-cadherin expression was significantly correlated with nuclear expression of Snail (P = 0.016, r = 0.249) (Table 2). These analyses indicate that the invasive breast carcinoma tissues had undergone the EMT process. Surprisingly, we found that high VEGFR-1 expression was strongly associated with low E-cadherin expression (P<0.001, r = −0.575), high N-cadherin expression (P<0.001, r = 0.426), and high Snail expression (P<0.001, r = 0.641). These data suggest that VEGFR-1 may be involved in the regulation of EMT. 10.1371/journal.pone.0065217.t002Table 2 Association analysis between expression levels of VEGFR-1, E-cadherin, N-cadherin and Snail in 94 invasive breast carcinoma samples. Definition E-cadherin N-cadherin Snail Low High P Low High P Low High P VEGFR-1 Low 10 27 <0.001 25 12 <0.001 32 5 <0.001 High 48 9 14 43 12 45 Spearman correlation −0.575 0.426 0.641 E-cadherin Low 18 40 0.009 20 38 0.002 High 21 15 24 12 Spearman correlation −0.269 −0.314 N-cadherin Low 24 15 0.016 High 20 35 Spearman correlation 0.249 P<0.05 was considered statistically significant. Expression of VEGFR-1 and PlGF in breast cancer cell lines To test the hypothesis that VEGFR-1 may regulate EMT in breast cancer cells, we analyzed the expression of VEGFR-1 and its corresponding ligand, PlGF, in 6 breast cancer cell lines. Both VEGFR-1 and PlGF were detected by Western blot and ELISA analyses, respectively (Figure 2A and B). We should note that there was variation of PlGF presence in the conditioned media. PlGF expression in the conditioned media of tested breast cancer cell lines was not as high as other ligands of VEGFR-1, and human PlGF was not detectable in standard culture medium containing 10% FBS (data not shown). High levels of PlGF was secreted from MDA-MB-231 but not from MCF-7 cells, while both cell lines expressed comparable levels of VEGFR-1 (Figure 2A and B). Highly tumorigenic MDA-MB-231 cells can metastasize in immunodeficient mice; however, less aggressive MCF-7 cells are only able to form tumors but not metastasize in mice [24], [25]. Therefore, we chose both MDA-MB-231 and MCF-7 cell lines for further study. 10.1371/journal.pone.0065217.g002Figure 2 Analysis of VEGFR-1 and PlGF expression in human breast cancer cell lines. (A) Lysates from different breast cancer cell lines were subjected to Western blot analysis for VEGFR-1 protein expression. HUVECs were used as a positive control. β-actin is shown as a loading control. The bar graph below shows the relative protein expression levels (VEGFR-1/β-actin) among the cell lines. Data are presented as average ± s.d. from three independent experiments. P<0.05. (B) Breast cancer cell line-derived conditioned media were subjected to ELISA for PlGF expression. Results are shown as average ± s.d. from three independent experiments. P<0.05. Conditioned media from HUVECs were included as a positive control. VEGFR-1 facilitated the migration and invasion in MDA-MB-231 and MCF-7 cells MDA-MB-231 and MCF-7 cells were stably transfected with pGPU6/GFP/Neo vectors that express shRNAs against VEGFR-1 or a control sequence (NC). Neomycin-resistant cells were characterized by Western blot using an anti-VEGFR-1 antibody. As shown in Figure 3A, VEGFR-1 protein expression was significantly decreased in MDA-MB-231 and MCF-7 cells expressing VEGFR-1-shRNA. Next, we assessed the migration and invasion capability of these cells in the presence of PlGF because it is a VEGFR-1-specific ligand [26]. PlGF treatment led to a three-fold increase in migration of MDA-MB-231 cells compared with cells without PlGF (P<0.05) (Figure 3B). Down-regulation of VEGFR-1 prevented PlGF-induced migration of MDA-MB-231 cells (Figure 3B). In addition, in the Matrigel-coated transwell assay in response to PlGF, the invasion of MDA-MB-231 cells increased by 1.5 fold (P<0.05) (Figure 3C). Reduction of VEGFR-1 expression blocked PlGF-induced invasion of MDA-MB-231 cells (Figure 3C). These findings demonstrate that the ability of MDA-MB-231 cells to migrate and invade is dependent on VEGFR-1 expression and activation. Similarly, PlGF significantly induced the migration and invasion of MCF-7 cells (Figure 3B and C). However, while migration was abrogated by down-regulating VEGFR-1, a significant decrease in invasion was not observed (Figure 3B and C). 10.1371/journal.pone.0065217.g003Figure 3 VEGFR-1-dependent migration and invasion of MDA-MB-231 and MCF-7 cells. (A) Western blot analysis of VEGFR-1 expression in MDA-MB-231 and MCF-7 cells transfected with a shRNA against VEGFR-1. VEGFR-1 expression was significantly decreased in both MDA-MB-231 (left) and MCF-7 cells (right) expressing VEGFR-1-shRNA. The bar graph shows the relative protein expression levels among groups. β-actin was used as a loading control. Data are presented as average ± s.d. for three independent experiments. P<0.05. (B) VEGFR-1 activation increased the migration of MDA-MB-231 and MCF-7 cells in vitro. PlGF induced a 3-fold increase in migration of MDA-MB-231 cells and at least a 4-fold of MCF-7 cells compared with the controls. Reduction of VEGFR-1 expression inhibited the PlGF-mediated migration of MDA-MB-231 and MCF-7 cells. (C) VEGFR-1 activation increased the invasion of MDA-MB-231 and MCF-7 cells in vitro. PlGF induced a 1.5-fold increase in invasion of MDA-MB-231 and 5-fold of MCF-7 cells compared with the controls. Decreased VEGFR-1 expression blocked the PlGF-mediated invasion of MDA-MB-231, but not in MCF-7 cells. Data are presented as average ± s.d. for three independent experiments. P<0.05. VEGFR-1 activation/expression regulated EMT in MDA-MB-231 and MCF-7 cells Surprisingly, PlGF treatment led to a spindle-shaped fibroblastic morphology in MCF-7 cells, indicating a loss of cell polarity (Figure 4A). This morphological change suggested a reminiscent of the phenotypic change of EMT. In contrast, down-regulation of VEGFR-1 resulted in MET in MDA-MB-231 cells. Post-EMT MDA-MB-231 cells lost their fibroblast-like morphology, which was accompanied by a cobblestone-like epithelial morphology (Figure 4A). ZO-1 is an important organizational component of tight junctions between epithelial cells that are the major structure in maintaining cellular polarity [27], [28]. Consistent with the phenotypic changes of losing cellular polarity, PlGF-induced VEGFR-1 activation led to a decrease in immunofluorescent staining of ZO-1 in MCF-7 cells (Figure 4B). In contrast, MDA-MB-231 cells expressing shRNA against VEGFR-1 cells acquired ZO-1 expression, indicating that the reduction of VEGFR-1 expression results in the establishment of cell polarity in post-EMT cells (Figure 4B). Approximately 40% of the MCF-7 cells underwent EMT in response to PlGF, while 30% of the MDA-MB-231 cells underwent MET due to the down-regulation of VEGFR-1 (data not shown). 10.1371/journal.pone.0065217.g004Figure 4 VEGFR-1 expression and activation mediated EMT changes in MCF-7 and MDA-MB-231 cells. (A) PlGF treatment led to a morphological change from a cobblestone-like shape to a spindle shape in MCF-7 cells. Down-regulation of VEGFR-1 resulted in a loss of the fibroblast-like morphology in MDA-MB-231 cells. (B) Immunofluorescent analysis of ZO-1, a cell polarity protein, in MCF-7 and MDA-MB-231 cells. PlGF-activated VEGFR-1 led to a loss of cell polarity in MCF-7 cells, whereas a decrease in VEGFR-1 expression in MDA-MB-231 cells led to the re-establishment of cell polarity. (magnification 200×). (C) Immunofluorescent analysis of EMT-related regulators in MCF-7 and MDA-MB-231 cells with VEGFR-1 activation or expression. (magnification 200×). (D) Western blot analysis of mesenchymal cell markers and epithelial cell markers in MCF-7 cells when VEGFR-1 was activated by PlGF or down-regulated by shRNA. (E) Western blot analysis of mesenchymal cell markers and epithelial cell markers in MDA-MB-231 cells when VEGFR-1 was activated by PlGF or down-regulated by shRNA. β-actin was used as a loading control for whole cell lysate, while lamin B1 was used as a loading control for nuclear lysate. The bar graphs show the relative expression of proteins among each treatment groups. Data are presented as average ± s.d. for three independent experiments. P<0.05. To investigate whether the phenotypic change mediated by VEGFR-1 activation/expression was indeed EMT, we examined the expression of EMT-related regulators by immunofluorescent analysis. While MCF-7 cells normally expressed a high level of E-cadherin and an undetectable level of vimentin, PlGF-treated cells significantly reduced E-cadherin expression and increased vimentin expression. By contrast, MDA-MB-231 cells highly expressed vimentin but lacked E-cadherin expression. However, down-regulation of VEGFR-1 led to a decrease in vimentin and a concomitant increase in E-cadherin (Figure 4C). Consistently, Western blot analysis revealed that PlGF treatment led to an increase in the expression of mesenchymal cell markers and a decrease in the expression of epithelial cell markers in MCF-7 cells (Figure 4D). Furthermore, down-regulation of VEGFR-1 inhibited the PlGF-mediated expression changes (Figure 4D). Conversely, down-regulation of VEGFR-1 resulted in decreased expression levels of N-cadherin and vimentin proteins and increased expression levels of E-cadherin and occludin proteins in MDA-MB-231 cells (Figure 4E). However, PlGF treatment did not influence expression changes in these proteins (Figure 4E). Modulation of VEGFR-1 activation and expression led to an expression change of Snail in MDA-MB-231 and MCF-7 cells We next investigated whether Snail, an E-cadherin repressor, was involved in VEGFR-1 activation-induced EMT. Western blot analysis showed that PlGF treatment increased Snail expression in the nucleus of MCF-7 cells (Figure 5). Consistent with our prior observations, an increase in nuclear Snail expression could be reversed by the down-regulation of VEGFR-1, even in the presence of PlGF (Figure 5). Finally, down-regulation of VEGFR-1 resulted in a decrease in nuclear Snail expression in MDA-MB-231 cells (Figure 5). However, PlGF treatment could not rescue the decreased Snail expression level mediated by VEGFR-1 down-regulation in MDA-MB-231 cells (Figure 5). 10.1371/journal.pone.0065217.g005Figure 5 VEGFR-1 mediated an expression change of Snail protein in MCF-7 and MDA-MB-231 cells. Western blot analysis of Snail in nuclear extracts of MCF-7 and MDA-MB-231 cells, in which VEGFR-1 was activated by PlGF or down-regulated by shRNA. The bar graphs show the relative expression of proteins among each treatment groups. Lamin B1 was used as a loading control for nuclear lysate. Data are presented as average ± s.d. for three independent experiments. P<0.05. Down-regulation of VEGFR-1 inhibited tumor metastasis in a human breast carcinoma xenograft model Finally, we investigated the effect of down-regulation of VEGFR-1 expression on tumor growth and metastasis in female athymic nude mice. As shown in Figure 6A, tumors expressing shRNA against VEGFR-1 grew significantly slower than those in the control group (P<0.05). Immunohistochemical analysis showed that tumors derived from MDA-MB-231 cells expressing VEGFR-1 shRNA had an elevated level of E-cadherin, with modest levels of N-cadherin and Snail (Figure 6B). In vivo imaging showed that 50% (3/6) of the mice injected with MDA-MB-231/shNC had cervical metastasis, while mice treated with MDA-MB-231/VEGFR-1 shRNA did not have observable metastases (Figure 6C). 10.1371/journal.pone.0065217.g006Figure 6 VEGFR-1 down-regulation inhibited tumor growth and metastasis in a human xenograft model. (A) Tumor-growth curve. The tumor volume in mice injected with MDA-MB-231 cells expressing VEGFR-1-shRNA was significantly smaller than the control. *P<0.05. (B) Immunohistochemical analysis of EMT related regulators in tumor sections. VEGFR-1 down-regulation in xenografts resulted in up-regulation of E-cadherin expression, with down-regulation of N-cadherin and Snail expression. (magnification: 400×). (C) In vivo imaging analysis. Mice treated with MDA-MB-231/shNC (a) had cervical metastasis; however, the mice treated with MDA-MB-231/VEGFR-1 shRNA (b) exhibited no observable metastasis. The reporter used GFP to show the metastasis. Discussion VEGFR-1 was initially identified in vascular endothelial cells. Recently, it is apparent that VEGFR-1 is also present in several types of cancers, suggesting that VEGFR-1 plays a role in tumor invasiveness [17], [29]–[32]. However, in the literature, the percentage of positive VEGFR-1 expression in breast cancer is not consistent and its role in tumorigenesis or metastasis is not fully understood. For instance, Dale et al [33] reported that VEGFR-1 was weakly expressed in breast cancer specimens, while Schmidt et al [23] found that VEGFR-1 was expressed in 39% of breast cancer specimens, predominantly in the cytoplasm. Given that VEGFR-1 is involved in the invasiveness of other tumor types [23], [29]–[31], the purpose of the current study was to examine VEGFR-1 expression in invasive breast carcinoma samples and to examine its role in the aggressiveness of breast cancer. In the present study, we found that 60.6% of the breast carcinoma tissues were positive for VEGFR-1 and that a high expression level of cytoplasmic VEGFR-1 was associated with a lymph node positive status. This is consistent with previous studies showing that VEGFR-1 expression is correlated with high metastasis and recurrence risks [33], [34]. These studies suggest that in addition to its canonical role in angiogenesis, VEGFR-1 may play a role in tumor growth and metastasis and may be an unfavorable progression indicator for patients with breast carcinoma. EMT, a critical physiological process during development and wound healing, has been implicated in tumor progression and metastasis [35]-[39]. Through this complex process, epithelial–derived tumor cells lose intercellular tight adhesion and acquire a mesenchymal phenotype with increased migratory behavior [40]. Cadherin switching, down-regulation of epithelial cadherins (eg. E-cadherin) and up-regulation of mesenchymal cadherins (eg. N-cadherin) are necessary for increased motility and are characteristics for EMT [41]. Among the 94 breast cancer samples in our study, 61.7% showed loss or reduction of E-cadherin, while 58.5% showed positive N-cadherin expression. The correlation analysis data showed that VEGFR-1 expression was associated with low E-cadherin expression and high N-cadherin expression, suggesting that VEGFR-1 may be involved in EMT. Although VEGFR-1 activation was able to induce EMT in human pancreatic carcinoma cells [18], to our best knowledge, no studies have reported that VEGFR-1 is a possible mediator for EMT in breast cancer. In this study, since VEGFR-1 expression was strongly associated with cadherin switching during EMT, we hypothesized that VEGFR-1 could regulate EMT of breast cancer cells, leading to breast cancer progression and metastasis. Snail-1 is a zinc-finger transcriptional factor that is overexpressed during tumor development and is associated with EMT [12], [13], [16]. We found that VEGFR-1 expression was significantly associated with Snail expression in the nucleus. This finding suggests that VEGFR-1 may regulate Snail and may result in EMT. The immuohistological data from human breast carcinoma tissues supports the hypothesis that VEGFR-1 expression may contribute to the aggressive behavior of breast cancer cells, possibly in part by mediating EMT. Not only is VEGFR-1 involved in angiogenesis, it also directly contributes to tumor cell survival, and thus may attribute to the development of human breast cancer [24]. Consistent with this previous report, our data showed that all six of the breast cancer cell lines tested expressed VEGFR-1. In agreement with a prior study in which VEGF had no effect of breast cancer cell motility [3], we found that exogenous VEGF did not alter the invasion capacity of MDA-MB-231 and MCF-7 cells in the transwell assay (data not shown). Since PlGF, a ligand for VEGFR-1, acts as an autocrine factor to activate the VEGFR-1 signaling pathway [42], we detected PlGF expression in conditioned media derived from breast cancer cell lines. We found that MDA-MB-231 cells expressed a high level of PlGF, while MCF-7 cells expressed less, and this correlated with their metastatic capacity. Furthermore, we found that PlGF-mediated VEGFR-1 activation promoted the migration and invasion in breast cancer cell lines, which is consistent with the report using a pancreatic cancer model [29]. However, it remains to be elucidated how PlGF activates cytoplasmic VEGFR-1 and why VEGF does not cause activation. Consistent with these results, MDA-MB-231 breast xenografts treated with VEGFR-1/shRNA showed significant suppression of tumor growth and metastasis capacity in athymic nude mice. Interestingly, we found that in addition to facilitating migration and invasion, VEGFR-1 activation also led to morphologic and molecular changes related to EMT. Furthermore, down-regulation of VEGFR-1 in post-EMT MDA-MB-231 cells resulted in a partial MET morphologic change, suggesting a role of VEGFR-1 in regulating EMT-MET. VEGFR-1 activation led to an increase in nuclear translocation of Snail, suggesting that VEGFR-1 activation-induced EMT might be mediated in part by Snail. Moreover, histological examination of VEGFR-1/shRNA treated tumor xenografts showed molecular changes of MET. It has been reported that VEGFR-1 expression is significantly increased in breast cancer patients with a poor prognosis [43]. Consistent with the report, our data suggest that VEGFR-1 may be an unfavorable progression indicator for breast carcinoma patients. We provide evidence that a reduction of VEGFR-1 expression inhibits cancer cell migration and invasion in vitro and in vivo. These findings suggest that VEGFR-1 might be a potential target for neoadjuvant therapy in patients with invasive breast cancer. Specifically, our data suggests that inhibitors against intracellular VEGFR-1 may be more effective than those inhibiting extracellular VEGFR-1 because VEGFR-1 is expressed in the cytoplasm of tumor cells but not on the membrane. Furthermore, while EMT has been reported to be associated with metastasis in patients, our report is the first to suggest a possible mechanism by which VEGFR-1 may regulate EMT to promote breast cancer progression and metastasis. Therapies targeting VEGFR-1 may be a novel therapeutic approach for untreatable breast cancer patients. Additional studies are clearly needed to further understand how VEGFR-1 regulates EMT. We thank Dr. Dianzeng Zhang for his contribution to the immunohistochemical studies and Dr. Guohong Xin, for his advice for the modification of this paper. ==== Refs References 1 Linos E , Spanos D , Rosner BA , Linos K , Hesketh T , et al (2008 ) Effects of reproductive and demographic changes on breast cancer incidence in China: a modeling analysis . J Natl Cancer Inst 100 : 1352 –1360 .18812552 2 Starzec A , Vassy R , Martin A , Lecouvey M , Di Benedetto M , et al (2006 ) Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1 . Life Sci 79 : 2370 –2381 .16959272 3 Taylor AP , Goldenberg DM (2007 ) Role of placenta growth factor in malignancy and evidence that an antagonistic PlGF/Flt-1 peptide inhibits the growth and metastasis of human breast cancer xenografts . Mol Cancer Ther 6 : 524 –531 .17308051 4 Mylona E , Alexandrou P , Giannopoulou I , Liapis G , Sofia M , et al (2007 ) The prognostic value of vascular endothelial growth factors (VEGFs)-A and -B and their receptor, VEGFR-1, in invasive breast carcinoma . Gynecol Oncol 104 : 557 –563 .17150246 5 Kalluri R , Neilson EG (2003 ) Epithelial-mesenchymal transition and its implications for fibrosis . J Clin Invest 112 : 1776 –1784 .14679171 6 Kalluri R , Weinberg RA (2009 ) The basics of epithelial-mesenchymal transition . J Clin Invest 119 : 1420 –1428 .19487818 7 Cavallaro U , Christofori G (2004 ) Multitasking in tumor progression: signaling functions of cell adhesion molecules . Ann N Y Acad Sci 1014 : 58 –66 .15153420 8 Cavallaro U , Schaffhauser B , Christofori G (2002 ) Cadherins and the tumour progression: is it all in a switch? Cancer Lett 176 : 123 –128 .11804738 9 Knudsen KA , Wheelock MJ (2005 ) Cadherins and the mammary gland . J Cell Biochem 95 : 488 –496 .15838893 10 Prasad CP , Rath G , Mathur S , Bhatnagar D , Parshad R , et al (2009 ) Expression analysis of E-cadherin, Slug and GSK3beta in invasive ductal carcinoma of breast . BMC Cancer 9 : 325 .19751508 11 Nagi C , Guttman M , Jaffer S , Qiao R , Keren R , et al (2005 ) N-cadherin expression in breast cancer: correlation with an aggressive histologic variant–invasive micropapillary carcinoma . Breast Cancer Res Treat 94 : 225 –235 .16258702 12 Batlle E , Sancho E , Francí C , Domínguez D , Monfar M , et al (2000 ) The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells . Nat Cell Biol 2 : 84 –89 .10655587 13 Sugimachi K , Tanaka S , Kameyama T , Taguchi K , Aishima S , et al (2003 ) Transcriptional repressor snail and progression of human hepatocellular carcinoma . Clin Cancer Res 9 : 2657 –2664 .12855644 14 Carver EA , Jiang R , Lan Y , Oram KF , Gridley T (2001 ) The mouse snail gene encodes a key regulator of the epithelial-mesenchymal transition . Mol Cell Biol 21 : 8184 –8188 .11689706 15 Blanco MJ , Moreno-Bueno G , Sarrio D , Locascio A , Cano A , et al (2002 ) Correlation of Snail expression with histological grade and lymph node status in breast carcinomas . Oncogene 21 : 3241 –3246 .12082640 16 ElMoneim HM , Zaghloul NM (2011 ) Expression of E-cadherin, N-cadherin and snail and their correlation with clinicopathological variants: an immunohistochemical study of 132 invasive ductal breast carcinomas in Egypt . Clinics (Sao Paulo) 66 : 1765 –1771 .22012049 17 Bates RC , Goldsmith JD , Bachelder RE , Brown C , Shibuya M , et al (2003 ) Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids . Curr Biol 13 : 1721 –1727 .14521839 18 Yang AD , Camp ER , Fan F , Shen L , Gray MJ , et al (2006 ) Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells . Cancer Res 66 : 46 –51 .16397214 19 Wu Y , Hooper AT , Zhong Z , Witte L , Bohlen P , et al (2006 ) The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma . Int J Cancer 119 : 1519 –1529 .16671089 20 Hulit J , Suyama K , Chung S , Keren R , Agiostratidou G , et al (2007 ) N-cadherin signaling potentiates mammary tumor metastasis via enhanced extracellular signal-regulated kinase activation . Cancer Res 67 : 3106 –3116 .17409417 21 Onder TT , Gupta PB , Mani SA , Yang J , Lander ES , et al (2008 ) Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways . Cancer Res 68 : 3645 –3654 .18483246 22 Dong F , Zhang X , Wold LE , Ren Q , Zhang Z , et al (2005 ) Endothelin-1 enhances oxidative stress, cell proliferation and reduces apoptosis in human umbilical vein endothelial cells: role of ETB receptor, NADPH oxidase and caveolin-1 . Br J Pharmacol 145 : 323 –333 .15765100 23 Schmidt M , Voelker HU , Kapp M , Dietl J , Kammerer U (2008 ) Expression of VEGFR-1 (Flt-1) in breast cancer is associated with VEGF expression and with node-negative tumour stage . Anticancer Res 28 : 1719 –1724 .18630531 24 Lee TH , Seng S , Sekine M , Hinton C , Fu Y , et al (2007 ) Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR1/FLT1 . PLoS Med 4 : e186 .17550303 25 Yang S , Zhang JJ , Huang XY (2012 ) Mouse models for tumor metastasis . Methods Mol Biol 928 : 221 –228 .22956145 26 Fischer C , Mazzone M , Jonckx B , Carmeliet P (2008 ) FLT1 and its ligands VEGFB and PlGF: drug targets for anti-angiogenic therapy? Nat Rev Cancer 8 : 942 –956 .19029957 27 Yonemura S , Itoh M , Nagafuchi A , Tsukita S (1995 ) Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells . J Cell Sci 108 : 127 –142 .7738090 28 Anderson JM , Van Itallie CM (1995 ) Tight junctions and the molecular basis for regulation of paracellular permeability . Am J Physiol 269 : G467 –475 .7485497 29 Wey JS , Fan F , Gray MJ , Bauer TW , McCarty MF , et al (2005 ) Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines . Cancer 104 : 427 –438 .15952180 30 Ferrer FA , Miller LJ , Lindquist R , Kowalczyk P , Laudone VP , et al (1999 ) Expression of vascular endothelial growth factor receptors in human prostate cancer . Urology 54 : 567 –572 .10475375 31 Decaussin M , Sartelet H , Robert C , Moro D , Claraz C , et al (1999 ) Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-Flt1 and VEGF-R2-Flk1/KDR) in non-small cell lung carcinomas (NSCLCs): correlation with angiogenesis and survival . J Pathol 188 : 369 –377 .10440746 32 Graeven U , Fiedler W , Karpinski S , Ergün S , Kilic N , et al (1999 ) Melanoma-associated expression of vascular endothelial growth factor and its receptors FLT-1 and KDR . J Cancer Res Clin Oncol 125 : 621 –629 .10541969 33 Dales JP , Garcia S , Bonnier P , Duffaud F , Carpentier S , et al (2003 ) Prognostic significance of VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) in breast carcinoma . Ann Pathol 23 : 297 –305 .14597894 34 Dales JP , Garcia S , Carpentier S , Andrac L , Ramuz O , et al (2004 ) Prediction of metastasis risk (11 year follow-up) using VEGF-R1, VEGF-R2, Tie-2/Tek and CD105 expression in breast cancer (n = 905) . Br J Cancer 90 : 1216 –1221 .15026804 35 Blick T , Widodo E , Hugo H , Waltham M , Lenburg ME , et al (2008 ) Epithelial mesenchymal transition traits in human breast cancer cell lines . Clin Exp Metastasis 25 : 629 –642 .18461285 36 Trimboli AJ , Fukino K , de Bruin A , Wei G , Shen L , et al (2008 ) Direct evidence for epithelial-mesenchymal transitions in breast cancer . Cancer Res 68 : 937 –945 .18245497 37 Christiansen JJ , Rajasekaran AK (2006 ) Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis . Cancer Res 66 : 8319 –8326 .16951136 38 Kang Y , Massagué J (2004 ) Epithelial-mesenchymal transitions: twist in development and metastasis . Cell 118 : 277 –279 .15294153 39 Gravdal K , Halvorsen OJ , Haukaas SA , Akslen L (2007 ) A switch from E-cadherin to N-cadherin expression indicates epithelial to mesenchymal transition and is of strong and independent importance for the progress of prostate cancer . Clin Cancer Res 13 : 7003 –7011 .18056176 40 Thiery JP (2002 ) Epithelial-mesenchymal transitions in tumour progression . Nat Rev Cancer 2 : 442 –454 .12189386 41 Maeda M , Johnson KR , Wheelock MJ (2005 ) Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition . J Cell Sci 118 : 873 –887 .15713751 42 Taylor AP , Leon E , Goldenberg DM (2010 ) Placental growth factor (PlGF) enhances breast cancer cell motility by mobilising ERK1/2 phosphorylation and cytoskeletal rearrangement . Br J Cancer 103 : 82 –89 .20551949 43 van 't Veer LJ , Dai H , van de Vijver MJ , He YD , Hart AA , et al (2002 ) Gene expression profiling predicts clinical outcome of breast cancer . Nature 415 : 530 –536 .11823860	23776453	PMC3679135	CC BY	2021-01-05 17:29:25	yes	PLoS One. 2013 Jun 11; 8(6):e65217
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23776449PONE-D-13-0315410.1371/journal.pone.0065187Research ArticleBiologyModel OrganismsAnimal ModelsRatMedicineAnesthesiologyAnesthetic MechanismsPerioperative Critical CareCritical Care and Emergency MedicineNeurointensive CareNeurologyCerebrovascular DiseasesIschemic StrokeNeurointensive CareThe Early Stage Formation of PI3K-AMPAR GluR2 Subunit Complex Facilitates the Long Term Neuroprotection Induced by Propofol Post-Conditioning in Rats PI3K-AMPAR GluR2 and Propofol Post-ConditioningWang Haiyun 1 * Wang Guolin 1 Wang Chenxu 1 Wei Ying 1 Wen Zhiting 1 Wang Chunyan 1 Zhu Ai 1 1 Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin Research Institute of Anesthesiology, Tianjin, People's Republic of China Xie Zhongcong Editor Massachusetts General Hospital, United States of America * E-mail: wanghy819@hotmail.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: HW . Performed the experiments: CW YW. Analyzed the data: ZW. Contributed reagents/materials/analysis tools: AZ. Wrote the paper: HW GW. Revised submission critically for important intellectual content: CW. 2013 11 6 2013 8 6 e6518720 1 2013 22 4 2013 © 2013 Wang et al2013Wang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Previously, we have shown that the phosphoinositide-3-kinase (PI3K) mediated acute (24 h) post-conditioning neuroprotection induced by propofol. We also found that propofol post-conditioning produced long term neuroprotection and inhibited the internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit up to 28 days post middle cerebral artery occlusion (MCAO). However, the relationship between PI3K with AMPA receptor GluR2 subunit trafficking in propofol post-conditioning has never been explored. Here we showed that propofol post-conditioning promoted the binding of PI3K to the C-terminal of AMPA receptor GluR2 subunit and formed a complex within 1 day after transient MCAO. Interestingly, the enhanced activity of PI3K was observed in the hippocampus of post-conditioning rats at day 1 post ischemia, whereas the decrease of AMPA receptor GluR2 subunit internalization was found up to 28 days in the same group. Administration of PI3K selective antagonist wortmannin inhibited the improvement of spatial learning memory and the increase of neurogenesis in the dentate gyrus up to 28 days post ischemia. It also reversed the inhibition of AMPA receptor GluR2 internalization induced by propofol post-conditioning. Together, our data indicated the critical role of PI3K in regulating the long term neuroprotection induced by propofol post-conditioning. Moreover, this role was established by first day activation of PI3K and formation of PI3K-AMPA receptor GluR2 complex, thus stabilized the structure of postsnaptic AMPA receptor and inhibited the internalization of GluR2 subunit during the early stage of propofol post-conditioning. This work was supported by Natural Science Foundation of China (81071059, 81100984, 30972847), Science and Technology Supported Key Project of Tianjin (12ZCZDSY03000), Scientific Grant from Tianjin Health Bureau (09KZ106). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction During the cerebral ischemia, a rise in intracellular calcium ([Ca2+]i) is thought to initiate a cascade of events leading to the cell death, including activation of proteases and endonucleases, generation of free radicals that destroy cell membranes by lipid peroxidation, and induction of apoptosis [1]–[6]. Although α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are initially thought to be relatively impermeable to Ca2+, it is now clear that there is also AMPA receptor (AMPAR) exhibiting considerable Ca2+ permeability. AMPARs containing the GluR2 subunit exhibit low Ca2+ permeability, whereas AMPARs lacking GluR2 are much more Ca2+ permeable (Cp-AMPARs) [7]–[9]. AMPAR-mediated excitotoxicity is thought to play a critical role in CNS ischemic insults [10], [11]. We have showed that propofol post-conditioning inhibited AMPAR GluR2 subunit internalization in hippocampal neurons and provided neuroprotection to cerebral ischemia/reperfusion (I/R) injury. These effects were sustained to 28 days post-ischemia [12]. Therefore, the maintenance of the surface expression of Ca2+-impermeable AMPARs may play the key protective role during cerebral ischemia/reperfusion injury. The intracellular signaling pathways, which modulate AMPARs trafficking during such processes, are not fully understood. We have found that propofol post-conditioning displayed the acute (24 h) neuroprotection partly through the phosphorylation of Akt, one of the phosphoinositide-3-kinase (PI3K) effectors [13]. The PI3K/Akt pathway is an attractive target because it has been shown to be involved in the synaptic plasticity, neuroprotection during cerebral ischemia/reperfusion injury [14]–[16]. A previous study showed that a slow, but constant, turnover of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) at synapses is required for maintaining AMPA receptor clustering and synaptic strength under basal conditions [17]. PIP3 is the product of the reaction catalyzed by Class I PI3K, therefore we hypothesized that keeping the activity of PI3K plays a key role in maintenance the basal structure of the surface AMPARs, thus inhibiting the internalization of AMPAR GluR2 subunit during cerebral ischemia/reperfusion injury. In this context, we first studied the role of PI3K activation in long term neuroprotection induced by propofol post-conditioning; second, we explored whether the inhibition of PI3K activity can affect the internalization of AMPAR GluR2, and in which pattern them interacted. Materials and Methods Adult male Sprague–Dawley rats (250–280 g) were cared for according to the Guide for the Care and Use of Laboratory Animals. The committee of experimental animals of Tianjin Medical University approved all the surgical procedures. All rats were anesthetized by intraperitoneal injection of Inactin (thiobutabarbital, 100 mg/kg; RBI, Natick, MA) and ventilated with oxygen (35%) and air mixture [12]. Stroke Model and Grouping The reversible right side MCAO (60 min) was performed as previous published [13]. Regional cerebral blood flow (CBF) was monitored by laser-Doppler flowmeter (Periflux system 5000; Perimed Inc., Jarfalla, Sweden). The rats that did not show a cerebral blood flow reduction of at least 70% were excluded from the experimental group [18]. One hour after MCAO, the suture was removed to allow reperfusion, confirmed by the increase of CBF at the same area. Polyethylene catheters were inserted into the right femoral artery and vein for blood pressure monitoring, blood gases measurement, and drug administration. Physiological variables (mean arterial blood pressure, temperature, arterial blood gases and plasma glucose) were measured 30 min before ischemia, at the onset of ischemia and 30 min after reperfusion. Body temperature was monitored with a rectal probe and maintained at 37±0.5°C by warming blanket and lamps until the animals showed adequate motor activity. All rats were divided randomly into six groups: (i) sham-operated group (n = 65); (ii) Ischemia/Reperfusion (I/R) group: 60 min MCAO followed by reperfusion (n = 60); (iii) propofol Post-cond group: propofol 20 mg/kg/h was infused intravenously with syringe pump (Beijing Slgo Medical Technology Development Co., Ltd., Beijing, China) at the onset of reperfusion for 4 h (n = 64). The other three groups were (iv) Wort + sham-operated group (n = 65); (v) Wort + I/R group (n = 62); (vi) Wort + propofol Post-cond group (n = 64); received wortmannin 0.6 mg/kg intravenously 30 min before sham-operation, MCAO and propofol Post-cond procedures, respectively. In case of sham-operated, I/R, Wort + sham-operated and Wort + I/R group, equivalent dose of saline was administered in the same manner of propofol administration. Morris Water Maze Task On day 9 and 23 after MCAO, rats (n = 8–10/group) were tested for spatial learning memory using Morris Water Maze (MWM) procedure [19], [20]. The MWM training consisted of spatial acquisition and reference memory probe trials. For spatial acquisition, latency (time to reach the platform) and swim speed were recorded with a computerized tracking system (Ethovision 3.0; Noldus Information Technology, Wageningen, the Netherlands). Four trials from four different random start positions at north, east, south, and west were tested daily (each lasted 2 min with 30-s intervals) for 5 days. Rats that failed to find the platform within 2 min were guided and their maximum latency score was recorded as 120 s. At 24 h after the last training day, rats were tested for reference memory. The time period (s) when a rat stayed in the goal quadrant, where the hidden platform was previously located, was recorded and expressed as a percent of time in the 60 s total swimming period. In this study, swimming time and distance within only a 30 cm circular zone around the previous platform, that is, not in the whole quadrant, were recorded [21]. Neurogenesis in the Ipsilateral Dentate Gyrus (DG) of Hippocampus Bromodeoxyuridine (BrdU), a thymidine analog which replaces thymidine in newly synthesized DNA, was used to label endogenous proliferating cells. BrdU (100 mg /kg) was injected intraperitoneally on day 7 to 9 after MCAO. After 28 days, the animals were deeply anesthetized and transcardially perfused with 100 mL normal saline, followed by 50 mL 4% in 0.2 M phosphate buffer. The brains were removed, post-fixed for 24 h in paraformaldehyde–phosphate buffer and placed for 48 h in 30% sucrose. The 33 μm coronal sections of brain were prepared at the level of bregma –3.3±0.2 mm. Ten sections of each brain (n = 4–5 rats/group) with immunofluorescence-double staining were used to calculate the ration between BrdU (anti-BrdU-antibody, Abcam Biotechnology, Cambridge, UK) + neuron-specific nuclear protein (NeuN) (anti-neuron-specific nuclear protein, Chemicon International, Temecula, CA, USA)-positive cells and the total amount of BrdU positive cells. For each section, 50 BrdU positive cells in the ipsilateral dentate gyrus (DG) were analyzed for coexpression of BrdU and NeuN to determine the ratio of newly generated neurons (BrdU + NeuN) to the total amount of newborn cells (BrdU). Positive and negative controls and tests for excluding cross-reactions for the two secondary antibodies were performed. For the measurement of the dentate gyrus volume, we adapted the method from Engelhard et al. [22]. Briefly, 10 sections (n = 5 rats/group, 40 μm each section) were prepared at the level of bregma –3.3±0.2 mm and stained with hematoxylin and eosin (HE). We measured the dentate gyrus volume of 10 sections for each brain using the Image J software 1.42 (National Institutes of Health, USA). The volume of the dentate gyrus was calculated by multiplying the mean value of the 10 sections with the thickness of one slice (40 μm) and by 10. PI3K Activity Measurement To determine the true activity of PI3K, we preformed a PI3K enzyme-linked immunosorbent assay (ELISA) kit (Cat# K-1000 s, Echelon Biosciences, Salt Lake City, UT, USA) [23], [24]. The rat hippocampus (n = 4–5 rats/group for each time point, 14–15 rats per group) was harvested at day 1, 14 and 28 after MCAO. Saline 0.9% 500 μL was then added and centrifuged for 10 minutes at 12,000 rpm. The upper limpid liquid was aliquoted (∼20 aliquots per rat) and stored at −20°C. We added 25 μL of samples in kinase reaction buffer to an equal volume of 8 μM of phosphatidylinositol-(4,5)-bisphosphate (PIP2) substrate, incubated at 37°C for 2 h. An equal volume of stop solution, containing PIP3 detector and EDTA was then added to stop the reaction. A 100 μL aliquot of this mixture was added to the wells that was coated with PIP3, and then incubated at room temperature for 1 h. Transferred the mixture to corresponding wells of the Detection Plate (K-1001 s) and incubated for 1 h at room temperature, then sealed with the secondary detector (K-SEC1) for 30 min. The reaction was stopped by adding TMB solution (K-TMB1) and read absorbance at 450 nm. The sample values were extrapolated from a standard curve of O.D. vs. known PIP3 concentration, and the activity was expressed as the percentage of control. Detection of AMPA Receptor GluR2 Subunit Internalization and Immunoblotting Surface and intracellular AMPA receptor GluR2 levels were performed with a protein cross-linking assay [12], [25]. Briefly, On day 1, 14 and 28 after transient MCAO, rats (n = 4–5 rats/group for each time point, 14–15 rats per group) were decapitated, brains were removed rapidly, and the hippocampus was rapidly isolated on an ice-cold platform and chopped into 400 μm slices using a McIllwain tissue chopper (Vibratome, St. Louis, MO). Slices were then incubated with 2 mM bis (sulfosuccinimidyl) suberate (BS3; Pierce Biotechnology, Rockford, IL, USA) for 15 min at 4°C. Cross-linking was terminated by 100 mM glycine (10 min at 4°C) and pelleted by brief centrifugation. Samples were aliquoted (∼15 aliquots per rat) and stored at −80°C for further analysis. The protein (30 μg) was separated by SDS/PAGE, transferred to PVDF membranes and probed with primary antibodies for AMPAR subunits GluR2 (Cat# MAB 397, N terminus, 1:1000, Millipore, Billerica, MA) at 4°C overnight. Blots were washed and then incubated with goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (1:5000; Millipore). We compared identical amounts of cross-linked and non-cross-linked tissue probed with antibodies to GluR2 to rule out the concern that cross-linking might interfere with immunodetection of the surface band. We used Image J (NIH) to measure the band densities in blinded fashion. T (Total) protein level = surface (cross-linked) protein + intercellular (non-cross-linked) protein; S/T ratio = surface protein / (surface protein + intercellular protein). Coimmunoprecipitation and Western Blotting of PI3K and AMPAR GluR2 Subunit To determine the relationship between PI3K and AMPAR GluR2 subunit, we used coimmunoprecipitation and immunoblotting assay [26], [27]. At day 1, 14, 28 after MCAO, hippocampus slices (n = 4–5 rats/group for each time point, 14–15 rats per group) were collected and homogenized in lysis buffer with 0.5% Triton X-100, 150 mM NaCl, 5 mM EDTA, and 50 mM Tris supplemented containing protease and phosphatase inhibitors. Total protein were aliquoted (∼5 aliquots per rat) and stored at −80°C. Immunoprecipitations of equivalent protein (100 μg) amounts were performed at 4°C for 4 h by using 1 μg anti-GluR2 antibody (Cat# AB 10529, C terminus, 1:1000, Millipore, Billerica, MA) or an equal amount of control IgG. The antibody protein complexes were captured with protein A/G plus agarose (Santa Cruz Biotechnology). Proteins were eluted from the beads and subjected to SDS-PAGE and immunoblotting for anti-PI3K (Cat# 4292, p85, 1:1000, Cell Signaling, Beverly, MA). Blots were developed using enhanced chemiluminescence detection (Amersham Biosciences). Band intensities were quantified using Image J software 1.42 (National Institutes of Health, USA). Statistical Analyses Data are presented as mean ± SEM. Statistical calculations were performed in SPSS 16.0 (SPSS Science, Inc., Chicago, IL, USA). Probe test data of MWM and neurogenesis were analyzed by one-way ANOVA and followed by post-hoc Turkey test. The latency of MWM and physiological parameters were analyzed using a four factors (ischemia × propofol × wortmannin × time) repeated measures ANOVA, followed by post-hoc Turkey test. Separate univariate analyses of ELISA and Western blot data were performed with respect to ischemia/reperfusion injury exposure, propofol post-conditioning, wortmannin administration and days of recovery, followed by post-hoc Turkey test. A value of P<0.05 was considered as statistically significant. Results Physiological Variables All rats were anesthetized with Inactin (thiobutabarbital, 100 mg/kg) at the beginning of our experiment, and the long-lasting effect of Inactin maybe the reason for no or little movement in rats. The animals receiving the 20 mg/kg/h dose of propofol were heavily sedated with little or no movement and did not show any response to the withdrawal reflex resulting from pinching the hind paw. No statistical differences were observed in physiological parameters (mean arterial blood pressure, temperature, arterial blood gases and plasma glucose) between the groups at each time point (data not shown). Rats weighted from 250 to 280 g, with a median weight of 263.4 g. The rectal temperature was maintained at 37±0.5°C by warming blanket and lamps. Major bleeding was observed in four rats, which were removed from the study. Spatial Memory Outcome In the two sessions of training (day 9 to 13 and day 23 to 27, Figure 1A and B), rats in propofol Post-cond group required less time to find the platform than those in I/R group (from day 10 to 13, P<0.0001; from day 23 to 27, P<0.0001, respectively), although they spent more time than those in sham-operated group (from day 10 to 12, P<0.0001; from day 24 to 26, P<0.0001; P = 0.012 at day 13, P = 0.001 at day 23 and P = 0.014 at day 27, respectively). Pretreatment of wortmannin, which is a selective PI3K inhibitor, increased the escape latencies (P = 0.002 at day 10; P<0.0001 vs. propofol Post-cond group at the other days). As expected, the latency (the time to reach platform) and path length were significantly shortened during the two trials of 5-day acquisition period, suggesting that spatial acquisition had developed. Although the analysis of escape latency revealed significant differences between groups, there were no significant differences in the swimming speed between these groups (averaged 0.25±0.04 m/s). 10.1371/journal.pone.0065187.g001Figure 1 Propofol post-conditioning enhanced the spatial learning memory ability of rats after transient MCAO, whereas selective PI3K antagonist wortmannin decreased it. (A) Escape latency in the first session from day 9 to 13. (B) Escape latency in the second session from day 23 to 27. (C) Time in the target quadrant in probe trials (day 14 and 28). Data are expressed as mean ± SEM (n = 8–10/group), P<0.05, P<0.01. Sham, sham-operated group; I/R, I/R group; Pro, propofol Post-cond group; W + sham, Wort + sham-operated group; W + I/R, Wort + I/R group; W + Pro, Wort + propofol Post-cond group. In the two sessions (14 and 28 days after reperfusion) of probe test, rats in propofol Post-cond group spent significantly more time than those of I/R group in the quadrant where the platform had been (32.0±1.6 vs. 21.5±2.5, P<0.0001 at day 14; 37.8±1.8 vs. 32.5±2.1, P = 0.004 at day 28), but the time was decreased by administration of wortmannin (25.2±1.9 vs. 32.0±1.6, P = 0.003 vs. propofol Post-cond group at day 14; 25.2±1.9 vs. 21.5±2.5, P = 0.001 vs. I/R group at day 14; 31.4±3.4 vs. 37.8±1.8, P<0.0001 vs. propofol Post-cond group at day 28; 31.4±3.4 vs. 32.5±2.1, P = 0.515 vs. I/R group at day 28; Figure 1C). Neurogenesis in the Ipsilateral DG of Hippocampus The newly generated neurons in the ipsilateral DG after 28 days of survival are shown in Figure 2A and B. Twenty-eight days after transient MCAO, the amount of BrdU + NeuN positive neurons increased 1.5 folds in the DG of I/R group as compared with that of sham-operated group (252.3±22.1%, P<0.0001). Propofol administration for 4 h from the beginning of reperfusion stimulated neurogenesis in the ipsilateral DG as compared with that of I/R group (434.0±19.5% vs. 252.3±22.1%, P<0.0001). Wortmannin eliminated the stimulation of neurogenesis induced by I/R insult and propofol post-conditioning (for Wort + I/R group: 159.0±10.8% vs. 252.3±22.1%, P<0.0001 as compared with I/R group; for Wort + propofol Post-cond group: 267.4±32.9% vs. 434.0±19.5%, P<0.0001 as compared with propofol Post-cond group). The average volume of the ipsilateral DG was similar for all groups, independent of the drug usage or cerebral ischemia (Figure 2C). 10.1371/journal.pone.0065187.g002Figure 2 Effect of propofol post-conditioning and wortmannin on neurogenesis in the DG of hippocampus after transient MCAO. (A) BrdU and NeuN immunofluorescence-double staining of new generated neurons in the DG. Scale bar: 10 μm. (B) Quantification of neurogenesis at day 28 after focal ischemia/reperfusion. (C) Quantification of the ipsilateral DG volume in each group. Bar represents mean ± SEM (n = 4–5/group), P<0.05, *P<0.01. Sham, sham-operated group; I/R, I/R group; Pro, propofol Post-cond group; W + sham, Wort + sham-operated group; W + I/R, Wort + I/R group; W + Pro, Wort + propofol Post-cond group. PI3K Activity PI3K activity was shown in Figure 3. The cerebral ischemia/reperfusion injury inhibited the activity of PI3K at day 1 post MCAO as compared with that of sham-operated group (68.4±4.5% vs. 100.0±4.3%, P<0.01), whereas at day 14 and 28, it enhanced the PI3K activity (109.5±6.4%, P<0.01 at day 14; 111.2±5.3%, P = 0.03 at day 28 vs. sham-operated group, respectively). Propofol post-conditioning increased PI3K activity at day 1 as compared with that of I/R group (187.0±15.2% vs. 68.4±4.5%, P<0.01). There was no significant difference between these two groups at day 14 and 28. As compared with sham-operated group, propofol post-conditioning also elevated the activity of PI3K (187.0±15.2% vs. 100.0±4.3%, P<0.01 at day 1; 117.0±8.3% vs. 100.0±5.5%, P<0.01 at day 14; 111.6±7.4% vs. 100.0±4.3%, P<0.05 at day 28, respectively). Administration of PI3K selective inhibitor wortmannin reversed the enhancement of PI3K activity induced by propofol post-conditioning at day 1 (69.4±7.2% vs. 187.0±15.2%, P<0.01 vs. propofol post-Cond group, 69.4±7.2% vs. 68.4±4.5%, P = 0.842 vs. I/R group), but this effect disappeared at other time points. 10.1371/journal.pone.0065187.g003Figure 3 Effect of propofol post-conditioning and wortmannin on the activation of PI3K after transient MCAO. Bar represents mean±SEM (n = 4–5/group), P<0.05, *P<0.01. Sham, sham-operated group; I/R, I/R group; Pro, propofol Post-cond group; W + sham, Wort + sham-operated group; W + I/R, Wort + I/R group; W + Pro, Wort + propofol Post-cond group. AMPARs GluR2 Subunit Internalization The GluR2 S/T values in I/R group were significantly reduced as compared with those of sham-operated rats (day 1: 23.0±2.3% vs. 100.0±2.1%; day 14: 36.6±3.3% vs. 100.0±1.4%; day 28: 42.8±5.2% vs. 100.0±1.7%; P<0.01 at varies time points), whereas these were increased by propofol post-conditioning (day 1: 66.0±5.0% vs. 23.0±2.3%; day 14: 55.4±3.3% vs. 36.6±3.3%; day 28: 54.4±2.0% vs. 42.8±5.2%; P<0.05 vs. I/R group at day 1, 14 and 28 after MCAO, Figure 4A and B), indicating that propofol reversed the internalization of AMPARs GluR2 subunit induced by ischemia/reperfusion injury, and this trend was kept until 28 days after reperfusion. Administration of PI3K selective antagonist wortmannin eliminated the effect of propofol in restricting GluR2-containing AMPARs in the cell surface up to 1 day after transient MCAO (24.6±6.0% vs. 66.0±5.0%, P<0.01 vs. propofol Post-con group), whereas this effect disappeared at day 14 and 28 (55.6±3.3% vs. 55.4±3.3%, P = 0.913 at day 14; 55.4±6.0% vs. 54.4±2.0%, P = 0.505 at day 28 vs. propofol Post-con group, respectively; Figure 4A and B). There was no difference in total subunit protein expression between the six groups on days 1, 14 and 28 after transient MCAO (Figure 4C). 10.1371/journal.pone.0065187.g004Figure 4 Propofol post-conditioning maintained the surface expression of AMPAR GluR2 subunit during cerebral ischemia/reperfusion injury, whereas wortmannin reversed it on day 1 after transient MCAO. (A) Western blot analysis showed BS3 cross-linked surface and intracellular pools of AMPAR GluR2 subunit at day 1, 14 and 28 after reperfusion. (B) Quantification of surface/total GluR2 subunit expression after reperfusion. (C) Quantification of total GluR2 subunit expression. Bar represents mean ± SEM (n = 4–5/group), P<0.05, *P<0.01. Sham, sham-operated group; I/R, I/R group; Pro, propofol Post-cond group; W + sham, Wort + sham-operated group; W + I/R, Wort + I/R group; W + Pro, Wort + propofol Post-cond group. Coimmunoprecipitation and Immunoblotting of PI3K and AMPAR GluR2 subunit As shown in Figure 5A and B, the cerebral ischemia/reperfusion injury caused a significant reduction of PI3K p85 subunit which bound to C-terminal of AMPARs GluR2 (76.8±5.8% vs. 100.0±4.8%, P<0.01 vs. sham-operated) at day 1 after MCAO. Rats in propofol Post-cond group showed an increase in bound PI3K (180.4±5.1% vs. 100.0±4.8%, P<0.01 vs. sham-operated; 180.4±5.1% vs. 76.8±5.8%, P<0.01 vs. I/R group, P<0.001, respectively) at day 1. Wortmannin inhibited the binding of PI3K with C-terminal of GluR2 subunit in the other three groups (for Wort + sham-operated group: 91.6±6.0% of sham-operated group, P>0.05; for Wort + I/R group: 70.9±4.6% vs. 76.8±5.8%, P>0.05 as compared with I/R; for Wort + propofol Post-cond group: 120.4±1.9% vs. 180.4±5.1%, P<0.01 as compared with propofol Post-cond group). We can not detect such interaction between PI3K and AMPAR GluR2 subunit at day 14 and 28 after MCAO. 10.1371/journal.pone.0065187.g005Figure 5 Propofol promoted the formation of PI3K-AMPAR GluR2 complex in the early stage (1 day) after transient MCAO, whereas wortmannin inhibited it. (A) Immunoprecipitation and immunoblotting assays showed PI3K-AMPARs GluR2 binding after transient MCAO. (B) Quantification of the binding of PI3K to the C-terminal of AMPAR GluR2 subunit at day 1, 14 and 28 after MCAO. Bar represents mean ± SEM (n = 4–5/group), P<0.05, **P<0.01. Sham, sham-operated group; I/R, I/R group; Pro, propofol Post-cond group; W + sham, Wort + sham-operated group; W + I/R, Wort + I/R group; W + Pro, Wort + propofol Post-cond group. In the present study, we reported an important role of stimulation and maintenance the activity of PI3K during the early stage (24 h) of propofol post-conditioning, which could improve MWM performance, increase neurogenesis in the ipsilateral DG of hippocampus and inhibit the internalization of AMPAR GluR2 subunit (Ca2+ impermeable AMPARs) up to 28 days for cerebral ischemia/reperfusion injury rats. Our results supported this finding by administration of selective PI3K antagonist wortmannin, which reversed the improvement of spatial learning memory, the increase of neurogenesis and the inhibition of GluR2 internalization induced by propofol post-conditioning after transient MCAO. We also detected the formation of intracellular PI3K-AMPAR GluR2 subunit complex at the acute phrase of PI3K activation, indicating a critical protective PI3K-AMPAR GluR2 pathway which mediates propofol induced post-conditioning. Discussion In the adult brain, the persistent neuronal production suggested a potential ability for self-repair after injury [28], [29], especially following ischemic brain injury [30]. After ischemia, the proliferation of progenitors was upregulated several fold, and roughly half of postischemic precursors acquired neuronal phenotype in the granule cell layer of DG, while a few became astrocytes in CA4 sector [31]. About 80% of the initially proliferated cells disappear within 4 wk post ischemia, only the long-term surviving neurons may contribute to postischemia recovery [32]. This is the reason we chose 28 days to check the generation of neurons in the DG of hippocampus. In the current study, we found that cerebral ischemia/reperfusion injury stimulated neurogenesis in the ipsilateral DG to 252.3% of sham-operated group. Propofol post-conditioning promoted the increase of new neurons from 252.3% to 434.0%, whereas pretreatment of the selective PI3K antagonist wortmannin eliminated the stimulation of neurogenesis induced by ischemic insult (from 252.3±22.1% to 159.0±10.8%) and propofol post-conditioning (from 434.0±19.5% to 267.4±32.9%). We also assessed the learning ability and spatial memory by MWM, and found that the improved spatial acquisition were seen in propofol-treated rats, suggesting that propofol post-conditioning induced increases in post stroke neurogenesis may contribute to post stroke recovery. In our experiments, we could not detect the statistical differences in physiological parameters (mean arterial blood pressure, temperature, arterial blood gases and plasma glucose) between the groups at each time point. We still found that the blood pressure of rats in propofol used groups was a little bit lower than the other groups. The decreased blood pressure may negate the neuroprotection induced by propofol post-conditioning. Currently several molecular regulatory pathways are known to be involved in the neuroprotective mechanisms of ischemic post-conditioning, as it increased the expression of glutamine synthetase [33] and glutamate transporter-1 [34] in global ischemia; promoted the opening of mitochondrial potassium ATP-dependent channel (mitoK(ATP)), thus inhibited the opening of mitochondrial permeability transition pore (MPTP) in focal cerebral ischemia [35]. PI3K survival signaling pathway supports cell survival [36], including its role in blocking neuronal death after stroke [37], [38]. Previously, we observed that propofol post-conditioning established acute (24 h) neuroprotection partly through the activation of Akt, a central effector in the PI3K pathway [13]. However, the role of PI3K/Akt pathway in modulating the long term effect of propofol post-conditionig has never been explored and the phosphorylated level of Akt can not totally represent the activation of PI3K [39]. In the present study, we investigated the activity of PI3K with the competitive ELISA method which measures the catalyzed production of PIP3 produced by activated PI3K [23], [24]. We found that cerebral ischemia/reperfusion decreased PI3K activity from baseline (sham-operated group, 100.0%±4.3%) to 68.4%±4.5% (P<0.01), whereas propofol post-conditioning increased it to 187.0%±15.2% at day 1. At day 14 and 28, the activity of PI3K declined almost to the baseline level (117.7%±8.3% at day 14, 111.6%±7.4% at day 28), with propofol 20 mg/kg/h post-conditioning exposure. The selective PI3K antagonist wortmannin exposure inhibited the expected increase in PI3K activity following propofol post-conditioning at 1 day after transient MCAO in rat hippocampus, from the increase of 187.0%±15.2% dropped to 69.4%±7.2% of baseline. However, such effect disappeared at day 14 and 28 due to the half-life of of wortmannin was 57.8 h in PBS [40], [41]. AMPARs mediate fast synaptic transmission at excitatory synapses in the central nervous system (CNS) and are heteromeric complexes composed of glutamate receptor subunit 1–4 (GluR1-GluR4) [42], [43]. Of these, the GluR2 subunit plays a crucial role in controlling the calcium permeability of AMPA receptors. GluR2 mRNA ordinarily undergoes post-transcriptional editing so that the expressed protein contains a positively-charged arginine in place of the gene-encoded glutamate at a critical position in the M2 membrane loop that forms the lining of the AMPA receptor's pore [44]. Therefore, GluR2-lacking AMPAR-mediated excitotoxicity is thought to play a critical role in CNS ischemic insults [10], [45]. Our previous study showed that AMPAR GluR2 subunit in hippocampal neurons redistributed to the cell surface during propofol post-conditioning and this effect sustained to 28 days post-ischemia. However, when ischemic rats were challenged with saline, the AMPARs GluR2 subunit S/T ratio decreased, indicating a reduction in the cell surface expression. What are the intracellular signaling pathways that produce either net AMPAR insertion or internalization, thereby dictating the expression of these two opposing forms of AMPAR-dependent synaptic plasticity? A recent study showed that continuous synthesis and availability of PIP3 at the postsynaptic terminal was necessary for sustaining synaptic function in rat hippocampal neurons. This requirement was specific for synaptic, but not extrasynaptic, AMPA receptors [17]. As PIP3 is the catalyzed production of PI3K, we speculate that the activition of PI3K is necessary for the maintenance of AMPARs GluR2 subunit expression at postsynaptic membrane. Interestingly, we found here, the administration of selective PI3K antagonist wortmannin suppressed the AMPARs GluR2 subunit S/T ratio, thus reversed the inhibition of GluR2 internalization induced by propofol post-condittioning during the first day after cerebral ischemia/reperfusion injury, such effect of wortmannin disappeared at day 14 and 28 after transient MCAO duo to its metabolism in vivo [37], [38]. However, the pattern in which PI3K communicated with AMPARs GluR2 is largely unknown. The p85 regulatory subunit of PI3K contains multiple protein-protein interaction motifs [46]. Therefore, we hypothesize that this subunit directly binds to regions of the C-terminal of GluR2, and regulates its trafficking. Consistent with this hypothesis, we found that C-terminal of GluR2 antibody could specifically precipitate PI3K p85 subunit of hippocampal lysate using a coimmunoprecipitation assay (Figure 5A). We further found that ischemia/reperfusion injury decreased the formation of such PI3K-GluR2 complex, whereas propofol post-conditioning increased it. Furthermore, adding wortmannin significantly suppressed the elevated formation of this complex induced by propofol post-conditioning. In our present study, such changes of the PI3K-GluR2 complex could only be detected in hippocampus at day 1 after transient MCAO, suggesting that PI3K regulated the internalization of AMPARs GluR2 through the formation of intracellular complex with C-terminal of GluR2 at the early stage of propofol post-conditioning. However, in the post-conditioning group, the PI3K activity declined to the baseline at 14 and 28 days post ischemia, whereas the inhibition of AMPARs GluR2 subunit internalization sustained to 28 days implied that in addition to PI3K, there was other alternate pathways which could maintain the recruiting of AMPARs GluR2 to cellular membranes, thus minimize delayed cerebral injury during propofol post-conditioning. Another attractive scenario is that, in spit of the surface and synaptic GluR2 distribution altered, the total protein levels of GluR2 subunit were unaltered in hippocampal neurons between 6 groups at all times examined. The above results indicated that during ischemic insults, facts of GluR2-lacking AMPARs to be delivered and GluR2-containing AMPARs must be removed, were consistent with a role for placeholders or “slots” that specify (delimit) AMPAR number at synaptic sites [47]. Although the molecular identify of the slots is unknown, receptor-binding or scaffolding proteins such as stargazin are thought to participate in slot formation [48]. The activation of PI3K and the formation of PI3K-AMPAR GluR2 complex in propofol post-conditioning group within the first day post ischemia, whereas the improvement of spatial learning memory, enhanced neurogenesis in the ipsilateral DG and inhibited the internalization of AMPAR GluR2 subunit (Ca2+ impermeable AMPARs) up to 28 days in the same group. All these observations suggested that alternative pathways may regulate the long term neuroprotection of propofol post-conditioning after the function of PI3K disappeared in a PI3K-independent manner [39], [49]. In conclusion, we presently showed that propofol post-conditioning (20 mg/ kg/ h infused at the onset of reperfusion for 4 h) provided long term neuroprotection through enhancing the activity of PI3K, thereby promoted the binding of PI3K to the C-terminal of AMPA receptor GluR2 subunit within 1 day after transient MCAO, thus stabilized the structure of postsynaptic AMPA receptor and decreased the internalization of AMPA receptor GluR2 subunit during cerebral ischemia/reperfusion injury. Our data indicated the important role of maintenance PI3K activity in regulating the long term (28 days post ischemia) neuroprotection induced by propofol post-conditioning. Moreover, our study also showed that the decrease of AMPA receptor GluR2 subunit internalization, the improvement of spatial learning memory ability and the increase of neurogenesis in the ipsilateral DG of hippocampus up to 28 days in the same group, indicating that when the effect of PI3K disappeared, there will be other upstream what could provide sustained neuroprotection for propofol post-conditioning. ==== Refs References 1 Rothman SM , Olney JW (1986 ) Glutamate and the pathophysiology of hypoxic–ischemic brain damage . Ann Neurol 19 : 105 –111 .2421636 2 Choi DW (1990 ) Cerebral hypoxia: some new approaches and unanswered questions . J Neurosci 10 : 2493 –2501 .1974918 3 Choi DW (1995 ) Calcium: still center-stage in hypoxic–ischemic neuronal death . Trends Neurosci 18 : 58 –60 .7537408 4 Puttfarcken PS , Getz RL , Coyle JT (1993 ) Kainic acid-induced lipid peroxidation: protection with butylated hydroxytoluene and U78517F in primary cultures of cerebellar granule cells . Brain Res 624 : 223 –232 .8252395 5 Bredesen DE (1995 ) Neural apoptosis . Ann Neurol 38 : 839 –851 .8526456 6 Meldrum BS (1995 ) Excitatory amino acid receptors and their role in epilepsy and cerebral ischemia . Ann NY Acad Sci 757 : 492 –505 .7611707 7 Hollmann M , Hartley M , Heinemann S (1991 ) Ca2+ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition . Science 252 : 851 –853 .1709304 8 Hume RI , Dingledine R , Heinemann SF (1991 ) Identification of a site in glutamate receptor subunits that controls calcium permeability . Science 253 : 1028 –1031 .1653450 9 Burnashev N (1996 ) Calcium permeability of glutamate-gated channels in the central nervous system . Curr Opin Neurobiol 6 : 311 –317 .8794087 10 Liu B , Liao M , Mielke JG , Ning K , Chen Y , et al (2006 ) Ischemic insults direct glutamate receptor subunit 2-lacking AMPA receptors to synaptic sites . J Neurosci 26 : 5309 –5319 .16707783 11 Tanaka H , Grooms SY , Bennett MV , Zukin RS (2000 ) The AMPAR subunit GluR2: still front and center-stage . Brain Res 886(1–2) : 190 –207 .11119696 12 Wang H , Luo M , Li C , Wang G (2011 ) Propofol post-conditioning induced long-term neuroprotection and reduced internalization of AMPAR GluR2 subunit in a rat model of focal cerebral ischemia/reperfusion . J Neurochem 119 : 210 –219 .21790606 13 Wang HY , Wang GL , Yu YH , Wang Y (2009 ) The role of phosphoinositide-3-kinase/Akt pathway in propofol-induced postconditioning against focal cerebral ischemia-reperfusion injury in rats . Brain Res 1297 : 177 –184 .19703434 14 Jackson C , Welch HC , Bellamy TC (2010 ) Control of cerebellar long-term potentiation by P-Rex-family guanine-nucleotide exchange factors and phosphoinositide 3-kinase . PLoS One 5 : e11962 .20694145 15 Ohta H , Arai S , Akita K , Ohta T , Fukuda S (2011 ) Neurotrophic effects of a cyanine dye via the PI3K-Akt pathway: attenuation of motor discoordination and neurodegeneration in an ataxic animal model . PLoS One 6(2) : e17137 .21347252 16 Lu C , Liu L , Chen Y , Ha T , Kelley J , et al (2011 ) TLR2 ligand induces protection against cerebral ischemia/reperfusion injury via activation of phosphoinositide 3-kinase/Akt signaling . J Immunol 187 : 1458 –1466 .21709150 17 Arendt KL , Royo M , Fernández-Monreal M , Knafo S , Petrok CN , et al (2010 ) PIP3 controls synaptic function by maintaining AMPA receptor clustering at the postsynaptic membrane . Nat Neurosci 13 : 36 –44 .20010819 18 Xing B , Chen H , Zhang M , Zhao D , Jiang R , et al (2008 ) Ischemic post-conditioning protects brain and reduces inflammation in a rat model of focal cerebral ischemia/reperfusion . J Neurochem 105 : 1737 –1745 .18248611 19 Morris RG , Garrud P , Rawlins JN , O'Keefe J (1982 ) Place navigation impaired in rats with hippocampal lesions . Nature 297 : 681 –683 .7088155 20 Morris R (1984 ) Developments of a water-maze procedure for studying spatial learning in the rat . J Neurosci Methods 11 : 47 –60 .6471907 21 Yun YJ , Lee B , Hahm DH , Kang SK , Han SM , et al (2007 ) Neuroprotective effect of palmul-chongmyeong-tang on ischemia-induced learning and memory deficits in the rat . Biol Pharm Bull 30 : 337 –342 .17268076 22 Engelhard K , Winkelheide U , Werner C , Kluge J , Eberspächer E , et al (2007 ) Sevoflurane affects neurogenesis after forebrain ischemia in rats . Anesth Analg 104 : 898 –903 .17377103 23 Bhattacharyya S , O-Sullivan I , Katyal S , Unterman T , Tobacman JK (2012 ) Exposure to the common food additive carrageenan leads to glucose intolerance, insulin resistance and inhibition of insulin signalling in HepG2 cells and C57BL/6J mice . Diabetologia 55 : 194 –203 .22011715 24 Chuang CC , Yang RS , Tsai KS , Ho FM , Liu SH (2007 ) Hyperglycemia enhances adipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt, and peroxisome proliferator-activated receptor gamma signaling . Endocrinology 148 : 4267 –4275 .17540722 25 Boudreau AC , Wolf ME (2005 ) Behavioral sensitization to cocaine is associated with increased AMPA receptor surface expression in the nucleus accumbens . J Neurosci 25 : 9144 –9151 .16207873 26 Mayat E , Petralia RS , Wang YX , Wenthold RJ (1995 ) Immunoprecipitation, immunoblotting, and immunocytochemistry studies suggest that glutamate receptor delta subunits form novel postsynaptic receptor complexes . J Neurosci 5 : 2533 –2546 . 27 Gu Z , Jiang Q , Fu AK , Ip NY , Yan Z (2005 ) Regulation of NMDA receptors by neuregulin signaling in prefrontal cortex . J Neurosci 25 : 4974 –4984 .15901778 28 Parent JM (2003 ) Injury-induced neurogenesis in the adult mammalian brain . Neuroscientist 9 : 261 –272 .12934709 29 Lie DC , Song H , Colamarino SA , Ming GL , Gage FH (2004 ) Neurogenesis in the adult brain: new strategies for central nervous system diseases . Annu Rev Pharmacol Toxicol 44 : 399 –421 .14744252 30 Tonchev AB (2011 ) Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates . Arch Ital Biol 149 : 225 –231 .21701994 31 Liu J , Solway K , Messing RO , Sharp FR (1998 ) Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils . J Neurosci 18 : 7768 –7778 .9742147 32 Takasawa K , Kitagawa K , Yagita Y , Sasaki T , Tanaka S , et al (2002 ) Increased proliferation of neural progenitor cells but reduced survival of newborn cells in the contralateral hippocampus after focal cerebral ischemia in rats . J Cereb Blood Flow Metab 22 : 299 –307 .11891435 33 Zhang W , Miao Y , Zhou S , Jiang J , Luo Q , et al (2011 ) Neuroprotective effects of ischemic postconditioning on global brain ischemia in rats through upregulation of hippocampal glutamine synthetase . J Clin Neurosci 18 : 685 –689 .21371894 34 Zhang W , Miao Y , Zhou S , Wang B , Luo Q , et al (2010 ) Involvement of Glutamate Transporter-1 in Neuroprotection against Global Brain Ischemia-Reperfusion Injury Induced by Postconditioning in Rats . Int J Mol Sci 11 : 4407 –4416 .21151445 35 Robin E , Simerabet M , Hassoun SM , Adamczyk S , Tavernier B , et al (2011 ) Postconditioning in focal cerebral ischemia: role of the mitochondrial ATP-dependent potassium channel . Brain Res 1375 : 137 –146 .21182830 36 Franke TF , Hornik CP , Segev L , Shostak GA , Sugimoto C (2003 ) PI3K/Akt and apoptosis: size matters . Oncogene 22 : 8983 –8998 .14663477 37 Chan PH (2004 ) Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia . Neurochem Res 29 : 1943 –1949 .15662830 38 Zhao H , Shimohata T , Wang JQ , Sun G , Schaal DW , et al (2005 ) Akt contributes to neuroprotection by hypothermia against cerebral ischemia in rats. J Neurosci . 25 : 9794 –9806 . 39 Ripple MO , Kalmadi S , Eastman A (2005 ) Inhibition of either phosphatidylinositol 3-kinase/Akt or the mitogen/extracellular-regulated kinase, MEK/ERK, signaling pathways suppress growth of breast cancer cell lines, but MEK/ERK signaling is critical for cell survival . Breast Cancer Res Treat 93 : 177 –188 .16187238 40 Yuan H , Luo J , Weissleder R , Cantley L , Josephson L (2006 ) Wortmannin-C20 conjugates generate wortmannin . J Med Chem 49 : 740 –747 .16420059 41 Yuan H , Pupo MT , Blois J , Smith A , Weissleder R , et al (2009 ) A stabilized demethoxyviridin derivative inhibits PI3 kinase . Bioorg Med Chem Lett 19 : 4223 –4227 .19523825 42 Hollmann M , Heinemann S (1994 ) Cloned glutamate receptors . Annu Rev Neurosci 17 : 31 –108 .8210177 43 Dingledine R , Borges K , Bowie D , Traynelis SF (1999 ) The glutamate receptor ion channels. Pharmacol . Rev 51 : 7 –61 . 44 Gryder DS , Castaneda DC , Rogawski MA (2005 ) Evidence for low GluR2 AMPA receptor subunit expression at synapses in the rat basolateral amygdala . J Neurochem 94 : 1728 –1738 .16045445 45 Peng PL , Zhong X , Tu W , Soundarapandian MM , Molner P , et al (2006 ) ADAR2-dependent RNA editing of AMPA receptor subunit GluR2 determines vulnerability of neurons in forebrain ischemia . Neuron 49 : 719 –733 .16504947 46 Vanhaesebroeck B , Leevers SJ , Panayotou G , Waterfield MD (1997 ) Phosphoinositide 3-kinases: a conserved family of signal transducers . Trends Biochem Sci 22 : 267 –272 .9255069 47 Barry MF, Ziff EB (2002) Receptor trafficking and the plasticity of excitatory synapses. Curr Opin Neurobiol 12: 279 –286. 48 Schnell E , Sizemore M , Karimzadegan S , Chen L , Bredt DS , et al (2002 ) Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number . Proc Natl Acad Sci USA 99 : 13902 –13907 .12359873 49 Filippa N , Sable CL , Filloux C , Hemmings B , Van Obberghen E (1999 ) Mechanisms of protein kinase B activation by cyclic AMP-dependent protein kinase . Mol Cell Biol 19 : 4989 –5000 .10373549	23776449	PMC3679144	CC BY	2021-01-05 17:29:25	yes	PLoS One. 2013 Jun 11; 8(6):e65187
==== Front Gastroenterol Res PractGastroenterol Res PractGRPGastroenterology Research and Practice1687-61211687-630XHindawi Publishing Corporation 10.1155/2013/381616Research ArticleInfluence of Gastrectomy on Cortical and Cancellous Bones in Rats 0000-0002-3700-5337Iwamoto Jun 1 Sato Yoshihiro 2 Matsumoto Hideo 1 1Institute for Integrated Sports Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan2Department of Neurology, Mitate Hospital, Fukuoka 826-0041, JapanJun Iwamoto: jiwamoito@a8.keio.jpAcademic Editor: Sergio Morini 2013 28 5 2013 2013 3816166 2 2013 15 4 2013 17 4 2013 Copyright © 2013 Jun Iwamoto et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The aim of the present study was to examine the influence of gastrectomy (GX) on cortical and cancellous bones in rats. Twenty male Sprague-Dawley rats were randomized into the two groups of 10 animals each: a sham operation (control) group and a GX group. Seven weeks after surgery, the bone mineral content and density (BMC and BMD, resp.) and the mechanical strength of the femur were determined, and bone histomorphometric analyses were performed on the tibia. GX induced decreases in the BMC, BMD, ultimate force, work to failure, and stiffness of the femoral distal metaphysis and the BMC, BMD, and ultimate force of the femoral diaphysis. GX induced a decrease in cancellous bone mass, characterized by an increased osteoid thickness, osteoid surface, osteoid volume, and bone formation. GX also induced a decrease in cortical bone mass, characterized by increased endocortical bone resorption. The GX induced reductions in the bone mass and strength parameters were greater in cancellous bone than in cortical bone. The present study showed that the response of bone formation, resorption, and osteoid parameters to GX and the degree of GX-induced osteopenia and the deterioration of bone strength appeared to differ between cortical and cancellous bones in rats. ==== Body 1. Introduction Gastric surgery is mostly needed for the treatment of gastric cancer. After gastrectomy (GX), the daily dietary intake of calcium, magnesium, phosphorus, iron, zinc, vitamin D, vitamin B12, and folic acid is reported to be lower than the recommendations [1–3]. In particular, GX impairs calcium and vitamin D metabolism, leading to a risk of bone disease including not only osteoporosis, but also osteomalacia or a mixed pattern of osteoporosis osteomalacia with secondary hyperparathyroidism [4–7]. The risk for vertebral and hip fractures is increased in GX patients [7–12]. However, a strategy for preventing fractures in GX patients has not yet been established. Preclinical studies using animals could be useful for identifying available interventions to prevent fractures in GX patients. An animal model of osteopenia can be created by performing GX in rats. Total GX and resection of the acid-producing part of the stomach (fundectomy) induces gastrinemia and malnutrition [13, 14], thereby initiating osteopenia in rats [15–19]. However, although bone histomorphometry studies have demonstrated GX-induced decreases in cancellous bone volume per tissue volume and cortical bone area in rats [17, 18], the changes in bone formation, resorption, and osteoid parameters after GX have not been adequately studied. The difference in the responses of cortical and cancellous bones to GX remains to be established in rats. Understanding the differential influence of GX on cortical and cancellous bones is important for clarifying the effect of interventions on the skeleton in GX rats. The aim of the present study was to examine the influence of GX on cortical and cancellous bones in rats. 2. Materials and Methods 2.1. Handling of Animals Twenty male Sprague-Dawley rats (11-week old) were purchased from Charles River Japan (Kanagawa, Japan). The animals were fed a standard pellet diet containing 1.25% calcium (calcium carbonate) and 0.9% phosphorus (CRF-1; Oriental Yeast, Co., Ltd., Tokyo, Japan). The rats were housed in a local animal room at a temperature of 24°C, a humidity of 50%, and a 12 h on/off cycle for lighting. Free access to water and a pellet diet were allowed. After 1 week of adaptation to this environment, the rats (12-week old) were sorted into strata according to body weight and were then randomized using the stratified weight method into the following two groups of 10 animals each: a sham operation control (CON) group and a GX group. A total GX was performed under general anesthesia using 25–30 mg/kg of pentobarbital (Kyoritsu Seiyaku Co., Ltd., Tokyo, Japan) injected intraperitoneally together with 2%-3% isoflurane (Mylan Inc., Tokyo, Japan) delivered via a Table Top Laboratory Animal Anesthesia System (V1 Type VetEquip, Inc., CA, USA). A longitudinal incision was made on the abdomen to expose the stomach. The whole stomach was excised, and an anastomosis of the esophagus and duodenum was performed. The weight of the rats was monitored weekly, and the duration of observation was 7 weeks. This experiment was performed at the laboratory of Hamri Co., Ltd. (Ibaraki, Japan), which has been approved by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Hamri Co., Ltd. (Ibaraki, Japan). 2.2. Preparation of Specimens Rats were labeled by subcutaneous injection of 10 mg/kg of calcein (Dojindo Laboratories, Kumamoto, Japan) at 7 and 3 days before death. Seven weeks after surgery, the animals were sacrificed by exsanguination under general anesthesia with 2%-3% isoflurane (Mylan Inc., Tokyo, Japan) using a Table Top Laboratory Animal Anesthesia System (V1 Type VetEquip, Inc., CA, USA). The bilateral femora and the right tibia were harvested from each animal. The length of the left femur was measured using a dial caliper, and the weight was measured using an electronic balance (A&D Company, Tokyo, Japan). Then, the femur was preserved in saline, stored in a freezer (−60°C), and processed for the mechanical strength test. The right femur and tibia were preserved in 70% ethanol and were processed for peripheral quantitative computed tomography (pQCT) and the bone histomorphometric analysis, respectively. 2.3. pQCT of the Femur The distal metaphysis and diaphysis of the femur were scanned using pQCT (XCT-Research SA+; Stratec Medizintechnik GmbH, Pforzheim, Germany) in 70% ethanol/saline. The bones were placed horizontally in a polypropylene tube and were scanned at a voxel size of 0.12 mm. The scan line was adjusted using the scout view, and sites 3 mm and 14 mm proximal to the distal growth plate were scanned. For the analysis, a threshold of 395 mg/cm3 in contour mode 2 was used to separate the bone tissue from the marrow in the distal metaphysis. The total bone mineral content (BMC) and bone mineral density (BMD) were evaluated. A constant threshold of 690 mg/cm3 in contour mode 1 was used to separate the cortical bone from the trabecular bone in the diaphysis. The cortical thickness, BMC, and BMD were evaluated. 2.4. Bone Histomorphometry of the Tibia The tibia was cut into two parts (proximal metaphysis and diaphysis plus distal metaphysis) with a diamond band saw (EXAKT BS 3000; Norderstedt, Germany), and each part was stained according to the method of Villanueva [20]. After dehydration with ethanol and acetone, the bone tissue was embedded in methyl methacrylate (Wako Pure Chemical, Japan). Frontal sections of the tibial proximal metaphysis were cut at a thickness of 5 μm on a microtome (Leica RM 2065; Nussloch, Germany). Cross-sections of the tibial diaphysis were obtained 4 mm proximal to the tibiofibular junction at a thickness of 20–30 μm with a microgrinder (Exakt KG 4000; Norderstedt, Germany). Then, the specimens were observed under a fluorescence microscope (Zeiss Axioplan 2; Jena, Germany) coupled with a video camera (CCD Color Camera CS 5270 I; Tokyo Electronic Industry Co., Ltd., Tokyo, Japan). A bone morphometry software program (Winroof Version 3.5; Mitani Corporation, Japan) was used for the histomorphometric analysis. The histomorphometric parameters measured for the proximal metaphysis were the total tissue volume (TV), bone volume (BV), bone surface (BS), osteoid surface (OS), osteoid volume (OV), osteoid width (O.Wi), number of osteoid width measurements (N.O.Wi), eroded surface (ES), single- and double-labeled surfaces (sLS and dLS, resp.), interlabel width, osteoblast surface (Ob.S), osteoclast surface (Oc.S), and number of osteoclasts (N.Oc). Osteoclasts were identified as cells inside the resorption lacunae on the bone surface. The data were then used to calculate the percent cancellous bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), osteoid thickness (O.Th), OS/BS, OV/BV, ES/BS, mineralizing surface/BS (MS/BS), mineral apposition rate (MAR), adjusted MAR (Aj.AR), bone formation rate (BFR)/BS, Ob.S/BS, Oc.S/BS, N.Oc/BS, osteoid maturation time (Omt), and mineralization lag time (Mlt) in accordance with the standard nomenclature proposed by Parfitt et al. [21] and issues in modern bone histomorphometry [22]. Cancellous bone was assessed at a site 0.5–2.0 mm proximal to the lower margin of the growth plate and 150 μm deep from the endocortical surface at the proximal metaphysis, an area that consists of secondary spongiosa. The parameters assessed for the diaphysis were the total tissue area (Tt.Ar), marrow area (Ma.Ar), periosteal and endocortical BS (perimeter), sLS, dLS, interlabel width, and endocortical ES, in accordance with the method described by Chen et al. [23]. The endocortical OS, Oc.S, and N.Oc were also measured. Two large cavities with and without vessels, which were independent of osteocytes and lacunae [24, 25], were observed in the intracortical area of each specimen to measure the cavity area (porotic Ar). These data were then used to calculate the cortical area (Ct.Ar), the periosteal and endocortical MS/BS, MAR, and BFR/BS, and the endocortical OS/BS, ES/BS, Oc.S/BS, N.Oc/BS, and Aj.AR. 2.5. Mechanical Strength Test of the Femur Each specimen was submerged for 1 hour before testing to allow temperature equilibration. The mechanical strength of the femoral diaphysis was evaluated using the three-point bending test. A load was applied to the bone midway between two supports placed 18 mm apart. The femur was positioned so that the loading point was at the center of the femoral diaphysis, and bending occurred at the medial-lateral axis. Load-displacement curves were recorded at a crosshead speed of 5 mm/min using a material-testing machine (MZ-500S; Maruto Instrument, Tokyo, Japan). The parameters analyzed were the stiffness, work to failure, and ultimate force. Just after the three-point bending test of the femoral diaphysis, the distal metaphysis of the femur was isolated over a length of 10 mm from the joint surface of the femoral condyle. The mechanical strength of this segment was then measured using a compression test. A compressive load was applied using a rectangular parallelepiped crosshead (length, 2 cm; width, 2 cm; and height, 1 cm) on the femoral distal metaphysis from the lateral aspect to the medial aspect. The specimens were positioned so that the loading point was at the center of the femoral lateral condyle. Load-displacement curves were recorded at a crosshead speed of 5 mm/min and a compression depth of 2.5 mm using a materials-testing machine (MZ500D; Maruto). The parameters analyzed were the stiffness, work to failure, and ultimate force. 2.6. Statistical Analysis Data were expressed as the mean ± standard deviation (SD). Comparisons among the groups were performed using an unpaired t-test. All the statistical analyses were performed using the Stat View J-5.0 program on a Windows computer, and a P value < 0.05 was regarded as significant. 3. Results 3.1. Body Weight, Femoral Length, Femoral Weight, and Femoral BMC and BMD Table 1 shows the body weight, femoral length, femoral weight, and BMC and BMD. The initial body weight did not differ significantly between the two groups. The final body weight was lower in the GX group than in the CON group. The GX did not significantly influence the femoral length. However, the GX induced decreases in the femoral weight and the BMC and BMD of the femoral diaphysis and distal metaphysis. The GX also induced a decrease in the cortical thickness of the femoral diaphysis. The mean GX-induced reductions in the BMC and BMD of the femoral diaphysis were 22.9% and 3.2%, respectively. The mean GX-induced reductions in the BMC and BMD of the femoral distal metaphysis were 33.3% and 32.4%, respectively. 3.2. Bone Histomorphometric Analysis of the Tibia Figure 1 and Table 2 show the results of the bone histomorphometric analysis of the tibial proximal metaphysis. The GX induced decreases in the BV/TV and Tb.N and an increase in the Tb.Sp without significantly influencing the Tb.Th (Figure 1). The mean GX-induced reduction in the BV/TV was 48.6%. These changes were associated with increased bone formation (Ob.S/BS, MS/BS, MAR, and BFR/BS) and increased O.Th, OS/BS, OV/BV, and Mlt (Table 2). However, the GX did not significantly influence bone resorption (ES/BS, Oc.S/BS, and N.Oc/BS) or the Omt (Table 2). Figure 2 and Table 3 show the results of the bone histomorphometric analysis of the tibial diaphysis. GX induced a decrease in the Ct.Ar and increases in the Ma.Ar and endocortical BS (Figure 2). The mean GX-induced reduction in the Ct.Ar was 16.8%. These changes were associated with increased endocortical bone resorption (ES/BS, Oc.S/BS, and N.Oc/BS) without any significant changes in endocortical bone formation (MS/BS, MAR, and BFR/BS) (Table 3). The GX did not significantly influence the Tt.Ar or the periosteal BS (Figure 2), despite the significant but modest decrease in periosteal bone formation (MAR and BFR/BS) (Table 3). The GX did not significantly influence porotic Ar or endocortical OS/BS (Table 3). 3.3. Mechanical Strength of the Femur Figure 3 shows the results of the mechanical strength test of the femur. In the femoral diaphysis, the GX induced a decrease in the ultimate force but did not cause any significant changes in the stiffness or work to failure. In the femoral distal metaphysis, the GX induced decreases in the stiffness, work to failure, and ultimate force. The mean GX-induced reduction in the ultimate force was 21.4% for the femoral diaphysis and 36.2% for the femoral distal metaphysis. 4. Discussion The present study confirmed that GX induced cortical and cancellous osteopenia, leading to a deterioration in the bone strength of the metaphysis and diaphysis in the long bones of rats. The response of bone formation, resorption, and osteoid parameters to GX and the degree of GX-induced osteopenia and the deterioration of bone strength appeared to differ between cortical and cancellous bones. These results revealed the differential responses of cortical and cancellous bones to GX in rats. GX in rats has been reported to induce osteopenia [14, 17–19, 26–30]. In the present study, GX induced decreases in the BMC, BMD, ultimate force, work to failure, and stiffness of the femoral distal metaphysis, the BMC, BMD, and ultimate force of the femoral diaphysis, and the cancelous BV/TV and Ct.Ar in the tibia. The mean GX-induced reductions in the BMC, BMD, and ultimate force were greater in the femoral distal metaphysis (33.3%, 32.4%, and 36.2%, resp.) than in the femoral diaphysis (22.9%, 3.2%, and 21.4% resp.). The mean GX-induced reduction in BV/TV was also greater than that of Ct.Ar (48.6% and 16.8%, resp.). Thus, the GX-induced osteopenia and deterioration in bone strength were more severe at skeletal sites rich in cancellous bone, compared with those rich in cortical bone. In rats, cancellous osteopenia after GX appears to be similar to that which occurs after ovariectomy although the cortical osteopenia that occurs after GX may be more severe than that occurring after ovariectomy [17–19]. Although bone histomorphometry studies have demonstrated GX-induced decreases in cancellous BV/TV and Ct.Ar in rats [17, 18], the changes in bone formation, resorption, and osteoid parameters after GX have not been adequately studied. Furthermore, cortical bone has received less attention than cancellous bone in bone histomorphometry studies. In the present study, the GX-induced decrease in cancellous BV/TV was characterized by increases in O.Th, OS/BS, OV/BV, and bone formation, without any significant alteration in bone resorption. No significant influence on Tb.Th or Omt was seen, consistent with the results of a previous study showing that GX-induced cancellous osteopenia was characterized by a normal width but a decreased maturation time of the osteoid and an increased bone formation rate in rats [14]. As to cortical bone, a GX-induced decrease in Ct.Ar was characterized by increased endocortical bone resorption without any significant alterations in endocortical bone formation and OS/BS. The Tt.Ar and periosteal BS did not change significantly, consistent with the results of previous studies [28, 31]. These results suggested that the response of bone formation, resorption, and osteoid parameters to GX appeared to differ between cortical and cancellous bones. The risk of osteomalacia in cancellous bone must be considered after GX. GX might not influence the longitudinal bone growth [30], resulting in the nonsignificant influence of GX on the femoral length. Gastric acid is thought to facilitate the intestinal absorption of ingested calcium by mobilizing calcium from insoluble complexes in the diet [15]. After a GX in humans, pigs, and rats, the serum gastrin level decreases, calcium absorption is impaired, the serum calcium and 25(OH)D levels decrease, and the 1,25(OH)2D level increases [6, 7, 32, 33]. However, we did not evaluate the serum and urinary calcium and phosphorus levels, the serum 25(OH)D, 1,25(OH)2D, and parathyroid hormone (PTH) levels, or bone turnover markers. The influence of GX on these biochemical parameters may be important for translating the results of our preclinical study into clinical practice. Thus, further studies are needed to address this issue. Approximately 80–90% of BMC is comprised of calcium and phosphorus. Other dietary components, such as protein, magnesium, zinc, copper, iron, fluoride, vitamins D, A, C, and K are required for normal bone metabolism [34]. After GX, not only calcium, phosphorus, and vitamin D, but also magnesium, iron, zinc, vitamin B12, and folic acid become deficient [31–33]. Zinc is essential for normal development of the skeleton [35]. Magnesium, vitamin B12, and folic acid are related to bone quality [36, 37]. These dietary components could have played a role in the impairment of bone mass and quality in terms of bone strength in GX rats. Although gastric acid secretion and gastric acidity have been suggested to play an important role in the intestinal absorption of calcium from ingested food or calcium salts such as calcium carbonate, available evidence suggests that gastric acid secretion and gastric acidity do not normally play a role in the absorption of dietary calcium carbonate [38]. Further studies are needed to clarify the contribution of malabsorption of nutrients, trace elements, and other vitamins in GX-induced changes in bone parameters in rats. Therapy of GX-induced osteopenia may consist of improving impaired calcification indicated by increased osteoid parameters in cancellous bone and increasing periosteal bone formation as well as decreasing endocortical bone resorption in cortical bone. Primarily, calcium and vitamin D supplementations are required because GX impairs calcium and vitamin D metabolism. Clinical studies showed the effects of alendronate and teriparatide on BMD in patients with postgastrectomy osteoporosis [39–41]. Preclinical study demonstrated the effect of incadronate, alendronate, estrogen, and PTH on cancellous bone mass in GX rats [42, 43]. However, a strategy for the prevention of fractures in GX patient population remains to be established although bisphosphonates and teriparatide may be good candidates for therapy of GX-induced osteopenia based on the clinical data. Further studies are needed to clarify the best therapy for GX-induced osteopenia. 5. Conclusions The present study compared the influence of GX on cortical and cancellous bones in rats and found that the responses of bone formation, resorption, and osteoid parameters to GX appear to differ between cortical and cancellous bones. GX-induced reductions in BMC, BMD, and bone strength were greater at skeletal sites rich in cancellous bone than at sites rich in cortical bone, and the GX-induced decrease in bone mass was greater in cancellous bone than in cortical bone. These differential responses of cortical and cancellous bones should be taken into consideration when clarifying the effect of interventions on the skeleton in GX rats. Conflict of Interests All the authors state that they have no conflict of interests. Acknowledgments The authors thank Drs. Kiichi Nonaka and Kaori Shindo (ELK Corporation, Tokyo, Japan) for pQCT analysis, Dr. Toshimi Masaki (Mitani Institute for Bone Histomorphometry, Tokyo, Japan) for bone histomorphometric analysis, and Dr. Tsuyoshi Ishii (Maruto Instrument Co., Ltd., Tokyo, Japan) for biomechanical testing. Figure 1 Bone histomorphometric analysis of the tibial proximal metaphysis. Structural parameters of cancellous bone. Data are expressed as the mean ± SD. An unpaired t-test was used to compare data between the two groups. a P < 0.05, b P < 0.001, and c P < 0.0001 versus GX. CON: control, GX: gastrectomy. Figure 2 Bone histomorphometric analysis of the tibial diaphysis. Structural parameters of cortical bone. Data are expressed as the mean ± SD. An unpaired t-test was used to compare data between the two groups. a P < 0.001 and b P < 0.0001 versus GX. CON: control, GX: gastrectomy. Figure 3 Mechanical strength of the femoral diaphysis and distal metaphysis. Data are expressed as the mean ± SD. An unpaired t-test was used to compare data between the two groups. a P < 0.05, b P < 0.01, c P < 0.001, and d P < 0.0001 versus GX. CON: control, GX: gastrectomy. Table 1 Body weight and femoral length, weight, and BMC and BMD. Control (n = 10) GX (n = 10) P value Initial body weight (g) 390 ± 17 383 ± 14 NS Final body weight (g) 516 ± 35 404 ± 39 <0.0001 Femoral length (mm) 39.9 ± 0.9 39.3 ± 0.9 NS Femoral weight (g) 1.36 ± 0.09 1.21 ± 0.09 0.0001 Femoral diaphysis Cortical thickness (mm) 0.807 ± 0.041 0.614 ± 0.048 <0.0001 BMC (mg/mm) 10.9 ± 0.9 8.4 ± 0.6 <0.0001 BMD (mg/cm3) 1372 ± 14 1328 ± 17 <0.0001 Femoral distal metaphysis BMC (mg/mm) 13.2 ± 1.1 8.8 ± 1.1 <0.0001 BMD (mg/cm3) 525 ± 39 355 ± 38 <0.0001 Data are expressed as the mean ± SD. An unpaired t-test was used to compare data between the two groups. GX: gastrectomy, NS: not significant. Table 2 Bone histomorphometric analysis of the tibial proximal metaphysis (formative and resorptive parameters of cancellous bone). Control (n = 10) GX (n = 10) P value O.Th (μm) 2.85 ± 0.30 5.54 ± 1.21 <0.0001 OS/BS (%) 8.49 ± 4.04 35.39 ± 8.84 <0.0001 OV/BV (%) 0.85 ± 0.43 6.64 ± 3.12 <0.0001 Ob.S/BS (%) 4.49 ± 2.19 25.29 ± 8.05 <0.0001 MS/BS (%) 23.9 ± 6.6 40.1 ± 4.8 <0.0001 MAR (μm/day) 1.80 ± 0.31 2.97 ± 0.43 <0.0001 Aj.AR (μm/day) 5.70 ± 1.61 3.51 ± 0.87 <0.01 BFR/BS (μm3/μm2/year) 162 ± 63 436 ± 95 <0.0001 ES/BS (%) 26.9 ± 5.0 30.9 ± 5.5 NS Oc.S/BS (%) 5.59 ± 2.47 8.27 ± 3.56 NS N.Oc/BS (#/mm) 1.90 ± 0.81 2.63 ± 1.11 NS Omt (day) 1.61 ± 0.25 1.91 ± 0.54 NS Mlt (day) 0.55 ± 0.24 1.74 ± 0.93 0.001 Data are expressed as the mean ± SD. An unpaired t-test was used to compare data between the two groups. GX: gastrectomy, NS: not significant. Table 3 Bone histomorphometric analysis of the tibial diaphysis (formative and resorptive parameters of cortical bone). Control (n = 10) GX (n = 10) P value Periosteal MS/BS (%) 76.6 ± 8.0 66.4 ± 15.0 NS MAR (μm/day) 1.94 ± 0.21 1.65 ± 0.16 <0.01 BFR/BS (μm3/μm2/year) 546 ± 98 405 ± 121 <0.05 Endocortical OS/BS (%) 22.3 ± 8.9 24.9 ± 10.9 NS MS/BS (%) 25.6 ± 7.6 23.0 ± 10.3 NS MAR (μm/day) 1.70 ± 0.58 2.00 ± 0.62 NS Aj.AR (μm/day) 1.99 ± 0.57 1.99 ± 0.91 NS BFR/BS (μm3/μm2/year) 165 ± 97 176 ± 127 NS ES/BS (%) 15.5 ± 12.1 53.9 ± 8.9 <0.0001 Oc.S/BS (%) 1.61 ± 2.06 5.92 ± 2.50 0.001 N.Oc/BS (#/mm) 0.55 ± 0.71 2.03 ± 0.83 <0.001 Data are expressed as the mean ± SD. An unpaired t-test was used to compare data between the two groups. GX: gastrectomy, NS: not significant. ==== Refs 1 Moizé V Andreu A Flores L Long-term dietary intake and nutritional deficiencies following sleeve gastrectomy or Roux-En-Y gastric bypass in a mediterranean population Journal of the Academy of Nutrition and Dietetics 2013 113 400 410 23438491 2 Pech N Meyer F Lippert H Manger T Stroh C Complications and nutrient deficiencies two years after sleeve gastrectomy BMC Surgery 2012 12, article 13 3 Pech N Meyer F Lippert H Manger T Stroh C Complications, reoperations, and nutrient deficiencies two years after sleeve gastrectomy Journal of Obesity 2012 2012 9 pages 828737 4 Gennari C Martini G Nuti R Secondary osteoporosis Aging 1998 10 3 214 224 2-s2.0-0031717111 9801731 5 Tovey FI Hall ML Ell PJ Hobsley M A review of postgastrectomy bone disease Journal of Gastroenterology and Hepatology 1992 7 639 645 1486191 6 Davies M Heys SE Selby PL Berry JL Mawer EB Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels. Implications for metabolic bone disease Journal of Clinical Endocrinology and Metabolism 1997 82 1 209 212 2-s2.0-0031014998 8989260 7 Zittel TT Zeeb B Maier GW High prevalence of bone disorders after gastrectomy American Journal of Surgery 1997 174 4 431 438 2-s2.0-0030861747 9337169 8 Melton LJ III Crowson CS Khosla S O’Fallon WM Fracture risk after surgery for peptic ulcer disease: a population-based cohort study Bone 1999 25 1 61 67 2-s2.0-0033035171 10423023 9 Kanis J Johnell O Gullberg B Risk factors for hip fracture in men from southern europe: The MEDOS Study Osteoporosis International 1999 9 1 45 54 2-s2.0-6544261972 10367029 10 Mellstrom D Johansson C Johnell O Osteoporosis, metabolic aberrations, and increased risk for vertebral fractures after partial gastrectomy Calcified Tissue International 1993 53 6 370 377 2-s2.0-0027431052 8293349 11 Glatzle J Piert M Meile T Prevalence of vertebral alterations and the effects of calcium and vitamin D supplementation on calcium metabolism and bone mineral density after gastrectomy British Journal of Surgery 2005 92 5 579 585 2-s2.0-18844361831 15779069 12 Lim JS Kim SB Bang HY Cheon GJ Lee JI High prevalence of osteoporosis in patients with gastric adenocarcinoma following gastrectomy World Journal of Gastroenterology 2007 13 48 6492 6497 2-s2.0-38349030795 18161918 13 Gepp H Koch M Schwille PO Vagus-sparing gastric fundectomy in the rat: development of osteopenia, relationship to urinary phosphate and net acid excretion, serum gastrin and vitamin D Research in Experimental Medicine 2000 200 1 1 16 2-s2.0-0034532921 11197917 14 Rümenapf G Schwille PO Erben RG Osteopenia following total gastrectomy in the rat—state of mineral metabolism and bone histomorphometry European Surgical Research 1997 29 3 209 221 2-s2.0-0030898081 9161838 15 Persson P Gagnemo-Persson R Chen D Gastrectomy causes bone loss in the rat: is lack of gastric acid responsible? Scandinavian Journal of Gastroenterology 1993 28 4 301 306 2-s2.0-0027583706 8488363 16 Surve VV Andersson N Alatalo S Does combined gastrectomy and ovariectomy induce greater osteopenia in young female rats than gastrectomy alone? Calcified Tissue International 2001 69 5 274 280 2-s2.0-0035207803 11768197 17 Surve VV Andersson N Lehto-Axtelius D Håkanson R Comparison of osteopenia after gastrectomy, ovariectomy and prednisolone treatment in the young female rat Acta Orthopaedica Scandinavica 2001 72 5 525 532 2-s2.0-0034768783 11728082 18 Andersson N Surve VV Lehto-Axtelius D Pharmacological treatment of osteopenia induced by gastrectomy or ovariectomy in young female rats Acta Orthopaedica Scandinavica 2004 75 2 201 209 2-s2.0-2342526556 15180236 19 Andersson N Surve VV Lehto-Axtelius D Drug-induced prevention of gastrectomy- and ovariectomy-induced osteopaenia in the young female rat Journal of Endocrinology 2002 175 3 695 703 2-s2.0-0036932756 12475380 20 Villanueva AR A bone stain for osteoid seams in fresh, unembedded, mineralized bone Stain Technology 1974 49 1 1 8 2-s2.0-0015979432 4131619 21 Parfitt AM Drezner MK Glorieux FH Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee Journal of Bone and Mineral Research 1987 2 6 595 610 2-s2.0-0023488581 3455637 22 Dempster DW Compston JE Drezner MK Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee Journal of Bone and Mineral Research 2013 28 2 17 23197339 23 Chen MM Yeh JK Aloia JF Tierney JM Sprintz S Effect of treadmill exercise on tibial cortical bone in aged female rats: a histomorphometry and dual energy x-ray absorptiometry study Bone 1994 15 3 313 319 2-s2.0-0027947457 8068453 24 Iwamoto J Matsumoto H Takeda T Sato Y Liu X Yeh JK Effects of vitamin K2 and risedronate on bone formation and resorption, osteocyte lacunar system, and porosity in the cortical bone of glucocorticoid-treated rats Calcified Tissue International 2008 83 2 121 128 2-s2.0-51449090899 18543014 25 Iwamoto J Matsumoto H Takeda T Sato Y Yeh JK Effects of vitamin K2 on cortical and cancellous bone mass, cortical osteocyte and lacunar system, and porosity in sciatic neurectomized rats Calcified Tissue International 2010 87 3 254 262 2-s2.0-79952277373 20556371 26 Stenström M Olander B Lehto-Axtelius D Erik Madsen J Nordsletten L Alm Carlsson G Bone mineral density and bone structure parameters as predictors of bone strength: an analysis using computerized microtomography and gastrectomy-induced osteopenia in the rat Journal of Biomechanics 2000 33 3 289 297 2-s2.0-0033622801 10673112 27 Ohta A Motohashi Y Sakai K Hirayama M Adachi T Sakuma K Dietary fructooligosaccharides increase calcium absorption and levels of mucosal calbindin-D9k in the large intestine of gastrectomized rats Scandinavian Journal of Gastroenterology 1998 33 10 1062 1068 2-s2.0-0031764109 9829361 28 Morohashi T Ohta A Yamada S Dietary fructooligosaccharides prevent a reduction of cortical and trabecular bone following total gastrectomy in rats Japanese Journal of Pharmacology 2000 82 1 54 58 2-s2.0-0033982647 10874589 29 Lehto-Axtelius D Stenström M Johnell O Osteopenia after gastrectomy, fundectomy or antrectomy: an experimental study in the rat Regulatory Peptides 1998 78 1–3 41 50 2-s2.0-0032583130 9879745 30 Mühlbauer RC Schenk RK Chen D Lehto-Axtelius D Hâkanson R Morphometric analysis of gastrectomy-evoked osteopenia Calcified Tissue International 1998 62 323 326 9504957 31 Stenström M Olander B Carlsson CA Carlsson GA Håkanson R Methodologic aspects of computed microtomography to monitor the development of osteoporosis in gastrectomized rats Academic Radiology 1995 2 9 785 791 2-s2.0-0029361349 9419640 32 Wojtyczka A Bergé B Rümenapf G Gastrectomy osteopenia in the rat: the role of vitamin B12 deficiency and the type of reconstruction of the digestive tract Clinical Science 1998 95 6 735 744 2-s2.0-0032439620 9831699 33 Maler GW Kreis ME Zittel TT Becker HD Calcium regulation and bone mass loss after total gastrectomy in pigs Annals of Surgery 1997 225 2 181 192 2-s2.0-0031045364 9065295 34 Ilich JZ Kerstetter JE Nutrition in bone health revisited: a story beyond calcium Journal of the American College of Nutrition 2000 19 6 715 737 2-s2.0-0034510590 11194525 35 Aaseth J Boivin G Andersen O Osteoporosis and trace elements—an overview Journal of Trace Elements in Medicine and Biology 2012 26 149 152 22575536 36 Iwamoto J Takeda T Sato Y Menatetrenone (vitamin K2 ) and bone quality in the treatment of postmenopausal osteoporosis Nutrition Reviews 2006 64 12 509 517 2-s2.0-33846059877 17274493 37 Sato Y Honda Y Iwamoto J Kanoko T Satoh K Effect of folate and mecobalamin on hip fractures in patients with stroke: a randomized controlled trial Journal of the American Medical Association 2005 293 9 1082 1088 2-s2.0-14144253560 15741530 38 Bo-Linn GW Davis GR Buddru DJ Morawsk SG Santa Ana C Fordtran JS An evaluation of the importance of gastric acid secretion in the absorption of dietary calcium The Journal of Clinical Investigation 1984 73 640 647 6707197 39 Iwamoto J Uzawa M Sato Y Takeda T Matsumoto H Effect of alendronate on bone mineral density and bone turnover markers in post-gastrectomy osteoporotic patients Journal of Bone and Mineral Metabolism 2010 28 2 202 208 2-s2.0-77952498232 19690798 40 Bacun T Kibel A Pavić R Impressive results of teriparatide treatment of postgastrectomy osteoporosis Med Glas Ljek Komore Zenicko-Doboj Kantona 2011 8 305 308 41 Suzuki Y Ishibashi Y Omura N Alendronate improves vitamin D-resistant osteopenia triggered by gastrectomy in patients with gastric cancer followed long term Journal of Gastrointestinal Surgery 2005 9 7 955 960 2-s2.0-24044538885 16137591 42 Suzuki Y Fukushima S Iwai T Bisphosphonate incadronate prevents total gastrectomy-induced osteopenia in rats Bone 2004 35 6 1346 1352 2-s2.0-13844315149 15589215 43 Andersson N Surve VV Lehto-Axtelius D Pharmacological treatment of osteopenia induced by gastrectomy or ovariectomy in young female rats Acta Orthopaedica Scandinavica 2004 75 2 201 209 2-s2.0-2342526556 15180236	23781240	PMC3679695	CC BY	2021-01-05 10:59:05	yes	Gastroenterol Res Pract. 2013 May 28; 2013:381616
==== Front Radiat OncolRadiat OncolRadiation Oncology (London, England)1748-717XBioMed Central 1748-717X-8-1252369759510.1186/1748-717X-8-125ResearchSNPs in genes implicated in radiation response are associated with radiotoxicity and evoke roles as predictive and prognostic biomarkers Alsbeih Ghazi 14galsbeih@kfshrc.edu.saEl-Sebaie Medhat 2medhatelsebaie@kfshrc.edu.saAl-Harbi Najla 1nharbi@kfshrc.edu.saAl-Hadyan Khaled 1khadyan@kfshrc.edu.saShoukri Mohamed 3shoukri@kfshrc.edu.saAl-Rajhi Nasser 2nrajhi@kfshrc.edu.sa1 Radiation Biology Section, Biomedical Physics Department, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia2 Radiation Oncology Section, Oncology Centre, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia3 National Biotechnology Center, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia4 Radiation Biology Section, Biomedical Physics Department, KFSHRC, MBC-03, P.O. Box 3354, Riyadh 11211, Saudi Arabia2013 22 5 2013 8 125 125 2 2 2013 14 5 2013 Copyright © 2013 Alsbeih et al.; licensee BioMed Central Ltd.2013Alsbeih et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Biomarkers are needed to individualize cancer radiation treatment. Therefore, we have investigated the association between various risk factors, including single nucleotide polymorphisms (SNPs) in candidate genes and late complications to radiotherapy in our nasopharyngeal cancer patients. Methods A cohort of 155 patients was included. Normal tissue fibrosis was scored using RTOG/EORTC grading system. A total of 45 SNPs in 11 candidate genes (ATM, XRCC1, XRCC3, XRCC4, XRCC5, PRKDC, LIG4, TP53, HDM2, CDKN1A, TGFB1) were genotyped by direct genomic DNA sequencing. Patients with severe fibrosis (cases, G3-4, n = 48) were compared to controls (G0-2, n = 107). Results Univariate analysis showed significant association (P < 0.05) with radiation complications for 6 SNPs (ATM G/A rs1801516, HDM2 promoter T/G rs2279744 and T/A rs1196333, XRCC1 G/A rs25487, XRCC5 T/C rs1051677 and TGFB1 C/T rs1800469). In addition, Kaplan-Meier analyses have also highlighted significant association between genotypes and length of patients’ follow-up after radiotherapy. Multivariate logistic regression has further sustained these results suggesting predictive and prognostic roles of SNPs. Conclusions Univariate and multivariate analysis suggest that radiation toxicity in radiotherapy patients are associated with certain SNPs, in genes including HDM2 promoter studied for the 1st time. These results support the use of SNPs as genetic predictive markers for clinical radiosensitivity and evoke a prognostic role for length of patients’ follow-up after radiotherapy. Single nucleotide polymorphism (SNP)RadiosensitivityLate reactions to radiotherapyFibrosisFollow upNasopharyngeal carcinoma ==== Body Background Genetic variations are frequent in humans, and the challenge of radiogenomic studies is to determine which polymorphisms influence individual radiosensitivity and the risk to develop severe complications following radiotherapy [1]. Single nucleotide polymorphism (SNP) is the largest type of inherited genetic variation [2]. These polymorphic variations can influence the stability of mRNA, rate of transcription, the protein translation and/or the protein-protein interactions leading to sub-optimum function and expression of different degrees of clinical radiation sensitivity [3]. To identify these variations, many investigators followed an intuitive approach of targeting SNPs in candidate genes arbitrarily involved in radiation response [4-6]. Although many studies, carried out often on limited number of RT patients, have reported significant associations, results were globally inconsistent between studies [7]. The pathways involved in radiation response encompass multitude of genes of which we have selected 11 candidate genes for their presumed or demonstrated influence on radiosensitivity [3,7-9]. These include CDKN1A (p21), TP53, ATM, HDM2, TGFB1, XRCC1, XRCC3, XRCC4, XRCC5 (Ku80), PRKDC, and LIG4 which are involved in various pathways. Since SNPs in these genes are likely to affect the outcome to radiation treatment [1], in this study we have genotyped 45 (12 primary and 33 neighboring) SNPs in 155 Head and Neck cancer patients treated with curative radiotherapy, and have been associated with follow-up and the grade of fibrosis in normal tissues. Methods Patients’ population and clinical data A total of 155 Head and Neck cancer patients had consented to be enrolled in this study during treatment or regular follow-up of their disease. The patients were treated by standardized 3D conformal RT, before the implementation of IMRT, for nasopharyngeal carcinoma as described elsewhere [10,11]. Total radiation dose to the upper neck was 66 Gy. Where possible, patients (n = 47) received a boost of 2 additional fractions to the nasopharynx to bring the dose received to 70 Gy. Locally advanced stages (II to IVB, n = 74) received neoadjuvant and concurrent chemotherapy consisting of cisplatinum and epirubicin [10]. The grade (G) of subcutaneous and deep tissue fibrosis, a late radiation-induced complication, was jointly evaluated by two participating physicians according to the RTOG/EORTC grading system. The maximum grade of fibrosis recorded over the length of the patients’ follow-up has been reported. For groups comparison, patients with major toxicity [12], severe fibrosis (G3-4), were referred to as the radiosensitive group (cases, n = 48) and were compared to patients with minor (G0-2) fibrotic reactions (controls, n = 107). The KFSHRC Research Ethics Committee has approved the study (RAC # 2000 031 and 2040 025) and all patients had signed informed consent. DNA extraction, amplification, sequencing and genotyping of polymorphisms During the regular follow-up of the patients, a 5 ml blood sample was drawn from consenting patients. DNA was extracted using Puregene DNA Purification Kit (Gentra System, USA) according to the manufacturer’s instruction. The selected 12 primary SNPs were: CDKN1A (p21, Cip1) codon 31 C/A (Ser/Arg) rs1801270, TP53 (p53) codon 72 G/C (Arg/Pro) rs1042522, HDM2 (MDM2) promoter position 309 rs2279744, ATM codon 1853 G/A (Asp/Asn) rs1801516, XRCC1 codon 399 G/A (Arg/Gln) rs25487, XRCC3 codon 241 C/T (Thr/Met) rs861539, XRCC4 codon 247 G/T (Ala/Ser) rs3734091, XRCC5 (Ku80) 3′ UTR A/G rs1051685, LIG4 (DNA Ligase IV) codon 591 A/G (Ile/Val) rs2232641, LIG4 codon 9 C/T (Thr/lle) rs1805388, PRKDC (DNA-PKcs) codon 3434 A/G (Ile/Thr) rs7830743, TGFB1 (TGFß1) codon 10 C/T (Leu/Pro) rs1982073. The PCR primers used are available upon request. Relevant segments of DNA were amplified by thermal cycling, sequenced using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences) and genotyped using SeqManII sequence analysis software (DNASTAR Inc.). Data analysis The univariate analysis of the association between SNPs allelic frequencies and grade of fibrosis were measured by the odds ratio (OR) with its 95% confidence interval. Significance of OR was assessed by the Chi-square (χ2) test, continuity uncorrected. A P-value of 0.05 or less is considered statistically significant. Kaplan-Meier analysis was used to evaluate the potential relationship between genotypes and length of patients follow-up. Multivariate logistic regression was used to assess the joint effect of all potential risk factors. Statistical analysis was carried out using the IBM SPSS Statistics platform (Version 19, SPSS Science, IL, USA). Results Patients and treatment The age of patients at RT ranged between 15 and 77 years/old with a median of 47. There were 39 females and 116 males. All patients had completed at least 24 months of follow-up after RT (range: 24–180 months, median: 40 months). Late normal tissue reactions to radiotherapy (xerostomia, skin atrophy and subcutaneous and deep tissue fibrosis) were scored, however, only grade of fibrosis is reported here because it was completed for all patients. There were 17, 54, 36, 38, and 10 patients who had exhibited fibrotic reactions of grade 0, 1, 2, 3 and 4, respectively. Patients with major toxicity (G3 and G4, cases) were compared to those having minor reactions (G0, G1 and G2, controls) [12]. Therefore, radiosensitive patients with severe subcutaneous and/or deep tissue fibrosis (G3-4, cases, n = 48) were compared to patients having no, mild or moderate fibrosis (G0-2, controls, n = 107). The distribution of controls and radiosensitive patients according to chemotherapy and radiation boost received is given in Figure 1. Briefly, patients who received chemotherapy (79) and RT boost (54) were proportionally distributed between controls and cases. Thus, the ratio of patients who received chemotherapy to the patients who did not, were comparable in the control and the radiosensitive groups (0.50 vs. 0.52, P = 0.80). Similarly, the average total doses received (with and without boost) in controls (67.50 Gy, SD = 1.94) and in the radiosensitive groups (67.17 Gy, SD = 1.84) were not significantly different (P = 0.35). Associated diseases were uncommon (25 patients) who were proportionally distributed between cases and controls. Figure 1 Distribution of the 155 nasopharyngeal carcinoma patients according to the grade of subcutaneous and deep tissue fibrosis following radiotherapy. The patients developed either minimal to moderate (controls: G0-2) or severe (cases: G3-4) fibrotic reactions. Patients who received chemotherapy (CT) or radiotherapy boost (RT-B) are indicated. Genotyping analysis Between the 45 SNPs genotyped, 16 were all majority (wild-types) alleles that have been omitted from the analysis. There were wide variations in the distribution of the different genotypes according to the grade of fibrosis. The allelic frequencies are given in Table 1. As compared to the controls (G0-2), the radiosensitive group (G3-4) harbored relatively higher number of variant ATM rs1801516 A allele which appeared to be a risk factor (OR = 2.86, CI 95%: 1.18-6.48, P < 0.01), and lower numbers of the variants HDM2 rs2279744 G (OR = 0.49, CI 95%: 0.29-0.84, P < 0.01), the rare HDM2 rs1196333 A (OR = 0.13, CI 95%: 0.02-0.99, P = 0.02), TGFB1 rs1800469 T (OR = 0.57, CI 95%: 0.34-0.96, P = 0.03), XRCC1 rs25487 A (OR = 0.41, CI 95%: 0.21-0.79, P < 0.01), and XRCC5 rs1051677 C (OR = 0.39, CI 95%: 0.17-0.91, P = 0.02) alleles which appeared to have protective effect; therefore, the majority alleles were the risk factors. In addition, we have computed the False Discovery Rate (FDR) for all P-values of the SNPs tested. The FDR-values of the 6 significant SNPs were between 0.14 and 0.009. This indicates that the expected proportion of false positive discovery satisfies the significance threshold allowed for this test (<0.20). Table 1 Allele frequencies of the assessed polymorphisms in 155 Head and Neck cancer patients who either developed minimal (controls: G0-2) or severe (cases: G3-4) late reactions (fibrosis) after radiotherapy Gene and SNP Allele 1a Allele 2b Odds ratio P-value Cases Controls Cases Controls (95% CI) n (%) n (%) n (%) n (%) CDKN1A C/A rs1801270 74 (77) 157 (73) 22 (23) 57 (27) 0.82 (0.47–1.44) 0.49 TP53 G/C rs1042522 52 (54) 112 (52) 44 (46) 102 (48) 0.93 (0.57–1.51) 0.76 TP53 C/T rs1800371 96 (100) 213 (99.5) 0 (0) 1 (0.5) NI NI ATM G/A rs1801516 82 (85) 202 (94) 14 (15) 12 (6) 2.86 (1.18–6.48) <0.01 HDM2 T/G rs2279744 71 (74) 125 (58) 25 (26) 89 (42) 0.49 (0.29–0.84) <0.01 HDM2 T/A rs1196333 95 (99) 198 (93) 1 (1) 16 (7) 0.13 (0.02–0.99) 0.02 TGFB1 G/A rs9282871 95 (99) 213 (99.5) 1 (1) 1 (0.5) NI NI TGFB1 C/T rs1982073 40 (42) 102 (48) 56 (58) 112 (52) 1.28 (0.78–2.07) 0.32 TGFB1 G/C rs1800471 92 (96) 207 (97) 4 (4) 7 (3) NI NI TGFB1 C/T rs1800469 67 (70) 122 (57) 29 (30) 92 (43) 0.57 (0.34–0.96) 0.03 TGFB1 G/A rs11466314 96 (100) 213 (99.5) 0 (0) 1 (0.5) NI NI TGFB1 del rs8179182 94 (98) 214 (100) 2 (2) 0 (0) NI NI TGFB1 C/T rs1800472 92 (96) 199 (93) 4 (4) 15 (7) 0.58 (0.19–1.79) 0.33 TGFB1 C/T rs11466334 95 (99) 213 (99.5) 1 (1) 1 (0.5) NI NI XRCC1 G/A rs25487 83 (86) 155 (72) 13 (14) 59 (28) 0.41 (0.21–0.79) <0.01 XRCC1 C/T rs3213368 87 (91) 193 (90) 9 (9) 21 (10) 0.95 (0.42–2.16) 0.90 XRCC1 G/A rs2139720 88 (92) 190 (89) 8 (8) 24 (11) 0.72 (0.31–1.67) 0.44 XRCC1 C/T rs3213369 96 (100) 213 (99.5) 0 (0) 1 (0.5) NI NI XRCC3 G/A rs861539 55 (57) 133 (62) 41 (43) 81 (38) 1.22 (0.75–1.99) 0.42 XRCC3 A/C rs3212112 95 (99) 213 (99.5) 1 (1) 1 (0.5) NI NI XRCC4 G/T rs3734091 95 (99) 213 (99.5) 1 (1) 1 (0.5) NI NI XRCC5 A/G rs41296835 96 (100) 213 (99.5) 0 (0) 1 (0.5) NI NI XRCC5 T/C rs1051677 89 (93) 178 (83) 7 (7) 36 (17) 0.39 (0.17–0.91) 0.02 XRCC5 A/G rs1051685 85 (89) 195 (91) 11 (11) 19 (9) 1.33 (0.61–2.91) 0.48 PRKDC T/C rs7830743 93 (97) 197 (92) 3 (3) 17 (8) 0.37 (0.11–1.31) 0.11 LIG4 T/C rs1805384 90 (94) 200 (93) 6 (6) 14 (7) 0.95 (0.36–2.56) 0.92 LIG4 C/T rs4987182 93 (97) 209 (98) 3 (3) 5 (2) NI NI LIG4 C/T rs1805389 95 (99) 206 (96) 1 (1) 8 (4) NI NI LIG4 C/T rs1805388 91 (95) 195 (91) 5 (5) 19 (9) 0.56 (0.20–1.56) 0.26 Significantly associated SNPs are highlighted in bold. a Allele 1: majority or wild-type allele. b Allele 2: minority or variant allele. NI: Not Informative because of low frequency. The relationship between the genotypes of the 6 significantly-associated-SNPs and the length of follow-up following RT, a surrogate measure of patients’ survival assuming that most absence to follow-up are due to death, was evaluated using Kaplan-Meier analysis (Figure 2). Results showed significant difference in the median follow-up with respect to genotypes for ATM rs1801516, HDM2 rs2279744 and XRCC1 rs25487 (Log Rank Mantel-Cox test: P = 0.001, 0.03 and 0.04, respectively). For these SNPs, the protective genotype, whether majority or minority allele, correspond to longer patients’ follow-up. Thus, the estimated medians follow-up in months were: ATM rs1801516 A/A = 24, A/G = 80, G/G = 114; HDM2 rs2279744 T/T = 75, T/G = 140, G/G = 114; XRCC1 rs25487 G/G = 79, G/A = 112, A/A = 180. Figure 2 Kaplan-Meier analysis of the relationship between the genotypes of the 6 significantly-associated-SNPs and length of patients’ follow-up following radiotherapy. Symbols represent censored data points. The joint effect of all potential risk factors (age, gender, total radiation dose, chemotherapy, follow-up, associated disease and genotypes), was assessed using multivariate logistic regression (Table 2). Results showed that HDM2 rs2279744, HDM2 rs1196333, TGFB1 rs1800469, XRCC1 rs25487 and Follow-Up (P = 0.03, 0.03, 0.007, 0.002 and 0.007, respectively) were significantly associated with the group of fibrotic reaction (G0-2 versus G3-4). Table 2 Multivariate logistic regression analysis of various risk factors that might affect patients’ risk to develop severe fibrosis following radiotherapy Risk factors P value ATM G/A rs1801516 0.55 HDM2 T/G rs2279744 0.03 HDM2 T/A rs1196333 0.03 TGFB1 C/T rs1800469 <0.01 XRCC1 G/A rs25487 <0.01 XRCC5 (Ku80) T/C rs1051677 0.42 Gender 0.79 Age at Radiotherapy 0.56 Total Radiation Dose 0.39 Chemotherapy 0.21 Follow Up <0.01 Associated Diseases 0.48 Significant associations are highlighted in bold. Discussion The aim of this study was to evaluate in our local cancer patients whether various risk factors including genetic polymorphic variations in candidate genes involved in radiation response are associated with the severity of RT-induced fibrotic reactions in normal tissues. The 155 Head and Neck cancer patients included in this report had nasopharyngeal carcinoma. This cancer site is prevalent in Saudi Arabia and is ideal for this type of study because patients follow standardized treatment with curative radiation without surgery [10]. This could be considered contribution to the radiogenomic consortium that contains mainly breast, prostate and gynecologic cancer patients [13]. Among the 45 genetic variations scored, univariate analysis showed significant association between grade of fibrosis and allelic frequency of 6 SNPs (ATM rs1801516, HDM2 rs2279744, HDM2 rs1196333, TGFB1 rs1800469, XRCC1 rs25487 and XRCC5 rs1051677; Table 1) and therefore, could be considered candidate for predictive markers testing. These results were further sustained as the values of the False Discovery Rate (FDR) of these SNPs (0.14 - 0.009) have satisfied the significance threshold allowed for this test (<0.20). Interestingly, apart from ATM where the variant A allele was associated with increased risk, the variant alleles of the remaining significantly associated SNPs showed decreased risk (Odds or Risk Ratios < 1) to develop severe fibrosis, and therefore, they exhibit protective effect. In addition, Kaplan-Meier analysis on these 6 SNPs suggested that the protective alleles of 3 of these SNPs (ATM rs1801516 A, HDM2 rs2279744 G, and XRCC1 rs25487 A) significantly correlate with longer follow-up of patients. Our results suggest that on average, the presence of protective allele at the heterozygous status would increase patients’ follow-up by 51 months while homozygous status would increase this index by 77 months. Thus, these SNPs could be used as prognostic biomarkers for length of follow-up following radiotherapy, as patients harboring protective alleles have higher probability to live longer (Figure 2). Estimated median follow-up suggests that harboring one protective allele of each of these SNPs would increase survival by about 4 years, while having the 2nd protective allele would add 2 more years. Multivariate logistic regression, that assesses the joint effect of various risk factors, has confirmed the association between HDM2 T/G rs2279744, HDM2 T/A rs1196333, TGFB1 C/T rs1800469, XRCC1 G/A rs25487, follow-up and radiosensitivity (Table 2). These are interesting results that plaid in favor of the potential use of genetic markers as predictors of normal tissue response and prognostic of follow-up. This is an important conclusion since the subject is currently a hot topic debate [7]. For instance, a large prospective study has failed to replicate previously reported associations between individual SNP genotype and radiation toxicity [7]. On the contrary, genome-wide associations study evaluating erectile dysfunction following radiotherapy has showed significant association not only in a gene that plays a role in male gonad function, but also in genes that relate to specific African ancestry [14]. This is the first study on the association between HDM2 T309G promoter (rs2279744) and radiosensitivity; previous studies were only concerned with its cancer predisposing potential [15]. The functional polymorphic variant in the HDM2 promoter at position 309 (rs2279744) have been suggested to affect the transcriptional activator SP1 binding, thereby modulating HDM2 transcription level. The G variant has been shown to increase the affinity for Sp1, resulting in higher levels of HDM2 mRNA and protein and the subsequent attenuation of the TP53 pathway [16]. Results presented here showed that the same variant G allele, and also the variant G allele in the neighboring HDM2 rs2279744 SNP, is associated with reduced risk to develop late normal tissues complications, a phenomenon that is dependent on the amount of cell depletion following radiotherapy. Therefore, in line with our results, it is conceivable that this HDM2 G variant allele could promote cell survival following irradiation and thus, cells would appear more radioresistant, despite the probable high risk of genomic instability due to presumably attenuated TP53. This may also have implication for the promotion of secondary cancers following radiotherapy. In addition, this is also the 1st study to report association between XRCC5 (KU80) polymorphisms and clinical radiosensitivity. XRCC5 is a component of the non-homologous end-joining (NHEJ) to repair DNA double-strand breaks. Previously, SNPs in XRCC5 have been shown to influence cancer risk and chromosomal radiosensitivity [15]. Our study showed that, although uncommon, the variant XRCC5 rs1051677 C allele was more frequent in the controls, thus it has a protective effect. The variant ATM rs1801516 A allele (Asn) was previously reported to be significantly associated with increased radiation sensitivity [17]. Other studies have also showed similar association with enhanced risk of various adverse reactions after RT [18]. On the other hand, the majority XRCC1 rs25487 allele (Arg) was associated with increased risk to develop late reactions to radiotherapy (Reviewed in [19]). This suggests that the variant (or minority) allele could confer higher radioresistance in favor of normal tissues involved in the radiation treatment [5]. TGFB1 encodes for the versatile cytokine TGFB1 assumed to be involved in response to tissue injuries. Therefore, SNPs that alter protein production can results in excessive deposition of scar tissue and fibrosis. Therefore, many SNPs have been studied in the literature and the effect of haplotype needs to be clarified as co-segregation of polymorphic variations in TGFB1 gene has been suggested to play a role in radiation response [3,5,20]. Results presented here are encouraging and illustrates that radiation response requires the concerted action of multiple genes and, therefore, it is a complex genetically controlled trait with the outcome being determined by multitude of additive effects. In addition, the original demonstration of an association between certain SNPs and length of followup after radiotherapy suggests prognostic role for patients’ survival. The results also indicate that not all variant SNPs are risky, and some of them could be advantageous from a radiosensitivity point of view. The SNPs studied were synonymous, non-synonymous and in non-coding regions of the genome. From an evolutionary perspective, the genome is in consistent development due to environmental interactions and, in general, natural selection favors the allele of the SNP that constitutes the most advantageous genetic adaptation. It is possible that the substitutions observed frequently are likely to be neutral or favorable, whereas those observed rarely are likely to be deleterious [21]. Although a large prospective study has failed to replicate previously reported associations between individual SNPs genotype and radiation toxicity [7], a genome-wide associations study evaluating erectile dysfunction following radiotherapy for prostate cancer has showed significant association not only in a gene that plays a role in male gonad development and function, but also in genes that relate to specific African ancestry that would not have been identified in a cohort of European ancestry [14]. The genomic revolution with the advent of high-throughput techniques can help uncovering the panoply of these interacting factors at the DNA (genome), RNA (transcriptome) or protein (proteome) level. Research using genome-wide analysis tools heralds the future of individualized radiation treatment in broadly personalized medicine. In addition to predictive and prognostic testing, the products of the identified genes could become targets for innovative therapies in susceptible individuals. Conclusions Univariate analysis showed that between 45 SNPs in 11 genes involved in cell cycle control and DNA repair, 6 were significantly associated with radiation toxicity in radiotherapy patients. Kaplan-Maier analysis has highlighted a significant association between genotype and follow-up of patients. Multivariate logistic analysis has sustained these conclusions. Larger cohort, independent replication of these findings and genome wide association studies (GWAS) are required to confirm these results and validates the use of SNPs as predictive and prognostic biomarkers to individualize radiotherapy on genetic basis. Competing interests We declare no competing interest. Authors’ contributions GA designed the study, analyzed results and drafted manuscript. NAH and KAH processed and genotyped samples. MES and NAR collected samples and followed patients. MS performed statistical analysis. All authors read and approved the final manuscript. Acknowledgments We wish to thank Dr. B. Meyer, Mr. M. Rajab for helping in DNA sequencing, Ms. M. Al-Buhairi, L. A. Venturina S. Al-Qahtani and N. Venturina for technical help and Dr. B. Moftah for continuous support. Funded by KFSHRC grant 2000 031 and 2040 025. ==== Refs Parliament MB Radiogenomics: associations in all the wrong places? Lancet Oncol 2012 13 7 8 10.1016/S1470-2045(11)70331-X 22169270 Cargill M Altshuler D Ireland J Sklar P Ardlie K Patil N Shaw N Lane CR Lim EP Kalyanaraman N Characterization of single-nucleotide polymorphisms in coding regions of human genes Nat Genet 1999 22 231 238 10.1038/10290 10391209 Andreassen CN Alsner J Overgaard M Overgaard J Prediction of normal tissue radiosensitivity from polymorphisms in candidate genes Radiother Oncol 2003 69 127 135 10.1016/j.radonc.2003.09.010 14643949 Andreassen CN Alsner J Overgaard M Sorensen FB Overgaard J Risk of radiation-induced subcutaneous fibrosis in relation to single nucleotide polymorphisms in TGFB1, SOD2, XRCC1, XRCC3, APEX and ATM–a study based on DNA from formalin fixed paraffin embedded tissue samples Int J Radiat Biol 2006 82 577 586 10.1080/09553000600876637 16966185 Alsbeih G Al-Harbi N Al-Hadyan K El-Sebaie M Al-Rajhi N Association between normal tissue complications after radiotherapy and polymorphic variations in TGFB1 and XRCC1 genes Radiat Res 2010 173 505 511 10.1667/RR1769.1 20334523 Talbot CJ Tanteles GA Barnett GC Burnet NG Chang-Claude J Coles CE Davidson S Dunning AM Mills J Murray RJ A replicated association between polymorphisms near TNFalpha and risk for adverse reactions to radiotherapy Br J Cancer 2012 107 748 753 10.1038/bjc.2012.290 22767148 Barnett GC Coles CE Elliott RM Baynes C Luccarini C Conroy D Wilkinson JS Tyrer J Misra V Platte R Independent validation of genes and polymorphisms reported to be associated with radiation toxicity: a prospective analysis study Lancet Oncol 2012 13 65 77 10.1016/S1470-2045(11)70302-3 22169268 Chang-Claude J Popanda O Tan XL Kropp S Helmbold I von Fournier D Haase W Sautter-Bihl ML Wenz F Schmezer P Ambrosone CB Association between polymorphisms in the DNA repair genes, XRCC1, APE1, and XPD and acute side effects of radiotherapy in breast cancer patients Clin Cancer Res 2005 11 4802 4809 10.1158/1078-0432.CCR-04-2657 16000577 West CM Elliott RM Burnet NG The genomics revolution and radiotherapy Clin Oncol (R Coll Radiol) 2007 19 470 480 10.1016/j.clon.2007.02.016 17419040 Al-Amro A Al-Rajhi N Khafaga Y Memon M Al-Hebshi A El-Enbabi A El-Husseiny G Radawi A Belal A Allam A El-Sebaie M Neoadjuvant chemotherapy followed by concurrent chemo-radiation therapy in locally advanced nasopharyngeal carcinoma Int J Radiat Oncol Biol Phys 2005 62 508 513 10.1016/j.ijrobp.2004.09.050 15890594 Alsbeih GA Al-Harbi NM El-Sebaie MM Al-Rajhi NM Al-Hadyan KS Abu-Amero KK Involvement of mitochondrial DNA sequence variations and respiratory activity in late complications following radiotherapy Clin Cancer Res 2009 15 7352 7360 10.1158/1078-0432.CCR-09-0960 19920115 Cox DJ Stetz J Pajak FT Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer (EORTC) Int J Radiat Oncol Biol Phys 1995 31 1341 1346 10.1016/0360-3016(95)00060-C 7713792 West C Rosenstein BS Alsner J Azria D Barnett G Begg A Bentzen S Burnet N Chang-Claude J Chuang E Establishment of a radiogenomics consortium Int J Radiat Oncol Biol Phys 2010 76 1295 1296 10.1016/j.ijrobp.2009.12.017 20338472 Kerns SL Ostrer H Stock R Li W Moore J Pearlman A Campbell C Shao Y Stone N Kusnetz L Rosenstein BS Genome-wide association study to identify single nucleotide polymorphisms (SNPs) associated with the development of erectile dysfunction in African-American men after radiotherapy for prostate cancer Int J Radiat Oncol Biol Phys 2010 78 1292 1300 10.1016/j.ijrobp.2010.07.036 20932654 Al-Hadyan KS Al-Harbi NM Al-Qahtani SS Alsbeih GA Involvement of single-nucleotide polymorphisms in predisposition to head and neck cancer in Saudi Arabia Genet Test Mol Biomarkers 2012 16 95 101 10.1089/gtmb.2011.0126 21877955 Bond GL Hu W Bond EE Robins H Lutzker SG Arva NC Bargonetti J Bartel F Taubert H Wuerl P A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans Cell 2004 119 591 602 10.1016/j.cell.2004.11.022 15550242 Alsbeih G El-Sebaie M Al-Harbi N Al-Buhairi M Al-Hadyan K Al-Rajhi N Radiosensitivity of human fibroblasts is associated with amino acid substitution variants in susceptible genes and correlates with the number of risk alleles Int J Radiat Oncol Biol Phys 2007 68 229 235 10.1016/j.ijrobp.2006.12.050 17331670 Andreassen CN Overgaard J Alsner J Overgaard M Herskind C Cesaretti JA Atencio DP Green S Formenti SC Stock RG Rosenstein BS ATM sequence variants and risk of radiation-induced subcutaneous fibrosis after postmastectomy radiotherapy Int J Radiat Oncol Biol Phys 2006 64 776 783 10.1016/j.ijrobp.2005.09.014 16338099 Andreassen CN Can risk of radiotherapy-induced normal tissue complications be predicted from genetic profiles? Acta Oncol 2005 44 801 815 10.1080/02841860500374513 16332587 Azria D Ozsahin M Kramar A Peters S Atencio DP Crompton NE Mornex F Pelegrin A Dubois JB Mirimanoff RO Rosenstein BS Single nucleotide polymorphisms, apoptosis, and the development of severe late adverse effects after radiotherapy Clin Cancer Res 2008 14 6284 6288 10.1158/1078-0432.CCR-08-0700 18829510 Zhu Y Spitz MR Amos CI Lin J Schabath MB Wu X An evolutionary perspective on single-nucleotide polymorphism screening in molecular cancer epidemiology Cancer Res 2004 64 2251 2257 10.1158/0008-5472.CAN-03-2800 15026370	23697595	PMC3679989	CC BY	2021-01-05 01:58:55	yes	Radiat Oncol. 2013 May 22; 8:125
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23776688PONE-D-13-0161010.1371/journal.pone.0066611Research ArticleBiologyAnatomy and PhysiologyImmune PhysiologyCytokinesMusculoskeletal SystemCartilageImmunologyImmune SystemCytokinesMolecular Cell BiologyToxicologyChemistryChemical ReactionsMaillard ReactionMedicineAnatomy and PhysiologyImmune PhysiologyCytokinesMusculoskeletal SystemCartilageNon-Clinical MedicineRheumatologyOsteoarthritisAdvanced Glycation End Products Induce Peroxisome Proliferator-Activated Receptor γ Down-Regulation-Related Inflammatory Signals in Human Chondrocytes via Toll-Like Receptor-4 and Receptor for Advanced Glycation End Products AGEs Downregulate Chondrocyte PPARγ via TLR4/RAGEChen Ying Ju 1 Sheu Meei Ling 2 Tsai Keh Sung 3 Yang Rong Sen 4 * Liu Shing Hwa 1 * 1 Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan 2 Institute of Biomedical Sciences, National Chung Hsing University and Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan 3 Department of Laboratory Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 4 Department of Orthopaedics, College of Medicine, National Taiwan University, Taipei, Taiwan Zissel Gernot Editor University Medical Center Freiburg, Germany * E-mail: shinghwaliu@ntu.edu.tw (SHL); rsyang@ntuh.gov.tw (RSY)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SHL RSY. Performed the experiments: YJC MLS KST. Analyzed the data: YJC MLS KST RSY SHL. Contributed reagents/materials/analysis tools: MLS. Wrote the paper: YJC SHL. 2013 12 6 2013 8 6 e666114 1 2013 6 5 2013 © 2013 Chen et al2013Chen et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Accumulation of advanced glycation end products (AGEs) in joints is important in the development of cartilage destruction and damage in age-related osteoarthritis (OA). The aim of this study was to investigate the roles of peroxisome proliferator-activated receptor γ (PPARγ), toll-like receptor 4 (TLR4), and receptor for AGEs (RAGE) in AGEs-induced inflammatory signalings in human OA chondrocytes. Human articular chondrocytes were isolated and cultured. The productions of metalloproteinase-13 and interleukin-6 were quantified using the specific ELISA kits. The expressions of related signaling proteins were determined by Western blotting. Our results showed that AGEs enhanced the productions of interleukin-6 and metalloproteinase-13 and the expressions of cyclooxygenase-2 and high-mobility group protein B1 and resulted in the reduction of collagen II expression in human OA chondrocytes. AGEs could also activate nuclear factor (NF)-κB activation. Stimulation of human OA chondrocytes with AGEs significantly induced the up-regulation of TLR4 and RAGE expressions and the down-regulation of PPARγ expression in a time- and concentration-dependent manner. Neutralizing antibodies of TLR4 and RAGE effectively reversed the AGEs-induced inflammatory signalings and PPARγ down-regulation. PPARγ agonist pioglitazone could also reverse the AGEs-increased inflammatory signalings. Specific inhibitors for p38 mitogen-activated protein kinases, c-Jun N-terminal kinase and NF-κB suppressed AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Taken together, these findings suggest that AGEs induce PPARγ down-regulation-mediated inflammatory signalings and reduction of collagen II expression in human OA chondrocytes via TLR4 and RAGE, which may play a crucial role in the development of osteoarthritis pathogenesis induced by AGEs accumulation. This study was supported by the grant from the National Science Council of Taiwan (NSC97-2314-B-002-052-MY3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Osteoarthritis (OA) is a progressive degenerative joint disease with signs and symptoms of inflammation, including joint pain, swelling, and stiffness leading to significant functional impairment and disability in older adults [1]. Cartilage damage in OA is caused by the disruption of a shift in the balance between catabolic and anabolic capacities of chondrocytes. Catabolic activities of OA chondrocytes are related to the elevated release of cartilage degrading enzymes, such as matrix metalloproteinases (MMPs), while anabolic activities result in the productions of type II collagen and aggrecan [2]. Several risk factors including obesity, increasing age, trauma, genetic predisposition, and endocrine factors are known to affect the progression of OA [3]. Aging has been considered to be a major risk factor for OA [4]. Advanced glycation end products (AGEs) produced irreversibly by the non-enzymatic glycation of proteins have been observed to accumulate with aging in various organs, especially in articular cartilage [5], [6]. Accumulation of AGEs in cartilage chondrocytes shows the decreased proteoglycan and collagen synthesis, which leads to stiffness and brittleness of the articular cartilage [7]. Furthermore, AGEs can also up-regulate the production of MMPs that mediate cartilage degradation leading to the joint destruction [8]. In chondrocytes of OA, AGEs has been shown to trigger the expressions of interleukin (IL)-6 and IL-8 through receptor for AGEs (RAGE) [9]. Activation of mitogen-activated protein kinase (MAPK)-regulated NF-κB signaling was involved in this AGEs/RAGE-induced expressions of IL-6 and IL-8 in chondrocytes [9]. On the other hands, toll-like receptor 4 (TLR4) has been shown to be up-regulated in the diabetic kidneys that the up-regulation of TLR4 is associated with the TLR4 ligands AGEs and high-mobility group protein B1 (HMGB1) in diabetic nephropathy [10]. HMGB1 has also been found to induce the amplification of inflammation and angiogenesis through TLRs and RAGE [11]. However, the role of TLR4 and RAGE in AGEs-induced inflammatory signalings in human chondrocytes remains to be clarified. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily [12], [13]. PPARγ was originally identified to play an important role in adipocyte differentiation and lipid metabolism [14], [15]. It has been shown that PPARγ signaling is involved in the metabolic disorders [16] and cardiovascular diseases [17]. PPARγ is known to be expressed in many cell types including immune cells, endothelial cells, synoviocytes, and chondrocytes [18]–[20]. PPARγ expression has been found to be decreased in human OA cartilage and down-regulated in IL-1β-treated chondrocytes [21]. PPARγ agonist pioglitazone has also been demonstrated to be capable of decreasing the progression of guinea pig OA [22]. Activation of PPARγ lead to the inhibition of various inflammatory signalings, such as COX-2, IL-1β, IL-6 and TNFα, and MMP-1 expression in monocytes as well as synoviocytes [18], [19]. PPARγ activators have ability to prevent the inflammation-induced expressions of iNOS, COX-2, and MMP-13 in human chondrocytes [20], [23]. AGEs has recently been shown to down-regulate PPARγ expression in rabbit chondrocytes [24]. However, little is known about the relationship among AGEs, RAGE, TLR4, and PPARγ in the pathogenesis of OA. Here, we tried to investigate the roles of PPARγ, TLR4, and RAGE in AGEs-induced inflammatory signalings in human OA chondrocytes. Materials and Methods Ethics Statement The samples of cartilage specimens were collected with written approvals from the institutional Ethics Committee at National Taiwan University Hospital, Taipei, Taiwan, and also from the patients. Reagents Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for RAGE, TLR4, IκBα and Histone H1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibodies specific for COX-2, collagen II, NF-κB p65, phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38MAPK, p38MAPK, PPARγ, β-actin, α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody specific for phospho-IKKα/β (Ser180/181) and phospho-p65 (Ser536) were purchased from Cell Signaling (Danvers, MA, USA). Mouse monoclonal antibody specific for RAGE was purchased from R&D Systems (Minneaplis, MN, USA). MMP-13 and IL-6 ELISAs and mouse monoclonal antibody specific for TLR4 were purchased from eBioscience. NF-κB (p65) Transcription Factor Assay Kit was purchased from Cayman Chemical Company (U.S.A). SB203580, SP600125, PD98059, bovine serum albumin (BSA), pioglitazone, pyrrolidine dithiocarbamate (PDTC) and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Isolation and culture of chondrocytes from human articular cartilage Human articular chondrocytes were isolated from healthy femoral head articular resected cartilage specimens and obtained from 10 patients aged 50–76 years (mean age, 63.7±2.51 years) who were generally healthy undergoing joint replacement surgery. Cartilage pieces were minced finely, and chondrocytes were isolated by sequential enzymatic digestion at 37°C with 0.2% collagenase (type II; Sigma-Aldrich) for four hours in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY). Isolated chondrocytes were filtered through 100 µM nylon filters. The cells were grown in the plastic cell culture dishes in 95% air–5% CO2 with DMEM medium, which was supplemented with 20 mM HEPES and 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) (pH adjusted to 7.4). Experiments were performed using cells from passages 3–7. In experiments, chondrocytes were plated in 6-well plates in complete DMEM medium and serum-starved for 12 hours overnight and then treated with 5–100 µg/ml AGEs for various time intervals in the presence or absence of pharmacological inhibitors for MAPKs or NF-κB or neutralizing antibodies for RAGE or TLR4. Chondrocytes cultured without AGEs or with BSA alone were served as controls. Preparation of AGEs BSA (1 mg/ml) was incubated under sterile conditions with D-glucose (1 mg/ml) in 0.2 M phosphate buffer (pH 7.4) at 37°C for 8 weeks. After incubation, AGEs were dialyzed against PBS for 24 hours to remove unbound sugars and filter-sterilized using a 0.22 µM Millipore filter (Millipore, Billerica, MA, USA). AGEs were identified by Ultraflex III MALDI-TOF/TOF (Bruker) and the AGEs protein concentration was measured by BCA protein assay. Cell viability assay Cell viability was determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay. After treatment of cells with or without AGEs or with BSA alone for 24 hours, cells were washed with PBS. MTT (0.2 mg/ml) was then added to each well and the mixture was incubated for four hours at 37°C. Culture medium was then replaced with an equal volume of DMSO to dissolve blue formazan crystals. After the mixture was shaken at room temperature for 10 minutes, the absorbance was measured at 550 nm. Measurement of IL-6 and MMP-13 productions Human OA chondrocytes (1×106/ml) cultured in 6-well plates were stimulated with or without AGEs for 24 hours in the presence or absence of pioglitazone. IL-6 and MMP-13 productions in the culture media were quantified by using the commercially available IL-6 or MMP-13 specific ELISA kits (eBioscience) according to the manufacturer's instructions. The plates were read at 450 nm. Western blot analysis The cellular lysates were prepared. Equal proteins (20–40 µg) were resolved on SDS-PAGE and transferred to immobilon polyvinyl difluoride (PVDF) membranes. The blots were blocked with 4% BSA for one hour at room temperature and then probed with the primary antibodies against COX-2, HMGB1, IκB kinase (IKK)α/β, phospho-IKKα/β, IκBα, phospho-IκBα, p65, phospho-p65, TLR4, phospho-ERK, phospho-p38MAPK, phospho-JNK, collagen II, NF-κB p65, (1∶1000, Santa Cruz) overnight at 4°C. After three washes, the blots were subsequently incubated with the secondary goat anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase (1∶1000) for one hour at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY). Preparation of nuclear extracts and NF-κB activation measurement The binding activity of NF-κB to DNA was measured in nuclear extracts using NF-κB (p65) Transcription Factor Assay kit (Cayman Chemical Company). In brief, cells were lysed in a hypotonic buffer on ice for 15 minutes and centrifuged for 30 seconds to pellet nuclei. Then the pellet was re-suspended in nuclear extract buffer on ice for 15 min. The lysates were centrifuged at 14,000 × g for 10 minutes, and supernatants containing the nuclear proteins were collected. NF-κB activation was measured according to the manufacturer’s instruction using 10 µg of nuclear protein per well. Following color development, absorbance was read at 450 nm within 5 minutes. Statistics The results are presented as mean±SEM. Each experiment was performed four times or more to ensure reproducibility. The significant difference from the respective controls for each experimental test condition was assessed by one-way analysis of variance (ANOVA) and two-tailed Student's t-test. The difference is significant if the P-value is less than 0.05. Software used:SigmaPlot 10.0 and GraphPad Prism 5. Results AGEs induce inflammatory responses in human OA chondrocytes Human OA chondrocytes were treated with increasing doses of AGEs (5, 25, 50, and 100 µg/ml) for 24 hours and no significant cytotoxic effect was found as compared with normal control or BSA control (Figure 1A). AGEs effectively induced the productions of MMP-13 and IL-6 (Figures 1B and 1C) and resulted in the reduction of collagen II expression (Figure 1D) in a dose-dependent manner. Moreover, AGEs significantly up-regulated the expressions of COX-2 (Figures 2A and 2B) and HMGB1 (Figures 2C and 2D) in a dose- and time-dependent manner. On the other hand, AGEs (50 µg/ml) markedly induced the phosphorylations of IKKα/β, IκBα, and NF-κB p65 (Figure 3A, 3B and 3C) and the degradation of IκBα (Figure 3B) and the translocation of NF-κB p65 from cytosol to nucleus (Figure 4A and 4B) in a time-dependent manner. Also, AGEs could significantly activate NF-κB activity. (Figure 4D). Pretreatment with NF-κB inhibitor PDTC (20 µM) could effectively reverse the reduction of collagen II expression induced by AGEs (Figure 3D). These results indicate that AGEs are capable of inducing inflammatory signalings and reducing collagen II expression in human OA chondrocytes. 10.1371/journal.pone.0066611.g001Figure 1 AGEs induce inflammatory signalings in human OA chondrocytes. Human OA chondrocytes (1×106/ml) were incubated with AGEs (5–100 µg/ml) for 24 hours and cytotoxic effect was determined by MTT assay (A). Productions of MMP-13 (B) and IL-6 (C) were quantified by the ELISA kits. Protein expressions of collagen II (D) were determined by Western blotting. Densitometric analysis for collagen levels corrected to β-actin is shown. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. 10.1371/journal.pone.0066611.g002Figure 2 AGEs induce inflammatory signalings in human OA chondrocytes. Human OA chondrocytes (1×106/ml) were incubated with AGEs (5–100 µg/ml) for 24 hours (A, C) or 0.5–24 hours (B, D). Protein expressions of COX-2 (A, B) and HMGB1 (C, D) were determined by Western blotting. Densitometric analysis for COX-2 and HMGB1 levels corrected to α-tubulin is shown. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. 10.1371/journal.pone.0066611.g003Figure 3 AGEs activate NF-κB signaling in human OA chondrocytes. Human OA chondrocytes (1×106/ml) were incubated with AGEs (50 µg/ml) for indicated time intervals. The phosphorylations of IKKα/β (A), IκBα (B) and p65 (C) and the degradation of IκBα (B) were determined by Western blotting. In D, Chondrocytes were pretreated with PDTC (20 µM) for 1 hour followed by treatment with AGEs for 2 hours. Protein expression of collagen II was determined by Western blotting. Densitometric analysis for p-IKKα/β, p-IκBα, IκBα, p-p65, and collagen II levels corrected to α-tubulin is shown. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. #: P<0.05 versus AGEs alone. 10.1371/journal.pone.0066611.g004Figure 4 AGEs activate NF-κB activity in human OA chondrocytes, which can be reversed by pioglitazone. Human OA chondrocytes (1×106/ml) were incubated with AGEs (50 µg/ml) for indicated time intervals. The expressions of nuclear p65 (A) and cytosol IκBα degradation (B) were determined by Western blotting. In C, chondrocytes were pretreated with PDTC (20 µM) for 1 hour followed by treatment with AGEs for 2 hours. Protein expression of PPARγ was determined by Western blotting. Densitometric analysis for nuclear p65, cytosolic IκBα, and PPARγ levels corrected to Histone H1, α-tubulin, and β-actin, respectively, is shown. In D, chondrocytes (1×106/ml) were pretreated with pioglitazone (10 and 50 µg/ml) for 1 hour followed by stimulating with AGEs (50 µg/ml) for 24 hours. NF-κB activity was measured using NF-κB (p65) Transcription Assay kit and quantified with a spectrophotometric plate reader at wavelengths of 450 nm. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. #: P<0.05 versus AGEs alone. Involvement of TLR4 and RAGE in AGEs-increased inflammatory responses in human OA chondrocytes It has been reported that TLR4 and RAGE are presented in articular cartilage and are increased with aging and OA [25], [26]. We next investigated whether RAGE and TLR4 are involved in AGEs-induced increase of inflammatory responses in human OA chondrocytes. With AGEs (5–100 µg/ml) treatment, the expressions of TLR4 (Figures 5A and 5B) and RAGE (Figures 5C and 5D) were up-regulated in a dose- and time-dependent manner. Moreover, pretreatment with neutralizing antibodies for TLR4 and RAGE could effectively suppress the AGEs (50 µg/ml)-increased COX-2 (Figures 5E and 5F) and HMGB1 (Figures 5G and 5H) expressions. Quentification and statistical analysis were performed in Figure 6. These results indicate that TLR-4 and RAGE are involved in the AGEs-up-regulated COX-2 and HMGB1 expressions in human OA chondrocytes. 10.1371/journal.pone.0066611.g005Figure 5 Involvement of TLR4 and RAGE in AGEs-induced COX-2 and HMGB1 protein expressions in human OA chondrocytes. In A-D, human OA chondrocytes (1×106/ml) were treated with AGEs (5–100 µg/ml) for 24 hours (A, C) or 0.5–24 hours (B, D). Protein expressions of TLR4 (A, B) and RAGE (C, D) were measured by Western blotting. In E–H, human OA chondrocytes (1×106/ml) were pretreated with neutralizing antibodies of TLR4 (20 µg/ml) and RAGE (10 µg/ml) for 1 hour followed by treatment with AGEs (50 µg/ml) for 24 hours. Protein expressions of COX-2 (E, F) and HMGB1 (G, H) were determined by Western blotting. Results shown are representative of at least three independent experiments. 10.1371/journal.pone.0066611.g006Figure 6 Densitometric analysis for TLR4, RAGE, COX-2, and HMGB1 levels. Values are corrected to α-tubulin or β-actin levels. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. #: P<0.05 versus AGEs alone. AGEs down-regulate PPARγ expression via TLR4 and RAGE in human OA chondrocytes Previous evidence suggested that PPARγ plays a crucial role in the development of OA progression [27]. The decreased expression of PPARγ in OA cartilage might result in the increased inflammatory and catabolic responses [21]. We next tested whether AGEs affect the expression of PPARγ and the involvement of TLR4 and RAGE in human OA chondrocytes. AGEs (5–100 µg/ml) effectively decreased the expression of PPARγ in a dose- and time-dependent manner (Figures 7A and 7B). Chondrocytes pretreated with NF-κB inhibitor PDTC showed no effect on AGEs-induced down-regulation of PPARγ expression (Figure 4C). Moreover, pretreatment with neutralizing antibodies for TLR4 and RAGE could effectively suppress the AGEs (50 µg/ml)-decreased PPARγ expression (Figures 7D and 7E). These results indicate that TLR-4 and RAGE are involved in the AGEs- down-regulated PPARγ expression in human OA chondrocytes. 10.1371/journal.pone.0066611.g007Figure 7 AGEs down-regulate PPARγ protein expression in human OA chondrocytes. Human OA chondrocytes (1×106/ml) were treated with AGEs (5–100 µg/ml) for 24 hours (A) or 0.5–24 hours (B). In C and D, human OA chondrocytes were pretreated with neutralizing antibodies of RAGE (10 µg/ml; C) and TLR4 (20 µg/ml; D) for 1 hour and then stimulated with AGEs (50 µg/ml) for 24 hours. PPARγ protein expression was measured by Western blotting. Densitometric analysis for PPARγ level corrected to β-actin is shown. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. #: P<0.05 versus AGEs alone. PPARγ agonist pioglitazone reverses the AGEs-increased inflammatory responses in human OA chondrocytes We next evaluated the effect of PPARγ agonist pioglitazone on AGEs-increased inflammatory responses in human chondrocytes. As shown in Figure 8, pioglitazone (10 and 50 µM) significantly reversed the AGEs (50 µg/ml)-increased MMP-13 (Figure 8A) and IL-6 (Figure 8B) productions. Moreover, AGEs-induced COX-2 and HMGB1 expressions could also be inhibited by pioglitazone (Figure 8C and 8D). Pioglitazone also inhibited the AGEs-down-regulated collagen II expression (Figure 8E). On the other hand, the AGEs-increased NF-κB activity could be decreased by pioglitazone (Figure 4D). These results provide the further evidence that PPARγ down-regulation is involved in the AGEs-induced inflammatory signalings and collagen II reduction in human OA chondrocytes. 10.1371/journal.pone.0066611.g008Figure 8 Effects of pioglitazone on inflammatory signalings in human OA chondrocytes. Human OA chondrocytes (1×106/ml) were pretreated with pioglitazone (10 and 50 µg/ml) for 1 hour followed by stimulating with AGEs (50 µg/ml) for 24 hours. Productions of MMP-13 (A) and IL-6 (B) were quantified by specific ELISA kits. Protein expressions of COX-2, HMGB1, and collagen II were determined by Western blotting (C). Densitometric analysis for COX-2, HMGB1, and collagen II levels corrected to β-actin is shown. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. #: P<0.05 versus AGEs alone. Involvement of MAPK signaling in AGEs-induced PPARγ down-regulation MAPK signaling pathway has been shown to be involved in the AGEs-induced IL-6 and IL-8 expressions in chondrocytes [9]. We next investigated whether MAPK signaling is involved in the AGEs-mediated PPARγ down-regulation and collagen II reduction in human OA chondrocytes. AGEs (50 µg/ml) effectively increased the phosphorylations of JNK and p38MAPK in a time-dependent manner (Figures 9A and 9B). Pretreatment with SP600125 (a selective inhibitor of JNK) and SB203580 (a specific inhibitor of p38MAPK) effectively reversed AGEs-induced PPARγ down-regulation and collagen II reduction (Figure 9C). Moreover, AGEs (50 µg/ml) slightly increased the phosphorylation of ERK at 0.5 h after AGEs treatment (Figure 10A). However, PD98059 (an ERK inhibitor) could not affect the AGEs-induced PPARγ down-regulation (Figure 10B) and collagen II reduction (Figure 10C). These results indicate that MAPK signaling including JNK and p38MAPK is involved in the AGEs-mediated PPARγ down-regulation and collagen II reduction in human OA chondrocytes. 10.1371/journal.pone.0066611.g009Figure 9 Involvement of MAPK signaling in AGEs-induced PPARγ down-regulation and reduction of collagen II expression. Human OA chondrocytes (1×106/ml) were incubated with AGEs (50 µg/ml) for 0.5–24 hours (A, B) or 24 hours (C). The phosphorylations of JNK (A) and p38MAPK (B) were determined by Western blotting. In C, chondrocytes were pretreated with SP600125 (10 and 20 µM) or SB203580 (1 and 10 µM) for 1 hour followed by treatment with AGEs for 24 hours. Protein expressions of PPARγ and collagen II was determined by Western blotting. Densitometric analysis for p-JNK, p-p38MAPK, PPARγ, and collagen II levels corrected to JNK, p38MAPK, β-actin, and β-actin, respectively, is shown. All data are presented as mean ± SEM for three independent experiments. : P<0.05 versus control. #: P<0.05 versus AGEs alone. 10.1371/journal.pone.0066611.g010Figure 10 ERK signaling is not involved in the effects of AGEs on chondrocytes. Human OA chondrocytes (1 ×106/ml) were incubated with AGEs (50 µg/ml) for 0.5–24 hours (A) or 24 hours (B,). The phosphorylation of ERK (A) were determined by Western blotting. In B and C, chondrocytes were pretreated with PD98059 (10 and 20 µM) for 1 hour followed by treatment with AGEs for 24 hours. Protein expressions of PPARγ and collagen II was determined by Western blotting. Densitometric analysis for p-ERK, PPARγ and collagen II levels corrected to ERK, β-actin, and β-actin, respectively, is shown. All data are presented as mean ± SEM for three independent experiments. *: P<0.05 versus control. Discussion AGEs are a group of compounds that are formed mainly via the Maillard reaction, which happens when reducing sugar reacts with macromolecules such as amino acids in proteins, lipids or DNA in a non-enzymatic way. Several studies suggested that accumulation of AGEs may be a mechanism for the age-related development of OA [5]–[7]. In patients with focal degeneration of cartilage, the increased levels of AGEs have been found in their healthy cartilage [28]. Moreover, AGEs formation has also been shown to be accelerated in diabetic patients [29]. Diabetes has recently been suggested to be an independent risk factor for OA [30], [31]. Previous studies have shown that PPARγ signaling plays a potent anti-inflammatory role by negatively regulating the expressions of several pro-inflammatory genes [18], [19]. Several studies have also found that PPARγ agonists can reduce the expression and synthesis of cartilage degradation products in vitro and in vivo, and suggested that activation of PPARγ is capable of reducing the progression of OA [22], [20], [27], [32]. In the present study, we demonstrated for the first time that AGEs down-regulate the PPARγ expression and induce the productions of IL-6 and MMP-13, which result in the reduction of the expression of type II collagen (major cartilage matrix macromolecules) in human OA chondrocytes. PPARγ agonist pioglitazone significantly inhibited the productions of IL-6 and MMP-13 and reversed the reduction expression of collagen II. These results suggest that PPARγ signaling plays an important role in AGEs accumulating human OA chondrocytes. The maintenance of structural and functional integrity of articular cartilage is known to depend on the balance between catabolic and anabolic of matrix components. The doublet effects of AGEs on synthesis and degradation of matrix constituents implicate that extracellular matrix turnover in articular cartilage is affected by accumulation of AGEs [33]. Of various types of collagens, type II collagen is related to build up the structural backbone of the extracellular matrix in human articular cartilage [34]. The degradation of collagen II by interstitial collagenases, MMPs, has been demonstrated to be a crucial step resulting in the destruction of the joints in OA patients [35]. MMP-13 (collagenase-3) is an important enzyme that preferentially cleaves collagen II in OA cartilage [35]. Moreover, IL-6 and HMGB-1 are two important mediators of inflammation. IL-6 is a multifunctional cytokine with a wide range of biological activities, including mediation of acute-phase responses and effects on bone metabolism [36]. Patients with OA exhibits elevated IL-6 levels [36]. HMGB-1 is a ubiquitous cytokine acting as a potent promoter of inflammation. HMGB-1 has been considered to be an important trigger of arthritis [37]. It has also been reported that HMGB-1 is involved in the pathogenesis of cartilage destruction in OA [38]. In the present study, the results showed that AGEs not only induce the productions of MMP-13 and IL-6 and the reduction of collagen II, but also increase the expressions of COX-2 and HMGB-1 in human OA chondrocytes in a dose- and time-dependent manner. In addition, PPARγ agonist pioglitazone could also effectively reverse these AGEs-induced effects in human chondrocytes, indicating that AGEs interfere with the extracellular matrix turnover in cartilage may through a down-regulation of PPARγ. TLRs are known to evolutionarily recognize the conserved products unique to microbial metabolism involved in innate immune responses and the pathology of a number of inflammatory diseases [39]. Previous study has revealed that TLR4 is capable of regulating the early onset of joint inflammation and cartilage destruction in a murine model of immune complex-mediated arthritis [40]. On the other hand, RAGE is a member of the immunoglobulin superfamily and involved in homeostasis, development, and inflammation. RAGE interacts with diverse ligands, including AGEs, several members of the S100 protein family, and HMGB1, which has been shown to be present in articular cartilage [8], [41]. Increased expression of RAGE has been suggested to be related to various acute and chronic inflammatory diseases including OA [42]. A recent report has shown that both TLR and RAGE signaling systems are activated in preterm birth and suggested that the interactions between TLR-mediated acute inflammation and RAGE-mediated chronic inflammation may contribute to increase the preterm birth risk [43]. The study of Qin et al. has also suggested that the cross-talk between TLR4 and RAGE contributes an increase in inflammatory signalings in macrophages [44]. In the present study, we used neutralizing antibodies for TLR4 and RAGE to investigate the roles of TLR4 and RAGE in AGEs-induced inflammatory signalings in human OA chondrocytes. Our data showed that AGEs can up-regulate both TLR4 and RAGE expressions in a dose- and time- dependent manner. Both neutralizing antibodies for TLR4 and RAGE effectively suppressed the AGEs-increased COX-2 and HMGB-1 expressions and reversed the AGEs-induced PPARγ down-regulation. These findings indicate that both TLR4- and RAGE-mediated inflammatory signalings implicate in the AGEs accumulation-related OA pathogenesis. TLR ligands have been found to be capable of leading the activations of MAPKs and NF-κB in chondrocytes [45]. Several studies have shown that MAPKs signals (p38, JNK, and ERK) are activated and involved in the increased inflammatory signalings and MMPs expressions in AGEs (100–400 µg/ml)-treated chondrocytes [9], [46], [47]. In these studies, the authors found that SB202190 (p38 inhibitor) could inhibit the AGEs-induced responses in chondrocytes; however, the effects of SP600125 (JNK inhibitor) and PD98059 (ERK inhibitor) are controversial [9], [46], [47]. In the present study, we have found that AGEs (50 µg/ml) markedly enhance the phosphorylations of JNK and p38MAPK, but induce a slight and transient increase in ERK phosphorylation, in human OA chondrocytes. Specific inhibitors of JNK and p38MAPK, but not ERK, effectively inhibited AGEs-induced down-regulation of PPARγ and the reduction of collagen II. These results suggest that JNK and p38MAPK are involved in the AGEs-mediated down-regulation of PPARγ in human OA chondrocytes. This finding is consistent with the findings in IL-1β-treated human chondrocytes [21] as well as AGEs-treated rabbit chondrocytes [24]. Besides, we have also found that PDTC, a NF-κB inhibitor, is capable of inhibiting the AGEs-induced reduction of collagen II expression, but can not abolish the AGEs-induced PPARγ down-regulation in human OA chondrocytes. Moreover, pioglitazone could decrease the AGEs-increased NF-κB activity and collagen II reduction. These findings suggest that the destruction of collagen II by AGEs in human OA chondrocytes may be through a JNK/p38MAPK-activated PPARγ down-regulation-triggered NF-κB activation signaling pathway. In conclusion, as indicated in Figure 11, our results demonstrated for the first time that AGEs induce the inflammatory signalings, productions of MMP-13 and IL-6, and collagen II reduction in human OA chondrocytes via a TLR4 and RAGE-regulated p38MAPK/JNK-activated PPARγ down-regulation-triggered NF-κB activation signaling pathway. In addition, these findings implicate that the accumulation of AGEs is correlated to the erosion of human OA cartilage and stimulates chondrocytes to produce more catabolic factors (MMPs and cytokines) and less anabolic factors (collagen II). The TLR4 and RAGE-regulated down-regulation of PPARγ is important in the net catabolic effect of AGEs on cartilage and may play a crucial role in the development of OA pathogenesis induced by AGEs accumulation. The clinical significance of these findings needs to be clarified in the future. 10.1371/journal.pone.0066611.g011Figure 11 The proposed schematic representation of AGEs-induced inflammatory signalings and resulted reduction of collagen II expression mediated by the down-regulation of PPARγ via TLR4 and RAGE in human OA chondrocytes is shown. ==== Refs References 1 Glass GG (2006 ) Osteoarthritis . Dis Mon 52 : 343 –362 .17142123 2 Goldring MB , Goldring SR (2007 ) Osteoarthritis . J Cell Physiol 213 : 626 –634 .17786965 3 Blagojevic M , Jinks C , Jeffery A , Jordan KP (2010 ) Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis . Osteoarthritis Cartilage 18 : 24 –33 .19751691 4 Felson DT , Zhang Y (1998 ) An update on the epidemiology of knee and hiposteoarthritis with a view to prevention . Arthritis Rheum 41 : 1343 –1355 .9704632 5 Verzijl N , DeGroot J , Oldehinkel E , Bank RA , Thorpe SR , et al (2000 ) Age-related accumulation of Maillard reaction products in human articular cartilage collagen . Biochem J 350 : 381 –387 .10947951 6 DeGroot J (2004 ) The AGE of the matrix: chemistry, consequence and cure . Curr Opin Pharmacol 4 : 301 –305 .15140424 7 Verzijl N , DeGroot J , Ben ZC , Brau-Benjamin O , Maroudas A , et al (2002 ) Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis . Arthritis Rheum 46 : 114 –123 .11822407 8 Yammani RR , Carlson CS , Bresnick AR , Loeser RF (2006 ) Increase in production of matrix metalloproteinase 13 by human articular chondrocytes due to stimulation with S100A4: Role of the receptor for advanced glycation end products . Arthritis Rheum 54 : 2901 –2911 .16948116 9 Rasheed Z , Akhtar N , Haqqi TM (2011 ) Advanced glycation end products induce the expression of interleukin-6 and interleukin-8 by receptor for advanced glycation end product-mediated activation of mitogen-activated protein kinases and nuclear factor-κB in human osteoarthritis chondrocytes . Rheumatology 50 : 838 –851 .21172926 10 Wada J , Makino H (2013 ) Inflammation and the pathogenesis of diabetic nephropathy . Clin Sci (Lond) 124 : 139 –152 .23075333 11 van BeijnumJR , Buurman WA , Griffioen AW (2008 ) Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1) . Angiogenesis 11 : 91 –99 .18264787 12 Braissant O , Foufelle F , Scotto C , Dauca M , Wahli W (1996 ) Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-α, -β, and -γ in the adult rat . Endocrinology 137 : 354 –366 .8536636 13 Barish GD , Narkar VA , Evans RM (2006 ) PPAR δ: a dagger in the heart of the metabolic syndrome . J Clin Invest 116 : 590 –597 .16511591 14 Tontonoz P , Hu E , Spiegelman BM (1994 ) Stimulation of adipogenesis in fibroblasts by PPAR 2, a lipid-activated transcription factor . Cell 79 : 1147 –1156 .8001151 15 Chawla A , Schwarz EJ , Dimaculangan DD , Lazar MA (1994 ) Peroxisome proliferator-activated receptor (PPAR)γ: adipose-predominant expression and induction early in adipocyte differentiation . Endocrinology 135 : 798 –800 .8033830 16 Cho MC , Lee K , Paik SG , Yoon DY (2008 ) Peroxisome proliferators activated receptor (PPAR) modulators and metabolic disorders . PPAR Res 2008 : 679137 .18566691 17 Takano H , Komuro I (2009 ) Peroxisome proliferator-activated receptor γ and cardiovascular diseases . Circ J 73 : 214 –220 .19129679 18 Jiang C , Ting AT , Seed B (1998 ) PPARγ agonists inhibit production of monocyte inflammatory cytokines . Nature 391 : 82 –86 .9422509 19 Ji JD , Cheon H , Jun JB , Choi SJ , Kim YR , et al (2001 ) Effects of peroxisome proliferator activated receptor-γ (PPARγ) on the expression of inflammatory cytokines and apoptosis induction in rheumatoid synovial fibroblasts and monocytes . J Autoimmun 17 : 215 –221 .11712859 20 Fahmi H , di Battista JA , Pelletier JP , Mineau F , Ranger P , et al (2001 ) Peroxisome proliferator-activated receptor γ activators inhibit interleukin-1β–induced nitric oxide and matrix metalloproteinase 13 production in human chondrocytes . Arthritis Rheum 44 : 595 –607 .11263774 21 Afif H , Benderdour M , Mfuna-Endam L , Martel-Pelletier J , Pelletier JP , et al (2007 ) Peroxisome proliferator-activated receptor γ1 expression is diminished in human osteoarthritic cartilage and is down-regulated by interleukin-1β in articular chondrocytes . Arthritis Res Ther 9 : R31 .17386086 22 Kobayashi T , Notoya K , Naito T , Unno S , Nakamura A , et al (2005 ) Pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, reduces the progression of experimental osteoarthritis in guinea pigs . Arthritis Rheum 52 : 479 –487 .15692987 23 Fahmi H , Pelletier JP , Mineau F , Martel-Pelletier J (2002 ) 15d-PGJ2 is acting as a 'dual agent' on the regulation of COX-2 expression in human osteoarthritic chondrocytes . Osteoarthritis Cartilage 10 : 845 –848 .12435328 24 Yang Q , Chen C , Wu S , Zhang Y , Mao X , et al (2010 ) Advanced glycation end products down-regulates peroxisome proliferator-activated receptor γ expression in cultured rabbit chondrocyte through MAPK pathway . Eur J Pharmacol 649 : 108 –114 .20863825 25 Loeser RF , Yammani RR , Carlson CS , Chen H , Cole A , et al (2005 ) Articular chondrocytes express the receptor for advanced glycation end products: Potential role in osteoarthritis . Arthritis Rheum 52 : 2376 –2385 .16052547 26 Schelbergen RF , Blom AB , van den Bosch MH , Slöetjes A , Abdollahi-Roodsaz S , et al (2012 ) Alarmins S100A8 and S100A9 elicit a catabolic effect in human osteoarthritic chondrocytes that is dependent on Toll-like receptor 4 . Arthritis Rheum 64 : 1477 –1487 .22127564 27 Fahmi H , Martel-Pelletier J , Pelletier JP , Kapoor M (2011 ) Peroxisome proliferator-activated receptor γ in osteoarthritis . Mod Rheumatol 21 : 1 –9 .20820843 28 Steenvoorden MM , Huizinga TW , Verzijl N , Bank RA , Ronday HK , et al (2006 ) Activation of receptor for advanced glycation end products in osteoarthritis leads to increased stimulation of chondrocytes and synoviocytes . Arthritis Rheum 54 : 253 –256 .16385542 29 McCance DR , Dyer DG , Dunn JA , Bailie KE , Thorpe SR , et al (1993 ) Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus . J Clin Invest 91 : 2470 –2478 .8514859 30 Berenbaum F (2011 ) Diabetes-induced osteoarthritis: from a new paradigm to a new phenotype . Ann Rheum Dis 70 : 1354 –1356 .21474484 31 Schett G, Kleyer A, Perricone C, Sahinbegovic E, Iagnocco A, et al.. (2012) Diabetes is an independent predictor for severe osteoarthritis: results from a longitudinal cohort study. Diabetes Care, doi: 10.2337/dc12-0924. 32 Fahmi H , Pelletier JP , di Battista JA , Cheung HS , Fernandes JC , et al (2002 ) Peroxisome proliferator-activated receptor activators inhibit MMP-1 production in human synovial fibroblasts likely by reducing the binding of the activator protein 1 . Osteoarthritis Cartilage 10 : 100 –108 .11869069 33 DeGroot J , Verzijl N , Jacobs KM , Budde M , Bank RA , et al (2001 ) Accumulation of advanced glycation endproducts reduces chondrocyte-mediated extracellular matrix turnover in human articular cartilage . Osteoarthritis Cartilage 9 : 720 –726 .11795991 34 Shoulders MD , Raines RT (2009 ) Collagen structure and stability . Annu Rev Biochem 78 : 929 –958 .19344236 35 Billinghurst RC , Dahlberg L , Ionescu M , Reiner A , Bourne R , et al (1997 ) Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage . J Clin Invest 99 : 1534 –1545 .9119997 36 Akira S , Taga T , Kishimoto T (1993 ) Interleukin 6 in biology and medicine . Adv Immunol 54 : 1 –78 .8379461 37 Andersson U , Erlandsson-Harris H (2004 ) HMGB1 is a potent trigger of arthritis. . J Intern Med . 255 : 344 –350 .14871458 38 Terada C , Yoshida A , Nasu Y , Mori S , Tomono Y , et al (2011 ) Gene expression and localization of high-mobility group box chromosomal protein-1 (HMGB-1) in human osteoarthritic cartilage . Acta Med Okayama 65 : 369 –377 .22189477 39 Medzhitov R (2001 ) Toll-like receptors and innate immunity . Nat Rev Immunol 1 : 135 –145 .11905821 40 Van Lent PL , Blom AB , Grevers L , Sloetjes A , van den Berg WB (2007 ) Toll-like receptor 4 induced FcR expression potentiates early onset of joint inflammation and cartilage destruction during immune complex arthritis: Toll-like receptor 4 largely regulates FcR expression by interleukin 10 . Ann Rheum Dis 66 : 334 –340 .17068066 41 Wolff DA , Stevenson S , Goldberg VM (1992 ) S-100 protein immunostaining identifies cells expressing a chondrocytic phenotype during articular cartilage repair . J Orthop Res 10 : 49 –57 .1370178 42 Lin L , Park S , Lakatta EG (2009 ) RAGE signaling in inflammation and arterial aging . Front Biosci 14 : 1403 –1413 . 43 Noguchi T , Sado T , Naruse K , Shigetomi H , Onogi A , et al (2010 ) Evidence for activation of Toll-like receptor and receptor for advanced glycation end products in preterm birth . Mediators Inflamm 2010 : 490406 .21127710 44 Qin YH , Dai SM , Tang GS , Zhang J , Ren D , et al (2009 ) HMGB1 enhances the proinflammatory activity of lipopolysaccharide by promoting the phosphorylation of MAPK p38 through receptor for advanced glycation end products . J Immunol 183 : 6244 –6250 .19890065 45 Kim HA , Cho ML , Choi HY , Yoon CS , Jhun JY , et al (2006 ) The catabolic pathway mediated by Toll-like receptors in human osteoarthritic chondrocytes . Arthritis Rheum 54 : 2152 –2163 .16802353 46 Nah SS , Choi IY , Yoo B , Kim YG , Moon HB , et al (2007 ) Advanced glycation end products increases matrix metalloproteinase-1, -3, and -13, and TNF-alpha in human osteoarthritic chondrocytes . FEBS Lett 581 : 1928 –1932 .17434489 47 Rasheed Z , Haqqi TM (2012 ) Endoplasmic reticulum stress induces the expression of COX-2 through activation of eIF2α, p38-MAPK and NF-κB in advanced glycation end products stimulated human chondrocytes . Biochim Biophys Acta 1823 : 2179 –2189 .22982228	23776688	PMC3680452	CC BY	2021-01-05 17:29:29	yes	PLoS One. 2013 Jun 12; 8(6):e66611
==== Front PLoS PathogPLoS PathogplosplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 23785291PPATHOGENS-D-12-0239610.1371/journal.ppat.1003435Research ArticleBiologyPlant SciencePlant PathologyPlant PathogensExtreme Resistance as a Host Counter-counter Defense against Viral Suppression of RNA Silencing Suppressor of RNA Silencing and Extreme ResistanceSansregret Raphaël 1 Dufour Vanessa 1 Langlois Mathieu 1 Daayf Fouad 2 Dunoyer Patrice 3 Voinnet Olivier 3 4 * Bouarab Kamal 1 * 1 Centre SEVE, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada 2 Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada 3 Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, Strasbourg, France 4 Department of Biology, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Ding Shou-Wei Editor University of California Riverside, United States of America * E-mail: voinneto@ethz.ch (OV); Kamal.Bouarab@USherbrooke.ca (KB)The authors have declared that no competing interests exist. Conceived and designed the experiments: RS PD OV KB. Performed the experiments: RS VD ML PD FD. Analyzed the data: RS VD ML FD PD OV KB. Contributed reagents/materials/analysis tools: FD PD OV KB. Wrote the paper: RS OV KB. 6 2013 13 6 2013 22 9 2015 9 6 e100343525 9 2012 6 5 2013 © 2013 Sansregret et al2013Sansregret et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.RNA silencing mediated by small RNAs (sRNAs) is a conserved regulatory process with key antiviral and antimicrobial roles in eukaryotes. A widespread counter-defensive strategy of viruses against RNA silencing is to deploy viral suppressors of RNA silencing (VSRs), epitomized by the P19 protein of tombusviruses, which sequesters sRNAs and compromises their downstream action. Here, we provide evidence that specific Nicotiana species are able to sense and, in turn, antagonize the effects of P19 by activating a highly potent immune response that protects tissues against Tomato bushy stunt virus infection. This immunity is salicylate- and ethylene-dependent, and occurs without microscopic cell death, providing an example of “extreme resistance” (ER). We show that the capacity of P19 to bind sRNA, which is mandatory for its VSR function, is also necessary to induce ER, and that effects downstream of P19-sRNA complex formation are the likely determinants of the induced resistance. Accordingly, VSRs unrelated to P19 that also bind sRNA compromise the onset of P19-elicited defense, but do not alter a resistance phenotype conferred by a viral protein without VSR activity. These results show that plants have evolved specific responses against the damages incurred by VSRs to the cellular silencing machinery, a likely necessary step in the never-ending molecular arms race opposing pathogens to their hosts. Author Summary Multiple and complex layers of defense help plants to combat pathogens. A first line of defense relies on the detection, via dedicated host-encoded receptors, of signature molecules (so called pathogen-associated molecular patterns, PAMPs) produced by pathogens. In turn, this PAMP-triggered immunity (PTI) may be itself antagonized by adapted pathogens that have evolved virulence effectors to target key PTI components. Host plants react to PTI suppression by producing disease resistance (R) proteins that recognize virulence effectors and activate highly specific resistance called Effector Triggered Immunity (ETI). It has been noted that RNA silencing, a sequence-specific antiviral defense response based on the production of virus-derived 21–24 nt small RNAs on the one hand, and its suppression by virulence effectors, called viral suppressors of RNA silencing (VSRs) on the other, are conceptually similar to PTI. Here we provide strong support to this hypothesis by showing that extreme resistance is indeed activated following detection, in specific host species, of the VSR activity of a viral virulence effector. The ensuing antiviral immunity displays many characteristics of ETI, suggesting that one or several R proteins must sense the integrity of the host silencing machinery. RS is a fellow of the FQRNT. KB and FD are supported by Discovery grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). OV was supported by an award from the Bettencourt Foundation and the Louis D. Prize from the French Academy of Science. PD was supported by the Agence National pour la Recherche (ANR-08-JCJC-0063 and ANR-10-LABX-36). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Plants fight microbial attacks using both constitutive and induced defenses, which include basal and highly specific resistance [1]. Basal resistance, or PTI (for PAMP-Triggered Immunity), often relies on the detection of highly conserved signature molecules that include fungal polysaccharides or bacterial flagellin, collectively termed pathogen-associated molecular patterns (PAMPs; [1], [2]). To circumvent this first layer of defense, many host-adapted microbes produce effector proteins that suppress various steps of PTI [3]. As a counter-response, plants have, in turn, evolved classes of specialized receptors called resistance (R) proteins that directly detect pathogen's encoded suppressors of PTI, or that sense the molecular consequences of their adverse action on defense-related host factors. R protein activation triggers potent defense responses collectively named Effector Triggered Immunity (ETI) that often –albeit not always (see below) culminate in Hypersensitive Response (HR), a rapid and localized cell death process thought to limit or preclude pathogens' growth [1], [2]. As a consequence of the gene-for-gene type of interaction linking these two components, plant R genes and their corresponding pathogen-encoded virulence factors evolve constantly and rapidly, so that HR, a common and ultimate manifestation of ETI, is usually only observed in specific plant species infected with specific pathogen strains. The plant hormones salicylic acid (SA), ethylene and jasmonic acid (JA) are crucially implicated in signaling networks underpinning both PTI and ETI [1], [2], [4], [5]; antimicrobial pathogenesis-Related Proteins (PRs), which include taumatine-like proteins and chitinases, are also often induced by both pathways and constitute, therefore, typical molecular markers of pathogen-induced defenses [6]. Although the occurrence of HR is classically used to discern PTI from ETI during bacterial or fungal infections [7], an HR-independent process known as Extreme Resistance (ER) is activated by a number of R proteins during ETI against viruses; ER is characterized by the lack of detectable accumulation of the triggering virus, and is accompanied by the onset of a broad-spectrum antiviral state in the absence of macroscopic or microscopic cell death lesions [8]–[11]. RNA silencing is a conserved regulatory process that has evolved as an antiviral and antimicrobial defense mechanism in plants and animals [12]–[17]. Common features of RNA silencing across organisms include the involvement of double-stranded (ds)RNA as an initiator molecule, and accumulation of 21–24 nt small (s)RNAs that are processed from dsRNA by the RNAse III-like enzyme Dicer [18]–[20]. sRNAs are then incorporated into Argonaute (AGO)-containing effector complexes termed RNA-induced Silencing Complexes (RISCs) and, in case of extensive sequence complementarity between sRNA guide and target, AGO catalyses cleavage of the target RNA. Arabidopsis thaliana possesses four Dicer-like (DCLs) and ten AGO proteins [21], among which DCL4 and its surrogate DCL2, as well as AGO1 and AGO2, play essential roles as processors and effectors of virus-derived short interfering (si)RNAs, respectively [22]–[29]. DCL1- and AGO1-dependent micro (mi)RNAs produced from endogenous loci regulate the expression of many transcripts displaying miRNA sequence-complementarity, including mRNAs for transcription factors, enzymes, and regulators of PTI induced, notably, by bacteria [15]–[17], [30], [31]. As a consequence of these multiple RNA silencing-based defense layers, plant viruses, pathogenic bacteria, oomycetes and, possibly, fungi, have evolved suppressors of RNA silencing (SRs) that apparently target many steps of the siRNA and miRNA pathways [14], [32]–[37]. SRs are highly diverse in sequence, structure, and activity, and single SRs may target multiple points in RNA silencing pathways [14], [31]. Several viral SRs (VSRs) are known to affect AGO1 function [14]. For example, The Beet western yellows virus P0 protein was suggested to act as an F-box protein targeting AGO proteins for degradation, thereby preventing RISC assembly [38]–[40]. Turnip crinckle virus P38 was recently shown to bind directly and specifically AGO1 through mimicry of host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms [41], [42]. Physical sequestration of siRNAs is another common property of VSRs in vitro [43]–[47], although the extent to which this specific feature contributes to effective RNA silencing suppression in vivo remains unclear [42]. The most compelling example of active silencing suppression mediated by siRNA binding is provided by the tombusvirus P19 protein, of which the closely related Tomato bushy stunt virus (TBSV) and Carnation Italian Ringspot virus (CIRV; 97% identity) are the type representatives. Following its original discovery as a VSR [35], P19 was co-crystalized as a head-to-tail homodimer in direct association with an siRNA duplex [43], [48]. Supporting a direct and critical contribution of homodimerization and siRNA binding to the P19 VSR activity, stable point mutant alleles of the proteins lacking either property display complete loss-of-VSR-function phenotypes in both virus-infected and transgenic plants [43], [49]–[51]. sRNA binding by P19 also explains why its constitutive expression in Arabidopsis promotes developmental defects resembling those of plants carrying mutations in miRNA pathway components. Indeed, it was shown that P19 binds endogenous siRNAs and miRNAs, incurring, in the process, misregulation of the cognate endogenous targets of these molecules [42], [52]. Remarkable parallels can be drawn between the general framework of silencing activation and its suppression by pathogens on the one hand, and the classical PTI-ETI scheme for resistance, on the other. This has prompted the suggestion that the two processes might be, in fact, manifestations of similar, if not identical, phenomena [31], [53]. In the case of (+)-stranded RNA viruses, for example, viral-derived dsRNA can be assimilated to a PAMP because this molecule is a mandatory product of viral replication. Similarly, the Dicer/AGO consortium orchestrating the antiviral reaction may be conceptually compared to the first defense layer underlying PTI [31]. Pursuing the comparison one step further and taking into account that VSRs are virulence effectors, it can be anticipated that the damages incurred by VSRs to the cellular silencing machinery may be sensed by host-encoded functions comprising, perhaps, dedicated R genes; the effects of such functions would thus be diagnosed, at least partly, by the typical outputs of ETI, including HR [31]. Supporting this notion, at least three VSR proteins from distinct virus families are known to trigger HR-like lesions in a host-specific manner [53]–[58]. It remains largely unknown, however, if these responses are stimulated by intrinsic silencing suppression properties or by other, unrelated functions of the viral proteins involved. Also unclear is whether virus resistance is effectively triggered upon recognition of these VSRs in these specific hosts, and to what extent the output of the induced defense compares with that of classical ETI. The present series of experiments was aimed at addressing these various issues using the well-characterized P19 VSR in tobacco. The results support the idea that RNA silencing and its suppression by viruses can be effectively rationalized within the frame of PTI-ETI, since we demonstrate, in authentic infection contexts, that (i) tombusviral virulence (ii) suppression of RNA silencing and (iii) induction of an ER-type of resistance with molecular features of ETI are all dependent upon the ability of P19 to bind sRNAs. Collectively, the data support the existence of host-encoded sensors that monitor the status/integrity of key RNA silencing components in plants. We propose, consequently, that perturbation of these components by pathogen-encoded SRs may activate potent ETI-like resistance responses. This proposed host counter-counter defensive layer likely constitutes an important driver in the evolution and diversification of SRs from viruses and perhaps other parasites. Results P19 is required to trigger an ETI-like resistance against TBSV in N. tabacum TBSV P19 was shown to induce a HR-like response in N. tabacum and other Nicotiana species; a host-specific response strongly evocative of R gene-mediated ETI [54]–[57], To ascertain further if, indeed, P19 acts as an elicitor of immune responses, we generated transgenic N. tabacum cv. Xanthi lines expressing P19 under the GVG glucocorticoide inducible promoter, which is activated by dexamathazone (Dex::P19; [59]). The expression of P19 was quantified in two independent lines 0, 12 and 24 hours post Dex application (hpp); non-transgenic plants sprayed with DEX provided a negative control. While very low P19 transcript accumulation was observed before DEX treatment in the two transgenic lines, it was up to 4000 times higher following DEX application, at 12 and 24 hpp, compared to 0 hpp and to DEX-treated non-transgenic plants (Figure 1A). Accumulation of the P19 protein, mostly under homodimeric form, was also detected by Western analysis in the DEX-induced transgenic lines, but not in non-transgenic lines, using a polyclonal P19 antibody (Figure 1B). Accumulation of P19 following DEX induction correlated with the onset of three key markers of plant defense responses: (i) the progressive development of HR-like lesions in the sprayed areas of leaves, (ii) the accumulation of distinct PR proteins, PR1, PR2 and PR3, at 24 and 48 hpp (Figures 1C–D), and (iii) the accumulation of salicylic acid (SA) which was 4–5 times higher following DEX application at 24 hpp in the DEX-induced transgenic lines compared to 0 hpp and to DEX-treated non-transgenic plants (Figure S1). Collectively, therefore, the results presented in Figure 1 and Figure S1 suggest that in N. tabacum, P19 effectively acts as an elicitor of plant defense responses displaying at least superficial characteristics of ETI. 10.1371/journal.ppat.1003435.g001Figure 1 DEX::P19 transgenic plants display defense responses following DEX application. (A–B) Leaves of five week old DEX::P19 transgenic and wild type plants (N. tabacum cv. Xanthi) were sprayed with DEX and the kinetics of P19 accumulation at transcript (A) and protein (B) levels was subsequently analyzed by qPCR and Western analysis, respectively. Actin was used as an internal control. (C) DEX::P19 transgenic or wild type plants were sprayed with DEX, and appearance of HR was assessed 5 days post-DEX application. We observed two and sometimes three bands for P19 dimers. These additional bands appear when P19 is expressed in N. tabacum but not in N. benthamiana. We believe that these additional bands are due to post-translational regulation of P19 by N. tabacum; this regulation might have a biological significance but evidence of this is not known yet. (D) PR protein accumulation at 0, 1 and 2 days post DEX application in wild type and DEX::P19 transgenic lines. Western analysis was conducted using anti-PR1, -PR2 and -PR3 antibodies. Coomassie or ponceau staining of the same extracts is shown to demonstrate equal protein loading. Experiments were repeated three times and showed similar results. To test if P19 effectively induces resistance against TBSV in N. tabacum, Agrobacterium strains expressing either TBSV-GFP or TBSVΔP19-GFP, which is unable to express P19 [26], were used to inoculate leaves of 5-week old N. tabacum. At 5 days post-infiltration (dpi), virus accumulation was monitored under UV light via the appearance of green fluorescence in infiltrated leaves, and by Western analysis using an anti-GFP antibody. Viral replication was assessed directly in parallel by Northern analysis, using a GFP DNA fragment as a probe, which detects both genomic and sub-genomic RNAs of TBSV-GFP. We found that the presence or absence of P19 expression from TBSV-GFP had dramatically contrasted consequences on virus replication. Thus, GFP was not observed (Figure 2A–B) and the viral RNAs were below detection limits of Northern analyses (Figure 2C) in TBSV-GFP-inoculated leaves. In sharp contrast, however, both GFP accumulation and viral RNA replication were readily detectable in TBSVΔP19-GFP-infiltrated leaves at 5 dpi (Figures 2A–C). To further characterize the P19-mediated defense response, we used trypan blue staining as a diagnostic of cell death. Leaves were thus inoculated either with TBSV-GFP, P50 from Tobacco mosaic virus (TMV), which induces an HR in N. tabacum carrying the resistance gene N (as a positive control), or GUS as a negative control. We found that the visible and microscopic HR observed in P50-treated plants was absent from TBSV-GFP-infected and control leaves (Figure S2). These results strongly suggest that extreme resistance (ER) was triggered in TBSV-GFP-inoculated leaves of N. tabacum, and implicate, therefore, P19 as the elicitor of this defense. In fact, the results obtained here with P19 in tobacco are highly reminiscent of the well-studied interaction between Potato virus X coat protein (CP) and the Rx resistance protein in Solanum tuberosum or tobacco [8]. Indeed, while Rx typically confers ER to PVX in the context of authentic virus infections, isolated and prolonged production of CP, for instance via Agrobacterium-mediated transient expression, does trigger an HR in Rx potato genotypes [8], [9], as seen previously and here upon transient and transgenic expression of P19 in specific Nicotiana species (Figure 1B–C, [56]). With both PVX and TBSV, the potent antiviral state accompanying the ER (e.g. Figure 2C) probably stops virus replication before the CP or P19 have reached the levels required to trigger an HR [8], [9], a phenomenon presumably bypassed when both elicitors are produced in a virus replication-independent manner. 10.1371/journal.ppat.1003435.g002Figure 2 P19 is required for extreme resistance of N. tabacum against TBSV. (A) Leaves of 5 weeks old N. tabacum cv. Xanthi plants were infiltrated with Agrobacterium expressing TBSV-GFP or TBSVΔP19-GFP. Pictures of infiltrated leaves were taken 5 dpi under transmitted light and UV. (B) GFP accumulation in infiltrated leaves from three independent plants. Western analysis was carried out using an anti-GFP antibody. Coomassie staining of the same extracts is shown to demonstrate equal protein loading. (C) Northern analysis of TBSV-GFP and TBSVΔP19-GFP RNA accumulation in infected plants at 5 dpi, using a GFP DNA fragment as a radioactive probe. Viral genomic and subgenomic RNAs are indicated; ribosomal RNA was used to demonstrate equal RNA loading. Experiments were repeated three times and showed similar results. Salicylic acid and ethylene are required for extreme resistance induced by P19 The potent (Figure 2C) and broad-spectrum [8] antiviral state triggered by ER is suspected to underlie the production of defense-related hormones, including SA, which possesses demonstrated antiviral activities [27], [60], [61]. The gaseous hormone ethylene is also important for induction of plant immunity [4]. To investigate the possible roles of these compounds in the ER-like resistance induced by P19 against TBSV, SA-deficient transgenic tobacco plants expressing NahG (Salicylate hydroxylase; [62]) and plants insensitive to Ethylene (ETR; [63]) were inoculated with TBSV-GFP using Agrobacterium-mediated delivery. At 5 dpi, leaves were observed under UV and samples were harvested for Western analysis using the anti-GFP antibody. Unlike WT plants, both transgenic plants failed to display resistance against TBSV (Figure 3A–B) and, accordingly, the P19-dependent induction of PR proteins was compromised in NahG plants ([64], [65]; Figure S3). Overall, these results indicate that SA and ethylene are required for the ER induced by P19 against TBSV. We then investigated if the HR-like lesions induced by P19 in tobacco leaves (Figure 1C) were SA- and/or ethylene-dependent. As seen in Figure 3C, necrosis was as extensive in leaves of NahG and ETR plants as it was in their non-transgenic counterparts at 5 dpi (Figure 3C), indicating that the HR triggered by P19, unlike the induced antiviral state, is neither SA- nor ethylene-dependent. 10.1371/journal.ppat.1003435.g003Figure 3 Salicylic acid and ethylene are required for extreme resistance induced by P19 against TBSV. (A) Leaves of SA-deficient and ethylene-insensitive plants, or their corresponding WT counterparts, were infiltrated with Agrobacterium tumefaciens expressing TBSV-GFP. Leaves were observed under optical light and GFP fluorescence was visualized under UV at 5 dpi. (B) Western analysis was conducted to detect TBSV-GFP accumulation in the infiltrated leaves depicted in (A), using an anti-GFP antibody. Ponceau staining of the membrane is shown to demonstrate equal protein loading. (C) A. tumefaciens expressing P19 triggers an HR response is all depicted genotypes at 5 dpi. Experiments were repeated three times and showed similar results. sRNAs binding by P19 is necessary for P19-mediated elicitation of defense Resolving the crystal structure of the P19-siRNA complex granted the identification of point mutations that debilitate the protein's VSR function without impacting its stability [43]. It was notably shown that a double mutation affecting tryptophan residues 39 and 42 (W39-42R) was sufficient to abolish siRNA binding by P19 in vitro, with the resulting stable mutant allele being unable to suppress RNA silencing in planta [43]. Using the same allele, we thus tested if the capacity of P19 to sequester siRNAs was required for the elicitation of ER in N. tabacum. We generated transgenic N. tabacum cv. Xanthi lines expressing CIRV P19W39-42R under the DEX inducible promoter (DEX::P19W39-42R). Expression of P19W39-42R was quantified in two independent lines 0, 12 and 24 hours after DEX application; transgenic line Dex::P19#1, expressing WT P19 (Figure 1B), was used as a reference for functional P19 levels in these experiments. Upon DEX application onto leaves of five week old plants, quantification of both mRNA (Figure 4A) and protein (Figure 4B) levels showed that accumulation of the P19 mRNA and of P19 homo-dimers was similar in the two independent DEX::P19W39-42R tobacco lines tested and in the Dex::P19#1 reference line (Figure 4A–B). Remarkably, P19W39-42R was neither able to induce SA accumulation, HR-like symptoms nor to promote accumulation of PR1, PR2 and PR3 compared to WT P19 (Figure 3C–D and Figure S1), suggesting that small RNA binding by P19 is necessary to trigger the onset of defense in N. tabacum. The results also show that defense elicitation can occur independently of virus infection, suggesting that binding of endogenous sRNAs by P19 is prerequisite for elicitation. 10.1371/journal.ppat.1003435.g004Figure 4 Binding of small RNAs is mandatory for induction of plant immune responses by P19. (A–B) Leaves of five week old Dex::P19, Dex::P19W39-42R transgenic lines (N. tabacum cv. Xanthi) were sprayed with DEX and the kinetics of P19W39-42R accumulation at transcript (A) and protein (B) levels was analysed by qPCR and Western analysis, respectively. Actin was used as an internal control. (C) The transgenic lines described above were sprayed with DEX and appearance of an HR was assessed 5 day post-DEX application. (D) PR proteins accumulation at 0, 1 and 2 days post DEX application in Dex::P19 and Dex::P19W39-42R transgenic lines. Western analysis was conducted using anti-PR1, -PR2 and -PR3 antibodies. Coomassie or ponceau staining of the same extracts is shown to demonstrate equal protein loading. Experiments were repeated three times and showed similar results. RNA silencing suppression and sRNAs binding are not sufficient, per se, to trigger HR-like lesions in N. tabacum The above results prompted us to investigate if silencing suppression via sRNA binding was sufficient, per se, to trigger the HR-associated defense response elicited by P19 in N. tabacum. To that aim, we used Agrobacterium strains producing various VSRs unrelated to P19. HcPro from Tobacco etch virus, P15 from Peanut clump virus and P21 from Beet yellows virus are all known to bind sRNAs in vitro, with high affinity for 21 nt-long species (Figure 5A; [45]). In the same in vitro assay, P14 from Pothos latent virus was shown to bind different sizes of sRNAs ranging from 21 nt to 26 nt, while P25 from PVX was, by contrast, devoid of sRNA binding activity (Figure 5A; [45]). 10.1371/journal.ppat.1003435.g005Figure 5 Effects of VSRs unrelated to P19. (A) List of VSRs used in this study alongside their preferential sRNA binding sizes, as established in vitro. P25 is unable to bind sRNAs in vitro. Leaves of five week-old N. tabacum cv. Xanthi were transiently infiltrated with Agrobacterium tumefaciens expressing either P19, P14, P15, P21, P25 or HcPro. (B) HR response as evaluated 5 days post infiltration of the various VSRs lsited in (A). (C–D) Leaves of N. tabacum cv. Xanthi were infiltrated with a mixture of Agrobacteria containing either P14, P15, P21 or HcPro together with a GFP transgene used as a visual and molecular reporter of the onset of RNA silencing in the co-infiltrated tissues. GFP fluorescence was visualized 4 days post-infiltration under UV light (C) and by Western analysis using an anti-GFP antibody (D). Ponceau staining of the same extracts is depicted to demonstrate equal protein loading. (E) Leaves of five week-old N. tabacum cv. Xanthi were infiltrated with A. tumefaciens strains expressing P19 in combination with either P14, P15, P21, HcPro, P25 or the GUS reporter gene. Appearance of P19-triggered HR lesions was monitored at 40 hpi (Left panel) and 96 hpi (Right panel). Experiments were repeated three times with similar results. (F) Western analysis of P19 protein levels in P19-VSR co-treatments. Proteins extracts from P19-VSRs or P19-GUS co-treated leaves were subjected to anti-P19 immunoblotting after 48 h. Ponceau staining of the membrane is shown to demonstrate equal protein loading. Experiments were repeated three times and showed similar results. We found that, unlike P19, neither of the above VSRs was able to trigger the HR-like response at 5 dpi following their transient expression in leaves of N. tabacum (Figure 5B). Nonetheless, in a well-established silencing suppression assay based on transient co-expression of a silencing GFP target transgene with VSRs [66], all of these proteins were clearly able to stabilize GFP accumulation, as assessed under UV illumination (Figure 5C) and by Western analysis (Figure 5D). By contrast, GFP accumulation remained low in tissues co-infiltrated with a control Agrobacterium strain expressing the GUS reporter gene (Figure 5C–D). Thus, all the VSRs tested were able to suppress GFP RNA silencing in this assay. The results indicate that the failure of the P19-unrelated VSRs to trigger an HR-like response cannot be explained by their inability to suppress RNA silencing in N. tabacum. Therefore, RNA silencing suppression is, in itself, insufficient to trigger this response. Moreover, given the documented high affinity of some of the VSRs used for siRNAs [42], [45], [67] the data suggest that sRNA binding per se is also insufficient to promote defense in N. tabacum. The most parsimonious interpretation of these results entails, therefore, that P19-mediated elicitation of host defenses in Nicotiana species involves the specific recognition of P19-sRNA complexes, or of downstream molecular events triggered by the specific association of both components. Co-expression of unrelated VSRs compromise the onset of HR elicited by P19, but not resistance conferred by Rx against PVX Even though none of the above-tested VSRs triggered, on its own, a defense response in N. tabacum, the intrinsic abilities of most of these proteins to bind sRNAs predicted that their co-expression with P19 would compromise the onset of HR-like lesions observed in Agrobacterium-infiltrated tissues (Figure 1C). As shown in Figure 5E, this was indeed the case: the appearance of necrotic tissues was significantly delayed and less extensive at 96 hours in leaf patches that had received the P19-VSR co-treatments compared to leaves co-treated with P19 and GUS as a negative control (Figure 5E). Remarkably, the delayed onset of HR was not observed in co-treatments involving P19 and the P25 protein of PVX, which, unlike all the other VSRs tested, does not bind sRNAs in vitro ([45]; Figure 5E). Western analyses employing a P19 antibody also confirmed that the delayed onset of HR was unlikely to be a consequence of altered levels of P19 homodimers in the P19-VSR co-treated leaves, compared to control leaves (Figure 5F). Given that P14, P15, P21 and Hc-Pro are all known to bind sRNA, at least in vitro [45], we assessed whether the compromised HR-like cell death phenotype observed upon concomitant expression of P19 with these VSRs resulted from a direct competition for sRNA binding, potentially decreasing the amount of P19-siRNA complexes. To address this point we transiently expressed, in N. benthamiana, a HA-tagged version of P19 (P19HA), either alone or in combination with P15 or P21 (Figure 6). As a source of siRNAs, we used a 35S promoter-driven inverted-repeat (IR) construct, corresponding to the 5′ part (‘GF’) of the GFP sequence, which is processed into 21 nt- and 24 nt-long siRNAs. Northern analysis of the sRNA fraction of P19HA immunoprecipitates showed that, as expected, P19 specifically bound the 21 nt-long GF siRNAs. Both P15HA and P21HA displayed the same 21 nt siRNA size preference as P19 for binding. However, P21 sequestered 21 nt siRNAs significantly more efficiently than the two other VSRs, as shown by the much stronger signal detected in P21HA immunoprecipitates (Figure 6). This most likely explains the decreased GF siRNA levels observed in P19HA and P15HA immunoprecipitated fractions when these VSRs were concomitantly expressed with P21 (Figure 6). However, in contrast to P21, P15 did not alter the amount of siRNA bound by P19 whereas P19 prevented P15 siRNA binding and competed with P21 siRNA binding (Figure 6). Therefore, in the case of P15, the compromised P19-triggered HR-like cell death phenotype is unlileky to result from a reduction in the amount of formed P19-siRNA complexes. Overall, these results show that, although necessary, the sRNA binding capacity of P19 is not sufficient for host defense elicitation in N. tabacum, suggesting that the onset of ER is intrinsically linked to the VSR function of P19 and not just the formation of P19-siRNA complexes per se (Figure 3). 10.1371/journal.ppat.1003435.g006Figure 6 Differential effects of co-expressed VSRs on P19 siRNA-binding capacity. (A) RNA gel blot analysis of GF siRNA accumulation (@GF) in total RNA and HA immunoprecipitated fractions from Nicotiana benthamiana infiltrated leaves expressing HA-tagged P15, P19 or P21 VSRs, either alone (-) or in combination with untagged VSRs. Ethidium bromide staining of ribosomal RNA (rRNA) is used as loading control. (B) Protein blot analysis of HA-tagged VSRs accumulation (@HA) in total (input) or immunoprecipitated fractions (IP@HA) of the samples described in (A). Coomassie staining of the membrane was used to verify equal loading after western blotting. EV: empty vector. Experiments were repeated three times and gave similar results. To further ascertain this idea, we took advantage of the fact that Rx-mediated ER is triggered by the PVX-encoded CP protein, which does not display any intrinsic VSR activity [68]. Moreover, Rx-mediated ER can be recapitulated in transgenic N. tabacum upon inoculation of PVX-GFP using leaf-infiltration of Agrobacterium. We reasoned that, unlike in the above example where resistance was highly dependent upon the VSR function of the P19 elicitor, Rx-mediated resistance would remain unaffected by co-expression of VSRs with PVX-GFP. As shown in Figure 7, accumulation of Agrobacterium-delivered PVX-GFP was abolished in leaves of plants expressing transgenic Rx, compared to non-transgenic plants. Furthermore, this pattern remained unaffected by transient co-expression of HcPro, P21, P15 P14 VSRs, or a control GUS transgene (Figure 7). 10.1371/journal.ppat.1003435.g007Figure 7 VSRs that bind small RNAs in vitro do not compromise resistance mediated by Rx against PVX. Leaves of five week-old N. tabacum expressing the Rx gene and its corresponding counterpart lacking this R gene was infiltrated with of A. tumefaciens strains expressing PVX-GFP together with a strain expressing P14, P15, P21, P25, HcPro or the GUS reporter gene. GFP accumulation in infiltrated leaves was detected by Western analysis using an anti-GFP antibody. Ponceau staining of the membrane is shown to demonstrate equal protein loading. Experiments were repeated three times and gave similar results. Discussion Cross-talk between RNA silencing pathways and both PTI and ETI pathways has been established experimentally in the case of bacterial pathogens [12], [15]. In all cases so far, PAMP recognition activates endogenous RNA silencing pathways to target negative regulators of disease resistance, leading to potentiation of basal defense [12], [15], [31]. Bacterial-encoded SRs, in turn, target this basal defense by inhibiting various, and perhaps multiple, steps of host silencing pathways. The work presented here describes how the activity of the viral suppressor P19 is sensed in specific Nicotiana species to induce immunity against the P19-producing virus. This immunity displays several key attributes of ETI, including the involvement of SA and ethylene, as well as the production of PR proteins. The timing of P19 homodimers accumulation correlates with the extent of cell death and PR proteins production; this is in agreement with data showed previously in which the authors used the same inducible promoter as the one we used in this study [69]. Remarkably, antiviral immunity is also accompanied by a lack of visible HR-like lesions, at least in the context of authentic tombusvirus infection, a phenomenon highly reminiscent of extreme resistance (ER) observed, for instance, during the CP-Rx interaction in PVX-infected plants. Further supporting the analogy between P19-mediated defense and the ER triggered by Rx, strong and isolated expression of their respective elicitors (i.e. P19 or CP, respectively) promotes the appearance of HR-like lesions in both cases. Nonetheless, a marked difference between the Rx-CP and the P19 systems is the reliance of the latter upon RNA silencing suppression, a function not associated with the CP of PVX [68]. Our findings were, in fact, not completely unprecedented. Hence, the P38 capsid protein of Turnip crinkle virus binds AGO to inhibit its loading with sRNAs [41], [42], [70]. P38 was also shown to induce HR-associated defense responses in the Arabidopsis ecotype Dijon-0 and its inbred derivative Dijon-17 [71], [72], a level of host specificity that strongly evokes an ETI-type of response. The elicitor of the N resistance gene, which confers ETI to TMV, had been also mapped to the p50 helicase subunit of the viral replicase, p126. Remarkably, the same domain of p126 was recently identified as being sufficient to suppress RNA silencing in N. benthamiana [73]. Moreover, the helicase enzymatic activity of p50 was found dispensable for both N-mediated resistance and silencing suppression, suggesting that the VSR activity of P50 might stimulate ETI via the activation of N. Seminal work carried out more than a decade ago also provided key insights into the potential contribution of the 2b protein from Tomato aspermy cucumovirus (TAV2b) to the induction of ETI, possibly through its VSR activity. Indeed, when expressed from recombinant TMV, TAV2b was found to activate strong host resistance in tobacco, typical of the gene-for-gene interaction linking R proteins to their elicitors [53]. Moreover, the N-terminal region of TAV2b was found critical for both VSR activity and resistance elicitation, suggesting that the same or overlapping domains of the protein are involved [53]. Interestingly, Chen et al. [74] recently showed that Tav2b effectively binds sRNAs, highly reminiscent of the situation presented here with P19. The seminal observation made with TAV2b led the authors to suspect that RNA silencing and its suppression on the one hand, and ETI on the other, were probably linked phenomena, at least in some cases; this view became strongly substantiated through subsequent work conducted with plant pathogenic bacteria (reviewed in [31]). The data obtained in this manuscript add further strength to this idea by showing the importance of RNA silencing suppression in the resistance mediated by P19, because immunity to TBSV was only achieved if the protein retained its capacity to suppress gene silencing, for which sRNA binding is a prerequisite. We suspect that the reported ETI-like response triggered by P19 in the absence of visible HR might also strongly contribute to its additional, albeit poorly understood, role as a host-specific determinant of systemic viral movement [51], [75]. This hypothesis is particularly appealing given the involvement of SA and ethylene in the P19-elicited response in N. tabacum. Indeed both hormones are known to mediate, directly or indirectly, systemic, in addition to localized, defense responses. Immune signaling pathways seem to be widely conserved across fungal, bacterial and viral interactions that lead to ETI in plants. The fact that P19-mediated resistance was compromised by many unrelated VSRs, unlike resistance activated by Rx argues, therefore, against an interference at the level of disease resistance signaling. Moreover, the PVX coat protein (elicitor of Rx) does not possess VSR function [68]. The fact that the integrity of the P19 binding domain is required for defense elicitation, together with the failure of PVX P25, among the VSR tested here, to alter the P19-mediated HR response, suggests that sRNA binding, required for VSR function, is a key component for defense activation in N. tabacum. It is, however unlikely to be sufficient, because none of the other VSRs tested was able to recapitulate, on its own, the defense phenotype induced by P19 when transiently expressed, despite that many of them bind sRNA in vitro and probably in vivo. Additionally, P15 could suppress the P19-mediated HR even though it did not outcompete P19 for siRNA binding in the N. benthamianan transient expression assay. The simplest interpretation of these results, therefore, is that P19 dimers complexed with sRNAs initiate a signal that is specifically sensed in N. tabacum to trigger extreme resistance against TBSV or that a conserved motif or structure important for sRNA binding by P19 is sensed in the plant. A non-mutually exclusive possibility holds that sensing occurs downstream, as a consequence of specific P19-sRNA association in a manner suppressed by the action of VSRs such as P15, which may share downstream silencing targets with P19 including AGOs. Interestingly, HR-like lesions and PR proteins accumulation could be triggered by P19 in the absence of a viral infection, suggesting that endogenous sRNAs, including siRNAs and miRNAs, which are effectively bound by P19 together with viral-derived siRNAs during infection [43], [49], [67], [76], form one component of the trigger. Hence, a recent study in transgenic Arabidopsis shows that binding of endogenous miRNAs by VSRs is much less widespread than was originally anticipated. In fact, P19 was, among many VSRs tested (including several used in the present study), the only protein to prevent loading of miRNAs into AGO1. By contrast, all of the VSRs tested could effectively prevent loading of exogenous siRNAs into AGO1 [42]. This peculiarity may contribute to explain the specific ability of P19 to trigger HR-like lesions and ER in N. tabacum. It is also possible that the binding of P19 to si/miRNAs promotes a specific change in the integrity or conformation of silencing effector proteins, including AGOs, and that these changes are sensed in a host-specific manner. miRNAs have roles in plant basal and race-specific resistance against bacterial pathogens [15], [16], [77]. Furthermore, some plant miRNAs appear to have evolved to control R gene expression presumably to prevent the known fitness cost of their constitutive expression in the absence of pathogens [78]–[80]. For example, nta-miR6019 (22-nt) and nta-miR6020 (21-nt) guide the cleavage of the TIR-NB-LRR N transcript from tobacco, which confers resistance to Tobacco mosaic virus [79]. Likewise, Sl-miR482 attenuates expression of a large family of NBS-LRR genes from tomato and its accumulation is decreased in plants infected with Turnip crinkle virus, Cucumber mosaic virus, Tobacco rattle virus and Pst DC3000 [80]. Therefore, given the above context, miRNA sequestration by P19 might generally enhance host immune responses induced by virulent and avirulent pathogens. Interestingly, however, miR168, which targets the antiviral silencing effector AGO1, is specifically not sequestered and, in fact, induced by P19, suggesting that, in this case, miRNA binding by P19 favours viral infection without activating immune responses [81]. We have shown here, with the P19-N. tabacum model, that the general scheme of silencing induction and suppression by plant viruses can be readily accommodated within the classical frame of ETI/PTI. In particular, our study sheds light on an additional layer of defense, whereby hosts can sense and respond to the damages caused by VSRs to the cellular silencing machinery. The existence of this additional layer is also consistent with the fast evolving and highly diverse nature of VSRs. Indeed, potent host counter-counter-defense measures probably impose strong selective pressure on pathogens to accelerate or refine the modeling of their virulence factors, thereby contributing further to the never-ending arms race opposing parasites to their hosts. A future challenge will be to assess the extent to which the phenomenon described here is shared not only among plant-virus, but also plant-bacteria, plant-fungal and plant-oomycetes interactions, and how elucidation of its biochemical and genetic underpinnings might improve our understanding of PTI and ETI at large. Finally, and most importantly, a strong -albeit still speculative- implication of our results is the existence of dedicated host-encoded R proteins that should monitor the status of key RNA silencing components in plants, and perhaps other organisms. Identifying these elusive silencing-associated R proteins and their guardees would certainly constitute a major breakthrough in the field. Materials and Methods Plant conditions and transgenic lines Wild type and transgenic plants were grown under conditions of 8 h darkness at 19°C, 16 h light at 22°C, with 70% relative humidity. Independent tobacco (N. tabacum) transgenic lines carrying the wild type P19 and its mutant P19W39-42R under Dex inducible promoter [59] were generated using the Agrobacterium tumefaciens leaf disc transformation method [82]. The disarmed pTA7001-Dex-P19, pTA7001-Dex-P19W39-42R were used for transformation. The transgenic plants generated were named Dex::P19 and Dex::P19W39-42R. Transient expression A. tumefaciens strains containing the constructs P19 [35], P25 [68], P15 [83], HcPro [67], P14 and P21 [45] were grown overnight at 28°C in Lauria Bertani (LB) broth supplemented with 50 µg/ml kanamycine, 10 µg/ml rifampicine and 25 µg/ml gentamycine. Bacterial cultures were then pelleted at 4 500× g for 15 min and the supernatant was discarded. Pellets were resuspended in 10 mM MgCl2 supplemented with 200 µM acetosyringone and brought to an OD 0.5. These bacterial suspensions were infiltrated in the plant leaves using a syringe. Co-agroinfiltration of mGFP and VSRs were done at 0.5 OD. Protein extraction and gel blot analysis For transgenic plants, a solution of 25 µg/ml Dexametasone supplemented with 0.1% v/v Silwet L-77 was sprayed onto leaves of 5 week-old transgenic plants. Samples were harvested at 0, 12, 24 and 48 hours after DEX application, immediately frozen in liquid nitrogen, and kept at −80°C before extraction. Total proteins were extracted from 100 to 200 mg of homogeny of frozen leave 200 µl of extraction buffer [25 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% glycérol, 5 mM dithiothreitol (DTT)] and a protease inhibitor cocktail (Sigma). The crude extract was centrifuged at 12000 g for 15 min. The supernatant was kept and total proteins were quantified by Bradford assay (Bio-Rad Laboratories, Ontario). Samples were diluted in Leammli buffer and boiled for 5 minutes before separation on 12% SDS-PAGE. 50 µg of proteins of each sample was used for Western analysis. Proteins were subjected to gel blot analysis using a rabbit polyclonal PR1, PR2 or PR3 antibodies, at a dilution of 1 : 8000 [84]. For detection of the GFP, a rabbit polyclonal IgG antibody was used at 1/3000 (GFP (FL), sc-8334, Santa Cruz Biotechnology). For detection of P19, we used an affinity purified rabbit polyclonal IgG antibody obtain from GeneScript and raised against a synthetic peptide of the P19 protein (GNDAREQANSERWDC). It was used at 1/300. Coomassie Blue or red ponceau staining were used to confirm equal protein loading. Horse Radish Peroxidase-conjugated anti-rabbit IgG was used as secondary antibody at 1 : 14500 (Sigma Aldrich). Immunodetection was conducted with chemiluminescent substrate (Bio-Rad, immun-star kit) followed by X-ray film exposure. Quantitative PCR Total RNA was extracted from tobacco tissues using the RNeasy Plant Mini Kit (Qiagen Science, Maryland, USA) according to the manufacturer's instructions. 2 µg of each RNA samples were reverse transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen). Samples were diluted 1/5 in DEPC water and qPCR was performed using POWER SYBR Green (Applied Biosystems, Warrington, UK) according to the manufacturer's instruction. Primers used were: qPCR NTAC1F 5′-CTGTACTACTCACTGAAGCACCTC-3, qPCR NTAC1R 5′- GGCGACATATCATAGCAGGA -3, qPCR P19F 5′-TTGGTTTCAAGGAAAGCTG-3, qPCR P19R 5′-GATCCAAGGACTCTGTGCA-3, qPCR1. Virus infections A. tumefaciens strains containing the constructs 35S::TBSV-GFP or 35S::TBSVΔP19-GFP (Kindly provided by Herman B. Scholthof [26]) were grown overnight at 28°C in Lauria Bertani (LB) broth supplemented with 50 µg/ml kanamycine, 10 µg/ml rifampicine and 25 µg/ml gentamincin. Bacterial cultures were then pelleted at 4 500× g for 15 min and the supernatant was discarded. Pellets were resuspended in 10 mM MgCl2 brought to 0.5 OD and supplemented with 200 µM acetosyringone. Bacterial suspensions were then incubated at room temperature for 1–3 hours before being infiltrated into young leaves of 5 week-old Nicotiana tabacum plants, using a syringe. Inoculated plants were grown under conditions of 8 h darkness at 18°C, 16 h light at 20°C with 70% relative humidity. Viral infection was monitored over time under U.V. illumination and samples were collected at 6 dpi, frozen in liquid nitrogen and kept at −80°C before extraction of protein or RNA. A. tumefaciens strain containing the construct 35S::PVX-GFP [85] was used for PVX assays. Infections were conducted as described above, except that the final OD used was 0.25. VSR were co-agroinfiltrated at final OD of 0.25. Northern analysis Total RNA was extracted using TRI reagent (Sigma), precipitated with isopropanol and the RNA pellet was resuspended in 50% deionized Formamide. Analysis was performed as described [66]. The signal was detected using X-ray films. Trypan blue staining Sample were boiled for 5 minutes in the staining solution [10 ml of lactic acid, 10 g of phenol, 10 ml of glycerol, 10 ml of water, 10 mg of trypan blue, mixed 1∶1 with ethanol]. Samples were then destained using chloral hydrate as previously described [86], [87]. Immunoprecipitation experiments The cassettes for transient expression of GFFG dsRNA and silencing suppressors have been described previously [66], [67]. Agrobacterium-mediated transient expression in N. benthamiana leaves was as described previously [88]. For immunoprecipitation experiments, 400 mg of frozen tissue harvested 5 days post-infiltration was ground in liquid nitrogen and homogenized in 3 ml/g of extraction buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% NP-40 and complete protease inhibitor cocktail (Roche) for 30 min at 4°C. Cell debris was removed by centrifugation at 12000 g at 4°C for 30 min. Extracts were pre-cleared by incubation with Protein A-agarose (Roche) at 4°C for 1 h. Pre-cleared extracts were then incubated with anti-HA polyclonal antibody (Sigma) and protein A-agarose overnight at 4°C. Immunoprecipitates were washed three times (15 min each) in extraction buffer. Aliquots of the inputs and immunoprecipitates were collected for protein blot analysis. For RNA analysis, immune complex were subjected to Tri-Reagent extraction (Sigma). Salicylic acid quantification An amount of one to one v/w of cold 80% MeOH was added to finely ground plant tissue (300–500 mg) for extraction of phenolic compounds. Samples were vortexed then shaken overnight at 4°C. The following morning, the samples were vortexed and centrifuged 16,000 g for 10 min. The supernatant was transferred to a new Eppendorf tube, filtered through a 0.22-µm syringe filter and 50 to 100 µl injected into HPLC. Samples were injected using Waters 2695 separation module (Waters Corp.) and a Lichrospher RP-18 (5 µm) column (4 mm×250 mm) at 30°C, and compounds detected with a Waters 996 diode array scanning 200 nm–400 nm, followed, in tandem, by a Waters 2475 Fluorescence detector, with an excitation wavelength of 290 nm emission and a scan of 300–500 nm. The maximum expected emission for free salicylic acid using this excitation wavelength was at 390–400 nm. The HPLC system was controlled and data analysed with the Empower2 software. Standard free salicylic acid (Sigma 84210) standards were prepared at 100 ng/ml, 250 ng/ml, 500 ng/ml, 1000 ng/ml and injected under the same conditions. The solvents were acidified water (solvent A: 0.1% Phosphoric acid in nanopure water) and acetonitrile HPLC grade (solvent B) with an elution flow rate of 1 mL/min. The gradient used was as follows: time (min)/%A/%B: 0/100/0, 5/95/5, 10/95/5, 14/90/10, 20/80/20, 23/80/20, 30/65/35, 35/65/35, 43/50/50, 48/25/75, 55/0/100 and 60/0/100. The injected volume was 50 µL for each sample. Three biological replicates for each treatment/time point were extracted and injected independently into the HPLC. Linear regressions were generated between compound concentration (independent variable) and peak areas (dependent variable). The equations obtained were used to calculate the concentration of each phenolic compound in the analyzed samples. Every sample was also spiked with 0.8 µg/ml free salicylic acid and injected independently to confirm the quantities determined by the software. Supporting Information Figure S1 P19-mediated accumulation of SA in N. tabacum requires its capacity to bind sRNAs. Five-week-old WT, Dex::P19 and Dex::P19W39-42 plants were sprayed with Dex and samples were harvested at 0 and 24 hours post treatment (hpt) for SA quantification. Error bars represent the SD (n = 3). Experiments were repeated two times and gave similar results. (TIF) Click here for additional data file. Figure S2 TBSV does not induce microscopic HR in N. tabacum . (A–B) Five week-old N. tabacum cv. Xanthi plants were transiently infiltrated with a solution of A. tumefaciens expressing either GUS, TBSV-GFP or P50 and 3 and 5 dpi leaves were stained with trypan blue to see macroscopic (A) and microscopic HR using an Zeiss Axioskop microscope (Zeiss AxioCam MRc with the Axiovision Rel. 4.8 program) under bright-field illumination (B). Experiments were repeated at least three times and gave similar results. (TIF) Click here for additional data file. Figure S3 P19-mediated, SA-dependent immune responses are compromised in NahG plants. Leaves of five weeks old tobacco wild type and NahG plants were transiently infiltrated with a solution of A. tumefaciens expressing P19 (0.25 OD in MgCl2 10 mM). The accumulation of acidic PR3 and PR5 proteins was monitored by western blot 3 days post agroinfiltration of both, wild type and nahG plants. Lower panel shows Ponceau Red staining of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) for confirmation of equal loading. Experiments were repeated three times and showed similar results. (TIF) Click here for additional data file. We thank Prof. David Baulcombe and Sainsbury Laboratory for providing suppressors of RNA silencing, Dr Chua for pTA plasmid, and Drs Dietrich and Linthorst, respectively, for seeds of NahG and ETR plants. We also thank Dr Kauffmann for providing antibodies for PR proteins, Dr Herman Scholtoff for TBSV constructs and Dr Moffett for PVX constructs. ==== Refs References 1 Jones JD , Dangl JL (2006 ) The plant immune system . Nature 444 : 323 –329 .17108957 2 Zipfel C (2009 ) Early molecular events in PAMP-triggered immunity . Curr Opin Plant Biol 12 : 414 –420 .19608450 3 Dodds PN , Rathjen JP (2010 ) Plant immunity: towards an integrated view of plant–pathogen interactions . Nat Rev Genet 11 : 539 –548 .20585331 4 van Loon LC , Rep M , Pieterse CMJ (2006 ) Significance of Inducible Defense-related Proteins in Infected Plants . Annu Rev Phytopathol 44 : 135 –162 .16602946 5 Bent AF , Mackey D (2007 ) Elicitors, effectors, and R genes: The new paradigm and a lifetime supply of questions . Annu Rev Phytopathol 45 : 399 –436 .17506648 6 Fritig B , Heitz T , Legrand M (1998 ) Antimicrobial proteins in induced plant defense . Curr Opin Immunol 10 : 16 –22 .9523105 7 Oh H-S , Park DH , Collmer A (2010 ) Components of the Pseudomonas syringae Type III Secretion System Can Suppress and May Elicit Plant Innate Immunity . Mol Plant Microbe Interact 23 : 727 –739 .20459312 8 Bendahmane A , Kanyuka K , Baulcombe DC (1999 ) The Rx Gene from Potato Controls Separate Virus Resistance and Cell Death Responses . Plant Cell 11 : 781 –792 .10330465 9 Kang B-C , Yeam I , Jahn MM (2005 ) Genetics of Plant Virus Resistance . Annu Rev Phytopathol 43 : 581 –621 .16078896 10 Eggenberger AL , Hajimorad MR , Hill JH (2008 ) Gain of Virulence on Rsv1-Genotype Soybean by an Avirulent Soybean mosaic virus Requires Concurrent Mutations in Both P3 and HC-Pro . Mol Plant Microbe Interact 21 : 931 –936 .18533833 11 Wen RH , Khatabi B , Ashfield T , Maroof MAS , Hajimorad MR (2013 ) The HC-Pro and P3 Cistrons of an Avirulent Soybean mosaic virus Are Recognized by Different Resistance Genes at the Complex Rsv1 Locus . Mol Plant Microbe Interact 26 : 203 –215 .23051173 12 Katiyar-Agarwal S , Morgan R , Dahlbeck D , Borsani O , Villegas A , et al (2006 ) A pathogen-inducible endogenous siRNA in plant immunity . Proc Natl Acad of Sci USA 103 : 18002 –18007 .17071740 13 Katiyar-Agarwal S , Jin H (2007 ) Discovery of Pathogen-Regulated Small RNAs in Plants . Methods Enzymol 215 –227 .17720487 14 Ding SW , Voinnet O (2007 ) Antiviral immunity directed by small RNAs . Cell 130 : 413 –426 .17693253 15 Navarro L , Dunoyer P , Jay F , Arnold B , Dharmasiri N , et al (2006 ) A Plant miRNA Contributes to Antibacterial Resistance by Repressing Auxin Signaling . Science 312 : 436 –439 .16627744 16 Katiyar-Agarwal S , Jin H (2010 ) Role of Small RNAs in Host-Microbe Interactions . Annu Rev Phytopathol 48 : 225 –46 .20687832 17 Zhang Y , Jiang W-k , Gao L-z (2011 ) Evolution of MicroRNA Genes in Oryza sativa and Arabidopsis thaliana: An Update of the Inverted Duplication Model . PLoS ONE 6 : e28073 .22194805 18 Hamilton AJ , Baulcombe DC (1999 ) A Species of Small Antisense RNA in Posttranscriptional Gene Silencing in Plants . Science 286 : 950 –952 .10542148 19 Elbashir SM , Lendeckel W , Tuschl T (2001 ) RNA interference is mediated by 21- and 22-nucleotides RNAs . Genes Dev 15 : 188 –200 .11157775 20 Bernstein E , Caudy AA , Hammond SM , Hannon GJ (2001 ) Role for a bidentate ribonuclease in the initiation step of RNA interference . Nature 409 : 363 –366 .11201747 21 Hammond SM , Bernstein E , Beach D , Hannon GJ (2000 ) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells . Nature 404 : 293 –296 .10749213 22 Deleris A , Gallego-Bartolome J , Bao J , Kasschau K , Carrington J , et al (2006 ) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense . Science 313 : 68 –71 .16741077 23 Qu F , Ye X , Morris JT (2008 ) Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1 . Proc Natl Acad Sci USA 105 : 14732 –14737 .18799732 24 Harvey JJ , Lewsey MG , Patel K , Westwood J , Heimstadt S , et al (2011 ) An antiviral defense role of AGO2 in plants . PLoS One 6 : e14639 .21305057 25 Jaubert M , Bhattacharjee S , Mello AFS , Perry KL , Moffett P (2011 ) ARGONAUTE2 Mediates RNA-Silencing Antiviral Defenses against Potato virus X in Arabidopsis . Plant Physiol 156 : 1556 –1564 .21576511 26 Scholthof HB , Alvarado VY , Vega-Arreguin JC , Ciomperlik J , Odokonyero D , et al (2011 ) Identification of an ARGONAUTE for Antiviral RNA Silencing in Nicotiana benthamiana . Plant Physiol 156 : 1548 –1555 .21606315 27 Wang XB , Jovel J , Udomporn P , Wang Y , Wu Q , et al (2011 ) The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana . Plant Cell 23 : 1625 –1638 .21467580 28 Garcia D , Garcia S , Pontier D , Marchais A , Renou Jean P , et al (2012 ) Ago Hook and RNA Helicase Motifs Underpin Dual Roles for SDE3 in Antiviral Defense and Silencing of Nonconserved Intergenic Regions . Mol Cell 48 : 109 –20 .22940249 29 Bucher E , Lohuis D , van Poppel PMJA , Geerts-Dimitriadou C , Goldbach R , et al (2006 ) Multiple virus resistance at a high frequency using a single transgene construct . J Gen Virol 87 : 3697 –3701 .17098987 30 Voinnet O (2009 ) Origin, biogenesis, and activity of plant microRNAs . Cell 136 : 669 –687 .19239888 31 Ruiz-Ferrer V , Voinnet O (2009 ) Roles of Plant Small RNAs in Biotic Stress Responses . annu rev plant biol 60 : 485 –510 .19519217 32 Anandalakshmi R , Pruss GJ , Ge X , Marathe R , Mallory AC , et al (1998 ) A viral suppressor of gene silencing in plants . Proc Natl Acad Sci USA 95 : 13079 –13084 .9789044 33 Brigneti G , Voinnet O , Li W-X , Ji L-H , Ding S-W , et al (1998 ) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana . EMBO J 17 : 6739 –6746 .9822616 34 Baulcombe DC , Molnar A (2004 ) Crystal structure of p19 - a universal suppressor of RNA silencing . Trends Biochem Sci 29 : 279 –281 .15276178 35 Voinnet O , Pinto YM , Baulcombe D (1999 ) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses . Proc Natl Acad Sci USA 96 : 14147 –14152 .10570213 36 Navarro L , Jay F , Nomura K , He SY , Voinnet O (2008 ) Suppression of the MicroRNA Pathway by Bacterial Effector Proteins . Science 321 : 964 –967 .18703740 37 Qiao Y , Liu L , Xiong Q , Flores C , Wong J , et al (2013 ) Oomycete pathogens encode RNA silencing suppressors . Nat Genet 45 : 330 –333 .23377181 38 Baumberger N , Tsai CH , Lie M , Havecker E , Baulcombe DC (2007 ) The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation . Curr Biol 17 : 1609 –1614 .17869110 39 Bortolamiol D , Pazhouhandeh M , Marrocco K , Genschik P , Ziegler-Graff V (2007 ) The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing . Curr Biol 17 : 1615 –1621 .17869109 40 Csorba T , Lozsa R , Hutvagner G , Burgyan J (2010 ) Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1 . Plant J 62 : 463 –472 .20128884 41 Azevedo J , Garcia D , Pontier D , Ohnesorge S , Yu A , et al (2010 ) Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein . Genes Dev 24 : 904 –915 .20439431 42 Schott G , Mari-Ordonez A , Himber C , Alioua A , Voinnet O , et al (2012 ) Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1 . EMBO J 31 : 2553 –2565 .22531783 43 Vargason J , Szittya G , Burgyan J , Tanaka Hall T (2003 ) Size selective recognition of siRNA by an RNA silencing suppressor . Cell 115 : 799 –811 .14697199 44 Lakatos L , Csorba T , Pantaleo V , Chapman E , Carrington J , et al (2006 ) Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors . EMBO J 25 : 2768 –2780 .16724105 45 Merai Z , Kerenyi Z , Kertesz S , Magna M , Lakatos L , et al (2006 ) Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing . J Virol 80 : 5747 –5756 .16731914 46 Csorba T , Bovi A , Dalmay T , Burgyan J (2007 ) The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways . J Virol 81 : 11768 –11780 .17715232 47 Hemmes H , Lakatos L , Goldbach R , Burgyán J , Prins M (2007 ) The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs . RNA 13 : 1079 –1089 .17513697 48 Ye K , Malinina L , Patel DJ (2003 ) Recognition of small interfering RNA by a viral suppressor of RNA silencing . Nature 426 : 874 –878 .14661029 49 Lakatos L , Szittya G , Silhavy D , Burgyan J (2004 ) Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses . EMBO J 23 : 876 –884 .14976549 50 Omarov R , Sparks K , Smith L , Zindovic J , Scholthof HB (2006 ) Biological Relevance of a Stable Biochemical Interaction between the Tombusvirus-Encoded P19 and Short Interfering RNAs . J Virol 80 : 3000 –3008 .16501109 51 Scholthof HB (2006 ) The Tombusvirus-encoded P19: from irrelevance to elegance . Nat Rev Micro 4 : 405 –411 . 52 Jay F , Wang Y , Yu A , Taconnat L , Pelletier S , et al (2011 ) Misregulation of AUXIN RESPONSE FACTOR 8 underlies the developmental abnormalities caused by three distinct viral silencing suppressors in Arabidopsis . PLoS Pathog 7 : e1002035 .21589905 53 Li H-W , Lucy AP , Guo H-S , Li W-X , Ji L-H , et al (1999 ) Strong host resistance targeted against a viral suppressor of the plant gene silencing defense mechanism . EMBO J 18 : 2683 –2691 .10329615 54 Scholthof H , Scholthof K , Jackson A (1995 ) Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector . Plant Cell 7 : 1157 –1172 .7549478 55 Chu M (2000 ) Genetic dissection of tomato bushy stunt virus p19-protein-mediated host-dependent symptom induction and systemic invasion . Virology 266 : 79 –87 .10612662 56 Angel CA , Schoelz JE (2013 ) A Survey of Resistance to Tomato bushy stunt virus in the Genus Nicotiana Reveals That the Hypersensitive Response Is Triggered by One of Three Different Viral Proteins . Mol Plant Microbe Interact 26 : 240 –248 .23075040 57 Angel CA , Hsieh Y-C , Schoelz JE (2011 ) Comparative Analysis of the Capacity of Tombusvirus P22 and P19 Proteins to Function as Avirulence Determinants in Nicotiana species . Mol Plant Microbe Interact 24 : 91 –99 .20977306 58 Choi CW , Qu F , Ren T , Ye X , Morris TJ (2004 ) RNA silencing-suppressor function of Turnip crinkle virus coat protein cannot be attributed to its interaction with the Arabidopsis protein TIP . J Gen Virol 85 : 3415 –3420 .15483259 59 Aoyama T , Chua N-H (1997 ) A glucocorticoid-mediated transcriptional induction system in transgenic plants . Plant J 11 : 605 –612 .9107046 60 Ji L-H , Ding S-W (2001 ) The Suppressor of Transgene RNA Silencing Encoded by Cucumber mosaic virus Interferes with Salicylic Acid-Mediated Virus Resistance . Mol Plant Microbe Interact 14 : 715 –724 .11386367 61 Alamillo JM , Saénz P , García JA (2006 ) Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco . Plant J 48 : 217 –227 .17018032 62 Friedrich L , Vernooij B , Gaffney T , Morse A , Ryals J (1995 ) Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene . Plant Mol Biol 29 : 959 –968 .8555459 63 Knoester M , van Loon LC , van den Heuvel J , Hennig J , Bol JF , et al (1998 ) Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi . Proc Natl Acad Sci USA 95 : 1933 –1937 .9465120 64 Cordelier S , de Ruffray P , Fritig B , Kauffmann S (2003 ) Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin . Plant Mol Biol 51 : 109 –118-118 .12602895 65 Ménard R , Alban S , de Ruffray P , Jamois F , Franz G , et al (2004 ) β-1,3 Glucan Sulfate, but Not β-1,3 Glucan, Induces the Salicylic Acid Signaling Pathway in Tobacco and Arabidopsis . Plant Cell 16 : 3020 –3032 .15494557 66 Himber C , Dunoyer P , Moissiard G , Ritzenthaler C , Voinnet O (2003 ) Transitivity-dependent and -independent cell-to-cell movement of RNA silencing . EMBO J 22 : 4523 –4533 .12941703 67 Dunoyer P , Lecellier CH , Parizotto EA , Himber C , Voinnet O (2004 ) Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing . Plant Cell 16 : 1235 –1250 .15084715 68 Voinnet O , Lederer C , Baulcombe DC (2000 ) A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana . Cell 103 : 157 –167 .11051555 69 Kim MG , Geng X , Lee SY , Mackey D (2009 ) The Pseudomonas syringae type III effector AvrRpm1 induces significant defenses by activating the Arabidopsis nucleotide-binding leucine-rich repeat protein RPS2 . Plant J 57 : 645 –653 .18980653 70 Dunoyer P , Brosnan CA , Schott G , Wang Y , Jay F , et al (2010 ) An endogenous, systemic RNAi pathway in plants . EMBO J 29 : 1699 –1712 .20414198 71 Oh JW , Kong Q , Song C , Carpenter CD , Simon AE (1995 ) Open reading frames of turnip crinkle virus involved in satellite symptom expression and incompatibility with Arabidopsis thaliana ecotype Dijon . Mol Plant Microbe Interact 8 : 979 –987 .8664506 72 Zhao Y , DelGrosso L , Yigit E , Dempsey DA , Klessig DF , et al (2000 ) The amino terminus of the coat protein of Turnip crinkle virus is the AVR factor recognized by resistant arabidopsis . Mol Plant Microbe Interact 13 : 1015 –1018 .10975658 73 Wang L-Y , Lin S-S , Hung T-H , Li T-K , Lin N-C , et al (2012 ) Multiple Domains of the Tobacco mosaic virus p126 Protein Can Independently Suppress Local and Systemic RNA Silencing . Mol Plant Microbe Interact 25 : 648 –657 .22324815 74 Chen H-Y , Yang J , Lin C , Yuan YA (2008 ) Structural basis for RNA-silencing suppression by Tomato aspermy virus protein 2b . EMBO Rep 9 : 754 –760 .18600235 75 Silhavy D , Molnar A , Lucioli A , Szittya G , Hornyik C , et al (2002 ) A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs . EMBO J 21 : 3070 –3080 .12065420 76 Papp I , Mette MF , Aufsatz W , Daxinger L , Schauer SE , et al (2003 ) Evidence for Nuclear Processing of Plant Micro RNA and Short Interfering RNA Precursors . Plant Physiol 132 : 1382 –1390 .12857820 77 Zhang X , Zhao H , Gao S , Wang W-C , Katiyar-Agarwal S , et al (2011 ) Arabidopsis Argonaute 2 Regulates Innate Immunity via miRNA393*-Mediated Silencing of a Golgi-Localized SNARE Gene, MEMB12 . Mol Cell 42 : 356 –366 .21549312 78 Zhai J , Jeong D-H , De Paoli E , Park S , Rosen BD , et al (2011 ) MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs . Genes Dev 25 : 2540 –2553 .22156213 79 Li F , Pignatta D , Bendix C , Brunkard JO , Cohn MM , et al (2012 ) MicroRNA regulation of plant innate immune receptors . Proc Natl Acad Sci USA 109 : 1790 –1795 .22307647 80 Shivaprasad PV , Chen H-M , Patel K , Bond DM , Santos BACM , et al (2012 ) A MicroRNA Superfamily Regulates Nucleotide Binding Site–Leucine-Rich Repeats and Other mRNAs . Plant Cell 24 : 859 –874 .22408077 81 Varallyay E , Valoczi A , Agyi A , Burgyan J , Havelda Z (2010 ) Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation . EMBO J 29 : 3507 –3519 .20823831 82 Horsh RB , Fry JE , Hoffman NL , Eichholtz D , Rogers SG , et al (1985 ) A simple and general method for transferring genes into plants . Science 227 : 1229 –1231 .17757866 83 Dunoyer P , Pfeffer S , Fritsch C , Hemmer O , Voinnet O , et al (2002 ) Identification, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus . Plant J 29 : 555 –567 .11874569 84 Cordelier S , de Ruffray P , Fritig B , Kauffmann S (2003 ) Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin . Plant Mol Biol 51 : 109 –118-118 .12602895 85 Peart JR , Cook G , Feys BJ , Parker JE , Baulcombe DC (2002 ) An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus . Plant J 29 : 569 –579 .11874570 86 Bouarab K , Melton R , Peart J , Baulcombe D , Osbourn A (2002 ) A saponin-detoxifying enzyme mediates suppression of plant defences . Nature 418 : 889 –892 .12192413 87 El Oirdi M , Bouarab K (2007 ) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogen Botrytis cinerea . New Phytol 175 : 131 –139 .17547673 88 Hamilton A , Voinnet O , Chappell L , Baulcombe D (2002 ) Two classes of short interfering RNA in RNA silencing . EMBO J 21 : 4671 –4679 .12198169	23785291	PMC3681747	CC BY	2021-01-05 12:54:30	yes	PLoS Pathog. 2013 Jun 13; 9(6):e1003435
==== Front Front Oncol Front Oncol Front. Oncol. Frontiers in Oncology 2234-943X Frontiers Media S.A. 23785667 10.3389/fonc.2013.00153 Oncology Review Article The Role of microRNAs in the Tumorigenesis of Ovarian Cancer Di Leva Gianpiero 1 Croce Carlo M. 1* 1Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, The Ohio State University Columbus, OH, USA Edited by: Angeles Alvarez Secord, Duke University Medical Center, USA Reviewed by: Angeles Alvarez Secord, Duke University Medical Center, USA; Reuven Reich, Hebrew University of Jerusalem, Israel Correspondence: Carlo M. Croce, Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Biological Research Tower, 400W 12th Avenue, Columbus, OH 43210, USA e-mail: carlo.croce@osumc.eduThis article was submitted to Frontiers in Women’s Cancer, a specialty of Frontiers in Oncology. 23 3 2013 13 6 2013 2013 3 15301 3 2013 29 5 2013 Copyright © 2013 Di Leva and Croce.2013This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Epithelial ovarian cancer (EOC) is a complex disease, with multiple histological subtypes recognized. There have been major advances in the understanding of the cellular and molecular biology of this human malignancy, however the survival rate of women with EOC has changed little since platinum-based-treatment was introduced more than 30 years ago. Since 2006, an increasing number of studies have indicated an essential role for microRNAs (miRNAs) in ovarian-cancer tumorigenesis. Several miRNA profiling studies have shown that they associate with different aspects of ovarian cancer (tumor subtype, stage, histological grade, prognosis, and therapy resistance) and pointed to a critical role for miRNAs in the pathogenesis and progression of EOC. In this review, we discuss the current data concerning the accumulating evidence of the modulated expression of miRNAs in EOC, their role in diagnosis, prognosis, and prediction of response to therapy. Given the heterogeneity of this disease, it is likely that increases in long-term survival might be also achieved by translating the recent insights of miRNAs involvement in EOC into novel targeted therapies that will have a major impact on the management of ovarian cancer. microRNAovarian cancernoncoding RNAmiRNA profilingmiRNA profiles ==== Body Introduction Among United States women, ovarian cancer is the sixth most common cancer and the second most common gynecologic cancer (after endometrial cancer) (Parkin et al., 2002). Ovarian-cancer occurs in 1 of 2500 postmenopausal women in the United States and accounts for 5–6% of all cancer-related deaths (Jemal et al., 2008). The 5-year survival rate of ovarian-cancer ranges from 30 to 90%, depending on the spread of disease at diagnosis. When ovarian cancer is diagnosed at early stages, the survival rate is close to 90%; unfortunately, the vast majority of patients is identified when they have late-stage disease (Goff et al., 2000). This is primarily because ovarian cancer has few early or specific symptoms shared with many more common gastrointestinal, genitourinary, and gynecological conditions and have not yet proved useful for early diagnosis. Patients diagnosed with advanced disease are managed with surgical cytoreduction and chemotherapy, but many experience resistance to chemotherapy and relapse, yielding an overall 5-year survival rate of 10–30%. At the cellular and molecular levels, ovarian cancers are remarkably heterogeneous (Boxes 1 and 2). The normal ovary is a complex tissue with several distinct components. Although ovarian cancers can develop from germ cells or granulosa-theca cells, more than 90% of ovarian cancers have an epithelial histology and are thought to arise from cells that cover the ovarian surface or that line subsurface inclusion cysts (Feeley and Wells, 2001). One of the major disappointments in the field of ovarian-cancer research is the failure of currently established therapies to induce a cure at diagnosis, even in chemosensitive tumors. Efforts have been made to cure ovarian cancer over the past decade using different classes of chemotherapeutic agents in various combinations, dosages, and schedules to overcome chemoresistance following front-line paclitaxel–platinum treatment. Our improved understanding of the underlying biology of ovarian tumor etiology and chemoresistance has led to the development of molecular targeted therapies. Many small-molecule inhibitors and monoclonal antibodies that target multiple crucial cancer characteristics, including cell growth and survival, angiogenesis, and metastases are now entering clinical trials (Figure 1). However, new efforts are needed to identify new and better markers/therapeutics to aid the diagnostic and curative process of ovarian cancer. Box 1 Ovarian-cancer subtypes. Despite the controversy surrounding the histogenesis of ovarian cancers, it is widely believed today that most OECs (90%) arise from the ovarian surface epithelium. OECs are now classified in several histologic subtypes with distinctive risk factors, genetic abnormalities, and oncologic pathways that partly determine biologic behavior, response to chemotherapy, and prognosis. OECs develop from simple flattened epithelial cells into four different main histotypes that resemble the well-differentiated cells of the fallopian tube (serous, the most common with 7 out of every 10 epithelial ovarian cancers), endometrium (endometrioid, 1 in 20 epithelial ovarian cancers), endocervix (mucinous, 1 in 10 epithelial ovarian cancers), and cells that form nests within the vagina (clear cells, the least common subtype of epithelial ovarian cancer with just 3 in 100 cases). A significant proportion of endometrioid carcinomas and clear-cell carcinomas is associated with preexisting endometriosis. Serous carcinoma can be classify in high-grade (HGSC) and low-grade tumors (LGSC). Most HGSCs are biologically aggressive neoplasms, and they often manifest at an advanced stage, with up to 85% of patients with ovarian serous carcinoma presenting with widespread peritoneal metastases. Up to 80% of HGSCs show initial response to platinum-based chemotherapy, but about 70% may demonstrate recurrence. LGSC behaves like a slow-growing indolent neoplasm (presumably due to lack of p53 mutations) and has a better prognosis. Unlike HGSCs, recurrent LGSCs have high extreme drug resistance to paclitaxel, carboplatin, and cisplatin (33–69% of cases), but low drug resistance to etoposide and doxorubicin. Endometrioid carcinomas are platinum-sensitive tumors and, owing to low-grade early-stage disease and better chemotherapy sensitivity, are associated with the most favorable prognosis among all subtypes of EOCs. Mucinous neoplasms account for 10–15% of all ovarian tumors and up to 80% of mucinous neoplasms are benign cystadenomas. Clear-cell carcinomas are biologically aggressive neoplasms despite their being diagnosed at early stages. In this case, recurrence following surgery is common. Clear-cells and mucinous cancers generally do not respond as well as serous and endometrioid cancers to platinum- and taxane-based chemotherapy. Box 2 Ovarian-cancer genomic alteration. At molecular levels, the gene expression profiles of the different ovarian-cancer histotypes correlate with their morphological counterparts in normal tissues. HGSC is frequently characterized by mutations or loss of heterozygosity of p53 and genetic-epigenetic silencing of BRCA genes (∼80% of cases). Instead, LGSCs are characterized by frequent mutations of K-ras, BRAF, and erb-B2 (HER-2) genes. In endometrioid carcinomas CTNNB1 mutations (38–50% of cases), PTEN mutations (20%), and microsatellite instability (up to 19%) are commonly seen. In mucinous carcinomas, K-ras mutations are common and constitute an early event in tumorigenesis. Moreover, HER-2 gene amplification is seen in 15–20% of tumors. Clear-cell carcinomas are the most common histologic subtype characterized by the highest frequency of PI3KCA mutations: 33% of these carcinomas demonstrate activating mutations of PI3KCA. Mutations involving the PIK3CA gene lead to activation of the PI3K/AKT pathway, resulting in improved cell survival and invasion. The different histotypes of EOCs have also been correlated with the abnormal re-expression of homeobox genes (HOX) that are normally expressed during the formation of gynecological organs. HOXA9 is highly expressed in serous, HOX 10 in endometrioid, and HOXA11 in mucinous ovarian cancer. The vast majority of OECs (90%) are sporadic, with the remainder being inherited as part of hereditary ovarian-cancer syndromes. Germline mutations of BRCA1 and BRCA2 tumor-suppressor genes are responsible for most hereditary ovarian cancers (90–95%). The lifetime risk of developing ovarian cancer is about 20–50% in patients carrying BRCA mutations. Figure 1 Signaling pathways and new therapeutic targets for ovarian cancer. Despite many efforts in these last few years, clinical responses to curative chemotherapy in ovarian cancer remain very low and have merely led to improvements in the survival of patients. Therefore, new different drugs and combination treatments have been identified and are now entering clinical trials to test new therapeutic possibilities. In the figure, we depicted in blue some of the most important new or promising clinical targets in ovarian cancers. Many other different therapeutic approaches have also been taken and here not depicted, such as new cytotoxic agents (trabectedin, patupilone, canfosfamide), anti-folate transporter 1 inhibitor (farletuzumab), PARP inhibitor (olaparib). MicroRNAs (miRNAs) are single-stranded RNAs (ssRNAs) ∼19–25 nt in length that negatively regulate gene expression by translation inhibition or messenger RNA (mRNA) degradation, through base-pairing to partially complementary sites on the target mRNAs, usually in the 3′ untranslated region (UTR) (Garofalo and Croce, 2011; Mendell and Olson, 2012). Bioinformatic analyses predict the existence of ∼1000 miRNAs in the human genome capable to regulate up to 60% of all human transcriptome. Due to their highly abundance, conservation and tissue specific expression miRNAs play key roles in almost all biological processes, including gene regulation, cell developmental control, and the development of various diseases, such as cancer. Dysregulation of miRNA expression appears to play a fundamental role in the onset, progression and dissemination of cancers, and replacement of down-regulated miRNAs in tumor cells results in a positive therapeutic response (Croce, 2009). Accumulating data have shown that miRNAs are also aberrantly expressed in ovarian cancer and can be associated with tumor subtypes, clinical outcomes, and chemoresistance, indicating that miRNAs may be involved in ovarian tumorigenesis and progression. In the following review, we will discuss recent progresses in understanding the functions of miRNAs in ovarian cancer and their possible use as diagnostic and prognostic markers, and eventually as new targets or tools of a specific therapy. MicroRNA in Cancer Over the last 10 years, we have seen an enormous number of studies concerning the role of miRNAs in cancer (8461 PubMed hits on February 2013). Despite the large variety both in terms of the diseases and the experimental approaches taken, many essential and general principles of miRNA activity in human cancer have been defined. miRNAs activity is firstly related to redundancy: each miRNA exerts its full functional effects by regulating hundreds or thousands of targets (Mendell and Olson, 2012). To complicate the scenario, many miRNAs exist in families with similar seed sequences, and many of them from different families are co-expressed as a large clusters to amplify and maximize their inhibitory activity. Redundancy can also be found at the level of the target mRNAs where different miRNAs may also repress the same target. The second important aspect of miRNA/cancer relationship has been revealed by the application of the high-throughput technologies which have allowed the identification of cancer-specific miRNA fingerprints in all type of human cancer (Calin and Croce, 2006; Croce, 2009). In general, the expression of miRNAs in malignant cells is significantly different from that of normal counterpart cells and facilitate the stratification of cancer and the identification of the tissue of origin for poorly differentiated tumors. Some of the most commonly dysregulated miRNAs in cancer are summarized in Table 1. The third essential aspect of miRNA implication in cancer is represented by the fact that miRNAs can be up- or down-regulated in malignancies and, therefore, respectively referred to as oncogenes or tumor suppressors, sometimes even if there is no evidence for their causative role in tumorigenesis (Esquela-Kerscher and Slack, 2006; Croce, 2009). The more characterized and first identified tumor-suppressor miRNA is represented by the miR-15/16 cluster, deleted in 68% of Chronic Lymphocytic Leukemia (CLL) patients carrying a 13q14.3 translocation (Calin et al., 2002). A mouse model that mimics the minimal deletion region 13q14.3 or that specifically deletes the miR-15a/16-1 cluster exhibited a full spectrum of CLL-associated phenotypes, consistent with the miR-15a/16-1 locus having a tumor-suppressor role in the B-cell lineage (Klein et al., 2010). An example of oncogenic miRNA is miR-155, which is overexpressed in different types of B-cell malignancies, including pediatric Burkitt’s lymphoma, Hodgkin’s lymphoma, aggressive CLL, and diffuse large B-cell lymphoma (DLBCL) (Croce, 2009). Transgenic expression of miR-155 is sufficient to initiate lymphomagenesis in mice (Costinean et al., 2006). Another aspect to be considered is the high context-dependent activity of miRNAs. The same miRNA can act as an oncogene in one type of cells and as a tumor suppressor in another due to different targets and mechanisms of action (Croce, 2009). Accordingly, this miRNA may be found upregulated in some cancer types, where it acts as oncogene, but downregulated in other cancers, indicative of tumor-suppressor function. An example is represented by miR-222/221 cluster that is overexpressed in breast, lung, or liver cancers where targets important tumor suppressors such as PTEN, p27, p57, while the same cluster is downregulated in erythroblastic leukemias where target c-KIT oncogene (Garofalo et al., 2012). The causes of the widespread misexpression of miRNAs in cancers can be explained by different mechanisms including: (a) chromosomal alterations of the miRNA genes, (b) DNA point mutations, (c) epigenetic mechanisms, (d) transcriptional modulation, or (e) genetic and epigenetic alterations in the transcriptional and post-transcriptional machinery responsible for miRNA production (Di Leva and Croce, 2010). Despite the large scientific interest for the understanding of miRNA dysregulation in cancer, the most daunting task is still the discovery of the biological functions of the dysregulated miRNAs in cancer. One main rule is emerged from the vast literature about the biological activity of miRNAs in cancer: oncogenic miRNAs repress the expression of tumor-suppressor genes, while tumor-suppressor miRNAs repress the expression of oncogenes (Esquela-Kerscher and Slack, 2006). For example, overexpression of the oncogene, miR-21, frequently highly expressed in solid and hematologic malignancies, represses strong tumor suppressors as PTEN or programed cell death 4 (PDCD4) while loss of the tumor-suppressor miR-15a/miR-16-1 in CLL induces the overexpression of the anti-apoptotic BCL2 (Cimmino et al., 2005; Meng et al., 2007; Asangani et al., 2008). Another common mechanism of miRNA activity in cancer cells is their involvement in negative and positive feedback loops. This is well illustrated by miR-146a, which is transactivated by the NF-κB pathway and negatively feeds back on this signaling cascade by targeting two upstream activators of the pathway, TRAF6 and IRAK1 (Taganov et al., 2006). Interestingly, deletion of miR-146a in mice results in increased activity of NF-κB in splenocytes and the consequent development of NF-κB-dependent myeloid sarcomas (Zhao et al., 2011). Finally, a particularly intriguing, but poorly understood, aspect of the biology of miRNAs is their presence in numerous body fluids, including serum, plasma, saliva, and amniotic fluid (Cortez et al., 2011). miRNAs in serum correlate with the presence of hematologic malignancies and solid tumors and have been reported to be of value for early detection of various types of cancer, preceding diagnosis by conventional methods (Boeri et al., 2011). The question of whether tumor-associated miRNAs detected in circulation results from tumor cell death and lyses, or instead from secretion by tumor cells remains unanswered. It has been reported that the profile of secreted miRNAs does not reflect the miRNA composition of the primary tumor, suggesting active regulation of miRNA release or cellular retention (Collino et al., 2010). Generally, miRNAs are present in biological fluids as lipoprotein complexes, small membranous vesicles known as exosomes or as non-vesicular Ago2 ribonucleoprotein complexes (Arroyo et al., 2011). Table 1 Common miRNAs altered in human cancers. MicroRNA Expression in cancer Function Mechanism of deregulation Targets Let-7a-2 Down in breast, lung, colon, ovarian, and stomach cancer Tumor suppressor Repressed by MYC KRAS, HMGA2, MYC, DICER, BCLXL, IMP-1, CDC34, IL6 miR-15/16 Down in CLL, prostate cancer, and pituitary adenomas Tumor suppressor Genomic loss, mutated, activated by p53 BCL2, COX2, CHECK1, CCNE1, CCND1, CCND2, BMI-1, FGF2, FGFR1, VEGF, VEGFR2, CDC25a miR-29 family Down in AML, CLL, lung and breast cancer, lymphoma, hepatocarcinoma, rhabdomyosarcoma Tumor suppressor Genomic loss, activated by p53, repressed by MYC CDK6, MCL1, TCL1, DNMT1, DNMT3a, DNMT3b miR-34 family Down in colon, lung, breast, kidney, and bladder cancer Tumor suppressor Repressed by MYC SIRT1, BCL2, NOTCH, HMGA2, MYC, MET, AXL. NANOG, SOX2, MYCN, SNAIL miR-26a Down in liver cancer Tumor suppressor Repressed by MYC CCND2, CCNE2 miR-200 family Down in aggressive breast and ovarian cancer Tumor suppressor Repressed by ZEB1/2 ZEB1, ZEB2, BMI-1, SUZ-12, FN1, LEPR, CTNNB1, JAG1, MALM2, MALM3, p38 alpha miR-155 Up in high risk CLL, AML, breast, lung, colon cancer, and lymphoma Oncogene Activated by NF-KB SOCS1, BACH1, MEIS1, ETS1, FOXO3A, hMSH2, hMSH6, hMLH1, SMAD5, WEE1, SHIP1, CEBPB miR-21 Up in lung, breast, pancreas stomach, ovary prostate cancer, and CLL, AML, glioblastoma, myeloma Oncogene Activated by IL6, GF1alpha PTEN, TPM1, PDCD4, SPRY1, TIMP3, RECK miR-221/-222 Up in invasive ductal carcinoma, lung cancer, hepatocellular carcinoma, papillary thyroid cancer Oncogene Activated by MET in lung cancer; repressed by ERalpha in breast; activated by PLZF in melanoma; activated by NF-Kb and cJun in prostate cancer and glioblastoma cells p27(Kip1), p57(Kip2), PTEN, TIMP3, FOXO3A, ERalpha, KIT, TRSP1, DICER, APAF1, PUMA, PTPμ miR-17/92 Up in lung, breast, colon Oncogene Activated by E2F1 and MYC PTEN, BIM, HIF1, PTPRO, p63, E2F2, E2F3, TSP-1, CTGF, p21(WAF1), JAK1, SMAD4, TGFbetaII, MnSOD, GPX2, TRXR2 miRNA as Diagnostic Tools in Ovarian Cancer Genome-wide miRNA profiling techniques have shown their utility to classify tumors based on their origin and differentiation state and to help in diagnosis and prognosis. As discussed above, several reports have already shown this miRNA feature for several tumor types. The first report of miRNA dysregulation in ovarian cancer came from our laboratory in 2007: miRs expression profiles were shown to discriminate between ovarian-cancer specimens and normal ovaries (Iorio et al., 2007) (Table 2). miR-200a and miR-141 were identified as highly upregulated in cancer, whereas miR-199a, miR-140, miR-145, and miR-125b1 were most significantly downregulated. In this study, specific miRNA deregulation was also used to differentiate the histological subtypes of ovarian carcinomas. For example, miR-200a and miR-200c were upregulated in all subtypes (mucinous, endometrioid, and clear cells), miR-200b and miR-141 were upregulated in serous as well as endometrioid carcinomas, and miR-21, miR-203, and miR-205 were upregulated only in endometrioid carcinomas. Indeed, miR-145 was downregulated in serous and clear-cell carcinomas, while miR-222 was downregulated in both endometrioid and clear-cell carcinomas. Later, Coukos laboratory highlighted a large down-regulation of miRNAs in ovarian-cancer cell lines and cancer specimens (Zhang et al., 2008). The authors identified a different expression of 44 miRNAs between early- and late-stage ovarian cancer with a complete down-regulation for all miRNAs in late-stage tumors. These signatures included three known tumor-suppressors miRNAs, mir-15a, mir-34a, and mir-34b. The authors also showed that the region containing the up-regulated miR-182 was amplified in 28.9% of ovarian carcinomas, whereas, miR-15a was deleted in 23.9% of ovarian carcinomas, as previously also shown for CLL (Calin et al., 2002). Further genome-wide miRNA studies were performed and all defined a large miRNA dysregulation in ovarian tumors. To date, Nam et al. (2008) identified 23 aberrantly expressed miRNAs in at least 60% of ovarian-cancer samples with miR-21 as the most upregulated (85% samples) and miR-125b (95% samples) most down-regulated miRNAs. Yang et al. (2008a) identified 36 miRNAs differentially expressed between normal ovarian cells and tumors, including miR-199a, miR-214, miR-200a which were found upregulated in 53, 56, and 43% tumor tissues respectively, and associated with high-grade and late-stage tumors. miR-100 was identified instead as downregulated in 76% of tumors. In contrast with these data, Eitan et al. (2009) identified miR-200a, miR-34a, and miR-449b as the most down-regulated miRNAs in the advanced (stage III) ovarian tumors with miR-200a associated in the early-stage disease to an improved overall survival. miR-200a was also identified as predictor of favorable outcome in another profiling study of a cohort of 55 advanced ovarian tumors (Hu et al., 2009). Another member of the miR-200 family, miR-200c, was also identified by Marchini et al. (2011) as associated with progression-free survival, overall survival, or both in multivariate analysis of stage I ovarian cancers. In a later study in 2011, The Cancer Genome Atlas (TCGA) provided the first comprehensive molecular classification of a large cohort of high-grade serous ovarian carcinomas by integrated analyses of multidimensional data, including miRNA expression profiles, to identify molecular abnormalities that influence ovarian-cancer pathophysiology, affect outcome, and constitute therapeutic targets. Transcriptomic analyses identified four molecular subtypes of ovarian cancer, not significantly associated to survival, named Immunoreactive, Differentiated, Proliferative, and Mesenchymal based on the enrichment of specific genes in the subtype. miRNA expression instead differentiated the tumor specimens in three main subtypes where miRNA subtype 1 overlapped with the mRNA Proliferative subtype and miRNA subtype 2–3 overlapped with the mRNA Mesenchymal subtype. The subtype 1 was associated with worse patient survival as compared to the other two subtypes. A recent elaboration of the TCGA data has been performed by Dr. Zhang laboratory (Yang et al., 2013). The authors showed that integrated analysis of miRNAs and transcriptome is able to group the transcriptional subtypes into two more clinically relevant subtypes, one mesenchymal and one epithelial. The analyses highlights the important role of a miRNA regulatory network consisting of eight key miRNAs for the mesenchymal subtype including miR-141 and miR-200, miR-29c, miR-101, miR-506, and miR-128. Table 2 miRNA profiling studies in human epithelial ovarian cancers. Reference Number of samples/subtypes Method of analyses Main findings Iorio et al. (2007) 15 Normals/69 tumors 31 Serous/8 endometrioid/4 clear cells/9 poorly differentiated/1 mucinous miRNA microarray Ovarian cancer-specific miRNA signature Subtypes specific miRNA signature Epigenetic mechanism responsible for their aberrant expression Yang et al. (2008a) 10 Tumors and 10 “normal” HIOSE cell line miRNA microarray Ovarian cancer-specific miRNA signature miR-214 induces cell survival and cisplatin resistance through targeting PTEN Laios et al. (2008) 3 Primary serous/3 recurrent serous tumors qRT-PCR miR-9 and miR-223 can be biomarkers in recurrent ovarian cancer Nam et al. (2008) 22 Serous tumors/8 normals miRNA microarray Ovarian cancer-specific miRNA signature Zhang et al. (2008) 106 Tumors 109 Tumors 76 Tumors 504 Tumors miRNA microarray, aCGH, affymetrix cDNA microarray, tissue array, miRNAs are downregulated in malignant transformation and tumor progression Genomic copy number loss and epigenetic silencing account for miRNA dysregulation 96 Tumors qPCR validation Dahiya et al. (2008) 34 Tumors and HOSE-B cell line miRNA microarray Ovarian cancer-specific miRNA signature Sorrentino et al. (2008) Drug-resistant vs. wild-type cancer cell lines miRNA microarray Paclitaxel and cisplatin resistance is associated with a specific miRNA fingerprint Yang et al. (2008b) 69 Tumors (42 sensitive/27 resistant) miRNA microarray Let-7i is a modulator of platinum-based chemotherapy Let-7i is a biomarker to predict chemotherapy response and survival Boren et al. (2009) 16 Ovarian cancer cell lines miRNA microarray miRNA signature associates to cell line drug response Wyman et al. (2009) 33 Tumors/HOSE-B cell line Deep sequencing Ovarian cancer-specific miRNA signature Subtypes specific miRNA signature Eitan et al. (2009) 19 Tumors (stage I)/38 tumors (stage III) miRNA microarray miRNA signature during progression miRNA expression associated with response to platinum-chemotherapy Hu et al. (2009) 55 Advanced-stage tumors miRNA microarray miR-200b-429 are biomarkers for ovarian cancer outcome Lee et al. (2009) 33 High-grade serous tumors 2 Low-grade serous tumors miRNA microarray No abnormalities in miRNA expression correlated to BRCA1/2 status 2 Serous borderline tumors miR-34c and miR-422b are prognostic biomarkers 3 Normal fallopian tubes Nagaraja et al. (2010) 10 Human clear-cell ovarian cancer cell lines and 1 normal ovarian surface epithelial cultures Deep sequencing Clear-cell ovarian cancer-specific miRNA signature miR-101 inhibits mTOR pathway and increases rapamycin sensitivity Creighton et al. (2010) 8 Serous tumors 4 Serous cancer cell lines 4 NOSE cell lines Deep sequencing miR-31 is downregulated in cancer Reduced levels of miR-31 are correlated with defects in the p53 pathway Vaksman et al. (2011) 21 Tumors (13 effusions/8 primary tumors) miRNA microarray miRNA signatures for the primary tumors and effusions Kim et al. (2010) 103 Tumors miRNA microarray miRNA signature is correlated with clinico-pathological parameters (subtype, grade, survival) Marchini et al. (2011) 144 Tumors (stage I) miRNA microarray Ovarian cancer-specific miRNA signature miR-200c is a predictor of survival and relapse Cancer Genome Atlas Research Network (2011) 489 Serous tumors miRNA microarray Global analyses of mRNA expression, miRNA expression, promoter methylation, and DNA copy number MicroRNAs signatures have demonstrated their potential as diagnostic tool also in the ability to predict the clinical response to chemotherapy. In 2008, again in collaboration with our laboratory, Dr. Zhang’s group identified the involvement of let-7i in response to cisplatin for the treatment of ovarian cancer (Yang et al., 2008b). Another study performed by Eitan et al. (2009) analyzed the miRNA correlation to platinum-based chemotherapy response in stage III patients. By comparing the miRNA expression between patients that achieved complete response with no recurrence within 6 months of the end of treatment and patients that progressed really rapidly after treatment, the authors identified seven miRNAs to be significantly differentially expressed, including hsa-miR-27a, 23a, miR-378. The differential expression of these three miRNAs between sensitive and resistant tumors was also observed in the subset of stage III patients treated by the combined paclitaxel/carboplatin treatment. Leskelä et al. (2010) demonstrated instead that miR-200 family showed a significant association with treatment response to paclitaxel–carboplatin regimen: women lacking complete response to paclitaxel–carboplatin regimen had tumors with significantly lower miR-200c levels than the ones who had achieved complete response; in addition, higher expression of miR-200c was associated with lower relapse/progression rates. Currently, a combined treatment of carboplatin/paclitaxel has been adapted as the standard treatment for women with advanced epithelial ovarian cancer (EOC) (Hiro et al., 2010). However, many patients with ovarian cancer which initially respond to chemotherapy eventually relapse with drug-resistant disease (Hassan et al., 2011). Acquired chemoresistance is a major obstacle for successful cancer treatment. Based on the correlation between drug response and miRNA expression, many studies have tried to manipulate the mechanisms responsible for chemoresistance by altering the levels of miRNAs. For example, Liu et al. (2012) have demonstrated that a chimera composed of a MUC1 aptamer and let-7i can efficiently deliver let-7i into paclitaxel/cisplatin resistant ovarian tumor cells and subsequently resensitize to the apoptotic role of the drugs the ovarian malignant cells. Prislei et al. (2013) showed that overexpression of miR-200c repressed the expression of level of class III β-tubulin (TUBB3), a factor associated with drug resistance and poor prognosis in ovarian cancer, and increased sensitivity to paclitaxel and cisplatin. Same results were also obtained by Cochrane et al. (2010) which showed that restoration of miR-200c increases sensitivity to microtubule-targeting agents and mitigates invasiveness by 85%. Two other studies reported that miR-182 and miR-125b conferred resistance to cisplatin, possibly by their anti-apoptotic activity due to the repression of two important tumor suppressors, PDCD4 and Bcl-2 antagonist killer 1 (Bak1), respectively (Kong et al., 2011; Wang et al., 2013). Because the majority of ovarian-cancer patients are diagnosed with advanced-stage disease, identification of early tumor is essential to improve prognosis and therapy. One of the best ways to early diagnose, aid prognosis, and predict therapeutic response is by using diagnostic or prognostic serum and tissue biomarkers. Unfortunately, there are not many reliable serum biomarkers for ovarian-cancer currently used in the clinic, and tissue-based markers require an invasive procedure to obtain samples. Therefore, new efforts are needed to identify new serum markers to aid in the screening process. The ability to profile miRNAs in circulation have shown a non-invasive opportunity to identify promising alternative approaches to the current strategies for ovarian-cancer surveillance and early diagnosis. In this context, by using serum samples from patients with various stages of ovarian cancer, Taylor and Gercel-Taylor (2008) showed that 46% of miRNAs in the exosomes were the same as those in the primary tumors. Twelve miRNAs were present at a higher proportion in malignant cells (es: mir-155, -29), while 31 were present at elevated levels exclusively in exosomes (es: miR-203, -205). A study from our laboratory identified 23 miRNAs differentially expressed in serum of patients with ovarian cancer; only 10 of these miRNAs were in common with miRNAs that have been previously published in the literature as part of the miRNA signatures of ovarian cancer (including mir-21) (Resnick et al., 2009). In another study, the levels of four miRNAs (miR-200a, b, c, and miR-182) were identified as differentially expressed between the serum of 28 patients with serous ovarian cancer and healthy age-matched volunteers (Kan et al., 2012). miR-200c was the most differentially expressed and a combination of miR-200b and miR-200c gave the best predictive power for serous ovarian cancer. A different approach was instead taken by Vaksman et al. (2011) who analyzed the miRNA expression profiles of primary ovarian cancers and effusions from patients with disease spread beyond the ovary in order to define a new parameter for metastatic dissemination. The authors identified three groups of miRNAs: (1) highly expressed in primary ovarian cancer and effusions; (2) overexpressed only in primary ovarian cancer; and (3) overexpressed only in effusions. miR-210, -182, and -99a were significantly overexpressed in effusions compared to primary tumors, whereas hsa-miR-145 was significantly overexpressed in primary carcinomas. Overall, the above studies provided useful biomarkers information for ovarian cancer. The large heterogeneity of the results, based on differences in the samples analyzed and technology applied, highlights that additional studies are needed to define predictive and reliable miRNA signatures that can find a clinico-pathological application. Cause of miRNA Dysregulation in Ovarian Cancer An important aspect of miRNA biology in ovarian cancer that has been taken in consideration during the last few years is the cause of the large dysregulation of miRNAs in cancer that profiling studies have shown. First, chromosomic alterations have been identified for several miRNA genes. As previously described, Coukos’s laboratory identified a consistent amplification of miR-182 region and deletion of miR-15 in ovarian carcinomas (Zhang et al., 2008). The same authors also showed a remarkably high frequency of miR-210 gene copy deletions in ovarian-cancer patients, and that gene copy number correlated with miR-210 expression levels (Giannakakis et al., 2008). Loss of heterozygosity at the let-7a-3/let-7b and mir-143/mir-145 loci was also detected in 50 and 22% of 90 ovarian carcinomas, respectively (Bearfoot et al., 2008). The authors analyzed also the somatic mutations status of a ∼500-bp genomic region surrounding 10 ovarian-cancer-implicated miRNA genes (let-7a-2, let-7a-3, let-7b, miR-10b, miR-125b-1, miR-125b-2, miR-143, miR-145, miR-200c, miR-206). Despite analyzing a large cohort of primary tumors, no somatic mutations were detected at the genomic regions corresponding to the primary, precursor, or mature miRNAs in any of the miRNA genes studied, suggesting that somatic mutations may not be a common mechanism of miRNA inactivation in ovarian cancer. Another mechanism that has been analyzed to explain the miRNA dysregulation in ovarian cancer is the altered levels or presence of mutations in the main enzymes of the miRNA biogenesis machinery, such as Dicer and Drosha. In fact, aberrations of the main proteins that are involved in miRNA processing will lead to changes in miRNA expression and ultimately to alterations of miRNA-mediated gene regulation. A work published in 2010 evaluated a decrease of 60 and 51% in the levels of Dicer and Drosha mRNAs, respectively, in 39% of ovarian-cancer tissues analyzed (Merritt et al., 2008). The authors also showed that, neither Dicer nor Drosha mRNA levels were significantly associated with age, tumor grade, or response to chemotherapy but low Dicer and Drosha mRNA levels were, however, significantly associated with advanced tumor stage or suboptimal cytoreductive surgery, respectively. Moreover, women whose tumor had low levels of Dicer and Drosha mRNA showed a reduced overall survival, indicating a clinical relevance for an alteration in the levels of Dicer and Drosha mRNAs in ovarian-cancer cells. Same conclusion were also obtained by another study performed by Pampalakis et al. (2010) who observed a marked down-regulation of Dicer expression in ovarian tumors of higher grade and higher stage. Unfortunately, the basis of the observed down-regulation of Dicer or Drosha expression in ovarian tumors is unknown. We may speculate that the presence of a large CpG island at Dicer genomic loci indicates that genomic DNA methylation may account for Dicer down-regulation in ovarian tumors, but further investigations are needed. An intriguing report came from Dr. Huntsman laboratory which discovered the presence of recurrent somatic missense mutations of DICER1 in non-epithelial ovarian tumors (Heravi-Moussavi et al., 2012). These mutations were predominantly restricted to a specific subset of non-epithelial ovarian tumors and highly prevalent (60%) in Sertoli–Leydig cell tumors. The recurrent and focal nature of these mutations and their restriction to non-epithelial ovarian tumors suggested a common oncogenic mechanism associated with a specific altered DICER1 function that is selected during tumor development in these cell types. The localized and focal pattern of mutations is typical of dominantly acting oncogenes, like KRAS and BRAF. The absence of loss of heterozygosity that is seen in association with germline DICER1 mutations provides further evidence against a role for DICER1 as either a haploinsufficient or a two-hit recessive tumor suppressor in this non-EOC. We can imagine that the new mutated DICER1 alleles produce viable protein that is able to create a different miRNA profile with oncogenic potentials. As well as for Dicer and Drosha, Vaksman, and collaborators suggested a role in tumor progression of ovarian cancer for the Ago family members, Ago1 and Ago2. Specifically, the authors identified an overexpression of Ago1 and Ago2 in the peritoneal effusions compared with primary ovarian carcinomas, and high levels of Ago2 mRNA in solid metastases compared with primary tumors Vaksman et al., 2012). Based on high-throughput results, Zhang et al. (2008) showed that if deletions occur in up to 15% of genomic loci harboring miRNAs that are downregulated, at least one-third of down-regulated miRNAs may be silenced by epigenetic alterations. Iorio et al. (2007) showed that 11 miRNAs, including miR-21, miR-203, miR-146b, miR-205, miR-30-5p, and miR-30c as the most significantly induced, are differentially expressed after treatment with the demethylating agent 5-AZA. Lu et al. (2007) showed that let-7a-3 is methylated in EOC, and low expression of let-7a is associated with poor prognosis. Epigenetic silencing of miR-130b through hypermethylation of the adjacent CpG island has been also identified and low expression of miR-130b was correlated to ovarian cancer with high stage and multidrug resistance (Yang et al., 2012). In fact, treatment of ovarian-cancer cells with demethylating agents increased miR-130b levels and decreased the IC50 of paclitaxel and cisplatin treatment. Complementary mechanisms, including transcriptional regulation, may also cooperate in miRNA deregulation of ovarian cancer. An important example of miRNA transcriptional control in ovarian cancer is represented by the miR-200 family, which has been shown highly modulated in ovarian cancer. The miR-200 family contains miR-200a, miR-200b, miR-200c, miR-141, and miR-429 which are arranged in two clusters in the human genome. miR-200a, miR-200b, and miR-429 are located on chromosome 1, while miR-200c and miR-141 are on chromosome 12. ZEB1/2, two transcription factors involved in the mediation of the epithelial to mesenchymal transition, can inhibit the expression of miR-200 family members by binding to the promoter of both miR-200 clusters thereby blocking transcription (Gregory et al., 2008). In turn, over expression of miR-200 family members repress ZEB1/2 levels, and leads to higher levels of E-cadherin and an epithelial phenotype (Burk et al., 2008). In fact, Park et al. (2008) have shown a positive correlation in the expression of E-cadherin with the expression of miR-200c in ovarian-cancer tissues. We can summarize that cancer cells after being triggered by molecular signaling, such as TGF-β or PDGF-D, increases their levels of ZEB1/2 which in turn decrease the expression of miR-200 and induce EMT. An interesting example of multi mechanism control of miRNA expression in ovarian cancer is represented by the miR-34 family. miR-34 clusters are part of the transcriptional program activated by p53 (He et al., 2007). Mutational and loss of TP53 function is one of the most frequent genetic abnormalities in ovarian cancer and is observed in 60–80% of both sporadic and familial cases (Bast et al., 2009). In accordance with the loss of TP53, miR-34 members (miR-34a, b, c) have been found strongly repressed in ovarian cancer: miR-34a expression is decreased in 100% and miR-34b*/c in 72% of EOC with p53 mutation (Corney et al., 2010). However, miR-34a was also downregulated in 93% of tumors with wild-type p53, indicating the presence of other mechanisms implicated in the suppression of miR-34a gene. In fact, the authors identified methylation and reduced copy number at the mir-34a gene in 27 and 39% of ovarian-cancer tissues, respectively. Conclusion In the next 5 years we could witness a very exciting periods for drug development in ovarian cancer since the introduction of cisplatin into chemotherapy. The discovery of aberrantly expressed miRNAs in ovarian cancer have defined new pathways in ovarian tumorigenesis and progression. miRNA expression profiles in tissues and biological fluids can potentially be used for the detection and surveillance of ovarian cancer. But miRNAs have also shown their efficacy in altering the sensitivity to traditional drugs. Therefore, targeted therapies should be accelerated by the delivery of specific miRNAs or miRNA inhibitors, which have been shown to be effective in ovarian-cancer cells. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ==== Refs References Arroyo J. D. Chevillet J. R. Kroh E. M. Ruf I. K. Pritchard C. C. Gibson D. F. (2011 ). Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma . Proc. Natl. Acad. Sci. U.S.A. 108 , 5003 –5008 .10.1073/pnas.1019055108 21383194 Asangani I. A. Rasheed S. A. Nikolova D. A. Leupold J. H. Colburn N. H. Post S. (2008 ). MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer . Oncogene 27 , 2128 –2136 .10.1038/sj.onc.1210856 17968323 Bast R. C. Jr.Hennessy B. Mills G. B. (2009 ). The biology of ovarian cancer: new opportunities for translation . Nat. Rev. Cancer 9 , 415 –428 .10.1038/nrc2644 19461667 Bearfoot J. L. Choong D. Y. Gorringe K. L. Campbell I. G. (2008 ). Genetic analysis of cancer-implicated microRNA in ovarian cancer . Clin. Cancer Res. 14 , 7246 –7250 .10.1158/1078-0432.CCR-08-1348 19010840 Boeri M. Verri C. Conte D. Roz L. Modena P. Facchinetti F. (2011 ). MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer . Proc. Natl. Acad. Sci. U.S.A. 108 , 3713 –3718 .10.1073/pnas.1100048108 21300873 Boren T. Xiong Y. Hakam A. Wenham R. Apte S. Chan G. (2009 ). MicroRNAs and their target messenger RNAs associated with ovarian cancer response to chemotherapy . Gynecol. Oncol. 113 , 249 –255 .10.1016/j.ygyno.2009.01.014 19237188 Burk U. Schubert J. Wellner U. Schmalhofer O. Vincan E. Spaderna S. (2008 ). A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells . EMBO Rep. 9 , 582 –589 .10.1038/embor.2008.74 18483486 Calin G. A. Croce C. M. (2006 ). MicroRNA signatures in human cancers . Nat. Rev. Cancer 6 , 857 –866 .10.1038/nrc1997 17060945 Calin G. A. Dumitru C. D. Shimizu M. Bichi R. Zupo S. Noch E. (2002 ). Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia . Proc. Natl. Acad. Sci. U.S.A. 99 , 15524 –15529 .10.1073/pnas.242606799 12434020 Cancer Genome Atlas Research Network . (2011 ). Integrated genomic analyses of ovarian carcinoma . Nature 474 , 609 –615 .10.1038/nature10166 21720365 Cimmino A. Calin G. A. Fabbri M. Iorio M. V. Ferracin M. Shimizu M. (2005 ). miR-15 and miR-16 induce apoptosis by targeting BCL2 . Proc. Natl. Acad. Sci. U.S.A. 102 , 13944 –13949 .10.1073/pnas.0506654102 16166262 Cochrane D. R. Howe E. N. Spoelstra N. S. Richer J. K. (2010 ). Loss of miR-200c: a marker of aggressiveness and chemoresistance in female reproductive cancers . J. Oncol. 2010 , 821717 .10.1155/2010/821717 20049172 Collino F. Deregibus M. C. Bruno S. Sterpone L. Aghemo G. Viltono L. (2010 ). Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs . PLoS ONE 5 :e11803 10.1371/journal.pone.0011803 20668554 Corney D. C. Hwang C. I. Matoso A. Vogt M. Flesken-Nikitin A. Godwin A. K. (2010 ). Frequent downregulation of miR-34 family in human ovarian cancers . Clin. Cancer Res. 16 , 1119 –1128 .10.1158/1078-0432.CCR-09-2642 20145172 Cortez M. A. Bueso-Ramos C. Ferdin J. Lopez-Berestein G. Sood A. K. Calin G. A. (2011 ). MicroRNAs in body fluids – the mix of hormones and biomarkers . Nat. Rev. Clin. Oncol. 8 , 467 –477 .10.1038/nrclinonc.2011.76 21647195 Costinean S. Zanesi N. Pekarsky Y. Tili E. Volinia S. Heerema N. (2006 ). Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice . Proc. Natl. Acad. Sci. U.S.A. 103 , 7024 –7029 .10.1073/pnas.0602266103 16641092 Creighton C. J. Benham A. L. Zhu H. Khan M. F. Reid J. G. Nagaraja A. K. (2010 ). Discovery of novel microRNAs in female reproductive tract using next generation sequencing . PLoS ONE 5 :e9637 10.1371/journal.pone.0009637 20224791 Croce C. M. (2009 ). Causes and consequences of microRNA dysregulation in cancer . Nat. Rev. Genet. 10 , 704 –714 .10.1038/nrg2634 19763153 Dahiya N. Sherman-Baust C. A. Wang T. L. Davidson B. Shih IeM. Zhang Y. (2008 ). MicroRNA expression and identification of putative miRNA targets in ovarian cancer . PLoS ONE 3 :e2436 10.1371/journal.pone.0002436 18560586 Di Leva G. Croce C. M. (2010 ). Roles of small RNAs in tumor formation . Trends Mol. Med. 16 , 257 –267 .10.1016/j.molmed.2010.04.001 20493775 Eitan R. Kushnir M. Lithwick-Yanai G. David M. B. Hoshen M. Glezerman M. (2009 ). Tumor microRNA expression patterns associated with resistance to platinum based chemotherapy and survival in ovarian cancer patients . Gynecol. Oncol. 114 , 253 –259 .10.1016/j.ygyno.2009.04.024 19446316 Esquela-Kerscher A. Slack F. J. (2006 ). Oncomirs – microRNAs with a role in cancer . Nat. Rev. Cancer 6 , 259 –269 .10.1038/nrc1840 16557279 Feeley K. M. Wells M. (2001 ). Precursor lesions of ovarian epithelial malignancy . Histopathology 38 , 87 10.1046/j.1365-2559.2001.01042.x 11207821 Garofalo M. Croce C. M. (2011 ). MicroRNAs: master regulators as potential therapeutics in cancer . Annu. Rev. Pharmacol. Toxicol. 51 , 25 –43 .10.1146/annurev-pharmtox-010510-100517 20809797 Garofalo M. Quintavalle C. Romano G. Croce C. M. Condorelli G. (2012 ). miR221/222 in cancer: their role in tumor progression and response to therapy . Curr. Mol. Med. 12 , 27 –33 .10.2174/156652412798376170 22082479 Giannakakis A. Sandaltzopoulos R. Greshock J. Liang S. Huang J. Hasegawa K. (2008 ). miR-210 links hypoxia with cell cycle regulation and is deleted in human epithelial ovarian cancer . Cancer Biol. Ther. 7 , 255 –264 .10.4161/cbt.7.2.5297 18059191 Goff B. A. Mandel L. Muntz H. G. Melancon C. H. (2000 ). Ovarian carcinoma diagnosis . Cancer 89 , 2068 –2075 .10.1002/1097-0142(20001115)89:10<2068::AID-CNCR6>3.0.CO;2-Z 11066047 Gregory P. A. Bert A. G. Paterson E. L. Barry S. C. Tsykin A. Farshid G. (2008 ). The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1 . Nat. Cell Biol. 10 , 593 –601 .10.1038/ncb1722 18376396 Hassan M. K. Watari H. Christenson L. Bettuzzi S. Sakuragi N. (2011 ). Intracellular clusterin negatively regulates ovarian chemoresistance: compromised expression sensitizes ovarian cancer cells to paclitaxel . Tumour Biol. 32 , 1031 –1047 .10.1007/s13277-011-0207-0 21761117 He L. He X. Lim L. P. de Stanchina E. Xuan Z. Liang Y. (2007 ). A microRNA component of the p53 tumour suppressor network . Nature 447 , 1130 –1134 .10.1038/nature05939 17554337 Heravi-Moussavi A. Anglesio M. S. Cheng S. W. Senz J. Yang W. Prentice L. (2012 ). Recurrent somatic DICER1 mutations in nonepithelial ovarian cancers . N. Engl. J. Med. 366 , 234 –242 .10.1056/NEJMoa1102903 22187960 Hiro J. Inoue Y. Toiyama Y. Yoshiyama S. Tanaka K. Mohri Y. (2010 ). Possibility of paclitaxel as an alternative radiosensitizer to 5-fluorouracil for colon cancer . Oncol. Rep. 24 , 1029 –1034 .20811685 Hu X. Macdonald D. M. Huettner P. C. Feng Z. El Naqa I. M. Schwarz J. K. (2009 ). A miR-200 microRNA cluster as prognostic marker in advanced ovarian cancer . Gynecol. Oncol. 114 , 457 –464 .10.1016/j.ygyno.2009.05.022 19501389 Iorio M. V. Visone R. Di Leva G. Donati V. Petrocca F. Casalini P. (2007 ). MicroRNA signatures in human ovarian cancer . Cancer Res. 67 , 8699 –8707 .10.1158/0008-5472.CAN-07-1936 17875710 Jemal A. Siegel R. Ward E. Hao Y. Xu J. Murray T. (2008 ). Cancer statistics . CA Cancer J. Clin. 58 , 71 –96 .10.3322/CA.2007.0010 18287387 Kan C. W. Hahn M. A. Gard G. B. Maidens J. Huh J. Y. Marsh D. J. (2012 ). Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer . BMC Cancer 12 :627 .10.1186/1471-2407-12-627 23272653 Kim T. H. Kim Y. K. Kwon Y. Heo J. H. Kang H. Kim G. (2010 ). Deregulation of miR-519a, 153, and 485-5p and its clinicopathological relevance in ovarian epithelial tumours . Histopathology. 57 , 734 –743 .10.1111/j.1365-2559.2010.03686.x 21083603 Klein U. Lia M. Crespo M. Siegel R. Shen Q. Mo T. (2010 ). The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia . Cancer Cell 17 , 28 –40 .10.1016/j.ccr.2009.11.019 20060366 Kong F. Sun C. Wang Z. Han L. Weng D. Lu Y. (2011 ). miR-125b confers resistance of ovarian cancer cells to cisplatin by targeting pro-apoptotic Bcl-2 antagonist killer 1 . J. Huazhong Univ. Sci. Technol. Med. Sci. 31 , 543 –549 .10.1007/s11596-011-0487-z 21823019 Laios A. O’Toole S. Flavin R. Martin C. Kelly L. Ring M. (2008 ). Potential role of miR-9 and miR-223 in recurrent ovarian cancer . Mol. Cancer 7 :35 10.1186/1476-4598-7-35 18442408 Lee C. H. Subramanian S. Beck A. H. Espinosa I. Senz J. Zhu S. X. (2009 ). MicroRNA profiling of BRCA1/2 mutation-carrying and non-mutation-carrying high-grade serous carcinomas of ovary . PLoS ONE 4 :e7314 10.1371/journal.pone.0007314 19798417 Leskelä S. Leandro-García L. J. Mendiola M. Barriuso J. Inglada-Pérez L. Muñoz I. (2010 ). The miR-200 family controls beta-tubulin III expression and is associated with paclitaxel-based treatment response and progression-free survival in ovarian cancer patients . Endocr. Relat. Cancer 18 , 85 –95 .10.1677/ERC-10-0148 21051560 Liu N. Zhou C. Zhao J. Chen Y. (2012 ). Reversal of paclitaxel resistance in epithelial ovarian carcinoma cells by a MUC1 aptamer-let-7i chimera . Cancer Invest. 30 , 577 –582 .10.3109/07357907.2012.707265 22812695 Lu L. Katsaros D. de la Longrais I. A. Sochirca O. Yu H. (2007 ). Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis . Cancer Res. 67 , 10117 –10122 .10.1158/0008-5472.CAN-07-2544 17974952 Marchini S. Cavalieri D. Fruscio R. Calura E. Garavaglia D. Nerini I. F. (2011 ). Association between miR-200c and the survival of patients with stage I epithelial ovarian cancer: a retrospective study of two independent tumour tissue collections . Lancet Oncol. 12 , 273 –285 .10.1016/S1470-2045(11)70012-2 21345725 Mendell J. T. Olson E. N. (2012 ). MicroRNAs in stress signaling and human disease . Cell 148 , 1172 –1187 .10.1016/j.cell.2012.02.005 22424228 Meng F. Henson R. Wehbe-Janek H. Ghoshal K. Jacob S. T. Patel T. (2007 ). MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer . Gastroenterology 133 , 647 –658 .10.1053/j.gastro.2007.05.022 17681183 Merritt W. M. Lin Y. G. Han L. Y. Kamat A. A. Spannuth W. A. Schmandt R. (2008 ). Dicer, Drosha, and outcomes in patients with ovarian cancer . N. Engl. J. Med. 359 , 2641 –2650 .10.1056/NEJMoa0803785 19092150 Nagaraja A. K. Creighton C. J. Yu Z. Zhu H. Gunaratne P. H. Reid J. G. (2010 ). A link between mir-100 and FRAP1/mTOR in clear cell ovarian cancer . Mol. Endocrinol. 24 , 447 –463 .10.1210/me.2009-0295 20081105 Nam E. J. Yoon H. Kim S. W. Kim H. Kim Y. T. Kim J. H. (2008 ). MicroRNA expression profiles in serous ovarian carcinoma . Clin. Cancer Res. 14 , 2690 –2695 .10.1158/1078-0432.CCR-07-1731 18451233 Pampalakis G. Diamandis E. P. Katsaros D. Sotiropoulou G. (2010 ). Down-regulation of dicer expression in ovarian cancer tissues . Clin. Biochem. 43 , 324 –327 .10.1016/j.clinbiochem.2009.09.014 19782670 Park S. M. Gaur A. B. Lengyel E. Peter M. E. (2008 ). The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2 . Genes Dev. 22 , 894 –907 .10.1101/gad.1640608 18381893 Parkin D. M. Bray F. Ferlay J. Pisani P. (2002 ). Global cancer statistics . CA Cancer J. Clin. 55 , 74 –108 .10.3322/canjclin.55.2.74 15761078 Prislei S. Martinelli E. Mariani M. Raspaglio G. Sieber S. Ferrandina G. (2013 ). miR-200c and HuR in ovarian cancer . BMC Cancer 13 :72 .10.1186/1471-2407-13-72 23394580 Resnick K. E. Alder H. Hagan J. P. Richardson D. L. Croce C. M. Cohn D. E. (2009 ). The detection of differentially expressed microRNAs from the serum of ovarian cancer patients using a novel real-time PCR platform . Gynecol. Oncol. 112 , 55 –59 .10.1016/j.ygyno.2008.08.036 18954897 Sorrentino A. Liu C. G. Addario A. Peschle C. Scambia G. Ferlini C. (2008 ). Role of microRNAs in drug-resistant ovarian cancer cells . Gynecol. Oncol. 111 , 478 –486 .10.1016/j.ygyno.2008.08.017 18823650 Taganov K. D. Boldin M. P. Chang K. J. Baltimore D. (2006 ). NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses . Proc. Natl. Acad. Sci. U.S.A. 103 , 12481 –12486 .10.1073/pnas.0605298103 16885212 Taylor D. D. Gercel-Taylor C. (2008 ). MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer . Gynecol. Oncol. 110 , 13 –21 .10.1016/j.ygyno.2008.04.033 18589210 Vaksman O. Hetland T. E. Trope’ C. G. Reich R. Davidson B. (2012 ). Argonaute, Dicer, and Drosha are up-regulated along tumor progression in serous ovarian carcinoma . Hum. Pathol. 43 , 2062 –2069 .10.1016/j.humpath.2012.02.016 22647351 Vaksman O. Stavnes H. T. Kaern J. Trope C. G. Davidson B. Reich R. (2011 ). miRNA profiling along tumour progression in ovarian carcinoma . J. Cell. Mol. Med. 15 , 1593 –1602 .10.1111/j.1582-4934.2010.01148.x 20716115 Wang Y. Q. Guo R. D. Guo R. M. Sheng W. Yin L. R. (2013 ). MicroRNA-182 promotes cell growth, invasion and chemoresistance by targeting programmed cell death 4 (PDCD4) in human ovarian carcinomas . J. Cell. Biochem. 114 , 1464 –1473 .10.1002/jcb.24488 23296900 Wyman S. K. Parkin R. K. Mitchell P. S. Fritz B. R. O’Briant K. Godwin A. K. (2009 ). Repertoire of microRNAs in epithelial ovarian cancer as determined by next generation sequencing of small RNA cDNA libraries . PLoS ONE 4 :e5311 .10.1371/journal.pone.0005311 19390579 Yang C. Cai J. Wang Q. Tang H. Cao J. Wu L. (2012 ). Epigenetic silencing of miR-130b in ovarian cancer promotes the development of multidrug resistance by targeting colony-stimulating factor 1 . Gynecol. Oncol. 124 , 325 –334 .10.1016/j.ygyno.2011.10.013 22005523 Yang D. Sun Y. Hu L. Zheng H. Ji P. Pecot C. V. (2013 ). Integrated analyses identify a master microRNA regulatory network for the mesenchymal subtype in serous ovarian cancer . Cancer Cell 23 , 186 –199 .10.1016/j.ccr.2012.12.020 23410973 Yang H. Kong W. He L. Zhao J. J. O’Donnell J. D. Wang J. (2008a ). MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN . Cancer Res. 68 , 425 –433 .10.1158/0008-5472.CAN-07-2488 18199536 Yang N. Kaur S. Volinia S. Greshock J. Lassus H. Hasegawa K. (2008b ). MicroRNA microarray identifies Let-7i as a novel biomarker and therapeutic target in human epithelial ovarian cancer . Cancer Res. 68 , 10307 –10314 .10.1158/0008-5472.CAN-08-1954 19074899 Zhang L. Volinia S. Bonome T. Calin G. A. Greshock J. Yang N. (2008 ). Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer . Proc. Natl. Acad. Sci. U.S.A. 105 , 7004 –7009 .10.1073/pnas.0801615105 18458333 Zhao J. L. Rao D. S. Boldin M. P. Taganov K. D. O’Connell R. M. Baltimore D. (2011 ). NF-kappaB dysregulation in microRNA-146a-deficient mice drives the development of myeloid malignancies . Proc. Natl. Acad. Sci. U.S.A. 108 , 9184 –9189 .10.1073/pnas.1105398108 21576471	23785667	PMC3682193	CC BY	2021-01-06 07:44:55	yes	Front Oncol. 2013 Jun 13; 3:153
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23840605PONE-D-12-3800710.1371/journal.pone.0067149Research ArticleBiologyMolecular Cell BiologySignal TransductionSignaling CascadesAkt Signaling CascadeMedicineDrugs and DevicesDrug Research and DevelopmentDrug DiscoveryOncologyCancers and NeoplasmsGenitourinary Tract TumorsProstate CancerBreast TumorsBasic Cancer ResearchFunctional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines Cell-Specific Role of ILK as PDK2 in Cancer CellsLee Su-Lin 1 ¤ Chou Chih-Chien 1 Chuang Hsiao-Ching 1 Hsu En-Chi 1 Chiu Po-Chen 1 Kulp Samuel K. 1 Byrd John C. 1 2 Chen Ching-Shih 1 3 * 1 Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America 2 Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, United States of America 3 Institute of Basic Medical Sciences, National Cheng-Kung University, Tainan, Taiwan Agoulnik Irina U. Editor Florida International University, United States of America * E-mail: chen.844@osu.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SLL CCC SKK JCB CSC. Performed the experiments: SLL CCC HCC ECH PCC. Analyzed the data: SLL CCC HCC SKK JCB CSC. Wrote the paper: CSC SKK. ¤ Current address: Laboratory of Bioorganic Chemistry, Natural Products Chemistry Section, NIDDK, National Institutes of Health, Bethesda, Maryland, United States of America 2013 19 6 2013 8 6 e671496 12 2012 13 5 2013 © 2013 Lee et al2013Lee et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression. This work was supported by Public Health Service Grants R01CA112250 from the National Cancer Institute, the Stefanie Spielman Fund for Breast Cancer Research to C.S.C, and a Specialized Center of Research grant from the Leukemia and Lymphoma Society to C.S.C. and J.C.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The phosphatidylinositol-3-kinase (PI3K)/Akt signaling axis plays a pivotal role in regulating multiple cellular events including cell growth, survival, metabolism, and motility through the modulation of a plethora of downstream effectors. In response to growth factor or cytokine stimulation, activated PI3K facilitates the production of phosphatidylinositol 3,4,5-trisphosphate, leading to the membrane recruitment and subsequent activating phosphorylation of Akt at Thr308 and Ser473 by phosphoinositide-dependent kinase (PDK)1 and PDK2, respectively. In contrast to the well-characterized PDK1 [1], the molecular identity of PDK2 remains elusive [2]. Although recent evidence has demonstrated that the rictor-mTOR complex (mTORC2) acts as the PDK2 in many types of nonmalignant and tumor cells [3], [4], a number of other kinases have also been implicated in mediating Akt-Ser473 phosphorylation in different cell types [2]. These Ser-473-Akt kinases include integrin-linked kinase (ILK) [5], [6], [7], MAPKAP kinase (MK)2 [8], DNA-dependent kinase (DNA-PK) [9], ataxia telangiectasia mutated (ATM) [10], protein kinase C (PKC)α [11], PKCβII [12], and p21-activated kinase (PAK)1 and PAK2 [13]. Among these putative PDK2s, ILK has received much attention in light of the mechanistic link between aberrant ILK upregulation and tumor progression in many types of human malignancies including those of breast, colon, liver, ovary, pancreas, prostate, stomach, and thyroid [14], [15], [16], [17], [18], [19], [20], [21]. In addition to its ability to mediate the phosphorylation of Akt and glycogen synthase kinase (GSK)3β [5], [6], [7], [22], ILK has been shown to serve as a scaffold protein linking integrins with the actin cytoskeleton [23], and to mediate growth factor/integrin-induced activation of ERKs [24], [25], [26], [27] or p38 [28], [29], [30], [31]. Equally important, ILK exhibits a unique ability to modulate the expression of growth factor receptors, including human epidermal growth factor receptor (HER)2 and epidermal growth factor receptor (EGFR), through the oncoprotein Y box-binding protein (YB)-1 [32], providing a link with growth factor receptor signaling. However, despite recent advances in understanding the tumor-promoting function of ILK, an issue that remains in dispute is whether ILK has kinase activity [33], [34]. For example, genetic studies in various nonmalignant cell types, including chondrocytes [35], fibroblasts [36], and keratinocytes [37], and, more recently, in mice [38] indicate that ILK deletion or mutation did not alter Akt or GSK-3β phosphorylation. In contrast, other studies have demonstrated the suppressive effect of targeted ILK excision on Akt-Ser473 phosphorylation in macrophages [22], the heart [39], skeletal muscle [40], and the peripheral nervous system [41]. Moreover, siRNA-mediated silencing of ILK in MDA-MB-231, PC-3, and other cell lines examined resulted in inhibition of Ser473-Akt phosphorylation and induction of apoptosis [42], [43], and the small-molecule inhibitors of ILK, QLT0267 [21], [32], [42], [43], [44], [45], [46], [47], [48], [49], [50] and T315 [compound 22 in ref. [51]], exhibited in vitro and/or in vivo antitumor efficacy in various types of cancer cells, in part, by targeting Akt activation. Equally important, recent evidence indicates that ILK forms complexes with rictor in PC-3 and MDA-MB-231 cells, and that this complex formation might play a role in regulating the ability of ILK to promote Akt phosphorylation and cancer cell survival and aggressive phenotype [42], [52]. Together, these seemingly contradictory data raise a possibility that ILK is responsible for Ser473-Akt phosphorylation in a cell line- and/or cellular context-specific manner. In this study, we used small-molecule inhibitors and genetic knockdown to examine the role of mTORC2 versus ILK as the PDK2 in PTEN-negative LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cell lines. As Akt phosphorylation is constitutively upregulated in these cell lines, they provided a suitable model to study the regulation of Ser473-Akt phosphorylation independent of growth factor or other external stimuli. Evidence indicates that, while mTORC2 acts as the PDK2 in LNCaP cells, ILK plays a major role in facilitating Ser473-Akt phosphorylation in PC-3 and MDA-MB-468 cells. In addition, we obtained evidence that ILK formed complexes with rictor in PC-3 and MDA-MB-468, but not in LNCaP cells, and that both mTORC2 and ILK might play a role in mediating epidermal growth factor (EGF)-induced Ser473-Akt phosphorylation in PTEN-positive MDA-MB-231 cells. Together, these data indicate the complexity in the cellular regulation of Akt activation in different cell lines. Materials and Methods Cell culture and reagents LNCaP and PC-3 prostate cancer and MDA-MB-468 breast cancer cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA), and maintained at 37°C in a humidified incubator with 5% CO2 in the following culture media: PC-3 and LNCaP, RPMI 1640; MDA-MB-468, DMEM, all of which contained 10% fetal bovine serum (FBS). T315, an ILK inhibitor recently developed in the authors’ laboratory, was synthesized according to an established procedure [51], and its identity and purity were confirmed by nuclear magnetic resonance spectroscopy (300 MHz), high-resolution mass spectrometry, and elemental analysis. Ku-0063794 and doxycycline were purchased from Chemdea (Ridgewood, NJ) and Sigma-Aldrich (St. Louis, MO), respectively. For in vitro studies, stock solutions of T315 and Ku-0063794 were made in DMSO and diluted in culture medium to a final DMSO concentration of 0.1%. Antibodies against various target proteins were purchased from the following commercial sources: Akt, p-473S-Akt, p-308T-Akt, GSK-3β, p-9S-GSK-3β, ILK, p70S6K, p-389T-p70S6K, mTOR, rictor, raptor, ATM, DNA-PK, PAK1, PAK2 (Cell Signaling Technology, Inc., Danvers, MA); MK2, PKCα, PKCβII (Santa Cruz Biotechnology, Inc, Santa Cruz, CA); β-actin (MP Biomedicals, Irvine, CA). shRNA for ILK in the pLKO.1 vector was purchased from Sigma-Aldrich. Human ILK full-length cDNA in the pCMV-SPORT6 vector and lentiviral pTRIPz vectors encoding Tet/ON inducible control shRNA or ILK shRNA with a red fluorescent protein (RFP) reporter gene (V2THS_48753) were purchased from Thermo Scientific (Rockford, IL). Control siRNA and siRNAs for ILK, ATM, PAK1, and PAK2 were purchased from Cell Signaling Technology, Inc. The siRNA for MK2, DNA-PK, PKCα, and PKCβII were purchased from Santa Cruz Biotechnology, Inc. The shRNA for rictor in the pLKO.1 vector was purchased from Addgene (Cambridge, MA). Cell viability assay Effect of test agents on cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in six replicates. Cells were seeded and incubated in 96-well plates in the respective medium with 10% FBS for 24 h, and then exposed to various concentrations of test agents dissolved in DMSO in 5% FBS-supplemented medium. The medium was removed from each well and replaced by 200 µl of 0.5 mg/ml MTT in 10% FBS-containing medium, and cells were incubated in the CO2 incubator at 37°C for 2 h. Supernatants were removed from the wells, and the MTT dye was dissolved in 120 µl/well DMSO. Absorbance at 570 nm was determined on a plate reader. Cell viabilities are expressed as percentages of that in the corresponding vehicle-treated control group. Immunoblotting Cells were seeded in 10-cm plates (1×106 cells/plate), incubated in 10% FBS-supplemented medium (10 mL/plate) for 24 h, and exposed to individual test agents at the indicated concentrations for 24 h. Cells were collected by scraping followed by centrifugation, washed with cold phosphate-buffered saline, and lysed in SDS lysis buffer (1% SDS, 50 mM Tris buffer pH 8.1, 10 mM EDTA) containing cocktails of protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (0.625 mM glycerophosphate, 1.25 mM NaF, 0.25 mM sodium pyrophosphate, 0.5 mM Na3VO4). Lysates were sonicated for 10 s to disrupt cellular organelles and genomic DNA, and then centrifuged at 13,200 rpm for 15 min. Equal amounts of proteins, as determined by a colorimetric bicinchoninic acid assay (Pierce, Rockford, IL), were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% non-fat milk for 40 min, membranes were probed with primary antibodies at 1∶1000 or 1∶500 dilution in TBST at 4°C for 16 h, followed by goat anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugates at 1∶5,000 and 1∶3,000 dilutions, respectively, for 1 h at room temperature. Proteins were visualized by enhanced chemiluminescence. Transfection via nucleofection Cells (1×106) were harvested by trypsinization, re-suspended in culture medium, centrifuged at 100 rpm for 10 min, and transfected with various siRNA or plasmids using an Amaxa Nucleofection system (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer’s instructions. Expression of various siRNA or plasmids was confirmed by immunoblotting analysis of the target proteins. Lentiviral transfection To produce infectious lentiviruses encoding ILK shRNA, 293T cells were co-transfected with a lentiviral pTRIPz vector encoding Tet-inducible ILK shRNA together with 2 packaging vectors (pMD2.G and psPAX) using the calcium phosphate transfection protocol. The virus-containing medium was collected 5 days after transfection. To generate MDA-MB-468 stable clones that express inducible ILK shRNA, cells were seeded in 6-well plates (1×106 cells/well) for 24 h, and infected with 1 mL virus-containing medium in the presence of 10 µg/mL polybrene. After 1 h incubation, spin infection was performed by centrifugation of the plates at 2600 rpm for 1.5 h at 25°C followed by overnight incubation. The transfected cells were re-seeded onto T75 flasks with fresh medium containing puromycin (0.3 µg/mL) for selection of stable clones. After 7 days of selection, the stable cell pools were established, and their purities were monitored by RFP expression via fluorescence microscopy. The expression of ILK shRNA was verified by Western blot analysis of lysates from cells incubated in the absence or presence of doxycycline. For ILK knockdown experiments, MDA-MB-468 stable clones with Tet/ON inducible ILK shRNA were treated with doxycycline (2 µg/mL) for 5 days with replacement of medium every 2 days to induce the expression of shRNAs. Successful silencing of ILK in response to doxycycline-induced shRNA expression was verified by Western blot analysis. Co-immunoprecipitation of ILK-Akt and ILK-rictor complexes PC-3 cells (2×106) were seeded in 15-cm culture dishes in 10% FBS-supplemented RPMI 1640 medium, incubated for 24 h, and then treated with 2.5 μM T315 or DMSO vehicle control in 5% FBS-containing medium for 24 h. Cells were harvested and lysed in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1mM Na3VO4) at 4°C for 30 min. Cell lysates were incubated with protein A/G agarose beads (Santa Cruz Biotechnology) to eliminate nonspecific binding, and aliquots containing equal amounts of proteins were directly analyzed for levels of ILK, p-Ser473-Akt, Akt and/or rictor by immunoblotting (input), or incubated overnight with goat anti-Akt or anti-ILK antibody- or IgG-conjugated agarose beads (Santa Cruz Biotechnology) for immunoprecipitation. Agarose beads were washed three times with wash buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100) and resuspended in SDS sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, and 0.1% bromophenol blue) for immunoblotting analysis using antibodies against the relevant target proteins. ILK kinase assay The effect of T315 on the kinase activity of immunoprecipitated ILK was determined in an in vitro kinase assay using bacterially expressed glutathione S-transferase (GST)-tagged Akt (GST-Akt) as substrate. GST-Akt fusion protein was expressed in Escherichia coli strain BL21 (DE3; ATCC) by isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich) induction for 3 h at 37°C. Bacterial cells were lysed in STE buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, and 1 mM PMSF) containing 1 mg/ml lysozyme via sonication on ice for 5 min, and centrifuged at 16,000 rpm for 20 min. The supernatant was exposed to glutathione-Sepharose beads (GE Healthcare) with gentle rocking at 4°C for 2 h, and the glutathione beads were then washed three times with ice-cold PBS buffer. The GST-Akt fusion protein was eluted by reduced glutathione in elution buffer (10 mM glutathione, 50 mM Tris-HCl pH 8.0, 5% glycerol) at room temperature for 15 min. For the preparation of immunoprecipitated ILK, PC-3 cells were lysed as described in the previous section, and equivalent amounts of lysates were immunoprecipitated with ILK antibody or rabbit IgG in the presence of protein A/G agarose at 4°C for 12 h. The resulting immunocomplexes were washed three times with lysis buffer, incubated with DMSO vehicle or T315 at indicated concentrations at room temperature for 10 min, and exposed to 2 mg of GST-Akt and 200 mM of ATP in kinase buffer (50 mM HEPES pH 7.0, 10 mM MgCl2, 10 mM MnCl2, 2 mM NaF, 1 mM Na3VO4, 2 mM DTT). After 20 min of incubation at 30°C, the reaction was quenched by adding SDS sample buffer, and equal amounts of proteins were subjected to immunoblotting analysis using antibodies against Akt, p-473S-Akt, and ILK. Matrix colony formation assay Cells were cultured in growth factor–depleted three-dimensional Cultrex Basement Membrane Extract (BME) (Trevigen, Gaithersburg, MD), as previously reported [53]. In brief, cell culture dishes (24-well plates) were pre-coated with undiluted phenol red-free BME. PC-3 cells (104 cells per well) were suspended in 200 µl of serum-free medium, and then mixed with 100 µl of cold BME. The cell suspension was added dropwise to each well. After this bottom cell-containing layer was set, serum-free medium containing DMSO or T315 at indicated concentrations was added on top. Medium was changed every three days. After culture for 6 days, cells were fixed with 4% paraformaldehyde for 20 min. Fixation was quenched by three 10-min washes with 0.75% glycine in PBS, and cells were examined microscopically for colony formation. Statistical analysis Quantitative data from in vitro experiments are presented as mean ± SD. All experiments were performed using 3–6 replicates/group in at least three independent experiments. Differences between group means were analyzed by ANOVA followed by Dunnett’s post-hoc test for multiple comparisons. Results Differential susceptibility of different prostate and breast cancer cell lines to the effect of mTORC2 and ILK inhibitors on Ser473-Akt phosphorylation To shed light onto the contentious issue of the role of mTORC2 versus ILK as PDK2, we examined the effects of the 2nd-generation mTORC inhibitor Ku-0063794 [54] versus the ILK inhibitor T315 [51] on cytotoxicity and Ser473-Akt phosphorylation in three PTEN-negative cell lines, i.e., LNCaP, PC-3, and MDA-MB-468. Ku-0063794 was reported to inhibit the kinase activities of mTORC1/2 with IC50 values of ∼10 nM, and, at 100 nM, to effectively block Ser473-Akt phosphorylation [54]. T315 inhibited the kinase activity of immunoprecipitated ILK with IC50 of 0.6 µM, and showed in vitro efficacy in suppressing the phosphorylation of other ILK downstream targets, including GSK3β and myosin light chain, without affecting that of the mTORC2 substrates serum- and glucocorticoid-induced protein kinase-1 and protein kinase Cα [51]. Moreover, T315 exhibited no appreciable inhibition of a series of signaling kinases, including PDK1, Akt, GSK3β, focal adhesion kinase, cKit, and EGFR [51]. A more thorough kinase profiling analysis is currently underway. Despite the high potency of Ku-0063794 in inhibiting mTORC1/2 signaling, its cytotoxicity varied to a great extent among the three cell lines examined (Fig. 1A). Among them, LNCaP cells were most susceptible to the suppressive effect of Ku-0063794 on cell viability (IC50, 0.4 µM); however, its antitumor activity leveled off at ≥1 µM with a maximum inhibition of 75% of cell viability. This phenomenon might be attributable to the mTOR inhibition-mediated induction of autophagy, which promotes survival in cancer cells [55]. Compared to LNCaP cells, PC-3 and MDA-MB-468 cells were less sensitive to the antiproliferative effect of Ku-0063794. The maximal levels of inhibition attained by the drug were 50% and 30% for PC-3 and MDA-MB-468, respectively, at concentrations ≥2 µM. 10.1371/journal.pone.0067149.g001Figure 1 Effects of mTORC1/2 inhibition on cytotoxicity and Ser-473-Akt phosphorylation in PTEN-negative cancer cell lines. (A) Differential susceptibility of LNCaP, PC-3, and MDA-MB-468 (468) cells to Ku-0063794-mediated suppression of cell viability in 5% FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Dose-dependent inhibitory effects of Ku-0063794 on mTOR signaling, as indicated by p70SK phosphorylation, and Akt phosphorylation at Ser-473 versus Thr-308. Representative blots from three independent experiments are shown. Mechanistic evidence suggests that this differential sensitivity to Ku-0063794-induced cytotoxicity was attributable to differences between the sensitive and resistant cell lines in their Akt phosphorylation status in response to Ku-0063794 exposure. As shown in Fig. 1B, exposure to Ku-0063794, at 200–800 nM, led to a robust, concentration-dependent decrease in the phosphorylation level of p70S6K in all three cell lines, indicative of its effectiveness in inhibiting mTOR signaling. While this mTOR inhibition was associated with the selective repression of Akt phosphorylation at Ser-473 in LNCaP cells, no appreciable changes were noted in PC-3 or MDA-MB-468 cells, which refutes the link between mTOR signaling and Akt phosphorylation status in these two cell lines. In contrast, T315 was effective in suppressing the viability of all three cell lines with IC50 values in the range of 1.5–2 µM (MDA-MB-468, 1.5 µM; PC-3 and LNCaP, 2 µM) (Fig. 2A). The ability of T315 to inhibit ILK in these cell lines was evident in the dose-dependent suppression of relevant biomarkers, including the phosphorylation of GSK3β [5], [6], [7], [22] and the expression of the oncogenic factor Y-box binding protein (YB)-1 [32], [51], [56] (Fig. 2B). However, the selective downregulation of Akt phosphorylation at Ser-473 by T315 was observed in PC-3 and MDA-MB-468 cells, but not in LNCaP cells (Fig. 2B). These data suggest that mTORC2 and ILK might play mutually exclusive roles in mediating the phosphorylation of Ser473-Akt in these cell lines. This premise was supported by the inability of T315 to enhance Ku-0063794-mediated suppression of Ser473-Akt phosphorylation in LNCaP cells relative to Ku-0063794 alone (Fig. 1B), and vice versa in PC-3 cells (Fig. 2C versus 2B). 10.1371/journal.pone.0067149.g002Figure 2 Evidence that ILK acts as a Ser-473-Akt kinase in PC-3 and MDA-MB-468 cells. (A) Dose-dependent effect of T315 on the viability of LNCaP, PC-3, and MDA-MB-468 (468) cells in 5% FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Dose-dependent effects of T315 on the phosphorylation of Akt at Ser473 versus Thr308 and GSK3β. Suppression of GSK3β phosphorylation served as a marker for T315-mediated ILK inhibition. (C) Left panel, co-treatment with 3 µM T315 did not enhance the ability of Ku-0063793 to inhibit Ser473-Akt phosphorylation in LNCaP cells. Right panel, co-treatment with 0.8 µM Ku-0063793 did not enhance the ability of T315 to inhibit Ser473-Akt phosphorylation in PC-3 cells. Cells were exposed to test agents at the indicated concentrations for 24 h in 5% FBS-supplemented medium. (D) Left, representative Western blot of the phosphorylation and/or expression levels of Akt, ILK, and the components of mTORC complexes mTOR, raptor, and rictor in PC-3, LNCaP, and MDA-MB-468 (468) cancer cell lines. Right, relative expression levels of p-Ser-473- and p-Thr-308-Akt, Akt, ILK, mTOR, raptor, and rictor in PC-3 and MDA-MB-468 cells relative to those in LNCaP cells. Amounts of immunoblotted proteins from three independent experiments were quantitated by densitometry and normalized to β-actin. Signals from phosphorylated Akt were first normalized to that of total Akt and then to that of β-actin. The abundance of each protein in PC-3 and MDA-MB-231 cells was expressed as a percentage of that in LNCaP cells. Values, means; bars, S.D. (n = 3). All immunoblots are representative of three independent experiments. P<0.05, compared to LNCaP cells. Differential expression of ILK and mTOR Pursuant to these findings, we assessed the relative expression levels of ILK versus mTOR, as well as the mTORC components raptor and rictor, in these cell lines. While all three cell lines exhibited upregulated Akt phosphorylation, at both Ser-473 and Thr-308, consistent with their PTEN-negative functional status, ILK and mTOR were differentially expressed among these cell lines (Fig. 2D). ILK expression levels in PC-3 and MDA-MB-468 cells were approximately threefold to fourfold higher than that in LNCaP cells, while the expression of mTOR in PC-3 was about 60% lower than that in LNCaP and MDA-MB-468 cells (P<0.05). It is especially noteworthy that PC-3 and LNCaP cells exhibited an inverse expression profile of ILK versus mTOR, i.e., PC-3 cells were high in ILK but low in mTOR expression, while LNCaP cells showed the opposite pattern. With regard to the mTORC components, while raptor was consistently expressed across these cell lines, the expression levels of rictor were lower in LNCaP cells than in PC-3 and MDA-MB-468 cells (P<0.05). mTORC2 is responsible for Ser473 Akt phosphorylation in LNCaP cells, while ILK acts as the PDK2 in PC-3 and MDA-MB-468 cells The cell line-specific roles of mTORC2 and ILK as PDK2 were further verified by genetic knockdown experiments in LNCaP and PC-3 cells. In addition to ILK and the rapamycin-insensitive component of the mTORC2 complex rictor, we also carried out si/shRNA-mediated knockdown of a series of other kinases that have been implicated in mediating Ser473-Akt phosphorylation, including MK2 [8], DNA-PK [57], ATM [10], PKCα [11], PKCβII [12], and PAK1/2 [13], in different cell systems in response to growth factor or other external stimuli. Western blot analysis indicates the high efficiency of these si/shRNAs in reducing the expression of the respective target proteins in both cell lines (Fig. 3A and 4A). More importantly, the consequent effects of these knockdowns on Ser473-Akt phosphorylation confirmed the cell line specificity of mTORC2 versus ILK in mediating the PDK2 function in LNCaP and PC-3 cells. As shown, only the knockdown of rictor, but not ILK or any of the other targeted kinases, resulted in a reduction in p-Ser473-Akt expression in LNCaP cells (Fig. 3A). Conversely, this downregulation of Ser473-Akt phosphorylation was highly specific for the siRNA-mediated knockdown of ILK in PC-3 cells (Fig. 4A). 10.1371/journal.pone.0067149.g003Figure 3 Evidence that mTORC2 is the Ser-473-Akt kinase in LNCaP cells. (A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of siRNA-mediated ILK knockdown, alone or in combination with Ku-0063794 (0.4 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Dose-dependent effect of ectopic expression of ILK, alone (left panel) or in combination with Ku-0063794 (0.4 µM, 24 h), on the phosphorylation of Ser473-Akt and GSK3β. All immunoblots are representative of three independent experiments. 10.1371/journal.pone.0067149.g004Figure 4 Evidence that ILK is the Ser473-Akt kinase in PC-3 and MDA-MB-468 cells. (A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of shRNA-mediated rictor knockdown, alone or in combination with T315 (2 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Western blot analysis of the effect of shRNA-mediated repression of ILK (left) or rictor (right) on the phosphorylation of Akt at Ser473 versus Thr308, GSK3β (ILK target) and p70S6K (mTOR target) in MDA-MB-468 cells. For induction of ILK shRNA, cells stably transfected with a lentiviral vector encoding tetracycline-inducible ILK shRNA (TRE-ILKi) were exposed to 2 µg/ml doxycycline for 5 days. All immunoblots are representative of three independent experiments. In addition to these si/shRNA data, the involvement of ILK in mediating Ser473-Akt phosphorylation in LNCaP cells was refuted by three lines of evidence. First, as we have already shown, LNCaP cells express a low abundance of ILK relative to the other cell lines examined (Fig. 2D). Second, the siRNA-mediated knockdown of ILK alone in LNCaP cells had no appreciable effect on Ser473-Akt phosphorylation, which, however, was markedly reduced by co-treatment with Ku-0063794 (0.4 µM) (Fig. 3B), consistent with the effect observed after combination treatment with T315 and Ku0063794 (Fig. 2C). Third, ectopic expression of ILK, as evidenced by dose-dependent elevation in GSK3β phosphorylation, had a modest effect on Ser473-Akt phosphorylation in LNCaP cells, but could not rescue cells from Ku-0063794-mediated suppression of Akt phosphorylation (Fig. 3C). Similarly, in PC-3 cells, phosphorylation of Ser473-Akt was unaffected by shRNA-mediated knockdown of rictor, but was inhibited by co-treatment with T315 (Fig. 4B), thereby ruling out the involvement of mTORC2 as PDK2 in this cell line. Although we employed a similar approach to interrogate the role of ILK in mediating the phosphorylation of Ser473-Akt in MDA-MB-468 cells, this cell line was refractory to siRNA-mediated knockdown of ILK after introduction of the siRNA via nucleofection. Therefore, we generated a stable clone that expressed ILK shRNA from a TRE (tetracycline response element) promoter by transfecting cells with a lentiviral Tet-inducible ILK-shRNA vector. As shown, doxycycline induced the expression of ILK shRNA, as indicated by reduced ILK expression and GSK3β phosphorylation, and downregulated Ser473-specific phosphorylation of Akt (Fig. 4C, left panel). In contrast, silencing of rictor, as indicated by loss of rictor expression and reduced p70S6K phosphorylation, had no effect on the p-Ser473-Akt level (right panel). Evidence that ILK binds and phosphorylates Akt in PC-3 cells We obtained two lines of evidence that Akt is targeted by ILK for phosphorylation in PC-3 cells. First, the physical interaction between these two kinases was verified by co-immunoprecipitation. Lysates of PC-3 cells that had been treated for 24 h with either T315 or vehicle were analyzed directly for levels of ILK, phospho-Ser473-Akt, and Akt by immunoblotting (input), or were subjected to immunoprecipitation with anti-Akt antibodies or IgG. The levels of ILK, phospho-Ser473-Akt, and Akt in the resulting immunocomplexes were then analyzed by immunoblotting, which showed that ILK recognized and bound Akt, and T315 was able to attenuate the interaction of ILK with Akt. Second, we demonstrated the ability of ILK to directly phosphorylate Akt at Ser-473. Specifically, the kinase activity of ILK was assayed using ILK immunoprecipitated from PC-3 cells and bacterially expressed, GST-tagged Akt as substrate. As shown in Fig. 5B, ILK phosphorylated Akt at Ser-473. Equally important, this ILK-mediated phosphorylation of Ser473-Akt was dose-dependently inhibited by T315 (P<0.05). 10.1371/journal.pone.0067149.g005Figure 5 Evidence that ILK binds and phosphorylates Akt in PC-3 cells. (A) Co-immunoprecipitation analysis reveals the association of ILK with Akt in PC-3 cells, which can be attenuated by T315. PC-3 cells were treated with 2.5 µM T315 or DMSO control for 24 h. Equal amounts of cell lysates were immunoblotted (WB) with antibodies against ILK, p-Ser473-Akt, or Akt (Input; lower panel), or were immunoprecipitated (IP) with anti-Akt antibody and protein A/G agarose followed by immunoblotting with antibodies against ILK, p-Ser473-Akt, or Akt (upper panel). (B) Effect of T315 on the kinase activity of ILK. Equal amounts of immunocomplexes obtained by immunoprecipitation of ILK from PC-3 cell lysates were incubated with bacterially expressed GST-Akt and 200 mM of ATP in the presence of DMSO or T315 at indicated concentrations for 20 min, and were then subjected to Western blotting with antibodies against p-Ser-473-Akt, Akt, or ILK. The immunoprecipitation procedure using IgG was performed as control. Left panel, Western blot analysis of the dose-dependent effect of T315 on the kinase activity of ILK. Right panel, relative expression levels of phosphorylated Ser-473-Akt after normalization to total Akt and subtraction from control (time zero). Amounts of immunoblotted proteins from three independent experiments were quantitated by densitometry. The abundance of phosphorylated Ser-473-Akt in T315-treated cells was expressed as a percentage of that in the DMSO control cells (0 µM). Values, means; bars, S.D. (n = 3). All immunoblots are representative of three independent experiments. P<0.05, compared to DMSO control (0 µM). ILK interacts with rictor in PC-3 and MDA-MB-468, but not in LNCaP cells Recent evidence revealed an interaction between ILK and rictor in MDA-MB-231 and PC-3 cells, which played a pivotal role in regulating the Ser-473-Akt kinase activity of ILK [42], [52]. Pursuant to this finding, we investigated this complex formation in MDA-MB-468, PC-3 and LNCaP cells. Co-immunoprecipitation analysis indicated that ILK formed complexes with rictor in MDA-MB-468 and PC-3 cells, but not in LNCaP cells (Fig. 6). Moreover, in line with the effect noted with QLT0267 [52], T315 reduced the formation of this complex in both cell lines (Fig. 6). 10.1371/journal.pone.0067149.g006Figure 6 ILK interacts with rictor in PC-3 and MDA-MB-468, but not in LNCaP cells. Co-immunoprecipitation analysis reveals that ILK associates with rictor in MDA-MB-468 and PC-3 cells, which can be attenuated by T315. No such association was observed in LNCaP cells. Cells were treated with 2.5 µM T315 or DMSO control for 24 h. Equal amounts of cell lysates were immunoblotted with antibodies against ILK, rictor or β-actin (Input; lower panels), or were immunoprecipitated (IP) with anti-ILK antibody and protein A/G agarose followed by immunoblotting (WB) with antibodies against ILK or rictor (upper panels). Putative role of ILK in promoting invasive phenotype in cancer cells The cell line-specific role of ILK as PDK2 in PC-3 and MDA-MB-468 cells raised a question with regard to its biological function beyond the regulation of Akt signaling in these cells. As ILK is known to promote the aggressive phenotype of cancer cells, in part, by facilitating epithelial-to-mesenchymal transition (EMT) through Snail upregulation [58], we examined the effect of ILK inhibition by genetic knockdown and T315 (2 µM) on the expression of Snail and the EMT markers E-cadherin and vimentin in PC-3 and MDA-MB-468 cells. As shown, both genetic silencing and pharmacological inhibition of ILK suppressed Snail expression and EMT in these two cell lines, as manifested by the concomitant increase in E-cadherin expression and decrease in vimentin expression (Fig. 6A). This is consistent with our earlier finding that T315 blocked the expression of YB-1 in PC-3 cells [51], which plays a pivotal role in facilitating EMT through translational activation of Snail and other EMT-inducing transcriptional factors [59]. Moreover, the suppressive effect of T315 on the invasive phenotype of PC-3 cells was investigated using the basement membrane matrix colony formation assay, which has been used frequently to assess the metastatic capacity of cancer cells [60]. As shown, T315 inhibited the ability of the aggressive PC-3 cells to form colonies in the basement membrane matrix (Fig. 5C). PC-3 cells formed large colonies with stellate morphology, indicative of invasiveness [60], in the control group (DMSO), but T315, at 1 and 2 µM, was effective in blocking this invasive colony formation. Together, these findings suggest a potential functional role of ILK in promoting survival signaling and aggressive phenotype in cancer cells through distinct pathways. Role of ILK versus mTORC2 in mediating epidermal growth factor (EGF)-induced Ser473-Akt phosphorylation in MDA-MB-231 cells As the results above were generated using PTEN-deficient cell lines, one might raise a question of whether the cell line-specific function of ILK versus mTORC2 as PDK2 was associated with the loss of PTEN function in these cells. To address this issue, we investigated the effects of T315 and Ku-0063794 on EGF-induced Ser473-Akt phosphorylation in the PTEN-functional MDA-MB-231 cells. Earlier studies have demonstrated the key role of ILK in mediating endogenous Ser473-Akt phosphorylation in this cell line [42], [43] as it was susceptible to inhibition by QLT-0267 [42], [43] and T315 [51]. As shown in Fig. 7C, exposure of MDA-MB-231 cells to EGF (150 ng/ml) gave rise to robust increases in the phosphorylation of Akt at both Ser473 and Thr308. Ku-0063793 and T315 effectively reduced the EGF-induced phosphorylation of Ser473-Akt to the basal level, while Thr308-Akt phosphorylation was unaffected by either agent. These data suggest the involvement of both ILK and mTORC2 in facilitating Ser473-Akt phosphorylation in response to EGF in MDA-MB-231 cells, indicating that their PDK2 activity is not dependent upon the lack of PTEN function. 10.1371/journal.pone.0067149.g007Figure 7 Pharmacologic inhibition or genetic silencing of ILK suppresses EMT and invasive phenotype. (A) Inhibition of ILK by si/shRNA-mediated silencing or treatment with T315 reduces the expression of Snail in association with an increase in that of E-cadherin and concomitant reduction in that of vimentin in PC-3 (left panel) and MDA-MB-468 (right panel) cells. Cells were treated with T315 for 24 h. For induction of ILK shRNA, MDA-MB-468 cells stably transfected with a lentiviral vector encoding tetracycline-inducible ILK shRNA (TRE-ILKi) were exposed to 2 µg/ml doxycycline for 5 days. Immunoblots are representative of three independent experiments. (B) Images of invasive colonies after growth of PC-3 cells on basement membrane matrix for 6 days in the presence of DMSO control versus T315 at the indicated concentrations. Bars, 100 μm. (C) Dose-dependent effects of T315 and Ku-0063794 on the EGF-induced phosphorylation of Akt at Ser-473 versus Thr-308 in the PTEN-functional MDA-MB-231 cell line. Cells were co-treated with inhibitor and EGF for 24 h. Representative blots from three independent experiments are shown. Discussion Although substantial evidence indicates the pivotal role of mTORC2 in mediating Ser473-Akt phosphorylation in different types of nonmalignant and cancer cells, other kinases have also been implicated to function as the Ser473-specific Akt kinase in a cellular context- or cell line-dependent manner. For example, DNA-PK has been shown to act as the Ser473-Akt kinase in the nucleus in response to DNA double-strand breaks in human umbilical vascular endothelial cells [9]. In contrast, in MCF-7 cells, while mTORC2 mediated β1 integrin-induced Ser473-Akt phosphorylation, PAK1/2 was responsible for this phosphorylation in response to other stimuli, including lysophosphatidic acid and platelet-derived growth factor [13]. In this study, we interrogated the role of mTORC2 versus ILK in mediating the phosphorylation of Ser473-Akt in PTEN-negative LNCaP, PC-3, and MDA-MB-468 cells in light of their constitutive activation of Akt. Relative to PTEN-functional cells, these cell lines require no external stimulation to induce Akt activation, which allowed the cell context-independent study of Akt regulation by upstream kinases. Moreover, these cell lines differ with respect to their invasive phenotypes, i.e., non-invasive LNCaP cells versus moderately invasive MDA-MB-468 and highly invasive PC-3 cells. By using pharmacological inhibitors and genetic knockdown, we demonstrated the cell line specificity of mTORC2 and ILK in facilitating Ser473-Akt phosphorylation in these cell lines. Moreover, reminiscent of that reported in MDA-MB-231 cells [42], [52], our data indicate the interaction of ILK with rictor in MDA-MB-468 and PC-3 cells, which, however, was not found in LNCaP cells. This discrepancy might reflect a low abundance of rictor in LNCaP cells (Fig. 2D), which could underlie the inability of ILK to regulate Ser-473-Akt phosphorylation in these cells. Nevertheless, despite lack of Akt inactivation, LNCaP cells were still susceptible to T315-mediated inhibition of cell proliferation, in part, due to the drug’s ability to target other downstream signaling effectors of ILK, including the dephosphorylating activation of GSK3β and reduction of the expression of the oncoprotein YB-1 (Fig. 2B). GSK3β activation in response to various cellular stresses has been shown to induce apoptosis by facilitating the proteasomal degradation of key signaling proteins governing survival and cell cycle progression, including β-catenin and cyclin D1 [61]. In addition, GSK3β has been reported to function as a repressor of androgen receptor-mediated transactivation and cell growth in LNCaP cells [62]. Moreover, the role of YB-1 in regulating cancer cell growth survival is well recognized as siRNA-mediated depletion of YB-1 has been shown to induce apoptosis in many cancer cell lines [63]. Thus, the effects on these two signaling effectors might underlie the high in vitro efficacy of T315 in suppressing the proliferation of LNCaP cells. Although this investigation was conducted in a limited number of PTEN-negative cell lines, the proposed function of ILK in promoting cancer cell aggressiveness is consistent with recent reports that link upregulation of ILK expression with tumor metastasis and progression in prostate cancer, breast cancer, and many other types of human malignancies [14], [15], [16], [17], [18], [19], [20], [21]. Moreover, ILK upregulation has been reported to be a cellular response to various stimuli in the tumor environment leading to activation of Akt signaling in cancer cells, including human leukemic cells co-cultured with bone marrow-derived stromal cells [27], hepatocellular or renal carcinoma cells exposed to hypoxia [64], ovarian cancer cells treated with peritoneal tumor fluids [17], and MDA-MB-231 cells undergoing transforming growth factor β-1-induced EMT [52]. Together, these findings raise a possibility that in the course of tumor progression, cancer cells upregulate the expression of ILK in response to microenvironmental stimuli, such as hypoxia and inflammatory cytokines, to gain growth advantage and metastatic capacity mediated through various downstream signaling effectors, including Akt, GSK3β, and YB-1 [23], [32], [65], [66]. For example, as GSK3β inhibition upregulates Snail, a transcriptional repressor of E-cadherin, ILK activation leads to the transition of cancer cells from an epithelial to mesenchymal phenotype [58]. Moreover, ILK activates the expression of YB-1, which facilitates EMT through the translational activation of Snail and other EMT-inducing transcription factors [59]. This premise is supported by the finding that genetic knockdown or pharmacological inhibition of ILK could inhibit EMT, in part, through the suppression of Snail expression in PC-3 and MDA-MB-468 cells. In conclusion, the present study demonstrates that ILK mediates Ser473-Akt phosphorylation in PTEN-negative PC-3 and MDA-MB-468 cells. As ILK plays an intricate role in diverse cellular functions associated with survival, proliferation, motility, EMT, and angiogenesis [65], [66], [67], the adoption of ILK as PDK2 might bestow growth advantage and metastatic potential on cancer cells in the course of tumor progression. From a therapeutic perspective, ILK represents an important target for the treatment of metastatic cancer, of which the proof-of-concept is being investigated by using the ILK inhibitor T315 in different tumor models. ==== Refs References 1 Mora A , Komander D , van Aalten DM , Alessi DR (2004 ) PDK1, the master regulator of AGC kinase signal transduction . Semin Cell Dev Biol 15 : 161 –170 .15209375 2 Dong LQ , Liu F (2005 ) PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle . Am J Physiol Endocrinol Metab 289 : E187 –196 .16014356 3 Hresko RC , Mueckler M (2005 ) mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes . J Biol Chem 280 : 40406 –40416 .16221682 4 Sarbassov DD , Guertin DA , Ali SM , Sabatini DM (2005 ) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex . Science 307 : 1098 –1101 .15718470 5 Delcommenne M , Tan C , Gray V , Rue L , Woodgett J , et al (1998 ) Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase . Proc Natl Acad Sci U S A 95 : 11211 –11216 .9736715 6 Persad S , Attwell S , Gray V , Delcommenne M , Troussard A , et al (2000 ) Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells . Proc Natl Acad Sci U S A 97 : 3207 –3212 .10716737 7 Persad S , Attwell S , Gray V , Mawji N , Deng JT , et al (2001 ) Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343 . J Biol Chem 276 : 27462 –27469 .11313365 8 Lynch DK , Ellis CA , Edwards PA , Hiles ID (1999 ) Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism . Oncogene 18 : 8024 –8032 .10637513 9 Bozulic L , Surucu B , Hynx D , Hemmings BA (2008 ) PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival . Mol Cell 30 : 203 –213 .18439899 10 Viniegra JG , Martinez N , Modirassari P , Hernandez Losa J , Parada Cobo C , et al (2005 ) Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM . J Biol Chem 280 : 4029 –4036 .15546863 11 Partovian C , Simons M (2004 ) Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells . Cell Signal 16 : 951 –957 .15157674 12 Kawakami Y , Nishimoto H , Kitaura J , Maeda-Yamamoto M , Kato RM , et al (2004 ) Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion . J Biol Chem 279 : 47720 –47725 .15364915 13 Riaz A , Zeller KS , Johansson S (2012 ) Receptor-specific mechanisms regulate phosphorylation of AKT at Ser473: role of RICTOR in beta1 integrin-mediated cell survival . PLoS One 7 : e32081 .22384145 14 Pontier SM , Huck L , White DE , Rayment J , Sanguin-Gendreau V , et al (2010 ) Integrin-linked kinase has a critical role in ErbB2 mammary tumor progression: implications for human breast cancer . Oncogene 29 : 3374 –3385 .20305688 15 Bravou V , Klironomos G , Papadaki E , Taraviras S , Varakis J (2006 ) ILK over-expression in human colon cancer progression correlates with activation of beta-catenin, down-regulation of E-cadherin and activation of the Akt-FKHR pathway . J Pathol 208 : 91 –99 .16278819 16 Chan J , Ko FC , Yeung YS , Ng IO , Yam JW (2011 ) Integrin-linked kinase overexpression and its oncogenic role in promoting tumorigenicity of hepatocellular carcinoma . PLoS One 6 : e16984 .21347395 17 Ahmed N , Riley C , Oliva K , Stutt E , Rice GE , et al (2003 ) Integrin-linked kinase expression increases with ovarian tumour grade and is sustained by peritoneal tumour fluid . J Pathol 201 : 229 –237 .14517840 18 Schaeffer DF , Assi K , Chan K , Buczkowski AK , Chung SW , et al (2010 ) Tumor expression of integrin-linked kinase (ILK) correlates with the expression of the E-cadherin repressor snail: an immunohistochemical study in ductal pancreatic adenocarcinoma . Virchows Arch 456 : 261 –268 .20091050 19 Graff JR , Deddens JA , Konicek BW , Colligan BM , Hurst BM , et al (2001 ) Integrin-linked kinase expression increases with prostate tumor grade . Clin Cancer Res 7 : 1987 –1991 .11448915 20 Ito R , Oue N , Zhu X , Yoshida K , Nakayama H , et al (2003 ) Expression of integrin-linked kinase is closely correlated with invasion and metastasis of gastric carcinoma . Virchows Arch 442 : 118 –123 .12596061 21 Younes MN , Kim S , Yigitbasi OG , Mandal M , Jasser SA , et al (2005 ) Integrin-linked kinase is a potential therapeutic target for anaplastic thyroid cancer . Mol Cancer Ther 4 : 1146 –1156 .16093430 22 Troussard AA , Mawji NM , Ong C , Mui A , St -Arnaud R , et al (2003 ) Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation . J Biol Chem 278 : 22374 –22378 .12686550 23 Legate KR , Montanez E , Kudlacek O , Fassler R (2006 ) ILK, PINCH and parvin: the tIPP of integrin signalling . Nat Rev Mol Cell Biol 7 : 20 –31 .16493410 24 Huang Y , Li J , Zhang Y , Wu C (2000 ) The roles of integrin-linked kinase in the regulation of myogenic differentiation . J Cell Biol 150 : 861 –872 .10953009 25 Su JL , Chiou J , Tang CH , Zhao M , Tsai CH , et al (2010 ) CYR61 regulates BMP-2-dependent osteoblast differentiation through the {alpha}v{beta}3 integrin/integrin-linked kinase/ERK pathway . J Biol Chem 285 : 31325 –31336 .20675382 26 White DE , Cardiff RD , Dedhar S , Muller WJ (2001 ) Mammary epithelial-specific expression of the integrin-linked kinase (ILK) results in the induction of mammary gland hyperplasias and tumors in transgenic mice . Oncogene 20 : 7064 –7072 .11704830 27 Tabe Y , Jin L , Tsutsumi-Ishii Y , Xu Y , McQueen T , et al (2007 ) Activation of integrin-linked kinase is a critical prosurvival pathway induced in leukemic cells by bone marrow-derived stromal cells . Cancer Res 67 : 684 –694 .17234779 28 Esfandiarei M , Yazdi SA , Gray V , Dedhar S , van Breemen C (2010 ) Integrin-linked kinase functions as a downstream signal of platelet-derived growth factor to regulate actin polymerization and vascular smooth muscle cell migration . BMC Cell Biol 11 : 16 .20178627 29 Ishii T , Satoh E , Nishimura M (2001 ) Integrin-linked kinase controls neurite outgrowth in N1E-115 neuroblastoma cells . J Biol Chem 276 : 42994 –43003 .11560928 30 Leung-Hagesteijn C , Hu MC , Mahendra AS , Hartwig S , Klamut HJ , et al (2005 ) Integrin-linked kinase mediates bone morphogenetic protein 7-dependent renal epithelial cell morphogenesis . Mol Cell Biol 25 : 3648 –3657 .15831470 31 Sawai H , Okada Y , Funahashi H , Matsuo Y , Takahashi H , et al (2006 ) Integrin-linked kinase activity is associated with interleukin-1 alpha-induced progressive behavior of pancreatic cancer and poor patient survival . Oncogene 25 : 3237 –3246 .16407822 32 Kalra J , Sutherland BW , Stratford AL , Dragowska W , Gelmon KA , et al (2010 ) Suppression of Her2/neu expression through ILK inhibition is regulated by a pathway involving TWIST and YB-1 . Oncogene 29 : 6343 –6356 .20838384 33 Wickstrom SA , Lange A , Montanez E , Fassler R (2010 ) The ILK/PINCH/parvin complex: the kinase is dead, long live the pseudokinase! EMBO J . 29 : 281 –291 . 34 Fukuda K , Knight JD , Piszczek G , Kothary R , Qin J (2011 ) Biochemical, proteomic, structural, and thermodynamic characterizations of integrin-linked kinase (ILK): cross-validation of the pseudokinase . J Biol Chem 286 : 21886 –21895 .21524996 35 Grashoff C , Aszodi A , Sakai T , Hunziker EB , Fassler R (2003 ) Integrin-linked kinase regulates chondrocyte shape and proliferation . EMBO Rep 4 : 432 –438 .12671688 36 Sakai T , Li S , Docheva D , Grashoff C , Sakai K , et al (2003 ) Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation . Genes Dev 17 : 926 –940 .12670870 37 Lorenz K , Grashoff C , Torka R , Sakai T , Langbein L , et al (2007 ) Integrin-linked kinase is required for epidermal and hair follicle morphogenesis . J Cell Biol 177 : 501 –513 .17485490 38 Lange A , Wickstrom SA , Jakobson M , Zent R , Sainio K , et al (2009 ) Integrin-linked kinase is an adaptor with essential functions during mouse development . Nature 461 : 1002 –1006 .19829382 39 White DE , Coutu P , Shi YF , Tardif JC , Nattel S , et al (2006 ) Targeted ablation of ILK from the murine heart results in dilated cardiomyopathy and spontaneous heart failure . Genes Dev 20 : 2355 –2360 .16951252 40 Wang HV , Chang LW , Brixius K , Wickstrom SA , Montanez E , et al (2008 ) Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage . J Cell Biol 180 : 1037 –1049 .18332223 41 Pereira JA , Benninger Y , Baumann R , Goncalves AF , Ozcelik M , et al (2009 ) Integrin-linked kinase is required for radial sorting of axons and Schwann cell remyelination in the peripheral nervous system . J Cell Biol 185 : 147 –161 .19349584 42 McDonald PC , Oloumi A , Mills J , Dobreva I , Maidan M , et al (2008 ) Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival . Cancer Res 68 : 1618 –1624 .18339839 43 Troussard AA , McDonald PC , Wederell ED , Mawji NM , Filipenko NR , et al (2006 ) Preferential dependence of breast cancer cells versus normal cells on integrin-linked kinase for protein kinase B/Akt activation and cell survival . Cancer Res 66 : 393 –403 .16397254 44 Edwards LA , Thiessen B , Dragowska WH , Daynard T , Bally MB , et al (2005 ) Inhibition of ILK in PTEN-mutant human glioblastomas inhibits PKB/Akt activation, induces apoptosis, and delays tumor growth . Oncogene 24 : 3596 –3605 .15782140 45 Younes MN , Yigitbasi OG , Yazici YD , Jasser SA , Bucana CD , et al (2007 ) Effects of the integrin-linked kinase inhibitor QLT0267 on squamous cell carcinoma of the head and neck . Arch Otolaryngol Head Neck Surg 133 : 15 –23 .17224516 46 Kalra J , Warburton C , Fang K , Edwards L , Daynard T , et al (2009 ) QLT0267, a small molecule inhibitor targeting integrin-linked kinase (ILK), and docetaxel can combine to produce synergistic interactions linked to enhanced cytotoxicity, reductions in P-AKT levels, altered F-actin architecture and improved treatment outcomes in an orthotopic breast cancer model . Breast Cancer Res 11 : R25 .19409087 47 Eke I , Leonhardt F , Storch K , Hehlgans S , Cordes N (2009 ) The small molecule inhibitor QLT0267 Radiosensitizes squamous cell carcinoma cells of the head and neck . PLoS One 4 : e6434 .19649326 48 Muranyi AL , Dedhar S , Hogge DE (2009 ) Combined inhibition of integrin linked kinase and FMS-like tyrosine kinase 3 is cytotoxic to acute myeloid leukemia progenitor cells . Exp Hematol 37 : 450 –460 .19302919 49 Koul D , Shen R , Bergh S , Lu Y , de Groot JF , et al (2005 ) Targeting integrin-linked kinase inhibits Akt signaling pathways and decreases tumor progression of human glioblastoma . Mol Cancer Ther 4 : 1681 –1688 .16275989 50 Edwards LA , Woo J , Huxham LA , Verreault M , Dragowska WH , et al (2008 ) Suppression of VEGF secretion and changes in glioblastoma multiforme microenvironment by inhibition of integrin-linked kinase (ILK) . Mol Cancer Ther 7 : 59 –70 .18202010 51 Lee SL , Hsu EC , Chou CC , Chuang HC , Bai LY , et al (2011 ) Identification and characterization of a novel integrin-linked kinase inhibitor . J Med Chem 54 : 6364 –6374 .21823616 52 Serrano I , McDonald PC , Lock FE , Dedhar S (2013 ) Role of the integrin-linked kinase (ILK)/Rictor complex in TGFbeta-1-induced epithelial-mesenchymal transition (EMT) . Oncogene 32 : 50 –60 .22310280 53 Debnath J , Muthuswamy SK , Brugge JS (2003 ) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures . Methods 30 : 256 –268 .12798140 54 Garcia-Martinez JM , Moran J , Clarke RG , Gray A , Cosulich SC , et al (2009 ) Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR) . Biochem J 421 : 29 –42 .19402821 55 Fan QW , Weiss WA (2011 ) Autophagy and Akt promote survival in glioma . Autophagy 7 : 536 –538 .21266843 56 Stratford AL , Habibi G , Astanehe A , Jiang H , Hu K , et al (2007 ) Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapy . Breast Cancer Res 9 : R61 .17875215 57 Feng J , Park J , Cron P , Hess D , Hemmings BA (2004 ) Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase . J Biol Chem 279 : 41189 –41196 .15262962 58 Zhou BP , Deng J , Xia W , Xu J , Li YM , et al (2004 ) Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition . Nat Cell Biol 6 : 931 –940 .15448698 59 Evdokimova V , Tognon C , Ng T , Ruzanov P , Melnyk N , et al (2009 ) Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition . Cancer Cell 15 : 402 –415 .19411069 60 Koblinski JE , Kaplan-Singer BR , VanOsdol SJ , Wu M , Engbring JA , et al (2005 ) Endogenous osteonectin/SPARC/BM-40 expression inhibits MDA-MB-231 breast cancer cell metastasis . Cancer Res 65 : 7370 –7377 .16103089 61 Jacobs KM , Bhave SR , Ferraro DJ , Jaboin JJ , Hallahan DE , et al (2012 ) GSK-3beta: A Bifunctional Role in Cell Death Pathways . Int J Cell Biol 2012 : 930710 .22675363 62 Wang L , Lin HK , Hu YC , Xie S , Yang L , et al (2004 ) Suppression of androgen receptor-mediated transactivation and cell growth by the glycogen synthase kinase 3 beta in prostate cells . J Biol Chem 279 : 32444 –32452 .15178691 63 Lasham A , Samuel W , Cao H , Patel R , Mehta R , et al (2012 ) YB-1, the E2F pathway, and regulation of tumor cell growth . J Natl Cancer Inst 104 : 133 –146 .22205655 64 Abboud ER , Coffelt SB , Figueroa YG , Zwezdaryk KJ , Nelson AB , et al (2007 ) Integrin-linked kinase: a hypoxia-induced anti-apoptotic factor exploited by cancer cells . Int J Oncol 30 : 113 –122 .17143519 65 Hannigan G , Troussard AA , Dedhar S (2005 ) Integrin-linked kinase: a cancer therapeutic target unique among its ILK . Nat Rev Cancer 5 : 51 –63 .15630415 66 Persad S , Dedhar S (2003 ) The role of integrin-linked kinase (ILK) in cancer progression . Cancer Metastasis Rev 22 : 375 –384 .12884912 67 McDonald PC , Fielding AB , Dedhar S (2008 ) Integrin-linked kinase – essential roles in physiology and cancer biology . J Cell Sci 121 : 3121 –3132 .18799788	23840605	PMC3686768	CC BY	2021-01-05 17:30:01	yes	PLoS One. 2013 Jun 19; 8(6):e67149
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23840429PONE-D-12-2848410.1371/journal.pone.0066246Research ArticleBiologyBiochemistryMolecular cell biologySignal transductionSignaling cascadesAkt signaling cascadeERK signaling cascadeMedicineDrugs and DevicesDrug Research and DevelopmentObstetrics and GynecologyBreast CancerOncologyCancer TreatmentChemotherapy and Drug TreatmentCancers and NeoplasmsBreast TumorsBasic Cancer ResearchChemotherapeutic Potential of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2H-Benzo(b)pyran in Estrogen Receptor- Negative Breast Cancer Cells: Action via Prevention of EGFR Activation and Combined Inhibition of PI-3-K/Akt/FOXO and MEK/Erk/AP-1 Pathways Benzopyran Derivative Inhibits EGFR SignalingSaxena Ruchi 1 Chandra Vishal 1 Manohar Murli 1 Hajela Kanchan 2 Debnath Utsab 2 Prabhakar Yenamandra S. 2 Saini Karan Singh 1 Konwar Rituraj 1 Kumar Sandeep 3 Megu Kaling 3 Roy Bal Gangadhar 4 Dwivedi Anila 1 * 1 Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India 2 Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India 3 Department of Surgery, King George’s Medical University, Lucknow, Uttar Pradesh, India 4 Institute of Nuclear Medicine and Allied Sciences, DRDO, Delhi, India Agoulnik Irina U. Editor Florida International University, United States of America * E-mail: anila.dwivedi@rediffmail.comCompeting Interests: The details of patent are as follows: United States Patent no. 5254,568; Inventors: RS Kapil, S Durani, JD Dhar, BS Setty; Title: Benzopyrans as anti-estrogenic agents (reference # 17 in the manuscript). The authors confirm that this does not alter their adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Conceived and designed the experiments: RS AD. Performed the experiments: RS VC MM KSS. Analyzed the data: RS UD YSP. Contributed reagents/materials/analysis tools: BGR RK KM SK KH AD. Wrote the paper: RS AD. 2013 19 6 2013 8 6 e6624614 9 2012 7 5 2013 © 2013 Saxena et al2013Saxena et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Inhibition of epidermal growth factor receptor (EGFR) signaling is considered to be a promising treatment strategy for estrogen receptor (ER)-negative breast tumors. We have investigated here the anti-breast cancer properties of a novel anti-proliferative benzopyran compound namely, 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b)pyran (CDRI-85/287) in ER- negative and EGFR- overexpressing breast cancer cells. The benzopyran compound selectively inhibited the EGF-induced growth of MDA-MB 231 cells and ER-negative primary breast cancer cell culture. The compound significantly reduced tumor growth in xenograft of MDA-MB 231 cells in nude mice. The compound displayed better binding affinity for EGFR than inhibitor AG1478 as demonstrated by molecular docking studies. CDRI-85/287 significantly inhibited the activation of EGFR and downstream effectors MEK/Erk and PI-3-K/Akt. Subsequent inhibition of AP-1 promoter activity resulted in decreased transcription activation and expression of c-fos and c-jun. Dephosphorylation of downstream effectors FOXO-3a and NF-κB led to increased expression of p27 and decreased expression of cyclin D1 which was responsible for decreased phosphorylation of Rb and prevented the transcription of E2F- dependent genes involved in cell cycle progression from G1/S phase. The compound induced apoptosis via mitochondrial pathway and it also inhibited EGF-induced invasion of MDA-MB 231 cells as evidenced by decreased activity of MMP-9 and expression of CTGF. These results indicate that benzopyran compound CDRI-85/287 could constitute a powerful new chemotherapeutic agent against ER-negative and EGFR over-expressing breast tumors. This work was supported by Council of Scientific and Industrial Research, New Delhi and Ministry of Health and Family Welfare, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Breast cancer is the most common cancer diagnosed in women and the second most common cause of female cancer- related deaths [1]. The anti-hormones are suitably used for therapy of ER- positive breast cancer patients [2], [3]. In contrast to ER- positive, the ER- negative (ER-) breast cancers that constitute about 30% of the total, lack the E2-ER-ERE mediated hormone-dependent cell-proliferation pathway [4]. In ER- negative tumors, overexpression of EGFR or HER-2, leading to increased growth factor signaling, is observed [5]. A subgroup of ER-negative tumors is also negative for the expression of progesterone receptor and HER-2 protein [6]. Such tumors are designated as ‘triple-negative’ and are characterized by their unique molecular profile, aggressive behavior and distinct patterns of metastasis. Overexpression of the epidermal growth factor family of receptors (EGFR) in ER- ve cells has been the basis for the implication of EGF-induced mitogenic signal for the enhanced proliferation of these cancer cells [7], [8]. Hence, EGFR could serve as a target for therapeutic intervention in a subgroup of triple-negative breast cancer patients. Major pathways associated with EGFR signaling include the Ras/MAPK pathway, the phosphatidyl inositol 3-kinase (PI3K)/Akt pathway, the Janus kinase (JAK)/signal transducers and activators of transcription. These signaling pathways ultimately affect cell proliferation, survival, motility, and adhesion [1]. Thè key survival pathway modulated by EGFR is the MEK/Erk kinase cascade [9] which exerts its mitogenic and invasive effects via AP-1 transcriptional complex [10]. AP-1 transcription factors are believed to be responsible for cellular proliferation as well as invasion of ER-negative breast cancer cells [11]. EGF also exerts mitogenic effects by activating NF-κB and deactivating Forkhead transcripton factor FOXO-3a via activation of PI-3-K/Akt - dependent signal transduction pathway. The activated NF-κB up-regulates the expression of the cell cycle regulatory ccD1 gene that induces phosphorylation of Rb and cell-cycle progression and it also upregulates anti-apoptotic genes BclxL and XIAP [12]. Studies in mammalian cells have shown that the activation of FOXO-3a induces cell cycle arrest and/or apoptosis through the up-regulation of its key target genes such as p27Kip1, Bim, Bclxl and XIAP [13]. EGF has been shown to stimulate the migration of both normal and tumor cells [14]. It has been established that both Akt and Erk cell survival pathways mediate EGF- induced invasion of breast cancer cells via induction of MMP-9 activity [15]. At CDRI, based on structure-activity relationship, 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo (b) pyran (CDRI-85/287) was synthesized as possible anti-cancer and antiestrogenic agent [16], [17]. The compound displays significant anti-estrogenic activity and inhibits uterine growth, as is evident from earlier studies carried out in ovariectomized rats [18], [19] and also interferes with the formation of estrogen- ER complex [20]. The compound has also shown anti-estrogenic potential at uterine level in rhesus monkeys [21]. Further, CDRI-85/287 has shown significant anti-proliferative activity in endometrial cancer cells via inhibition of estrogen receptor pathway and cell survival pathway [22]. Cytotoxic effects of benzopyran based platinum II complexes have been reported earlier in ER- negative breast cancer cells but their mechanism of action have not been explored in these cells [23].We hypothesized that compound may interfere with various signaling mechanisms and inhibit growth in breast cancer cells which do not express ER. In this context, we report here the anti-proliferative activity of benzopyran derivative CDRI-85/287 in ER- negative breast cancer cells. The study demonstrates that CDRI-85/287 exerts its anti-proliferative and anti-invasive properties in ER negative breast cancer cells via preventing EGFR activation and subsequently inhibiting the tumor growth via inhibition PI-3-K/Akt and MEK/Erk pathways. The potent in vitro and in vivo anti-tumor activity of this compound in ER- negative breast cancer cells showed that CDRI-85/287 can serve as a lead candidate molecule for development of efficacious anti-breast cancer agent for the management of disease in patients with EGFR - overexpressing breast tumors. Materials and Methods Compound 2-[piperidinoethoxyphenyl]-3-phenyl-2H-benzo(b) pyran (CDRI-85/287), was synthesized according to the methods as described earlier [18]. Fig. 1A shows the chemical structure of the compound. 10.1371/journal.pone.0066246.g001Figure 1 Anti-proliferative activity of CDRI-85/287 in ER-negative breast cancer cells. (A) Structure and molecular formula of CDRI-85/287. (B) CDRI-85/287 suppresses the growth of ER-negative and EGFR over-expressing breast cancer cells MDA-MB231 and primary breast adenocarcinoma cells. Cells were treated with indicated concentrations of compound for 48h in the absence or presence of 100 ng/ml of EGF. Cell viability was measured using MTT cell viability assay. The percentage of viable cells was calculated as the ratio of treated cells to the control cells. Results are expressed as mean ± SEM, n = 5. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control and e-p<0.001, f-p<0.01, g-p<0.05, h-p>0.05 vs. EGF. Cell lines and cell culture Human breast cancer cell line, MDA-MB 231 and normal kidney cell lines HEK-293 and VERO were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). Cells were cultured at 37°C and 5% CO2. Prior to experiments, cells were cultured in phenol red-free DMEM supplemented with 10% charcoal stripped fetal bovine serum. Primary cell culture of breast adenocarcinoma Breast carcinoma samples were collected in the operating room of the Department of Surgery, King George’s Medical University, Lucknow. A specific written informed consent was obtained from patients, and the study was approved by the Institutional Ethics Committee, King George’s Medical University, Lucknow, India. The breast cancer tissue was collected from 60-65 year old post-menopausal women suffering from infiltrating ductal carcinoma in breast with high grade lesion and metastasis. Pathological report showed that the samples were negative for ER and PR expression and positive for EGFR. The cell isolation was based on the methods of Bresch et al., 1983 [24]. Briefly, tissue were collected in DMEM, minced in 1-mm pieces, and incubated with 1 mg/ml collagenase and DNase (2 mg/ml) in DMEM for 2 h at 37°C with periodic mixing. Digested tissue was mechanically dissociated through a 1-ml tip and resuspended in 10 ml of fresh DMEM. The cells were separated from tissue clumps and debris by decantation and centrifugation, washed twice with DMEM containing 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, and 2% of antibiotic-antimycotic solution (Sigma-Aldrich, USA) and then transferred into plastic culture flasks (75 cm2, Corning, USA). Cells were incubated at 37 °C with saturating humidity and 5% CO2. Prior to experiments, cells were cultured in phenol red-free DMEM supplemented with 10% charcoal stripped fetal bovine serum and 1% antibiotic antimycotic solution. Cell viability assay Cell viability was determined by MTT assay. Cells were seeded (2.5×103 cells/well) into 96-well plate and treated with indicated concentrations of CDRI-85/287 for 48 h. At the end of incubation, MTT [3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (0.5 mg/ml) (Sigma) was added and incubated for 2h at 37°C. After 2h of incubation, supernatants were removed and 100 µl of DMSO was added. The formazan crystals formed inside the viable cells were solubilized in DMSO and the OD was read with Microquant (Biotech, USA) at 540 nm. The IC50 value for the compound was determined by Compusyn software. The experiments were performed three times with five replicates in each. Co-immunoprecipitation assay Interaction between EGF-EGFR proteins was studied by co-immunoprecipitating the complex followed by immunoblotting. Briefly, 2 µg anti-EGFR antibody was added to 500 µg of cell lysate and samples were incubated for overnight at 4°C with constant rocking. In negative control, cell lysate was incubated with corresponding non-immune serum instead of anti-EGFR. Following incubation, 100 µl of Protein A-Sepharose beads suspension was added and samples were incubated for 1 h at 4 °C. Immunoprecipitated complexes were collected by centrifugation at 3000×g for 2min at 4°C and washed three times with RIPA buffer. Immunoprecipitates were resuspended in Laemmli sample buffer to a final concentration of and heated for 5min at 95°C. The supernatants were collected by centrifugation at 12,000×g for 30 s at room temperature. Equal volume of immunoprecipitated proteins were run on 8% SDS-PAGE and transferred on PVDF membrane. The proteins were probed with anti-EGF, followed by the corresponding peroxidase conjugated secondary antibody. Bands were detected and analyzed by densitometry using Quantity One Software (v. 4.5.1) and a Gel Doc imaging system (Bio-Rad). ELISA for EGFR activation Levels of phosphorylated and total EGFR were quantified by using ELISA kit (Invitrogen). In brief, MDA-MB 231 or primary culture cells were treated as indicated in figure 2B. At the end of incubation, cell lysate was prepared by lysing the cells in buffer containing 10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMNaF, 20 mM Na4P2O7, 2 mMNa3VO4, 1%TritonX-100, 10% glycerol, 0.1% SDS and 0.5% deoxycholate supplemented with protease and phosphatase inhibitors. EGFR activation was measured by following the manufacturer’s instructions. OD was taken with Microquant ELISA reader (Biotech, USA) at 450 nm. The experiments were performed three times with three replicates in each. 10.1371/journal.pone.0066246.g002Figure 2 CDRI-85/287 inhibits EGF- EGFR interaction and antagonizes EGF- induced EGFR activation. (A) Effect on EGF-EGFR complex formation as determined by co-immunoprecipitation in MDA-MB 231 cells. Cells were incubated as shown in the figure for 48 h. Cell lysates were immunoprecipitated with anti-EGFR antibody and subsequently immunoblotted with anti-EGF. NC is the negative control. Left panel shows the representative blot showing EGF-EGFR complex and right panel shows the densitometric analysis of bands. The results are presented as mean± SEM of three independent experiments. p values are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>0.05 vs. control. (B, C) Effect of CDRI-85/287 on EGFR activation was determined by ELISA. Cells were treated as indicated in the figure and analyzed for the expression of phosphorylated EGFR and total EGFR using ELISA kit as per manufacturer’s instructions. Activation of EGFR at different concentrations (left panel) and at various time points (right panel) in (B) MDA-MB 231 and (C) primary breast adenocarcinoma cells was determined by normalizing the expression of p-EGFR with EGFR. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. Molecular Docking Analysis The docking study was performed in AUTODOCK [25]. Prior to the docking, tyrphostin (AG-1478) and CDRI-85/287 were generated in SYBYL [26] and were subjected to optimization for their geometry using the Powell energy minimization algorithm, Gasteiger-Huckel charges, and 0.001 kcal/(mol.Å) as convergence criteria. The protein coordinates of EGFR from the EGFR - lapatinib (GW572016) co-crystal (PDB code: 1XKK) [27] were considered for investigating the binding modes of AG-1478 and CDRI-85/287. For docking study, the ligand-free protein was prepared in SYBYL by making use of the same procedure as adopted in case of study molecules. In SYBYL, using fit atom method, AG-1478 and CDRI-85/287 were positioned in 3D-space matching with lapatinib of co-crystal. The prepared EGFR protein and the corresponding study molecule were considered in AUTODOCK for the docking experiments. The grid size for the search of docking space was set at 60×60×60 distributed around the binding domain with a default grid spacing of 0.375 Å. The Lamarckian genetic algorithm was used for docking the molecules. For each molecule the docking was done with 100 runs. The molecules were allowed to flexibly dock into the protein coordinates (1XKK) to take their final conformation. The best docked conformers were collected from a population of 150 samples from 2.5 million energy evaluations. Along with AUTODOCK, Pymol [28] and MOE [29] software were used to visualize the interactions of protein and docked molecules. In order to validate the docking experiments lapatinib was also docked into the protein coordinates independent of its co-crystal structure. It has occupied almost the same position as that of lapatinib in co-crystal structure. Western blot analysis CDRI-85/287 and vehicle treated cells and tumors from xenograft model were lysed in RIPA buffer (50 mM Tris pH-7.4, 150 mM NaCl, 1% nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS, 1 µM sodium orthovanadate) supplemented with protease inhibitor cocktail (Sigma) and 1 mM PMSF. Supernatant was collected by centrifugation at 13,000 rpm for 10 minutes. Equal amounts of protein were separated by gel electrophoresis and then transferred to Immuno-Blot™ PVDF membrane (Millipore). The membrane was blocked with 5% skimmed milk and then incubated with appropriate primary antibody overnight at 4 °C. The membrane was then washed and incubated with a secondary peroxidase conjugated antibody for 1 h. Antibody binding was detected using enhanced chemiluminescence detection system (GE Healthcare). After developing, the membrane was stripped and re-probed using another primary antibody of interest or β-actin to confirm equal loading. Each experiment was repeated three times to assess for consistency of results. Quantitation of band intensity was performed by densitometry using Quantity One® software (v.4.5.1) and a Gel Doc imaging system (Bio-Rad). Anti-PCNA, p-PI-3-K, PI-3-K, p-Akt, Akt, pNF-κB, NF-κB, p27, p-FOXO-3a, FOXO-3a, p-Rb, Rb, cyclin D1, EGF and anti- β-actin antibodies were purchased from Santa Cruz, CA, USA. Antibodies for EGFR, Bcl2, Bax, Bclxl, XIAP, p-MEK, MEK, p-Erk, Erk, c-fos, c-jun, cleaved caspase-3, -8, -9, and cleaved PARP were procured from Cell Signalling Technology, USA. Transient transfection and transactivation assay MDA-MB 231 cells were seeded in 24 well plate and allowed to attain a confluency of 80–90%. Cells were then transfected with 100 ng of pAP1-Luc or p-c-fos-Luc or p-c-jun-Luc (Stratagene La Jolla, CA) using Lipofectamine -2000™ transfection reagent (Invitrogen) as per manufacturer’ protocol. To normalize for transfection efficiencies, 50 ng of pRL-SV40-luc (Promega, USA) was co-transfected. After 5 h of transfection, medium was changed and cells were treated with EGF, different concentrations of compound CDRI-85/287 or EGFR inhibitor, AG1478. After 18 h cells were lysed with passive lysis buffer. Luciferase activity was measured using Dual Luciferase Assay System (Promega) according to the manufacturer’s protocol to detect the transcriptional activity of the transfected promoter. The firefly luciferase intensity for each sample was normalized with transfection efficiency measured by renilla luciferase activity [30]. The experiments were performed three times with three replicates in each. Immunoflouresence imaging and confocal microscopy Cells were grown on coverslips in 12-well plate and treated with vehicle, 3 and 7.5 µM of CDRI-85/287 in MDA-MB 231 and primary breast adenocarcinoma cells respectively for 24h. Cells were then fixed in methanol and acetone in 1∶1 ratio at 4°C and permeabilized with 0.1% Triton X-100. Cells were washed with PBS and blocked with 1% BSA and incubated with NF-κB antibody for overnight followed by 1h incubation with fluorescence-tagged secondary antibody, then counterstained with DAPI for 5 min. Images were captured at 63X using Carl Zeiss LSM 510 META microscope and analyzed using LSM Image Examiner Software to detect fluorescence and DAPI emissions. Apoptosis analysis by flow cytometry Human breast cancer MDA-MB 231 cells and primary breast cancer cells (2×105 cells/ml) were cultured in 6-well plates and treated either with ethanol(vehicle) or with different concentrations of the compound. Adherent and detached cells were then harvested after 48 h, centrifuged and resuspended in 1 ml phosphate buffered saline (PBS). The Annexin V—FITC (fluorescein isothiocyanate)-labeled apoptosis detection kit (Sigma Chemical Co.) was used to detect and quantify apoptosis by flow cytometry as per manufacturer's instructions. Cells were analyzed using a flow cytometer (Becton Dickinson), and data were analyzed with CellQuest software. Cell cycle analysis by flow cytometry Cells were seeded (2×105cells/ml) in 6-well plates and treated with varying concentrations of CDRI-85/287 for 48h. After treatment, cells were washed with Phosphate buffered saline (PBS), fixed in 75% chilled ethanol, treated with RNase and then stained with propidium iodide (PI) (Sigma) solution (50 µg/ml). Cell cycle distribution was analyzed with fluorescence-activated cell sorter (Model FACS Calibur, BD biosciences, USA) and Cell- Quest software. The percentage of DNA content at different phases of the cell cycle was analyzed with Modfit-software (Verity Software House, ME, USA). The experiments were performed three times with three replicates in each. Caspase-3 colorimetric assay Caspase-3 activity was measured using the colorimetric Caspase-3 assay kit (Sigma). Briefly, treated cells were trypsinized and centrifuged for 5min at 600 xg at 4°C. Then cell pellets were resuspended in ice-cold cell lysis buffer and incubated on ice for 20 min. At the end of the incubation, cell lysates were centrifuged at 20,000 x g for 10 min at 4°C. Supernatants were then incubated with 1 mM caspase-3 substrate (DEVD-pNA) for 2h at 37°C and the OD was measured at 405 nm. The experiments were performed three times with three replicates in each. Measurement of mitochondrial membrane potential (Δψm) Δψm was estimated using JC-1 (cationic mitochondrial vital dye) as a probe using method as described previously [21]. Briefly, treated cells were collected and incubated for 20min with 5 µM JC-1 at 37°C, washed, and resuspended in media, and Δψm was measured at 590 nm for J-236 aggregates and at 530 nm for J-monomer. The ratio of 590/530 nm was considered as the relative Δψm value. The experiments were performed three times with three replicates in each. Transwell migration assay Cell invasion assays were conducted in a 24-well format using matrigel-coated invasion chambers (BD Matrigel™Invasion Chambers; Cat # 354480) having a membrane pore size of 8.0 µm. Briefly, 2×105 cells (MDA-MB-231) were seeded into the top chamber. The next day vehicle, EGF, 3 µM of compound, compound along with EGF and EGFR inhibitor was added into the bottom well for 12h. At the end of the treatment, the cells were post-stained with 0.1% crystal violet stain. The cells that invaded the BD Matrigel Matrix and passed through the pores of the BD FluoroBlok membrane were detected, photographed and counted under an inverted microscope (Nikon ECLIPSE TE2000-S, Nikon, Singapore). Gelatin zymography Zymography was performed in 10% polyacrylamide gels that had been cast in the presence of gelatin [15]. Briefly, samples (culture media) were resuspended in loading buffer and run on a 10% SDS-PAGE gel containing 0.5 mg/mL gelatin without prior denaturation. After electrophoresis, gels were washed to remove SDS and incubated for 30 min at room temperature in a renaturing buffer (50 mM Tris, 5 mM CaCl2, 0.02% NaN3, 1% Triton X-100). In the next step, gels were incubated for 48 h at 37°C in developing buffer [50 mM Tris-HCl (pH 7.8) 5 mM CaCl2, 0.15 M NaCl and 1% Triton X-100] and then subsequently stained with Coomassie Brilliant Blue G-250 and destained in 30% methanol, 10% acetic acid to detect gelatinase secretion. Tumour xenografts preparation in mice All experimental procedures of this study were approved by Institutional Animal Ethics Committee (IAEC) of CDRI, Lucknow and IAEC of INMAS (DRDO), Delhi. Briefly, MDA-MB 231cells in normal saline were implanted subcutaneously into 6-week-old athymic nude mice bearing the nu/nu gene [NIH(s) (nu/nu)]. After the tumors attained the size of approx 2000 mm3, mice were treated with CDRI-85/287 (10 mg/kg body weight doses, per day for 16 days, p.o.) Animals of control group were treated with vehicle only. Xenograft tumor size was measured using Vernier callipers (major and minor axis) and tumor volume was calculated according to the equation: (L X W2)/2 (mm3), where L = length and W = width. After euthanasia, animals were dissected for removal of tumors and various other organs for fixation in 4% formaldehyde for routine histology. Tissues were processed as per standard protocol. Statistical Analysis Results are expressed as mean ±SEM for at least three separate determinations for each experiment. Statistical significance was determined by ANOVA and Newmann Keul’s test (for in vitro) or Student’s t-test (for in vivo studies). p values < 0.05 were considered significant. Results CDRI-85/287 strongly impaired the growth of EGFR over-expressing breast cancer cells and primary breast adenocarcinoma cells CDRI-85/287 selectively inhibited the growth of estrogen receptor-negative and EGFR over-expressing MDA-MB 231 breast cancer cells while being non-toxic to normal human cells. (Fig. 1B). Treatment with the compound led to decrease in cell viability in a concentration dependent manner with IC50 of 3.7 µM in MDA-MB 231 cells (p<0.001). We also assessed the effect of compound on primary cells obtained from human breast carcinoma over-expressing EGFR. The compound inhibited the growth of these cells with IC50 of 7.9 µM (Fig. 1B). The compound was also found to significantly inhibit EGF-induced proliferation (p<0.001vs. EGF at 1 µM in MDA-MB 231 and p<0.01 vs. EGF at 2.5 µM in primary breast cancer cells) suggesting that it acts by antagonising EGF action in inhibiting proliferation of these cells. In normal kidney cell lines HEK-293 and VERO cells, the compound treatment did not cause significant inhibition of cell viability, upto 40 µM concentration (Fig. S1). CDRI-85/287 inhibits EGF- EGFR interaction and antagonises EGF- induced EGFR activation To identify the molecular characteristics responsible for compound’s toxicity, we sought to investigate the diverse biological responses triggered in ER-ve breast cancer cell lines by the compound. Since the compound was found to inhibit EGF- induced proliferation of breast cancer cells, we went onto see if the compound also prevents binding of EGF to EGFR. Co-immunoprecipitation studies indicated that CDRI-85/287 like AG1478 significantly reduced the formation of EGF-EGFR complex both per se (p<0.01) and in presence of EGF (p<0.01) (Fig. 2A). Further, we analyzed the effect of compound on EGFR activation using ELISA (Fig. 2B,C). After 30 min of incubation, the compound significantly decreased phospho form of EGFR which further decreased with time upto 3 h in both MDA-MB 231 cells(p<0.05 to p<0.001) and primary breast adenocarcinoma cells (p<0.01 to p<0.001) while 100 ng/ml of EGF induced tyrosine phosphorylation of EGFR (p<0.01). In a dose-dependent study, the compound inhibited both basal as well as EGF-induced activation of EGFR. At 2 µM concentration, the compound significantly inhibited (p<0.05) the phosphorylation of EGFR and the decrease was more significant at 3 µM (p<0.01) in presence of EGF in case of MDA-MB 231 cells. In primary breast cancer cells, significant inhibition of EGFR activation was observed at 5 µM (p<0.01) both in absence and in presence of EGF. Significant change in the level of total EGFR was not observed in the two cell lines on treatment with indicated concentrations of the compound. CDRI-85/287 displays a higher binding affinity for EGFR than AG1478 The docking experiments indicate that AG-1478 and CDRI-85/287 occupy a position in ATP-binding pocket of EGFR which is comparable to that of lapatinib. In the binding pocket, the quinazolin moiety of lapatinib occupied the central place with benzyloxyphenyl and furan-methylamine moieties extending to its (quinazolin) either side (binding energy -11.61 Kcal/mol) (Fig. 3A). However, in the binding pocket, AG-1478 has shown only partial occupancy (binding energy: −8.47 Kcal/mol; IC50 = 10 µM). The docking poses have indicated that the AG-1478’s quinazolin and 3-chloro aniline moieties have respectively occupied the positions of quinazolin and benzyloxyphenyl moieties of lapatinib. AG-1478 has no structural moiety to satisfy the space corresponding to furan-methylamine moiety of lapatinib (Fig. 3B). This may be a reason for AG-1478’s weak binding to EGFR. Interestingly the docking pose as well as binding pocket occupancy of CDRI-85/287 is comparable to that of lapatinib (binding energy: -10.01 Kcal/mol; IC50 = 2.5 µM) (Fig. 3C). Here the chroman and N-ethyl-piperidinyl moieties of CDRI-85/287 have occupied the binding areas of benzyloxyphenyl and furan-methylamine moieties of lapatinib, respectively. The aryl-bridge between the chroman and N-ethyl-piperidinyl moieties of CDRI-85/287 has served the purpose of quinazolin moiety of lapatinib. The differences in the binding energy of AG-1478 and CDRI-85/287 to EGFR have corroborated their activity. 10.1371/journal.pone.0066246.g003Figure 3 Molecular Docking analysis of CDRI-85/287 with EGFR. (A) Schematic partitioning of Lapatinib in the ATP-binding pocket. In the binding pocket quinazolin moiety of lapatinib has occupied the central place with benzyloxyphenyl and furan-methylamine moieties extending to its either side (wing I & II respectively). (B) Schematic partitioning of AG 1478 in the ATP-binding pocket. The chlorobenzene moiety of AG 1478 are designated as wing-I & dimethoxy moiety partially represent wing II site. The quinazoline moiety of AG 1478 has acted as central part of the scaffold. (C) Comparison of CDRI 85/287 structural moieties with AG 1478. (D) Docked conformation of AG 1478 with wild type EGFR protein (PDB ID. 1XKK). Red dash indicate H-bond interaction site with of EGFR where cyan, magenta & salmon colour specify alpha-helix, beta-sheet & loop regions of protein correspondingly. (E) Docked conformation of CDRI 85/287 analogue with EGFR protein (PDB ID 1XKK). Red dash shows H-bond interaction site with same protein site. (F) Superimposition of AG 1478 & CDRI 85/287 in a same protein binding pocket shows H-bond interaction with ASP855 & MET793 residues respectively. (G) Superimposition of Lapatinib (magenta), AG 1478 (green) and CDRI 85/287 derivative (Cyan). (H) Schematic 2D-representation of AG 1478. (I) proposed binding mode of CDRI 85/287 analogue with surrounding residues in the ATP-binding pocket of EGFR. CDRI-85/287 interferes with MEK/Erk activation and downstream signaling Next, we sought to study the effect of compound on MEK/Erk pathway involved in cell proliferation and invasion. In MDA-MB 231 cells, EGF significantly induced the activation of MEK which in turn led to activation of downstream MAPK, Erk (Fig. 4A). The compound at 3 µM significantly led to inhibition of MEK (p<0.001vs. control) and Erk (p<0.05vs. control) activation as evidenced by decreased expression of phospho forms of the proteins. Likewise, in primary breast adenocarcinoma cells the compound interfered with Erk activation leading to decreased levels of p-Erk (p<0.05 vs control) both in presence and in absence of EGF (Fig. 4A). 10.1371/journal.pone.0066246.g004Figure 4 CDRI-85/287 interferes with MEK/Erk activation to inhibit downstream signaling. (A) Western blot analysis to see the expression of p-MEK, MEK, p-Erk and Erk. MDA-MB 231 (left panel) and primary breast cancer cells (right panel) were treated with the indicated concentrations of CDRI-85/287 for 24h and 30 µg of whole cell lysate in each lane was probed for the expression of different proteins using specific antibodies. Representative blots are shown in the upper panel and densitometric quantitation of protein expression levels are shown as fold changes in the lower panel. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. (B) Transcriptional activation of the AP-1 promoter complex in transiently transfected MDA-MB 231 cells in response to CDRI-85/287 alone or in the presence of 100 ng/ml EGF. MDA-MB 231 cells were transfected with pAP1-Luc or p-c-fos-Luc or p-c-jun-Luc reporter plasmids and incubated with various concentrations of compound for 24h. pRL-luc plasmid was used as internal control. Results are described as % of normalized relative luciferase unit (RLU). Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>0.05 vs. control and e-p<0.001, f-p<0.01, g-p<0.05 and h-p>0.05 vs. EGF. (C) Effect of compound on AP-1 dependent proliferation markers. MDA-MB 231 (left panel) and primary breast cancer cells (right panel) were treated with the indicated concentrations of compound for 48h. 30 µg of whole cell lysate in each lane was probed for the expression of different proteins using specific antibodies. Representative blots are shown in the upper panel and densitometric quantitation of protein expression levels are shown as fold changes in the lower panel. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. Since Erk exerts part of its proliferative activity via AP-1 transcriptional complex, we next studied the effect on AP-1 transcriptional complex. For this we analyzed AP-1 transcriptional activation in MDA-MB 231 cells transiently transfected with AP-1 reporter plasmid. EGF increased the promoter activity of AP-1 transcriptional complex while the compound inhibited AP-1 promoter activity (Fig. 4B). The compound also inhibited the transcriptional activity of c-fos and c-jun, components of AP-1 transcriptional assembly in MDA-MB 231 cells transiently transfected with the respective reporter constructs. The compound also inhibited EGF induced transcriptional activation of AP-1 transcriptional machinery. AG1478 decreased the transcriptional activation via AP-1 complex since it blocks EGFR activation upstream. The compound also significantly reduced the levels of AP-1 dependent proliferation markers including PCNA, IGF-1, c-fos and c-jun in both MDA-MB 231 cells and primary breast adenocarcinoma cells (Fig. 4C). Modulation of PI-3-K/Akt, NF-κB and FOXO signaling and downstream effectors We further analyzed several major components of signaling pathways downstream of EGFR. We analyzed the effect of the compound on activation of PI-3-K and its downstream Akt. EGF significantly induced phosphorylation of PI-3-K which in turn increased phosphorylation of Akt (p<0.01vs.control) while the compound decreased the phosphorylated forms of the proteins, PI-3-K (p<0.01) and Akt (P<0.05). The compound significantly decreased the phosphorylation of the proteins even in the presence of EGF (p<0.05 vs control) thus showing that the compound acts by antagonizing EGF in deactivating these proteins and preventing further downstream signaling in both MDA-MB 231 and primary breast cancer cells (Fig. 5). The next objective was to study the effect of compound on NF-κB activation. The compound significantly reduced the phosphorylated form of NF-κB protein as evidenced by western blotting. Another downstream target of Akt is the Forkhead transcription factor FOXO-3a, which in its active form leads to transcription of genes involved in cell cycle arrest and prevents cellular proliferation [13]. The compound CDRI-85/287 decreased the expression of phosphorylated FOXO-3a in both MDA-MB 231 and primary breast cancer cells in contrast to EGF which in turn prevented its proteasomal degradation leading to increased expression of FOXO-3a protein confirmed by immunoblotting studies (Fig. 5). This unphosphorylated FOXO-3a remains in active form and would lead to modulation of cell cycle regulatory genes. 10.1371/journal.pone.0066246.g005Figure 5 CDRI-85/287 modulates PI-3-K/Akt, NF-κB and FOXO-3a pathway. Analysis of PI-3-K, Akt, NF-kB and FOXO phosphorylation in (A) MDA-MB 231 and (B) primary breast adenocarcinoma cells. Cells were treated with the indicated concentrations of compound for 24h and 30 µg whole cell lysate in each lane was probed for the expression of different proteins using specific antibodies. β-actin was used as a control to correct for loading. Representative blots are shown in left panel and densitometric analysis of bands is shown in right panel. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. Next, we analyzed the localization pattern of NF-κB under the effect of compound. Immunoblotting experiment displayed that the compound decreased both basal as well as EGF-induced expression of nuclear NF-κB while increased expression in cytosolic fraction (Fig. 6A). These results were validated by confocal microscopy that clearly indicates that 3 µM of CDRI-85/287 significantly decreased the nuclear pool of NF-κB protein (Fig. 6B). 10.1371/journal.pone.0066246.g006Figure 6 Effect of compound on downstream effectors of NF-κB and FOXO-3a. (A) Western blot analysis to see the nuclear and cytosolic expression of NF-κB in MDA-MB 231 cells. Cells were treated as shown in the figure for 48h. Nuclear and cytosolic proteins were extracted following manufacturer’s instructions and subjected for immunoblotting using anti-NF-κB antibody. Representative blots are shown in the left panel and densitometric quantitation of protein expression levels are shown as fold changes in the right panel. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. (B) Confocal microscopy to demonstrate the effect of compound on localization pattern of NFκB (p65). MDA-MB 231 and primary breast cancer cells were treated with vehicle or indicated concentration of compound for 24 hrs. Cells were fixed, permeabilized, incubated with NFκB antibody for overnight, and incubated with FITC-conjugated anti-rabbit antibody for 1 h. The preparations were washed and counterstained with DAPI and images were captured at 63X using Carl Zeiss LSM 510 META microscope. Representative micrographs demonstrating the subcellular distribution of NFκB are shown. (C) Transcriptional activation of the NF-κB promoter in transiently transfected MDA-MB 231 cells in response to compound either alone or in the presence of 100 ng/ml EGF. MDA-MB 231 cells were transfected with pNF-κB-Luc reporter plasmids and incubated with various concentrations of CDRI-85/287 for 24h. pRL-luc plasmid was used as internal control. Results are described as % of normalized relative luciferase unit (RLU). Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>0.05 vs. control and e-p<0.001, f-p<0.01, g-p<0.05 and h-p>0.05 vs. EGF. (D) Western blot analysis for the expression of cell cycle regulatory and anti-apoptotic proteins (Bclxl and XIAP) in MDA-MB 231 (left panel) and primary breast cancer cells (right panel). Cells were treated with the indicated concentrations of compound for 48h and 30 µg whole cell lysate in each lane was probed for the expression of different proteins using specific antibodies. β-actin was used as a control to correct for loading. Representative blots are shown. Densitometric analysis of bands is shown in Fig. S2. Further, the basal as well as EGF-induced promoter activity of NF-κB determined by transient transfection and transactivation assay in MDA-MB 231 cells was also found to decrease with increasing concentrations of the compound (p<0.001) (Fig. 6C). Next, we observed the effect of compound on downstream effectors of NF-κB and FOXO-3a. The compound led to increase in the expression of cell cycle dependent kinase inhibitor p27 in both MDA-MB231 and primary breast cancer cells (Fig. 6D). Increased levels of p27 led to decrease in the expression of cyclin D1 by the compound which in turn prevented phosphorylation of Rb protein maintaining it in unphosphorylated state as evidenced by the expression of total Rb. Unphosphorylated Rb sequesters and inactivates E2F preventing cell cycle progression from G1 to S phase by inhibiting the transcription of E2F dependent genes. CDRI-85/287 also decreased the expression of Bclxl and XIAP, the anti-apoptotic members of Bcl2 family. The results suggest that the compound exerts its effect via modulation of cell cycle progression and apoptosis induction. CDRI-85/287 caused G1/S phase arrest in EGFR-overexpressing breast cancer cells We next studied the effect of CDRI-85/287 on cellular distribution at different stages of the cell cycle in breast cancer cells. For this, flow cytometric analysis of PI-stained cells was done. CDRI-85/287 led to increased accumulation of cells in G1 phase of the cycle in both MDA-MB 231 and primary breast cancer cells (p<0.01 at 3 µM and 7.5 µM in MDA-MB 231 and primary breast cancer cells respectively), delaying progression into synthesis phase (Fig. 7A). G1/S phase arrest was further confirmed by studying the expression of cyclins, cdks and CKIs. The compound significantly reduced the expression of cyclin D1, cyclin E1, cdk4 and increased the expression of cell cycle inhibitory proteins p21 and p27 in a concentration dependent manner in MDA-MB231 cells (Fig. 7B). However, the expression of cyclin A1 was not significantly altered by the compound. The results clearly indicated that CDRI-85/287 inhibits growth of ER-negative breast cancer cells by inhibiting cell cycle progression from G1 to S phase. 10.1371/journal.pone.0066246.g007Figure 7 Inhibition of cell cycle progression by CDRI-85/287. (A) Cell cycle analysis by flow cytometry. MDA-MB 231 and primary breast cancer cells were treated with the indicated concentrations of compound for 48h. The cells stained with propidium iodide (PI) were subjected to flow cytometric analysis to determine the percentage of cells at each phase of the cell cycle. Representative images of flow cytometry of vehicle and compound treated cells are shown in the upper panel and the percentage of cell with SEM based on three independent experiments is shown in the lower panel. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. (B) Western blot analysis to see the expression of cell cycle regulatory proteins cyclin D1, A1, B1, cdk4, p21 and p27. MDA-MB 231 and primary breast cancer cells were treated with the indicated concentrations of compound for 48h and 30 µg whole cell lysate in each lane was probed for the expression of different proteins using specific antibodies. β-actin was used as a control to correct for loading. Representative blots are shown in the upper panel and densitometric quantitation of protein expression levels are shown as fold changes in the lower panel. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. CDRI-85/287 induced apoptosis in MDA-MB 231and primary breast adenocarcinoma cells via mitochondrial pathway Decrease in the expression of anti-apoptotic proteins XIAP and Bclxl prompted us to check if the loss in cell viability on treatment with the compound is due to induction of apoptosis. For this we did flow cytometric analysis of Annexin V/PI stained cell treated with vehicle or different concentration of the compound. The compound significantly increased the percentage of apoptotic cells in a concentration-dependent manner, and more than 50% of the cells displayed apoptosis at 5 µM in MDA-MB 231(p<0.001) and at 7.5 µM in primary breast cancer cells (p<0.001) corresponding with the results of cell proliferation assay (Fig. 8A). 10.1371/journal.pone.0066246.g008Figure 8 Analysis of apoptosis in MDA-MB 231 and primary breast adenocarcinoma cells. (A) Cells treated with vehicle and CDRI-85/287 were analyzed by flow cytometry of annexin-V/PI stained cells after 48h culture. AV+/PI− - intact cells; AV−/PI+ -nonviable/necrotic cells; AV+/PI− and AV+/PI+ - apoptotic cells. Representative images are shown in the upper panel and the percentage of cell fraction with SEM based on three independent experiments is shown in the lower panel. p values are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>0.05 vs. control. (B) Western blot analysis to see the expression of pro- and anti- apoptotic proteins. MDA-MB 231 and primary breast cancer cells were treated with the indicated concentrations of compound for 48h, and 30 µg whole cell lysate in each lane was probed for the expression of different proteins using specific antibodies. β-actin was used as a control to correct for loading. Representative blots are shown in the upper panel and densitometric quantitation of protein expression levels are shown as fold changes in the lower panel. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. Apoptosis was also evident upon examination of caspase-3 activity in MDA-MB 231 cells which was determined by incubating the cellular extracts with caspase-3 substrate (DEVD-pNA). Caspase 3 activity was significantly up-regulated by 1.5- fold and 2.5-fold in MDA-MB 231 cells that were treated with 2 and 3 µM of CDRI-85/287 respectively (p<0.05 at 1 µM to p<0.001 at 3 µM) (Fig. S3A). However in presence of caspase-3 inhibitor, no change in caspase-3 activity was observed on treatment with the compound. These results indicated that compound exerts its anti-proliferation activity in tumor cell-specific manner by activating the caspase pathway followed by induction of apoptosis. Further we went on to explore the pathway involved in execution of apoptosis. Alterations in the mitochondrial transmembrane potential (ψm) has been shown to be important for the release of mitochondrial proteins such as cytochrome c, which when in the cytosol can lead to activation of the caspase cascade and subsequent death [31]. Therefore, we measured Δψm using the JC-1 dye and the results showed a significant drop (p<0.001) in Δψm in the presence of 3 µM of CDRI-85/287 in MDA-MB 231 and at 7.5 µM in primary cancer cells (Fig. S3B). These results indicate that the apoptotic-signaling pathway activated by compound is likely to be mediated via the mitochondrial pathway. Next, we evaluated the levels of the pro- and anti-apoptotic proteins. While the level of Bax increased at protein level (Fig. 8B), compound triggered a dose-dependent decrease in the levels of anti-apoptotic Bcl2 in both cancer cell lines. CDRI-85/287 elicited a dose-dependent increase in the Bax/Bcl2 ratio, reaching upto 15-fold (p<0.001) and 9-fold (p<0.001) at IC50 concentration in MDA-MB231 and primary cancer cells respectively, thus mediating induction of apoptosis via intrinsic pathway. To further elucidate the role of CDRI-85/287 in inducing apoptosis, cells were treated with indicated concentrations of the compound and the whole cell lysates were probed for the expression of cleaved caspase-8, -9, -3 and cleaved PARP. Compound treatment caused a dose-dependent increase in the level of cleaved caspase-9, -3 and cleaved PARP in MDA-MB 231 and primary cancer cells. The expression of cleaved caspase-8 was not observed excluding the possibility of involvement of extrinsic pathway in the action of compound. Inhibition of EGF-induced MDA-MB 231 cell invasion by CDRI-85/287 We next studied the effect of compound on EGF-induced invasion of MDA-MB 231 (Fig. 9A). We found that EGF significantly increased the invasion of MDA-MB 231 cells (p<0.001). The level of EGF-induced cell invasion was 157% of control group. However, both basal as well as EGF- induced cell invasion were suppressed by treatment with 3 µM of CDRI-85/287 (p<0.01). EGF-induced cell invasion was decreased by 72% of control level in response to 3 µM of compound. 10.1371/journal.pone.0066246.g009Figure 9 Effect of compound CDRI-85/287 on the invasive properties of MDA-MB 231 cells. (A) Transwell migration assay. MDA-MB231 cells were seeded in the upper chamber with vehicle, EGF, CDRI-85/287, EGF along with the compound CDRI-85/287 or AG1478 added in the lower chamber. Cells that migrated to the low chamber were fixed, stained, and counted by light microscopy as described in Methods (left). These results are representative images of three independent experiments. The graph in the right panel shows the average number of migrated cells in three wells from three independent experiments (random fields were scanned and number of cells from four fields per well were counted). Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. (B) Inhibition of MMP-9 activity by CDRI-85/287 in breast cancer cells. MDA-MB 231 cells were treated as indicated for 12 h, conditioned media was collected and MMP activity was analyzed by gelatin zymography. Upper panel shows the representative gel image and lower panel shows the densitomentric analysis of band intensity. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. (C) Western blot analysis to demonstrate the effect of CDRI-85/287 on the expression of CTGF in conditioned media. Cells were treated with increasing concentrations of CDRI-85/287 for 24 h, conditioned media was collected and subjected to western blotting for the expression of CTGF. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control Next, we investigated if the compound suppresses MMP-9 activity in MDA-MB 231. MMP-9 (92 kDa gelatinase) is particularly known to play a critical role in cancer progression, such as angiogenesis as well as tumor growth, invasion and distant metastasis of various tumors and breast cancer as well [10], [15]. We found that EGF induced MMP-9 activity (p<0.05) while the compound decreased it significantly both in absence and in presence of EGF (p<0.001 and p<0.01 respectively in comparison to the control group) (Fig 9B). We also analyzed the expression of CTGF in conditioned media of MDA-MB 231 cells and found a concentration dependent decrease in the expression of CTGF secreted by these cells (p<0.05 to p<0.01 vs. control). Therefore, we demonstrated that CDRI-85/287 suppresses EGF-induced cell invasion through inhibition of MMP-9 activity and CTGF expression in MDA-MB 231 cells (Fig 9C). Benzopyran derivative CDRI-85/287 suppresses the growth of tumor xenograft in nude mice In vivo studies demonstrated that the compound at 10 mg/kg body weight/day significantly reduced tumor volume in xenograft mice model (Fig. 10A). The average tumor size in this group was 66% lower after 16 days of treatment compared with vehicle treated group (Fig. 10C). Histological assessment showed that tumors from vehicle-treated mice were primarily composed of tumor epithelial cells with small amounts of mouse-derived stroma and frequent blood vessels. Tumors from mice treated with CDRI-85/287 presented with large areas of stroma where deletion of epithelial cells had occurred. Cellular apoptosis was also evident in compound treated group (Fig. 10B) in comparison to the tumor from the vehicle treated group. Significant change was not observed in body weight in the vehicle and treated group (Fig 10D). 10.1371/journal.pone.0066246.g010Figure 10 Effect of CDRI-85/287 on tumor regression in MDA-MB 231 xenograft mouse model. (A) Representative images of MDA-MB 231 xenograft nude mice showing regression in tumor volume after treatment with CDRI-85/287(10 mg/kg, p.o.) in comparison to vehicle treated (VT) group. (B) Upper panel shows the dissected out tumors from vehicle treated and CDRI-85/287 treated xenograft mice. Lower panel shows representative images of hematoxylin and eosin stained tissue sections from vehicle- and CDRI-85/287 treated groups. (C) Graph showing tumor volume changes within 16 days after initiation of treatment (marked by arrow). Number of animals per group = 6 to 8. (D) The average body weights of the mice during tumor development and treatment (marked by arrow). Number of animals per group = 6 to 8. Histomorphology did not show any marked change in liver, lungs, spleen, uterus and kidney of the compound treated mice as compared to vehicle treated control group (Fig. S4). CDRI-85/287 inhibits EGFR activation, MEK/Erk and PI-3-K/Akt pathway in xenograft tissue Treatment with the compound led to significant reduction in phosphorylated forms of EGFR (p<0.001) and Erk (p<0.01) in comparison to vehicle treated group. A significant decrease was also observed in the expression of PCNA (p<0.01), c-fos (p<0.05), c-jun (p<0.01)and IGF-1(p<0.01) in compound treated group in comparison to vehicle treated mice (Fig. 11A). 10.1371/journal.pone.0066246.g011Figure 11 Effect of CDRI-85/287 on EGFR pathway in xenograft tissue. Western blot analysis to determine the effect of CDRI-85/287 on (A) activation of EGFR and MEK/Erk pathway, (B) activation of PI-3-K/Akt and expression of downstream effectors, and (C) the expression of pro- and anti- apoptotic proteins in vehicle and CDRI-85/287 treated xenograft tissue. 30 µg whole cell lysate in each lane was probed for the expression of different proteins using specific antibodies. β-actin was used as a control to correct for loading. Representative blots are shown in the left panel and densitometric quantitation of protein expression levels are shown as fold changes in the right panel. Results are expressed as mean ± SEM, n = 3. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. Western blot analysis indicated that the compound led to a decrease in the phosphorylated forms of Akt, NF-κB and FOXO-3a leading to decreased activation of NF-κB and increased activation of FOXO-3a in tumors from CDRI-85/287 treated xenograft mice in comparison to vehicle treated group. This in turn resulted in increased levels of p27 and decreased levels of cyclin D1, phosphorylated Rb and decreased expression of total Rb in compound treated xenograft tumor (Fig. 11B). Further, significant decrease was observed in the expression of anti-apoptotic protein Bclxl, XIAP and Bcl2 whereas the expression of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP was increased in compound treated mice (Fig. 11C). These results showed that compound caused the induction of apoptosis via intrinsic pathway and reduced the tumor growth. Discussion In search for novel agents for successful therapies of breast cancer, EGFR has long been used as a target for therapeutic intervention in ER-ve as well as triple negative breast tumors that do not respond to endocrine therapy [1]. In the present study, we have examined the effects of 2-[piperidinoethoxyphenyl] -3- phenyl- 2H-benzo (b) pyran (CDRI-85/287) on EGFR -mediated signaling and cell survival/apoptosis in ER- negative human breast cancer cells and in xenograft mice model. Initial findings suggest that CDRI-85/287 showed cytotoxic effects in ER- negative breast cancer cells MDA-MB231 and in human primary culture and caused regression in tumor size in MDA-MB 231 xenograft in mice. While exploring the mechanistic action of compound, we found that the compound exerts anti-proliferative and anti-invasive activity via interfering with EGF binding to EGFR and inhibition EGFR activation as observed in MDA-MB 231 and primary breast cancer cells. Docking studies revealed that CDRI-85/287 displayed better binding affinity to EGFR in comparison to the EGFR inhibitor AG1478. It has also been demonstrated by several investigators that transient activation of ERK1/2, a downstream effector of EGFR, also plays a pivotal role in cellular proliferation and leads to cell cycle arrest and causes induction of apoptosis [32]. The MEK/Erk pathway exerts its growth promoting effects via AP-1 transcriptional complex. Thus inhibition of Erk activation caused by the compound led to inhibition of activation of AP-1 transcriptional complex as evidenced by decrease in the EGF -induced transcriptional activation via AP-1, c-fos and c-jun. This in turn led to decreased expression of proliferation markers and prevented growth of breast cancer cells. These findings co-related well with the results obtained from in vivo studies in MDA-MB 231 tumor xenograft where anti-tumor response was observed in 85/287- treated mice. The mitogenic effect of EGF is also mediated via activation of PI-3-kinase and IκK-dependent activation of NF-κB and inhibition of FOXO activity [12]–[14]. Our results indicated that the compound CDRI-85/287 inhibited the activation of PI-3-K and Akt which led to inhibition of activation of downstream effector NF-κB and enhanced activation of FOXO-3a. The transcription factor NF-κB is well established as a regulator of genes encoding cytokines, cytokine receptors, and cell adhesion molecules that drive immune and inflammatory responses. It can regulate genes involved in both proliferation and apoptosis including anti-apoptotic genes BclxL and XIAP and growth inducible ErbB2 and cyclin D1 [33]. On the other hand FOXO-3a is known to suppress the activity of NF-κB by inducing IκB-Ras1 [34]. Upon treatment with our compound, the activation of FOXO-3a further led to increased expression of p27kip, cyclin dependent kinase inhibitor and decreased expression of cyclin D1. Decreased cyclin D1 expression led to decreased phosphorylation of tumor suppressor Rb protein thus maintaining it in active state. Active Rb sequesters E2F preventing transcription of genes involved in cell cycle progression from G1 to S phase. This was confirmed by concentration dependent decrease in the expression of cyclin E1, A1, D1, cdk4 and increased expression of cell cycle inhibitory proteins p21 and p27. The decreased activity of NF-κB and increased activity of FOXO-3a led to decrease in the expression of anti-apoptotic proteins Bclxl and XIAP both in vitro and in vivo leading to induction of apoptosis as evidenced by flow cytometry in MDA-MB 231 and primary breast adenocarcinoma cells. Further, the compound led to decreased Bax: Bcl2 ratio which might be responsible for significant decrease in mitochondrial membrane potential as observed with compound treatment. The reduced expression of Bcl2 and the presence of cleaved fragments of caspases-9,-3 and PARP in treated cells and in xenograft tumors confirmed the involvement of mitochondrial pathway of apoptosis triggered by these compounds. The expression of cleaved caspase-8 was not detectable which confirmed that caspase-8 pathway does not participate in CDRI-85/287 induced apoptosis in ER- negative MDA-MB 231 cells and primary breast cancer cells (data not shown). Finally, CDRI-85/287 arrested tumor growth in a MDA-MB 231 xenograft model of human estrogen receptor-negative breast cancer, showing that the potent effect of this compound could also be manifested in vivo, with concomitant inhibition of EGFR pathway in the treated tumors. It is reported that PI-3-K pathway and MAP kinase pathway are also involved in the EGFR-mediated modulation of cell invasion [35], [36]. Tumor cell migration and invasion is a critical factor for malignant tumor metastasis, which is a multiple process that requires the degradation of the extracellular matrix both at the primary tumor site and at the secondary colonization site. This degradation process is dependent on the activity of specific endopeptidases, the matrix metalloproteinases (MMPs). MMP-9 expression has been related to the invasive property of a variety of cancers including breast carcinoma [15]. MDA-MB 231 is a highly metastatic breast cancer cell line and expresses high level of MMP-9. Our data showed that compound prevented invasion of MDA-MB 231 cells via matrigel membrane and also significantly suppressed the MMP-9 activity in MDA-MB 231 cells. The expression of connective tissue growth factor, CTGF which again is an indicator of the invasive properties of the cell [37] was found to decrease in conditioned media on treatment with the compound. Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, and the regulatory role of CTGF in invasive breast cancer cell phenotypes has already been reported [38]. In conclusion, the present study demonstrates the anti-proliferative and anti-tumorigenic effect of CDRI-85/287 in ER-negative breast cancer cells and in xenograft mouse model, respectively. The study provides evidence that CDRI-85/287 exerts its anti-proliferative and anti-invasive properties via preventing EGFR activation and subsequently inhibits the tumor growth by inhibiting PI-3-K/Akt and MEK/Erk pathways. Inhibition of cell invasion through matrigel membrane and reduced MMP-9 activity confirms that apart from anti-proliferative properties, compound can efficiently block EGF- stimulated invasion of breast cancer cells. Based on these observations, we have hypothesised the working model of CDRI-85/287 as an anti-cancer agent in ER-negative tumors (Fig 12). In total, our observations strongly suggest that benzopyran derivative CDRI-85/287, due to its anti-proliferative and anti-invasive properties, can be rated as a promising candidate for future development as a novel therapeutic treatment strategy for more aggressive forms of breast cancer. In addition, such compounds may be effective in blocking the growth factor – mediated signaling in cases where resistance to endocrine therapy has occurred, although this needs to be proven under specific experimental conditions. 10.1371/journal.pone.0066246.g012Figure 12 Schematic representation of the action of compound on inhibition of EGFR signaling. The compound competes with EGF in activating EGFR and leads to subsequent induction of apoptosis, inhibition of cell cycle progression and cell invasion. Supporting Information Figure S1 Cytotoxicity of CDRI-85/287 in non-cancerous cells determined by MTT assay. Values are mean± SEM, n = 5; p value: d- p>0.05 (insignificant). (TIF) Click here for additional data file. Figure S2 Effect of compound on the expression of downstream effectors of NF-κB and FOXO-3a. Densitometric analysis of the western blots showing effect of compound on the expression of p27, cyclin D1, p-Rb/Rb, XIAP and Bclxl in MDA-MB 231(left panel) and primary breast adenocarcinoma cells (right panel). Results are expressed as mean ± SEM, n = 5. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. (TIF) Click here for additional data file. Figure S3 CDRI-85/287 induces apoptosis via intrinsic pathway in breast cancer cells. (A) Induction of caspase-3 proteolytic activity in MDA-MB 231 cells treated with CDRI-85/287 for 48 h. Proteolytic activity was measured by cleavage of the caspase-3 substrate DEVD-pNA. Results are expressed as mean ± SEM, n = 5. p values are a-p<0.001, b<0.01, c-p<0.05 and d-p>0.05 vs. control. (B) MDA-MB 231 and primary breast cancer cells were treated with 3 and 7.5 µM of CDRI-85/287 for 24h. Mitochondrial membrane potential was measured by normalization of the 590:530 nm JC-1 emission ratios and then normalized to untreated cells. Results are expressed as mean ± SEM, n = 3. a-p<0.001 vs. control. (TIF) Click here for additional data file. Figure S4 Representative sections of kidney, liver, lung, spleen and uterus of vehicle treated and CDRI-85/287 treated mice. Photomicrographs of histological sections were captured. Number of animals per group = 6 to 8. (TIF) Click here for additional data file. Authors thank Mr AL Vishwakarma and Dr. K. Mitra, SAIF facility, CDRI for help in flowcytometric analysis and confocal microscopy, Dr. C. Nath and Dr. P.K. Agnihotri, Toxicology Division for providing histology facility. The authors are grateful to Director, INMAS-DRDO, Delhi for permitting to conduct experiments on nude mice. This manuscript carries CDRI communication number 8457. ==== Refs References 1 Saxena R , Dwivedi A (2012 ) ErbB family receptor inhibitors as therapeutic agents in breast cancer: current status and future clinical perspective . Med Res Rev 32 (1) : 166 –215 .22183797 2 Jordan VC (1992) The strategic use of antiestrogens to control the development and growth of breast cancer. Cancer 70 (4 Suppl) : :977–982. 3 Hanahan D , Weinberg RA (2000 ) The hallmarks of cancer . Cell 100 (1) : 57 –70 .10647931 4 Rochefort H , Glondu M , Sahla ME , Platet N , Garcia M (2003 ) How to target estrogen receptor-negative breast cancer? Endocr Relat Cancer 10 (2) : 261 –266 .12790787 5 Anders C , Carey LA (2008 ) Understanding and treating triple-negative breast cancer . Oncology (Williston Park) 22 (11) : 1233 –1239 .18980022 6 Griffiths CL , Olin JL (2012 ) Triple negative breast cancer: a brief review of its characteristics and treatment options . J Pharm Pract 25 (3) : 319 –323 .22551559 7 Liu D , He J , Yuan Z , Wang S , Peng R , et al (2011 ) EGFR expression correlates with decreased disease-free survival in triple-negative breast cancer: a retrospective analysis based on a tissue microarray . Med Oncol 29 (2) : 401 –405 .21264531 8 Badve S , Dabbs DJ , Schnitt SJ , Baehner FL , Decker T , et al (2011 ) Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists . Mod Pathol 24 (2) : 157 –167 .21076464 9 Roberts PJ , Der CJ (2007 ) Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer . Oncogene 26 (22) : 3291 –3310 .17496923 10 Han H , Du B , Pan X , Liu J , Zhao Q , et al (2010 ) CADPE inhibits PMA-stimulated gastric carcinoma cell invasion and matrix metalloproteinase-9 expression by FAK/MEK/ERK-mediated AP-1 activation . Mol Cancer Res 8 (11) : 1477 –1488 .21047770 11 Shen Q , Uray IP , Li Y , Zhang Y , Hill J , et al (2008 ) Targeting the activator protein 1 transcription factor for the prevention of estrogen receptor-negative mammary tumors . Cancer Prev Res (Phila) 1 (1) : 45 –55 .19138935 12 Biswas DK , Cruz AP , Gansberger E , Pardee AB (2000 ) Epidermal growth factor-induced nuclear factor kappa B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells . Proc Natl Acad Sci U S A 97 (15) : 8542 –8547 .10900013 13 Krol J , Francis RE , Albergaria A , Sunters A , Polychronis A , et al (2007 ) The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells . Mol Cancer Ther 6 (12 Pt 1) : 3169 –3179 .18089711 14 Zhang YG , Du Q , Fang WG , Jin ML , Tian XX (2008 ) Tyrphostin AG1478 suppresses proliferation and invasion of human breast cancer cells . Int J Oncol 33 (3) : 595 –602 .18695891 15 Kim S , Choi JH , Kim JB , Nam SJ , Yang JH , et al (2008 ) Berberine suppresses TNF-alpha-induced MMP-9 and cell invasion through inhibition of AP-1 activity in MDA-MB-231 human breast cancer cells . Molecules 13 (12) : 2975 –2985 .19052522 16 Saeed A , Sharma AP , Durani N , Jain R , Durani S , et al (1990 ) Structure-activity relationship of antiestrogens. Studies on 2,3-diaryl-1-benzopyrans . J Med Chem 33 (12) : 3210 –3216 .2258906 17 United States Patent no. 5254,568; Inventors: RS Kapil, S. durani, JD Dhar, BS Setty; Title: Benzopyrans as anti-estrogenic agents. 18 Sharma AP , Saeed A , Durani S , Kapil RS (1990 ) Structure-activity relationship of antiestrogens. Effect of the side chain and its position on the activity of 2,3-diaryl-2H-1-benzopyrans . J Med Chem 33 : 3216 –3222 .2258907 19 Dhar JD , Setty BS , Durani S , Kapil RS (1991 ) Biological profile of 2-[4-(2-N-piperidinoethoxy) phenyl]-3-phenyl(2H)benzo(b) pyran- a potent anti-implantation agent in rat . Contraception 44 : 461 –72 .1824558 20 Sreenivasulu S , Singh MM , Dwivedi A , Setty BS , Kamboj VP (1992 ) CDRI-85/287, a novel antiestrogen and antiimplantation agent: biological profile and interaction with the estrogen receptors in immature rat uterus . Contraception 45 : 81 –92 .1591925 21 Dwivedi A , Bansode FW , Setty BS , Dhar JD (1999 ) Endometrial steroid receptors during decidualization in rhesus monkey (Macaca mulatta); their modulation by anti-oestrogen CDRI-85/287 . Hum Reprod 14 : 1090 –1095 .10221246 22 Fatima I , Chandra V , Saxena R , Manohar M , Sanghani Y , et al (2012 ) A 2,3-Diaryl-2H-1-benzopyran derivatives interfere with classical and non-classical estrogen receptor signaling pathways, inhibit Akt activation and induce apoptosis in human endometrial cancer cells . Mol Cell Endocrinol 348 (1) : 198 –210 .21878365 23 Gupta A , Mandal SK , Leblanc B , Descôteaux V , Asselin E , et al (2008 ) Synthesis and cytotoxic activity of benzopyran-based platinum(II) complexes . Bio Med Chem Lett 18 : 3982 –3987 . 24 Bresch GJ , Wolberg WH , Gilchrist KW , Voelkel JG , Gould MN (1983 ) A comparison of methods for the production of monodispersed cell suspensions from human primary breast carcinomas . Breast Cancer Res Treat 3 (1) : 15 –22 .6307435 25 Morris GM , Huey R , Lindstrom W , Sanner MF , Belew RK , et al (2009 ) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility . J Comput Chem 30 (16) : 2785 –2791 .19399780 26 SYBYL, version 7.3 (2006) Tripos Associates, St. Louis, MO, USA. 27 Wood ER , Truesdale AT , McDonald OB , Yuan D , Hassell A , et al (2004 ) A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells . Cancer Res 64 (18) : 6652 –6659 .15374980 28 Seeliger D , de Groot BL (2010 ) Ligand docking and binding site analysis with PyMOL and Autodock/Vina . J Comput Aided Mol Des 24 (5) : 417 –422 .20401516 29 MOE: The Molecular Operating Environment from Chemical Computing Group Inc, 1255 University St, Suite 1600, Montreal, Quebec, Canada H3B 3X3 (2012) Journal of Computer -Aided Molecular Design 26(6): 775-786 (last issue). Available: https://www.chemcomp.com/MOE-Structure_Based_Design.html. 30 Blesson CS , Awasthi S , Kharkwal G , Daverey A , Dwivedi A (2006 ) Modulation of estrogen receptor transactivation and estrogen-induced gene expression by ormeloxifene-a triphenylethylene derivative . Steroids 71 (11–12) : 993 –1000 .16965798 31 Xu YZ , Zheng RL , Zhou Y , Peng F , Lin HJ , et al (2011 ) Small molecular anticancer agent SKLB703 induces apoptosis in human hepatocellular carcinoma cells via the mitochondrial apoptotic pathway in vitro and inhibits tumor growth in vivo . Cancer Lett 313 (1) : 44 –53 .21944661 32 Meloche S , Pouyssegur J (2007 ) The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition . Oncogene 26 (22) : 3227 –3239 .17496918 33 Dolcet X , Llobet D , Pallares J , Matias-Guiu X (2005 ) NF-κB in development and progression of human cancer . Virchows Arch 446 (5) : 475 –482 .15856292 34 Hu MC , Lee DF , Xia W , Golfman LS , Ou-Yang F , et al (2004 ) IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a . Cell 117 (2) : 225 –237 .15084260 35 Ellerbroek SM , Halbleib JM , Benavidez M , Warmka JK , Wattenberg EV , et al (2001 ) Phosphatidylinositol 3-kinase activity in epidermal growth factor-stimulated matrix metalloproteinase-9 production and cell surface association . Cancer Res 61 (5) : 1855 –1861 .11280738 36 Ling H , Zhang Y , Ng KY , Chew EH (2011 ) Pachymic acid impairs breast cancer cell invasion by suppressing nuclear factor-kappaB-dependent matrix metalloproteinase-9 expression . Breast Cancer Res Treat 126 (3) : 609 –620 .20521099 37 Chen PS , Wang MY , Wu SN , Su JL , Hong CC , et al (2007 ) CTGF enhances the motility of breast cancer cells via an integrin-alphavbeta3-ERK1/2-dependent S100A4-upregulated pathway . J Cell Sci 120 (Pt 12) : 2053 –2065 .17550972 38 Shimo T , Kubota S , Yoshioka N , Ibaragi S , Isowa S , et al (2006 ) Pathogenic role of connective tissue growth factor (CTGF/CCN2) in osteolytic metastasis of breast cancer . J Bone Miner Res 21 (7) : 1045 –1059 .16813525	23840429	PMC3686794	CC BY	2021-01-05 17:30:01	yes	PLoS One. 2013 Jun 19; 8(6):e66246
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23840320PONE-D-13-0750210.1371/journal.pone.0065218Research ArticleBiologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCytometryFlow CytometryGene ExpressionProteomicsSpectrometric Identification of ProteinsMathematicsStatisticsBiostatisticsMedicineOncologyBasic Cancer ResearchMetastasisCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaModification of Glycosylation Mediates the Invasive Properties of Murine Hepatocarcinoma Cell Lines to Lymph Nodes Modification of Glycosylation and Tumor InvasionZhang Zhaohai 1 Sun Jie 1 2 Hao Lihong 3 Liu Chunqing 1 Ma Hongye 1 Jia Li 1 * 1 Department of Basic Laboratory Medicine, College of Laboratory Medicine, Dalian Medical University, Dalian, Liaoning Province, China 2 Liaoning International Travel Health Care Center, Dalian, Liaoning Province, China 3 Department of Histology and Embryology, Dalian Medical University, Dalian, Liaoning Province, China St-Pierre Yves Editor INRS, Canada * E-mail: jiali@dlmedu.edu.cnCompeting Interests: JS is an employee of Liaoning International Travel Health Care Center. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: LJ ZZ JS. Performed the experiments: ZZ JS LH. Analyzed the data: ZZ JS CL HM. Contributed reagents/materials/analysis tools: LH CL HM. Wrote the paper: LJ ZZ JS. 2013 20 6 2013 8 6 e6521818 2 2013 24 4 2013 © 2013 Zhang et al2013Zhang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Among the various posttranslational modification reactions, glycosylation is the most common, and nearly 50% of all known proteins are thought to be glycosylated. In fact, changes in glycosylation readily occur in carcinogenesis, invasion and metastasis. This report investigated the modification of glycosylation mediated the invasive properties of Hca-F and Hca-P murine hepatocarcinoma cell lines, which have high, low metastatic potential in the lymph nodes, respectively. Analysis revealed that the N-glycan composition profiling, expression of glycogenes and lectin binding profiling were different in Hca-F cells, as compared to those in Hca-P cells. Further analysis of the N-glycan regulation by tunicamycin (TM) application or PNGase F treatment in Hca-F cells showed partial inhibition of N-glycan glycosylation and decreased invasion both in vitro and in vivo. We targeted glycogene ST6GAL1, which was expressed differently in Hca-F and Hca-P cells, and regulated the expression of ST6GAL1. The altered levels of ST6GAL1 were also responsible for changed invasive properties of Hca-F and Hca-P cells both in vitro and in vivo. These findings indicate a role for glycosylation modification as a mediator of tumor lymphatic metastasis, with its altered expression causing an invasive ability differentially. This work was supported by grants from National Natural Science Foundation of China (81071415, 81271910), and supported by Program for Liaoning Excellent Talents in University (LR2011025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction It is well known that glycosylation affects many physicochemical properties of glycoproteins. Thus, modified oligosaccharides affect protein folding and stability, and as a result, regulate many physiological and pathological events, including cell growth, migration, differentiation, and tumor metastasis [1]–[4]. Therefore, it is not surprising that aberrant glycosylation patterns can be considered to have great potential as therapeutic targets or clinical biomarkers for early detection, diagnosis and monitoring of cancer treatment. Cancer metastasis shows a multi-step process. Specific changes in the glycosylation pattern of cell surface glycoproteins have been shown to correlate with the enhancement of the metastatic efficiency of tumor cells [5]. In particular, N-glycans cover the most outer surface and thus should be the first molecules to be contacted when cells interact with each other. There is ample evidence that altered N-glycosylation patterns are present on tumor cells and these findings have sparked the search for glycan based biomarkers for the detection of different types of cancer [6]–[9]. In human hepatocarcinoma cells, the alterations of N-glycan structure in surface of glycoproteins contribute to the alterations in metastasis-associated phenotypes [10], [11]. Sakuma et al reported that an α1, 6-fucosylated biantennary N-glycan structure correlates with pulmonary metastatic ability of cancer cells [12]. In the work of Granovsky et al, the importance of specific glycosylation events in mammary cancer metastases has been clearly demonstrated in vivo using genetically altered mice [13]. These findings suggest that glycosylation may be involved in the regulation of multiple aspects of tumor metastasis. The murine hepatocarcinoma cell lines Hca-F with a high lymphatic metastasis rate over 80% and Hca-P with a low lymphatic metastasis rate less than 30% have been derived from 615-mice ascites-type hepatocarcinoma cell lines H22 [14]. Hca-F and Hca-P cells metastasize only to the lymph nodes, and not to other organs [15]. However, the relationship between glycosylation modification and lymphatic metastasis of murine hepatocarcinoma cells is still not clear. In the current study, we compared the N-glycan composition profiling, expression of glycogenes and lectin binding profiling between Hca-F and Hca-P cell lines. Meanwhile, we mainly focused on the modification of N-glycan of cell surface to further address the important roles of glycosylation in lymphatic metastasis of murine hepatocarcinoma cells. Results MALDI-MS Analysis of N-glycan Composition Profiling from Hca-F and Hca-P Cells MALDI-TOF MS analysis was utilized to evaluate the N-glycan composition profiling of Hca-F and Hca-P cell lines. Fig. 1 showed the MS spectra of N-glycans released from cell membranes and the observed MS signals of the N-glycans (peaks 1–34 in Fig. 1A) and the assigned N-glycan signals as were summarized in Table 1. The observed signal intensities in the mass spectra are presented as a histogram (Fig. 1B), with the estimated monosaccharide compositions. High mannoses analyzed in both cell lines were detected at peak 5, 7, 11, 15, and 17 (Table 1). Several major N-glycan differences of cell membrane derived from Hca-F and Hca-P were detected. Peak 10, 26 were detected exclusively in the Hca-F cell line. Peak 29 was detected exclusively in Hca-P cell line. Furthermore there were some differences regarding the intensities of all peaks in the spectra recorded from pools of Hca-F and Hca-P samples. Among those oligosaccharides, peak 1, 2, 3, 4, 5, 6, 7 and 34 increased in high lymphatic metastasis cell line Hca-F, and peak 25, 30 increased in low lymphatic metastasis cell line Hca-P. These data indicated that differential N-glycan composition might be associated with tumor lymphatic metastasis. 10.1371/journal.pone.0065218.g001Figure 1 N-glycans composition profiling of Hca-F and Hca-P cell lines using Mass spectrometry analysis. (A) MALDI-TOF MS spectra of N-glycans released from membrane protein of Hca-F and Hca-P cell lines. (B) Histograms of relative intensities of the N-glycan signals observed. Values are the mean ± S.D of three permethylated samples from N-glycan samples. The signal numbers correspond to those described in Table 1. 10.1371/journal.pone.0065218.t001Table 1 Summary of N-glycan in N-glycan in Hca-F and Hca-P cell lines identified by MALDI-TOF MS. Peak no. Observed m/z Composition Hca-F Hca-P Hex HexNAc Man GlcNAc NeuAc Deoxyhexose 1 1171.69 1171.84 0 0 3 2 0 0 2 1345.85 1345.86 0 0 3 2 0 1 3 1375.89 1375.87 1 0 3 2 0 0 4 1416.89 1416.91 0 1 3 2 0 0 5 1579.94 1579.97 0 0 5 2 0 0 6 1591.01 1591.03 0 1 3 2 0 1 7 1784.07 1784.10 0 0 6 2 0 0 8 1795.18 1795.10 1 1 3 2 0 1 9 1836.12 1836.20 0 2 3 2 0 1 10 1982.22 No 1 1 3 2 1 0 11 1988.15 1988.20 0 0 7 2 0 0 12 1999.17 1999.28 2 1 3 2 0 1 13 2156.28 2156.27 1 1 3 2 1 1 14 2186.29 2186.30 2 1 3 2 1 0 15 2192.26 2192.30 0 0 8 2 0 0 16 2244.28 2244.34 2 2 3 2 0 1 17 2396.29 2396.37 0 0 9 2 0 0 18 2431.29 2431.41 2 2 3 2 1 0 19 2448.31 2448.42 3 2 3 2 0 1 20 2478.54 2478.44 4 2 3 2 0 0 21 2605.39 2605.47 2 2 3 2 1 1 22 2635.37 2635.44 3 2 3 2 1 0 23 2652.39 2652.50 4 2 3 2 0 1 24 2779.33 2779.57 2 2 3 2 1 2 25 2809.42 2809.56 3 2 3 2 1 1 26 2817.27 No 0 6 3 2 0 1 27 2839.35 2839.57 4 2 3 2 1 0 28 2897.47 2897.60 4 3 3 2 0 1 29 No 2938.68 3 4 3 2 0 1 30 2966.47 2966.65 2 2 3 2 2 1 31 2996.56 2996.64 3 2 3 2 2 0 32 3102.34 3101.89 5 3 3 2 0 1 33 3305.43 3305.77 6 3 3 2 0 1 34 3463.61 3463.19 5 3 3 2 1 1 The N-glycan were observed as [M+Na]+. Hex, hexose; HexNAc, N-acetylhexosamine; Man, mannose; GlcNAc, N-actylglucosamine; NeuAc, N-acetylneuraminic acid. Differential Expression of Glycogenes in Hca-F and Hca-P Cell Lines To evaluate the expression profile of glycogenes in high (Hca-F) and low (Hca-P) metastatic potential cells, a real-time RT-PCR analysis was performed. 9 genes (out of 62) were differentially expressed between the two cell lines. Six glycogenes, ST6GAL1, MGAT5, FUT8, B4GALT1, B3GALT1, and B3GNT8 were expressed at a higher level in Hca-F compared with those in Hca-P cells (i.e., >3-fold higher, Fig. 2A). Conversely, three glycogenes, CHST13, MGAT3, and ST8SIA were expressed at an elevated level (i.e., >3-fold higher, Fig. 2A) in Hca-P compared with the ones in Hca-F cells. Western blot analysis further confirmed the enzyme expression on Hca-F and Hca-P cells at protein level (Fig. 2B). These data indicated that differential glycogene expressions might be associated with lymphatic metastasis of murine hepatocarcinoma cells. 10.1371/journal.pone.0065218.g002Figure 2 Differential expression of glycogenes in Hca-F and Hca-P cell lines. (A) The mRNA levels of glycogenes analyzed by real-time RT-PCR. The relative amount of glycogenes mRNA levels was normalized to GAPDH levels. Relative intensities ratio (>3-fold) of the glycogenes signals were observed. (B) Western blot analysis for enzyme was assessed. Data are the means ± SD of triplicate determinants. Differential FITC–lectin Binding Profiles of Hca-F and Hca-P Cell Lines with Flow Cytometry To investigate the glycan profiles of cell surface from Hca-F and Hca-P cell lines, flow cytometry analysis was used observing fluorescence intensity. Fig. 3 shows that the obvious differences in fluorescence intensity for glycosylation were evident by comparison between the Hca-F and Hca-P cell lines as summarized below: (1) higher signals of SNA (Siaα 2-6Gal/GalNAc) in Hca-F cells, (2) higher fucosylation at the innermost GlcNAc in Hca-F cells as revealed by fluorescence intensity on LCA, (3) increased level of branching of N-glycans in Hca-F cells estimated by fluorescence intensity of L-PHA (Tri- and tetra-antennary complex oligosaccharides), ABA (Galβ1-3GalNAcα-Thr/Ser (T) and sialyl-T), and DSA ((GlcNAc)n, polyLacNAc and LacNAc (NA3, NA4)), (4) higher signals of E-PHA (NA2 and bisecting GlcNAc) in Hca-P cells (Fig. 3A, 3B). These results correlated well with the real-time PCR analysis of glycogene expression. High expressions of glycogenes are corresponding with high fluorescence intensity of lectins in both cell lines (Fig. 3C). 10.1371/journal.pone.0065218.g003Figure 3 Differential FITC–lectin binding profiles of Hca-F and Hca-P cell lines using flow cytometry. (A) Histograms of fluorescence intensities of cells with specific carbohydrate expression as determined by flow cytometry using 7 different lectins. (B) The data are means ± SD of 3 independent assays of Hca-F and Hca-P cell lines, P<0.05. (C) List of glycogenes responsible for lectin signals in Hca-F and Hca-P cell lines. N-glycosylation Modification Mediates the Invasive Ability of Hca-F Cells both in vitro and in vivo To test directly whether the N-glycosylation of Hca-F cells influenced cells invasive ability, the modification of N-glycosylation was finished. Tunicamycin (TM), an inhibitor of endogenous N-glycosylation of newly synthesized proteins, has effect on N-glycan of Hca-F cells. CD147 is a highly N-glycosylated immunoglobulin superfamily transmembrane protein that is composed of two extracellular Ig domains, which contributes to a highly N-glycosylated form, HG-CD147 (∼40–60 kDa) and lowly glycosylated form, LG-CD147 (∼32 kDa) [19]. Treatment of Hca-F cells, with TM at a dose dependent (0, 1, 5, and 10 mg/ml) for 12 h, showed that N-glycosylation was highly sensitive to inhibition by TM. The CD147 (40 kDa) completely disappeared, and the level of the CD147 (40–60 kDa) was decreased. The 27 kDa band that appeared was consistent with the size of the core protein. The fraction of CD147 remaining at 60 kDa was likely synthesized before TM treatment. In addition, aliquots of membrane fractions of Hca-F cells were also exposed to exogenous PNGase F (Fig. 4A) to achieve deglycosylation. The CD147 (40–60 kDa) completely disappeared, and a 27–33 kDa band appeared. These results suggested that the N-glycosylation process in Hca-F cells was highly sensitive to inhibition by TM and PNGase F. 10.1371/journal.pone.0065218.g004Figure 4 N-glycosylation modification mediates the invasive ability of Hca-F cells both in vitro and in vivo. (A) Western blot analysis of CD147 was performed using total membrane protein extracts. Hca-F cells were exposed to TM or PNGase F and then harvested for western blot analysis. Controls are Na+/K+-ATPase. Hca-F cells were treated with TM (B) or PNGase F (C) and thereafter the cell invasive ability was assessed by ECMatrix gel analysis in vitro. Hca-F cells were treated with TM (D, fluorescence; ×100) or PNGase F (F, fluorescence; ×100) and thereafter the cell invasive ability to peripheral lymph nodes was analyzed in vivo. The number of TM pre-treated (E) or PNGase F pre-treated (G) CFSE+Hca-F cells invasion to peripheral lymph nodes was measured by flow cytometry. Surface labeling was expressed as the percentage of positive cells in CFSE+Hca-F cells relative to the total number of analyzed cells (P<0.05). The data were obtained from three independent experiments. To examine whether the modification of N-glycosylation in Hca-F cells affected the invasive ability, we performed an in vitro ECMatrix gel analysis. Under TM and PNGase F treatment, the Hca-F cells showed decreased invasive ability, as compared with the Hca-F groups in the absence of TM and PNGase F (Fig. 4B, 4C). The influence of glycosylation modification on the invasive ability of Hca-F cells to peripheral lymph nodes in vivo was determined in order to explore the potential involvement of glycosylation during tumor cells invasion. Under TM and PNGase F treatment, the Hca-F cells were labeled with CFSE. The invasive ability of CFSE-tagged cells in TM or PNGase F-treated groups to lymph nodes was reduced obviously, as compared with the Hca-F groups in the absence of TM and PNGase F in vivo (Fig. 4D, 4F). To further investigate the positive ratio of CFSE-tagged cells in whole lymph node digest mixture, a flow cytometry analysis was carried out. As shown in Fig. 4E, 4G, the number of CFSE-tagged Hca-F cells in control, TM or PNGase F-treated groups were quite different. The Hca-F in the absence of TM and PNGase F -treated positive cells were 17.55% and 17.28%, but TM (10 µg/ml) and PNGase F-treated (24 hour) positive cells were only 10.81% and 9.57%. These observations supported that the modification of N-glycosylation could be associated with the invasive ability of murine hepatocarcinoma cells. Silence of ST6GAL1 Effects on the Invasive Ability of Hca-F Cells both in vitro and in vivo ST6GAL1 glycogene encodes the β-galactoside α-2, 6-sialyltransferase 1, which catalyzes the transfer of sialic acid residue in α-2, 6-linkage to terminal galactose of glycan chains. Fig. 2A has showed that glycogene ST6GAL1 was expressed at a higher level (4.66-fold) in Hca-F compared with those in Hca-P cells. We silenced, by siRNA, in order to elucidate the direct implication glycogene in the lymphatic metastasis-related phenotypes of Hca-F cells. As shown in Fig. 5A, ST6GAL1 expression at protein level was down-regulated in ST6GAL1 transfectants compared with Hca-F-control siRNA transfectants. The cell invasion was determined using the Transwell assay. Interestingly, knockdown of ST6GAL1 expression significantly inhibited Hca-F-ST6GAL1 siRNA cells invasion relative to the Hca-F-control siRNA cells (Fig. 5B). 10.1371/journal.pone.0065218.g005Figure 5 Silence of ST6GAL1 effects on the invasive ability of Hca-F cells both in vitro and in vivo. (A) Silencing of ST6GAL1 in Hca-F cells was analyzed by RNAi approach. After Hca-F cells were transfected with ST6GAL1 siRNA for 30 h, western blot analysis for ST6GAL1 was assessed. GAPDH was also examined and served as controls for sample loading. Relative signal intensities of ST6GAL1 protein levels were normalized against those of GAPDH by LabWorks (TM ver4.6, UVP, BioImaging systems) analysis, respectively (P<0.05). (B) In vitro ECMatrix gel analysis is performed. The average number of cells that invaded through the filter was counted. Hca-F-ST6GAL1 siRNA cells were significantly less invasive (P<0.05) than the Hca-F and Hca-F-control siRNA cells. (C, fluorescence; ×100) The results of ST6GAL1 siRNA-transfected CFSE+Hca-F cells invasion to lymph nodes were analyzed. The number of Hca-F-ST6GAL1 siRNA cells was decreased, compared with the Hca-F-control siRNA, Hca-F cells after 24 h. (D) The number of ST6GAL1 siRNA-transfected CFSE+Hca-F cells invasion to lymph nodes was measured by flow cytometry. Surface labeling was expressed as the percentage of positive cells relative to the total number of analyzed cells (P<0.05). (E) FITC-SNA binding profiles of Hca-F cells using flow cytometry. Histograms of fluorescence intensities of cells with specific carbohydrate expression as determined. Data are the average ± SD of triplicate determinants. The influence of glycogene on the invasive ability of Hca-F cells to peripheral lymph nodes in vivo was determined. Hca-F cells were labeled with CFSE, a green fluorescence dye, which can be transported across plasma membrane to react covalently with free amino group of intracellular macromolecules. The invasive ability of CFSE-tagged cells in ST6GAL1 siRNA-treated groups to lymph nodes was reduced obviously, as compared with control groups in vivo (Fig. 5C). To further investigate the positive ratio of CFSE-tagged cells in whole lymph node digest mixture, a flow cytometry analysis was carried out. As shown in Fig. 5D, the number of CFSE-tagged Hca-F cells in control, siRNA-treated groups were quite different. The Hca-F, control siRNA-treated positive cells were 17.21% and 17.52%, but ST6GAL1 siRNA-treated positive cells were only 8.95%. These observations supported that ST6GAL1 on Hca-F cells could play an important role in invasion to peripheral lymph nodes in vivo, and might therefore contribute to tumor lymphatic metastasis. In order to evaluate whether ST6GAL1 silencing could modify the N-glycosylation profile in terms of α-2, 6-linked sialic acid using a flow cytometry, each cell group was bind with SNA lectin. Fig. 5E showed that the ST6GAL1 knockdown resulted in a decrease of fluorescence intensity compared with the control cells. Overexpression of ST6GAL1 Influences the Invasive Ability of Hca-P Cells both in vitro and in vivo To explore the effect of ST6GAL1 on invasive ability, an Hca-P cell line transient expressing ST6GAL1 was established. It was found that the level of ST6GAL1 protein was notably increased in Hca-P transfectants (Fig. 6A). Furthermore, over-expression of ST6GAL1 significantly promoted Hca-P/ST6GAL1 cells invasion relative to the Hca-P/mock cells in vitro (Fig. 6B). 10.1371/journal.pone.0065218.g006Figure 6 Overexpression of ST6GAL1 influences the invasive ability of Hca-P cells both in vitro and in vivo. (A) Hca-P cells were transfected with a pcDNA3.1 expression vector, and western blot analysis for ST6GAL1 was assessed. GAPDH was also examined and served as controls for sample loading. Relative signal intensities of ST6GAL1 protein levels were normalized against those of GAPDH by LabWorks (TM ver4.6, UVP, BioImaging systems) analysis, respectively (P<0.05). (B) In vitro ECMatrix gel analysis is performed. The average number of cells that invaded through the filter was counted. Hca-P/ST6GAL1 cells were significantly more invasive (P<0.05) than the Hca-P and Hca-P/mock cells. (C, fluorescence; ×100) The results of ST6GAL1-transfected CFSE+Hca-P cells invasion to lymph nodes were analyzed. The number of Hca-P/ST6GAL1 cells was increased, compared with the Hca-P/mock, Hca-P cells after 24 h. (D) The number of ST6GAL1-transfected CFSE+Hca-P cells invasion to lymph nodes was measured by flow cytometry. Surface labeling was expressed as the percentage of positive cells relative to the total number of analyzed cells (P<0.05). (E) FITC-SNA binding profiles of Hca-P cells using flow cytometry. Histograms of fluorescence intensities of cells with specific carbohydrate expression as determined. Data are the average ± SD of triplicate determinants. The effect of glycogene ST6GAL1 on the invasive ability of Hca-P cells to peripheral lymph nodes in vivo was also analyzed. The invasive ability to peripheral lymph nodes in vivo of CFSE-tagged cells in Hca-P/ST6GAL1 groups to lymph nodes was increased obviously, as compared with Hca-P/mock groups in vivo (Fig. 6C). The flow cytometry analysis showed that the positive ratio of CFSE-tagged cells in whole lymph node digest mixture was different. Hca-P/ST6GAL1 positive cells showed increased ratio, as compared with the Hca-P/mock groups (Fig. 6D). Fig. 6E showed that the ST6GAL1 over-expression resulted in an increase of fluorescence intensity compared with the Hca-P/mock cells. These results clearly showed that glycogene ST6GAL1 was associated with lymphatic metastasis of murine hepatocarcinoma cells, thus suggesting the involvement of the lymphatic metastasis in the altered glycosylation profiles. Discussion In the present study, we investigated the possible correlation of glycosylation modification and the tumor lymphatic metastasis in murine hepatocarcinoma cell lines Hca-F and Hca-P with high, low metastatic potential to lymph nodes. The structural scheme of glycans is dependent on their compositions. MALDI-MS technology as a novel methodology provides high sensitivity and more rapid glycan analysis [20], [21], [22]. Zhang et al indicated that MS technology could facilitate the discovery of a novel and quantitative prognostic biomarker for gastric cancer with lymph node involvement [23].Three glycans were shown to provide good sensitivity and specificity for the separation of serum samples from patients with hepatocellular carcinoma and controls by MS technology [24]. In the current study, we compared the total N-glycans from Hca-F and Hca-P cell lines, and found dramatic differences in N-glycan profiles between these two groups (Fig. 1, Table 1). Peak 2, 6, 26, 34 corresponded to fucosylated oligosaccharides, and peak 10, 34 corresponded to sialylated oligosaccharides originating from Hca-F cells showed a significant increase. Moreover, major peaks 25, 30 corresponded to fucosylated and sialylated oligosaccharides originating from Hca-P cells also showed a significant increase. Therefore, monitoring of the N-glycan profile would be an important step in the prevention of tumor metastasis and would increase our understanding of metastasis mechanisms. Oligosaccharides on glycoproteins are altered in tumorigenesis, and they often play a role in the regulation of the biological characteristics of tumors [25], [26]. Each oligosaccharide is synthesized by a specific glycosyltransferase. Glycogenes, which encode glycosyltransferases, are involved in glycan synthesis and modification. The current study clearly showed that the glycogene expressions were highly regulated, with 9 (out of 62) glycogenes (at least 3-fold, Table 1) significantly differentially expressed among the two cell lines. For example, ST6GAL1, which is significantly expressed at an elevated level (4.66-fold higher) in Hca-F compared with Hca-P cells, encodes β-galactosamide: α-2, 6-sialyltranferase 1 that is a key enzyme in the formation of sialic acid residue in α-2, 6-linkage to terminal galactose of glycan chains [27]. The result was consistent with the MALDI-TOF MS analysis, and a higher level of sialylated oligosaccharides in Hca-F cells was displayed (Fig. 1 and Table1). Therefore, the major altering expressions of glycogenes in the two cell lines may be more important as indicators and functional contributors of tumor lymphatic metastasis. Lectin binding approache has been used for the analysis of glycoproteins and N-glycans [28]. Previous report [29] revealed lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces using flow cytometry assay. In this study, the obtained datasets could be statistically compared to identify lectins that show significant differences between the two cell lines (Fig. 3A, 3B). For example, SNA specifically recognized α-2, 6-linked sialic acid and a higher signal of SNA for N-glycosylation in Hca-F cells was shown. High expression of glycogene ST6GAL1 was responsible for high fluorescence intensity of SNA FITC-lectin (Fig, 3C). The result was consistent with the MALDI-TOF MS and glycogene expression analysis. The alterations in glycosylation can be associated with tumor either as a product of the tumor or as reaction to the disease [30], [31]. Inhibition of N-glycan processing disrupts normal cell adhesion and reduces the tumourigenic and metastatic capacity in vivo of rhabdomyosarcoma cell line S4MH [32]. Deglycosylation of Hca-F cells by tunicamycin influences on cells adhesion in vitro [33]. The CD147 gene has garnered attention because of its high expression in many malignant tumor cells, and its key role is in the processes of tumor progression [34]. In this study, we modified the N-glycosylation of Hca-F cells by tunicamycin or PNGase F treatment. Both treatments resulted in similar effects on the occurrence of a defective N-glycosylation in Hca-F cells. The altered N-glycosylation of CD147 were found in Hca-F cells, and further suggested a link between defective N-glycosylation of Hca-F cells and tumor invasion both in vitro and in vivo (Fig. 4). The detection of such strongly correlated glycosylation modification showed that not only glycan structure might alter, but that often tumor biological processes were affected. Increased expression of ST6GAL1 is reported in carcinomas of the colon, breast, ovarian and gastric cancer, acute myeloid leukemia, and in some brain tumors [35]–[40]. ST6GAL1 is also correlated with invasion in cancers [41], [42], [43]. Altered expression of ST6GAL1 mediates the adhesive capability of Hca-F cells to fibronectin [44]. Our data proved the glycogene ST6GAL1 as potential target for tumor lymphatic metastasis. In addition, we further tested directly that the silencing of ST6GAL1 in Hca-F cells resulted in decreased the invasive ability both in vitro and in vivo through modifying the N-glycosylation profile in terms of α-2, 6-linked sialic acid in murine hepatocarcinoma cells (Fig. 5). ST6GAL1 product also decreased remarkably in Hca-F- ST6GAL1 siRNA cells labeled with SNA lectin. Conversely, over expression of ST6GAL1 in Hca-P cells could increase the invasive ability both in vitro and in vivo (Fig. 6). These observations clearly demonstrate that the changes in glycogene expression levels have impact in the remodeling of cell surface glycosylation, which may consequently affect the biological functions of tumor cells such as tumor lymphatic metastasis. In conclusion, by analyzing the glycomics of Hca-F and Hca-P lines and detecting the quantitative changes of glycosylation, at least in this system, altered glycosylation showed the unusual property of association with tumor lymphatic metastasis. Although we feel that the modification of glycosylation effects remain the best explanation for the phenotype, there could be other potential effects on multiple glycomic alterations. Therefore, the molecular bases of tumor lymphatic metastasis-associated phenotype remains to be further investigated. Materials and Methods Cells Murine hepatocarcinoma cell lines Hca-F and Hca-P, which have high, low metastatic potential in the lymph nodes, (established and stored by Department of Pathology, Dalian Medical University, Dalian) were implanted in mouse abdominal cavity [16]. After 7 days, all mice were sacrificed and cells were retrieved from the abdominal cavity by 10 mL syringe. Then cells were cultured 1 day in 90% RPMI 1640 (Gibco) supplemented with antibiotics (1× penicillin/streptomycin 100 U/ml, Gibco), 10% fetal bovine serum (Gibco), at 37°C in a humidified atmosphere containing 5% CO2. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Dalian Medical University, China (Permit Number: 12-569). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Membrane Protein Extract A total of 1×107 cells were washed with phosphate buffered saline (PBS) and lysed on a plate with lysis and separation buffer containing a protease inhibitor cocktail. Cell membrane proteins were extracted from the cell suspension using a CelLytic MEM Protein Extraction kit (Sigma, St Louis, MO, USA). The membrane protein concentration was measured with a Micro BCA Protein Assay kit (PIERCE, Rockford, IL) and used for further experiments as described below. Release of N-glycans from Cell Membrane Proteins Three 100 µg aliquots of lyophilized samples (cell membrane proteins) were first digested with trypsin (10 µg) and chymotrypsin (10 µg) dissolved in 25 mM ammonium bicarbonate (25 µL) at 37°C for 18 h. The digest was left in a water bath (85°C, 5 min) and after cooling N-linked oligosaccharides were released from peptides by treatment with PNGaseF enzyme (2 µL; 6 U) at 37°C (18 h) followed by Pronase digestion (10 µg) at 37°C (8 h). During the incubation time, the reaction sample was mixed occasionally. The released N-glycans were purified using an Oasis HLB cartridge (60 mg/3 ml; Waters) and then were lyophilized. MS Analysis The mass spectra were carried out in reflectron positive ion mode with MALDI-TOF MS ((Bruker Corp., Billerica, MA, USA). To increase sensitivity and provide more informative fragmentation, the released glycans were permethylated [17], [18] and further characterized by MALDI-TOF MS. For the type of MALDI analysis of the permethylated glycans, 2, 5-DHB was used as the matrix. Values are the mean ± S.D of three permethylated samples from N-glycan samples. All MS spectra were obtained from Na+ adduct ions. Analysis of Glycogenes Total RNA were isolated from Hca-F and Hca-P cells using an RNeasy Mini Kit (QIAGEN) and cDNA was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN) from 5 µg of total RNA according to the manufacturer’s instruction. Real-time PCR amplification and analysis were performed on 7500 fast Real-time PCR System (Applied Biosystems) for 40 cycles (15 seconds at 95°C, 15 seconds at 60°C and 30 seconds at 72°C). All reactions were performed with QuantiTect SYBR Green PCR Kit (QIAGEN) according to the manufacturer's instruction. The sequences of the upstream and downstream primers were as follows: 5′-TGCGTGTGGAAGAAAGGGAGC-3′ and 5′-CTCCTGGCTCTTCGGCATCTG-3′ for ST6Gal 1; 5′-CCCTGGAAGTTGTCCTCTCA-3′ and 5′-TCCTCTGCCAGTGCC TTAAT-3′ for MGAT5; 5′-GGATTGCAAATTCCTGCCATTC-3′ and 5′-AACGTTGT CCCGGGTGTCA-3′ for FUT8; 5′-GGCGTCACCCTCGTCTATTA-3′ and 5′-GCCC TGCAGTGTAGAGGAGA-3′ for B4GALT1; 5′-ACCGTGACATCGTGGAAGTGG- 3′ and 5′-GGATGGTGGGCAGCGTGTC-3′ for B3GALT1; 5′-CGGCGCTATGGTG ACCTACTG-3′ and 5′-TCAGCAGCAGCAGGTCCTTG-3′ for B3GNT8; 5′-TACGA CTCACTATAGGGGCGACTA-3′ and 5′-CCCTCACTAAAGGGAGTCCTAGGA-3′ for CHST13; 5′-GCGTGATGGTGTGCTGTTCC-3′ and 5′-ACAGGGACTTCCGC A TGTGG-3′ for MGAT3; 5′-CCTAGCACAGGTCTCCTCATG-3′ and 5′-GGAAAT GGCCAGAATCCATA-3′ for ST8SIA4; 5′-ATTGCCCTCAACGACCACTT-3′ and 5′ -AGGTCCACCACCCTGTTGCT-3′ for GAPDH. Expression levels of each glycogene were normalized using either the expression level of GAPDH mRNA and compared between Hca-F and Hca-P cell lines. Real-time RT-PCR analysis was performed in triplicate. Flow Cytometry Assay Hca-F and Hca-P cell lines were washed thrice with fluorescence-activated cell sorting (FACS) buffer (PBS containing 20 g/L bovine serum), and then centrifuged at 1000 r/min for 5 min in a 1.0 mL eppendorf tube for collecting cells. The cells were blocked for 30 min 37°C in 5% powdered skim milk and then were washed between each step with FACS buffer. Cells were placed in sterile conical tubes in aliquots of 500,000 cells each and stained with one of the 7 FITC-lectins at a final concentration of 10 µg/ml for 40 min at 4°C in the dark. Residual unbound FITC–lectin was then discarded by repeat centrifugation of samples at 1000 r/min. After removal of supernatant, cells were resuspended in 0.2 ml PBS. The control, which was negative, cells and FITC–lectins were alone. Fluorescence and light scatters were analyzed in a BD Biosciences fluorescence-activated cell sorter (FACSCalibur) equipped with an argon laser tuned at 488 nm and a 635-nm diode, and Cell Quest software was used for acquisition. Ten thousand cells were analyzed for each sample. Three independent assays were carried out using both Hca-F and Hca-P cell lines. RNAi Assay Hca-F cells were incubated in appropriate antibiotic-free medium with 10% fetal bovine serum (Gibco), transferred to a 6 well tissue culture and incubated at 37°C, in a CO2 incubator to obtain 60–80% confluens. The cell cultures were transfected with ST6GAL1 siRNA Transfection Reagent Complex, respectively (Santa Cruz Biotech, Inc, sc-42805), which was prepared according to the protocol. Scrambled siRNA was used as the negative control. Transfer cells were cultured and incubated at 37°C for 6 hours, followed by incubation with complete medium for additional 24 h. Then cells were harvested and experimented as described for western blot analysis, in vitro and in vivo invasion assay. The cell transfection efficiency was 79% and the survival rate was 86%, respectively. Transfection To generate ST6GAL1-transfected mouse hepatocarcinoma cell line Hca-P the coding region of wild-type ST6GAL1 was subcloned into pcDNA3.1 expression vector (Invitrogen) to generate pcDNA3.1/ST6GAL1. The plasmid was mixed with Lipofectamine™ 2000 (Invitrogen) according to manufacturer’s instructions and added to Hca-P cells. The transient transfectants were selected and used as a population assigned Hca-P/ST6GAL1. The empty vector was used as a transfection control and resulting transfeatants were assigned Hca-P/Mock. The cell transfection efficiency was 72% and the survival rate was 86%. Tunicamycin Treatment To inhibit N-linked glycosylation of newly synthesized proteins, Hca-F cells were washed once with PBS and cultivated for 12 h in fresh culture media (90% DMEM supplemented with antibiotics) in the absence or presence of TM (Sigma Aldrich, St. Louis, MO) in a dose-dependent manner (0, 1, 5, or 10 µg/ml). The cells were washed with PBS again and then were determined by western blot analysis and invasion assay. The cell survival rates were 89%, 90%, 87% and 85% by trypan blue dye exclusion assay, respectively. PNGase F Treatment To remove N-glycans, membrane protein fractions (100 µg) from Hca-F cells were deglycosylated with 25 units of PNGase F (PNGase F from Elizabethkingia meningoseptica; Sigma Aldrich, St. Louis, MO) in lysis buffer. The probes were incubated for 8, 16, 24 hours at 37°C. Afterwards, reactions were stopped with sample buffer and the proteins were separated in a gel as described earlier. Besides, for the deglycosylation of membrane proteins, intact Hca-F cells were incubated with 25 units of PNGase for 24 hour, washed and subsequently treated as described for western blot analysis, in vitro and in vivo invasion assay. The cell survival rate was 88%, 89%, 85% and 87% by trypan blue dye exclusion assay, respectively. Western Blot Analysis Whole cell proteins were electrophoresed under reducing conditions in 10% polyacrylamide gels. The separated proteins were transferred to a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBST), the membrane was incubated with antibody (Santa Cruz Biotech or Abcam) and then with peroxidase-conjugated anti-rabbit IgG (1/10000 diluted; GE Healthcare UK Ltd., Little Chalf-ont, U.K.). A Na+/K+-ATPase α1 antibody (1/200 diluted; Santa Cruz Biotech) or GAPDH (1/200 diluted; Santa Cruz Biotech) was used as a control. All bands were detected using ECL Western blot kit (Amersham Biosciences, UK), according to the manufacturer’s instruction. The bands were analyzed with LabWorks (TM ver4.6, UVP, BioImaging systems). In vitro ECM Invasion Assay Cells invasion in vitro was demonstrated using 24-well transwell units (Corning, NY, USA) with 8 µm pore size polycarbonate filter coated with ECMatrix gel (Chemicon) to form a continuous thin layer (Zhu et al., 2005). Cells (3×105) were harvested in serum-free medium containing 0.1% BSA and added to the upper chamber. The lower chamber contained 500 µl RPMI 1640. Cells were incubated for 24 h at 37°C, 5% CO2 incubator. At the end of incubation, the cells on the upper surface of the filter were completely removed by wiping with a cotton swab. Then the filters were fixed in methanol and were stained with Wright-Giemsa. Cells that had invaded the Matrigel and reached the lower surface of the filter were counted under a light microscope at a magnification of 400×. Triplicate samples were acquired and the data were expressed as the average cell number of 5 fields. In vivo Invasion Assay Cells (5×106) were labeled with the vital dye carboxyfluorescein diacetate succinimidyl ester (CFSE, Sigma), respectively (Chen et al., 2005). Cells were incubated with 5 µM CFSE at 37°C for 10 min. Labeled cells were washed once and counted. After 1 day, the cells were harvested, and inoculated into the footpad of mice subcutaneously. After 12 h, lymph nodes were removed from mice. The frozen sections of lymph nodes were analyzed under fluorescence microscope with the Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA). The lymph nodes were incubated with 0.1% (w/v) collagenase IV (Sigma), in 90% RPMI 1640 (Gibco) 10% FBS (Gibco) for 30 min at 37°C. The ratio of green fluorescence positive Hca-F cells in whole lymph node digest mixture was detected by flow cytometry. Experiments were approved by the Committee on the Ethics of Animal Experiments of the Dalian Medical University, China (Permit Number: 12-569). Statistical Analysis Each experiment was performed at least in triplicate, and the measurements were performed in three independent experiments. Data are expressed as means ± standard deviation (SD). Student’s t-test was used to compare the means of two groups. P<0.05 was considered statistically significant. All analyses were performed using SPSS 13.0 statistical packages (SPSS Inc., Chicago, IL). ==== Refs References 1 Dwek RA (1995 ) Glycobiology: “towards understanding the function of sugars” . Biochem Soc Trans 23 : 1 –25 .7758655 2 Taniguchi N , Ekuni A , Ko JH , Miyoshi E , Ikeda Y , et al (2001 ) A glycomic approach to the identification and characterization of glycoprotein function in cells transfected with glycosyltransferase genes . Proteomics 1 : 239 –247 .11680870 3 Saxon E , Bertozzi CR (2001 ) Annu. Chemical and biological strategies for engineering cell surface glycosylation . Rev Cell Dev Biol 17 : 1 –23 . 4 Ohtsubo K , Marth JD (2006 ) Glycosylation in cellular mechanisms of health and disease . Cell 126 : 855 –867 .16959566 5 Dennis JW , Granovsky M , Warren CE (1999 ) Glycoprotein glycosylation and cancer progression . Biochim Biophys Acta 1473 : 21 –34 .10580127 6 Balog CI , Stavenhagen K , Fung WL , Koeleman CA , McDonnell LA , et al (2012 ) N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation . Mol Cell Proteomics 11 : 571 –585 .22573871 7 Dube DH Bertozzi CR (2005 ) Glycans in cancer and inflammation-potential for therapeutics and diagnostics . Nat Rev Drug Discov 4 : 477 –488 .15931257 8 Mehta A , Norton P , Liang H , Comunale MA , Wang M , et al (2012 ) Increased Levels of Tetra-antennary N-Linked Glycan but Not Core Fucosylation Are Associated with Hepatocellular Carcinoma Tissue . Cancer Epidemiol Biomarkers Prev 21 : 925 –933 .22490318 9 de Leoz ML , Young LJ , An HJ , Kronewitter SR , Kim J , et al (2011 ) High-mannose glycans are elevated during breast cancer progression . Mol Cell Proteomics 10 : M110.002717 . 10 Guo HB , Zhang Y , Chen HL (2001 ) Relationship between metastasis-associated phenotypes and N-glycan structure of surface glycoproteins in human hepatocarcinoma cells . J Cancer Res Clin Oncol 127 : 231 –236 .11315257 11 Guo R , Cheng L , Zhao Y , Zhang J , Liu C , et al (2013 ) Glycogenes mediate the invasive properties and chemosensitivity of human hepatocarcinoma cells . Int J Biochem Cell Bio 45 : 347 –358 .23103836 12 Sakuma K , Fujimoto I , Hitoshi S , Tanaka F , Ikeda T , et al (2006 ) An N-glycan structure correlates with pulmonary metastatic ability of cancer cells . Biochem Biophys Res Commun 340 : 829 –835 .16380076 13 Granovsky M , Fata J , Pawling J , Muller WJ , Khokha R , et al (2000 ) Suppression of tumor growth and metastsis in Mgat5-deficient mice . Nat Med 6 : 306 –312 .10700233 14 Hou L , Li Y , Jia YH , Wang B , Xin Y , et al (2001 ) Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice . World J Gastroenterol 7 : 532 –536 .11819823 15 Li HF , Ling MY , Xie Y , Xie H (1998 ) Establishment of a lymph node metastatic model of mouse hepatocellular carcinoma Hca-F cells in C3H/Hej mice . Oncol Res 10 : 569 –573 .10367938 16 Jia L , Wang S , Zhou H , Hu Y , Zhang J (2006 ) Caveolin-1 upregulates CD147 glycosylation and the invasive capability of murine hepatocarcinoma cell lines . Int J Biochem Cell Biol 38 : 1584 –1593 .16702020 17 Costello CE , Contado-Miller JM , Cipollo JF (2007 ) A glycomics platform for the analysis of permethylated oligosaccharide alditols . J Am Soc Mass Spectrom 18 : 1799 –1812 .17719235 18 Viseux N , Costello CE , Domon B (1999 ) Post-source decay mass spectrometry: optimized calibration procedure and structural characterization of permethylated oligosaccharides . J Mass Spectrom 34 : 364 –376 .10226363 19 Tang W , Chang SB , Hemler ME (2004 ) Links between CD147 Function, Glycosylation, and Caveolin-1 . Molecular Biology of the Cell 15 : 4043 –4050 .15201341 20 Dell A , Morris HR (2001 ) Glycoprotein Structure Determination by Mass Spectrometry . Science 291 : 2351 –2356 .11269315 21 Harvey DJ (2005 ) Proteomic analysis of glycosylation: structural determination of N-and O-linked glycans by mass spectrometry . Expert Rev Proteomics 2 : 87 –101 .15966855 22 Zaia J (2004 ) Mass spectrometry of oligosaccharides . Mass Spectro Rev 3 : 161 –227 . 23 Zhang MH, Xu XH, Wang Y, Ling QX, Bi YT, et al.. (2013) A Prognostic Biomarker for Gastric Cancer With Lymph Node Metastases. Anat Rec (Hoboken) Feb 4 doi: 10.1002/ar.22642. 24 Goldman R , Ressom HW , Varghese RS , Goldman L , Bascug G , et al (2009 ) Detection of Hepatocellular Carcinoma Using Glycomic Analysis. Clin . Cancer Res 15 : 1808 –1813 . 25 Dwek RA (1996 ) Glycobiology: toward understanding the function of sugars . Chem Rev 96 : 683 –720 .11848770 26 Varki A (1993 ) Biological roles of oligosaccharides: all of the theories are correct . Glycobiology 3 : 97 –130 .8490246 27 Harduin-Lepers A. Vallejo-Ruiz V , Krzewinski-Recchi MA , Samyn-Petit B , Julien S , et al (2001 ) The human sialyltransferase family . Biochimie 83 : 727 –737 .11530204 28 Hirabayashi J (2004 ) Lectin-based structural glycomics: glycoproteomics and glycan profiling . Glycoconj J 21 : 35 –40 .15467396 29 Venable A , Mitalipova M , Lyons I , Jones K , Shin S , et al (2005 ) Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces . BMC Dev Biol 21 : 5 –15 . 30 Ruhaak LR, Miyamoto S, Lebrilla CB (2013) Developments in the identification of glycan biomarkers for the detection of cancer. Mol Cell Proteomics Jan 30. [Epub ahead of print]. 31 Kim EH , Misek DE (2011 ) Glycoproteomics-based identification of cancer biomarkers . Int J Proteomics 2011 : 601937 .22084691 32 Calle Y , Palomares T , Castro B , del Olmo M , Bilbao P , et al (2000 ) Tunicamycin treatment reduces intracellular glutathione levels: effect on the metastatic potential of the rhabdomyosarcoma cell line S4MH . Chemotherapy 46 : 408 –428 .11053907 33 Jia L , Zhou H , Wang S , Cao J , Wei W , et al (2006 ) Deglycosylation of CD147 down-regulates Matrix Metalloproteinase-11 expression and the adhesive capability of murine hepatocarcinoma cell HcaF in vitro . IUBMB Life 58 : 209 –216 .16754299 34 Muramatsu T , Miyauchi T (2003 ) Basigin (CD147): a multifunctional transmembrane protein involved in reproduction, neural function, inflammation and tumor invasion . Histol and Histopathol 18 : 981 –987 . 35 Gessner P , Riedl S , Quentmaier A , Kemmner W (1993 ) Enhanced activity of CMP-NeuAc:Galh 1–4GlcNAc:a 2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients . Cancer Lett 75 : 143 –149 .8313349 36 Recchi MA , Harduin-Lepers A , Boilly-Marer Y , Verbert A , Delannoy P (1998 ) Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells . 5 : 19 –27 . 37 Li J , Wang Y , Xie Y , Xu Z , Yang J , et al (2012 ) Altered mRNA expressions of sialyltransferases in human gastric cancer tissues . Med Oncol 29 : 84 –90 .21140242 38 Wang PH , Lee WL , Juang CM , Yang YH , Lo WH , et al (2005 ) Altered mRNA expressions of sialyltransferases in ovarian cancers . Gynecologic Oncology 99 : 631 –639 .16112178 39 Skacel PO , Edwards AJ , Harrison CT , Watkins WM (1991 ) Enzymic control of the expression of the X determinant (CD15) in human myeloid cells during maturation: the regulatory role of 6′-sialyltransferase . Blood 78 : 1452 –1460 .1679356 40 Kaneko Y , Yamamoto H , Kersey DS , Colley KJ , Leestma JE , et al (1996 ) The expression of Galh1,4GlcNAc a2,6 sialyltransferase and a2,6-linked sialoglycoconjugates in human brain tumors . Acta Neuropathol (Berl) 91 : 284 –292 .8834541 41 Lin S , Kemmner W , Grigull S , Schlag PM (2002 ) Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells. Exp Cell Res . 276 : 101 –110 . 42 Christie DR , Shaikh FM , Lucas JA , Bellis SL (2008 ) ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function . J Ovarian Res 1 : 3 .19014651 43 Zhu Y , Srivatana U , Ullah A , Gagneja H , Berenson CS , et al (2001 ) Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro . Biochim Biophys Acta 1536 : 148 –160 .11406350 44 Yu S , Zhang L , Li N , Fan J , Liu L , et al (2012 ) Caveolin-1 up-regulates ST6Gal-I to promote the adhesive capability of mouse hepatocarcinoma cells to fibronectin via FAK-mediated adhesion signaling . Biochem Biophys Res Commun 427 : 506 –512 .23022190	23840320	PMC3688732	CC BY	2021-01-05 17:30:08	yes	PLoS One. 2013 Jun 20; 8(6):e65218
==== Front PLoS One PLoS One plos PLoS ONE 1932-6203 Public Library of Science San Francisco, USA 23824286 PONE-D-13-00766 10.1371/journal.pone.0065651 Research Article Biology Genetics Population Genetics Genetic Polymorphism Medicine Clinical Research Design Meta-Analyses Non-Clinical Medicine Health Care Policy Ethnic Differences Oncology Cancers and Neoplasms Genitourinary Tract Tumors Prostate Cancer Urology Association of Polymorphism rs198977 in Human Kallikrein-2 Gene (KLK2) with Susceptibility of Prostate Cancer: A Meta-Analysis A Meta-Analysis for Prostate Cancer Wang Lishan 1 2 1 Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, P.R. China 2 FengHe (ShangHai) Information Technology Co., Ltd, Shanghai, P.R. China Medeiros Rui Editor IPO, Inst Port Oncology, Portugal Competing Interests: The author Lishan Wang is employed by Fenghe (ShangHai) Information Technology Co., whose company provided funding towards this study. There are no patents, products in development or marketed products to declare. This does not alter the author's adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: LSW. 2013 18 6 2013 8 6 e6565129 12 2012 26 4 2013 © 2013 Lishan Wang 2013 Lishan Wang https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Objectives To assess the association of polymorphism rs198977 in the human kallikrein-2 gene (KLK2) and risk of prostate cancer (PCa). Methods Two investigators independently searched the PubMed, Elsevier, EMBASE, Web of Science, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI). Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) for rs198977 and PCa were calculated in a fixed-effects model (the Mantel-Haenszel method) and a random-effects model (the DerSimonian and Laird method) when appropriate. Results Six studies met the inclusion criteria in this meta-analysis, which included 5859 PCa cases and 4867 controls. Overall, rs198977 was associated with the PCa risk (TT+CT vs. CC, pooled OR = 1.163, 95% CI = 1.076–1.258, P-value <0.0001). When stratified by ethnicity, significant association was observed in Caucasian samples under both allele comparison (T vs. C, pooled OR = 1.152, 95% CI = 1.079–1.229, P-value <0.0001) and dominant model (TT+CT vs. CC, pooled OR = 1.197, 95% CI = 1.104–1.297, P-value <0.0001). In the overall analysis, a comparably significant increase in the frequency of allele T for rs198977 was detected between cases and controls in Caucasian. Conclusion This meta-analysis suggests that rs198977 of KLK2 was associated with susceptibility of PCa in Caucasian and the allele T might increase the risk of PCa in Caucasian. No current external funding sources for this study. ==== Body pmcIntroduction Prostate cancer (PCa) is the most commonly diagnosed visceral malignancy accounting for more than one-third of all incident cancers and is the second-leading cause of cancer deaths in men in the United States and the western world [1] [2]. Prostate-specific antigen (PSA) has been used for detecting PCa since 1994 [3]. The wide availability of total PSA revolutionized PCa screening and ushered in the PSA era resulting in a decrease of PCa metastasis and death. Several novel blood-based biomarkers, such as human glandular kallikrein 2 (hK2) may also help PCa diagnosis, staging, prognostication, and monitoring [4]. There is considerable evidence for a genetic basis underlying risks for PCa [5], and it is among the most heritable of the common cancers with a heritability of 42% estimated from a twin study [6].The kallikrein (KLK) gene family, consisting of 15 genes spanning a region of approximately 300 kb on 19q13.4, encodes the largest known cluster of serine-proteases in the human genome [7] [8]. Two of the classical KLK genes, KLK3 and KLK2, that are contiguously located, are expressed almost exclusively in prostate tissue. KLK3 encodes PSA [9], which is produced primarily by prostate epithelium and is widely used as a tumor marker for early detection and monitoring of PCa [10] [11]. KLK2 encodes hK2 that is specific for the prostate and regulates the expression of PSA [12]. They share an 80% sequence homology and are both primarily expressed in the prostate gland [13]. Using a set of single nucleotide polymorphisms (SNPs) that span the KLK2 region, Nam et al. have demonstrated that KLK2 variants correlate with hK2 serum levels [14]. In addition, Stenman et al. showed that hK2 mRNA expression was significantly higher in PCa tissue compared with benign tissue [15]. Moreover, hK2 is expressed at higher level in poorly differentiated cancers and is a likely candidate for prostate carcinogenesis [16]. All of these observations strongly implicate KLK2 as a plausible candidate gene involved in PCa susceptibility. During this decade, a number of studies have assessed the association between polymorphism rs198977 in KLK2 and risk of PCa in different populations; however, the results are inconsistent and inconclusive [17] [18]. Different methodologies have been used, and in particular, samples are collected from all over the world. Therefore, it is not surprising that there has been a lack of replication in different studies. By using all the available published data to increase the statistical power, it was hypothesized that a meta-analysis might allow plausible candidate genes to be excluded and causative genes to be identified with reliability. To confirm whether the polymorphism rs198977 in KLK2 is associated with susceptibility of PCa, we have taken a meta-analysis in which all the published case-control studies are processed. Materials and Methods Literature Search Published reports assessing the association between polymorphism of KLK2 and risk of PCa were collected through a comprehensive search of six databases, including PubMed, Elsevier, EMBASE, Web of Science, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI). The search terms were as follows: (‘KLK2’ OR ‘kallikrein-2’ OR ‘hK2’) AND (‘prostate cancer’). Searching was completed on Nov 1, 2012. Publication date and publication language were not restricted in our search. Meanwhile, reference lists were examined manually to further identify potentially relevant studies. Unpublished reports were not considered. If more than one article was published by the same author using the same case series, the one investigating the most individuals was selected. Inclusion and Exclusion Criteria Abstracts of all citations and retrieved studies were reviewed. Studies meeting the following criteria were included: (1) Using a case–control design; (2) Detecting the relationship between the polymorphism rs198977 and PCa; (3) Providing available genotype data of rs198977. Studies were excluded if one of the following existed: (1) The design was based on family or sibling pairs; (2) The genotype frequency of rs198977 was not reported; (3) The association of rs198977 with susceptibility of PCa (e.g. cancer progression and mortality) was not detected, or (4) There was insufficient information for extraction of data. Data Extraction All data were extracted independently by two reviewers (lishan wang and weidong zang) according to the inclusion criteria listed above. The results were compared and disagreements were discussed and resolved with consensus. Evaluation was based on title and abstract whenever available. Full text articles of potentially relevant studies were obtained and re-evaluated for inclusion. The following characteristics were collected from each study using an Excel data extraction form: first author, year of publication, country of sample, ethnicity, numbers of cases and controls, main background of samples, and genotyping methods (Table 1). 10.1371/journal.pone.0065651.t001 Table 1 Characteristics of studies included in the meta-analysis. Author Year Country Ethnicity No.(cases/controls) Matching criteria Genotyping methods Ref. Nam, R. K. 2003 Canada Caucasian 616/671 Age, race PCR-RFLP [17] Chiang,C.H 2005 China Asian 254/168 Age, race PCR-RFLP [18] Nam, R. K. 2005 Canada Caucasian 996/1092 Age, race PCR-RFLP [19] Mittal,R. D. 2007 India Asian 135/142 Age, race PCR-RFLP [20] Ahn, J. 2008 USA Caucasian 1172/1157 Age, race Sequencing [21] Klein,R.J.(a)* 2010 Sweden Caucasian 1397/724 Age, race Sequencing [22] Klein,R.J.(b)* 2010 Sweden Caucasian 1219/842 Age, race Sequencing [22] * Klein’s study included two separate groups of samples, data was extracted according to the groups (a and b) as they were independent with each other and analyzed respectively in the initial study. PCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism. Statistical Analysis The statistical analysis was conducted using STATA 11.0 (Stata Corp LP, College Station, TX, United States); P-value <0.05 was considered statistically significant. Hardy-Weinberg equilibrium (HWE) in the controls was tested by the chi-square test for goodness of fit, and a P –value <0.05 was considered as significant disequilibrium. Pooled odds ratio (ORs) were calculated for allele comparison (T vs. C), dominant model (TT+CT vs. CC), and recessive model (TT vs. CC+CT), respectively. The significance of pooled ORs was determined by Z-test and P-value <0.05 was considered as statistically significant. The OR and 95% CI were estimated for each study in a random-effects model or in a fixed-effects model. Heterogeneity among studies was examined with the χ2 -based Q testing and I2 statistics [19]. P-value <0.1 was considered significant for the χ2-based Q testing and I2 was interpreted as the proportion of total variation contributed by between-study variation. If there was a significant heterogeneity (P-value <0.1), we selected a random-effects model (the DerSimonian and Laird method) to pool the data. If not, we selected a fixed-effects model (the Mantel-Haenszel method) to pool the data. Heterogeneity was also quantified using the I2 metric (I2<25%, no heterogeneity; I2 = 25–50%, moderate heterogeneity; I2>50%, large or extreme heterogeneity) [25]. Publication bias was examined with funnel plots and with the Egger’s tests [20] [21]. If there is evidence of publication bias, the funnel plot is noticeably asymmetric. For the Egger’s tests the significance level was set at 0.05. Results Study Characteristics A total of 121 papers were retrieved after the first search, and 115 of these were excluded from the analysis for reasons detailed in Figure 1. Only 6 case-control studies met the inclusion criteria in this meta-analysis, which included 5859 PCa cases and 4867 controls [17] [18]. Characteristics of studies included in the meta-analysis were presented in Tables 1 and 2. The qualities of the studies were considered acceptable for our meta-analysis. We calculated HWE for all six publications and found that only Klein’s study [18] was inconsistent with Hardy-Weinberg disequilibrium (P-value = 0.01). The flow chart of selection of studies and reasons for exclusion was presented in Figure 1. Studies had been carried out in Canada (n = 2), Sweden (n = 1), USA (n = 1), China (n = 1) and India (n = 1). Four studies [17][22] [23] used Caucasian samples while two studies [24][25] used Asian samples. 10.1371/journal.pone.0065651.g001 Figure 1 Flow chart of selection of studies and specific reasons for exclusion from the meta-analysis. 10.1371/journal.pone.0065651.t002 Table 2 Genotype frequencies of rs198977 in studies included in the meta-analysis. Author Year Case genotypea Control genotypeb HWEc Ref. CC CT TT CC CT TT Nam, R. K. 2003 315 251 50 394 240 37 0.873 [17] Chiang,C.H 2005 169 83 2 94 52 9 0.195 [18] Nam, R. K. 2005 522 394 80 621 413 58 0.782 [19] Mittal,R. D. 2007 78 50 7 72 52 18 0.211 [20] Ahn, J. 2008 621 469 82 660 428 69 0.719 [21] Klein,R.J.(a) 2010 788 515 94 443 233 48 0.052 [22] Klein,R.J.(b) 2010 670 460 89 483 287 72 0.010 [22] a Absolute number of patients; b Absolute number of controls; c HWE: Hardy-Weinberg equilibrium, it was evaluated using the goodness-of-fit chi-square test. P-values were presented. P<0.05 was considered representative of a departure from HWE. Evaluation of rs198977 and Association with PCa There were six case-control studies [17] [18]which had been performed to study the polymorphism rs198977 and PCa risk. Results of the meta-analysis were shown in Table 3. Overall, when all the eligible studies were pooled into the meta-analysis, we found that a significant PCa risk was associated with rs198977 polymorphism in a dominant model (TT+CT vs. CC, pooled OR = 1.163, 95% CI = 1.076–1.258, P-value <0.0001); while no significant association was observed in either allele comparison (T vs. C, pooled OR = 1.077, 95% CI = 0.957–1.212, P-value = 0.216) or recessive model (TT vs. CT+CC, pooled OR = 0.993, 95% CI = 0.718–1.373, P-value = 0.964). 10.1371/journal.pone.0065651.t003 Table 3 Pooled odds ratio for rs198977 in meta-analyses. Population Genetic Model Pooled OR(95% CI) P-valuea P-valueb (Publication bias) P-valuec (heterogeneity) I2 All Allele T vs.C 1.077(0.957–1.212) 0.216 0.047 0.006 67.0% Dominant 1.163(1.076–1.258) <0.0001 0.050 0.143 37.4% Recessive 0.993(0.718–1.373) 0.964 0.093 0.001 72.2% Caucasian Allele T vs.C 1.152(1.079–1.229) <0.0001 0.328 0.319 14.9% Dominant 1.197(1.104–1.297) <0.0001 0.129 0.703 0.0% Recessive 1.173(0.930–1.480) 0.177 0.328 0.077 52.5% Asian Allele T vs.C 0.701(0.542–0.906) 0.007 – 0.928 0.0% Dominant 0.765(0.560–1.045) 0.092 – 0.924 0.0% Recessive 0.278(0.128–0.600) 0.001 – 0.239 27.8% a Random-effects model was used when the p-value for heterogeneity test<0.10, otherwise the fixed-effect model was used. b Egger’s test to evaluate publication bias, P –value <0.05 is considered statistically significant. c P-value <0.1 is considered statistically significant for Q statistics. When studies were stratified by ethnicity, significant associations were observed in Caucasian group in both allele comparison (T vs. C, pooled OR = 1.152, 95% CI = 1.079–1.229, P-value <0.0001) and dominant model (TT+CT vs. CC, pooled OR = 1.197, 95% CI = 1.104–1.297, P-value <0.0001) (Figure 2); while no significant association was observed in Caucasian group in recessive model (TT vs. CT+CC, pooled OR = 1.173, 95% CI = 0.930–1.480, P-value = 0.177). In addition, significant associations were observed in Asian group in both allele comparison (T vs. C, pooled OR = 0.701, 95% CI = 0.542–0.906, P-value = 0.007) and recessive model (TT vs. CT+CC, pooled OR = 0.278, 95% CI = 0.128–0.600, P-value = 0.001); while no significant association was observed in Asian group in dominant model (TT+CT vs. CC, pooled OR = 0.765, 95% CI = 0.560–1.045, P-value = 0.092). 10.1371/journal.pone.0065651.g002 Figure 2 Forest plots of studies with Caucasian samples under dominant model (a) and Allele comparison model (b). Sensitivity Analysis The influence of a single study on the overall meta-analysis was investigated by omitting one study at a time, and the omission of any study made no significant difference, indicating that our results were statistically reliable. Evaluation of Heterogeneity For all samples, statistically significant heterogeneity was observed under both allele comparison (T vs. C, P-value by χ2 -based Q testing = 0.006 and I2 = 67.0%) and recessive model (TT vs. CT+CC, P-value by χ2 -based Q testing = 0.001 and I2 = 72.2%), but no significant heterogeneity was observed under dominant model (TT+CT vs. CC, P-value by χ2 -based Q testing = 0.143 and I2 = 37.4%). Then subgroup analysis was carried out. When studies were stratified by ethnicity, no statistically significant heterogeneity was observed in Caucasian under either allele comparison (T vs. C, P-value by χ2 -based Q testing = 0.319 and I2 = 14.9%) or dominant model (TT+CT vs. CC, P-value by χ2 -based Q testing = 0.703 and I2 = 0.0%), but there was significant heterogeneity under recessive model (TT vs. CT+CC, P-value by χ2 -based Q testing = 0.077 and I2 = 52.5%). For Asian, no statistically significant heterogeneity was observed under any model (all P-values by χ2 -based Q testing >0.1 and I2<50%). Results of heterogeneity were shown in Table 3. Publication Bias Funnel plot and Egger’s test were performed to assess the publication bias of the literature. Results publication bias was shown in Table 3. For all samples, publication bias was observed under allele comparison (T vs. C, P-value of Egger’s test = 0.047). After samples were stratified by ethnicity, no publication bias was observed under any model (all P-value of Egger’s test >0.05). Symmetrical funnel plots were obtained for Caucasian (Figure 3), but Funnel plot and Egger’s test were not available for Asian samples because of the small sample size. 10.1371/journal.pone.0065651.g003 Figure 3 Funnel plots of studies with Caucasian samples under dominant model (a) and Allele comparison model (b). Discussion The data from this meta-analysis showed a significant increase in frequency of genotype TT+CT of rs198977 polymorphism in patients with PCa than controls, which suggested that genotype TT+CT might increase the risk of PCa with pooled OR of 1.163. When stratified the samples by their ethnicity, the frequencies of both the allele T and genotype TT+CT in Caucasian had significant increase in cases than controls with pooled OR of 1.152 and 1.197, respectively; but things became different for Asian, there was a significant decrease in frequency of allele T in cases than control with pooled OR of 0.701. Though results from Asian samples indicated allele C of rs198977 was the risk factor, it still be under discussion because of the small sample size in our analysis. But the results clearly suggested that allele T of rs198977 was a moderate risk factor of PCa for Caucasian. In addition, Mikolajczyk et al. showed that increased hK2 expression in PCa tissues could influence cancer biology not only by activating uPA but also by inactivating its primary inhibitor, plasminogen activator inhibitor [26]. And results from transgenic mice demonstrate, with biologically relevant models, that KLK2 is the protease responsible for activating PSA [27]. Variants on KLK2 gene may affect the expression of hK2, and thus its biological function might be altered. These previous findings support our results and give us possible explanation to the mechanism. The degree of heterogeneity is one of the major concerns in meta-analysis as non-homogeneous data are liable to result in misleading results. In the present study, the Q testing and I2 statistics were carried out to test the significance of heterogeneity. For all samples, significant heterogeneity existed under allele comparison and recessive model. After stratifying the samples according to their ethnicity, the heterogeneity decreased. For Caucasian samples, significant heterogeneity was observed under recessive model, while no statistically significant heterogeneity was observed under any model in Asian samples. The results indicated ethnicity might play an important role in genetic heterogeneity of rs198977. In other words, there exist heterogeneity of rs198977 between Caucasian and Asian according to our results. Publication bias is another important factor affecting the quality of meta-analysis. In order to assess publication bias, funnel plot and Egger’s test were performed. Publication bias was observed under allele comparison when all studies were included. After removing Asian samples, no publication bias was observed under any model and symmetrical funnel plots were obtained for Caucasian (all P-value of Egger’s test >0.05) (Table 3 and Figure 3). This indicated that two studies with Asian samples might be responsible for the publication bias, but the reason could not be exactly determined as Funnel plot and Egger’s test were not available for only two studies. Moreover, we performed a sensitivity analysis by removing one study each time and re-running the model to determine the effect on each overall estimate. The estimates changed little, which implied that our results were statistically reliable. However, there are still some limitations in this meta-analysis. (1) In six studies included for our analysis, two of them are Asian samples occupied only 6.52% of whole samples, so such results should be interpreted with caution; (2) Because the samples from 5 countries and controls were not uniform, as in most meta-analyses, results should be interpreted with caution; and (3) meta-analysis is retrospective research that is subject to methodological limitations. In order to minimize the bias, we used explicit methods for study selection, data extraction and data analysis. Nevertheless, our results should be interpreted with caution. This meta-analysis suggests that the polymorphism rs198977 of KLK2 was associated with susceptibility of prostate cancer in Caucasian and the allele T might increase the risk of prostate cancer. The pooled ORs in this study suggest that allele T and genotype TT+CT both have modest but definite genetic effect on prostate cancer in Caucasian. Larger and well-designed studies based on different ethnic groups are needed to confirm our results, especially for Asian samples. Supporting Information Checklist S1 PRISMA checklist. (DOC) Click here for additional data file. ==== Refs References 1 Jemal A , Siegel R , Xu J , Ward E (2010) Cancer statistics, 2010. CA: a cancer journal for clinicians 60 : 277–300.20610543 2 Siegel R , Ward E , Brawley O , Jemal A (2011) Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA: a cancer journal for clinicians 61 : 212–236.21685461 3 Nogueira L , Corradi R , Eastham JA (2010) Other biomarkers for detecting prostate cancer. BJU int 105 : 166–169.19930175 4 Shariat SF , Semjonow A , Lilja H , Savage C , Vickers AJ , et al . (2011) Tumor markers in prostate cancer I: blood-based markers. Acta Oncol 50 Suppl 161–75. 5 Langeberg WJ , Isaacs WB , Stanford JL (2007) Genetic etiology of hereditary prostate cancer. Frontiers in bioscience : a journal and virtual library 12 : 4101–4110.17485361 6 Lichtenstein P , Holm NV , Verkasalo PK , Iliadou A , Kaprio J , et al . (2000) Environmental and heritable factors in the causation of cancer–analyses of cohorts of twins from Sweden, Denmark, and Finland. The New Engl J Med 343 : 78–85.10891514 7 Yousef GM , Luo LY , Diamandis EP (1999) Identification of novel human kallikrein-like genes on chromosome 19q13.3-q13.4. Anticancer research 19 : 2843–2852.10652563 8 Diamandis EP , Yousef GM , Luo LY , Magklara A , Obiezu CV (2000) The new human kallikrein gene family: implications in carcinogenesis. TEM 11 : 54–60.10675891 9 Riegman PH , Vlietstra RJ , van der Korput JA , Romijn JC , Trapman J (1989) Characterization of the prostate-specific antigen gene: a novel human kallikrein-like gene. Biochem biophy res commun 159 : 95–102. 10 Catalona WJ , Smith DS , Ratliff TL , Basler JW (1993) Detection of organ-confined prostate cancer is increased through prostate-specific antigen-based screening. JAMA 270 : 948–954.7688438 11 Catalona WJ , Richie JP , deKernion JB , Ahmann FR , Ratliff TL , et al . (1994) Comparison of prostate specific antigen concentration versus prostate specific antigen density in the early detection of prostate cancer: receiver operating characteristic curves. The J urol 152 : 2031–2036.7525994 12 Partin AW , Catalona WJ , Finlay JA , Darte C , Tindall DJ , et al . (1999) Use of human glandular kallikrein 2 for the detection of prostate cancer: preliminary analysis. Urology 54 : 839–845.10565744 13 Yousef GM , Diamandis EP (2001) The new human tissue kallikrein gene family: structure, function, and association to disease. Endocrine rev 22 : 184–204.11294823 14 Nam RK , Zhang WW , Klotz LH , Trachtenberg J , Jewett MA , et al . (2006) Variants of the hK2 protein gene (KLK2) are associated with serum hK2 levels and predict the presence of prostate cancer at biopsy. Clin cancer res 12 : 6452–6458.17085659 15 Lintula S , Stenman J , Bjartell A , Nordling S , Stenman UH (2005) Relative concentrations of hK2/PSA mRNA in benign and malignant prostatic tissue. The Prostate 63 : 324–329.15599939 16 Tremblay RR , Deperthes D , Tetu B , Dube JY (1997) Immunohistochemical study suggesting a complementary role of kallikreins hK2 and hK3 (prostate-specific antigen) in the functional analysis of human prostate tumors. The Am J pathol 150 : 455–459.9033261 17 Nam RK , Zhang WW , Trachtenberg J , Diamandis E , Toi A , et al . (2003) Single nucleotide polymorphism of the human kallikrein-2 gene highly correlates with serum human kallikrein-2 levels and in combination enhances prostate cancer detection. J clin oncol 21 : 2312–2319.12805332 18 Klein RJ , Hallden C , Cronin AM , Ploner A , Wiklund F , et al . (2010) Blood biomarker levels to aid discovery of cancer-related single-nucleotide polymorphisms: kallikreins and prostate cancer. Cancer Prev Res (Phila) 3 : 611–619.20424135 19 Higgins JP , Thompson SG (2002) Quantifying heterogeneity in a meta-analysis. Statist med 21 : 1539–1558. 20 Light RJ, Pillemer DB (1984) Summing up : the science of reviewing research. Harvard University Press, Cambridge, Mass. 21 Egger M , Davey Smith G , Schneider M , Minder C (1997) Bias in meta-analysis detected by a simple, graphical test. BMJ 315 : 629–634.9310563 22 Nam RK , Zhang WW , Jewett MA , Trachtenberg J , Klotz LH , et al . (2005) The use of genetic markers to determine risk for prostate cancer at prostate biopsy. Clin cancer res 11 : 8391–8397.16322300 23 Ahn J, Berndt SI, Wacholder S, Kraft P, Kibel AS et al .. (2008) Variation in KLK genes, prostate-specific antigen and risk of prostate cancer. Nature genetics 40: 1032–1034; author reply 1035–1036. 24 Chiang CH , Hong CJ , Chang YH , Chang LS , Chen KK (2005) Human kallikrein-2 gene polymorphism is associated with the occurrence of prostate cancer. J urology 173 : 429–432. 25 Mittal RD , Mishra DK , Thangaraj K , Singh R , Mandhani A (2007) Is there an inter-relationship between prostate specific antigen, kallikrein-2 and androgen receptor gene polymorphisms with risk of prostate cancer in north Indian population? Steroids 72 : 335–341.17257635 26 Mikolajczyk SD , Millar LS , Kumar A , Saedi MS (1999) Prostatic human kallikrein 2 inactivates and complexes with plasminogen activator inhibitor-1. Int J cancer 81 : 438–442.10209959 27 Williams SA , Xu Y , De Marzo AM , Isaacs JT , Denmeade SR (2010) Prostate-specific antigen (PSA) is activated by KLK2 in prostate cancer ex vivo models and in prostate-targeted PSA/KLK2 double transgenic mice. The Prostate 70 : 788–796.20058238	23824286	PMC3688805	CC BY	2023-06-01 23:50:08	yes	PLoS One. 2013 Jun 18; 8(6):e65651
==== Front Malar Res TreatMalar Res TreatMRTMalaria Research and Treatment2090-80752044-4362Hindawi Publishing Corporation 10.1155/2013/426040Research ArticleA Study on Course of Infection and Haematological Changes in falciparum-Infected in Comparison with Artemisinin(s)-Treated Mice Kuthala Kalyan Kumar Meka Sowjanya Kanikaram Sunita Department of Zoology and Aquaculture, Acharya Nagarjuna University, Nagarjunanagar, Guntur, Andhra Pradesh 522 510, IndiaSunita Kanikaram: sunitamichael@yahoo.comAcademic Editor: Mats Wahlgren 2013 11 6 2013 2013 42604029 12 2012 12 5 2013 17 5 2013 Copyright © 2013 Kalyan Kumar Kuthala et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.To find out the efficacy and effect of artemisinin derivatives on haematological indices, C57BL/6J mice were challenged with Plasmodium falciparum and treated with therapeutic doses of AS, AE, and AL. Course of infection was studied in the infected and treated groups up to day 42. Peak level of parasitaemia (38%) was observed on day 11 in infected group. Haematological indices indicated significant (P < 0.05) decrease in RBC, WBC, haemoglobin, packed cell volume, mean cell volume, and platelet counts in infected mice. But all the parameters were restored to normal values, and significant (P < 0.05) changes were observed in all drug-treated groups. Insignificant changes were observed for MCHC (P > 0.05) in all drug-treated groups. Percent of peak parasitaemia was much reduced in AL- (3.2% on day 3) treated group in comparison with AE- (2.4% on day 4) and AS- (4% on day 2) treated groups. Parasites were completely cleared on day 6 in AS group, day 5 in AE group, and day 4 in AL group. Hence, our results strongly support that combination therapy has high efficacy rates than monotherapy. No adverse effects were observed on haematological parameters when animals were treated with therapeutic dosages. ==== Body 1. Introduction Despite advances in knowledge, malaria continues to cause significant morbidity and mortality worldwide. Over 40% of the world population lives in malaria-endemic areas and it is very high (20%) in severe malaria (parasitaemia > 5%). Today malaria is the most important problem for which an estimated 300–500 million cases were recorded and 1.5–2.7 million deaths occur each year [1]. Among them 19,500 death cases due to malaria have been recorded in India [2]. Mortality rate usually depends on the management of malaria which involves antimalarial drug resistance of Plasmodium falciparum and occurrence of systemic complications. Most of the systemic complications from malaria are mainly because of hyperparasitaemia [3]. Blood is the most easily accessible diagnostic tissue. Variations in haematological parameters are influenced by any disease condition which affects the haemopoietic physiology. This is likely to happen with an endemic disease such as malaria that affects the host homeostasis [4]. The target of malaria parasite is RBC so that peripheral blood smear examination is the major diagnostic tool of the disease. Microscopic diagnosis is the “imperfect gold standard” for malaria parasite detection and species identification. This technique requires technical expertise and time consuming in repeated smear examinations [5]. However, it is a valuable technique when performed correctly in the right hands but can be unreliable and perceived as useless when poorly executed [6, 7]. Haematological changes associated with malaria infection are well recognized, but specific changes may differ from the level of malaria endemicity, background haemoglobinopathy, nutritional status, demographic factors, and immunity to malaria [8]. Hence, haematological changes are the most common and important complications encountered and are considered a hallmark of malaria, playing a major role in the fatality. Prediction of the haematological changes enables the clinician to establish an effective and early therapeutic intervention in order to prevent the occurrence of major complications [3]. The haematological abnormalities that have been reported include anaemia, thrombocytopenia, lymphocytosis and rarely disseminated intravascular coagulation [9], leucopenia, leucocytosis, neutropenia, neutrophilia, and eosinophilia, and monocytosis also have been reported [10, 11]. Artemisinin(s) monotherapy and combination therapy (ACTs) are currently used as first line treatment for uncomplicated malaria. Artesunate is a semisynthetic derivative of artemisinin consisting of sodium succinyl salt of dihydroartemisinin, a potent blood schizonticide highly effective against multidrug resistant strains of P. falciparum. Hence it is widely used for the treatment of malaria [12]. α, β arteether (30 : 70 mixture of enantiomers) is a fast-acting blood schizonticide completed multicentric clinical trials in P. falciparum endemic areas and was found to be effective and marketed in 1997 as E Mal. Arteether has a higher safety margin compared to other artemisinin derivatives, has longer half-life, and produces high cure rates when administered in a short 3-dose (i.m.) regimen [13]. Artemether + lumefantrine combination combines the benefits of a rapid short-lived schizonticidal effect of arteether with a slower but longer acting schizonticidal effect of Lumefantrine, a highly lipophilic aryl amino alcohol. The present study was aimed to reveal the effect of 3 frequently used artemisinin derivatives, namely, artesunate (oral), arteether (i.m.), and artemether + lumefantrine (oral) on haematological parameters. The present investigation was also aimed to study the course of infection in falciparum-infected and drug-treated mice to know the efficacy of Artemisinin derivatives based on parasite clearance time (PCT). Thus the aim of our study was to investigate the different hematological changes with P. falciparum malaria and to define the possible role of Plasmodium species in the pathogenesis related to haematological changes. 2. Materials and Methods 2.1. Animals C57BL/6J male mice of age of 10-week-old were purchased from National Centre for Laboratory Animal Sciences (N.C.L.A.S), National Institute of Nutrition (NIN), Hyderabad, India, and allowed to acclimatize for 15 days. Animals were fed with standard feed daily and water was given adlibitum at room temperature of 24 ± 5°C with 12 hrs. light and dark cycle. The study was conducted in accordance with guidelines in the Guide of the Care and Use of Laboratory Animals [14]. 2.2. Diagnosis, Collection, and Storage of Blood For the present experiment the species of P. falciparum was collected from an infected person from Government General Hospital in Guntur, Andhra Pradesh. Patient is tested with SD BIOLINE Malaria rapid test (P.f/p.v) and blood smear examination by 10 to 15 years experienced microbiologist who confirmed P. falciparum with high parasitaemia. At the time of blood collection the patient does not receive any preantimalarial treatment even paracetamol. The blood containing P. falciparum was extracted from peripheral vein of hand of the infected individual using 5 mL sterile syringe. Immediately blood was transferred to EDTA vacutainer (BD Franklin, USA); kept in thermocol ice box, and transferred to the laboratory. The obtained antigen was inoculated into the mice within half an hour of collection. 2.3. Preparation and Inoculation of Antigen The collected blood was washed several times in phosphate buffered saline (PBS, pH 7.0) by centrifugation at 1000 rpm/15 min. The washed erythrocytes were suspended in PBS and packed by centrifugation, and supernatant was removed by a pipette. The sediment with P. falciparum-infected erythrocytes was diluted with PBS. After obtaining P. f. antigen, the parasites were maintained experimentally in three C57BL/6J male mice, and the level of parasitaemia was monitored after the next day of inoculation by smear preparation. After the achievement of high level of parasitaemia (stock blood), blood samples were collected and diluted in normal saline at the ratio of 75% parasitized blood and 25% PBS. The diluted parasitized blood was then inoculated into different experimental mice groups on day “0” via intraperitoneal (i.p.) route because malaria parasite penetrates the peritoneal wall into the blood stream within one minute of inoculation. Mice of control group were inoculated with distilled water on day “0” and maintained as control. 2.4. Drug Administration A total number of 40 C57BL/6J male mice were distributed into 5 groups, namely, control (CON), infected (INF), drug treated with artesunate (DT AS), drug treated with arteether (DT AE) and drug treated with artemether + lumefantrine (DT AL) of 8 animals each. For all drug-treated groups, therapeutic dosages according to WHO recommendation were administered. Doses were calculated according to the average body weight of the mice (approximately 30 gm/mouse). For monotherapy, artesunate (AS) tablets (Falcigo) from Zydus Cadila Health Care Limited, India, and arteether (AE) (E Mal) from Themis Chemicals Limited, Mumbai, India, were obtained. For combination therapy, artemether + lumefantrine (AL) (Lumerax-20 DT) from Ipca Laboratories Limited, India, was obtained. Artesunate: through oral gavage in 4 (double divided dose), 2, 2, 2, and 2 mg/kg body weight for five days. Arteether: intramuscularly in 3 mg/kg body weight for three days. Artemether + Lumefantrine: through oral gavage in 3.5 mg/kg body weight for three days. 2.5. Course of Infection Course of infection was studied in all the 12-week C57BL/6J experimental mice. Parasitaemia was monitored daily up to day “42” by making peripheral blood smears. Comparison of parasitaemia between infected mice and drug-treated mice revealed the efficacy of each drug used for the study. Thin blood films were made on the prelabelled slide with free flowing whole blood directly from the mouse tail snips after the first drop was wiped off with cotton wool. The blood films were stained with JSB I and JSB II for the detection of malarial parasites and for estimation of parasitaemia. After staining, slides were washed with tap water to remove excess stain and allowed to drain in a vertical position and to air dry. A field was selected where the RBCs are in an evenly distributed monolayer and observed under 100x oil immersion objective. A minimum of 1000 RBCs were counted from 10 fields under microscope, and the number of infected RBCs will be recorded. The percent of parasitaemia was determined by enumerating the number of infected RBCs per total number of RBCs counted (5): (1) parasitaemia%=no. of infected RBCsno. of RBCs counted×100. 2.6. Evaluation of Responses Parasite clearance time (PCT) was defined as the time from the start of treatment until first negative blood smear for asexual stages which remained negative for an additional 24 hours. For our study we followed 42-day followup for all treatment groups. Thus PCT was observed in all the experimental groups. 2.7. Estimation of Haematological Parameters At the end of the series of experiments (i.e., after achieving first negative blood smear), 6 animals in all the drug-treated groups were sacrificed using chloroform anaesthesia. Six animals in infected and six animals in control group were sacrificed on day 28. Two animals in all the groups kept for 42 days, and one animal is used to calculate parasitaemia on each day. Blood samples were collected by cardiac puncture into EDTA vacutainer to see the frequency of haematological abnormalities especially anaemia, thrombocytopenia, and reduced blood counts in malaria. Whole blood was immediately analyzed for complete blood picture (CBP), that is, red blood cell (RBC) count, haemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), white blood cell (WBC) count, and platelet (PLT) count using the fully automated ABX Pentra 60 + Analyser (Horiba ABX, Montpellier, France). Briefly, 53 µL of blood was aspirated into a needle divided and distributed to the various chambers for sample analysis. 2.8. Statistical Analysis The means, standard deviations of normally distributed data were compared between control versus infected group and infected versus drug-treated groups using Student's t-test with MINITAB 11.12.32. Bit statistical package and graphs were drawn from MS Excel 2010. The values were given as mean ± SD and are statistically significant at t > 2.306, P < 0.05* (significant), P < 0.001 (more significant), and P < 0.0001* (highly significant). P value more than 0.05 was considered as statistically not significant (NS). 3. Results 3.1. Observation of Plasmodium falciparum Erythrocytic Stages (Asexual Forms) In the present study, P. falciparum erythrocytic stages were developed in the blood of C57BL/6J mouse model after inoculation of 0.3 mL. of P. f antigen intramuscularly to the experimental mice. Various stages of parasite are seen in the blood films stained with JSB-I and JSB-II solutions. The blood films were scanned under high power objective (×40) and examined closely under oil immersion objective (×100). The various erythrocytic stages of P. falciparum were seen such as trophozoites (immature and mature), schizonts, and gametocyte. Appearance of ring forms at the edge of RBC is the characteristic feature of Plasmodium falciparum species (Figure 5). The erythrocytic stages observed were as follows. Immature Trophozoite: undivided nucleated cells with blue-coloured cytoplasm or a ring of cytoplasm within the red cell. Mature Trophozoite: compact cytoplasm, enlarged amoeboid shape, and no ring structure. Schizont: individual nucleated cell distributed throughout the red cell in a circle. 3.2. Study of Course of Infection in Plasmodium falciparum-Infected Mice After inoculation of P. f. antigen on day “0” into the mice of infected (INF) group, the parasites started developing in the peripheral blood from day “1” onwards and reached to peak level on 11th day with 38% parasitaemia. Then the parasitaemia gradually decreased and completely disappeared by day 28 (Figure 1). 3.3. Effect of Artesunate on Plasmodium falciparum in Experimental Mice After inoculating P. f. antigen to mice of DT AS group, parasitic ring stags have appeared on 1st day. Then artesunate drug was administered orally for the next 5 days successively with 2 mg/kg body weight with a double divided dose on the first day. After treating the mice with drug, the peak level of infection was observed on day “3” with 3.5% of parasitaemia only. Then the parasitaemia gradually decreased and disappeared on the 6th day. But the blood smears were examined till the 42nd day and no parasites were observed (Figure 2). The Parasite Clearance Time (PCT) was 144 hours (6 days) with artesunate. 3.4. Effect of Arteether on Plasmodium falciparum in Experimental Mice After inoculation of P. f. antigen to mice of DT AE group, parasitic ring stags have appeared on the 1st day. Arteether was administered (i.m.) on successive 3 days. After Arteether treatment, the peak level of infection was observed on day “2” with 4.2% parasitaemia. Then the parasites were completely disappeared by the 5th day (Figure 3). The Parasite Clearance Time (PCT) was 120 hours (5 days) with arteether. 3.5. Effect of Artemether + Lumefantrine on Plasmodium falciparum in Experimental Mice After giving P. f. antigen to the mice in DT AL group, parasitic ring stages have appeared on the 1st day. Then artemether + lumefantrine (artemisinin based combination therapy) was administered orally for the next three days. With the combination therapy, peak level of infection was observed on day “3” with 3.2% parasitemia, and no parasites were observed on day “4” till the 42nd day (Figure 4). The Parasite Clearance Time (PCT) was 96 hours (4 days) with artemether + lumefantrine. 3.6. Changes in Haematological Values of Experimental C57BL/6J Mice due to Plasmodium falciparum Table 1 shows changes in haematological values between control versus infected group. In the infected group the mean value for HGB is 7.93 ± 0.175 (P < 0.0001), RBC is 5.9 ± 0.201 (P < 0.0001), PCV is 2892 ± 1.46 (P < 0.0001), MCV is 81.07 ± 2.19 (<0.0001), MCH is 27.67 ± 1.17 (0.0001), PLT is 516 ± 23.4 (<0.0001), WBC is 6957 ± 102 (<0.0001), neutrophils is 28.3 ± 0.724 (P < 0.0001), lymphocytes is 69.70 ± 2.00 (P < 0.0001), and eosinophils is 1.0 ± 0.669 (P < 0.014). And these values were significantly decreased when compared to the control values (12.85 ± 0.321, 8.0 ± 0.141, 38.67 ± 1.97, 91.48 ± 1.64, 32.33 ± 1.26, 685 ± 20.7, 9080 ± 98.9, 36.15 ± 0.693, 59.85 ± 2.00, and 2.0 ± 0.187), respectively. MCHC (33.17 ± 1.08, P > 0.05) and monocytes (1.0 ± 0.190, P > 0.05) have not shown significant change in the infected group when compared to control values (34.42 ± 1.12, 1.0 ± 0.228), respectively. Table 2 shows changes in haematological values in infected (INF) versus artesunate drug-treated (DT AS) group. Significant changes were observed in HGB (P < 0.0001), RBC (P < 0.0001), PCV (P < 0.01), MCV (P < 0.01), PLT (P < 0.0001), WBC (P < 0.001), neutrophils (P < 0.0001), lymphocytes (P < 0.0001), and eosinophil (P < 0.05) in artesunate-treated group when compared with control. MCH (P > 0.05), MCHC (P > 0.05), and monocytes (P > 0.05) did not show statistically significant difference between INF and DT AS groups. Table 3 shows changes in haematological values in infected (INF) versus arteether drug-treated (DT AE) group. HGB (P < 0.0001), RBC (P < 0.0001), PCV (P < 0.05), PLT (P < 0.0001), WBC (P < 0.001), neutrophils (P < 0.0001), lymphocytes (P < 0.0001), and eosinophil (P < 0.05) values were significantly increased in DT AE group when compared to INF group. MCV (P > 0.05), MCH (P > 0.05), MCHC (P > 0.05), and monocytes (P > 0.05) have not shown significant change in DT AE group when compared to INF group. Table 4 shows changes in haematological values in infected (INF) versus artemether + lumefantrine drug-treated (DT AL) group. HGB (P < 0.0001), RBC (P < 0.0001), PCV (P < 0.01), MCV (P < 0.001), PLT (P < 0.0001), WBC (P < 0.001), neutrophils (P < 0.0001), lymphocytes (P < 0.01), and eosinophil (P < 0.05) in artemether + lumefantrine-treated group have significantly increased when compared to the infected group. Insignificant changes were observed for MCH (P > 0.05), MCHC (P > 0.05), and monocytes (P > 0.05) between INF and DT AL groups. 4. Discussion Severe falciparum malaria is associated with large parasite burdens. The peripheral blood parasite count is the prognostic indicator that correlates with the total parasite biomass and thus the severity of falciparum malaria. Haematological abnormalities are considered a hallmark of malaria and reported to be the most pronounced in P. falciparum infection, probably as a result of the higher levels of parasitaemia [15]. This study was conducted to assess and compare the incidence and severity of haematological changes in falciparum-infected C57BL/6J mice using an experimental model. In this study we observed that there is a marked reduction in RBC count, WBC count, haemoglobin (HGB), packed cell volume (PCV), mean cell volume (MCV), and platelet counts in falciparum-infected mice when compared to control animals. The reduction was more evident in falciparum infection. It is thought to result from a combination of haemolysis of parasitized red blood cells, accelerated removal of both parasitized and innocently unparasitized red cells, depressed as well as ineffective erythropoiesis with dyserythropoietic changes, and anaemia of chronic disease [15]. Other factors contributing to anaemia in malaria include decreased red blood cell deformability, splenic phagocytosis, and/or pooling. So they have an increased rate of clearance from the circulation [16]. Malaria parasite within RBCs, ingest and digest haemoglobin more than it needs for its own metabolism. Destruction of RBC following parasitisation cannot account for the degree of anaemia observed during malaria infection, suggesting that the destruction of uninfected RBC (uRBC) is the major cause of haemoglobin (HGB) loss [17]. The low PCV value with a correspondent high density of malaria parasite was suggested to be due to excessive destruction of red blood cells by the malaria parasites. As noted in this study, it was established that the more malaria parasites in the blood circulation cause more destruction of the red blood cells as demonstrated by the low PCV. The low PCV may necessitate the need for blood transfusion which in turn has a high risk of transmitting viral hepatitis, HIV, and other associated risks. The significant reduction in PCV level indicates a relationship between malaria parasite and anaemia [18]. Statistically insignificant changes were observed in MCHC (P > 0.05) and monocyte (P > 0.05) values in infected mice when compared to control mice. MCHC levels were not significantly changed which is consistent with the earlier reports [19]. In our study we observed that there was a significant reduction in total WBC counts (P < 0.0001). Leucopenia appears to be a common finding in both nonimmune patients with falciparum malaria and semi-immune children living in malaria-endemic regions [20]. The differential leucocyte count showed normal monocytic, eosinophilic counts and decreased neutrophilic counts. Similarly in the majority of cases, either neutropenia or neutrophilia was reported [20]. Phagocytosis of malaria pigment is by monocyte/macrophages and less frequently by neutrophils [15, 20, 21]. Monocytes and rarely neutrophils contained malaria pigment and in very rare cases, erythrophagocytosis by monocytes was also observed in some studies. Our findings also showed that artemisinin(s) given at the therapeutic doses may not cause neutropenia, which is consistent with prior studies [22, 23]. Lymphocytes, particularly T cells, play a major role in immunity to falciparum malaria by releasing proinflammatory cytokines. However excessive secretion of proinflammatory cytokines has been shown to contribute to the severity in humans [24–26]. Our study showed that mice infected with falciparum had a higher lymphocytic count, and this may represent overstimulation of the proinflammatory pathway. Further studies on the role of lymphocytes are required to determine the significance of our findings. It is a general consensus that thrombocytopenia is very common in falciparum malaria [27, 28] and usually believed that a significant reduction in platelet counts (P < 0.0001) than control animals. Thrombocytopenia seems to be due mainly to a reduced platelet life span and splenic pooling. The reduced platelet life span may be caused by binding of malaria antigen onto platelets followed by antibody-mediated phagocytosis [29] or to platelet activation in vivo. Macrophage activation and hyperplasia especially in the spleen may also play a role [30]. The release of platelet contents can activate the coagulation cascade and contributes to decreased inhibitors concentration and consequently further thrombocytopenia [31]. Artemisinin derivatives are most effective against Plasmodium parasite (as monotherapies); combination therapies consisting of artemisinin(s) and other antimalarial drugs have been demonstrated to have better parasite clearance and efficacies [32–34]. Antimalarial treatment with artemisinin or one of its derivatives is associated with a more rapid decline in parasitaemia than with other antimalarial drugs [35]. Artemisinins induce a decrease in parasitized RBC deformability. In the presence of heme Fe++, these drugs generate carbon centered free radicals that could damage the RBC membrane or cytoskeleton and thereby increase the rigidity of the infected RBC. Artesunate, by acting on young ring forms, attenuated the reduction in deformability parasite, prevented their development to more rigid mature trophozoites, and thereby attenuated the reduction in deformability associated with continued parasite growth. Artesunate induces changes either in the parasite or in the RBC directly and led to increased antigenicity and thus increased opsonization. Terminal half-lives of the orally administered drugs are usually less than 2 h. We observed that once daily administration with artemisinin derivatives provides equivalent cure rates to more frequent administration which is consistent with prior studies [36, 37]. In artesunate group peak of parasitaemia on day 3 with 3.5% and parasites were completely cleared on day 6. First negative blood smear observed on day 6. The peak level of parasitaemia was reduced when compared to AE-treated group (4.2% on day 2). Insignificant changes were observed in MCH (P > 0.05), MCHC (P > 0.05), and monocytes (P > 0.05) for artesunate-treated group. Alpha beta arteether is an ethyl derivative of artemisinin which is an efficient schizonticidal drug in mild malaria. The clinical efficacy of arteether is characterized by an almost immediate onset and rapid reduction in parasitaemia, with complete clearance in most cases within 48 hours. But in our study we observed delayed parasite clearance time in AE-treated groups when compared to previous studies. In arteether group parasites cleared on day 5. We observed positive blood smears for one additional day after the completion of 3-day drug course. The peak level of parasitaemia (4.2% on day 2) was increased when compared to AS-treated group (4 on day 2). Insignificant changes were observed in MCV (P > 0.05), MCH (P > 0.05), MCHC (P > 0.05), and monocytes (P > 0.05) for arteether-treated group. No adverse effects were observed on hematological parameters when animals-treated with α, β arteether which are consistent with prior studies [38, 39]. AE showed lower efficacy than AL and AS. The levels of efficacy are similar to the findings of the recent studies on ACTs in Africa which revealed that AL has higher efficacy rates when compared to other antimalarials [40–43]. In our study we observed rapid parasite clearance in artemether + lumefantrine-treated group in comparison with artesunate- and arteether-treated animals which are in agreement with previous studies. Our results strongly support that combination therapies have high efficacy rates than monotherapy. Artemisinin(s) can be used alone, but this leads to high rate of recrudescence (return of parasites), and there is a possibility of emergence of resistant strains to the single drug treatment on repeated and inappropriate use and other drugs are required to all parasites and to prevent recrudescence [44]. But in our study no parasite recrudescence was observed in monotherapy and combination therapy. But there is a marked variation in peak level parasitaemia and clearance time. In AL group parasites cleared on day 4. We observed positive blood smears for one additional day after the completion of 3-day drug course. The peak level of parasitaemia (3.2% on day 3) was decreased when compared to AS- (4%) and AE- (4.2%) treated groups. The rate of parasite clearance was used as a measure of the artemisinin pharmacodynamic effect in vivo [35]. Artemisinin resistance is characterized by prolongation in clearance times [18]. There is clear evidence that combinations improve efficacy without increasing toxicity. We found higher efficacy rates when animals were treated with combination (AL) drug than monotherapy. We observed positive blood smears on day 3 for all treatment groups. When compared to AS and AE groups, AL group showed higher efficacy rates by clearing parasites on day 4. Insignificant changes were observed in MCHC (P > 0.05) 0 and monocytes (P > 0.05) for artemether + lumefantrine-treated group. 5. Conclusion Considering the higher efficacy rates of artemether +lumefantrine (AL) as compared with artesunate (AS) and arteether (AE), we conclude that AL is clinically more effective than AS and AE. No adverse effects were observed on haematological parameters when animals were treated with artemisinin derivatives. Artemisinin resistance is characterized by prolongation in clearance times which we observed in the present study. The result of falciparum positive blood smear in all treatment groups on day 3 (72 h) was a good predictor for treatment failure and considered as a simple screening measure for artemisinin resistance. Conflict of Interests The authors declare that they do not have conflict of interests. Acknowledgments The authors are extremely thankful to the University Grants Commission, New Delhi, India, for providing financial assistance in the form of Major Research Project (2010–2013) to carry out this work. My Special thanks to Professor Y. Prameela Devi, Department of Zoology, Kakatiya University, Warangal, India, for helping me to write up the Major Research Project. Also thankful to Professor V. Viveka Vardhani (Former Head) and Dr. K. Veeraiah, Head of the Department of Zoology and Aquaculture, for providing facilities through UGC-SAP-DRS and their cooperation during the course of work. Figure 1 Course of infection to Plasmodium falciparum in experimental C57BL/6J mice. Figure 2 Effect of artesunate on Plasmodium falciparum in experimental C57BL/6J mice. Figure 3 Effect of arteether on Plasmodium falciparum in experimental C57BL/6J mice. Figure 4 Effect of artemether + lumefantrine on Plasmodium falciparum in experimental C57BL/6J mice. Figure 5 Developmental stages of Plasmodium falciparum in C57BL/6J mice blood. ((a) and (b)) Ring form or early trophozoite. ((c) and (d)-A) Developing trophozoite. (d)-B Early schizont. (e) Mature trophozoite showing clumped pigment. (f) Trophozoite. Table 1 Changes in haematological values between control versus infected mice. Parameter Experimental group (N = 6) t value P value Control (CON) Infected (INF) (Day 28) (Day 28) HGB (g/dL) 12.85 ± 0.321 7.93 ± 0.175 32.94 <0.0001* RBC (106/mm3) 8.0 ± 0.141 5.90 ± 0.201 20.84 <0.0001* PCV (%) 38.67 ± 1.97 28.92 ± 1.46 9.74 <0.0001* MCV (fL) 91.48 ± 1.64 81.07 ± 2.19 9.34 <0.0001* MCH (pg) 32.33 ± 1.26 27.67 ± 1.17 6.65 <0.0001* MCHC (g/dL) 34.42 ± 1.12 33.17 ± 1.08 1.96 >0.05 NS PLT (103/mm3) 685 ± 20.7 516.7 ± 23.4 13.19 <0.0001* WBC (cells/mm3) 9080 ± 98.9 6957 ± 102 36.61 <0.0001* Neutrophils (%) 36.15 ± 0.693 28.3 ± 0.724 21.65 <0.0001* Lymphocytes (%) 59.85 ± 2.00 69.70 ± 2.00 8.53 <0.0001*** Monocytes (%) 1.0 ± 0.228 1.0 ± 0.117 0.00 >0.05 NS Eosinophils (%) 2.0 ± 0.187 1.0 ± 0.669 3.70 <0.05* The values are given as mean ± SD are statistically significant at t > 2.306, P < 0.05, P < 0.001, and P < 0.0001; not significant at P > 0.05; HGB: hemoglobin; RBC: red blood cells; PCV: packed cell volume; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration; PLT: platelets; WBC: white blood cells. Table 2 Changes in haematological values between infected versus artesunate drug-treated mice. Parameter Experimental group (N = 6) (at the end of the experiment after parasite clearance) t value P value Infected (INF) Drug-treated artesunate (DT AS) HGB (g/dL) 7.93 ± 0.175 11.20 ± 0.846 9.28 <0.001 RBC (106/mm3) 5.90 ± 0.201 7.95 ± 0.0894 22.73 <0.0001*** PCV (%) 28.92 ± 1.46 33.67 ± 2.94 3.54 <0.05* MCV (fL) 81.07 ± 2.19 86.83 ± 3.19 3.65 <0.05* MCH (pg) 27.67 ± 1.17 28.43 ± 0.625 1.42 >0.05 NS MCHC (g/dL) 33.17 ± 1.08 32.75 ± 1.73 0.50 >0.05 NS PLT (103/mm3) 516.7 ± 23.4 675 ± 11.0 15.02 <0.0001* WBC (cells/mm3) 6957 ± 102 8700 ± 522 8.04 <0.001 Neutrophils (%) 28.3 ± 0.724 40 ± 0.754 27.43 <0.0001* Lymphocytes (%) 69.70 ± 2.00 57.67 ± 2.80 8.56 <0.0001* Monocytes (%) 1.0 ± 0.117 1.0 ± 0.126 0.00 >0.05NS Eosinophils (%) 1.0 ± 0.669 2.0 ± 0.518 2.69 <0.05* The values are given as mean ± SD are statistically significant at t > 2.306, P < 0.05, P < 0.001, and P < 0.0001; not significant at P > 0.05; HGB: hemoglobin; RBC: red blood cells; PCV: packed cell volume; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration; PLT: platelets; WBC: white blood cells. Table 3 Changes in haematological values between infected versus arteether drug-treated mice. Parameter Experimental group (N = 6) (at the end of the experiment after parasite clearance) t value P value Infected (INF) Drug-treated arteether (DT AE) HGB (g/dL) 7.93 ± 0.175 11.08 ± 0.770 29.53 <0.0001* RBC (106/mm3) 5.90 ± 0.201 7.73 ± 0.154 17.66 <0.0001*** PCV (%) 28.92 ± 1.46 31.67 ± 1.86 2.84 <0.05* MCV (fL) 81.07 ± 2.19 86.17 ± 5.49 2.11 >0.05NS MCH (pg) 27.67 ± 1.17 27.6 ± 1.23 0.10 >0.05NS MCHC (g/dL) 33.17 ± 1.08 33.35 ± 0.985 0.30 >0.05NS PLT (103/mm3) 516.7 ± 23.4 650 ± 22.8 10.00 <0.0001* WBC (cells/mm3) 6957 ± 102 8983 ± 475 10.22 <0.001* Neutrophils (%) 28.3 ± 0.724 37.01 ± 0.708 21.08 <0.0001* Lymphocytes (%) 69.70 ± 2.00 60.67 ± 1.63 8.57 <0.0001* Monocytes (%) 1.0 ± 0.117 1.0 ± 0.210 8.57 >0.05NS Eosinophils (%) 1.0 ± 0.669 2.0 ± 0.729 8.57 <0.05* The values are given as mean ± SD are statistically significant at t > 2.306, P < 0.05, P < 0.001, and P < 0.0001; not significant at P > 0.05; HGB: hemoglobin; RBC: red blood cells; PCV: packed cell volume; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration; PLT: platelets; WBC: white blood cells. Table 4 Changes in haematological values between infected versus artemether-lumefantrine drug-treated mice. Parameter Experimental group (N = 6) (at the end of the experiment) t value P value Infected (INF) Drug-treated artemether-lumefantrine (DT AL) HGB (g/dL) 7.93 ± 0.175 11.08 ± 0.770 9.77 <0.001 RBC (106/mm3) 5.90 ± 0.201 7.78 ± 0.317 12.23 <0.0001*** PCV (%) 28.92 ± 1.46 33.67 ± 2.94 3.54 <0.05* MCV (fL) 81.07 ± 2.19 87.0 ± 1.79 5.14 <0.001 MCH (pg) 27.67 ± 1.17 28.7 ± 1.16 1.54 >0.05NS MCHC (g/dL) 33.17 ± 1.08 33.33 ± 1.34 1.54 >0.05NS PLT (103/mm3) 516.7 ± 23.4 680 ± 11.4 15.38 <0.0001* WBC (cells/mm3) 6957 ± 102 8033 ± 378 6.74 <0.001 Neutrophils (%) 28.3 ± 0.724 41.00 ± 1.41 19.58 <0.0001* Lymphocytes (%) 69.70 ± 2.00 58.33 ± 4.55 5.61 <0.001** Monocytes (%) 1.0 ± 0.117 1.0 ± 0.297 0.00 >0.05NS Eosinophils (%) 1.0 ± 0.669 2.0 ± 0.566 0.00 <0.05* The values are given as mean ± SD are statistically significant at t > 2.306, P < 0.05, P < 0.001, and P < 0.0001**; not significant at P > 0.05; HGB: hemoglobin; RBC: red blood cells; PCV: packed cell volume; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration; PLT: platelets; WBC: white blood cells. ==== Refs 1 Abro AH Ustadi AM Younis NJ Abdou AS Al Hamed D Saleh AA Malaria and hematological changes Pakistan Journal of Medical Sciences 2008 24 2 287 291 2-s2.0-44849111829 2 Sharma VP Roll back of malaria Current Science 1998 75 8 756 757 3 Taha K Zein El-Dein S Idrees M Haematological changes in malaria: relation to Plasmodium species Kuwait Medical Journal 2007 39 3 262 267 4 Maina RN Walsh D Gaddy C Impact of Plasmodium falciparum infection on haematological parameters in children living in Western Kenya Malaria Journal 2010 9 3, supplement, article S4 2-s2.0-78650476373 5 World Health Organization, New perspective, malaria diagnosis Geneva, Switzerland, 2000 6 Lathia TB Joshi R Can hematological parameters discriminate malaria from nonmalarious acute febrile illness in the tropics? Indian Journal of Medical Sciences 2004 58 6 239 244 2-s2.0-3242673623 15226575 7 Wever PC Henskens YMC Kager PA Dankert J Van Gool T Detection of imported malaria with the Cell-Dyn 4000 hematology analyzer Journal of Clinical Microbiology 2002 40 12 4729 4731 2-s2.0-18744391361 12454179 8 Price RN Simpson JA Nosten F Factors contributing to anemia after uncomplicated falciparum malaria American Journal of Tropical Medicine and Hygiene 2001 65 5 614 622 2-s2.0-0035208249 11716124 9 Facer CA Haematological aspect of malaria Infection and Haematology 1994 Oxford Butterworth Heineman Limited 259 294 10 Murphy GS Oldfield EC Falciparum malaria Infectious Disease Clinics of North America 1996 10 4 747 775 2-s2.0-0030468714 8958167 11 Jandle JH Hemolytic anaemias caused by infection of red blood cells Blood 1996 2nd edition New York, NY, USA Little brown company 473 501 12 Van Agtmael MA Cheng-Qi S Qing JX Mull R Van Boxtel CJ Multiple dose pharmacokinetics of artemether in Chinese patients with uncomplicated falciparum malaria International Journal of Antimicrobial Agents 1999 12 2 151 158 2-s2.0-0033067324 10418761 13 Dutta GP Tripathi R New antimalarial drug development in India: arteether α , β-a blood schizontocide Proceedings of the Indian National Science Academy 2003 69 6 861 870 14 Guide for the Care and Use of Laboratory Animals 1996 Washington, DC, USA Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council 15 Perrin LH Mackey LJ Miescher PA The hematology of malaria in man Seminars in Hematology 1982 19 2 70 82 2-s2.0-0020120892 7041265 16 Dondorp AM Angus BJ Chotivanich K Red blood cell deformability as a predictor of anemia in severe falciparum malaria American Journal of Tropical Medicine and Hygiene 1999 60 5 733 737 2-s2.0-0032957732 10344643 17 Ekvall H Malaria and anemia Current Opinion in Hematology 2003 10 2 108 114 2-s2.0-0037370755 12579035 18 Dondorp AM Nosten F Yi P Artemisinin resistance in Plasmodium falciparum malaria New England Journal of Medicine 2009 361 5 455 467 2-s2.0-68049123592 19641202 19 Inocent G Djuidje Marceline N Pankoui Bertrand MJ Fotso Honore K Iron status of malaria patients in Douala-Cameron Pakistan Journal of Nutrition 2008 7 5 620 624 20 Abdalla SH Peripheral blood and bone marrow leucocytes in Gambian children with malaria: numerical changes and evaluation of phagocytosis Annals of Tropical Paediatrics 1988 8 4 250 258 2-s2.0-0024270028 2467614 21 Amodu OK Adeyemo AA Olumese PE Gbadegesin RA Intraleucocytic malaria pigment and clinical severity of malaria in children Transactions of the Royal Society of Tropical Medicine and Hygiene 1998 92 1 54 56 2-s2.0-0031907433 9692152 22 Aprioku JS Obianime AW Biochemical, haematological and reproductive indices in some biochemical systems Insight Pharmaceutical Sciences 2011 1 1 1 10 23 Maiteki-Sebuguzi C Jagannathan P Yau VM Safety and tolerability of combination antimalarial therapies for uncomplicated falciparum malaria in Ugandan children Malaria Journal 2008 7, article 106 2-s2.0-46149101225 24 Ho M Schollaardt T Snape S Looareesuwan S Suntharasamai P White NJ Endogenous interleukin-10 modulates proinflammatory response in Plasmodium falciparum malaria Journal of Infectious Diseases 1998 178 2 520 525 2-s2.0-0031871881 9697735 25 Day NPJ Hien TT Schollaardt T The prognostic and pathophysiologic role of pro- and antiinflammatory cytokines in severe malaria Journal of Infectious Diseases 1999 180 4 1288 1297 2-s2.0-0033370714 10479160 26 Biemba G Gordeuk VR Thuma P Weiss G Markers of inflammation in children with severe malarial anaemia Tropical Medicine and International Health 2000 5 4 256 262 2-s2.0-0034067075 27 Akhtar MN Jamil S Amjad SI Butt AR Farooq M Association of malaria with thrombocytopenia Annals of King Edward Medical College 2005 11 536 537 28 Rehman ZU Alam M Mahmood A Mubarik A Sattar A Karamat KA Thrombocytopenia in acute malarial infection Pakistan Journal of Pathology 1999 10 9 11 29 Essien EM Ebhota MI Platelet hypersensitivity in acute malaria (Plasmodium falciparum ) infection in man Thrombosis and Haemostasis 1981 46 2 547 549 2-s2.0-0019404380 6458113 30 Horstmann RD Dietrich M Bienzle U Rasche H Malaria-induced thrombocytopenia Blut 1981 42 3 157 164 2-s2.0-0019472153 7011445 31 Mohanty D Ghosh K Nandwani SK Fibrinolysis, inhibitors of blood coagulation, and monocyte derived coagulant activity in acute malaria American Journal of Hematology 1997 54 1 23 29 8980257 32 Olliaro PL Taylor WRJ Developing artemisinin based drug combinations for the treatment of drug resistant falciparum malaria: a review Journal of Postgraduate Medicine 2004 50 1 40 44 2-s2.0-1942504778 15047998 33 Adjuik M Babiker P Garner P Olliaro P Taylor W White N Artesunate combinations for treatment of malaria: meta-analysis Lancet 2004 363 9402 9 17 2-s2.0-0347285290 14723987 34 Nosten F White NJ Artemisinin-based combination treatment of falciparum malaria The American Journal of Tropical Medicine and Hygiene 2007 77 6 181 192 2-s2.0-40049110830 18165491 35 White NJ Assessment of the pharmacodynamic properties of antimalarial drugs in vivo Antimicrobial Agents and Chemotherapy 1997 41 7 1413 1422 2-s2.0-0030858470 9210658 36 Bunnag D Viravan C Looareesuwan S Karbwang J Harinasuta T Double blind randomised clinical trial of oral artesunate at once or twice daily dose in falciparum malaria Southeast Asian Journal of Tropical Medicine and Public Health 1991 22 4 539 543 2-s2.0-0026406093 1820641 37 Luxemburger C Ter Kulle FO Nosten F Single day mefloquin-artesunate combination in the treatment of multi-drug resistant falciparum malaria Transactions of the Royal Society of Tropical Medicine and Hygiene 1994 88 2 213 217 2-s2.0-0028182993 8036679 38 Asthana OP Srivastava JS Kamboj VP A multicentric study with arteether in patients of uncomplicated Plasmodium falciparum malaria Journal of Association of Physicians of India 2001 49 692 696 2-s2.0-0035405071 11573553 39 Sethi N Srivastava R Singh RK Murthy PSR Systamic toxicity study of a new schizontocidal antimalarial drug arteether in rats and monkeys Indian Journal of Parasitology 1998 12 2 223 235 40 WHO Susceptibility of Plasmodium falciparum to antimalarial drugs. Report on global monitoring 1996–2004 WHO, Geneva, Switzerland, 2005 41 Falade C Makanga M Premji Z Ortmann C-E Stockmeyer M Ibarra de Palacios P Efficacy and safety of artemether-lumefantrine (Coartem) tablets (six-dose regimen) in African infants and children with acute, uncomplicated falciparum malaria Transactions of the Royal Society of Tropical Medicine and Hygiene 2005 99 6 459 467 2-s2.0-17144379315 15837358 42 Guthmann J-P Ampuero J Fortes F Antimalarial efficacy of chloroquine, amodiaquine, sulfadoxine-pyrimethamine, and the combinations of amodiaquine + artesunate and sulfadoxine-pyrimethamine + artesunate in Huambo and Bié provinces, central Angola Transactions of the Royal Society of Tropical Medicine and Hygiene 2005 99 7 485 492 2-s2.0-21044459137 15876443 43 Piola P Fogg C Bajunirwe F Supervised versus unsupervised intake of six-dose artemether-lumefantrine for treatment of acute, uncomplicated Plasmodium falciparum malaria in Mbarara, Uganda: a randomised trial Lancet 2005 365 9469 1467 1473 2-s2.0-17844383710 15850630 44 WHO Guidelines for the Treatment of Malaria 2006 Geneva, Switzerland World Health Organization	23841019	PMC3693172	CC BY	2021-01-05 11:33:48	yes	Malar Res Treat. 2013 Jun 11; 2013:426040
==== Front Front GenetFront GenetFront. Genet.Frontiers in Genetics1664-8021Frontiers Media S.A. 10.3389/fgene.2013.00123Plant ScienceOpinion ArticleGMO debate: inconclusive Dronamraju Krishna Foundation for Genetic ResearchHouston, TX, USACorrespondence: kdronamraj@aol.comThis article was submitted to Frontiers in Plant Genetics and Genomics, a specialty of Frontiers in Genetics Edited by: Richard A. Jorgensen, University of Arizona, USA 01 7 2013 2013 4 12322 5 2013 06 6 2013 Copyright © 2013 Dronamraju.2013This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. ==== Body In his Foreword to my book Haldane's Daedalus Revisited, Nobel laureate Joshua Lederberg (1995) correctly emphasized the difficulty of making accurate scientific predictions. Nevertheless, I agree with M. S. Swaminathan (2012) that the current concerns of biosafety will soon give way to an appreciation of the potential benefits of the new genetics including the GMO technology. I have reviewed the subject of GMOs in agriculture in my book: “Emerging Consequences of Biotechnology” (2009). A recent debate in Science (3 May, 2013) highlighted the views of the two opposing groups with respect to the desirability of introducing GM food crops in India. Two eminent scientists, P. M. Bhargava and G. Padmanaban, argued opposing and favoring GM crops, respectively. They were discussing, in particular, the moratorium imposed by India's Minister for environment and forests Jairam Ramesh in February 2010 on the cultivation of GM eggplant. Bhargava supported the indefinite moratorium which he termed “perfectly justified,” whereas Padmanaban considered it “very unfortunate” because that decision was more populistic than science-based. Bhargava pointed out that there is a close association between the consumption of GM food and increased incidence of allergies, childhood cancers, and gastrointestinal disorders amongst Americans during the last several years. Monsanto obtained crude data on the impact of feeding transgenic corn MON 863 for one mammalian species, instead of the three used for evaluations of pesticides or drugs. This study was first classified as confidential by the Company (2002). The data was then used to obtain commercial release agreements all over the world. After heated discussions in Europe concerning the possible physiological effects provoked by this GMO, a decision in the German Appeal Court allowed public access to the crude data in June 2005. Monsanto then published its own interpretation of the data (Hammond et al., 2006) in which it was concluded that the MON 863 was safe to eat. However, after careful analysis of the crude data, Seralini et al. (2007, 2009) applied appropriate statistical methodology to test the effects of the Bt maize on mammalian health. First, GM fed rats were compared to their closest isogenic controls, and then to the six reference groups which were fed various other maize-based diets that Monsanto added in the study. Data were compiled by organ, dose and timing of dietary exposure. In addition, the effects on the rat metabolism of the diet composition without GM maize was studied, comparing only control and reference groups between them to avoid systematically linking these effects to the GM diet. Monsanto did not conduct such a statistical study (Hammond et al., 2006). It is important to note that in order to isolate the effect of the GM transformation process from other variables it is only valid if we compare the GMO (in this case MON 863) with its isogenic non-GM equivalent. The inclusion in the analysis of unrelated feeding groups serves to confuse rather than clarify the effect of the MON 863. After the consumption of MON863, rats showed slight but dose-related significant variations in growth for both sexes, resulting in 3.3% decrease in weight for males and 3.7% increase for females. Chemical measurements revealed signs of hepatorenal toxicity, marked also by differential sensitivities in males and females. Triglycerides increased by 24–40% in females (either at week 14, dose 11% or at week 5, dose 33%, respectively); urine phosphorus and sodium excretions diminished in males by 31–35% (week 14, dose 33%). Long-term experiments are essential in order to establish the real nature and extent of the possible pathology. Present data cannot support the conclusion that GM corn MON 863 is a safe product. The goal of the statistical analysis is to decide whether the consumption of GMOs can be considered to have no effect. This most important issue is totally overlooked in the experimental design and the statistical report made by Monsanto on MON 863. Moreover, any hypothesis which is not statistically significant with their reductive method is always excluded. This disturbing oversight runs false negative results and a risk of health consequences for millions of people and animals. Health risk assessment of genetically modified organisms (GMOs) cultivated for food or feed is under debate throughout the world, and very little data have been published on mid- or long-term toxicological studies with mammals. I thank Prof. M. S. Swaminathan and Ms. Michele Wambaugh for helpful comments. ==== Refs References Alberts B. (2013 ). Science news and analysis . Science 340 , 539 –540 23641086 Dronamraju K. R. (1995 ). Haldane's Daedalus Revisited . Oxford : Oxford University Press. Dronamraju K. R. (2009 ). Emerging Consequences of Biotechnology . New Jersey, NJ : World Scientific Publishing Co. Hammond B. Lemen J. Dudek R. Ward D. Jiang C. Nemeth M. (2006 ). Results of a 90-day safety assurance study with rats fed grain from corn rootworm-protected corn . Food Chem. Toxicol . 44 , 147 –160 10.1016/j.fct.2005.06.008 16084637 Lederberg J. (1995 ). Foreword , in Haldane's Daedalus Revisited , ed Dronamraju K. R. (Oxford : Oxford University Press ), vii–ix. Seralini G-E. Cellier D. de Vendemois J. S. (2007 ). New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity . Arch. Environ. Contam. Toxicol . 52 , 596 –602 10.1007/s00244-006-0149-5 17356802 Seralini G-E. de Vendemois J. S. Cellier D. Sultan C. Buiatti M. Gallagher L. (2009 ). How subchronic and chronic health effects can be neglected for GMOs, pesticides or chemicals . Int. J. Biol. Sci . 5 , 438 –443 10.7150/ijbs.5.438 19584953 Swaminathan M. S. (2012 ). This I believe: agricultural science and genetics . Front. Genet . 3 :282 10.3389/fgene.2012.00282 23227031	23847650	PMC3696726	CC BY	2021-01-04 22:53:30	yes	Front Genet. 2013 Jul 1; 4:123
==== Front J Anal Methods ChemJ Anal Methods ChemJAMCJournal of Analytical Methods in Chemistry2090-88652090-8873Hindawi Publishing Corporation 10.1155/2013/951319Research ArticleAntitumor Molecular Mechanism of Chlorogenic Acid on Inducting Genes GSK-3β and APC and Inhibiting Gene β-Catenin Xu Ruoshi 1 Kang Qiumei 2 Ren Jie 2 Li Zukun 2 Xu Xiaoping 2 1West China School of Stomatology, Sichuan University, Chengdu, Sichuan 610041, China2West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, ChinaXiaoping Xu: 371748507@qq.comAcademic Editor: Yu-Ming Fan 2013 16 6 2013 2013 95131924 1 2013 6 3 2013 Copyright © 2013 Ruoshi Xu et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Inhibiting gene β-catenin and inducting genes GSK-3β and APC, promoting the tumor cell apoptosis in Wnt pathway, by chlorogenic acid were discussed (CGA). Method. The different genes were scanned by the 4∗44K mouse microarray chips. The effect of the three genes was confirmed by RT-PCR technique with CGA dosage of 5, 10, and 20 mg/kg. Result. The expression of GSK-3β and APC was upregulated in group of 20 mg/kg dosage (P < 0.05) and the expression of β-catenin was downregulated in the same dosage (P < 0.05). Conclusion. The results infer that the multimeric protein complex of β-catenin could be increased by CGA upregulated genes GSK-3β and APC, which could inhibit the free β-catenin into the nucleus to connect with TCF. So the transcriptional expression of the target genes will be cut to abnormal cell proliferation. It is probably one of the ways that can stop the tumor increase by CGA. ==== Body 1. Introduction Chlorogenic acid is the ester of caffeic acid and quinic acid in shikimate pathway, which is commonly found in some plants, such as honeysuckle, Cortex Eucommiae, Semen Coffea Arabica, and green tea. Chlorogenic acid has antibacterial, antiviral, clear free radicals, and antitumor effects [1]. In recent years, the effective anticancer activity and low toxicity of chlorogenic acid were constantly confirmed and draw the attention of the people [2–4]. Kurata et al. [5] showed that the inhibition of tumor cell proliferation effect of chlorogenic acid was enhanced with increasing dose; they speculated that this inhibition of tumor cell proliferation may be obtained by enhancing the activity of the DNA ladder and caspase-3 as well as increasing the expression of c-Jun. Gmnado and Feng et al. showed that the in vitro experiments show that the anticancer mechanism of CGA contains inhibition of cell growth, regulation of cell cycle, and induction of apoptosis pathways, such as (1) to reduce ROS expression to reduce cell growth/reproduction signal transduction pathway of NF-κB, AP-l, and MAPKs to reduce cancer cell viability, (2) to improve the activity of the NAD (P)H and GST, (3) to inhibit the expression of tetradecanoyl method wave alcohol acetate (TPA), in order to reduce the c-Jun NH2-terminal kinase, p38 kinase, and MAPK kinase-4 to prevent cancer transformation, and (4) to stimulate the expression of NF-E2-related factor and the activity of GST regulated by Nrf2 downstream cascade links antioxidant response element (ARE) to inhibit the growth of cancer cells [6, 7]. Chlorogenic acid is considered to be an effective cancer chemical repellant because of its significant inhibitory effect on colorectal cancer, liver cancer, and laryngeal [8]. In this paper, the biological gene chip technology was used to detect the variation of a whole set of gene sequences in breast tumor-bearing mice cells in the dynamic treatment cycle with CGA. The differential genes were screened by using genomics profiling, which was also the potential target gene associated with tumor disease according to the gene GO characteristic quality. Further verification testing must be designed, such as accurate quantitative PCR technology and western blot technology, to verify the potential target point in the treatment of chlorogenic acid for tumors. 2. Instrument and Material 2.1. Tumor Lines and Animals EMT-6 mice breast tumor lines were preserved by Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University. Female BALB/C mice, weighing 17~18 g, and originally purchased from Experimental Animal Center of Sichuan University, have been bred in the ordinary small animal housing of the same center where the feeding conditions comply with GB 14925-2001 with the ambient temperature of 19 ± 3°C and humidity of 55 ± 15%. 2.2. Instruments and Regents CO2 incubator (MCO-15AC, SANYO, JP), biological safety cabinets (NU-425, NUAIRE, USA), electronic balance (JY12001, Sartorius, USA), high-speed centrifuge (TGL-16G, Shanghai, China), PCR (ABI-9700, ABI, USA), hybridization oven (G2545A, Agilent, USA), scanner (G2565BA, Agilent, USA), spectrophotometer (ND1000, NanoDrop, Autoclave), Quantitative PCR instrument (Bio-Rad, USA), IQ2-cryogenic high-speed centrifuge, standard 96-well plate, Clean Bench, Sujing Group Aetna, ChemiDoc XRS + system medium (Gibco, China), Calf serum (Minhai, Lanzhou, China), 0.25% trypsin (Gibco, USA), double antibiotics (penicillin and streptomycin sulfate) (North China), Cy3 NHS ester (lot no. PA13105), GE healthcare, aaUTP Ambion (AM8436), Low RNA Input Linear Amplification Kit (lot no. 5184–3523), Gene Expression Hybridization Kit (lot no. 5188–5242), 5188–5327 Gene Expression Wash Buffer Kit (including wash buffers 1 and 2), Stabilization and Drying Solution (lot no. 5185–5979), Gasket slide (lot no. G2534-60003a), 444K mouse microarray, Hybridization CGAmber (lot no. G2534A), Agilent USA, RNeasy Mini kit (lot no. 74106), QIAGEN, USA. Trizol reagent, Invitrogen. DEPC, Oligo, 5x reaction buffer, Riblock Rnase Inhibitor, dNTP MIX, RevertAid M-MulV, 2Taq Master Mix novoprotein, IQTM SYBR Green Supermix, Bio Rad. RevertAid First Strand cDNA Synthesis Kit, Fermentas, fluorescent imager DNA ladder, novoprotein, chlorogenic acid for injection (CGA, lot no. 061101, 30 mg), white powder for injection (content of 99.87%), offered by Sichuan Jiuzhang Biological Chemical Technology Development Co. Ltd. Docetaxel for injection (DX, lot no. 10112611, 20 mg), purchased from Jiangsu Hengrui Medicine Co. Ltd. IFNα-2b (20110908, 500 IU) purchased from Anhui Anke Biotechnology (Group) Co. Ltd. 0.9% saline (NS, lot no. A060418) (Kelun, Sichuan, China). 2.3. Preparation of Sample Solution To use CGA solution, CGA freeze-dried powder was dissolved with sterile saline to make concentrations of CGA sample solution that were 1, 0.5, and 0.25 mg·mL−1, respectively. To use DX solution, 20 mg of DX injection powder was diluted with 1.5 mL of special solvent. To this, about 8.0 mL of saline was added to be stock solution of 2 mg·mL−1. The stock solution was diluted to be sample solution with the concentration of 0.25 mg·mL−1 with saline before use. To use IFNα-2b solution, IFNα-2b was diluted with 20 mL sterilized saline. 3. Methods 3.1. Preparation of Balb/c-EMT-6 Mice Model 3.1.1. Preparation and Subcultivation The anabiotic EMT-6 breast tumor lines were implanted subcutaneously into the right forelimb of mice. The tumor-bearing mice were obtained until the tumor grew to 1 cm × 1 cm × 1 cm. Then took out the tumor, cleaned it with NS, weighed it, cut it into pieces, and placed it in a homogenizer. NS (about 1 : 4) was added to the homogenizer, and cell suspension was obtained after fast homogenate. 0.2 mL of the cell suspension was inoculated subcutaneously into the right forelimb of mice and was randomized. 3.1.2. Experiment Design 30 female BABL/C mice were randomly divided into 6 groups (n = 5), including normal saline (negative) group, CGA high-dose group (20 mg·Kg−1), CGA middle-dose group (10 mg·Kg−1), CGA low-dose group (5 mg·Kg−1), DX positive control group (5 mg·Kg−1), and IFNα-2b positive control group (5 million U·Kg−1). After being inoculated for 24 hours, the NS group, CGA groups, and IFNα-2b positive control group were continuously subcutaneously administered for 12 days. The DX group was administered interday for 6 times. The tumors were taken after 24 hrs of the last administration, then weighed, cut into pieces, frozen in liquid nitrogen, and stored at −70°C after segmentation package. 3.2. Detection of the Level Changes of Cell Gene with Gene Chip 3.2.1. Total RNA Extraction The lysis/binding buffer was added, 1 mL per 0.1 g tissue, for homogenate on ice to prevent RNA degradation. Adding homogenate additive (1/10) into the homogenate; the homogenate was vortexed, mixed, and kept on ice for 10 min. Then added phenol and chloroform mixture in the same volume as lysis, vortex them for 30 s, centrifuged them in 10,000 g for 5 minutes at room temperature. Added the absolute ethanol to the supernatant, then vortex and mixed them, made them going through the purification column repeatedly, centrifuged them at 10000 g for 15 seconds, discarded the supernatant. Added 350 μL Wash I, centrifuged them for 5 s to purify the column, then centrifuged at 10,000 g for 15 s, discarded filtrate. Added 10 μL DNase I and 70 μL buffer RDD to the film, placed them for 15 min, and then successively added 350 μL Wash 1 and 500 μL Wash 2, centrifuged them for 5 s, cleaned the purification columns two times. Each cleaning must be centrifugal at 10,000 g for 15 s and discard the filtrate. Added 100 μL 95°C preheated nuclease-free water to spin column and placed the spin column in a new collection tube after the second cleaning. The total RNA would be obtained after centrifuging at the maximum speed for 30 s and then stored at −70°C. 3.2.2. Purification of Total RNA QIAGEN RNeasy Kit was used for further extraction and purification of the total RNA. 100 μL RNase-free water was added to dissolve the total RNA, then mixed with 350 μL buffer RLT and 250 μL ethanol. (The preparation of RLT: 14 mL original RLT can be added to 140 μL of β-mercaptoethanol.) Transferred the sample to the RNeasy column, centrifuged at 10000 g for 30 s, and abandoned filtrate. Clean the RNeasy minicolumn twice with 500 μL buffer RPE, centrifuged at 10,000 g for 30 s and 2 min, respectively, and discard the filtrate. Add 40 μL RNase free water to the column, and centrifuge 10000 g for 1 min. Repeat the operation once more; the purified RNA was prepared. 3.2.3. Total RNA Quality Testing The quality of total RNA can be determined with agarose gel electrophoresis. Prepare the electrophoresis buffer 50x TAE, which was processed with DECP and autoclaves → a 1% agarose gel was prepared after adding on amount of agarose to lx TAE electrophoresis buffer → run on a gel for 15 min, then observe and picture the gel over the gel imager → lab-on-chip. 3.3. Validation of Protein Expression of Specific Genes by Fluorescence Quantitative PCR 3.3.1. Primers and Reaction Conditions The primers were designed through the 01190 software and synthesized by Sangon Biotech (Shanghai). The primer information of these four genes was shown in Table 1. With the template of sample cDNA, the differentially expressed genes were verified to use SYBR Green by real-time fluorescent quantitative PCR. The conditions of the q-PCR reactions were subjected to 94°C for 10 min, followed by 40 cycles at 94°C for 15 s and 54.5°C for 30 s, and finally 72°C for 45 s. The expression levels of GSK-3β, APC, and β-catenin in test samples were detected with GAPDH as the reference gene and calculated by 2−ΔΔCT relative quantification method, which showed the differential expression of different groups compared to the NS group. 3.3.2. RNA Extraction After isinfecting the reagent bottle, boxes, and gloves under UV light for 30 min, the tissues were homogenized in the trizol reagent. The homogenate was incubated at 15–30°C for 5 min, to which chloroform was added, then vortex them for 15 s, and centrifuged separating the mixture for 15 min in low temperature. Transferred the upper supernatant into another tube, added 0.5 mL isopropanol and mixed them at 15–30°C, incubated them for 10 min, 4°C, then centrifuged them at 12000 g for 10 min, discarded supernatant, and added 1 mL 75% ethanol into the tube, centrifuged them at 7500 g for 5 min, discarded the supernatant, and dried them in the air for 3−5 min. Added 20 μL DEPC deionized water to dissolve the RNA and stored them at −70°C. 3.3.3. RNA Quality Testing (1) OD Value Detection. A260/A280 value was determined by UV with 1 μL extracted from each of RNA which was diluted to 100 μL with TE buffer. (2) RNA Formaldehyde Electrophoresis. The voltage of conditions electrophoresis was 80 V, and running time was 40 min. The RNA samples prepared in Section 3.3.2 were diluted with DEPC water suitably and mixed with an equal volume of sample buffer, then heated for 4 min in boiling water, cooled on ice for 2 min, and then centrifuged in 1200 rpm for 5 s. The sample was spotted on plate to be carried on the electrophoresis. 3.3.4. The Synthesis of cDNA The RevertAid First Strand cDNA Synthesis Kit was used for reverse transcription. The total reaction volumes of RT-PCR reactions were 20 μL. The system consists of 2 μL RNA, 1 μL Oligo(dT)18 primer, 9 μL RNase-free water, 4 μL 5x reaction buffer, 1 μL Riblock Rnase Inhibitor, 2 μL 10 mM dNTP MIX, and 1 μL RevertAid M-MulV. The reverse transcription system liquids of the above were subjected to 42°C, 60 min, and 70°C, 5 min, in a PCR instrument. The reaction products were stored at −80°C for long-term preservation. 3.3.5. The Test of RT-PCR Amplification Products Briefly, the presence or absence of the only bands was observed at 496 bp as the standard showed the quality of cDNA. If there is one and only one band, the PCR product cDNA was qualified, and Q-PCR can be the next. The voltage was 130V, and the analysis time was 25 min. The RT-PCR amplification system had 25 μL liquid of total reaction volumes, in which consists of 10 μL 2x Taq Master Mix, 2 μL cDNA, 0.75 μL Forward GAPDH primer pair and 0.75 μL Reverse GAPDH primer, and 11.5 μL RNase-free water. PCR reactions were subjected to 94°C for 10 min, followed by 40 cycles at 94°C for 15 s and 54.5°C for 30 s and finally 72°C for 45 s. 3.3.6. Real-Time Fluorescent Quantitative PCR 1 μL cDNA was diluted to 100 μL with sterile water on standby. Gene primers, F and R, were diluted 20-fold, respectively. This reaction system was shown in Table 2. Each sample should go through the three parallel tests, taking the mean value to calculation. 3.3.7. SYBR Green I Reaction Designed and Optimized (1) Optimization of the Annealing Temperature. By setting a certain temperature range, we can screen the optimal annealing temperature by real-time quantitative PCR reactions. The melting curve can be used to assess the specificity of the reaction in the quantitative PCR instrument. If there are multiple peaks on the melting curve, which indicates nonspecific products such as primer dimer, they are amplified along with specific products simultaneously, which also indicates the primers of the reaction need to be redesigned. (2) Construction of the Standard Curve. cDNA was selected as the template and set eight 10-fold dilution series of points each dilution was repeated three times. The equation of the linear regression line has obtained the logarithmic value of template initial concentration as the abscissa and the CT value as the vertical coordinate. Standard curve correlation coefficient (r) or the coefficient of determination (R 2) can be used to evaluate the degree of linearization with the specific requirements of r > 0.990 and R 2 > 0.980. 4. Results 4.1. Results of CGA Suppression of the Mice Breast Cancer (EMT-6) See Table 3. 4.2. Electrophoresis Graph and Lab-on-Chip As is shown in Figure 1, the luminance ratios of 28 s and 18 s are greater than or equal to 2 in electrophoresis graph, which preliminary determines that total RNA was qualified. The result has shown that the quality of RNA extracted from each group of tumor tissue samples meets the requirement of further detection of gene chip. Then the differential genes closely related to the tumor cell suppression in the course of treatment were screened by the time series analysis, GO analysis, and pathway analysis and validated with q-PCR. 4.3. The Quality Results of RNA, cDNA (1) The Results of OD Values. As is shown in Table 4, the A260/A280 value of RNA was from 1.63 to 2.18, which meets the quality requirements of RNA. (2) Formaldehyde Electrophoresis of RNA. As is shown in Figure 4, the formaldehyde denaturing electrophoresis showing the 28S and 18S were clear without DNA impurity band and degradation RNA, which meets the requirements of the experiment. (3) Results of cDNA Quality Test. As is shown in Figure 5, there is only one band at 496 bp, which indicates that the quality of the PCR product meets the requirements. 4.4. PCR Expression of GSK-3β, APC, and β-Catenin The PCR expression of GSK-3β, APC, and β-catenin was shown in Figure 6 in the tumor tissue of Balc-b/EMT-6 tumor-bearing mice after the treatment of DX (5 mg/kg), IFN (5 × 106 U·kg-1), CGA (20 mg/kg), CGA (10 mg/kg), and CGA (5 mg/kg) for 12 days. Compared with the blank group, the expression of GSK-3β and APC was upregulated in group of 20 mg/kg dosage (P < 0.05) and the expression of β-catenin was downregulated in the same dosage group (P < 0.05). 5. Discussion (1) As shown in Table 3, compared to the positive control group of cytotoxic anticancer drugs DX, biological response modifier (BRM) IFN groups, and the negative group of NS, the three dosag of the CGA groups showed better antitumor effect (P < 0.05); especially the inhibition rate of the 20 mg·kg−1dose group was more than 50%, which was equivalent to 59.92% of the DX group and 40.80% of the IFN group. It is suggested that chlorogenic acid should be able to be a new good anticancer agent in future clinical application. (2) According to the data of the different time points in the inhibitory process acquired with the total RNA that was qualified in Figures 1, 2, and 3 the differential genes closely related to the Wnt pathway in the course of treatment were screened by the time series, GO, and pathway. The different genes which were connected with CGA treatment were extracted and were confirmed with q-PCR. The downward trends of the tumor genomes 18 and 21 were screened by the logarithm of standardization and fitting the change course of genes with similar trends; the genetic trend was closer to the fitted values of P smaller, such as Figures 7(a) and 7(b). (3) As shown in Figure 7(a), the trend of genome 18 was confirmed to the anticancer process of CGA. According to the analysis of KEGG signaling pathway and the characteristics of GO in genome 18, some downregulated genes were found which are related to the tumor suppression pathway, such as BdnF mediated MAPK, Cflar, Cln3 Ddit3 Notch2 Rps6, Sox9, Spn, and Ppp1r13l. The gene 21 time sequence diagram relates to the Wnt pathway, mTOR pathway, the Notch pathway, and some immune-related pathways, such as B cell receptor pathway, T-cell receptor pathway, and metabolic pathway. It was inferred that CGA had a multipathway to inhibit the tumor growing up. (4) Because our animal model was a breast cancer (EMT-6/BALB-C), we focused on the influence of Wnt pathway (shown in Figure 8) which was a special pathway that the breast cancer had. In particular, the gene glycogen synthase kinase 3(GSK-3β) and downstream gene ubiquitin ligase E3(APC) were upregulated. And the two kinds of genes had closed relationship, in which the gene APC was upregulated by upstream gene GSK-3β. And then, both genes could cause the down-stream gene β-catenin to downregulate directly in the Wnt pathway. As you know, the cancer will be developing when the gene β-catenin expression was upregulated. What is the gene β-catenin? From its GO searching revealed, β-catenin is a multifunctional protein, which can assist the cells react to extracellular signal and changes by interaction with the cytoskeleton. This protein acts as a transcription (transcription) factor in the nucleus to promote cell division genes. The accumulation of β-catenin would lead to abnormal activation of the downstream transcription factors after transferring to the nucleus, which mainly cause tumor. So, it is suggested that the β-catenin inhibited was one of the ways to inhibit tumor growth. On the other hand, the upregulation of GSK-3β is a multifunctional serine/threonine class of protein kinase, which plays an important role in the Wnt/wingless, PI3-kinase, and Hedgehog signaling pathway with the physiological functions including transcriptional activation, cell proliferation, and cell differentiation, cell movement. It can phosphorylate shaft protein and β-catenin, cause the degradation of β-catenin protein, and thereby inhibit the activation of the pathway. The activated APC plays an important role in promoting complexes degradation in a fast, efficient, and highly selective way in the anaphase cell cycle, which also can phosphorylate shaft protein and β-catenin, cause the degradation of β-catenin protein, and thereby inhibit the activation of the pathway. So the activation of GSK-3β and APC and downregulation of β-catenin in the group of CGA suggested that CGA could inhibit tumor by activating GSK-3β and APC. (5) As shown in Figure 6(a), the regulation confirmed by Q-PCR in treatment process of CGA for EMT-6 breast cancer indicates groups of CGA in each dose and DX can activate expression of GSK-3β, especially the CGA 20 mg/kg group and the DX group can significantly upregulate GSK-3β (P < 0.05). As shown in Figure 6(b), groups of CGA at 20 mg/kg and 10 mg/kg and DX can activate expression of APC, but groups of CGA at 5 mg/kg dose little to upregulation. As shown in Figure 6(c), DX group, IFN group and CGA 20 mg/kg group was inhibited gene β-catenin. 6. Conclusion It is suggested that affecting the gene expression of GSK-3β, APC, and β-catenin by chlorogenic acid in Wnt pathway was one of the targets in the multipathway to antitumor of CGA. Conflict of Interests All authors are researchers of Sichuan University and have no conflict of interests. Some parts of the experiment were entrusted to the third party but not sponsored by this party. This statement is made in the interest of full disclosure and not because the authors consider this to be a conflict of interests. Acknowledgments The Sichuan Province Science and Technology Support Project Fund 2011SZ0131 supported this work. The authors would like to show great thanks to Hongwei Liu for his great help. Figure 1 The EC of CGA 1–4. Figure 2 The EC of CGA 5-6, DX, and blank. Figure 3 The EC of DX3 and blank. Figure 4 RNA electrophoresis. Figure 5 cDNA electrophoresis. Figure 6 The average PCR expression of different genes in different groups. (a) GSK-3β, (b) gene APC, and (c) gene β-catenin. Figure 7 The expression trend graphs of gene 18 and gene 21 (P = 0). Figure 8 Wnt signaling pathway. Table 1 The Primer information of these four genes. Gene symbol Forward primer Reverse primer GSK-3β ACC ATC CTT ATC CCT CCA CAg Aag Cgg CgT TAT Tg APC CAC TgA gAA TAA ggC TgA C TTC CgT AAT ATC CCA CC β-Catenin ggT gCT ATT CCA CgA CT CCC TTC TAC TAT CTC CTC C GAPDH CAA GGT CAT CCA TGA CAA CTT TG GTC CAC CAC CCT GTT GCT GTA G Table 2 The reaction system of q-PCR (n = 3). Composition Plus the amount IQ SYBR Green Supermix 10 μL Forward primer 1 μL Reverse primer 1 μL cDNA 2 μL Nase-free water 6 μL Total volume 20 μL Table 3 Results of CGA suppression of the mice breast cancer. Groups Dose (mg·kg−1) Animals Average tumor weight (g) The average tumor inhibition rate (%) DX 5 5 0.780 ± 0.244 59.92 IFN 5 million U·kg−1 5 1.152 ± 0.254 40.80 CGA 5 5 1.326 ± 0.254 31.86 CGA 10 5 1.222 ± 0.363 37.20 CGA 20 5 0.968 ± 0.633** 50.26 Negative — 5 1.946 ± 0.233 — Table 4 OD value of RNA. No. Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 1 1.86 2.08 2.18 2.07 2.18 2.15 2 1.95 2.09 2.17 2.09 2.04 2.16 3 2.05 2.12 2.23 2.09 1.90 2.17 4 1.63 2.07 2.14 2.10 1.94 2.19 5 2.15 2.06 2.18 2.13 1.95 2.17 ==== Refs 1 Liu J-H Qiu A-Y Chlorogenic acid extraction and purification and application prospects Cereals & Oils 2003 9 44 446 2 Dorrell DG Chlorogenic acid content of meal from cultivatial sunflower Crop Science 1976 16 422 426 3 Shimizu M Yoshimi N Yamada Y Suppressive effects of chlorogenic acid on N-methyl-N-nitrosourea- induced glandular stomach carcinogenesis in male F344 rats Journal of Toxicological Sciences 1999 24 5 433 439 2-s2.0-0033374861 10656166 4 Matsunaga K Katayama M Sakata K Inhibitory effects of chlorogenic acid on azoxymethane- induced colon carcinogenesis in male F344 rats Asian Pacific Journal of Cancer Prevention 2002 3 163 166 12718596 5 Kurata R Adachi M Yamakawa O Yoshimoto M Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L. ) leaves Journal of Agricultural and Food Chemistry 2007 55 1 185 190 2-s2.0-33846105409 17199331 6 Rylova SN Amalfitano A Persaud-Sawin DA The CLN3 gene is a novel molecular target for cancer drug discovery Cancer Research 2002 62 1 801 808 11830536 7 Pereira RC Delany AM Canalis E CCAAT/enhancer binding protein homologous protein (DDIT3) induces osteoblastic cell differentiation Endocrinology 2004 145 4 1952 1960 2-s2.0-1642464760 14684614 8 Pellati F Benvenuti S Magro L Melegari M Soragni F Analysis of phenolic compounds and radical scavenging activity of Echinacea spp. Journal of Pharmaceutical and Biomedical Analysis 2004 35 2 289 301 2-s2.0-1942439725 15063463	23844319	PMC3697783	CC BY	2021-01-05 11:20:05	yes	J Anal Methods Chem. 2013 Jun 16; 2013:951319
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23840897PONE-D-13-0222310.1371/journal.pone.0068574Research ArticleRecombinant Human Adenovirus-p53 Injection Induced Apoptosis in Hepatocellular Carcinoma Cell Lines Mediated by p53-Fbxw7 Pathway, Which Controls c-Myc and Cyclin E rAd-p53 Induced Apoptosis by p53-Fbxw7 PathwayTu Kangsheng Zheng Xin Zhou Zhenyu Li Chao Zhang Jing Gao Jie Yao Yingmin Liu Qingguang * Department of Hepatobiliary Surgery, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi, China Koepp Deanna M Editor University of Minnesota, United States of America * E-mail: liuqingguang@vip.sina.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: KT QL. Performed the experiments: KT XZ ZZ CL JZ JG. Analyzed the data: KT XZ ZZ YY. Contributed reagents/materials/analysis tools: KT XZ ZZ CL JZ JG YY. Wrote the manuscript: KT QL. 2013 1 7 2013 8 7 e6857411 1 2013 29 5 2013 © 2013 Tu et al2013Tu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.F-box and WD repeat domain-containing 7 (Fbxw7/hAgo/hCdc4/Fbw7) is a p53-dependent tumor suppressor and leads to ubiquitination-mediated suppression of several oncoproteins including c-Myc, cyclin E, Notch, c-Jun and others. Our previous study has indicated that low expression of Fbxw7 was negatively correlated with c-Myc, cyclin E and mutant-p53 in hepatocellular carcinoma (HCC) tissues. But the role and mechanisms of Fbxw7 in HCC are still unknown. Here, we investigated the function of Fbxw7 in HCC cell lines and the anti-tumor activity of recombinant human adenovirus-p53 injection (rAd-p53, Gendicine) administration in vitro and in vivo. Fbxw7-specific siRNA enhanced expression of c-Myc and cyclin E proteins and increased proliferation in cell culture. rAd-p53 inhibited tumor cell growth with Fbxw7 upregulation and c-Myc and cyclin E downregulation in vitro and a murine HCC model. This effect could be partially reverted using Fbxw7-specific siRNA. Here, we suggest that the activation of Fbxw7 by adenoviral delivery of p53 leads to increased proteasomal degradation of c-Myc and cyclin E enabling growth arrest and apoptosis. Addressing this pathway, we identified that rAd-p53 could be a potential therapeutic agent for HCC. This study was supported by a grant from the National Natural Science Foundation of China (no. 81272645 and no. 81071897) and the Scholarship Award for Excellent Doctoral Student granted by Ministry of Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction HCC is one of the most common malignancies worldwide. It is a common cancer and occurring with increasing frequency in China [1]. Ubiquitination by the ubiquitin-proteasome system (UPS) is a post-translational modification that regulates diverse cellular processes, including cell proliferation, cell cycle progression, transcription, immune response, DNA damage repair and apoptosis [2,3]. The UPS consists of the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin-protein ligase (E3). Among the E3 ubiquitin ligase enzymes, the SKP1-CUL1-F-box (SCF) E3 ligase complex is one of the best characterized. Fbxw7 is a member of the F-box protein family, which determines the substrate specificity of the SCF-type E3 complex, and is able to target various oncogenic proteins for ubiquitination such as Cyclin E, c-Myc, c-Jun, Notch, Presenilin, Mcl-1, Sterol regulatory element-binding proteins (SREBP), mTOR, Krüppel-like factors (KLFs), c-Myb and Aurora A [4–6]. Because all these characterized substrates are well known oncogenic proteins that are frequently overexpressed in a variety of human cancers, Fbxw7 is believed to be a tumor suppressor that contributes to the negative regulation of these oncogenic proteins [6]. Our previous study has indicated that the mRNA and protein expression of Fbxw7 was significantly down-regulated in the HCC tumor tissues compared to the normal tumor-adjacent tissues. Fbxw7 protein was expressed at significantly lower levels in patients with high histological grade and advanced tumor-node-metastasis (TNM) stage [7]. But the role and mechanisms involved in Fbxw7 are still unclear in HCC. p53 gene functions as the cellular gatekeeper for cell growth, division and induction of cell death through apoptosis, mutations and deletions of p53 gene are detectable in about 50% of HCCs [8]. In addition, loss of functional p53 gene is associated with poorly-differentiated HCCs, a shorter tumor-free interval and survival time [9,10]. Hence, reconstitution of a wild-type p53 gene is an attractive therapeutic approach to treat HCC. Fbxw7 has been identified as a p53 target gene [11,12]. In support of this notion, Fbxw7 was dramatically up-regulated by infection with adenovirus-mediated transfer of wild-type p53 into the p53-deficient cells [4]. Moreover, p53-binding sites were discovered in the Fbxw7 exon, further emphasizing Fbxw7 as a direct target of p53 [11]. Fbxw7 targets for ubiquitination and degradation of c-Myc and Cyclin E, cell cycle regulators that are frequently deregulated in HCC [13,14]. Our previous study pointed out that Fbxw7 protein expression was negatively correlated with c-Myc, Cyclin E and mutant-p53 in HCC tissues [7]. We hypothesize that deregulation or mutations in p53 contribute to hepatocarcinogenesis through Fbxw7, c-Myc and Cyclin E pathway. In this report, we investigated the function of Fbxw7 in HCC, then treated HCC cells with rAd-p53 and evaluated p53, Fbxw7, c-Myc and Cyclin E expression and anti-tumor activities in vitro and in vivo. rAd-p53 is a gene therapy drug that is used to treat neck squamous cell carcinoma (HNSCC) [15]. It is composed of replication-incompetent adenovirus serotype 5 (Ad5) encoding for human wild-type p53 gene. Our results suggest that rAd-p53 administration induces HCC growth arrest and apoptosis by Fbxw7-dependent c-Myc and Cyclin E proteolysis. Materials and Methods Ethics statement All animal protocols were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University (Permit Number: 2012-0096). Cell culture The immortal human liver cell line LO2 and HCC cell lines SMMC-7721, HepG2, Hep3B, Huh7 and Bel-7402 were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). All the cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (FBS, Gibco BRL, USA) with 100 units/mL penicillin and 100 µg/mL streptomycin (Sigma, USA) and cultured in a humidified 5% CO2 incubator at 37°C. Fbxw7 sequence The Fbxw7 (α, β and γ) sequence was amplified using cDNA from LO2, SMMC-7721, HepG2, Hep3B, Huh7 and Bel-7402 cells with KOD-FX DNA polymerase (TOYOBO, Japan) and sequencing primers (Table 1). These PCR products were separated on 1% agarose gels containing ethidium bromide (EB) and purified with ethanol precipitation. Purified PCR products were sequenced by Shenzhen Huada Gene Technology Co., Ltd. (China). Table 1 Primers for Fbxw7 sequence and PCR amplification. Sequencing primers 5’–3’ Fbxw7α-1F primer ATGAATCAGGAACTGCTCTCTGTGGGC Fbxw7α-1R primer CTCAGAACCATGGTCCAACTTTCTTTTCA Fbxw7α-2F primer CATACACATACTAACAGTGTCACGAACTCCA Fbxw7α-2R primer CTCCAGTAGCGACATGTCTGAGCTGCT Fbxw7α-3F primer GATGCAAGTGATAGAACCCCAGTTTCAAC Fbxw7α-3R primer TCTCTGCATTCCACACTTTGAGTGTCC Fbxw7α-4F primer GTCTGAGAACATTAGTGGGACATACAGGT Fbxw7α-4R primer TCTTTGAGTTCCATTCCACTTGTTAACG Fbxw7α-5F primer TGATACATCAATCCGTGTTTGGGATGT Fbxw7α-5R primer TCACTTCATGTCCACATCAAAGTCCAG Fbxw7β-1F primer ATGTGTGTCCCGAGAAGCGGTTTGATACTG Fbxw7β-1R primer CTCAGAACCATGGTCCAACTTTCTTTTCA Fbxw7γ-1F primer ATGTCAAAACCGGGAAAACCTACTCTAAAC Fbxw7γ-1R primer CTCAGAACCATGGTCCAACTTTCTTTTCA PCR primers Fbxw7-F primer AAAGAGTTGTTAGCGGTTCTCG Fbxw7-R primer CCACATGGATACCATCAAACTG p53-F primer ATTCTGGGACAGCCAAGTC p53-R primer TAGTTGTAGTGGATGGTGGTA c-Myc-F primer CTTCTCTCCGTCCTCGGATTCT c-Myc-R primer GAAGGTGATCCAGACTCTGACCTT Cyclin E-F primer GTTATAAGGGAGACGGGGAG Cyclin E-R primer TGCTCTGCTTCTTACCGCTC GAPDH-F primer CAAGCTCATTTCCTGGTATGAC GAPDH-R primer CAGTGAGGGTCTCTCTCTTCCT Reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (qRT-PCR) Fbxw7-specific oligonucleotide primers were designed to amplify a 249-bp PCR product encoding the common region among three Fbxw7 isoforms. The primers in Table 1 were used for PCR. RT-PCR was performed as previous report [5]. PCR amplification for quantification of Fbxw7, c-Myc, Cyclin E and (glyceraldehyde-3-phosphate dehydrogenase) GAPDH mRNA was done in the ABI PRISM 7300 Sequence Detection System (Applied Biosystems, USA) using the SYBR® Premix Ex TaqTM ii(Perfect Real Time) Kit (Takara Bio, Japan). Three experimental replicates were performed. Colony formation assay 200 LO2, SMMC-7721, HepG2, Hep3B, Huh7 and Bel-7402 cells were placed in a fresh six-well plate in triplicate and maintained in DMEM containing 10% FBS for 2 weeks. Cell colonies were fixed with 20% methanol and stained with 0.1% coomassie brilliant blue R250 at room temperature for 15 min. The colonies were counted by ELIspot Bioreader 5000 (BIO-SYS, Karben, GE). Fbxw7 RNA interference Fbxw7-specific siRNA, sense 5-GGAGUAUGGUCAUCACAAAtt-3 and antisense 5-UUUGUGAUGACCAUACUCCac-3 (Silencer Predesigned siRNA, GenePharma Co. Ltd, Shanghai, China) and TurboFect Transfection Reagent (Thermo Scientific, USA) were then added in 6-well plates. After incubation, cells were seeded at 1.5x105 per well in a volume of 2 mL in 6-well plates and incubated at 37°C and 5% CO2. The RNA interference assay was done after 24 h incubation. MTT Assay Proliferation was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Roche Diagnostics, USA). After 24 h incubation following Fbxw7-specific siRNA or rAd-p53 (Gendicine, Sibiono GeneTech Co., Ltd, Shenzhen, China) addition, cells were cultured further for 0 to 72 h and the absorbance of the samples was measured using a model 550 microplate reader (Bio-Rad Laboratories, CA, USA), at a wavelength of 570 nm corrected to 655 nm. Three experimental replicates were performed. Western blot analysis Fbxw7 (ab74054, Abcam, Hong Kong) (1:1000), c-Myc (#5605, Cell Signaling Technology, Beverly, MA, USA) (1:1000), Cyclin E (#4129, Cell Signaling Technology, Beverly, MA, USA) (1:1000), p53 (S371, Bioworld Technology, St. Louis Park, MN, USA) (1:1000) and β-actin (sc-47778, SANTA CRUZ, CA, USA) (1:1000) antibodies were used for Western blot assay. Secondary horseradish peroxidase-conjugated goat anti-mouse or rabbit antibodies (Bio-Rad, USA) were used at a 1:5000 dilution and detected by the enhanced chemiluminescence reagent (Millipore, Billeria, MA, USA). Flow cytometry An Annexin-V-FLUOS Staining Kit (Roche, USA) was used to analyze apoptosis levels. Briefly, Hep3B cells were infected by rAd-p53 for various times and 106 treated cells were washed with PBS and centrifuged at 200×g for 5 min, then resuspended in 100µL of Annexin-V-FLUOS labeling solution (predilute 20 µL Annexin-V-Fluos labeling reagent in 1 mL Incubation buffer and add 20 µL Propidium iodide solution) and incubated 10-15 min at 15-25°C, the samples were analyze by BD FACS Canto II Flow Cytometer (Becton Dickinson, USA). Three experimental replicates were performed. In vivo treatments with rAd-p53 A nude mouse xenograft model was established using 4-6 week-old female BALB/c nude mice (Centre of Laboratory Animals, The Medical College of Xi’an Jiaotong University, Xi’an, China). Mice were housed in sterilized cage (2 animals/cage) with constant temperature and humidity and fed with regular autoclaved chow diet with water ad libitum. 107 Hep3B cells were inoculated subcutaneously into the left flank of each nude mouse. 1 week after inoculation, all mice were divided randomly into two groups (six mice per group) and treated every week with multiple-center intratumoral injection of rAd-p53 or saline per animal. The dose of rAd-p53 administered was 1×107 VP/mm3 tumor for each group, the anticancer agents were diluted to 0.1 mL for tumors with the largest diameters of 0.5–0.9 cm, 0.2 mL for 1.0–1.4 mL tumors, and 0.3 mL for those more than 1.5 cm [16]. The tumor volume for each mouse was determined by measuring in two dimensions and calculated as tumor volume = length× (width)2 /2. After 4 weeks treatment, all mice were sacrificed by cervical dislocation under anesthesia with ether and the xenograft tumor tissue was explanted for routine pathological examination. Tumor tissues were subjected to immunohistochemical analysis, and a fraction was homogenized for protein extraction and immunodetection of p53 and β-actin. Immunohistochemistry Tumor xenograft samples were fixed in 10% buffered formalin solution and embedded in paraffin. Fbxw7 (1:100), c-Myc (1:100) and Cyclin E (1:100) antibodies were in immunohistochemistry with streptavidin peroxidase conjugated method (SP–IHC). Immunohistochemistry was performed as previously reported [7]. Statistical analysis The SPSS 13.0 statistical package (SPSS, Chicago, USA) was used for all calculations. Differences between two groups were estimated with Student’s t test and ANOVA. One-way ANOVA was used for statistical comparisons among four groups. The correlation between Fbxw7 mRNA expression levels and colony formation numbers was analyzed with Linear regression and correlation analysis. The differences between groups were considered to be statistically significant when the P value was <0.05. Results Fbxw7 mRNA levels correlated negatively with colony formation ability of HCC cell lines Fbxw7 is a mediator of c-Myc and Cyclin E proteolysis modulating cell cycle regulation. We measured Fbxw7 mRNA expression level and assessed colony formation ability of LO2 cells, a non transformed human liver cell line, and of the HCC cell lines SMMC-7721, Bel-7402, Hep3B, Huh7 and HepG2. Cell lines expressing the highest Fbxw7 mRNA levels showed the poorest ability to form colonies eg. LO2 and Hep3B cells (Figure 1A and 1B). Thus, Fbxw7 mRNA expression level was negatively correlated with the number of cell colonies (t=-4.124, r = -0.900, P = 0.015, Figure 1C). 10.1371/journal.pone.0068574.g001Figure 1 Fbxw7 mRNA expression correlates negatively colony formation ability in HCC cell lines. A) The Fbxw7 mRNA expression level of HCC cell lines (n=3). B) Colony formation assay quantification with HCC cell lines. C) Correlation plot for Fbxw7 mRNA levels and colony number. Values are depicted as mean±standard error. Fbxw7 siRNA knockdown in LO2 and Hep3B increased c-Myc and Cyclin E expression as well as cell proliferation Since the presence of Fbxw7 reduces the ability of cell colony formation, we investigated whether the ablation of Fbxw7 mRNA affects cell proliferation and expression of cell cycle regulators as c-Myc and Cyclin E, both degradation targets of Fbxw7. We confirmed by qRT-PCR high levels of Fbxw7 mRNA in LO2 cells, and its successful knockdown by the administration of an Fbxw7 specific siRNA (Figure 2A). The Fbxw7 knockdown led to increased c-Myc and Cyclin E protein levels as shown by Western blot analysis (Figure 2B). Furthermore, we evaluated the proliferation of LO2 cells with and without Fbxw7-specific siRNA using MTT assay. The MTT activity of LO2 cells transfected with Fbxw7 siRNA was significantly increased compared control cells, suggesting higher proliferation rates (Figure 2C). The Hep3B cell line behaved similarly under Fbwx7 suppressing conditions (Figure 2A, 2B and 2C). 10.1371/journal.pone.0068574.g002Figure 2 Effect of Fbxw7 knockdown on LO2 and Hep3B cell lines. A) qRT-PCR confirmed knockdown in LO2 and Hep3B cells treated with Fbxw7 siRNA. B) Expression of c-Myc and Cyclin E proteins were enhanced by Fbxw7 siRNA, as confirmed by Western blot analysis. β-actin served as loading control. C) Proliferation rates were assessed by MTT assay. Cell viability was normalized to time point 0 h. The proliferation rate of LO2 and Hep3B cells treated with Fbxw7 siRNA was significantly greater than in control siRNA cells. Values are depict as mean±standard error, n=3. P<0.05 vs control siRNA. Expression of Fbxw7 is not compromised by mutations in HCC cell lines We have previously shown decreased Fbxw7 mRNA and protein levels in HCC tumor tissues [7]. These, together with the latter in vitro results, support the model of tumor suppressive activity of Fbxw7 in HCC. It has been shown that Fbxw7 is inactivated by mutations in diverse human cancer types with an overall mutation frequency of ~ 6% [17]. We examined Fbxw7 cDNA derived from LO2 cell lines and HCC cell lines SMMC-7721, Bel-7402, Hep3B, Huh7 and HepG2. No mutations, such as missense mutations, deletions or insertions were detected performing database alignments, suggesting that other mechanisms might be involved in Fbxw7 inactivation contributing to an oncogenic phenotype. rAd-p53 decreased c-Myc and Cyclin E levels, increased Fbxw7 expression and induced apoptosis Fbwx7 protein has been shown to be one target of the p53 tumor suppressor and mutations in this gene were detected in 27.8 to 50% of HCC cases in several studies involving patients of different ethnicities [8,18,19]. We have previously shown that Fbwx7 protein expression was negatively correlated with mutant p53 in human HCC tissues [7]. Here, we hypothesize that the exogenous expression of p53 using rAd-p53 would enhance Fbxw7 expression and thereby suppress c-Myc and Cyclin E protein expression reducing cell proliferation [20]. Initially, we performed a multiplicity of infection (MOI) gradient and MTT assay to determine the half-maximal inhibitory concentration (IC50) of the vector in Hep3B (Figure 3A). The p53 null cell line Hep3B was infected with rAd-p53 at an MOI of 723 (IC50 of rAd-p53). 48 hour after infection, p53 and Fbxw7 mRNA levels were significantly elevated compared to uninfected controls, whereas c-Myc and Cyclin E levels were slightly decreased, however not statistically significant (Figure 3B). We confirmed that rAd-p53 increased p53 and Fbwx7 protein levels not only in Hep3B cells but in HepG2 (wild type-p53) and Huh7 (mutant-p53) cells as well. In contrast, c-Myc and Cyclin E protein levels were significantly down-regulated in all three cell lines (Figure 3C). We next evaluated proliferation and apoptosis of infected Hep3B by MTT assay and flow cytometry. The proliferation of infected Hep3B cells was significantly decreased compared to uninfected control cells confirming our hypothesis (P<0.05, Figure 3D). Consistent with this result, 54% of Hep3B cells were AnnexinV positive and Propidium Iodide (PI) negative at 72 hours post infection (h.p.i.), indicating a significantly increase in apoptotic cells (P<0.05, Figure 3E). 10.1371/journal.pone.0068574.g003Figure 3 rAd-p53 induces apoptosis in HCC cell lines with increased Fbxw7 and decreased c-Myc and Cyclin E. A) MTT activity was measured after infecting Hep3B cells with rAd-p53 in an MOI gradient to determine their IC50 after 48 h. Mean OD values were used to calculate the IC50 via the modified Kou-type method: lgIC50 = Xm-I (P-(3-Pm-Pn)/4), in which Xm: lg maximum dose; I: lg (maximum dose/ adjacent dose); P: sum of positive response rate; Pm: the largest positive response rate; Pn: the smallest positive response rate. B) Over-expression of p53 24 h after infection as confirmed by RT-PCR. Quantification of RT-PCR for p53 and Fbxw7 indicated elevated mRNA levels for rAd-p53 infected Hep3B cells, whereas c-Myc and Cyclin E levels were not significantly decreased. C) Protein expression of p53 and Fbxw7 was enhanced, while c-Myc and Cyclin E expression was suppressed by rAd-p53 in Hep3B, HepG2 and Huh7 cells, as confirmed by Western blot analysis. β-actin served as loading control. D) Hep3B proliferation rate assessed by MTT assay was significantly lower in rAd-p53 infected cells compared to control cells. E) Quantification of apoptotic cell population (AnnexinV positive/ PI negative) by flow cytometry. rAd-P53 infected Hep3B cells were composed of a larger subset of apoptotic cells after 72 hours of infection compared to control. Values are depicted as mean±standard error, n=3. P<0.05 vs control. rAd-p53 induced apoptosis can be reverted by an Fbxw7-specific siRNA To determine whether Fbxw7-mediated c-Myc and Cyclin E degradation participate in rAd-p53 induced apoptosis in Hep3B cells, we infected with rAd-p53 at an MOI of 723 overnight, and subsequently transfected with an Fbxw7-specific siRNA. Fbxw7 knockdown in infected Hep3B cells reverted partially the effect of exogenous p53 overexpression (Figure 4A), leading to a significant two-fold reduction of apoptotic cells and increased proliferation rates (P<0.05, Figure 4B and 4C). In vitro, these results indicate that p53 induced growth arrest and apoptosis is somewhat mediated by Fbwx7 degradation of c-Myc and Cyclin E in Hep3B cells. 10.1371/journal.pone.0068574.g004Figure 4 rAd-p53 induced apoptosis was reverted by Fbxw7-specific siRNA. A) Fbxw7-siRNA treatment in Hep3B successfully down-regulated Fbxw7 protein but enhanced c-Myc and Cyclin expression as shown by immunostaining. Over-expression of rAd-p53 in the same cell line could enhance the levels of Fbxw7 and decrease c-Myc and Cyclin E expression. Fbxw7 knockdown in p53 over-expressing cells rescued partially the phenotype showing lower Fbxw7 levels with higher c-Myc and Cyclin E expression. β-actin served as loading control. B) The proliferation rate of rAd-p53 infected Hep3B cells was increased in the presence of Fbxw7-specific siRNA (MTT assay). C) The percentage of apoptotic Hep3B cells infected with rAd-p53 was decreased by Fbxw7-specific siRNA. Values are mean±standard error, n=3. * P<0.05 vs control; P<0.05 vs Fbxw7 siRNA; * P<0.05 vs rAd-p53. rAd-p53 inhibits tumor growth by p53-Fbxw7 pathway in vivo We next sought to determine, whether exogenous p53 affected tumor growth in Hep3B subcutaneous tumor model. Tumor bearing mice treated with 1x107 rAd-p53 VP/mm3 administered by multi-center intratumoral injection, showed 2.1 fold reduction in tumor volume and 2.3 fold reduction in tumor weight compared to controls (P<0.05, Figure 5). We confirmed the increased expression of p53 by Western blot (Figure 6A) and performed immunohistochemistry for Fbxw7, c-Myc and Cyclin E. We could not detect c-Myc or Cyclin E in rAd-p53 treated xenografts, whereas saline treated mice showed faint Fbwx7 staining but strong c-Myc and Cyclin E signal (Figure 6B), confirming our in vitro observations. 10.1371/journal.pone.0068574.g005Figure 5 rAd-p53 inhibits tumor growth in vivo. Multicenter intratumoral administration of rAd-P53 in Hep3B subcutaneous tumor in nude mice was monitored over time. Tumor size and weight was plotted in A), and B) respectively. C) Exemplarily, tumor bearing mice and explanted tumors are depicted. Values are mean±standard error. * P<0.05 vs rAd-p53. 10.1371/journal.pone.0068574.g006Figure 6 p53-Fbxw7 pathway involvement in rAd-p53 treated tumor bearing mice. A) Immonostaining of tissue homogenates confirm p53 over-expression in rAd-p53 treated mice, n=3. β-actin served as loading control. B) Fbxw7, c-Myc and Cyclin E were detected by SP–IHC on paraffin sections of both groups. Tumors treated with rAd-p53 showed strong signal for Fbsw7 protein (a), while c-Myc and Cyclin E were not detectable in the same tissue section (b and c). In saline treated tumors, weak Fbxw7 staining (d) with strong c-Myc and Cyclin E staining (e and f) was detected. Micrographs were acquired at 400 fold magnification, n=6. Discussion In this study, we first demonstrated that Fbxw7 is a key tumor suppressor that regulated cell proliferation in different HCC cell lines, given that Fbxw7 mRNA expression correlated negatively with colony formation ability. Ablation of Fbxw7 by RNAi in Hep3B and LO2, cell lines with high expression of the target gene, consistently showed accumulation of c-Myc and Cyclin E and enhanced cell proliferation. c-Myc protein plays crucial roles in mitogenic signaling and cell growth responses [13] and Cyclin E is a key component of the cell cycle machinery [14]. Both are frequently deregulated in HCC. This observation been reported for cell lines derived from gastric, colorectal, ovarian and breast cancer [21–24]. Here, we found a similar effect on HCC cell lines. Active Cyclin E-Cdk2 complex is required for G1-S phase transition, modulating pRb and thereby activating E2F transcription factors that enable DNA replication [25]. High levels of Fbxw7 might decrease the pool of available Cyclin E triggering arrest in G1 phase accounting for low proliferation rates. The regulation of Fbxw7 in HCC has not been yet clarified. In T-cell actue lymphoblastic leukemia (T-ALL), colorectal, breast and gastric cancer, abnormal levels of Fbxw7 have been associated with genomic mutations [22,24,26,27]. In the HCC cell lines here studied, we failed to identify any mutations, suggesting the involvement of further regulating factors. It has been shown that Fbxw7 is directly regulated by p53 [4]. Here, we showed over-expression of p53 using the commercial gene therapy drug Gendicine, which resulted in upregulation of Fbxw7 thereby preventing c-Myc and Cyclin E accumulation in vitro and in vivo. This and the observed increase in apoptosis and decrease in proliferation in vitro, could be partially reverted by treatment with a Fbxw7-specific siRNA. p53 is a key trigger of the intrinsic apoptotic pathway [28]. Although the levels of p53 remain unchanged in infected cells treated with Fbxw7 siRNA, a 2-fold reduction in apoptosis is observed, indicating a possible involvement of Fbxw7. We suggest that this protein contributes to trigger apoptosis during rAd-p53 administration likely by downstream effects of c-Myc degradation or unknown apoptotic effects of Fbxw7 or downstream targets. Alternatively, it has been shown that c-Myc can induce apoptosis by Caspase-3 dependent and caspase-independent signaling [29]. This mechanism might account for the significantly reduced tumor burden in Hep3B tumor model. Low proliferation rates and increase in apoptosis suggest that rAd-p53 has an anti-tumor effect as well in HCC. Intraepithelial delivery of p53 via adenoviral vectors has shown to increase apoptosis in oral leukoplakia, a well recognized precancerous lesion for squamous cell carcinoma [20]. In conclusion, we demonstrated that Fbxw7 can be activated by adenoviral delivery of p53, leading to increase proteasomal degradation of c-Myc and Cylin E. Low levels of the studied cell cycle regulators might attenuate the oncogenic phenotype of HCC cell lines by restricting G1-S transition and c-Myc mediated cell cycle reentry. Moreover, we suggest that Fbxw7 synergizes with p53 to trigger apoptosis in vitro. Altogether, we hypothesize that p53 contributes to hepatocarcinogenesis in part through downregulation of Fbxw7 activity and accumulation of c-Myc and Cyclin E. Addressing this pathway, we identified that rAd-p53 could be a potential therapeutic agent for HCC. Whether Fbxw7 itself is a druggable target in HCC, needs still to be clarified. We thank Egon Jacobus (German Cancer Research Center, University of Heidelberg) for the revision of manuscript. ==== Refs References 1 Forner Alejandro, Llovet Josep M, Bruix Jordi (2012 ) Hepatocellular carcinoma . Lancet 379 : 1245 -1255 . doi:10.1016/S0140-6736(11)61347-0. PubMed: 22353262.22353262 2 Wertz IE , Kusam S , Lam C , Okamoto T , Sandoval W et al. (2011 ) Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7 . Nature 471 : 110 -114 . doi:10.1038/nature09779. PubMed: 21368834.21368834 3 Crusio KM , King B , Reavie LB , Aifantis I (2010 ) The ubiquitous nature of cancer: the role of the SCF (Fbw7) complex in development and transformation . Oncogene 29 : 4865 -4873 . doi:10.1038/onc.2010.222. PubMed: 20543859.20543859 4 Wang Z , Fukushima H , Gao D , Inuzuka H , Wan L et al. (2011 ) The two faces of FBW7 in cancer drug Resistance . Bioessays 33 : 851 -859 . doi:10.1002/bies.201100101. PubMed: 22006825.22006825 5 Tu K , Zheng X , Yin G , Zan X , Yao Y et al. (2012 ) Evaluation of Fbxw7 expression and its correlation with expression of SREBP-1 in a mouse model of NAFLD. Mol Med Report 6: 525-530 6 Welcker M , Clurman BE (2008 ) FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation . Nat Rev Cancer 8 : 83 -93 . doi:10.1038/nrc2290. PubMed: 18094723.18094723 7 Tu K , Zheng X , Zan X , Han S , Yao Y et al. (2012 ) Evaluation of Fbxw7 expression and its correlation with the expression of c-Myc, cyclin E and p53 in human hepatocellular carcinoma . Hepatol Res 42 : 904 -910 . doi:10.1111/j.1872-034X.2012.01005.x. PubMed: 22548670.22548670 8 Hsu IC , Metcalf RA , Sun T , Welsh JA , Wang NJ et al. (1991 ) Mutational hotspot in the p53 gene in human hepatocellular carcinomas . Nature 350 : 427 -428 . doi:10.1038/350427a0. PubMed: 1849234.1849234 9 Piao Z , Kim H , Jeon BK , Lee WJ , Park C (1997 ) Relationship beween loss of heterozygosity of tumor suppressor genes and histologic differentiation in hepatocellular carcinoma . Cancer 80 : 865 -872 . doi:10.1002/(SICI)1097-0142(19970901)80:5. PubMed: 9307185.9307185 10 Honda K , Sbisà E , Tullo A , Papeo PA , Saccone C et al. (1998 ) p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation . Br J Cancer 77 : 776 -782 . doi:10.1038/bjc.1998.126. PubMed: 9514057.9514057 11 Mao JH , Perez-Losada J , Wu D , Delrosario R , Tsunematsu R et al. (2004 ) Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene . Nature 432 : 775 -779 . doi:10.1038/nature03155. PubMed: 15592418.15592418 12 Yada M , Hatakeyama S , Kamura T , Nishiyama M , Tsunematsu R et al. (2004 ) Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7 . EMBO J 23 : 2116 -2125 . doi:10.1038/sj.emboj.7600217. PubMed: 15103331.15103331 13 Grandori C , Cowley SM , James LP , Eisenman RN (2000 ) The Myc/Max/Mad network and the transcriptional control of cell behavior . Annu Rev Cell Dev Biol 16 : 653 -699 . doi:10.1146/annurev.cellbio.16.1.653. PubMed: 11031250.11031250 14 Hwang HC , Clurman BE (2005 ) Cyclin E in normal and neoplastic cell cycles . Oncogene 24 : 2776 -2786 . doi:10.1038/sj.onc.1208613. PubMed: 15838514.15838514 15 Peng Z (2005 ) Current status of gendicine in China: recombinant human Ad-p53 agent for treatment of cancers . Hum Gene Ther 16 : 1016 -1027 . doi:10.1089/hum.2005.16.1016. PubMed: 16149900.16149900 16 Xie Q , Liang BL , Wu YH , Zhang J , Chen MW et al. (2012 ) Synergistic anticancer effect of rAd/P53 combined with 5-fluorouracil or iodized oil in the early therapeutic response of human colon cancer in vivo . Gene 499 : 303 -308 . doi:10.1016/j.gene.2012.02.007. PubMed: 22441128.22441128 17 Akhoondi S , Sun D , von der Lehr N , Apostolidou S , Klotz K et al. (2007 ) FBXW7/hCDC4 is a general tumor suppressor in human cancer . Cancer Res 67 : 9006 -9012 . doi:10.1158/0008-5472.CAN-07-1320. PubMed: 17909001.17909001 18 Bressac B , Kew M , Wands J , Ozturk M (1991 ) Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa . Nature 350 : 429 -431 . doi:10.1038/350429a0. PubMed: 1672732.1672732 19 Hayashi H , Sugio K , Matsumata T , Adachi E , Takenaka K et al. (1995 ) The clinical significance of p53 gene mutation in hepatocellular carcinomas from Japan . Hepatology 22 : 1702 -1707 . doi:10.1002/hep.1840220614. PubMed: 7489977.7489977 20 Li Y , Li LJ , Zhang ST , Wang LJ , Zhang Z et al. (2009 ) In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for oral leukoplakia . Clin Cancer Res 15 : 6724 -6731 . doi:10.1158/1078-0432.CCR-09-1296. PubMed: 19861457.19861457 21 Kwak EL , Moberg KH , Wahrer DC , Quinn JE , Gilmore PM et al. (2005 ) Infrequent mutations of Archipelago (hAGO, hCDC4, Fbw7) in primary ovarian cancer . Gynecol Oncol 98 : 124 -128 . doi:10.1016/j.ygyno.2005.04.007. PubMed: 15913747.15913747 22 Ibusuki M , Yamamoto Y , Shinriki S , Ando Y , Iwase H (2011 ) Reduced expression of ubiquitin ligase FBXW7 mRNA is associated with poor prognosis in breast cancer patients . Cancer Sci 102 : 439 -445 . doi:10.1111/j.1349-7006.2010.01801.x. PubMed: 21134077.21134077 23 Iwatsuki M , Mimori K , Ishii H , Yokobori T , Takatsuno Y et al. (2010 ) Loss of FBXW7, a cell cycle regulating gene, in colorectal cancer: clinical significance . Int J Cancer 126 : 1828 –1837 . PubMed: 19739118.19739118 24 Yokobori T , Mimori K , Iwatsuki M , Ishii H , Onoyama I et al. (2009 ) p53-Altered FBXW7 expression determines poor prognosis in gastric cancer cases . Cancer Res 69 : 3788 -3794 . doi:10.1158/0008-5472.CAN-08-2846. PubMed: 19366810.19366810 25 Ohtsubo M , Theodoras AM , Schumacher J , Roberts JM , Pagano M (1995 ) Human cyclin E, a nuclear protein essential for the G1-to-S phase transition . Mol Cell Biol 15 : 2612 -2624 . PubMed: 7739542.7739542 26 Callens C , Baleydier F , Lengline E , Ben Abdelali R , Petit A et al. (2012 ) Clinical impact of NOTCH1 and/or FBXW7 mutations, FLASH deletion, and TCR status in pediatric T-cell lymphoblastic lymphoma . J Clin Oncol 30 : 1966 -1973 . doi:10.1200/JCO.2011.39.7661. PubMed: 22547598.22547598 27 Strohmaier H , Spruck CH , Kaiser P , Won KA , Sangfelt O et al. (2001 ) Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line . Nature 413 : 316 -322 . doi:10.1038/35095076. PubMed: 11565034.11565034 28 Haupt S , Berger M , Goldberg Z , Haupt Y (2003 ) Apoptosis - the p53 network . J Cell Sci 116 : 4077 -4085 . doi:10.1242/jcs.00739. PubMed: 12972501.12972501 29 Prendergast GC (1999 ) Mechanisms of apoptosis by c-Myc . Oncogene 18 : 2967 -2987 . doi:10.1038/sj.onc.1202727. PubMed: 10378693.10378693	23840897	PMC3698167	CC BY	2021-01-05 17:31:09	yes	PLoS One. 2013 Jul 1; 8(7):e68574
==== Front Diagn PatholDiagn PatholDiagnostic Pathology1746-1596BioMed Central 1746-1596-8-862368820910.1186/1746-1596-8-86Case ReportUlnar malignant peripheral nerve sheath tumour diagnosis in a mixed-breed dog as a model to study human: histologic, immunohistochemical, and clinicopathologic study Tavasoly Abbas 1atavasoli@ut.ac.irJavanbakht Javad 1javadjavanbakht@ut.ac.irKhaki Fariba 1khakifariba@ut.ac.irHosseini Ehsan 2hosseiniehsan460@gmail.comBahrami Alimohammad 2bahrami2222@gmail.comHassan Mehdi Aghamohammad 3aghamh@ut.ac.irMirabad Mohammadmehdi 3mehdi.mirabad@ut.ac.ir1 Department of Pathology, Faculty of Veterinary Medicines, Tehran University, Tehran, Iran2 Paraveterinary Faculty of Ilam, University of Ilam, Ilam, Iran3 Department of Clinical Science, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran2013 20 5 2013 8 86 86 3 5 2013 16 5 2013 Copyright © 2013 Tavasoly et al.; licensee BioMed Central Ltd.2013Tavasoly et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Canine Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are uncommonly reported in the ulnar, since they are underestimated relative to the more common spindle cell tumours of soft tissue. In dogs, MPNST accounts for 27% of nervous system tumours. In man, MPNST represents 5-10% of all soft tissue sarcomas and is often associated with neurofibromatosis type 1 (NF-1).An 8-year-old, 9 kg, female mixed-breed dog with a subcutaneous mass on the upper right side of the ulnar region was presented to the small animal research and teaching hospital of Tehran University. The dog was anorexic with general weakness. The mass (7 × 4 cm) was removed surgically and processed routinely. Microscopically, the mass was composed of highly cellular areas with a homogeneous population of round or spindle cells, high cellular pleomorphism, high mitotic index and various morphologic patterns. Furthermore, spindle cells arranged in densely or loosely sweeping fascicles, interlacing whorls, or storiform patterns together with wavy cytoplasm, nuclear palisades, and round cells were arranged in sheets or cords with a meshwork of intratumoral nerve fibers. In addition, in this case the presence of neoplastic cells within the blood vessels was observed. Immunohistochemically, tumor was positive for vimentin and S-100 protein. The histopathologic features coupled with the S-100 and vimentin immunoreactivity led to a diagnosis of malignant neurofibroma. To the best of our knowledge, primary ulnar MPNST has not been reported in animals. This is the first documentation of an ulnar malignant peripheral nerve sheath tumour in a dog. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1310907815984587 DogPathologyImmunohistochemistryTumorMarkers ==== Body Background Canine peripheral nerve sheath tumors (PNSTs) are uncommonly reported, and their clinical behavior has not been well documented [1,2]. These tumors are relatively common in humans but occur infrequently in domestic animals [3]. Based on the morphologic and biologic behavior, PNSTs are divided to benign PNST (BPNST) and malignant PNST (MPNST) forms with several morphologic features [1-3]. MPNST is an aggressive and uncommon neoplasm that develops within peripheral nerve [4]. In human MPNSTs, variable histologic patterns and heterogenous differentiation have been reported [5-7], including epithelioid MPNST [8,9] and MPNST with divergent differentiation such as rhabdomyoblastic (malignant Triton tumor) [10-12] cartilaginous, osseous, angiomatous [13] and glandular forms [14,15], or their complex [16]. Similar representation such as epithelioid type [17], melanotic type [18,19] cartilaginous and osteogenic [20] or glandular epithelial differentiation have been found in MPNSTs in dogs or other animals [3,21]. Histologically, in human MPNSTs, the malignant nature of these tumours were evident upon their microscopic inspection, as they were comprised of an infiltrative and cellular proliferation of atypical, mitotically active spindle cells. Most MPNSTs are high-grade tumours with a high mitotic rate and commonly induce necrosis. The most common histological patterns include a high-grade fibrosarcomatous mass composed of densely packed sheets of plump but relatively uniform spindle or oval cells [4,22-26]. Immunohistochemical studies have contributed to the definition of clear diagnostic criteria for PNSTs. In human medicine, the expression of S-100 protein is used to differentiate between spindle cell tumours of neural and non-neural origins [27]. In veterinary medicine, immunohistochemical detection of S-100 and vimentin has been able to distinguish conclusively between subsets of such spindle cell tumours [28]. This report describes the histologic and immunohistochemical diagnosis of a neurofibrosarcoma in the ulnar of a dog. Case presentation An 8-year-old, 9 kg, female, mixed-breed dog referred to the Small Animal Clinic of Tehran University. Clinical examination revealed a rapidly growing, nodular, subcutaneous mass, 7×4 cm in diameter, invading and strongly adhering to the underlying tissue. The mass was located on the upper right side of the ulnar region. The dog had anorexia, general weakness and inability to stand. Ultrasonography revealed a subcutaneous mass with a central depth of 5.4 cm. ventrally, the margins of the lesion appeared to be well defined and the abdominal organs were not visibly affected. The blood cell count was undertaken manually and calcium and phosphorus concentration were measured by commercial kits (Pars Azmoon, Alborz, Iran) using a semi-automatic analyzer (EMP 168 Vet Biochemical analyzer; Shenzhen Emperor Electronic technology Co. Ltd, Shenzhen, China). Haematological and biochemical profiles (including blood cell count and serum calcium and phosphorus concentrations) were within normal ranges. For partial excisional biopsy, an intravenous combination of diazepam (0.27 mg/kg) (Tamin, Tehran, Iran) and ketamine hydrochloride (5.50 mg/kg) (Alfasan, Woerden, the Netherlands) were used for induction and maintenance of anesthesia and the dog received normal saline solution 0.9% intravenously, to a total of 0.5 L (12 mL/kg/h) (Samen, Mashhad, Iran). Atropine sulphate (0.03 mg/kg) (Tamin, Tehran, Iran) was given subcutaneously as pre-medication. The mass was removed surgically and processed routinely. The sample was fixed in formalin and embedded in paraffin for sectioning. The sections were stained with haematoxylin and eosin. For further study, paraffin sections were stained immunohistochemically with S-100, vimentine markers (Abcam Co., Cambridge, USA). For immunohistochemistry, sections from tumor were mounted on adhesive-coated slides (Superfrost Plus, Menzel-Glaser, Braunschwaig, Germany), processed through xylene, and rehydrated in ethanol. Antigen retrieval was by boiling in a microwave oven (700 W) twice for 5 minutes in Tris–EDTA buffer—1.21 g Tris base (A 1379, Applichem, Darmstadt, Germany) and 0.372 g EDTA (8418, Merck, Darmstadt, Germany)—in 1 liter of distilled water and pH 9. Endogenous peroxidase was blocked with 0.6% (v/v) H2O2 in Tris-buffered saline (TBS; pH 7.6) for 15 minutes at 20°C before the antibodies used included those for vimentin (prediluted, monoclonal: V9), S-100 protein (1:4,800, polyclonal rabbit anti-S-100). Histopathology Histopathologically, neurofibrosarcoma tumour cells were not circumscribed by connective tissue and neoplastic cells often exhibited an aggressive behavior, high cellularity, cellular pleomorphism, and various morphologic patterns. Atypical mononuclear or multinucleated cells were consistently observed. More than three mitotic figures per high-power field (400×) were found in this case (Figure 1B and 1C). Necrotic foci accompanied by pseudopalisading (Figure 2A) and infiltrates of various numbers of lymphocytes, plasma cells, and macrophages were common. Figure 1 A: The cells are in disorganized fascicles with pleomorhism, nuclear atypia and neoplastic spindle cells with wavy eosinophilic cytoplasmic processes. (H & E; bar 20 μm; ×400). B: the neoplastic cells form dense bundles separated by scanty collagenous stroma. Notice bland nuclei lacking atypia. (H & E; bar 30 μm; × 400) C: Upon histopathological examination of the ulnar mass, neurofibroma with a mixture of round and spindle shaped cells, mitotic figures, infiltrates of various numbers of lymphocytes and hypercellularity arrangement was diagnosed. (H & E; bar 25 μm; × 400). Figure 2 A: Necrotic focus is accompanied by nuclear pseudopalisading in MPNST, 2B: Neoplastic cells with cytoplasm and round nuclei are arranged in a cordlike pattern, 2C: The neoplastic cells show diffuse expression of S-100. IHC, 2D: The neoplastic cells show strong expression of vimentin. IHC 2E: A number of neoplastic cells show a marked cytoplasmic and nuclear immunoreaction for S-100 protein 2F: Fusiform or spindle-shaped neoplasmic cells are arranged in interdacing fascicles. In some areas, neurofibrosarcoma was composed mainly of a homogeneous population of round cells (Figure 1C). These round cells were arranged in sheets or cords with a meshwork of intratumoral nerve fibers. The nuclei were round or oval (Figure 1A and 2B). Furthermore, in some areas, the spindle cells had long wavy nuclei with tapered ends. Some had oval, round, short spindle nuclei, or had nuclear pseudo-inclusions (Figure 3A, 3B and Figure 1A, 1C). Figure 3 Immunohistochemical staining with, vimentin (A) and S-100 (B and C) are positive cells; the neoplastic cells in parts of the neurofibroma express S-100 and vimentin strongly. However, in some regions of the tumor tissue, fusiform or spindle cells arranged in densely or loosely sweeping fascicles, interlacing whorls, or storiform patterns together with wavy cytoplasm, nuclear palisades were predominant (Figure 2F). In addition, the neoplastic cells within the blood vessels were observed as well. Immunohistochemical features of the spindle-shaped neoplastic cells were predominantly positive for S-100 and vimentin (Figure 3A, 3B and 3C) and the cytoplasm and/ or nucleus of the neoplastic cells were diffusely labeled for expression of vimentin (Figure 2D), S-100 (Figure 2C and 2E). Discussion PNSTs have also been reported in cats, dogs, cattle, rats, and horses [3,29,30]. In human, these tumours have been subclassified as neurinomas, neurilemmomas, schwannoma, neurofibromas, neurofibrosarcomas, and malignant peripheral nerve sheath tumors (MPNSTs), based on their presumed cell(s) of origin. In the dog, 2 groups of tumors have been referred to as PNSTs, 1 occurring in the cranial and spinal nerves and 1 occurring in the skin and subcutaneous [31]. Many of the first group are consistent with MPNSTs and have metastatic potential [3]. Those occurring in the skin and subcutaneous of dogs are usually of uncertain histogenesis and are referred to by some as hemangiopericytoma [32]. Palisading, as seen in classic PNSTs, is usually absent [31]. The histological differentiation between malignant and benign PNST can be difficult because both may show undefined edges and some degree of cellular pleomorphism [33]. It has been suggested that malignant PNST in dog have aggressive behavior to intratumoral tissue, extensive necrotic areas and cellular pleomorphism [34-36]. All of these characteristics were observed in this case; however, the presence of neoplastic cells within the blood vessels was observed that determined the classification of malignancy. A high level of mitosis is also indicative of a malignant PNST [34,36]. In present study, the malignant histological appearance of the lesion (mitotic index, cellular pleomorphism or necrosis) occurred in conjunction with infiltrative growth. In dogs and humans, divergent differentiation is usually associated with a poor prognosis [21,37]. The S100 protein is the primary marker in the diagnosis of MPNST (malignant schwannoma, neurofibrosarcoma, and neurogenic sarcoma) and may be used as a single diagnostic tool [38,39] or in combination with other markers such as vimentin [40,41]. The neoplastic cells in this study showed positivity for S100 and vimentin immunolabeling. In this study, based on their morphologic features diagnostic for human neurofibrosarcoma, i.e., growth pattern and microscopic features (such as areas of high cellularity, cellular pleomorphism, various morphologic patterns, high mitotic index and high number of undifferentiated neoplastic cells), together with the presence of intratumoral nerve fibers and the restriction of the S100 and vimentin immunostaining to a subpopulation of the neoplastic cells, the tumour was diagnosed as neurofibrosarcoma. But the cause of MPNST in domestic animals has not yet been determined. Due to its anatomical location and difficulties encountered in complete surgical removal. Canine MPNST often recurs after surgery and the prognosis is generally poor [42]. In addition, the prognosis of human patients with primary MPNST is poor and removal is often followed by recurrence, metastasis and death [43-45]. No definitive evidence of distant organ metastasis was found in this case. Neurofibrosarcoma in this report had marked morphologic variation. The round cell type found in this case was morphologically similar to the primitive neuroectodermal tumour described as the small round-cell type in human MPNST [46] or one of the malignant schwannomas [47], suggesting that the presence of round cells implies a differentiation toward immature neural cells. Immunohistochemical expression of S100 protein and vimentin by the neoplastic cells prompted consideration of peripheral nerve sheath tumour in the differential diagnosis. Various immunohistochemical markers have been used to define MPNST. S-100 is commonly expressed in normal nervous tissue and in most MPNST [48], but also in most rhabdomyosarcomas and neurofibrosarcoma [49]. The present case was positive for expression of vimentin and S-100. On the basis of gross morphology, histopathological and immunohistochemical features, the final tumour in this study, a diagnosis of neurofibrosarcoma was made. For understanding of these complex neoplasms and the development of the effective differential diagnosis, further investigation will be needed into the clinical features and the basic science. Conclusion This study described histopathology and immunohistochemical features of canine subcutaneous neurofibrosarcoma of the ulnar region. The histological features of these tumours would suggest that most should be classified as high-grade MPNST. A subcutaneous MPNST may be diagnosed on the basis of observing the histopathological pattern described in present study. In addition, S-100 and vimentin immunohistochemical expression may be used to help confirm the diagnosis of neurofibrosarcoma. Finally, the use of immunohistochemistry may be helpful in distinguishing this type of neoplasm from other malignancies with similar morphology. The incidence of neurofibrosarcoma in animals is unknown; we hope this will become clearer. To our knowledge, this is the first report of ulnar MPNST in a dog, suggesting that this tumour should be included as a differential diagnosis for ulnar spindle cell tumours. Competing interests The authors declare that they have no competing interests. Authors’ contributions AT and FK participated in the histopathological evaluation, performed the literature review, acquired photomicrographs and drafted the manuscript and gave the final histopathological diagnosis. JJperformed sequencing alignment and manuscript writing. EH and AB carried out the immunohistochemical stains evaluation. MAH and MMM edited the manuscript and made required changes. All authors have read and approved the final manuscript. Acknowledgements The authors thank staff of the Department of pathology, Faculty of Veterinary Medicine, Tehran University for their valuable technical assistance. ==== Refs Hendrick MJ Mahaffey EA Moore FM Vos JH Walder EJ World Health Organization, International Histological Classification of Tumors of Domestic Animals, Histological Classification of Mesenchymal Tumors of Skin and Soft Tissues of Domestic Animals, Second Series 1998 2 Washington, DC: Armed Forces Institute of Pathology American Registry of Pathology 26 27 Koestner A Bilzer T Fatzer R Schulman FY Summers BA Van Winkle TJ World Health Organization, International Histological Classification of Tumors of Domestic Animals, Histological Classification of Tumors of the Nervous System of Domestic Animals, Second Series 1999 5 Washington, DC: Armed Forces Institute of Pathology American Registry of Pathology 37 38 Summers BA Cummings JF De Lahunta A Veterinary Neuropathology 1995 St. Louis, MO: Mosby 472– 501 Kitamura M Wada N Nagata S Iizuka N Jin YF Tomoeda M Yuki M Naka N Araki N Yutani C Tomita Y Malignant peripheral nerve sheath tumor associated with neurofibromatosis type 1, with metastasis to the heart: a case report Diagn Pathol 2010 5 2 10.1186/1746-1596-5-2 20205747 Ducatman BS Scheithauer BW Malignant peripheral nerve sheath tumor with divergent differentiation Cancer 1984 54 1049 1057 10.1002/1097-0142(19840915)54:6<1049::AID-CNCR2820540620>3.0.CO;2-1 6432304 Enzinger FM Weiss SW Soft Tissue Tumors 2001 4 St. Louis, MO: Mosby 215 216 1209–1263, 1405–1417 Takeuchi A Ushigome S Diverse differentiation in malignant peripheral nerve sheath tumors associated with neurofibromatosis-1: an immunohistochemical and ultra-structural study Histopathology 2001 39 298 309 10.1046/j.1365-2559.2001.01212.x 11532041 Laskin WB Weiss SW Bratthauer GL Epithelioid variant of malignant peripheral nerve sheath tumor (malignant epithelioid schwannoma) Am J Surg Pathol 1991 15 1136 1145 10.1097/00000478-199112000-00004 1746681 McMenamin ME Fletcher CD Expanding the spectrum of malignant change in schwannomas: epithelioid malignant change, epithelioid malignant peripheral nerve sheath tumor, and epithelioid angiosarcoma: a study of 17 cases Am J Surg Pathol 2001 25 13 25 10.1097/00000478-200101000-00002 11145248 Brooks JS Freeman M Enterline HT Malignant “Triton” tumors: natural history and immunohistochemistry of nine new cases with literature review Cancer 1985 55 2543 2549 10.1002/1097-0142(19850601)55:11<2543::AID-CNCR2820551105>3.0.CO;2-4 3922610 Daimaru Y Hashimoto H Enjoji M Malignant “triton” tumors: a clinicopathologic and immunohistochemical study of nine cases Hum Pathol 1984 15 768 778 10.1016/S0046-8177(84)80169-0 6235165 Kiryu H Urabe H Malignant triton tumor: a case with protean histopathological patterns Am J Dermatopathol 1992 14 255 262 10.1097/00000372-199206000-00013 1510224 Morphopoulos GD Banerjee SS Ali HH Stewart M Vasudev KS Eyden BP Harris M Malignant peripheral nerve sheath tumour with vascular differentiation: a report of four cases Histopathology 1996 28 401 410 8735715 Robbins P Papadimitriou J Glandular peripheral nerve sheath tumours Pathol Res Pract 1994 190 412 415 10.1016/S0344-0338(11)80418-8 8078812 Woodruff JM Christensen WN Glandular peripheral nerve sheath tumors Cancer 1993 72 3618 3628 10.1002/1097-0142(19931215)72:12<3618::AID-CNCR2820721212>3.0.CO;2-# 8252477 Wong SY Teh M Tan YO Best PV Malignant glandular triton tumor Cancer 1991 67 1076 1083 10.1002/1097-0142(19910215)67:4<1076::AID-CNCR2820670435>3.0.CO;2-C 1991255 Pumarola M Anor S Borras D Ferrer I Malignant epithelioid schwannoma affecting the trigeminal nerve of a dog Vet Pathol 1996 33 434 436 10.1177/030098589603300411 8817843 Patnaik AK Erlandson RA Lieberman PH Canine malignant melanotic schwannoma: a light and electron microscopic study of two cases Vet Pathol 1984 21 483 488 6485208 Kameyama M Ishikawa Y Shibahara T Kadota K Melanotic neurofibroma in a steer J Med Sci 2000 62 125 128 10.1292/jvms.62.125 Anderson GM Dallaire A Miller LM Miller CW Peripheral nerve sheath tumor of the diaphragm with osseous differentiation in a one-year-old dog J Am Hosp Assoc 1999 35 319 322 Patnaik AK Zachos TA Sams AE Aitken ML Malignant nerve-sheath tumor with divergent and glandular differentiation in a dog: a case report Vet Pathol 2002 39 406 410 10.1354/vp.39-3-406 12014509 Sasaki K Desimone M Rao HR Huang GJ Seethala RR Adrenocortical carcinosarcoma: a case report and review of the literature Diagn Pathol 2010 5 51 20687934 Kandemir NO Barut F Ekinci T Karagülle C Ozdamar SO Intranodal palisaded myofibroblastoma (intranodal hemorrhagic spindle cell tumor with amianthoid fibers): a case report and literature review Diagn Pathol 2010 5 12 10.1186/1746-1596-5-12 20181136 Qiao J Patel KU López-Terrada D Fang H Atrophic dermatofibrosarcoma protuberans: report of a case demonstrated by detecting COL1A1-PDGFB rearrangement Diagn Pathol 2012 7 166 10.1186/1746-1596-7-166 23199263 Ohno J Iwahashi T Ozasa R Okamura K Taniguchi K Solitary neurofibroma of the gingiva with prominent differentiation of Meissner bodies: a case report Diagn Pathol 2010 5 61 10.1186/1746-1596-5-61 20858283 Shaktawat SS Golka D Floret-like multinucleated giant cells in neurofibroma Diagn Pathol 2007 2 47 10.1186/1746-1596-2-47 18067673 Scheithauer BW Woodruff JM Erlandson RA Tumors of the peripheral Nervous system Atlas of Tumor Pathology 1999 3 Washington, DC: Armed Forces Institute of Pathology 303 369 fascicle 24 Koestner A Higgins RJ Meuten DJ Tumors of the nervous system Tumors in domestic animals 2002 4 Ames, IA: Iowa State Press 717 723 Saunders MO Tumors of neural origin Equine Dermatology 2003 733 735 Weiss SW Goldblum JR Weiss SW, Goldblum JR Benign tumors of peripheral nerves Enzinger and Weiss's Soft Tissue Tumors 2008 5 Philadelphia, PA: Mosby Elsevier 825 902 Meuten DJ Tumors of the skin and soft tissues Tumors in Domestic Animals 2002 4 Ames, IA: Iowa State Press 45 117 Meuten DJ Tumors of the nervous system Tumors in Domestic Animals 2002 4 Ames, IA: Iowa State Press 697 737 Nielsen AB Jensen HE Leifsson PS Immunohistochemistry for 2',3'-cyclic nucleotide-3'-phosphohydrolase in 63 dog peripheral nerve sheath tumors Vet Pathol 2011 48 796 802 10.1177/0300985810388521 21123863 Murcia PR Delhon G González MJ Vilas M Ramos-Vara JA De Las Heras M Nordhausen RW Uzal FA Cluster of cases of malignant schwannoma in dog Vet Rec 2008 163 331 335 10.1136/vr.163.11.331 18791208 Yamada M Nakagawa M Yamamoto M Furuoka H Matsui T Taniyama H Histopathological and immunohistochemical studies of intracranial nervous-system tumors in four dog J Comp Pathol 1998 119 75 82 10.1016/S0021-9975(98)80073-X 9717129 Mandrioli L Gentile A Morini M Bettini G Marcato PS Malignant, solitary, nasopharyngeal schwannoma in a dog Vet Rec 2005 156 552 553 15849348 Kim D-Y Cho D-Y Kim DY Lee J Taylor HW Malignant peripheral nerve sheath tumor with divergent mesenchymal differentiations in a dog J Vet Diagn Invest 2003 15 174 178 10.1177/104063870301500214 12661730 Beytut E Multicentric malignant schwannoma in a crossbred cow J Comp Path 2006 34 260 265 16615938 Peek SF Del Piero F Rebhun WC Adamus C Multicentric schwannomas causing chronic ruminal tympany and forelimb paresis in a Holstein cow Vet Rec 1997 140 504 505 10.1136/vr.140.19.504 9172298 Omi K Kitano Y Agawa H Kadota K An immunohistochemical study of peripheral neuroblastoma, ganglioneuroblastoma, anaplastic ganglioglioma, schwannoma and neurofibroma in cattle J Comp Pathol 1994 111 1 14 10.1016/S0021-9975(05)80106-9 7962722 Santin EA Herrea GA Wilkins LP Wolfe DF Metastatic multicentric neurofibrosacoma of the lumbosacral plexus in a cow Vet Pathol 1996 33 362 365 10.1177/030098589603300319 8740716 Johnson RC Anderson WI Luther PB Ryan AM Multicentric schwannoma in a mature Holstein cow Vet Rec 1988 123 649 650 2464232 Lee RM Ong CP Jacobsen AS Chan MY Hwang WS Malignant peripheral nerve sheath tumor mimicking carotid body tumor–case report and review J Pediatr Surg 2011 46 3 554 8 10.1016/j.jpedsurg.2010.11.029 21376209 Ziadi A Saliba I Malignant peripheral nerve sheath tumor of intracranial nerve: A case series review Auris Nasus Larynx 2010 37 5 539 45 10.1016/j.anl.2010.02.009 20399579 Gulati N Rekhi B Suryavanshi P Jambhekar NA Epithelioid malignant peripheral nerve sheath tumor of the uterine corpus Ann Diagn Pathol 2011 15 6 441 5 10.1016/j.anndiagpath.2010.06.002 20952276 Abe S Imamura T Park P Nakano H Okita H Hata J Takeishi A Small round-cell type of malignant peripheral nerve sheath tumor Mod Pathol 1998 11 747 753 9720503 Woodruff JM Selig AM Crowley K Allen PW Schwannoma (neurilemmoma) with malignant transformation. A rare, distinctive peripheral nerve tumor Am J Surg Pathol 1994 18 882 895 10.1097/00000478-199409000-00003 8067509 Bergmann W Burgener IA Roccabianca P Rytz U Welle M Primary splenic peripheral nerve sheath tumour in a dog J Comp Path 2009 141 2–3 195 8 19477462 Chijiwa K Uchida K Tateyama S Immunohistochemical evaluation of canine peripheral nerve sheath tumors and other soft tissue sarcomas Vet Pathol 2004 41 307 318 10.1354/vp.41-4-307 15232130	23688209	PMC3699426	CC BY	2021-01-05 01:30:48	yes	Diagn Pathol. 2013 May 20; 8:86
==== Front SpringerplusSpringerplusSpringerPlus2193-1801Springer International Publishing Cham 2385375235410.1186/2193-1801-2-278ResearchRETRACTED ARTICLE: Improved treatment of Asthma by using natural sources of antioxidants Van Toan Nguyen nvtoan@hcmiu.edu.vn 12Hanh Tran Thi ydaiphu@gmail.com 31 School of Biotechnology, International University, Ho Chi Minh City, Vietnam 2 Faculty of Applied sciences, University of the West of England, Bristol, UK 3 Home Clinic, 345 D5 Street, Binh Thanh District, Ho Chi Minh City, Vietnam 26 6 2013 26 6 2013 2013 2 1 27825 5 2013 24 6 2013 © Van Toan and Thi Hanh; licensee Springer. 2013This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Clinical Trials with ACTR Number ACTRN12612000766819 Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-2-278) contains supplementary material, which is available to authorized users. Keywords AsthmaBronchial asthmaNatural antioxidantsPulmonary functionsissue-copyright-statement© The Author(s) 2013 ==== Body Introduction Asthma is a chronic disease characterized by inflammation of the airways (Buse and Lemanske 2001), a complex disorder characterized by variable and recurring symptoms, airflow obstruction, bronchial hyperresponsiveness, and an underlying inflammation. The interaction of these features determines the clinical manifestations and severity of asthma, and it has been reported as a disease of increasing prevalence (Expert Panel Report 3: Guidelines for the diagnosis and management of asthma. NIH Publication No. 07–4051. 2007). The pathogenesis of asthma is unknown but imbalances between oxidants and antioxidants are believed to play a fundamental role. One key component of the oxidant-antioxidant hypothesis centers on the huge burden of oxidants derived from inflammatory cell infiltration into the lung. The eosinophil, in particular, is implicated as a major source of oxidative injury, including protein nitration (MacPherson et al. 2011). Dysfunctional mitochondria in lung cells are another potential source of oxidants. Mitochondrial injury to airway epithelium occurs in murine models of allergic asthma (Aguilera-Aguirre et al. 2009; Mabalirajan et al. 2008). There is evidence to support its role in human asthma as well, including increased oxidative injury to mitochondrial epithelial cell superoxide dismutase (SOD)( Comhair et al. 2005), enhanced mitochondrial proliferation in bronchial smooth muscle (Trian et al. 2007), and mutations in mitochondrial DNA (Reddy 2011). Overall, this oxidative burden, generated by both inflammatory and lung cells, can overwhelm antioxidant defense to cause oxidant stress during asthma. Thisk stress can alter or inactivate the function of essential proteins, lipids and nucleic acids culminating in severe cell injury, dysfunction and death. Among many unknown and complicated mechanisms, involvement of airways inflammation with an oxidant/antioxidant imbalance such as reactive oxygen species (ROS) can lead to lung injury as a result of direct oxidative damage to epithelial cells and cells shedding. As inflammation is often associated with an increased generation of reactive oxygen species (ROS), it is rational to surmise that an oxidant stress could be mechanistically important in asthma. ROS have been shown to be associated with the pathogenesis of asthma by inducing bronchial hyperreactivity as well as directly stimulating histamine release from mast cells and mucus secretion from airway epithelial cells (Ryszard 2000). The great external surface area (1–2) m2 of the human airway epithelium plus its direct contact with the environment, makes the respiratory tract a major target for oxidative injury from inhaled oxidants such as cigarette smoke, ozone, hyperoxia, nitrogen and sulphur oxides and other airborne pollutants. It has been well recognized that biological systems are capable of forming highly reactive moieties, both free radicals and non-radicals named reactive oxygen species (ROS) and reactive nitrogen species (RNS). Free radicals can especially be generated in a wide variety of chemical and biological systems, including the formation of plastics, the ageing of paints, the combustion of fuels and in the human body. In living organisms, the levels of free radicals and other ‘reactive species’ are controlled by a complex web of antioxidant defences, which minimize (but do not completely prevent) oxidative damage to biomolecules (Roberfroid and Calderon 1995; Gaston et al. 1994; Halliwell (2005). These biologically active species serve in cell signaling as messenger molecules of the autocrine or paracrine system (Saran and Bors 1989; Suzuki et al. 1997 ) and also in host defense, (biocidal effects against microbial and tumor cells) (Babior 1978) but their excessive production may result in tissue injury and inflammation (Halliwell et al. 1992; Gutteridge and Halliwell 1994). Reportedly, any excessive production of oxidants is kept to a minimum by a well coordinated and efficient endogenous antioxidant defense mechanism. It has been proposed that a deficit in the precise balance between exposure to oxidants and endogenous antioxidants results in oxidative stress which appears to be involved in the pathogenesis of a growing number of diseases, including lung pathologies such as respiratory distress syndrome, asthma, idiopathic and iatrogenic pulmonary fibrosis, cystic fibrosis, HIV-associated lung disease, lung cancer and other pulmonary diseases and conditions (Clement and Housset 1996; Barnes 1995) . As excessive ROS levels damage lipids, proteins and nucleic acids through oxidation and thus are associated with various diseases, such as atherosclerosis, arthritis, neurodegenerative disorders, and cancers , a regular supplement of antioxidants can assist the endogenous defense systems to counterbalance the harmful effects of excessive ROS (Balsano and Alisi 2009; Kaur and Geetha 2006). There has recently been a remarkable increment in scientific articles dealing with oxidative stress (Urguiaga and Leighton 2000). Consequently, knowledge about reactive oxygen and nitrogen species metabolism; definition of markers for oxidative damage; evidence linking chronic diseases and oxidative stress; identification of flavonoids and other dietary polyphenol antioxidants present in plant foods as bioactive molecules; and data supporting the idea that health benefits associated with fruits, vegetables in the diet are probably linked to the polyphenol antioxidants they contain. In addition, more than 8,000 polyphenolic compounds have been identified in various plant species and reported to possess many useful properties including antiallergic, antiinflammatory, antimicrobial, antiviral, antioxidant, oestrogenic, enzyme inhibition, vascular and cytotoxic anti- tumor activity (Pandey and Rizvi 2009; Asha et al. 2012). According to Ji-Xiao Zhu et al., among many other contained phenolic compounds plants, Rhizoma Homalomenae has been shown to be positive linear correlations between total phenolic content and antioxidant activity of the extracts in the DPPH (R = 0.9817), ABTS (R = 0.9873), b-carotene/linoleic acid (R = 0.8347) and reducing power (R = 0.9876) tests, respectively. Another source of natural antioxidants is naturally occurring from traditional Chinese medicines sources, have been identified as free radical or active oxygen scavengers (Duh 1998; Pan et al. 2007). Natural antioxidants can protect the human body from free radicals and retard the progress of many chronic diseases as well as retard lipid oxidative rancidity in foods or medicinal materials (Kang et al. 2008). Among the many mentioned sources of naturally occurring antioxidant, Seahorse (Hippocampus kuda Bleeker) has been well known for its special medicinal composition. According to Zhong-Ji Qian et al., the methanol extracts of seahorse contained high amount of phenolic compounds and those extracts exhibited good antioxidant activity by effectively scavenging various free radicals such as DPPH radicals, hydroxyl radicals, superoxide anion radicals, alkyl radicals, and reducing the ferric to ferrous ion in different antioxidant systems. In a search for treatment that might stop the recurrent attacks of breathlessness and wheezing to make it more susceptible to at least, providing relief for asthmatic patients, and if possible to treat the asthmatic disease, a method has emerged that seems to be extremely useful for application of natural sources of antioxidants for treatment of asthma. The method was using finely extracted powders from the seahorse (Hippocampus kuda) and Rhizoma Homalomenae (with a ratio of 1: 1 w/w) in honey to form into pill of 500mg. All the hand-rolled pills were dried in an oven at 55°C until the moisture content of the pill was consistent. In this paper, successful application of extracted powders from the seahorse (Hippocampus kuda) and Rhizoma Homalomenae together with honey in the form of medical pill for treatment of asthma is reported. Materials and methods Preparation of extracts from seahorse (Hippocampus kuda) The antioxidant extracts were prepared by adopting the method of Zhong-Ji Qian to check the total phenolic content and determine the total antioxidant activity. Preparation of extracts from Rhizoma Homalomenae The plant materials of Rhizoma Homalomenae used in this study was donated by DUC- HUNG- a traditional medicine shop in Nhatrang City- a central part of Vietnam. The dried materials were ground to the fine powder and passed through a 20-mesh sieve for the preparation of extracts.The sieved powder was subjected to water distillation for 5 hrs by adopting the method of Zeng et al. 2011. Preparation of medicinal pills In this study, a dosage of 500mg pill was prepared and named as BRONAS, and described as below: BRONAS was prepared from 200 mg of dried extract powder of Hippocampus kuda, 200 mg of dried extract powder of Rhizoma Homalomenae* and 130 mg of honey**, and then hand – rolled into pills of around 500 mg, each. Notes The moisture content of the dried extract powder of Hippocampus kuda was 3%. The moisture content of the dried extract powder of Rhizoma Homalomenaee was 3%. The moisture content of the used honey was 17%. All the hand- rolled pills were dried in an oven at 55°C for 34 to 46 hours. Moisture content of the pill was determined by the standard AOAC method (AOAC 2000) Determination of total antioxidant activity of BRONAS The antioxidant activity of BRONAS was determined right after drying the pills to the consistent moisture content. The antioxidant potential of BRONAS was determined on scavenging activity of the DPPH Free- Radical by adopting the method described by Sadhu et al. (2003). The maximum absorption (λ max) of a stable DPPH in methanol is 520 nm and the results are expressed as IC50 values. The percent inhibition, radical scavenging capacity was calculated using the following equation: DPPHscavenged%=Acontrol−AsampleAcontrolx100 Where: A control = Absorbance of DPPH alone. A sample = Absorbance of DPPH along with different concentrations of extracted sample. IC50 was calculated from the slope obtained by plotting a graph of concentration versus % inhibition. According to Molyneux P (Molyneux 2004) DPPH is the molecule of 1,1-diphenyl-2-picrylhydrazyl (α, α-diphenyl β picrylhydrazyl) characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that the molecules do not dimerize, as would be the case with most other free radicals. The delocalization also gives rise to the deep violet colour, characterized by an absorption band in ethanol solution centered at about 520 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form. With the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present). Representing the DPPH radical by Z and the donor molecule by AH, the primary reaction is Z* + AH = ZH + A* Where: ZH is the reduced form A* is free radical produced in this first step. The latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorized) by one molecule of the reductant. In this study, stock solutions of the BRONAS sample were used for preparing various concentrations of 200, 400, 600, 800 and 1000 (mg/L). Patients This study was performed at the Home Clinic, 345 D5 Street, Binh Thanh District of Ho Chi Minh city, Vietnam between September 2011 to early April 2013. Ninety two asthmatic patients (55% males and 45% females) who come from different parts of Vietnam, were randomly selected and included in the study. Their age range was 12–65 years. All Patients were desperately suffering from disease of asthma and all of them were many times hospitalized and measured value of FEV1/FVC were from 50% to 60%, of which the shortest disease history was 3 years and the longest was 11 years. It is worth noting here that most of the participating patients experienced a set of clinical symptoms which result in the sensation of difficulty breathing such as Spasm of the bronchial muscles and shortness of breathing, cough, expectoration, and prolonged expiratory phase with wheezing. In addition, all of the participating patients were those, who had ever used high doses of inhaled corticosteroids and particularly long acting inhaled β2-adrenergic agonists as the relief of bronchial constriction to reduce the asthmatic episodes. , adopting the method of Faruk et al. 2010 for checking possible effects of the given medicines on the treated patients. By applying this, a total computerized spirometer (Discom-14 Autospiror, Chest Corporation Tokyo, Japan) that measures Forced Volume Capacity (FVC), Forced Expiratory Volume in First Second (FEV1), Peek Expiratory Flow Rate (PEFR), and Maximal Mid-expiratory Flow Rate (MMEFR) was extensively applied as it would provide predicted values. The patients were then randomly allocated to 2 groups of A and B. The group A of 40 (18 Males: 22 Females) patients was given PREDISONE with a single dose of 10 mg/kg of body weight, daily for each 21 days and stopped for pulmonary function check. The group B (24 Male: 28 Female) was given BRONAS with a single dose of 1200 mg/kg of body weight, daily for 21 days and stopped for pulmonary function check as well. The group A was then given the same dose of BRONAS as to the group B for 21 days for further comparing the anti-inflammatory effect of BRONAS with the anti-inflammatory effect of PREDISONE. Data analysis All data are expressed as means ± standard deviation representative of similar tests carried out in triplicate. Statistical differences were determined by student’s t-test in which, p<0.05 was considered as statistically significant. Ethics All participated patients gave their consent prior to participation in the study. Results Data on total antioxidant activity of BRONAS were shown in the Table 1 and Figure 1. The values obtained were also shown that the DPPH scavenging effect in percentage of BRONAS were three times higher than those extracted from green tea using the same method of extraction (Yen and Chen 1995)Figure 1 The DPPH free radical scavenging of the BRONAS sample. Table 1 OD values after being measured in triplicate Blank 200 mg/L 400 mg/L 600 mg/L 800 mg/L 1000 mg/L 0.53 0.40 0.38 0.28 0.24 0.19 0.51 0.41 0.33 0.25 0.22 0.10 0.53 0.42 0.37 0.30 0.23 0.21 Mean 0.52 0.41 0.36 0.28 0.23 0.20 SD 0.01 0.01 0.02 0.03 0.01 0. 01 CV (%) 2.10 1.72 6.30 9.54 5.51 4.30 DPPH (%) 0 22.91 32.21 47.32 56.62 62.70 The Forced Volume Capacity (FVC), Forced Expiratory Volume in First Second (FEV1), Peek Expiratory Flow Rate (PEFR), and Maximal Mid-expiratory Flow Rate (MMEFR) were extensively applied for determining the response to the therapy and monitoring the rate of progression. The results have been summarized and shown in the Table 2 and Figure 2.Table 2 The effects of BRONAS and PREDISONE on pulmonary functions test Drug FVC % FEV1 % MMEFR % PEFR Before After Before After Before After Before After PREDISONE 63.45 ± 11.94 65.55 ± 12. 50 51.14 ± 11.63 54.78 ± 12.20 50.28 ± 22.45 51.72 ± 11.60 2.96 ± 1.34 3.57 ± 2.32 BRONAS 61.38 ± 15.60 69.35 ± 5. 32 55.39 ± 15.63 65.32 ± 5.16 53.28 ± 22.45 62.05 ± 3.60 3.26 ± 1.54 5.94 ± 2.50 Results were significant at (p<0.05) Figure 2 Percentage increment in FVC, FEV 1 , MMEFR after treatment with PREDNISONE and BRONAS. The ratio of FEV1 to FVC (v/v) after treatment was calculated using the formula: FEV1aftertreatmentFVCaftertreatment=Valueinpercentage The ratio of FEV1 to FVC (v/v) after 21 days of treatment with PREDNISONE was 84.50%. The ratio of FEV1 to FVC (v/v) after treatment with BRONAS was 94%. As the ratio of FEV1 to FVC (v/v) after 21 days of treatment with PREDNISONE was improved but lower (84.50%) than the one obtained from the treatment with BRONAS (94%) in the same period of treatment process, the asthmatic patients in Group A was then continuously given BRONAS with a single dose of 1200 mg/kg daily for another 21 days of treatment. The collected data in this stage of treatment has been summarized and shown in the Table 3 and Figure 3.Table 3 The effects of BRONAS on pulmonary functions test in the post treatment of PREDISONE Drug FVC % FEV1% MMEFR % PEFR Before After Before After Before After Before After BRONAS 65.63 ± 12.44 * 73.80 ± 9.67 62.34 ± 14.21 * 71.95 ± 10.18 52.34 ± 19.16 * 60. 92 ± 11.06 3.15 ± 1.75 * 5.86 ± 2.40 *Results were significant at (p<0.05) Figure 3 Percentage increment in FVC, FEV 1 , MMEFR in the post treatment of PREDISONE with BRONAS. After all the all ninety two patients having had treatment with BRONAS as given dose in the treating process mentioned above, sets of pulmonary functions test were conducted and found that till the 83th day of treatment, all the asthmatic patients were fully or almost fully recovered. The result of pulmonary functions test has been summarized and shown in Figure 4.Figure 4 Percentage increment in FVC, FEV 1 , MMEFR in the 83 rd treatment with BRONAS. This statement has been consolidated by the fact that no clinical symptoms of the severe asthmatic disease such as Spasm of the bronchial muscles and shortness of breathing, cough, expectoration, and prolonged expiratory phase with wheezing were seen again. Discussion From the Table 2, it could be reasoned that there was no significant increase in FVC%, MMEFR% and PEFR but only FEV1% by using single PREDNISONE in group A. In addition, the ratio of FEV1 to FVC (v/v) after 21 days of treatment was 84.50%. In group B, a significant improvement in Pulmonary Function Tests associated with improvement in patients’ clinical conditions was observed. After taking the BRONAS for the same period of treatment, the ratio of FEV1 to FVC (v/v) was pretty high (94%). Data collected from the oral administration of BRONAS of patients in group A, of which shown in Table 3 and Figure 4 would again support and explain the beneficial effect of taking BRONAS in reducing the incidence of asthma when the ratio of FEV1 to FVC (v/v) was around 97.50%. FEV1 has been found to be a better physiologic index than PEFR in the measurement of airflow obstruction (Christophe et al. 1998). So in this study, the values of PEFR were used as a reference to possibly support and/or explain the beneficial effect of orally administering the prescribed and prepared medical tablet/ pill in reducing, even treating the incidence of asthma. The strong antioxidant properties of the BRONAS may be attributed to the presence of the combined bioactive antioxidant compounds from the extract of Hippocampus kuda and those of Rhizoma Homalomenae. Based on the report of Zhong et al. (2008), the extracted antioxidants from Hippocampus kuda contained effective reducing power, DPPH radical scavenging, hydroxyl radical scavenging, superoxide radical scavenging, alkyl radical scavenging and inhibitory intracellular ROS. Zheng and Wang (2001) reported that the strong antioxidant properties of the Rhizoma Homalomenae extracts may be due to the presence of phenolics compounds such as protocatechuic acid, vanillic acid, syringic acid, caffeic acid, p-coumaric acid, ferulic acid and apigenin. Phenolic acids are the main phenols consumed by humans (Ghasemzadeh and Ghasemzadeh 2011). The six phenolics mentioned above are all prominent and naturally occurring, and all of them individually possess potent antioxidant activity (3 Joskova et al. 2013; Gao et al. 2012). The honey was intentionally used with the dried extract powder of Rhizoma Homalomenae and that of Hippocampus kuda since it has been scientifically considered as an antimicrobial agent and chelating in traditional medicine. In addition, there is a great variety of minor components, including phenolic acids and flavonoids (Natella et al. 1999; Joskova et al. 2013), the enzyme glucose oxidase, ascorbic acid, carotenoids, organic acids, amino acids, proteins and α-tocopherol (Kesic et al. 2009). By and large, the results obtained from this study showed that BRONAS can protect cellular components from many types of oxidative stress by both free radical scavenging mechanism and stabilizing the cell membranes and this may explain the improvement that occurred in both dynamic performance of the lung in moving air and the elastic recoil force of the lung, especially when the ratio of FEV1 to FVC reached at least 94%. Conclusion The study demonstrates that the combined antioxidants from extracted powders of Hippocampus kuda and Rhizoma Homalomenae together with honey in a form of medical pill (BRONAS) can help improve the treatment of asthma. BRONAS can be used to replace synthetic antioxidants, which are being restricted due to their side effects such as carcinogenicity. In addition, BRONAS can be prepared at a lower cost and in a more friendly way. Ethical approval The ACTRN12612000766819 was the Retrospectively registered and approved by Australian New Zealand Clinical Registry. Authors’ original submitted files for images Below are the links to the authors’ original submitted files for images.Authors’ original file for figure 1 Authors’ original file for figure 2 Authors’ original file for figure 3 Authors’ original file for figure 4 The original version of this paper (Van Toan and Thi Hanh 2013) is retracted because of ethical concerns: the clinical trial was not approved by an ethical board and the authors did not provide evidence that patient consent was obtained. The scientific advisor for this clinical trial (ANZCTR 2012) at the University of the West of England UK (an affiliation of the corresponding author) indicated he was not aware of this study and that the university was not involved. It is a requirement that experimental research reported in SpringerPlus was performed with the approval of an appropriate ethics committee, and that research carried out on humans must be in compliance with the guidelines of the World Medical Association. Dr. Max Haring, Executive Editor for SpringerPlus An erratum to this article can be found online at 10.1186/2193-1801-3-558 A retraction note to this article can be found online at http://dx.doi.org/10.1186/2193-1801-3-558. Competing interest The authors declare that they have no competing interest. Authors’ contribution NVT has been responsible for the all technical matters, scientific issues/values and the manuscript preparation. TTH has been responsible for patients recruitment and proof reading. Both authors read and approved the final manuscript. Acknowledgements The authors would like to express their sincere thanks to the Nguyen’s family’s research funds. They would like to extend their thanks to the Buddhist Priest Thich Giac Lam from Dat Nhau ward, Bu Dang District, Binh Phuoc Province, Vietnam for the supply of raw materials of Rhizoma Homalomenae. ==== Refs References AOAC Determination of moisture content 2000 Washington, DC Association of Official Analytical Chemists Aguilera-Aguirre L Mitochondrial dysfunction increases allergic airway inflammation J Immunol 2009 183 8 5379 5387 10.4049/jimmunol.0900228 19786549 Asha KK Suseela M Lakshmaman PT Flavonoids and phenolic compounds in two mangrove species and their antioxidant property Indian J Geo Mar Sci 2012 41 3 259 264 Babior BM Oxygen-dependent microbial killing by phagocytes N Engl J Med 1978 298 658 9 Balsano C Alisi A Antioxidant effects of natural bioactive compounds Curr Pharm Des 2009 15 3063 3073 10.2174/138161209789058084 19754380 Barnes PJ Nitric oxide and airway disease Ann Med 1995 27 389 93 10.3109/07853899509002592 7546629 Buse WW Lemanske RF Jr Asthma N Engl J Med 2001 344 350 362 10.1056/NEJM200102013440507 11172168 Christophe L Luca P Carole T Comparison of Serial Monitoring of Peak Expiratory Flow and FEV1 in the Diagnosis of Occupational Asthma Am J Respir Crit Care Med 1998 158 3 827 832 10.1164/ajrccm.158.3.9707093 9731012 Clement A Housset B Chretien J Dusser D Role of free radicals in airway injury Environmental impact on the airways 1996 New York Dekker 355 79 Comhair SA Xu W Ghosh S Superoxide dismutase inactivation in pathophysiology of asthmatic airway remodeling and reactivity Am J Pathol 2005 166 3 663 674 10.1016/S0002-9440(10)62288-2 15743779 Duh PD Antioxidant activity of burdock (Arctium lappa Linné): Its scavenging effect on free-radical and active oxygen J Am Oil Chem Soc 1998 5 455 461 10.1007/s11746-998-0248-8 Faruk H Jawad A Hayder G Usefulness of antioxidant drugs in Bronchial asthma Pak J Physiol 2010 6 2 18 21 Gao F Wei D Bian T Genistein attenuated allergic airway inflammation by modulating the transcription factors T-bet, GATA-3 and STAT-6 in a murine model of asthma Pharmacology 2012 89 3–4 229 36 10.1159/000337180 22508471 Gaston B Drazen JM Loscalzo J The biology of nitrogen oxides in the airways Am J Respir Crit Care Med 1994 149 538 51 10.1164/ajrccm.149.2.7508323 7508323 Ghasemzadeh A Ghasemzadeh N Flavonoids and phenolic acids: Role and biochemical activity in plants and human J Med Plants Res 2011 5 31 6697 6703 Gutteridge JMC Halliwell B Antioxidants in nutrition, health and disease 1994 Oxford Oxford University Press Halliwell B Gutteridge JMC Cross CE Free radicals, antioxidants and human disease: where are we now? J Lab Clin Med 1992 119 598 620 1593209 Halliwell B Free radicals and other reactive species in disease eLS 2005 Chichester John Wiley Joskova M Sadlonova V Nosalova G Polyphenols and their components in experimental allergic asthma Adv Exp Med Biol 2013 756 91 8 10.1007/978-94-007-4549-0_12 22836623 Kang KS Kim ID Kwon RH Undaria pinnatifida fucoidan extract protects against CCl4-induced oxidative stress. Biotechnol Bioprocess Eng 2008 13 168 173 10.1007/s12257-007-0101-1 Kaur IP Geetha T Screening methods for antioxidants-a review Mini Rev Med Chem 2006 6 3 305 312 10.2174/138955706776073448 16515469 Kesic A Mazolovic M Crnkic A The Influence of L-Ascorbic Acid Content on Total Antioxidant Activity of Bee-Honey Eur J Sci Res 2009 32 1 95 101 Mabalirajan U Dinda AK Kumar S Mitochondrial structural changes and dysfunction are associated with experimental allergic asthma J Immunol 2008 181 5 3540 3548 10.4049/jimmunol.181.5.3540 18714027 MacPherson JC Comhair SA Erzurum SC Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species J Immunol 2011 166 9 5763 5772 10.4049/jimmunol.166.9.5763 11313420 Molyneux P The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating an antioxidant activity Songklanakarin J Sci Technol 2004 26 2 211 219 Natella F Nardini M Felice MD Scaccini C Benzoic and cinnamic acid derivatives as antioxidants: structure–activity relation J Agric Food Chem 1999 47 1453 1459 10.1021/jf980737w 10563998 Pan Y Zhu J Wang H Antioxidant activity of ethanolic extract of Cortex fraxini and use in peanut oil Food Chem 2007 103 913 918 10.1016/j.foodchem.2006.09.044 Pandey KB Rizvi SI Plant polyphenols as dietary antioxidants in human health and disease Oxid Med Cell Longev 2009 2 5 270 278 10.4161/oxim.2.5.9498 20716914 Reddy PH Mitochondrial Dysfunction and Oxidative Stress in Asthma: Implications for Mitochondria-Targeted Antioxidant Therapeutics Pharmaceuticals (Basel) 2011 4 3 429 456 10.3390/ph4030429 21461182 Roberfroid M Calderon PB Roberfroid M Calderon PB Biologically relevant radicals Free radicals and oxidation phenomena in biological systems 1995 New York Marcel Dekker Inc 33 90 Ryszard D Oxidant stress in asthma Thorax 2000 55 Suppl 2 S51 S53 Sadhu SK Separation of Leucas aspera, a medicinal plant of Bangladesh, guided by prostaglandin inhibitory and antioxidant activities Chem Pharm Bull 2003 51 595 98 10.1248/cpb.51.595 12736464 Saran M Bors M Oxygen radicals acting as chemical messenger: a hypothesis Free Rad Res Commun 1989 7 3–6 213 20 10.3109/10715768909087944 Suzuki YJ Forman HJ Sevanian A Oxidants as stimulators of signal transduction Free Rad Biol Med 1997 22 269 85 10.1016/S0891-5849(96)00275-4 8958153 Trian T Benard G Begueret H Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma J Exp Med 2007 204 13 3173 3181 10.1084/jem.20070956 18056286 Urguiaga I Leighton F Plant polyphenol antioxidants and oxidative stress Biol Res 2000 33 55 64 15693271 Yen GC Chen HY Antioxidant Activity of Various Tea Extracts in Relation to Their Antimut agenicity J. Agric Food Chem 1995 43 27 32 10.1021/jf00049a007 Zeng LB Antioxidant activity and chemical constituents of essential oil and extracts of Rhizoma Homalomenae Food Chem 2011 125 456 463 10.1016/j.foodchem.2010.09.029 Zheng W Wang SY Antioxidant activity and phenolic compounds in selected herbs J Agric Food Chem 2001 49 5165 5170 10.1021/jf010697n 11714298 Zhong JQ Free Radical and Reactive Oxygen Species Scavenging Activities of the Extracts from Seahorse, Hippocampus kuda Bleeler Biotechnol Bioprocess Eng 2008 13 705 715 10.1007/s12257-008-0093-5	23853752	PMC3701788	CC BY	2021-01-05 07:02:09	yes	Springerplus. 2013 Jun 26; 2(1):278
==== Front Diagn PatholDiagn PatholDiagnostic Pathology1746-1596BioMed Central 1746-1596-8-982377753710.1186/1746-1596-8-98Case ReportCervical type AB thymoma (Mixed) tumour diagnosis in a mynah as a model to study human: clinicohistological, immunohistochemical and cytohistopathological study Khaki Fariba 1khakifariba@ut.ac.irJavanbakht Javad 1javadjavanbakht@ut.ac.irSasani Farhang 1fsasani@ut.ac.irGharagozlou Mohammad Javad 1mjavad@ut.ac.irBahrami Alimohammad 2am.bahrami@ilam.ac.irMoslemzadeh Hemmat 3hemmat_m_1361@yahoo.comSheikhzadeh Reza 4korzan2008@gmail.com1 Department of Pathology, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran2 Faculty of Para-Veterinary Medicine, University of Ilam, Ilam, Iran3 Graduate, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran4 Faculty of Veterinary Medicine, Teharn University, Tehran, Iran2013 18 6 2013 8 98 98 20 5 2013 3 6 2013 Copyright © 2013 Khaki et al.; licensee BioMed Central Ltd.2013Khaki et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Abstract Thymoma is a primary mediastinal neoplasm arising from or exhibiting differentiation towards thymic epithelial cells, typically with the presence of non-neoplastic lymphocytes. A 13-year-old male Mynah bird (acridotheres tristis) was presented for evaluation of a 2.3 × 1.5 × 1.0 cm mass in the left ventrolateral cervical region. The clinical signs, radiology, cytohistopathology and immunohistochimy findings related to the thymoma are presented. These findings indicated that the tumor was a type AB thymoma according to the World Health Organization (WHO) and veterinary classification. Thymomas are rarely reported in avian species and this is the first report in a Mynah bird. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1159525819982779. ThymomaLymphocytic typeDiagnosisMynah,Neck ==== Body Background Thymomas are rare malignancies both in human and veterinary medicine. They have been described in a wide range of domestic and laboratory animals, such as rats and mice [1,2], and occur more frequently in older animals. In humans, thymomas usually present in the fourth to fifth decade but can occur at all ages without sex predisposition [3]. In dogs and cats, thymomas are also found in older animals [4], and they are rarely reported in avian species and have only been documented in an African grey parrot (Psittacus erithacus),[5] a finch (unspecified species), [6] domestic chickens,[7,8] a budgerigar (Melopsittacus undulatus), [9] and a Java sparrow (Padda oryzivora) [10]. The etiology of thymomas is not yet understood. In contrast to lymphomas, there is no evidence of a etiology of thymomas in humans and domestic animals. A single report exists that describes a family with high incidence of thymomas and might point to a genetic predisposition [11]. Thymomas are derived from thymic epithelium with variable benign lymphocytic infiltration and thus are classified as lymphocyte-predominant, epithelial-predominant, or mixed [12,13]. Type AB thymoma (also known as mixed thymoma) accounts for approximately 28% to 34% of all thymomas [14,15]. Approximately 16% of this type may be associated with myasthenia gravis [14]. Morphologically, type AB thymoma is a thymic tumor in which foci having the features of type A thymoma are admixed with foci rich in nonneoplastic lymphocytes.[16]. Histologically, these tumors are composed of neoplastic thymic epithelial cells and a variable number of lymphocytes leading to a myriad of histologic subtypes depending on the proportion of these elements [17]. This report describes the clinical signs, cytopathological and immunohistichemical characteristics of a thymoma in a Mynah bird and because there is no established classification of thymic neoplasms in veterinary medicine and the avian thymomas bore a striking resemblance to their human counterparts, an attempt to classify this neoplasm according to the new World Health Organization (WHO) classification of human thymic epithelial tumors was made. Case presentation In April 2013, a 13-year-old adult male red-tail common Mynah bird (Acridotheres tristis) was referred to the Avian and Exotics Service of the Tehran Veterinary College, University of Tehran, for evaluation of a firm, round and circular neck mass, off-white to gray, lightly encapsulated, moveable, attached to the underlying tissue and measured 2.3 × 1.5 × 1.0 cm diameter was palpated on the left ventrolateral side of the neck near the thoracic inlet. No evidence of pain or discomfort was noticed, and the bird did not experience any difficulty in prehension and swallowing its food or in breathing. It had been fed predominantly a seed-based and table-food diet. Its owner first noticed the mass 4 weeks before presentation and had progressively increased in size over that period. On gross examination, The bird had lost over 12% of its body weight over the 1 months, decreasing from 184 g to 162 g, because of the size of the mass and the bird's weight loss, and the bird appeared emaciated. On radiographs, degenerative changes in right tarsal joint and inflammation of its adjacent soft tissues accompanying mineralization of its external soft tissue were observed. In addition, hepatomegaly was recorded as well. A fine-needle aspirate of the mass was done, and a blood sample was collected for a complete blood cell count (CBC) and plasma biochemical analysis. Radiographs were taken, and the results showed that the mass was of soft-tissue density. Results of the plasma biochemical profile, including bile acids, were within reference ranges (Animal Health Laboratory, University of Tehran). The CBC results revealed a moderate monocytosis at 2.94 × 109 cells/L [2.94 × 103 cells/dL] (reference range for Amazon parrots: 0–0.4 × 109 cells/L [0–0.4 × 103 cells/dL], Animal Health Laboratory, University of Tehran). Aspiration of the mass revealed a cystic structure and results of cytologic examination of the fluid revealed large numbers of lymphocytes with occasional erythrocytes. The bird died during examination. Cytology this case demonstrated a mixture of round and oval cells. The cytoplasm was scanty and the cell borders were indistinct. Moderate to marked nuclear pleomorphism (Figure 1A and 1B) was noted in this case. These pleomorphic cells displayed anisonucleosis, irregular nuclear membranes, coarse clumping of chromatin, and prominent nucleoli. Mitoses ranged from scant to frequent. Figure 1 (A) Fine-needle aspiration of thymoma showing predominately round to oval cells with moderate nuclear atypia in type AB thymoma. Note thick nuclear membranes and more prominent nucleoli. (B): High power view of type AB thymoma .Marked pleomorphism characterized these cells. Few lymphocytes are present. A midline portion of the mass was processed routinely, embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin. For further study, paraffin sections were stained immunohistochemically with CK 14 and CK18 (Abcam Co., Cambridge, USA) for this case. For immunohistochemistry, sections from each tumor were mounted on adhesive-coated slides (Superfrost Plus, Menzel-Glaser, Braunschwaig, Germany), processed through xylene, and rehydrated in ethanol. Antigen retrieval was by boiling in a microwave oven (700 W) twice for 5 minutes in Tris–EDTA buffer—1.21 g Tris base (A 1379, Applichem, Darmstadt, Germany) and 0.372 g EDTA (8418, Merck, Darmstadt, Germany)—in 1 liter of distilled water, pH 9. Endogenous peroxidase was blocked with 0.6% (v/v) H2O2 in Tris-buffered saline (TBS; pH 7.6) for 15 minutes at 20°C before the monoclonal antibodies used included those for CK14 and CK18. WHO classification of human thymic epithelial tumors The classification of human thymic epithelial tumors has its origins in a histiogenetic classification originally proposed by Kirchner and Mueller-Hermelink and others [18-20]. The new WHO classification basically recognizes the same histologic entities but uses an alphanumerical system, i.e., combinations of letters and numbers. Thymomas with spindle, oval-shaped epithelial cells are designated type A, and those with small round epithelial components are designated type B. Tumors combining these features are designated type AB. Nonorganotypic thymic carcinomas are classified as type C thymomas. Type B is further subdivided into B1, B2, and B3 on the basis of increasing epithelial areas and the emergence of nuclear atypia [21,22]. Necropsy examination indicated a thin bird with a foul-smelling oral cavity. Dried serous discharge surrounded the incision. A large necrotic ulcer was present in the esophagus with food and caseous material in the subcutaneous space. No gross perforations of the esophagus were present. The intestines appeared necrotic. The resected mass was greatly expansible, but there was no evidence of local invasion or distant metastasis. Microscopic findings demonstrated that lobules of small polygonal cells with small round or oval vesicular nuclei and indistinct nucleoli accompanied by a variable amount of lymphocytes and the tumoral cells were circular or round and more comprised of lymphoblasts with extensive mitotic features together with and lymphocyte-poor spindle cells (Figure 2A,2B and 2D). The histiocytes with light nuclei and pink cytoplasms among tumoral lymphoid cells were observed (Figure 3D). The tissue was severely hyperemic and remarkable eosinophils existed among cells together with high number of undifferentiated neoplastic cells (Figure 2C).The necrosis was found in some parts with pyknosis and karyorrhexis. In vessels, the tumoral cells were seen. Numerous eosinophils in vessels indicated eosinophilia, (Figure 3C) and plenty of histiocytes contained hemosiderosis in the tumor. Figure 2 Mixed thymoma; A and B: the tumoral cells were circular or round and more comprised of lymphoblasts with extensive mitotic cells (H&E;20 μm×100), C: Hyperemic and remarkable eosinophils existed among cells (H&E;50 μm×400) D: Note type A and B components. (H&E;30 μm×200). Figure 3 Mix ed thymoma; Immunohistochemical staining mixed thymoma with, CK 14(A) and CK 18 (B) are positive for the round cells of the tumor. Hematoxylin and eosin staining of tissue and cells sections. (C): In vessels, the tumoral cells were seen. Numerous eosinophils in vessels indicated eosinophilia (H&E;20 μ×200). (D): The histiocytes with light nuclei and pink cytoplasms among tumoral lymphoid cells were observe. (H&E;25 μm×400). A microscopic examination showed the tumor to contain round shaped cells with a lymphocyte rich component. In the immunohistochemical study, the round cells of the tumor were all positive for CK 14 (Figure 3A) and CK18 (Figure 3B). On the basis of this case findings, tumour was classified as AB thymomas by analogy with the WHO classification for human neoplasms. Mixture of lymphocyte-associated small polygonal cells and lymphocyte-poor spindle cells areas in tumor correspond closely to AB areas in human tumuors. These cytohistopathology and immunohistochemical findings indicated that the tumor was a type AB thymoma according to the World Health Organization and veterinary medicine classification. Discussion Thymomas were tumours arising from or exhibiting differentiation toward thymic epithelial cells. It has been reported that different subtypes of thymoma have multifarious genetic characteristics, recent studies indicated that chromosomal 1 gain plays an significant role in molecular genetic mechanism of thymic epithelium tumors [3,12,23]. In a study, Yuqing et al., 2012 suggested that different genes on chromosome 1 might employ different functions in the generation and development of thymic epithelium tumors [12]. Thymic epithelial tumors are rare both in human and veterinary medicine. To our knowledge, this study describes the largest serial of mixed thymoma observed in Mynah bird. In addition to, a description of the morphologic findings, immunohistochemical and cytohistopathological investigations were performed on this tumor with their human counterparts. Because of the high resemblance of the avian thymomas to their human counterparts, the current human WHO classification for thymic epithelial tumors was used. The avian thymus gland consists of 7 flattened lobes of tissue that are located bilaterally in the subcutis of the neck adjacent to the trachea [24]. Thus, thymomas in birds occur cranial and ventrolateral to the thoracic inlet. In mammals, the thymus gland is located within the mediastinum in the thoracic cavity [25]. The biological behaviors of thymomas are benign and the neoplasia arises from the epithelial portion of the thymus [26]. In all avian species, the differential diagnosis for a cervical swelling is fairly extensive and includes foreign body reaction, trauma, fungal granuloma, abscess, and neoplasia [5]. In this mynah, cervical swelling was observed 4 weeks before presentation and had progressively increased in size over that period. Radiographs performed at that time demonstrated mineral opacities in the lateral cervical region, and degenerative changes in right tarsal joint and inflammation of its adjacent soft tissues accompanying mineralization of its external soft tissue were observed. In addition, hepatomegaly was recorded as well. In all previously reported avian thymomas, limited presurgical diagnostics were performed before attempted mass resection. Ideally, a complete diagnostic work-up consisting of complete blood count, plasma biochemical analysis, radiographs, and computed tomography would be performed before surgical exploration.Fine-needle aspirate (FNA) with cytologic evaluation or biopsy are useful diagnostic modalities that can be used for an accurate preoperative diagnosis. FNA biopsy has gained increasing acceptance as a rapid, noninvasive, and effective diagnostic procedure in the investigation of cervical masses [27,28]. Because thymomas are uncommon neoplasms, experience with the cytologic diagnosis of these tumors is limited. To our knowledge, the correlation between cytologic findings of thymomas and various histologic classification has not been well studied previously. Ali and Erozan [26] found that it was possible to correlate the FNA findings with histologic subtypes determined on resection with adequate well preserved material. In the current study, aspirates with a high L:E ratio had a tendency to belong to predominantly mixed. Tao et al. [28] suggested a classification based on the size, shape, and pleomorphism of the epithelial component, which has been shown to have prognostic value. Riazmontazer et al. [29] also reported a case of invasive thymoma with atypical cytologic features in the aspirate. They described invasive malignant thymomas with cytologic atypia. Our data generally are compatible with these observations. The more atypical the neoplastic cells are, the more likely the tumor will display aggressive behavior.The cytologic features of thymomas include dual lymphoid and epithelial cellular populations and unique neoplastic tissue fragments that reflect histology and allow their accurate identification. The cytologic diagnosis of thymoma can be extremely challenging. In part, this is because a technically proficient interventional radiologist is needed, epithelial cells may be difficult to recognize in lymphoid rich aspirate smears, and there is inherent sampling error in a tumor that frequently displays heterogeneous histopathology [16,30]. Immunohistochemistry for cytokeratin is helpful in this case, since the presence of rare epithelial cells in serial sections is suggestive of thymoma [31,32]. Cytokeratin profiles have been established in human medicine for the thymus and thymomas, and have been shown to be clinically useful in determining the invasive potential of these neoplasms [16]. Since a cytomorphologic and histologic classification of thymomas seems not to be a useful prognosticator in animals, the use of a pan-specific cocktail of antibodies for cytokeratins is sufficient for the diagnosis of these tumors in veterinary medicine. Thymoma should be differentiated from other anterior mediastinal neoplasms with epithelial and/or lymphoid differentiation, including Non-Hodgkin (NHL) and Hodgkin lymphomas, thymic carcinomas, and germ cell malignancies. NHL and Hodgkin lymphoma can be separated from thymoma by their dispersed cell population, distinctive cytologic features, and positive staining for CD45, CD20, CD15, and CD30, and negative staining for CK14,CK18, CK20 respectively. Helpful cytologic and immunocytochemical features in making the diagnosis of thymic carcinoma are clear-cut cytological atypia, absence of immature lymphocytes (CD3 + .CD1a+, CD99+), and expression of CD5 and CD70 by neoplastic epithelial cells [12]. Immunohistochemical markers can be used to differentiate the epithelial cells and lymphocytes, aside from the proportion of both cells type [31].In this case, the round cells of the tumor were all positive for CK 14, CK18 and negative for CK10. Based on the WHO and veterinary classification of thymomas [32], this particular case is categorized as mixed type. Overall, the present study confirms previous observations [29,30] that FNA of anterior mediastinal thymic lesions generally yields adequate cellular tissue with distinct cytologic and immunophenotypic features that enables thymoma diagnosis. In this case, lymphocytes were present individually or in small clump as lobules of small polygonal cells with small round or oval vesicular nuclei and indistinct nucleoli. The proportion of neoplastic cells and nonneoplastic lymphocytes varies widely between tumors and between different lobules of the same tumor [33-35]. Based on this study, thymomas may be categorized in veterinary medicine as lymphocyte predominant, epithelial predominant, or mixed [32].When lymphocytes predominate, the neoplasm must be differentiated from thymic lymphoma. Thymomas have been described in different sites of the body. The anterior mediastinum or thoracic inlet is their usual site of occurrence, but these neoplasms can also be seen elsewhere, including the cervical region and posterior mediastinum, with variable compression of adjacent structures such as trachea, esophagus, and mediastinal vessels [32,36]. The majority of the thymomas are benign. Local invasion and metastasis are considered by most authors to be uncommon, with metastases being reported in the pulmonary and pericardial pleura, [37] lung, [32,36] mediastinal lymph node, [35] cervical portion of the thymus, [38] kidney, [32] and uterus. Despite, Marx and Mueller-Hermelink considered human type A and AB thymoma as clinically benign tumours [20]. The thymomas examined in our study showed massive local growth with compression, albeit not invasion, of adjacent organs. Furthermore, lymphatic congestion was seen in the cervical lymph nodes and based on their microscopic features of malignancy tumor (such as areas of high cellularity, cellular pleomorphism, high mitotic index, necrotic foci accompanied by pyknosis and karyorrhexis and high number of undifferentiated neoplastic cells), in this case, the tumor was considered to be malignant in nature. Limitations of the cytological method include an unproven ability to definitively separate thymoma into specific WHO subtypes using cytology alone, and to determine capsular invasion [16]. Altogether, the present report confirms previous observations [26,27] that fine needle aspiration of cervical thymic lesions generally yields adequate cellular tissue with distinct cytologic, histopathologic and immunophenotypic features that enables thymoma diagnosis. Conclusion Based on this report, we recommend that thymomas be included in the differential diagnosis of neck masses in avian. Because it was highly similar to human thymomas, the new WHO classification for human epithelial thymic tumors was used. The tumor was diagnosed as AB type thymomas, and the WHO nomenclature was found to be highly applicable. The incidence of thymoma in avian is unknown; we hope this will become clearer. To our knowledge, this is the first report of cervical thymoma type AB in a mynah, suggesting that this tumour should be included as a differential diagnosis for neck round and spindle cell tumours. Finally, for understanding of these complex neoplasms and the development of the effective differential diagnosis, further investigation will be needed into the clinical features and the basic science. Competing interests The authors declare that they have no conflict of interest. Authors’ contributions FS and MJG participated in the histopathological evaluation, performed the literature review, acquired photomicrographs and drafted the manuscript and gave the final histopathological diagnosis. JJ performed sequencing alignment and manuscript writing. FK carried out the immunohistochemical stains evaluation. HM, AMB and RS edited the manuscript and made required changes. All authors have read and approved the final manuscript. Acknowledgements The authors thank staff of the Department of pathology, Faculty of Veterinary Medicine, Tehran University for their valuable technical assistance. ==== Refs Naylor DC Krinke GJ Ruefenacht HJ Primary tumours of the thymus in the rat J Comp Pathol 1988 99 187 203 10.1016/0021-9975(88)90071-0 2846660 Francis D Barnes RD Kramer JJ The thymoma-prone (NZB X CFW) F1 mouse J Pathol 1971 103 188 193 10.1002/path.1711030308 4935922 Zettl A Strobel P Wagner K Katzenberger T Ott G Rosenwald A Peters K Krein A Semik M Mueller-Hermelink HK Marx A Recurrent genetic aberrations in thymoma and thymic carcinoma Am J Pathol 2000 157 257 266 10.1016/S0002-9440(10)64536-1 10880395 Day MJ Review of thymic pathology in 30 cats and 36 dogs J Small Anim Pract 1997 38 393 403 10.1111/j.1748-5827.1997.tb03492.x 9322178 Diaz-Figueroa O Garner MM Tully J What is your diagnosis? J Avian Med Surg 2004 18 1 51 53 Feldman WH Thymoma in a chicken (Gallus domesticus) Am J Cancer 1936 26 576 580 Campbell JG Appleby EC Tumours in young chickens bred for rapid body growth (broiler chickens): a study of 351 cases J Pathol Bacteriol 1966 92 1 77 90 10.1002/path.1700920110 4289155 Zubaidy AJ An epithelial thymoma in a budgerigar (Melopsittacus undulatus) Avian Pathol 1980 9 4 575 581 10.1080/03079458008418445 18770299 Maeda H Ozaki K Fukui S Narama I Thymoma in a Java sparrow (Padda oryzivora) Avian Pathol 1994 23 2 353 357 10.1080/03079459408419003 18671100 Deminatti MM Ribet M Gosselin B Bauters F Mencier E Savary JB Lai JL Vasseur F Morel P Bisiau-Leconte S Familial thymoma and translocation t (14;20) (q24; p13) Ann Genet 1994 37 72 74 7985981 Sato J Fujiwara M Kawakami T Sumiishi A Sakata S Sakamoto A Kurata A Fascin expression in dendritic cells and tumor epithelium in thymoma and thymic carcinoma Oncol Lett 2011 2 6 1025 1032 22848263 Ma Y Li Q Cui W Miao N Liu X Zhang W Zhang C Wang J Expression of c-Jun, p73, Casp9, and N-ras in thymic epithelial tumors: relationship with the current WHO classification systems Diagn Pathol 2012 14; 7 120 23694694 Okumura M Ohta M Tateyama H The World Health Organization histologic classification system reflects the oncologic behavior of thymoma: a clinical study of 273 patients Cancer 2002 94 3 624 632 10.1002/cncr.10226 11857293 Chen G Marx A Wen-Hu C New WHO histologic classification predicts prognosis of thymic epithelial tumors: a clinicopathologic study of 200 thymoma cases from China Cancer 2002 95 2 420 429 10.1002/cncr.10665 12124843 Rosai J Histological Typing of Tumours of the Thymus 1999 New York, NY: Springer-Verlag, 2nd ed Alexiev BA Drachenberg CB Burke AP Thymomas a cytological and immunohistochemical study, with emphasis on lymphoid and neuroendocrine markers Diagn Pathol 2007 2 13 10.1186/1746-1596-2-13 17498299 Kirchner T Mueller-Hermelink HK New approaches to the diagnosis of thymic epithelial tumors Prog Surg Pathol 1989 10 167 189 Kirchner T Schalke B Buchwald J Ritter M Marx A Mueller-Hermelink HK Well-differentiated thymic carcinoma. An organotypical low-grade carcinoma with relationship to cortical thymoma Am J Surg Pathol 1992 16 1153 1169 10.1097/00000478-199212000-00003 1463094 Marino M Mueller-Hermelink HK Thymoma and thymic carcinoma. Relation of thymoma epithelial cells to the cortical and medullary differentiation of thymus Virchows Arch A Pathol Anat Histopathol 1985 407 119 149 10.1007/BF00737071 3927579 Marx A Mueller-Hermelink HK From basic immunobiology to the upcoming WHO-classification of tumors of the thymus. The second conference on biological and clinical aspects of thymic epithelial tumors and related recent developments Pathol Res Pract 1999 195 515 533 10.1016/S0344-0338(99)80001-6 10483582 Rosai J Histological Typing of Tumours of the Thymus (WHO International Classification of Tumours) 1999 2 Berlin, Germany: Springer-Verlag 9 13 Hodges RD The Histology of the Fowl 1974 London, UK: Academic Press 213 221 Sasaki H Ide N Fukai I Kiriyama M Yamakawa Y Fujii Y Gene expression analysis of human thymoma correlates with tumor stage Int J Cancer 2002 101 342 347 10.1002/ijc.10624 12209958 Moulton JE Harvey JW Moulton JE Tumors of the lymphoid and hematopoietic tissues Tumors in Domestic Animals 1990 3 Berkeley, CA: University of California Press 267 270 Zubaidy AJ An epithelial thymoma in a budgerigar (Melopsittacus undulatus) Avian Pathol 1980 9 575 581 10.1080/03079458008418445 18770299 Ali SZ Erozan YS Thymoma; cytopathologic features and differential diagnosis on fine needle aspiration Acta Cytol 1998 42 845 854 10.1159/000331958 9684567 Shin HJ Katz RL Thymic neoplasia as represented by fine needle aspiration biopsy of anterior mediastinal masses. A practical approach to the differential diagnosis Acta Cytol 1998 42 855 864 10.1159/000331959 9684568 Tao LC Pearson FG Cooper JD Sanders DE Weisbrod G Donat EE Cytopathology of thymoma Acta Cytol 1984 28 165 170 6583970 Riazmontazer N Bedayat C Izadi B Epithelial cytologic atypia in a fine needle aspirate of an invasive thymoma. A case report Acta Cytol 1992 36 387 390 1580123 Rieker RJ Schnabel PA Mechtersheimer G Thomas M Dienemann H Schirmacher P Kern MA COX-2 expression in thymomas and thymic carcinomas: a novel therapeutic target? Diag Patho 2007 2 3 10.1186/1746-1596-2-3 Quintanilla-Martinez L Wilkins JR Ferry JA Harris NL Thymoma--morphologic subclassification correlates with invasiveness and immunohistologic features: a study of 122 cases Hum Pathol 1993 24 9 958 969 10.1016/0046-8177(93)90109-T 8253462 Jacobs RM Messick JB Valli VE Meuten DJ Tumors of the hemolymphatic system Tumors in domestic animals 2002 Iowa State Press, Ames, IA 119 198 Aronsohn MG Schunk KL Carpenter JL King NW Clinical and pathologic features of thymoma in 15 dogs J Am Vet Med Assoc 1984 184 1355 1362 6735856 Catherine A Rae R Jacobs C Guillerm O A comparison between the cytological and histological characteristics in thirteen canine and feline thymomas Can Vet J 1989 35 342 346 Momotani E Nakamura N Shoya S Morphologic eviden ce of histogenesis of epithelial thymoma in a cow Am J Vet Res 1981 42 114 121 7224305 Patnaik AK Lieberman PH Erlandson RA Antonescu C Feline cystic thymoma: A clinicopathologic, immuno-histologic, and electron microscopic study of 14 cases J Feline Med Surg 2003 5 27 35 10.1053/jfms.2002.0187 12547620 Jackson C Thymoma The incidence and pathology of tumors of the domesticated animals in South Africa Onderstepoort J Vet Sci Anim Ind 1936 6 199 218 Pellegrini N Pierotti P Contributo alla conoscenza deitumori del mediastino anteriore Ann Fac Med Vet Univ Pisa 1961 14 73 95	23777537	PMC3702390	CC BY	2021-01-05 01:30:49	yes	Diagn Pathol. 2013 Jun 18; 8:98
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23861775PONE-D-12-3828810.1371/journal.pone.0067591Research ArticleBiologyBiotechnologyGeneticsEpigeneticsRNA interferenceGene expressionRNA interferenceModel OrganismsAnimal ModelsMouseMolecular Cell BiologyMedicineOncologyCancers and NeoplasmsEndocrine TumorsThyroid, Papillary CarcinomaCancer Risk FactorsmiR-26a and its Target CKS2 Modulate Cell Growth and Tumorigenesis of Papillary Thyroid Carcinoma miR-26a Modulate Cell Growth of PTCLv Mingli 1 Zhang Xiaoping 1 Li Maoquan 2 Chen Quanchi 1 Ye Meng 1 Liang Wenqing 3 Ding Lanbao 1 Cai Haidong 1 Fu Da 1 Lv Zhongwei 1 * 1 Department of Nuclear Medicine, Shanghai 10th People’s Hospital, School of Medicine, Tongji University, Shanghai, China 2 Department of Interventional Therapy, Shanghai 10th People’s Hospital, School of Medicine, Tongji University, Shanghai, China 3 Department of Orthopaedics, Shaoxing People’s Hospital, Shaoxing, China Cheng Jin Q. Editor H.Lee Moffitt Cancer Center & Research Institute, United States of America * E-mail: lvzhongweikxy@163.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: M. Lv ZL. Performed the experiments: XZ MY. Analyzed the data: XZ M. Li WL. Contributed reagents/materials/analysis tools: QC LD HC DF. Wrote the paper: M. Lv. 2013 5 7 2013 8 7 e675916 12 2012 22 5 2013 © 2013 Lv et al2013Lv et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background While many studies have shown that levels of miR-26a are lower in papillary thyroid carcinoma (PTC), the role and mechanism of miR-26a in PTC are unclear. Method We used database searches to select potential mRNA targets of miR-26a. Anti-miR-26a, miR-26a mimic, siRNA for CKS2 and their effects on cell growth, cell-cycle distribution and colony formation were evaluated. We also evaluate the over-expressed miR-26a in TPC-1 cells in severe combined immune-deficient mice. We used luciferase reporter assays, real-time PCR and western blot analysis to measure the expression and activity of miR-26a, CKS2, and related factors such as cyclin B1, cyclin A, cdk1, bcl-xl and Akt. Finally, we measured the relationship between the levels of miR-26a and CKS2 in PTC and normal thyroid tissues. Results Relative to normal thyroid tissues, miR-26a is consistently down-regulated in TPC specimens, and CKS2 was identified as a potential target. Up-regulated miR-26a expression or down-regulated CKS2 expression in TPC-1 and CGTH W3 cells lines caused G2 phase-arrest. Decreased miR-26a expression or increased CKS2 expression could have inverse function on PTC cell lines. CyclinB1, cyclinA, bcl-xl and AKt are indirectly regulated by miR-26a in a CKS2-dependent manner. Finally, CKS2 is overexpressed in PTC specimens relative to normal thyroid tissue, and a significant inverse correlation exists between miR-26a and CKS2 expression in clinical PTC specimens. Conclusion Our data indicate that miR-26a functions as a growth-suppressive miRNA in PTC, and that its suppressive effects are mediated mainly by repressing CKS2 expression. The study was funded by Shanghai Municipal Science and Technology commission (NO. 10JC1412700), the Natural Science Foundation of China (NO. 30901770 and NO. 81150110493) and General Research Plan B of Zhejiang province (2012KYB213). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Papillary thyroid carcinoma (PTC), which represents 70–80% of thyroid malignancies, generally has an excellent prognosis, even with cervical lymph node metastasis. Its prognosis is associated with age, tumor size, and histological parameters which include extracapsular extension, extrathyroidal extension, lymph node invasion distant metastasis and histological variants. These histological variants, which include conventional PTC, papillary thyroid microcarcinoma (PTMC), follicular variant (FVPC) and tall cell variant (TCVPC), among others, are related to familial adenomatous polyposis and tumor aggressiveness [1]. In addition, PTC is typically accompanied by gene rearrangement (e.g. RET/PTC) and/or sporadic mutation (e.g. BRAF) [2]. MicroRNAs (miRNAs) are ∼22 nucleotide length, non-coding single-stranded RNAs. To date, it has been estimated that miRNAs regulate the expression of about one-third of the mammalian protein-coding mRNAs, including mechanisms such as cleavage, resulting in 100% inhibition, as well as weaker forms of inhibition. Expression of miRNAs is subject to spatiotemporal regulation [3], and a number of miRNAs have been identified in various thyroid tumors, including PTC. Nikiforove et al. (4) used a ‘miRNACHIP’ micro-array approach to demonstrate that miR-187, −221, −222, −146b, −155, −122a, −31, −205 and −224 are up-regulated in PTCs compared with normal thyroid tissue. Chen et al. (5) also identified the upregulation of miR-146b, −221 and −222 in PTC. In total to date, 41 miRNAs have been shown to be over-expressed in thyroid carcinoma, including PTC, follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC) and anaplastic thyroid carcinoma (ATC) [4]. Conversely, a number of miRNAs which contribute to the development and progression of thyroid carcinoma are down-regulated in PTC, including miR-26a, −345,−138, −219, −218, −300, −292 and −30c [4], [5], [6], and in ATC, including miR-26a, −138, −219, −345, −30d and −125b [4], [7], [8]. As previously discussed, miRNA-microarray analysis has shown that miR-26a is down-regulated in PTC tissue relative to adjacent normal tissue by miRNA-microarray analysis [5]. miR-26a appears to have opposing functions in tumor growth in various types of cancer, acting as a tumor suppressor in renal cancer, lung cancer, breast cancer and liver cancer, but functioning as an oncogene in glioma [9], [10], [11], [12], [13]. Until now, the mechanism of miR-26a in PTC has been unclear. In order to arrive at a better understanding of PTC pathogenesis, we investigated the function of miR-26a in PTC. We used Internet databases (http://mirecords.biolead.org/, www.targetscan.org/, www.ncRNA.org/) to select potential mRNA targets, followed by dual-luciferase assays to validate these interactions. We tested the effects of miR-26a on cell growth, cell cycle distribution, colony formation and examined its role in PTC tumorigenesis in a mouse model. Finally, we examined the expression levels of miR-26a and its target gene, CKS2, in PTC and normal thyroid tissue. Materials and Methods Clinical Specimens The research was approved by the Ethics Committee of Shanghai Tenth People's Hospital. All participant of the article provide their written informed consent to participate in this study. All experiments on animal were performed according to the Animal Experimental Ethics Committee of Tongji University China and approved by the Ethics Committee of Shanghai Tenth People's Hospital. PTC tissues and normal thyroid tissues were obtained from patients undergoing resection of PTC or thyroid nodules at Shanghai Tenth People’s Hospital (Tongji University, Shanghai, China). Both tumor and normal tissues were histologically confirmed. No local or systemic treatment had been conducted prior to the operation. All tissues were snap frozen in liquid nitrogen at the time of surgical removal. Cell Culture and miRNA Transfection The human PTC cell lines TPC-1 and CGTH W3 provided by the Chinese Academy of Medical were cultured in RPMI-1640 containing 10% fetal bovine serum, 100 U/Ml penicillin and 100 µg/mL streptomycin. HEK-293T cells were grown in DMEM (Invitrogen) containing 10% fetal bovine serum from North American. Lipofectamine 2000 (Invitrogen) were used to transfect miRNAs and vectors according to the manufacturer’s instructions. The miR-26a mimic, a nonspecific miR control, anti-miR26a, a nonspecific anti-miR control, siRNA for CKS2 and a nonspecific siRNA control were all purchased from Sigma Company. Selected Targets mRNA for miR-26a Target mRNAs for miR-26a were predicted using Targetscan (www.targetscan.org/), ncRNA (www.ncRNA.org/) and mirecords (http://mirecords.biolead.org/). In order to qualify as a target, the target mRNA had to contain at least one predicted miR-26a target site in its 3′UTR according to all three prediction algorithms. Gene Ontology (GO) was used in a subsequent screening step. We determined that the 3′-UTR of CKS2 was the best candidate target mRNA. Vector Construction and Luciferase Reporter Assays Luciferase reporter assays were carried out using the Dual-Luciferase Reporter Assay System (psiCHECK-2 vector, Promega). A fragment of 3′UTR of CKS2 (278 bp) containing the putative miR-26a binding site was amplified by PCR using the following primers: wt-CKS2 (forward) 5′ AAA-CTCGAG-AAGATTTGACATTCCCCA 3′ wt-CKS2 (reverse) 5′ CCC-GCGGCC-GTTCGACTTTACCTGTACT 3′ The PCR product was subcloned into the psiCHECK-2 vector downstream of the luciferase gene sequence. At the same time, a psiCHECK-2 vector containing 3′UTR of CKS2 with a mutant seed sequence of miR-26a was synthesized using the following primers: Mut-CKS2 (forward) 5′ TCAGTGAATATCCGAGAAATG 3′ Mut-CKS2 (reverse) 5′ TTGTACATTTCTCGGCTATT 3′ A PLL3.7 vector (Invitrogen) containing the miR-26a sequence (200 bp) was also synthesized using the following primers, and named miR-26a- PLL3.7. Restriction enzyme sites were added at upstream (HpaI) and downstream (XhoI) positions. miR-26a (forward) 5′ AA-GTTAAC-GTGGCCTCGTTCAAGTAATCCAGGATA GGCTGT 3′ miR-26a (reverse) 5′ AA-CTCGAG-AGCCTATCCTGGATTACTTGAACGAGG CCACG 3′ All constructs were verified by DNA sequencing. HEK-293 cells were plated in 96-well clusters, after which psiCHECK-2 vector containing wt- or mut-CKS2 was co-transfected with 100 ng construct with or without miR-26a precursors. Forty-eight hours after transfection, luciferase activity was detected using a dual-luciferase reporter assay system and normalized to Renilla activity. In addition we also constructed the PMCB vector, which contains CKS2 (marked as CKS2-PMCB) using the following primers. The procedure was similar to that used for miR-26a-PLL3.7. Restriction enzyme sites were added at upstream (EcoRI) and downstream (BamHI) positions. CKS2-PMCB (forward): AAA-GAATTC-ACGAGGATGGCCCACAAG CKS2-PMCB (reversed): AAA-GGATCC-CATTTTTGTTGATCTTTTGG mRNA Expression Profiling RNA isolation: Total RNA was isolated using Trizol reagent (Invitrogen) following the manufacturer’s instructions, and stored at −80°C. Reverse transcription: For miR-26a expression, total RNA was polyadenylated and reverse-transcribed using an NCode miRNA First-Strand cDNA Synthesis kit (Invitrogen). For CKS2 and β-Actin, total RNA was reversely transcribed using ImProm-II Reverse Transcription System (Promega). Quantitative: Real-time PCR analysis: qRT-PCR was performed following a standard SYBR-Green PCR kit protocol on a Step One Plus system (Applied Biosystems). β-Actin was used as an endogenous control to normalize the amount of total mRNA or miRNA in each sample, and relative expression was calculated with the comparative CT method. Western Blot Analysis Equal amounts of cell lysates were separated by 10% SDS-PAGE, and electrophoretically transferred to PVDF membrane. The membrane was blocked and probed with mouse anti-human CKS2 monoclonal antibody (Abcam) followed by HRP (horseradish peroxidase)-labeled goat-antimouse IgG (Abcam). Chemiluminescence was used to analyze protein levels and β-Actin was used as a protein loading control. CCK-8 Cell Proliferation Assay Cell proliferation rates were measured using a Cell Counting Kit (CCK-8) (Beyotime, Hangzhou, China). Twenty-four hours after cells were transfected with vectors, miR-26a mimic, anti-miR-26a, siRNA and their nonspecific controls, the cells were plated in 96-well plates at 500-cells per well. The cells were incubated for 24, 48, 72 or 96 h. Ten microliters of CCK-8 reagent was added to each well 1 h prior to detection. The OD 450 nm value in each well was determined by a microplate reader. Cell Cycle Analysis The effects of miR-26a and its target gene on cell cycle progression were assessed using propidium iodide flow cytometry. Cells were plated in 6-well plates at 2×105 per well and transfected with miRNAs, siRNA or vector. Seventy-two hours later, the cells were washed with PBS, harvested and fixed in 70% ethanol. Cells were treated with DNase-free RNase and stained with propidium iodide. Cell samples were analyzed on a FACSCalilur (BD Biosciences) and all cell cycle phase fractions were determined. Spheroid Formation Assay Cells stably expressing miR-26a were transfected with anti-miR-26a, CKS2 siRNA or a nonspecific control. Twenty-four hours later, cells were seeded in 6-well plates at 200 per well. Two weeks later, colonies were washed twice using PBS, fixed with methanol/acetic acid, and stained in 1% crystal violet. The number of colonies was counted in four different field visions under a microscope and the mean value was taken. Apoptosis Flow Cytometry Analysis Annexin V-FITC and propidium iodide flow cytometry using ApoAlert Annexin V kit (Clontech, Mountain View, CA) were used to assess the effects of miR-26a on cell death. TPC-1 cells and CGTH W3 cells stably expressing miR-26a, EV-control vector, with/without anti-miR-26a, siRNA for CKS2 and their nonspecific controls, were seeded in 6-well plates at 2×104 per well. Cells were harvested 72 h later and stained with Annexin V-FITC and propidium iodide according to the manufacturer’s protocol. Cell samples were analyzed on a FACSCalibur and apoptotic fractions were determined. In vivo Tumorigenesis Assay Male BALB/c nude mice (4 to 5 weeks of age) were purchased from the Animal Services Centre of Fudan University, China. All experiments on animal were performed according to the Animal Experimental Ethics Committee of Tongji University China. A total of 1×106 miR-26a-TPC-1 cells or PLL3.7-TPC-1 cells were injected into the dorsal flank of nude mice. Each group contained 6 mice and the experiment was repeated three times. Tumor size was measured every 2 days. The formula volume = (D×d2)/2 was used to evaluated tumor volume, where “D” is the longest diameter and “d” is the shortest diameter. Three weeks later, mice were sacrificed and the tumors dissected. The rate of growth of the tumor was analyzed by immunohistochemistry (ICH) for Ki-67 and PCNA, which is related to the increase in tumor size. The biopsy tissues were deparaffinized, dehydrated in xylene and graded alcohols, rinsed in PBS and stained by monoclonal antibodies for Ki-67 and PCNA (invitrogen Co.) at room temperature for 1 h, and then blocked by 3% H2O2 for 10 min at room temperature and washed thoroughly in PBS. Each tumor had at least three biopsy sections. Statistical Analysis SPSS 15.0 software was used for statistical analysis. Data are expressed as the mean ± SEM, using a two-tail Student’s test to carry out comparisons of two independent groups. A p-value <0.05 was considered statistically significant. All experiments were performed in triplicate. Results Expression of miR-26a is Decreased in Clinical PTC Specimens We compared miR-26a expression in 40 PTC specimens and 40 normal thyroid tissues. There were no significant differences in age, gender and the TNM tumor stage between two groups (Table 1). The average level of miR-26a was significantly lower in PTC specimens than in normal tissues (Fig. 1, p = 0.007). 10.1371/journal.pone.0067591.g001Figure 1 Average expression levels of miR-26a and CKS2 in human PTC specimens (n = 40) vs. normal thyroid tissues (n = 40). A. miR-26a down-regulation in PTC specimens (n = 40) vs. normal thyroid tissues (n = 40) (p<0.001). B. Increased CKS2 expression increased in PTC specimens results shown by RT-PCR and western blot (p = 0.003). 10.1371/journal.pone.0067591.t001Table 1 The clinical date of analyzed tissue samples. PTC Normal p Average age 40.18±2.19 41.97±1.25 >0.05 Gender F 19 18 >0.05 M 21 22 mir-26a Inhibits Proliferation and Spheroid Formation, and Promotes Apoptosis, in TPC-1 and CGTH W3 Cells To characterize the effect of miR-26a on cell proliferation we performed overexpression studies using a miR-26a mimic and inhibition studies using miR-26a specific anti-sense oligonucleotide inhibitor (anti-miR-26a). miR-26a inhibited, and anti-miR-26a promoted, cell proliferation in both TPC-1 cells and CGTH W3 cells (p<0.01)(Fig. 2A, Fig. S1). The nonspecific micro-RNA and anti-miR controls had no effect on cell growth in either cell line. 10.1371/journal.pone.0067591.g002Figure 2 miR-26a inhibition of TPC-1 cell growth. A. Effect of miR-26a on TPC-1 cell proliferation measured by CCK8 assay. B. Effect of stable over-expression of miR-26a on TPC-1 cell proliferation measured by CCK8 assay. C. Expression of miR-26a in miR-26a-TPC-1 cells is higher than EV-TPC-1 cell and TPC-1 cell. D. Colony formation assay of TPC-1 cells transfected with miR-26a, miR control, anti-miR-26a, anti-miR control, and miR-26a-TPC-1 cell vs EV-TPC-1 cell. E. The relative numbers of colonies obtained by counting five vision fields. Error bars correspond to 95% confidence intervals. F. Cell-cycle distribution of TPC-1 cells transfected with miR-26a mimic, anti-mir-26a and their nonspecific controls for 48 h. G. Flow cytometry and Annexin V assays showing the number of cells in apoptosis in TPC-1 cells stably over-expressing miR-26a or control TPC-1 cells with blank PLL3.7 (EV-TPC-1 cell). Next, we stably over-expressed miR-26a in TPC-1 (miR-26a-TPC-1) and CGTH W3 (miR-26a-CGTH W3) cells to examine its effect on cell growth. We first verified that expression levels of miR-26a were higher in miR-26a-TPC-1 cell and miR-26a-CGTH W3 cell than in control or untreated cells (Fig. 2C, Fig. S2). The growth inhibition effected by lentivirally-expressed miR-26a was similar to that induced by miR-26a mimic transfection in both cell lines (p<0.001, Fig. 2B, Fig. S3). Collectively these studies indicated that miR-26a modulates PTC cell growth. Next we showed that miR-26a over-expression decreases colony formation efficiency in TPC-1 cells (Figs. 2D and E), and that anti-miR-26a increased colony formation capacity in TPC-1 cell lines (Figs. 2D and E). The control lentivirus vector had no effect on cell colony formation. This result demonstrates that miR-26a modulates PTC cell colony formation capacity. In order to explore the mechanism of miR-26a in cellular proliferation, we examined its effect on cell cycle distribution and apoptosis. First we transfected TPC-1 and CGTH W3 cells with a miR-26a mimic, or anti-miR-26a, and examined their effects on cell cycle distribution. Overexpression of miR-26a significantly increased the number of cells in G2 phase in both cell lines (p<0.01, Fig. 2F, Fig. S4) compared with its nonspecific miRNA control. The number of anti-miR-26a-transfected cells in the G2 phase was significantly decreased compared with the nonspecific anti-miRNA-transfected controls in both cell lines. Flow cytometry and Annexin V assay results demonstrated that apoptosis was significantly increased in miR-26a stably-overexpressed TPC-1 and CGTH W3 cells compared to cells transfected with empty PLL3.7 (p<0.01, Fig. 2G, Fig. S5). These data indicate that the growth-suppressive effect of miR-26a is due to G2 phase-arrest and increased apoptosis. CKS2 is a Target of miR-26a To explore mi-26a-regulated target gene(s) and pathway(s), we used three publicly-available miRNA target prediction tools: Targetscan, ncRNA and mirecords. To increase the stringency of the target prediction protocol, we searched for mRNAs simultaneously predicted by all the three target-prediction programs and selected CKS2 as a potential target. To assess whether miR-26a could directly alter the expression of CKS2, a fragment of the 3′UTR of CKS2 mRNA (wt 3′UTR) containing the putative miR-26a binding sequence (or the mutant sequence, mut 3′UTR), was cloned into a luciferase reporter vector (Fig. 3A). HEK-293T cells were then transfected with the wild type or mut 3′UTR of CKS2 and miR-26a mimic. As shown in Fig. 3A, luciferase expression was decreased by about 50% when the wt 3′UTR and miR-26a mimic were cotransfected, while the mut 3′UTR had no effect on luciferase activity. Moreover cotransfection of HEK-293T cells with the wt 3′UTR and anti-miR-26a reversed the decrease caused by the miR-26a mimic (Fig. 3B). Collectively, these results indicate that CKS2 is a direct target of miR-26a. 10.1371/journal.pone.0067591.g003Figure 3 CKS2 is a target of miR-26a and miR-26a regulates CKS2 downstream signaling molecules. A. Putative binding site of miR-26a on the CKS2 3′UTR along with the mutation in the predicted seed region. B. Reporter assays on HEK-293T cells transfected with the reporter vectors containing either the wildtype or mutated CKS2 3′UTR. C. Protein levels of CKS2 in TPC-1 cells transfected with miR-26a mimic, anti-miR-26a or their nonspecific controls. D. miR-26a mediated reduction of CKS2 levels is associated with decreased expression of cyclinB1 and cdk1, and increased expression of bcl-xl and Akt, shown by RT-PCR and western blot. E. siRNA for CKS2 decreases expression of cyclinB1 and cdk1, and increases expression of bcl-xl and Akt, shown by RT-PCR and western blot. F. Transfection with anti-miR-26a increases cyclin B1 and cdk1 expression, and decreases bcl-xl and Akt expression, as shown by RT-PCR and western blot. Since microRNAs regulate cell function in a spatiotemporal fashion, we next elucidated whether the growth-suppressive effect of miR-26a was mediated by repression of CKS2 in TPC-1 and CGTH W3 cells. We first verified whether expression of CKS2 changed in response to transfection with miR-26a mimic or anti-miR-26a. Compared to controls, expression of CKS2 was significantly decreased by miR-26a transfection and increased by anti-miR-26a transfection (Fig. 3C), indicating that miR-26a decreases CKS2 expression in TPC-1 cells. CKS2 siRNA Inhibits Proliferation and Spheroid Formation, and Promotes Apoptosis, in TPC-1 and CGTH W3 Cells Next we silenced CKS2 expression in TPC-1 and CGTH W3 cells to determine whether the effect of reduced expression of CKS2 was similar to that of miR-26a. TPC-1 cells and CGTH W3 cells were transfected with siRNA for CKS2 or a miR-26a mimic. In the CCK8 cell proliferation assay, CKS2 knockdown led to significant cell growth inhibition, similar to that induced by miR-26a transfection (Fig. 4A). Moreover, cell colony formation efficiency decreased after transfection with CKS2 siRNA to an extent comparable to that induced by miR-26a (Fig. 4B). Furthermore, knockdown of CKS2 resulted in a increase in the number of cells in the G2 phase and, like transfection with miR-26a, promoted TPC-1 cell apoptosis (Figs. 4C and D, Fig. S6 and S7). 10.1371/journal.pone.0067591.g004Figure 4 Knockdown of CKS2 inhibits TPC-1 cell growth. A. Effect of miR-26a transfection or CKS2 knockdown on TPC-1 cell proliferation measured by CCK8 assay. B. Colony formation assay of TPC-1 cells transfected with miR-26a mimic, miR control, CKS2 siRNA and siRNA control, and the relative numbers of colonies counted under a microscope in five vision fields. Error bars correspond to 95% confidence intervals. C. Cell-cycle distribution of TPC-1 cells transfected with miR-26a mimic, miR control, CKS2 siRNA and siRNA control for 48 h. D. Flow cytometry and annexin V assays reflect the number of apoptotic TPC-1 cells after transfection with miR-26a mimic, miR control, CKS2 siRNA or siRNA control. Next we determined whether up-regulation of CKS2 had a similar effect to that of anti-miR-26a. Similar to the effects of anti-miR-26a, CKS2 overexpression increased cell proliferation and colony formation efficiency, and decreased the number of cells in G2 phase and apoptosis (Fig. 5). In summary, these results indicate that CKS2 is a downstream target gene of miR-26a, and that the growth-suppressive effect of miR-26a is mediated by repression of CKS2 in TPC-1 cells. 10.1371/journal.pone.0067591.g005Figure 5 Stable over-expressison of CKS2 and anti-miR-26a promotes TPC-1 cell growth. A. Effects of CKS2 or anti-miR-26a over-expression on TPC-1 cell proliferation. CKS2 = TPC-1 cell transfected with CKS2-PMCB vector; EV = TPC-1 cell transfected with EV-PMCB vector. B. Colony formation assay of TPC-1 cells transfected with CKS2-PMCB, EV-PMCB, anti-miR-26a and anti-miR control, and the relative numbers of colonies counted under microscope in five vision fields. Error bars correspond to 95% confidence intervals. C. Cell-cycle distribution of TPC-1 cells transfected with CKS2-PMCB, EV-PMCB, anti-miR-26a and anti-miR control for 48 h. D. Flow cytometry and Annexin V assays reflect the number of apoptotic TPC-1 cells after transfection with CKS2-PMCB, EV-PMCB, anti-miR-26a and anti-miR control. CKS2 Overexpression Rescues the Growth Suppressive Effect of miR-26a In order to prove that miR-26a exerts its growth inhibitory effects by inhibiting CKS2 expression, we also performed the rescued experiment. In the experiment, miR-26a-TPC-1 cells which stably over-expressing miR-26a were transient transfected with CKS2-PMCB or EV-PMCB. The cells transfected with CKS2-PMCB had stronger ability of proliferation and monoclonal formation (Fig. 6A and Fig. 6B). Furthermore transfected with CKS2-PMCB resulted in a decrease in the number of cells in the G2 phase and inhibited both cells apoptosis (Fig. 6C and Fig. 6D). All these results indicated that CKS2 overexpression could rescue the growth suppressive effect of miR-26a. MiR-26a exerts its growth inhibitory effects and promoted apoptosis by inhibiting CKS2 expression. 10.1371/journal.pone.0067591.g006Figure 6 CKS2 overexpression rescues the growth suppressive effect of miR-26a. A. Effects of CKS2 over-expression on miR-26a-TPC-1 cell proliferation. MiR-26a+CKS2 = miR-26a-TPC-1 cell transfected with CKS2-PMCB vector; MiR-26a +EV = miR-26a-TPC-1 cell transfected with EV-PMCB vector. B. Colony formation assay of miR-26a-TPC-1 cells transfected with CKS2-PMCB or EV-PMCB, and the relative numbers of colonies counted under microscope in five vision fields. Error bars correspond to 95% confidence intervals. C. Cell-cycle distribution of miR-26a-TPC-1 cells transfected with CKS2-PMCB or EV-PMCB for 48 h. D. Flow cytometry and Annexin V assays reflect the number of apoptotic miR-26a-TPC-1 cells after transfection with CKS2-PMCB, or EV-PMCB. miR-26a Suppresses Tumor Growth of TPC-1 Cells in vivo TPC-1 cells stably infected with miR-26a or a control vector were injected into the dorsal flank of nude mice. About 1 week after inoculation, the tumors became palpable. All mice had developed tumors by the end of 3 weeks. Eleven days after implantation, the average volume of transplanted tumors between the two groups became statistically significant, and miR-26a-PLL3.7 infected cells produced smaller tumors than control cells (Fig. 7A). At 17 days post-implantation, the average tumor volume of the miR-26a-PLL3.7 treated group was reduced by more than 50% compared with those treated with control TPC-1 cells (Fig. 7A). When mice were sacrificed 3 weeks post-implantation, the average weight of tumors in the miR-26a treated group was significantly less than the control (Fig. 7B). IHC analysis showed that miR-26a-treated mice had lower levels of Ki-67 and PNCA than control mice, indicating a lower tumor growth rate in miR-26a-transfected tumor cells (Fig. 7C). In summary, miR-26a suppressed the tumor growth of TPC-1 cell in nude mice. 10.1371/journal.pone.0067591.g007Figure 7 miR-26a suppresses tumor growth of TPC-1 cells in nude mice. A. Growth curve of tumors volumes over 21 days of observation. Each data point represents the mean ± SEM of six mice. B. The tumors were dissected from the mice and measured. C. IHC Ki-67 and PNCA staining of sections of transplanted tumors formed by TPC-1 cells infected with miR-26a-PLL3.7 or EV-PLL3.7. miR-26a Regulates CKS2 Downstream Signaling Molecules Given that miR-26a suppressed tumor cell proliferation and promoted apoptosis through inhibition of CKS2 expression, we examined the effect of miR-26a on CKS2 downstream signaling molecules in our system. Cyclin B1, Cyclin A, cdk1, bcl-xl and AKt were selected as CKS2 downstream genes [14], [15]. miR-26a-mediated reduction of CKS2 levels was associated with decreased expression of cyclinB1, cdk1, bcl-xl and AKt expression (Fig. 3D). Similarly, these genes expression was increased,in CKS2 siRNA-transfected TPC-1 cells (Fig. 3E). Conversely, transfection of anti-miR-26a resulted in elevated expression levels of cyclinB1, cdk1, bcl-xl and AKt (Fig. 3F). These results implicate cyclin B1, cdk1, bcl-xl and AKt in miR-26a-mediated tumor growth suppression. CKS2 Expression is Increased in Clinical PTC Specimens and is Inversely Correlated with miR-26a Levels In order to evaluate the clinical implications of the relationship between miR-26a and CKS2 we identified in our in vitro experiments, we next measured the expression levels of CKS2 in PTC specimens and normal thyroid tissues. As showed in Fig. 1B, average expression levels of CKS2 were significantly higher in PTC specimens than in normal thyroid tissues (p<0.05). Moreover, as anticipated, we observed a significant inverse correlation between CSK2 and miR-26a levels (Fig. 1C, r = −0.964; p<0.001) in these tissues. Discussion PTC is the most common form of follicular-cell derived carcinomas and comprises 75% of all newly-diagnosed thyroid cancers [16]. While studies have shown that miR-26a is down-regulated in PTC [4], [5], [6], [7], [8], to date no data have been available regarding the mechanism of miR-26a in this disease. miR-26a has been shown to inhibit tumor cell growth in an EZH2-dependent manner in nasopharyngeal carcinoma [17], and a similar pathway has been demonstrated in lung cancer [10]. Moreover, mir-26a promotes cholangiocarcinoma growth through activation of β-catenin [13]. It is known however that a given miRNA may target several mRNAs, and that miRNAs have tissue specific functions, and these functions may not be conserved across different tumor types. In this study we first validated that miR-26a was down-regulated in PTC specimens compared to normal thyroid tissues. We then tested the effects on various parameters of cell growth of miR-26a transfection in our TPC-1 and CGTH W3 cell line PTC models, and found that miR-26a overexpression inhibited TPC-cell proliferation, decreased clonogenic formation, induced G2-arrest and promoted apoptosis. Conversely, in the same cell models, anti-miR-26a promoted cell proliferation and clonogenic formation, and inhibited apoptosis. IHC analysis of Ki-67 and PCNA demonstrated that overexpression of miR-26a suppressed tumorigenesis in a murine model of TPC-1 xenograft, echoing similar findings in nasopharyngeal carcinoma, lung cancer, liver cancer [18] and lymphoma [19]. In all these studies, miR-26a was down-regulated, and re-established expression of miR-26a inhibited cell proliferation and promoted apoptosis. In cholangiocarcinoma and glioma however, miR-26a acts as an oncogene to promote cell growth [11], [13]. These conflicting data indicate that the expression and action of miR-26a is tissue- and time- dependent, and its role in different tumor cells may potentially alter according to its downstream targets. Cyclin-dependent kinase subunit (Cks) proteins are small cyclin-dependent kinase-interacting proteins which are essential for cell cycle control [20], [21]. The mammalian Cks family consists of two well-conserved small proteins, Cks1 and Cks2 (also referred to as CDC28 protein kinase regulatory subuinit2, or CKS2). Many human malignancies are characterized by CKS2 overexpression, and it is generally known as an oncogene. In our study, a variety of results indicated that CKS2 is a direct target of miR-26a in TPC-1 cells: (i) a seed binding site sequence for miR-26a was identified in the 3′UTR of CKS2 mRNA; (ii) CKS2 3′UTR luciferase reporter activity was decreased by overexpression of miR-26a but not by mut-miR-26a, contains a mutation in the seed binding site of miR-26a; (iii) CKS2 mRNA and protein levels were decreased by miR26a overexpression in TPC-1 and CGTH W3 cells, and this effect was attenuated by anti-miR-26a; (iv) silencing CKS2 expression in TPC-1 cells had similar cell-growth suppressive effects to those of overexpression of miR-26a; (v) CKS2 overexpression rescued the growth suppressive effect of miR-26a to an extent similar to that of anti-miR-26a; and (vi) a significant inverse correlation exists between miR-26a and CKS2 in clinical PTC specimens. Collectively, these results suggest that the growth inhibitory effect of miR-26a is mediated via repression of CKS2 expression. In order to further explore the molecular mechanism of the growth inhibition induced by miR-26a, we examined its effect on the expression of a panel of CKS2 downstream genes, namely cyclinB1, cyclin A, cdk1, bcl-xl and Akt [14], [15]. Our results showed that cyclinB1, cyclin A, bcl-xl and AKt were regulated by miR-26a and CKS2. Cdk1 and cyclin B1 are known to be important players in the cell cycle. Many studies have demonstrated that Cyclin B1-cdk1 protein kinase, also known as mitosis promoting factor (MPF), is essential for mitosis and that in its absence, cells are unable to progress past the G2 phase of the cell cycle [22], [23]. Consistent with this, we found that the growth-suppressive effect of miR-26a was partly due to a G2 phase-arrest. Together, these data are consistent with a model in which miR-26a suppresses CKS2 expression, leading to suppressed of cyclin B1 and cdk1 expression and G2 arrested, similar to the findings of a previous study [15]. Moreover, consistent with the previous data [16], our results indicate that miR-26a induced cell apoptosis involves by Bcl-xl and Akt, which are mainly involved in programmed cell death. In summary, this study provides novel evidence that in a model of papillary thyroid carcinoma, miR-26a suppresses proliferation and colony formation efficiency, induces G2 cell cycle arrest, promotes cell apoptosis and inhibits tumor growth in vivo by targeting CKS2 and ultimately regulating the expression of cyclinB1, cdk1, bcl-xl and AKt. Moreover, there is a significant inverse correlation between miR-26a and CKS2 in clinical PTC specimens. Our findings suggest that signaling molecules in this pathway might represent potential targets for future prevention and treatment of human papillary thyroid carcinoma. Supporting Information Figure S1 Effect of miR-26a on CGTH W3 TPC-1 cell proliferation measured by CCK8 assay. (TIF) Click here for additional data file. Figure S2 Expression of miR-26a in miR-26a-transfected CGTH W3 cells is higher than in EV- CGTH W3 cell and CGTH W3 cell. (TIF) Click here for additional data file. Figure S3 The effect of stable over-expression of miR-26a on CGTH W3 cells proliferation measured by CCK8 assay. (TIF) Click here for additional data file. Figure S4 Cell-cycle distribution of CGTH W3 cells transfected with miR-26a mimic, anti-mir-26a and their nonspecific control for 48 h. (TIF) Click here for additional data file. Figure S5 Flow cytometry and Annexin V assays show the number of apoptotic CGTH W3 cells after stable over-expression of miR-26a (miR-26a-CGTH W3 cell) compared to mock-transfected CGTH W3 cells (PLL3.7- CGTH W3 cell). (TIF) Click here for additional data file. Figure S6 Cell-cycle distribution of CGTH W3 cells transfected with miR-26a mimic, miR control, CKS2 siRNA or siRNA control for 48 h. (TIF) Click here for additional data file. Figure S7 Flow cytometry and Annexin V assays show the number of apoptotic TPC-1 cells transfected with miR-26a mimic, miR control, CKS2 siRNA or siRNA control. (TIF) Click here for additional data file. ==== Refs References 1 Gonzalez-Gonzalez R , Bologna-Molina R , Carreon-Burciaga RG , Gomezpalacio-Gastelum M , Molina-Frechero N , et al (2011 ) Papillary thyroid carcinoma: differential diagnosis and prognostic values of its different variants: review of the literature . ISRN Oncol 2011 : 915925 .22432054 2 Bartel DP , Chen CZ (2004 ) Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs . Nat Rev Genet 5 : 396 –400 .15143321 3 Schmittgen TD , Livak KJ (2008 ) Analyzing real-time PCR data by the comparative C(T) method . Nat Protoc 3 : 1101 –1108 .18546601 4 Pallante P , Visone R , Croce CM , Fusco A (2010 ) Deregulation of microRNA expression in follicular-cell-derived human thyroid carcinomas . Endocr Relat Cancer 17 : F91 –104 .19942715 5 He H , Jazdzewski K , Li W , Liyanarachchi S , Nagy R , et al (2005 ) The role of microRNA genes in papillary thyroid carcinoma . Proc Natl Acad Sci U S A 102 : 19075 –19080 .16365291 6 Tetzlaff MT , Liu A , Xu X , Master SR , Baldwin DA , et al (2007 ) Differential expression of miRNAs in papillary thyroid carcinoma compared to multinodular goiter using formalin fixed paraffin embedded tissues . Endocr Pathol 18 : 163 –173 .18058265 7 Mitomo S , Maesawa C , Ogasawara S , Iwaya T , Shibazaki M , et al (2008 ) Downregulation of miR-138 is associated with overexpression of human telomerase reverse transcriptase protein in human anaplastic thyroid carcinoma cell lines . Cancer Sci 99 : 280 –286 .18201269 8 Visone R , Pallante P , Vecchione A , Cirombella R , Ferracin M , et al (2007 ) Specific microRNAs are downregulated in human thyroid anaplastic carcinomas . Oncogene 26 : 7590 –7595 .17563749 9 Chen HC , Chen GH , Chen YH , Liao WL , Liu CY , et al (2009 ) MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma . Br J Cancer 100 : 1002 –1011 .19293812 10 Dang X , Ma A , Yang L , Hu H , Zhu B , et al (2012 ) MicroRNA-26a regulates tumorigenic properties of EZH2 in human lung carcinoma cells . Cancer Genet 205 : 113 –123 .22469510 11 Huse JT , Brennan C , Hambardzumyan D , Wee B , Pena J , et al (2009 ) The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo . Genes Dev 23 : 1327 –1337 .19487573 12 Slaby O, Redova M, Poprach A, Nekvindova J, Iliev R, et al. Identification of MicroRNAs associated with early relapse after nephrectomy in renal cell carcinoma patients . Genes Chromosomes Cancer 51 : 707 –716 .22492545 13 Zhang J, Han C, Wu T (2012) MicroRNA-26a promotes cholangiocarcinoma growth by activating beta-catenin. Gastroenterology 143: 246–256 e248. 14 Lan Y , Zhang Y , Wang J , Lin C , Ittmann MM , et al (2008 ) Aberrant expression of Cks1 and Cks2 contributes to prostate tumorigenesis by promoting proliferation and inhibiting programmed cell death . Int J Cancer 123 : 543 –551 .18498131 15 Martinsson-Ahlzen HS , Liberal V , Grunenfelder B , Chaves SR , Spruck CH , et al (2008 ) Cyclin-dependent kinase-associated proteins Cks1 and Cks2 are essential during early embryogenesis and for cell cycle progression in somatic cells . Mol Cell Biol 28 : 5698 –5709 .18625720 16 Ban Y , Yamamoto G , Takada M , Hayashi S , Shimizu K , et al (2012 ) Proteomic profiling of thyroid papillary carcinoma . J Thyroid Res 2012 : 815079 .22518348 17 Lu J , He ML , Wang L , Chen Y , Liu X , et al (2011 ) MiR-26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2 . Cancer Res 71 : 225 –233 .21199804 18 Ji J , Shi J , Budhu A , Yu Z , Forgues M , et al (2009 ) MicroRNA expression, survival, and response to interferon in liver cancer . N Engl J Med 361 : 1437 –1447 .19812400 19 Sander S , Bullinger L , Klapproth K , Fiedler K , Kestler HA , et al (2008 ) MYC stimulates EZH2 expression by repression of its negative regulator miR-26a . Blood 112 : 4202 –4212 .18713946 20 Hayles J , Beach D , Durkacz B , Nurse P (1986 ) The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function . Mol Gen Genet 202 : 291 –293 .3010051 21 Richardson HE , Stueland CS , Thomas J , Russell P , Reed SI (1990 ) Human cDNAs encoding homologs of the small p34Cdc28/Cdc2-associated protein of Saccharomyces cerevisiae and Schizosaccharomyces pombe . Genes Dev 4 : 1332 –1344 .2227411 22 Doree M , Hunt T (2002 ) From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner? J Cell Sci 115 : 2461 –2464 .12045216 23 Vassilev LT , Tovar C , Chen S , Knezevic D , Zhao X , et al (2006 ) Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1 . Proc Natl Acad Sci U S A 103 : 10660 –10665 .16818887	23861775	PMC3702500	CC BY	2021-01-05 17:32:03	yes	PLoS One. 2013 Jul 5; 8(7):e67591
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23874414PONE-D-13-0425710.1371/journal.pone.0067342Research ArticleBiologyComputational BiologyMolecular GeneticsGene ExpressionGeneticsEpigeneticsGene ExpressionMicrobiologyBacterial PathogensGram NegativeModel OrganismsAnimal ModelsMouseMolecular Cell BiologyNucleic AcidsDNARNACell GrowthGene ExpressionGenomic Instability in Liver Cells Caused by an LPS-Induced Bystander-Like Effect Bacteria Cause Genome Instability in Liver CellsKovalchuk Igor * Walz Paul Thomas James Kovalchuk Olga Department of Biological Sciences, University of Lethbridge, Lethbridge, Canada Leng Fenfei Editor Florida International University, United States of America * E-mail: igor.kovalchuk@uleth.caCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: IK OK. Performed the experiments: PW JT. Analyzed the data: IK PW OK. Contributed reagents/materials/analysis tools: IK JT OK. Wrote the paper: IK PW. 2013 9 7 2013 8 7 e6734228 1 2013 15 5 2013 © 2013 Kovalchuk et al2013Kovalchuk et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Bacterial infection has been linked to carcinogenesis, however, there is lack of knowledge of molecular mechanisms that associate infection with the development of cancer. We analyzed possible effects of the consumption of heat-killed E. coli O157:H7 cells or its cellular components, DNA, RNA, protein or lipopolysaccharides (LPS) on gene expression in naïve liver cells. Four week old mice were provided water supplemented with whole heat-killed bacteria or bacterial components for a two week period. One group of animals was sacrificed immediately, whereas another group was allowed to consume uncontaminated tap water for an additional two weeks, and liver samples were collected, post mortem. Liver cells responded to exposure of whole heat-killed bacteria and LPS with alteration in γH2AX levels and levels of proteins involved in proliferation, DNA methylation (MeCP2, DNMT1, DNMT3A and 3B) or DNA repair (APE1 and KU70) as well as with changes in the expression of genes involved in stress response, cell cycle control and bile acid biosynthesis. Other bacterial components analysed in this study did not lead to any significant changes in the tested molecular parameters. This study suggests that lipopolysaccharides are a major component of Gram-negative bacteria that induce molecular changes within naïve cells of the host. The work was sponsored by NSERC and the CIHR Chair in Gender and Health to Olga Kovalchuk. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction There is clear evidence linking environmental exposures to the onset of carcinomas [1]. Viral infections such as HIV, HCV and HBV have a prominent effect on the development of carcinomas during and after infection. The influence of some bacteria on the effects of genome stability is significant but not widely accepted. Helicobacter pylori and its association with the development of gastric cancer is one of the best examples [2]. Presence of a common intestinal bacteria such as E. coli may facilitate the development of various malignancies [3]. Bacteria can promote carcinogenesis by induction of chronic infection, leading to disruption of the cell cycle and alterations in cell growth and DNA damage [4]. The association of chronic inflammation with a variety of epithelial malignant tumors has been recognized for many years. For example, squamous carcinoma may develop along the draining sinus in chronic osteomyelitis, and development of adenocarcinoma is a significant risk in patients with chronic inflammatory bowel disease [5]. Even though a link between cancer induction and bacterial infection exists, it is unclear if living or heat-killed cells, or even remnants of the bacteria can trigger genome instability and cancer. Yamamoto et al. (1992) conducted tests which exposed urinary bladders to heat killed E. coli, which resulted in a 40× enhancement of tumourigenesis in pre-initiated tumour sites [6]. Exposure to bacterial pathogens and/or their components most frequently occurs through consumption of contaminated food or water. Contamination is more frequently identified in rural communities with a high frequency of large livestock farms [7]. Boiling of contaminated water is intended to kill the bacteria and prevent infections, but bacterial remnants such as proteins and LPS may remain intact and have the capability to interact with cells of the gastro-intestinal tract. For example, the liver is exposed to bacterial determinants and/or toxins through its physiological role of detoxification of the blood; specifically, the hepatocytes are involved in clearance of endotoxins [8]. Even though, epidemiological evidence identifies links between bacterial infection and cancer induction, it is still unclear, which/if any, component of the heat-killed bacteria could produce a genomic instability response in naïve cells of the host. Based on the literature, it can be hypothesized that exposure to heat-killed bacteria or their components, causes genomic instability in cells that does not require direct contact with a bacterial cell or its constituents. Research conducted within recent years has identified that heat- killed bacteria (whether pathogenic E. coli O157:H7 or non-pathogenic DH5α) induce genome instability [9]. It was identified that water containing only heat-killed bacteria continued to have the capacity to induce genome instability in the host. The effect remained even after water contaminated with heat-killed bacteria was filtered through a 0.45 µm filter. This indicated that whole bacteria were not required to induce genetic and possibly epigenetic changes, but rather only a single component of the bacteria. DNA damage is sensed through several independent proteins and protein complexes, depending on the type of damage. Single- and double-strand breaks (SSB and DSB) in the DNA are sensed by three members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family, namely ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [10]. These checkpoint kinases phosphorylate histone variant H2AX at a serine 139, forming a γH2AX - a sensitive indicator of both DNA damage and DNA replication stress. Double strand breaks are then repaired by either non-homologous or homologous recombination pathways, represented by KU70/KU80 or RAD51/RAD54 protein groups. Another type of common DNA damage is the oxidation and alkylation of nucleotides, and apurinic/apyrimidinic endonuclease 1 (APE1) is the main abasic endonuclease involved in the base excision repair (BER) pathway in mammals [11]. Disruption of a cell cycle caused by bacteria may also lead to increased cell proliferation. One of the proteins, proliferating cell nuclear antigen or PCNA, forms a homotrimer clamp around DNA and acts as a processivity factor for DNA polymerase δ [12]. PCNA is also involved in post-replication repair as well as is able to recruit maintenance DNA methyltransferase 1, DNMT1 to hemimethylated DNA [13]. Thus the analysis of levels of PCNA may serve as a good indication of the activity of DNA replication and DNA repair. Although little is known about the effect the pathogen infection may have on the chromatin structure of the host, it can be hypothesized that bacterial infection may alter the DNA methylation and chromatin structure of the infected host cells. DNA methylation in animals occurs primarily via the action of de novo DNA methyltransferases DNMT3A and DNMT3B and maintenance methyltransferase DNMT1. Whereas hypomethylated DNA is mostly associated with higher gene expression, hypermethylated DNA is typically associated with lower gene expression activity and proteins like MeCP2 bind methylated DNA and recruit histone-modifying proteins and non-histone proteins that reinforce condensed chromatin structure [14]. The purpose of this study was to analyze the level of proteins involved in DNA damage recognition, DNA repair and DNA methylation in liver of animals that consumed heat-killed bacteria and its components DNA, RNA, protein or LPS. We analyzed immediate and delayed effects of consumption of heat killed bacteria and bacterial components and found multiple changes in the expression of aforementioned proteins in response to whole bacteria or LPS but not other components. Materials and Methods Animal model Four-week-old C57BL/6 male mice and all subjects were handled and cared for according to the requirements set by the Canadian Council for Animal Care and Use. The procedures have been approved by the University of Lethbridge Animal Welfare Committee. The mice were housed in cages in a virus-free facility with a 12 h light/dark cycle and provided water (with or without treatment) and food pellets ad libitum. Water consumption, food intake and body weight were monitored for any significant changes. Mice were housed in groups (8 animals per group) according to the determinant from the bacteria they were exposed to (e.g., all mice in one compartment would receive the LPS-rich solution only). The two and four week groups were housed within the same compartment with half of the mice removed for each temporal experimental endpoint. E. coli O157:H7 bacteria were grown to OD600 0.2, and then heat-killed. 1.25 ml of bacterial suspension was then added to one litre of water to get approximately 6×106 bacteria/litre. For this study, six treatment groups were created: group 1 received control, tap water; group 2 received heat-killed bacteria; group 3 received DNA prepared from group #2; group 4 received RNA prepared from group #2; group 5 received protein prepared from group #2; group 6 received LPS prepared from group #2. Animals were sacrificed either immediately after treatment (4 animals in each group) or two weeks later (4 animals in each group). The following concentrations were used: DNA at 430 µg/L, RNA at 72.7 µg/L, protein at 9.6 µg/L and LPS at 50 µg/L. DNA extracted from a single E. coli cell weighs ∼5×10−9 µg, thus 6×106 bacteria would weigh 30×10−3 µg, which is ∼15,000-fold less DNA than was used in the experiment. Also, typical bacterial cell contains 0.1 pg of RNA; thus 6×106 bacteria would yield 0.6 µg of RNA, which is 200-fold less than used in our experiment. Average bacterium contains approximately 200×10−9 µg of protein [15]; thus 6×106 bacteria would yield 1.2 µg of protein, or 8 times less than used in our experiment. The use of increased concentrations of DNA, RNA and protein was intentional to ensure a large concentration of bacterial components was present in the water to induce a response to the contaminant. Proportionally higher concentration of bacteria in water was not possible to achieve without causing the water to be turbid. The water consumption was comparable among all groups of animals. Consumption of contaminated water did not cause any physiological distress in animals. Animals in the four week test group received normal water for two weeks following initial treatment (Figure S1). Animals were sacrificed either 2 or 4 weeks (depending on the test group) after the start of the treatment. Liver and muscle tissue samples were harvested and processed for molecular testing or fixed in paraformaldehyde for immunohistochemical analysis. The liver was chosen as an indirect target organ, because of its capacity to detoxify the host blood from possible toxins and pathogens [8]. Muscle cells were used as a control that should be neutral to bacterial exposure. DNA, RNA and protein extraction DNA was extracted from the E. coli O157:H7 using a Qiagen DNAeasy kit (Qiagen) in accordance with the manufacturer's specifications. RNA was extracted from the E. coli O157:H7 using TRIzol® Reagent following the manufactures protocols. For protein extraction, 1 ml of bacterial suspension was centrifuged at 5,000×g for 20 min at 4°C. Next, 500 µl of Lysis buffer (1% Sodium Dodecyl Sulphate) was added and each sample was sonicated for 30 s. Cell debris were removed by centrifugation at 10,000×g for 30 min. Supernatants containing the proteins were transferred to new tubes. Lipopolysaccharide extraction Bacterial cells were harvested by centrifugation (Eppendorf ® 5415R Centrifuge) at the speed of 1,000 rpm for 15 min and LPS was purified as previously described [16]. The procedure for purification of LPS does not completely exclude addition of portions of the bacterial cell wall [16]. Some amounts of other components such as proteins may also be included in the LPS rich solution, therefore the extract is identified as a crude LPS-rich solution. mRNA expression analysis and RT-PCR Total RNA was extracted from 100 mg of the mouse liver tissue using 1 ml TRIzol® Reagent (Invitrogen, Burlington, ON) according to the manufacturer's instructions. Tissue from the four animals per experimental group - exposed for two weeks to LPS, exposed to whole heat-killed bacteria, as well as control animals – were used for the gene expression analysis. The mRNA expression analysis was performed by Genome Quebec (Montreal, QC) with an Illumina MouseWG-6 v2.0 Expression BeadChip. Data produced from the Chip assay was analysed using an Ingenuity IPA Network Analyser and significance was calculated with the use of ANOVA and Significance analysis of microarrays (SAM) test. RT-PCR was carried out on a Bio-Rad Laboratory's CFX96 Real-Time PCR Detection System (Mississauga, Ontario), using Taq DNA polymerase (Fermentas, Burlington, Ontario). Each reaction contained 2 µl of cDNA, prepared with RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, Ontario), 10 pM of forward and reverse primers, 2 mM MgCl2, Taq buffer with KCl, and 0.625 units of Taq DNA polymerase. Specific primers were designed using integrated DNA Technology primer design software (Oligo Perfect™ Designer) (Table S1). A heat-map showing ANOVA analysis of the mRNA expression, was produced with the assistance of IPA Network® program. Immunohistochemical analysis Paraffin embedding and sectioning of the tissue was conducted at Pantomics (Richmond, CA). Tissue sample labels were recorded and replaced with a random numbered system to ensure no predetermined knowledge was given to either Pantomics or the individual quantifying the data visualized by the fluorescent probes. Upon fixation, the slides were stained with DAPI and immunostained by using either antibodies against phosphorylated γH2AX or antibodies against PCNA (both probes acquired from Santa Cruz Biotechnology, Santa Cruz, CA), as previously described [17]. Specifically, the primary antibody (either PCNA or γH2AX at 1∶500 and 1∶350, respectively) was added to goat serum/1× PBS (1∶200) overnight at 4°C. The slides were washed (similarly to previous cycles) to remove excess antibodies and goat anti-mouse serum (1∶200) for PCNA antibody and anti-rabbit 594 serum (1∶200) for γH2AX antibodies was added. Samples were examined with a Zeiss confocal microscope and quantified without prior knowledge of the predetermined pattern created by an independent third party. Each tissue sample was digitally sectioned into several equal portions and cells expressing PCNA or γH2AX were recorded by counting. Foci were counted by eye in a blinded fashion by two independent investigators. For γH2AX, at least 100 cells from each sample were examined. The PCNA index was quantified by enumerating PCNA-positive cells in at least 30 high power fields. The data are presented as the fold difference between treated and non-treated cells ± a standard error. Western blot analysis Tissue samples for protein analysis were snap-frozen in liquid nitrogen immediately after animals were sacrificed. Tissues were sectioned (∼25 mg), washed thoroughly, sonicated in 1% SDS and small aliquots of extracts were isolated for protein analysis using Bradford dye reagents from BioRad (Hercules, CA). For western blot analysis, each sample aliquot was standardized to be 2 mg/ml. Western blot analysis was performed as described before [17]. Equal amounts of protein (∼20 µg) were used for SDS polyacrylamide gel electrophoresis at 150 V for 1 h. Smaller predicted proteins such as PCNA (36 kDa) and MeCP2 (53 kDa) were identified using a 12% polyacrylamide gel, whereas KU70 (70 kDa), DNMT3A (85 kDa) and DNMT3B (96 kDa) were identified using a 10% polyacrylamide gel and DNMT1 (138 kDa) with an 8% gel. Specific antibodies used: KU70 and PCNA (both 1∶1000, Santa Cruz Biotechnology, Santa Cruz, CA), POL β, APE1, MeCP2, DNMT1, DNMT3A and DNMT3B (all are 1∶1000, Abcam, Cambridge, MA). Antibody binding was revealed by incubating with horseradish peroxidase-conjugated secondary antibodies (Amersham, Baie d'Urfé, QC) and the ECL Plus immunoblotting detection system (Amersham, Baie d'Urfé, QC). Chemiluminescence was detected by Biomax MR films (Eastman Kodak, New Haven, CT). Unaltered PVDF membranes were stained with Coomassie Blue (BioRad, Hercules, CA), and the intensity of the Mr 50,000 protein band was assessed as a loading control. Signals were quantified using NIH ImageJ 1.63 software and normalized to both actin and the Mr 50,000 protein which gave consistent results. Each protein extract was analyzed three times to ensure significance of the results. Analysis of DNA methylation DNA methylation was analyzed by cytosine extension assay as previously described [18]. The assay utilizes the methylation-sensitive endonuclease HpaII which recognize CCGG sequence and is not able to cut when internal cytosine (at the CpG) is methylated. DNA digested with HpaII leaves a 5′ guanine overhang. Next, the single nucleotide extension reaction with labeled (3H)dCTP fills in the overhang and the degree of (3H)dCTP incorporation is evaluated by measuring radioactive counts. The incorporation of (3H)dCTP inversely correlates with methylation level. In brief, one aliquot of genomic DNA from each liver sample was digested with HpaII, whereas the second aliquot with undigested DNA was used as the background control. The single-nucleotide extension reaction was performed with subsequent measurement of radioactive count as described before [18]. Each reaction for each of 4 animals per single experimental group was repeated twice and the average and SE were calculated. Statistical analysis To identify significant alterations, statistical analysis of the data was conducted for every experiment with a significance confidence level of a minimum of 95% (p≤0.05). A comparison between different treatments was performed, using ANOVA for continuous responses and statistical tests for contingency tables such as Fisher's exact test. The analysis of data was performed using the software packages Stat View and Analyze It for Excel and checked using the statistical analysis program SPSS 15. Results Exposure to LPS from heat-killed bacteria leads to increased levels of γH2AX H2AX is a histone variant and its phosphorylated form, γH2AX, serves as an indicator of the DNA strand breaks in the cells [19]. Immunofluorescence analysis of liver cells identified increased levels of γH2AX in animals exposed for two weeks to whole heat-killed bacteria (2.7 fold increase, p<0.05) and LPS (1.95 fold increase, p<0.05) between test groups (Figure 1). Liver cells of animals that consumed tap water for another two weeks (4 weeks group) also exhibited high levels of γH2AX – a 3.25 and 3.3 fold increase was observed in response to whole bacteria and LPS, respectively. Exposure to other bacterial components did not result in an altered level of γH2AX (Table S2). This analysis indicated that liver cells accumulate DNA damage in response to whole heat-killed bacteria or LPS present in drinking water and that the DNA damage continues to accumulate even two weeks after exposure. 10.1371/journal.pone.0067342.g001Figure 1 Immunohistochemical analysis of liver tissue samples stained with DAPI staining and Green Fluorescent antibody for γH2AX. A. Images taken from animals in the group exposed to tap water (control), DNA, RNA, protein, LPS and whole heat-killed bacteria. B. Bars show the average (with SD) fold difference in number of γH2AX-positive cells between treated and control groups. Asterisks show significant difference from control (p<0.05). KU70 and APE1 protein levels change in response to whole bacteria and LPS Elevated level of γH2AX indicates an increase in the level of strand breaks in the liver cells of animals that consumed whole heat-killed bacteria or LPS. To further test whether these additional strand breaks stimulated the increase in amount of DNA repair enzymes, we performed Western blot analysis of KU70, a key protein in the non-homologous end-joining (NJEJ) repair pathway [20]. A 2.5 fold increase was observed upon exposure of the mice for two weeks to whole heat-killed bacteria and a 1.67 fold increase upon LPS exposure (Figure 2A). In contrast, levels of KU70 were not changed in animals exposed to DNA, RNA and protein purified from the heat-killed bacteria. Animals that consumed tap water for two weeks after being exposed to heat killed bacteria or LPS had KU70 levels comparable to the control group (Table S3). These data may be a further indication of the accumulation of the strand breaks in liver cells in response to heat-killed bacteria and LPS. 10.1371/journal.pone.0067342.g002Figure 2 Western blot analysis of KU70 (A), APE1 (B) and PCNA (C) protein levels in liver tissue of mice exposed to whole heat-killed E. coli O157:H7 bacteria and DNA, RNA, protein, and LPS extracted from heat-killed bacteria. Bars show the average protein levels (with SD) as compared to the control set at 100%. Asterisks and bars show significant increase from non-exposed controls through the analysis of data using one way ANOVA test (p<0.05). Lower panel shows representative Western blots in 2 and 4 weeks groups. To test whether exposure to bacteria or its components activates any other repair pathways, we tested the protein level of APE1, involved in base excision repair (BER). A significant decrease in expression of APE1 was identified when animals were exposed to LPS (1.19 fold decrease) and whole heat-killed bacteria (1.13 fold decrease) in the two week group (Figure 2B) as compared to the control. All other components did not result in any significant difference in expression compared to the control (Table S4). In the four week group, levels of expression returned to comparable levels to those seen in the control group (Figure 2B). These data may be an indication that BER activity is suppressed in the liver cells of animals that consumed heat-killed bacteria or LPS. Exposure to LPS from heat-killed bacteria leads to increased expression of PCNA in liver cells Next, we measured the level of PCNA protein. Western blot analysis identified a significant increase in the expression of PCNA in animals exposed for 2 weeks to heat-killed whole bacteria or LPS (p<0.05 in both cases) when compared to the control group (Figure 2C). Analysis of samples from the four weeks group (2 weeks exposure plus 2 weeks normal water) showed that PCNA levels in whole heat-killed bacteria and LPS groups were still increased, as compared to the control, albeit to a lower extent (p<0.05) (Table S5). Other treatment groups did not show any significant alteration in the expression of PCNA. To support the data obtained by Western Blot analysis, an immunofluorescence analysis of PCNA protein was performed. We also found a substantial increase in PCNA in the liver tissue of animals exposed for 2 weeks to the whole heat-killed bacteria or LPS (Figure 3A). PCNA remained high in the 4 weeks group as well, although the difference was less pronounced as compared to 2 weeks group (Figure 3B, C; Table S6). Exposure to other components of bacterial cell did not result in any change in PCNA levels. 10.1371/journal.pone.0067342.g003Figure 3 Immunohistochemical analysis of liver tissue samples stained with DAPI staining and Green Fluorescent antibody for PCNA. A. Images of liver samples taken from individual animals within 2 week and 4 week test groups. B. Quantification of PCNA-positive cells. Bars represent the average (with SD) number of PCNA cells. Asterisks show significant differences from control (p<0.05). Exposure to LPS from heat-killed bacteria leads to an increase in expression of maintenance and de novo DNA methylation enzymes and a decrease in global genome DNA methylation Genome stability in part depends on the degree of chromatin condensation, with the latter depending on changes in DNA methylation and histone modifications. Changes in DNA methylation lead to changes in gene expression as well as in alterations in genome stability. To analyze the activity of DNA methylation in liver cells, we tested expression level of maintenance methyl transferase (MTase) DNMT1, de novo MTases DNMT3A and DNMT3B and protein that binds methylated DNA, MeCP2. Exposure to whole heat-killed bacteria resulted in an increase in the level of DNMT1 protein: 1.71 fold and 1.67 fold increases (p<0.05 in both cases) were observed in the two and four week samples, respectively (Figure 4A, Table S7). Exposure to LPS showed a significant 1.25 fold increase in the two week sample, and a 1.24 fold increase in the four week sample (p<0.05 in both cases). Exposure to DNA, RNA or protein did not change the expression of DNMT1. 10.1371/journal.pone.0067342.g004Figure 4 Western blot analysis of DNMT1 (A), DNMT3A (B), DNMT3B (C) and MeCP2 (D) protein levels in liver tissue of mice exposed to whole heat-killed E. coli O157:H7 bacteria and DNA, RNA, protein, and LPS extracted from heat-killed bacteria. Bars show the average protein levels (with SD) as compared to the control set at 100%. Asterisks and bars show significant increase from non-exposed controls through the analysis of data using one way ANOVA test (p<0.05). Lower panel shows representative Western blots in 2 and 4 weeks groups. The protein level of de novo DNA MTase, DNMT3A, also increased upon exposure to whole bacteria and LPS samples. A two week exposure resulted in a 1.96 fold increase for whole bacterial exposure and a 1.25 fold for LPS exposure (p<0.05 in both cases). The analysis of four week samples showed that the levels of DNMT3A dropped and showed no significant alteration for whole heat-killed bacteria and LPS as compared to the control group (Figure 4B, Table S8). All other samples did not have any significant alterations in expression of DNMT3A. The level of DNMT3B protein, also involved in de novo DNA methylation was increased upon exposure to whole bacteria and LPS samples. A two week exposure resulted in a 1.57 and 1.18 fold increase for whole bacterial and LPS exposures, respectively (p<0.05 in both cases). In the four week samples, the expression of DNMT3B increased in response to LPS (1.25 fold, p<0.05) but not to whole heat-killed bacteria (Figure 4C, Table S9). Exposure to other bacterial components did not have any significant alterations in expression of DNMT3B. Finally, MeCP2 protein levels were significantly increased in response to whole bacteria and LPS. Exposure to the whole heat-killed bacteria resulted MeCP2 expression increased by 1.76 and 1.37 fold in the two and four week samples (p<0.05 in both cases) (Figure 4D, Table S10). LPS levels were also increased in two and four weeks samples –1.2 and 1.28 fold increase was found, respectively (p<0.05 in both cases). All other samples did not have any significant alterations in expression of MeCP2. To test whether the increase in the expression of methyltransferases would also result in the increase global genome DNA methylation, we performed cytosine extension assay. The analysis showed DNA hypomethylation in the 2 weeks groups exposed to LPS or whole heat killed bacteria (p<0.05 in all cases), but not in the other treatment groups (p>0.05) (Figure S2). Exposure to LPS or whole heat-killed bacteria leads to alterations in mRNA expression within liver tissues Since only exposure to whole bacteria and LPS triggered changes in the expression of genes involved in DNA repair, proliferation and DNA methylation, we performed the microarray analysis only using tissue from animals exposed to whole bacteria, LPS for two weeks and control. As a cut off, we utilized the p<0.05 and fold change of log2>1 (2 fold). Whole heat-killed bacteria exposure increased the expression of interleukin L1, 6, 4, 17B and Tumour Necrosis Factor, and decreased expression of Glycine C-Acetyltransferase genes. LPS exposure increased the expression of Ccl6, Fads2, Plin2, Pnrc1 and Rxra genes. Several transcripts were altered in similar manner upon the exposure to the heat-killed bacteria and LPS. Dusp1, Gadd45g, Tff3, Esm1, Mmd2, Gsta1, Cyp7a1 and Alas1 genes changed their transcription levels in response to both whole heat-killed bacteria and LPS (Figure 5). 10.1371/journal.pone.0067342.g005Figure 5 Exposure to LPS and Bacteria alters mRNA levels in mouse livers. A. Clustering of differential expression of genes with the use of control as a standard. Red denotes high expression levels, whereas green denotes low expression levels. RTPCR gene expression analysis of Dusp1 (B), Alas1 (C), Tff3 (D), Esm1 (E), Mmd2 (F), Gsta1 (G), Cyp7A1 (H), Gadd45g (I). Bars show normalized expression levels (average from three reactions, with SD) of aforementioned genes in control and two groups exposed for 2 weeks, whole heat-killed bacteria and LPS groups. Normalization was conducted with Actin transcription levels. Asterisks indicate significant difference (p<0.05). To confirm the changes in expression of the aforementioned genes, RTPCR analysis was performed. RTPCR confirmed upregulation of the Dusp1 gene, which was found to be upregulated by 2.7 fold in response to LPS and by 2.2 fold in response to whole bacteria. Results also confirmed an increased expression of the gene Alas1 in the LPS group but not in the whole bacteria group (Figure 5). Microarray analysis identified a significant decrease in transcription levels of the Gadd45g, Tff3, Esm1, Mmd2, Gsta1 and Cyp7a1 genes in both experimental groups. RTPCR analysis confirmed the decrease in the expression in all abovementioned genes, except Gsta1 expression in the whole bacteria group (Figure 5). Discussion Previously, it was shown that exposure to heat-killed bacteria resulted in an increase in cell proliferation and genome instability of non-exposed liver cells [9]. This research attempted to identify which component of bacteria triggers this response. Exposure to LPS and not to DNA, RNA or proteins resulted in an increase in the level of γH2AX, PCNA, KU70 and DNA methyltransferase proteins. Furthermore, it was identified that a set of 8 genes (Dusp1, Gadd45g, Tff3, Esm1, Mmd2, Gsta1, Cyp7a1 and Alas1) changed their expression upon exposure to whole bacteria and LPS. Below we discuss these findings in details. γH2AX, KU70 and PCNA protein levels increase in liver cells of animals exposed to whole bacteria or LPS Exposure to LPS and heat-killed bacteria caused an increase in the phosphorylation of the H2AX protein. The increase in the γH2AX levels signifies the increase in DNA alteration, mainly DNA strand breaks. Recruitment of γH2AX activates homologous recombination and non-homologous end joining DNA repair pathways [21]. Therefore, it was not surprising to find higher levels of KU70 protein in liver of animals in response to consumption of heat-killed bacteria to parallel higher levels of γH2AX. Our previous research has indicated that pathogenic and non-pathogenic heat-killed bacteria induced higher levels of γH2AX within the liver tissue [9]. Current work demonstrates that a specific component of heat-killed bacteria – LPS – triggers the increase in stand breaks, as reflected by elevated levels of γH2AX and KU70. It was surprising to see the decrease in the level of BER enzyme APE1 in response to heat-killed bacteria and LPS. Since liver is not an organ that is directly exposed to bacteria or LPS consumed with water (although certain amount of toxins may reach liver cells), it is hard to imagine that bacteria or LPS directly cause the DNA damage in cells. One of many ways the DNA damage may be induced in liver cells is through activation of various signalling molecules, leading to production of radicals and nucleotide oxidation. The fact that APE1 is downregulated in the liver cells in response to bacteria or LPS may suggest that it is unlikely that oxidized nucleotides are formed more frequently in these cells. The downregulation may suggest that there are other types of damages to DNA occur in these liver cells, such as strand breaks, or even that there is no extra damage to DNA. How then one explains the increased level of γH2AX and KU70 in the liver cells of exposed animals? LPS and other endotoxins increase expression of NF-κB that in turn correlates with expression of COX-2 [22]. NF-κB is an inducible transcription factor that regulates a wide variety of genes that have been identified to respond to inflammatory signals [23]. Um et al., (2001) has shown that KU70 expression correlates with the expression of NF-κB and COX-2, reflecting high level of cell proliferation [24]. Also, cells with inhibited COX-2 and/or NF-κB genes were identified to have limited ability to repair their DSBs and proliferate [25]. Thus, it is possible that increase levels of γH2AX and KU70 in the liver cells of exposed animals reflect higher levels of DNA replication. Indeed, the amount of PCNA, a co-factor of replicative polymerase δ, increased in liver tissue after two weeks of exposure to whole bacteria or LPS. It is important to note that PCNA levels remained significantly higher even after two weeks of recovery (four weeks sample). High levels of PCNA may be associated either with an increase in cell proliferation or with an increase in DNA damage. Exposure to LPS can result in direct or indirect damage to DNA via ROS or RNOS pathways [26]. Although we can not rule out the possibility that the increase in PCNA as well as γH2AX and KU70 was due to the increase in the level of DNA damage, the fact that the level of APE1 was not increased, suggests that increased levels of γH2AX, KU70 and PCNA may be due to the increase in cell replication. It is interesting to note that such a response may not necessarily be triggered by direct exposure of liver cells to LPS. In fact, in healthy mice, most of the bacteria and LPS molecules probably do not penetrate the mucous layer of the intestine. It cannot be excluded, however, that a small amount of LPS is absorbed into the portal blood and passed through the liver cells. Healthy intestine is exposed to millions of non-pathogenic E. coli cells and thus the host develops antibodies against them; bile and serum in human contain various antibodies against commensal E. coli strains [27]. These bacterial strains however do not seem to cause any significant harm, although it should be admitted that studies like the one reported here would be difficult to conduct as mammals are infected with commensal E. coli strains in the first hours-days after birth. In contrast, LPS from pathogenic strain does cause serious health problems. For example, LPS has been suggested to be one of the causative agents in inflammation-induced atherosclerosis. Using anti-O157 lipopolysaccharide antibodies, LPS from pathogenic bacteria has been detected in infected humans [28]. Thus, it is possible that the increase in PCNA, γH2AX and KU70 levels is in part due to direct contact of liver cells with LPS. It is likely that the increase in γH2AX, KU70 and PCNA levels could be due to both, high level of DNA damage and high level of cell proliferation. It should be noted that without continuous exposure to the pathogenic bacteria and/or its components, the levels of DNA damage and thus DNA repair should potentially decrease, requiring fewer proteins such as γH2AX, KU70 and PCNA. In this respect it is curious to note that the levels of γH2AX remained high in animals that received tap water for two weeks after exposure to bacteria or LPS. This may be a further indication that that upregulation of these proteins was largely due to higher cell proliferation activity. Indeed, γH2AX loci are known to form in response to replicative stress – stalled replication fork recruits phosphorylated H2AX [29]. KU70 and PCNA levels decreased in the four weeks group and became similar to control levels. Exposure to DNA, RNA and protein did not induce any significant alterations in γH2AX, KU70 or PCNA expression, suggesting that these components are unlikely triggering any DNA damage or influencing replication. Protein levels of de novo and maintenance methyltransferases increase in liver cells of animals exposed to whole bacteria or LPS The amount of proteins associated with methylation of the genome, whether due to the de novo synthesis (Dnmt3A and Dnmt3B) or maintenance (DNMT1), significantly increased with LPS and bacterial exposure but not in response to other molecules. The constitutive expression of MeCP2 is caused by its ability to perpetuate its own expression. This cycling of expression results in continual expression of the MeCP2 protein and potential to repress genes and manipulate chromatin structure [30]. In this experiment, the expression of DNMT1, DNMT3A and DNMT3B returned to normal levels in animals that were allowed to recover by consuming uncontaminated water for the additional two week period after the initial exposure. This may indicate reversibility of potential changes in DNA methylation. This may also suggest that constant presence of a causative agent, such as LPS, is required for triggering changes in DNA methylation. The results identify that the naïve cells, distant from the exposed tissue, can be affected by exposure to LPS and whole heat killed bacteria. This indicated that within the two week exposure to multiple components of the bacteria, LPS was identified to be a key bacterial component inducing a response in distal cells and responsible for potential genomic instability. Altered levels of PCNA and γH2AX, increased expression of Ku70 and proteins involved in DNA methylation, in response to bacteria and LPS supported the hypothesis that a bystander-like effect induced genomic instability. Increase in the expression of MTases could be due to several reasons, including DNA damage and increased DNA replication or/and cell proliferation. Repair of DNA damage as well as replication result in passive loss of DNA methylation that needs to be restored by maintenance DNA MTase DNMT1. Indeed, our analysis showed a decrease in global genome DNA methylation in 2 weeks samples from LPS and whole heat-killed bacteria treatments (Figure S2). Restoration of the methylation levels observed in 4 weeks group is most likely due to the activity of overexpressed methyltransferases. As far as activation of de novo DNA methylatransferases goes, it can be suggested that either exposure to bacteria or LPS activate methylation of new CG regions in unreplicated DNA, or these exposures lead to increase in replication that somehow co-regulates the maintenance and de novo methyltransferases. In fact, co-culturing of gastric cancer cells with H. pylori was shown to activate expression of both, DNMT3a and DNMT1 as well as lead to hypermethylation of tumor suppressor gene WWOX [31]. Another study showed hypermethylation of COX-2 promoter paralleled by DNMT1 overexpression in gastric cancers associated with H. pylori infection [32]. Response to viral infection is slightly different. Infection of chickens with Marek's disease virus (MDV) resulted in upregulation of DNMT1, but downregulation of DNMT3B genes [33]. Other papers however showed that viral infection leads to differential regulation of DNMT1, DNMT3a and DNMT3b, with those genes being up- or downregulated, depending on the cell type and the virus type used in the experiments [34], [35]. Exposure to DNA, RNA and protein did not induce any significant alterations in expression of methyltransferases or changes in DNA methylation levels, suggesting that these components are unlikely candidates to trigger changes in epigenetic regulation of response to bacteria. Exposure to LPS and whole bacteria result in changes in the expression of eight different genes Microarray analysis of liver cells in animals exposed to LPS or whole heat-killed bacteria showed differential expression of eight genes that were verified with RTPCR. Altered expression of the eight genes (Dusp1, Gadd45g, Tff3, Esm1, Mmd2, Gsta1, Cyp7a1 and Alas1) could have detrimental effects on the host. Dual specificity phosphatase 1 (Dusp1) expression was found to be altered in fibroblasts exposed to oxidative/heat stress and upon stimulation with growth factors [36]. In this study, Abraham and Clark identified DUSP1 as having a potential role in the cellular response to environmental stress as well as in the negative regulation of proliferation and an inflammatory response. An increase in expression of Dusp1 may have occurred to assist in the cells' ability to survive the shock of the LPS-induced reaction [37]. An increased expression level of the gene coding for aminolevulinic acid synthase 1 (ALAS1) protein was also identified. This nuclear encoded mitochondrial enzyme is the first and rate-limiting enzyme in the heme biosynthetic pathway (Red Blood Cell (RBC) production). The production of RBCs could accelerate with influx of this specific protein and any other in an eight step process [36]. Tumour cells may increase the expression of enzymes in this process, to assist in the production of RBCs, to oxygenate new tumour cells throughout the body. Growth arrest and DNA-damage-inducible 45 gamma (Gadd45g) gene encodes the stress sensor protein that modulates the response of mammalian cells to genotoxic/physiological stress and modulates tumour formation. The transcription of the aforementioned gene is changed in response to stressors inducing growth arrest [38]. GADD45G protein also responds to environmental stresses by mediating the activation of p38/JNK pathway via MTK1/MEKK4 kinase [39]. A decrease in transcription level of Gadd45g gene inhibits the production and dimerization of MEKK4, allowing cellular proliferation, differentiation, inflammation and tumourigenesis [40]. Another mRNA with decreased gene transcription level in response to bacteria or LPS was identified as Tff3 (Trefoil factor 3). The function of the encoded protein is not well defined; however it is predicted to stabilize the mucus layer and affect healing of the cells themselves. Recently, TFF3 protein has been identified to be involved in the immune response [41]. This research has identified very low levels of the TFF3 protein during liver and gastrointestinal tissue damage, and high levels of Tff3 gene transcription briefly after the tissue was repaired. Our analysis showed that the transcription level of Tff3 was increased within the two week of exposure to whole heat-killed bacteria of LPS. It remains to be shown whether similar changes would be found within the four week sample group. The expression of the endothelial cell-specific molecule 1 (Esm1) gene coding for ESM1 protein was found to be lower in liver cells from the LPS and whole bacteria group. ESM1 is regulated by cytokines, identifying potential involvement in pathogenic infections. Esm1 expression has been shown to be increased in the presence of pro-angiogenic growth factors, such as VEGF (vascular endothelial growth factor) or FGF-2 (fibroblast growth factor 2). A significant decrease in transcription of Esm1 gene, correlates with previously reported decrease in transcription level of pro-angiogenic growth factors such as VEGF or FGF2 genes [42]. Macrophage differentiation associated 2 (Mmd2) gene was reduced in expression for LPS and whole heat-killed bacteria test groups. MMD2 is involved in the immune response and in differentiation of monocytes to macrophages. Since the response to bacteria/LPS may trigger an immediate immune response upon which monocytes differentiate into macrophages, it can be suggested that the expression of Mmd2 is no longer required at two weeks post exposure. It is possible that Mmd2 expression was increased in the first two days of exposure and then decreased at two weeks post exposure. It remains to be shown whether Mmd2 levels would return to normal levels after a two weeks recovery period. Glutathione S-transferase alpha 1 (Gsta1) mRNA was downregulated in our experiments. GSTA1 has enzymatic functions associated with the detoxification of electrophilic compounds such as carcinogens, environmental toxins and products of oxidative stress. These highly polymorphic enzymes alter the susceptibility of the organism to carcinogens, toxins and alter the effectiveness of some pharmaceutical drugs. The decrease in expression identified in our experiment implicated that the liver tissue was highly susceptible to damage caused by ROS. Finally, analysis showed a decrease in the steady state RNA levels of the cytochrome p450 family 7, subfamily a, polypeptide 1 (CYP7A1) gene. CYP7A1 is involved in drug metabolism and synthesis of bile acid and steroids from cholesterol within liver tissue. Conversion of cholesterol into bile acid is controlled by this protein and is the main process of removing cholesterol from the body [43]. Removal of cholesterol from the body is important to the overall homeostatic state of organism. Even though there seems to be no connection with an immune response for this particular protein, altered levels may affect the entire organism through inhibition of elimination of cholesterol. Conclusion This work is the first to show that a heat-killed bacterial component known as LPS can lead to distinct molecular changes in the liver. It is important to note, that many changes in liver cells after a two week exposure to LPS returned to levels similar to the control group, indicating the recovery period for such alteration is short. Changes in the levels of protein and expression of mRNAs in liver samples after exposure to whole heat-killed bacteria were more pronounced than after the exposure to LPS. This indicates that LPS may contribute to genome instability caused by bacterial contaminants in the intestine or blood, but it is not the only component. Toxins released by the bacteria upon death may also have some negative effect. Since filtering the water would not remove released toxins (such as Stx1 and Stx2), it is possible that exposure to these toxins may have had an additive effect to changes in the stability of cells in direct contact with the toxins or distal naïve cells. Indeed several reports suggest that Stx1 and especially Stx2 may interact with LPS, thus dramatically increasing LPS toxicity to the cells; injection of mice with the combination of Stx2 and LPS resulted in a severe hemolytic-uremic syndrome (HUS) as compared to a milder effect caused by Stx2 only [43]. On the other hand, injections of LPS only did not lead to HUS and did not cause death in mice, thus suggesting that LPS alone does not cause the renal failure [43]. Since we cannot exclude that the crude LSP extract that we used has a low amount of Shiga toxins, it is possible that the effect of LPS on genome stability and proliferation of liver cells is not purely due to LPS alone but rather due to the potentiation effect of Shiga toxins. The more drastic effect of heat-killed bacteria on liver cells as compared to crude LPS could be due to higher concentrations of Shiga toxins and thus a more drastic potentiation effect between LPS and Stx2. Another possible product of the bacterial exposure that can impact host cells is circulating inflammatory and anti-inflammatory cytokines produced by affected cells themselves [44]. Further research with the expansion into the inflammatory or anti-inflammatory field is required to identify every component of the bacteria that could induce the effects of carcinogenesis on liver tissue. Another remaining question is whether the bystander effect can induce genomic instability in other organs and tissues throughout the body. Recently, while this paper was accepted for publication, our work demonstrating similar effects of LPS on spleen cells of treated animals was accepted for publication [45]. The study showed that not only liver, but other organs can also exhibit similar response to pathogenic bacterial determinants. Supporting Information Figure S1 Experimental design to analyze potential genomic alterations induced in the liver cells of mice. Four-week-old animals received treatment water for two weeks. First set of four animals (per group) was sacrificed immediately after this treatment, whereas the second set of four animals was sacrificed in two weeks, after receiving normal tap water. (TIF) Click here for additional data file. Figure S2 Analysis of global genome DNA methylation. Global genome DNA methylation was analyzed by cytosine extension assay. The data are shown as the average (from 4 biological and 2 technical repeats with SE) level of incorporation of radioactive dCTP nucleotides (dpm 3H). Radioactive counts are inversely proportional to the DNA methylation levels. Asterisks show significant difference between treatment and control groups (p<0.05). (TIF) Click here for additional data file. Table S1 Sequence of the forward and reverse primers for RT-PCR analysis from liver samples. All primers created with the DNA Technology primer design software (Oligo Perfect™ Designer). (DOCX) Click here for additional data file. Table S2 Imunohistochemical analysis of γH2AX protein expression quantified. Average±SD represents the average protein expression±standard deviation, compared to the control test detected via analysis of images. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. Table S3 Western blot analysis of Ku70 quantified with Image J. program. Average±SD represents the average protein expression±standard deviation, compared to the control test. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. Table S4 Western blot analysis of Ape1 quantified with Image J. program. Average±SD represents the average protein expression±standard deviation, compared to the control test. Asterisks identify significant decreases from non-exposed controls. (DOCX) Click here for additional data file. Table S5 Western blot analysis of PCNA quantified with Image J. program. Average±SD represents the average protein expression±standard deviation, compared to the control test. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. Table S6 Imunohistochemical analysis of PCNA protein expression quantified. Average±SD represents the average protein expression±standard deviation, compared to the control test detected via analysis of images. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. Table S7 Western blot analysis of Dnmt1 quantified with Image J. program. Average±SD represents the average protein expression±standard deviation, compared to the control test. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. Table S8 Western blot analysis of Dnmt3A quantified with Image J. program. Average±SD represents the average protein expression±standard deviation, compared to the control test. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. Table S9 Western blot analysis of Dnmt3B quantified with Image J. program. Average±SD represents the average protein expression±standard deviation, compared to the control test. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. Table S10 Western blot analysis of MeCP2 quantified with Image J. program. Average±SD represents the average protein expression±standard deviation, compared to the control test. Asterisks identify significant increases from non-exposed controls. (DOCX) Click here for additional data file. We thank Valentina Titova for proofreading the manuscript. ==== Refs References 1 Parkin DM , Bray F , Ferlay J , Pisani P (2005 ) Global cancer statistics, 2002 . CA Cancer J Clin 55 : 74 –108 .15761078 2 Suerbaum S , Michetti P (2002 ) Helicobacter pylori infection . N Engl J Med 347 : 1175 –1186 .12374879 3 Horie H , Kanazawa K , Kobayashi E , Okada M , Fujimura A , et al (1999 ) Effects of intestinal bacteria on the development of colonic neoplasm II. Changes in the immunological environment . Eur J Cancer Prev 8 : 533 –537 .10643943 4 Ferrero RL , Ave P , Radcliff FJ , Labigne A , Huerre MR (2000 ) Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions . J Pathol 191 : 333 –340 .10878557 5 Rutegard JN , Ahsgren LR , Janunger KG (1988 ) Ulcerative colitis. Colorectal cancer risk in an unselected population . Ann Surg 208 : 721 –724 .3196092 6 Yamamoto M , Wu HH , Momose H , Rademaker A , Oyasu R (1992 ) Marked enhancement of rat urinary bladder carcinogenesis by heat-killed Escherichia coli . Cancer Res 52 : 5329 –5333 .1394138 7 Gannon VP , Duke GD , Thomas JE , Vanleeuwen J , Byrne J , et al (2005 ) Use of in-stream reservoirs to reduce bacterial contamination of rural watersheds . Sci Total Environ 348 : 19 –31 .16162311 8 Jirillo E , Caccavo D , Magrone T , Piccigallo E , Amati L , et al (2002 ) The role of the liver in the response to LPS: experimental and clinical findings . J Endotoxin Res 8 : 319 –327 .12537690 9 Koturbash I , Thomas JE , Kovalchuk O , Kovalchuk I (2009 ) Heat-killed bacteria induce genome instability in mouse small intestine, liver and spleen tissues . Cell Cycle 8 : 1935 –1939 .19440049 10 Falck J , Coates J , Jackson SP (2005 ) Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage . Nature 434 : 605 –611 .15758953 11 Lirussi L , Antoniali G , Vascotto C , D'Ambrosio C , Poletto M , et al (2012 ) Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells . Mol Biol Cell 23 : 4079 –4096 .22918947 12 Naryzhny SN , Zhao H , Lee H (2005 ) Proliferating cell nuclear antigen (PCNA) may function as a double homotrimer complex in the mammalian cell . J Biol Chem 280 : 13888 –13894 .15805117 13 Hervouet E , Lalier L , Debien E , Cheray M , Geairon A , et al (2010 ) Disruption of Dnmt1/PCNA/UHRF1 interactions promotes tumorigenesis from human and mice glial cells . PLoS One 5 : e11333 .20613874 14 Majumder S , Ghoshal K , Datta J , Bai S , Dong X , et al (2002 ) Role of de novo DNA methyltransferases and methyl CpG-binding proteins in gene silencing in a rat hepatoma . J Biol Chem 277 : 16048 –16058 .11844796 15 Zubkov MV , Fuchs BM , Eilers H , Burkill PH , Amann R (1999 ) Determination of total protein content of bacterial cells by SYPRO staining and flow cytometry . Appl Environ Microbiol 65 : 3251 –3257 .10388732 16 Darveau RP , Hancock RE (1983 ) Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains . J Bacteriol 155 : 831 –838 .6409884 17 Koturbash I , Rugo RE , Hendricks CA , Loree J , Thibault B , et al (2006 ) Irradiation induces DNA damage and modulates epigenetic effectors in distant bystander tissue in vivo . Oncogene 25 : 4267 –4275 .16532033 18 Koturbash I , Baker M , Loree J , Kutanzi K , Hudson D , et al (2006 ) Epigenetic dysregulation underlies radiation-induced transgenerational genome instability in vivo . Int J Radiat Oncol Biol Phys 66 : 327 –330 .16965987 19 Blanco-Rodriguez J (2012 ) Programmed phosphorylation of histone H2AX precedes a phase of DNA double-strand break-independent synapsis in mouse meiosis . Reproduction 144 : 699 –712 .23035256 20 Kocazeybek B (2003 ) Chronic Chlamydophila pneumoniae infection in lung cancer, a risk factor: a case-control study . J Med Microbiol 52 : 721 –726 .12867569 21 Burma S , Chen BP , Murphy M , Kurimasa A , Chen DJ (2001 ) ATM phosphorylates histone H2AX in response to DNA double-strand breaks . J Biol Chem 276 : 42462 –42467 .11571274 22 Wadleigh DJ , Reddy ST , Kopp E , Ghosh S , Herschman HR (2000 ) Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages . The Journal of biological chemistry 275 : 6259 –6266 .10692422 23 Baeuerle PA , Baltimore D (1996 ) NF-kappa B: ten years after . Cell 87 : 13 –20 .8858144 24 Um JH , Kang CD , Lee BG , Kim DW , Chung BS , et al (2001 ) Increased and correlated nuclear factor-kappa B and Ku autoantigen activities are associated with development of multidrug resistance . Oncogene 20 : 6048 –6056 .11593412 25 Lim JW , Kim H , Kim KH (2001 ) Nuclear factor-kappaB regulates cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells . Lab Invest 81 : 349 –360 .11310828 26 Sanlioglu S , Williams CM , Samavati L , Butler NS , Wang G , et al (2001 ) Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa B. J Biol Chem . 276 : 30188 –30198 . 27 Jackson GD , Berkovic SF , Tress BM , Kalnins RM , Fabinyi GC , et al (1990 ) Hippocampal sclerosis can be reliably detected by magnetic resonance imaging . Neurology 40 : 1869 –1875 .2247236 28 Reymond D , Johnson RP , Karmali MA , Petric M , Winkler M , et al (1996 ) Neutralizing antibodies to Escherichia coli Vero cytotoxin 1 and antibodies to O157 lipopolysaccharide in healthy farm family members and urban residents . J Clin Microbiol 34 : 2053 –2057 .8862557 29 Gagou ME , Zuazua-Villar P , Meuth M (2010 ) Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1 . Mol Biol Cell 21 : 739 –752 .20053681 30 Chahrour M , Jung SY , Shaw C , Zhou X , Wong STC , et al (2008 ) MeCP2, a Key Contributor to Neurological Disease, Activates and Represses Transcription . Science 320 : 1224 –1229 .18511691 31 Yan J , Zhang M , Zhang J , Chen X , Zhang X (2011 ) Helicobacter pylori infection promotes methylation of WWOX gene in human gastric cancer . Biochem Biophys Res Commun 408 : 99 –102 .21466786 32 Huang L , Zhang KL , Li H , Chen XY , Kong QY , et al (2006 ) Infrequent COX-2 expression due to promoter hypermethylation in gastric cancers in Dalian, China . Hum Pathol 37 : 1557 –1567 .16949912 33 Luo J , Yu Y , Chang S , Tian F , Zhang H , et al (2012 ) DNA Methylation Fluctuation Induced by Virus Infection Differs between MD-resistant and -susceptible Chickens . Front Genet 3 : 20 .22363343 34 Leonard S , Wei W , Anderton J , Vockerodt M , Rowe M , et al (2011 ) Epigenetic and transcriptional changes which follow Epstein-Barr virus infection of germinal center B cells and their relevance to the pathogenesis of Hodgkin's lymphoma . J Virol 85 : 9568 –9577 .21752916 35 Tang B , Zhao R , Sun Y , Zhu Y , Zhong J , et al (2011 ) Interleukin-6 expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsRNA treatment . Mol Immunol 48 : 1001 –1008 .21353307 36 Abraham SM , Clark AR (2006 ) Dual-specificity phosphatase 1: a critical regulator of innate immune responses . Biochem Soc Trans 34 : 1018 –1023 .17073741 37 Hammer M , Mages J , Dietrich H , Servatius A , Howells N , et al (2006 ) Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock . J Exp Med 203 : 15 –20 .16380512 38 Abell AN , Granger DA , Johnson GL (2007 ) MEKK4 stimulation of p38 and JNK activity is negatively regulated by GSK3beta . J Biol Chem 282 : 30476 –30484 .17726008 39 Ip YT , Davis RJ (1998 ) Signal transduction by the c-Jun N-terminal kinase (JNK) – from inflammation to development . Curr Opin Cell Biol 10 : 205 –219 .9561845 40 Paulsen FP , Woon CW , Varoga D , Jansen A , Garreis F , et al (2008 ) Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds . J Biol Chem 283 : 13418 –13427 .18326859 41 Bechard D , Scherpereel A , Hammad H , Gentina T , Tsicopoulos A , et al (2001 ) Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1 . J Immunol 167 : 3099 –3106 .11544294 42 Holt JA , Luo G , Billin AN , Bisi J , McNeill YY , et al (2003 ) Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis . Genes Dev 17 : 1581 –1591 .12815072 43 Griener TP , Strecker JG , Humphries RM , Mulvey GL , Fuentealba C , et al (2011 ) Lipopolysaccharide renders transgenic mice expressing human serum amyloid P component sensitive to Shiga toxin 2 . PLoS One 6 : e21457 .21731756 44 Murata A , Shimazu T , Yamamoto T , Taenaka N , Nagayama K , et al (1998 ) Profiles of circulating inflammatory- and anti-inflammatory cytokines in patients with hemolytic uremic syndrome due to E. coli O157 infection . Cytokine 10 : 544 –548 .9702419 45 Kovalchuk I, Walz P, Thomas J, Kovalchuk O. (2013) The increased expression of proteins involved in proliferation, DNA repair and DNA methylation in spleen of mice exposed to E. coli O157:H7 lipopolysaccharide. Environ Mol Mutagen, in press.	23874414	PMC3706549	CC BY	2021-01-05 17:32:34	yes	PLoS One. 2013 Jul 9; 8(7):e67342
==== Front Biol OpenBiol OpenbiolopenbioBiology Open2046-6390The Company of Biologists Bidder Building, 140 Cowley Road, Cambridge, CB4 0DL, UK 23862016BIO2013474710.1242/bio.20134747Research ArticleTargeting of vasoactive intestinal peptide receptor 2, VPAC2, a secretin family G-protein coupled receptor, to primary cilia Soetedjo Livana Glover De'Vona A. Jin Hua Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA Author for correspondence (huajin@uic.edu)15 7 2013 23 5 2013 2 7 686 694 11 3 2013 24 4 2013 © 2013. Published by The Company of Biologists Ltd2013This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.Summary Primary cilia protrude from the cell surface of many cell types in the human body and function as cellular antennae via ciliary membrane localized receptors. Neurons and glial cells in the brain possess primary cilia, and the malfunction of primary cilia may contribute to neurological deficits present in many cilia-associated disorders. Several rhodopsin family G-protein coupled receptors (GPCRs) are specifically localized to a subset of neuronal primary cilia. However, whether other family GPCRs target to neuronal cilia and whether glial primary cilia harbor any GPCRs are not known. We conducted a screening of GPCRs to determine their ability to target to primary cilia, and identified a secretin family member, Vasoactive Intestinal Receptor 2 (VPAC2), as a novel ciliary GPCR. Here, we show that endogenous VPAC2 targets to primary cilia in various brain regions, including the suprachiasmatic nuclei and the thalamus. Surprisingly, VPAC2 not only localizes to neuronal cilia but also to glial cilia. In addition, we show that VPAC2's C-terminus is both necessary and sufficient for its ciliary targeting and we define a novel ciliary targeting signal: the tetrapeptide RDYR motif in the C-terminus of VPAC2. Furthermore, we demonstrate that VPAC2 ciliary targeting is dependent on Tubby, the BBSome (a complex of Bardet–Biedl syndrome proteins) and the BBSome targeting factor, Arl6. Primary ciliaVPAC2VIPR2Ciliary GPCRNeuronal ciliaGlial cilia ==== Body Introduction Primary cilia are typically solitary, immotile microtubule-based organelles present in many cell types in the mammalian body (Berbari et al., 2009). They harbor membrane receptors and their downstream targets, and therefore function as signaling hubs (Garcia-Gonzalo and Reiter, 2012; Nachury et al., 2010; Pazour and Bloodgood, 2008). Defects in the structure or function of primary cilia lead to ciliopathies with pleotropic phenotypes including cognitive impairment. The fact that clinical features of many ciliopathies include neurological deficits supports the notion that primary cilia play a role in brain function (Green and Mykytyn, 2010; Lee and Gleeson, 2011; Lee and Gleeson, 2010; Louvi and Grove, 2011). However, the lack of a complete catalog of ciliary components, especially membrane receptors, has impeded our understanding of signaling pathways mediated by cilia in the brain. Most neurons in the brain possess a primary cilium (Bishop et al., 2007). A subset of neuronal cilia harbor certain G-protein coupled receptors (GPCRs) including somatostatin receptor 3 (SSTR3) (Händel et al., 1999), serotonin receptor 6 (5HT6) (Brailov et al., 2000; Hamon et al., 1999), melanin-concentrating hormone receptor 1 (MCHR1) (Berbari et al., 2008a; Berbari et al., 2008b), and dopamine receptor 1 (Domire et al., 2011). A recent study showed that GPR161, an orphan rhodopsin family GPCR, targets to neuronal cilia in primary hippocampal neuron culture (Mukhopadhyay et al., 2013). Interestingly, all ciliary GPCRs identified thus far belong to the rhodopsin family; whether GPCRs from other families target to neuronal primary cilia is not currently known. Primary cilia in the brain are found in glia as well. Astrocytes (Bishop et al., 2007; Berbari et al., 2007; Yoshimura et al., 2011) and oligodendrocytes (Cenacchi et al., 1996; Louvi and Grove, 2011), but not microglia (Bishop et al., 2007; Sarkisian et al., 2013), have been shown to possess a primary cilium. Interestingly, SSTR3 proteins have so far only been detected in neuronal cilia (Berbari et al., 2007) and little is known regarding the expression and distribution of other ciliary GPCRs in glial cells (Sarkisian et al., 2013). To expand the catalog of ciliary GPCRs in the brain, we performed an initial screening to identify GPCRs that can target to primary cilia in their GFP-tagged form and identified six GPCRs with this ability: Vasoactive Intestinal Peptide Receptor 2 (VPAC2, also known as VIPR2), Gastric Inhibitory Polypeptide Receptor (GIPR), G-protein coupled receptor 45 (GPR45), GPR63, GPR75 and GPR83. We showed that endogenous VPAC2, a secretin family GPCR, localizes to primary cilia in various brain regions including the thalamus and the suprachiasmatic nuclei (SCN). VPAC2 plays important roles in the control of mammalian circadian rhythms in the SCN. Mice lacking VPAC2 show altered circadian rhythms in locomotor behavior, neuronal firing and clock gene expression (Aton et al., 2005; Cutler et al., 2003; Harmar et al., 2002; Maywood et al., 2006). Recent studies have also shown that duplication of the VPAC2 gene, and the resulting higher than normal VPAC2 signaling in patients confer a significant risk to schizophrenia (Beri et al., 2012; Levinson et al., 2011; Vacic et al., 2011). We demonstrate that VPAC2 localizes to primary cilia not only in neurons but also in glial cells, including astrocytes and oligodendrocyte lineage cells. In addition, we showed that the C-terminus of VPAC2 is sufficient to target a non-ciliary membrane protein to cilia, and that a single amino acid mutation of the tetrapeptide RDYR in the C-terminus completely abolishes VPAC2 ciliary targeting. Furthermore, we show that the BBSome (a complex of Bardet–Biedl syndrome proteins), the BBSome targeting factor Arl6 and Tubby are important for ciliary targeting of endogenous VPAC2. Results Identification of novel ciliary GPCRs To expand the catalog of ciliary GPCRs, we screened 122 non-olfactory GPCRs for their ability to target to primary cilia (for full list, see supplementary material Table S1). We transiently transfected GFP-tagged human or mouse GPCR constructs in mouse inner medullary collecting duct (IMCD3) cells and/or primary neuron cultures. Among those tested, we found two secretin family GPCRs (VPAC2 and GIPR) and four rhodopsin family orphan GPCRs (GPR45, GPR63, GPR75 and GPR83) that target to primary cilia (Fig. 1, Fig. 2A,B). Although known to couple with the same group of heterotrimeric G proteins, the secretin family GPCRs lack the classical signature sequences found in most members of the rhodopsin family GPCRs. By evaluating protein sequences, it has been proposed that the helical bundle of secretin family GPCRs is structurally distinct from that of rhodopsin family GPCRs (Foord et al., 2005; Fredriksson et al., 2003). Therefore, we suspected that VPAC2 and GIPR might have different ciliary targeting mechanisms compared to those of known ciliary GPCRs. Fig. 1. Identification of novel ciliary GPCRs. GIPR-GFP, GPR45-GFP, GPR63-GFP, GPR75-GFP and GPR83-GFP target to primary cilia in IMCD3 cells. IMCD3 cells were transfected with plasmids expressing GIPR-GFP, GPR45-GFP, GPR63-GFP, GPR75-GFP or GPR83-GFP and serum starved to induce ciliogenesis. Cells were immunostained for acetylated α-tubulin (AcTubulin, red). DNA was labeled with mounting medium containing 4′,6-diamidino-2-phenylinodole (DAPI). Insets show unmerged images of the region around cilia. Scale bars: 5 µm. Fig. 2. Endogenous VPAC2 localizes to primary cilia. (A,B) VPAC2-GFP and GFP-VPAC2 target to IMCD3 primary cilia. IMCD3 cells expressing VPAC2-GFP (A) or GFP-VPAC2 (B) were serum starved to induce ciliogenesis and immunostained for acetylated α-tubulin (AcTubulin, red). (C,D) Endogenous VPAC2 localizes to primary cilia in the thalamus and SCN regions of the rat brain. Rat thalamus (C) and SCN (D) cryosections were fixed and immunostained for VPAC2 (green) and Arl13B (red). Seventeen sections at 0.2 µm intervals were projected using sum over z-axis. (E) VPAC2 mRNA levels were reduced by ∼94% in thalamic cultures infected with lentivirus containing VPAC2 shRNAmiR #1 or VPAC2 shRNAmiR #2 compared to cells infected with control lentivirus. mRNA levels were measured by RT-qPCR. Error bars represent the standard deviation (SD) of triplicate PCR assays. **P<0.001. GAPDH levels were used as an internal control for normalization. (F,G) VPAC2 localizes to primary cilia in thalamic neuron culture. Thalamic neuron culture was infected with lentivirus containing control shRNAmiR (F) or shRNAmiRs against VPAC2 (G) and immunostained for VPAC2 (green) and Arl13B (red). (H) Thalamic neuron culture cells were prepared and stained as in (F,G). Approximately 450 cilia were counted and the percentage of VPAC2 positive cilia was plotted. Error bars represent the SD among three different coverslips. (I,J) VPAC2 localizes to primary cilia in SCN neuron culture. SCN neuron culture was infected with lentivirus containing control shRNAmiR (I) or shRNAmiRs against VPAC2 (J) and immunostained for VPAC2 (green) and Arl13B (red). (K) SCN neuron culture cells were prepared and stained as in (I,J). Approximately 200 cilia were counted and the percentage of VPAC2 positive cilia was plotted. Error bars represent the SD among microscopic fields. (A–D,F,G,I,J) DNA was labeled with DAPI. Insets show unmerged images of the region around a cilium. Scale bars: 5 µm. Endogenous VPAC2 localizes to primary cilia in the brain We first investigated whether endogenous VPAC2 and GIPR localize to primary cilia. We were unable to find an antibody against GIPR that could be validated in vivo, therefore we focused on VPAC2 and studied its ciliary trafficking exclusively. In the brain, VPAC2 is strongly expressed in several regions including the suprachiasmatic nuclei (SCN) and the thalamus (Sheward et al., 1995). We co-stained brain cryosections from 7-day-old rat pups with antibodies against VPAC2 and a ciliary marker, Arl13B (Caspary et al., 2007) and observed that the anti-VPAC2 antibody stained cilia in various brain regions including the thalamus and SCN (Fig. 2C,D). The anti-VPAC2 antibody also stained primary cilia in both thalamic and SCN neuron cultures (Fig. 2F,I). Cilium staining was lost when endogenous VPAC2 was depleted using lentivirus-mediated constructs that encode synthetic small hairpin RNA sequences within the context of a microRNA (shRNAmiR) (Fig. 2F–K). The efficiency of shRNAmiR-mediated VPAC2 knockdown was assessed using RT-qPCR (Fig. 2E). Both shRNAmiRs resulted in over a 94% knockdown of endogenous VPAC2 mRNA expression. Taken together, our results show that endogenous VPAC2 localizes to primary cilia in various brain regions and primary cultures. Endogenous VPAC2 localizes to primary cilia of neurons and glial cells To determine whether VPAC2 targets to neuronal cilia, we co-stained primary thalamic culture with antibodies against VPAC2, Arl13B and a neuronal marker, MAP2. We found that 42% of MAP2 positive cells had primary cilia and 93% of those cilia contained VPAC2 (Fig. 3A). Although our thalamic neurons were cultured under serum-free conditions, a significant number of cells were negative for MAP2, indicating that non-neuronal cells were present in our culture. Interestingly, we detected VPAC2-containing cilia from some of the MAP2 negative cells (Fig. 3A), suggesting that VPAC2 targets to primary cilia in non-neuronal cells. Fig. 3. Endogenous VPAC2 localizes to primary cilia of neurons and glial cells. (A) VPAC2 localizes to primary cilia in neuronal (MAP2+) and non-neuronal (MAP2−) cilia in primary thalamic culture. Primary thalamic culture (DIV9) was immunostained for VPAC2 (green), MAP2 (white) and Arl13B (red). (B) VPAC2 localizes to primary cilia of astrocytes (GFAP+) in primary thalamic culture. Primary thalamic culture (DIV9) was immunostained for VPAC2 (green), GFAP (white), and Alr13B (red). (C) VPAC2 localizes to primary cilia of astrocytes (Aldh1L1+/GFAP+) in primary thalamic culture. Primary thalamic culture (DIV9) was immunostained for VPAC2 (green), GFAP (white), and Arl13B (red). (D) VPAC2 localizes to primary cilia of astrocytes (Aldh1L1+) in vivo. Brain cryosections from 7-day-old rat pups were fixed and immunostained for VPAC2 (green) and Aldh1L1 (red). (E,F) VPAC2 localizes to primary cilia of oligodendrocytes (Sox10+ or NG2+) in primary thalamic culture. Primary thalamic culture (DIV7) was immunostained for VPAC2 (green), Sox10 (white) and Arl13B (red) in panel E and VPAC2 (green), NG2 (white) and Arl13B (red) in panel F. (G) Microglial cells labeled with Iba1 do not possess VPAC2-positive cilia in primary thalamic culture. Primary thalamic culture (DIV7) was immunostained for VPAC2 (green), Iba1 (white) and Arl13B (red). (A–G) DNA was labeled with DAPI. Scale bars: 5 µm. To determine whether VPAC2 targets to primary cilia of glia, we stained cells with various glial cell markers. For astrocytes, we used antibodies against the “traditional” marker GFAP as well as Aldh1L1, a highly specific antigenic marker for astrocytes (Cahoy et al., 2008; Yang, Y. et al., 2011). For oligodendrocyte lineage cells, we used antibodies against Sox10, which is only expressed by oligodendrocyte progenitor cells and oligodendrocytes in the brain (Maka et al., 2005; Kang et al., 2010) and NG2, which is a marker for oligodendrocyte progenitor cells (Stallcup et al., 1990; Kang et al., 2010). For microglia, we used an antibody against Iba1, a microglia-specific calcium-binding protein (Imai et al., 1996). Of the cells that were positive for GFAP (Fig. 3B) or for both GFAP and Aldh1L1 (Fig. 3C), approximately 53% of them possessed VPAC2-positive cilia. Furthermore, we detected VPAC2-positive cilia in Aldh1L1+ cells from brain cryosections of 7-day-old rat pups (Fig. 3D). Taken together, we concluded that VPAC2 targets to primary cilia of astrocytes in vivo and in vitro. Under our culture conditions, less than 1% of cells were Sox10 or NG2 positive, while ∼10% of those cells had primary cilia and 50% of those cilia were VPAC2-positive (Fig. 3E,F). We failed to detect any Arl13B-positive cilia in cells co-labeled with Iba1 (Fig. 3G), consistent with previous reports that microglia may not possess a primary cilium (Bishop et al., 2007; Sarkisian et al., 2013). Together, our results suggest that VPAC2 localizes to primary cilia in both neurons and glial cells such as astrocytes and oligodendrocyte lineage cells. The C-terminus is sufficient and necessary for VPAC2 ciliary targeting As VPAC2 belongs to the secretin family, which does not share sequence homology with the rhodopsin family, we suspected that VPAC2 might have a different ciliary targeting mechanism compared to known rhodopsin family GPCRs. To study the ciliary targeting of VPAC2, we first tested two VPAC2 homologs for their ability to target to the primary cilium: Vasoactive Intestinal Peptide Receptor 1 (VPAC1, also known as VIPR1) and Pituitary Adenylate Cyclase-activating Polypeptide type 1 Receptor (PAC1, also known as ADCYAP1R1). Neither human VPAC1-GFP (hVPAC1-GFP) nor hPAC1-GFP targeted to IMCD3 primary cilia (Fig. 4A). Since most of the differences between VPAC2 and its homologs are concentrated in their C-terminal tails (supplementary material Fig. S1), we tested whether the C-terminus of VPAC2 is sufficient to target the non-ciliary membrane protein CD8α to cilia (Fig. 4B). CD8α is a well-characterized non-ciliary membrane protein that has been used in chimeras to identify ciliary targeting domains (Follit et al., 2010). As shown in Fig. 4D, a chimera protein CD8α-hVPAC2-Cterm-GFP containing the extracellular and transmembrane domains of CD8α and the C-terminus of VPAC2 targeted to primary cilia, while CD8α-GFP, CD8α-hVPAC1-Cterm-GFP, and CD8α-hPAC1-Cterm-GFP failed to do so (Fig. 4C,F,G). The hVPAC2-Cterm contains 58 amino acids with the last 44 amino acids differing significantly from those of VPAC1 and PAC1 (supplementary material Fig. S1). Interestingly, a chimera of CD8α-hVPAC2-Cterm44-GFP also targeted to primary cilia (Fig. 4E). Together, these results strongly suggest that the last 44 amino acids of VPAC2 are sufficient for ciliary targeting. Fig. 4. The C-terminus is sufficient and necessary for VPAC2 ciliary targeting. (A) VPAC2-GFP (but not VPAC1-GFP and PAC1-GFP) targets to primary cilia in IMCD3 cells. IMCD3 cells were transduced with lentivirus vectors expressing VPAC2-GFP, VPAC1-GFP or PAC1-GFP and were serum starved to induce ciliogenesis. Cells were immunostained for acetylated α-tubulin (AcTubulin, red). (B) Schematic of CD8α chimeras. (C–G) The last 44 amino acids of VPAC2's C-terminus are sufficient to target the non-ciliary membrane protein CD8α to primary cilia. IMCD3 cells expressing GFP-tagged CD8α, CD8α-VPAC2-Cterm, CD8α-VPAC2-Cterm44, CD8α-PAC1-Cterm and CD8α-VPAC1-Cterm were serum starved and immunostained for AcTubulin (red). DNA was labeled with DAPI (C–G). Insets show an unmerged image of the region around the cilium. Scale bars: 5 µm. RDYR motif at the C-terminus is required for VPAC2 ciliary targeting We next sought to determine which residues of VPAC2's C-terminus are required for VPAC2 ciliary targeting. This region does not contain a known ciliary targeting signal (CTS): an Ax[S/A]xQ motif identified in most neuronal ciliary GPCRs including SSTR3, 5HT6, MCHR1 and D1 (Berbari et al., 2008a; Domire et al., 2011), an [I/V]KARK motif found in GPR161, or a VxP motif that has been proposed to be a general CTS (Deretic et al., 2005; Geng et al., 2006). To identify the CTS of VPAC2, we conducted alanine-scanning mutagenesis of the last 44 amino acids of VPAC2, and tested their subcellular localization in IMCD3 cells. Most alanine mutations did not affect ciliary targeting of VPAC2, with the exception of the RDYR to ADAA mutation that completely abolished VPAC2 ciliary targeting (Fig. 5A; supplementary material Fig. S2). RDYR is a highly conserved tetrapeptide motif in VPAC2 proteins, from zebrafish to human (supplementary material Fig. S1). Interestingly, we found that single mutations in the RDYR motif resulted in the complete failure of VPAC2 targeting to cilia (supplementary material Fig. S3). We further confirmed these results in SCN neuron culture (Fig. 5B–G). Thus, the RDYR motif is necessary for ciliary targeting of VPAC2. Fig. 5. The RDYR motif is necessary for VPAC2 ciliary targeting. (A) Summary of the alanine-scanning mutagenesis performed for the last 44 amino acids of the VPAC2 C-terminus. IMCD3 cells expressing GFP tagged VPAC2 harboring indicated alanine mutations (GFP tag is in the N-terminus) were serum starved (supplementary material Fig. S2). The RDYR→ADAA mutation but not others abolished ciliary localization of GFP tagged VPAC2. (B) VPAC2-GFP localizes to primary cilia in SCN cells. SCN cells expressing VPAC2-GFP were immunostained for MAP2 (white) and Arl13B (red). (C–G) RDYR motif is required for VPAC2 ciliary targeting. SCN cells expressing VPAC2 harboring the RDYR→ADAA mutation or single mutations in the RDYR motif were immunostained for MAP2 (white) and Arl13B (red). DNA was labeled with DAPI (B–G). Insets show an unmerged image of the region around the cilium. Scale bars: 5 µm. The BBSome, Arl6 and Tubby are important for ciliary localization of VPAC2 Several proteins and protein complexes including the BBSome, the BBSome targeting factor Arl6 and Tubby have been shown to play key roles in neuronal cilia localization of endogenous SSTR3 and/or MCHR1 (Berbari et al., 2008b; Jin et al., 2010; Sun et al., 2012). To test whether the same targeting machinery is used by VPAC2, we depleted BBS2 (a BBSome subunit), Arl6 and Tubby by lentivirus-mediated RNAi in thalamic neuron culture. BBS2 and Tubby shRNAmiRs showed more than a 93% reduction in mRNA levels (Fig. 6A,B). To knockdown Arl6 in neuronal cultures, we used mArl6shRNAs, which have been shown to effectively knockdown Arl6 in mouse neuronal cells (Jin et al., 2010), and confirmed that they lowered the Arl6 protein level (Fig. 6C). As shown in Fig. 6D–G, knocking down Arl6, BBS2 or Tubby abolished ciliary targeting of VPAC2. Thus, Tubby and the BBSome/Arl6 are important for ciliary targeting of endogenous VPAC2. Fig. 6. The BBSome, Arl6 and Tubby are required for VPAC2 ciliary targeting. (A,B) BBS2 and Tubby mRNA levels were reduced by more than 93% in rat thalamic cultures infected with lentivirus containing shRNAmiR against BBS2 (A) or Tubby (B) compared to cells infected with control lentivirus. mRNA levels were measured by RT-qPCR. Error bars represent the SD of triplicate PCR assays. *P<0.001. GAPDH levels were used as an internal control for normalization. (C) Protein extracts from mouse thalamic cultures infected with control shRNA or Arl6 shRNAs were immunoblotted for Arl6 and actin (loading control). (D) Tubby and BBS2 are required for VPAC2 ciliary targeting. Rat thalamic cells were infected with lentivirus containing control shRNAmiR and shRNAmiRs against Tubby or BBS2 and immunostained for VPAC2 (green) and Arl13B (red). (E) Thalamic neuron culture cells were prepared and stained as in panel D. Approximately 500 cilia were counted and the percentage of VPAC2 positive cilia was plotted. (F) Knockdown of Arl6 affects VPAC2 ciliary targeting. Thalamic cultures were infected with control shRNA or Arl6 shRNAs or immunostained for VPAC2 (green) and Arl13B (red). (G) Arl6 is required for VPAC2 ciliary targeting. Rat thalamic neuron culture cells were prepared and stained as in panel F. Approximately 600 cilia were counted and the percentage of VPAC2 positive cilia was plotted. Error bars represent the SD among three different coverslips (E,G). *P<0.001. DNA was labeled with DAPI <2?show=[to]?>(D,F). Insets show unmerged images of the region around a cilium highlighted with a white arrow. Scale bars: 5 µm. Discussion We expanded the catalog of ciliary GPCRs and demonstrated for the first time that a secretin family GPCR, VPAC2, localizes to primary cilia not only in neurons but also in glial cells including astrocytes and oligodendrocytes (Fig. 3), thereby implicating the involvement of both neuronal and glial primary cilia in VPAC2-mediated signaling. We showed that VPAC2 localizes to primary cilia in various brain regions including the SCN and the thalamus (Fig. 2). The fact that VPAC2 localizes to cilia in the SCN implies that ciliary VPAC2 signaling may play a role in regulating circadian rhythm. Although there has been no prior investigation of a circadian rhythm phenotype in BBS or Tubby mutant mice or mice lacking cilia in the brain, many ciliary defect-associated pathological conditions such as obesity, hypertension and psychotic conditions are associated with abnormalities in circadian rhythms. The fact that VPAC2 localizes to cilia in various brain regions suggests that abnormal ciliary VPAC2 signaling may contribute to schizophrenia in patients with a gene duplication of VPAC2. This is in line with recent findings suggesting psychiatric diseases such as schizophrenia may result from defects in primary cilia-dependent signaling (Doherty, 2009; Marley and von Zastrow, 2010; Marley and von Zastrow, 2012). Unlike SSTR3, 5HT6 and MCHR1, the C-terminus of VPAC2 is sufficient to target a non-ciliary membrane protein, CD8α, to primary cilia (Fig. 4). We further identified a novel ciliary targeting signal, the tetrapeptide RDYR motif in the C-terminus of VPAC2, and showed that single amino acid mutations in this motif abolish ciliary targeting (Fig. 5). Interestingly, although VPAC2 belongs to the secretin family, and contains a different CTS compared to that of SSTR3 and MCHR1, VPAC2 ciliary targeting is dependent on the same set of proteins including the BBSome, Arl6 and Tubby (Fig. 6). As Bardet–Biedl syndrome (BBS), is most likely the result of ciliary protein targeting failure (Jin et al., 2010), the failure to traffic VPAC2 to cilia might contribute to the pleiotropic phenotype exhibited by BBS patients. Whether ciliary targeting of other GPCRs (GIPR, GPR45, GPR63, GPR75 and GPR83) is also dependent on the BBSome, Arl6 or Tubby remains unknown, and requires further investigation. As VPAC2-mediated signaling may be important for maintaining circadian rhythms and normal brain function, our research on VPAC2 ciliary trafficking provides a new avenue for future investigations into the role of ciliary VPAC2 signaling in the control of circadian rhythms and in psychotic diseases such as schizophrenia. Materials and Methods Animals Mice and rats were purchased from Charles Rivers (Wilmington, MA, USA). Animal care and use was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and was approved by the Institutional Laboratory Animal Care and Use Committee at the University of Illinois at Chicago. Plasmids Human VPAC2 and PAC1 ORFs (BC010569, BC117116) were obtained from the human ORFeome collection (Open Biosystems, Waltham, MA, USA). VPAC1 (BC064424) was obtained from the human ORFeome v8.1 collection (Yang, X. et al., 2011). The CD8α chimeras were generated by replacing CD8α C-terminal amino acids (aa) 213–235 with the C-termini of VPAC2 (aa 380–438), VPAC1 (aa 394–457), PAC1 (aa 406–468) and the partial C-terminus of VPAC2 (aa 395–438) (for primers used to construct CD8α chimeras, please refer to supplementary material Table S2). VPAC2 alanine scanning mutants were generated with Phusion DNA Polymerase. All constructs for transient transfection were subcloned into pEF5α.FRT derivatives and all lentivirus expression constructs were subcloned into a pCDH.EF1 derivative (System Biosciences, Mountain View, CA, USA). DNA constructs were confirmed by restriction digestion and sequencing (RRC-DNAS, Chicago, IL, USA). Cell culture IMCD3 and 293FT cells were maintained in DMEM:F12 (IMCD3) or DMEM high glucose (293FT) supplemented with 10% Fetal Bovine Serum, 100 units per ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO2. Plasmid transfections in IMCD3 cells were performed with Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA). IMCD3 cells were serum starved in DMEM:F12 and supplemented with 0.2% Fetal Bovine Serum, 100 units per ml penicillin and 100 µg/ml streptomycin, 16–24 hours after transfection to induce ciliogenesis. Cells were examined between 40–48 hours post transfection. For SCN cultures, P1–P5 rat (Sprague Dawley) pups were decapitated, their brains dissected and placed in Hank's Balanced Salt Solution (Sigma, St. Louis, MO, USA). Three hundred µm coronal sections were cut using a tissue slicer from Stoelting (Wood Dale, IL, USA). Under a dissection microscope, the sections containing the mid-part of the SCN were selected and the SCN region was cut out using a scalpel. Cells were enzymatically digested with papain (Worthington, Lakewood, NJ, USA) for 45 min at 37°C. Cells were then triturated and 20 µl of the cell suspension containing 5×104 cells were placed over coverslips (12 mm diameter) coated with PDL and Laminin (BD Biosciences, San Jose, CA, USA). One ml of NBactiv4 (BrainBits, Springfield, IL, USA) was added after 30 min. The medium was changed once every two to three days. Cells were transfected with Lipofectamine 2000 on days in vitro 5 (DIV5) and examined on DIV7. For thalamic cultures, P1 mouse or rat pups (CD-1 mouse or Sprague Dawley rat) were decapitated and their thalami were dissected. Cells were enzymatically digested with papain for 45 min at 37°C. Cells were then triturated and 20 µl cell suspension containing 2×104 cells were placed over the coverslip (12 mm diameter) coated with PDL (Sigma P7405). One ml of NBactiv4 was added after 30 min. The medium was changed once every four to five days. Cells were transfected with Lipofectamine 2000 on DIV5 and examined on DIV7. Cryosection P7 rats (Wistar rat) were decapitated and their brains were dissected, then washed in phosphate buffered saline (PBS) briefly, and immersed in 4% paraformaldehyde at 4°C overnight. Fixed specimens were then immersed in 15% sucrose (w/v) diluted in PBS for 48 hours and 30% sucrose (w/v) for 24 hours. All tissues were placed in Tissue Tek OCT (Sakura Finetek, Torrance, CA) and frozen over liquid nitrogen. Seven µm coronal sections were cut at −20°C using a Vacutome Cryostat (Microm International GmbH, Waldorf, Germany) and collected on 3-Aminopropyltriethoxysilane coated slides. Immunofluorescence and microscopy IMCD3, SCN and thalamic cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then blocked with 5% normal donkey serum for IMCD3 cells (2% for neuron cultures) in buffer containing 0.1% Triton X-100 for 40 min at room temperature. Primary antibodies were then applied for one hour at room temperature, and secondary antibodies (Alexa Fluor 488, 546 or 647, Life Technologies) were applied for 30 min at room temperature. Coverslips were then mounted using Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Cryosections were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C. Sections were then blocked with 5% normal donkey serum in PBST (PBS with 0.2% Tween-20) for 40 min. Primary antibodies were then applied overnight at 4°C, and secondary antibodies were applied for 30 min at room temperature. Nuclei were stained with Vectashield containing DAPI (Vector Laboratories). Images were acquired on Delta Vision Elite (GE, Schenectady, NY, USA). Forty to fifty sections at 0.2 µm intervals were acquired using 60× oil NA 1.4 objective and the section containing cilia was selected for each figure panel unless described otherwise. shRNAmiR lentivirus production and knockdown shRNAmiR lentivirus constructs were prepared according to Allaire et al. (Allaire et al., 2010) with slight modifications. Target sequences for ratVPAC2, ratBBS2 and ratTubby were designed using the Block-iT RNAi Designer (Life Technologies, Carlsbad, CA, USA) and subcloned into pCDNA6.2/GW-EmGFP-miR (Life Technologies) to obtain the shRNAmiR knockdown constructs. The EmGFP-shRNAmiR cassette was then amplified by the polymerase chain reaction (PCR) and subcloned into a pCDH.EF1 derivative (For primers used in this experiment, please refer to supplementary material Table S3). For virus production, 293FT cells were co-transfected with the pCDH vector, pSPAX2 (packaging vector) and pMD2.G (envelope vector) using Polyethylenimine Max (Polysciences, Warrington, PA, USA). The medium was changed after 16–20 hours, and the supernatant was collected and filtered (0.45 µm) after 48–72 hours. The virus was then concentrated with PEG-it (System Biosciences). The precipitated lentivirus was suspended with cold PBS, aliquot and stored at −80°C until use. Thalamic cultures and SCN cultures were infected with lentivirus harboring shRNAmiRs on either DIV1 or DIV2. Immunostaining was conducted on DIV7. RNA isolation, cDNA synthesis and real time quantitative PCR (RT-qPCR) Cultured thalamic neurons were lysed and total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The QuantiTect Reverse Transcription Kit (Qiagen) was used to reverse transcribe the mRNA into complementary DNA (cDNA). Real-time quantitative PCR was performed to amplify VPAC2, BBS2 and Tubby with specific primer sets (refer to supplementary material Table S4 for primer sequences). The template (5 ng) was amplified in 20 µl reaction volumes using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Data collection was performed on the ABI ViiA 7 and normalized to GAPDH. Each sample was assayed in triplicate. The relative standard curve method was used for analyzing quantitative PCR (qPCR) data. Antibodies and dilutions The commercial antibodies used were against: VPAC2 (Rabbit, 1:100, ab28624, Abcam, Cambridge, MA, USA), actin (Rabbit, 1:1000, A2066, Sigma), Arl13B (Mouse, 1:2000, 75–287, UC Davis/NIH NeuroMab Facility, Davis, CA, USA), Aldh1L1 (Mouse, 1:1000, 75-164, UC Davis/NIH NeuroMab Facility), acetylated α-tubulin (Mouse, 1:5000, mAb 6-11B-1, Sigma), GFAP (Chicken, 1:5000, ab4674, Abcam), MAP2 (Chicken, 1:5000, ab92434, Abcam) and Iba1 (Goat, 1:500, ab5076, Abcam). Antibodies against Sox10 (Guinea pig, 1:1000), NG2 (Guinea pig, 1:1000) and Arl6 (Rabbit, 1:1000) were gifts from Drs Wegner, Stallcup and Nachury, respectively. Supplementary Material Supplementary Material We thank the Human ORFeome collection, DNASU (Tempe, AZ) and Drs Kelly E. Mayo (Northwestern University) and Weihong Pan (Pennington Biomedical Research Center) for providing GPCR clones. We thank Dr Michael Wegner (Friedrich Alexander University of Erlangen and Nuremberg, Erlangen, Germany) for providing us with the antibody against Sox10, Dr William Stallcup (Burnham Institute, La Jolla, CA) for the antibody against NG2 and Dr Maxence Nachury (Stanford University, Stanford, CA) for the antibody against Arl6. This work was supported by NSF (SBE-0546843) and the University of Illinois at Chicago. Author Contributions: H.J. and L.S. designed the experiments. L.S. and D.A.G performed the experiments. H.J. and L.S. wrote the paper. Competing interests: The authors have no competing interests to declare. ==== Refs References Allaire P. D. Marat A. L. Dall'Armi C. Di Paolo G. McPherson P. S. Ritter B. (2010 ). The connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes. Mol. Cell 37 , 370 –382 10.1016/j.molcel.2009.12.037 20159556 Aton S. J. Colwell C. S. Harmar A. J. Waschek J. Herzog E. D. (2005 ). Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons. Nat. Neurosci. 8 , 476 –483 10.1038/nn1419 15750589 Berbari N. F. Bishop G. A. Askwith C. C. Lewis J. S. Mykytyn K. (2007 ). Hippocampal neurons possess primary cilia in culture. J. Neurosci. Res. 85 , 1095 –1100 10.1002/jnr.21209 17304575 Berbari N. F. Johnson A. D. Lewis J. S. Askwith C. C. Mykytyn K. (2008a ). Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors. Mol. Biol. Cell 19 , 1540 –1547 10.1091/mbc.E07-09-0942 18256283 Berbari N. F. Lewis J. S. Bishop G. A. Askwith C. C. Mykytyn K. (2008b ). Bardet–Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia. Proc. Natl. Acad. Sci. USA 105 , 4242 –4246 10.1073/pnas.0711027105 18334641 Berbari N. F. O'Connor A. K. Haycraft C. J. Yoder B. K. (2009 ). The primary cilium as a complex signaling center. Curr. Biol. 19 , R526 –R535 10.1016/j.cub.2009.05.025 19602418 Beri S. Bonaglia M. C. Giorda R. (2012 ). Low-copy repeats at the human VIPR2 gene predispose to recurrent and nonrecurrent rearrangements. Eur. J. Hum. Genet. [Epub ahead of print] DOI 10.1038/ejhg.2012.235 10.1038/ejhg.2012.235 Bishop G. A. Berbari N. F. Lewis J. Mykytyn K. (2007 ). Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. J. Comp. Neurol. 505 , 562 –571 10.1002/cne.21510 17924533 Brailov I. Bancila M. Brisorgueil M. J. Miquel M. C. Hamon M. Vergé D. (2000 ). Localization of 5-HT(6) receptors at the plasma membrane of neuronal cilia in the rat brain. Brain Res. 872 , 271 –275 10.1016/S0006-8993(00)02519-1 10924708 Cahoy J. D. Emery B. Kaushal A. Foo L. C. Zamanian J. L. Christopherson K. S. Xing Y. Lubischer J. L. Krieg P. A. Krupenko S. A. (2008 ). A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28 , 264 –278 10.1523/JNEUROSCI.4178-07.2008 18171944 Caspary T. Larkins C. E. Anderson K. V. (2007 ). The graded response to Sonic Hedgehog depends on cilia architecture. Dev. Cell 12 , 767 –778 10.1016/j.devcel.2007.03.004 17488627 Cenacchi G. Giangaspero F. Cerasoli S. Manetto V. Martinelli G. N. (1996 ). Ultrastructural characterization of oligodendroglial-like cells in central nervous system tumors. Ultrastruct. Pathol. 20 , 537 –547 10.3109/01913129609016358 8940761 Cutler D. J. Haraura M. Reed H. E. Shen S. Sheward W. J. Morrison C. F. Marston H. M. Harmar A. J. Piggins H. D. (2003 ). The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro. Eur. J. Neurosci. 17 , 197 –204 10.1046/j.1460-9568.2003.02425.x 12542655 Deretic D. Williams A. H. Ransom N. Morel V. Hargrave P. A. Arendt A. (2005 ). Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4). Proc. Natl. Acad. Sci. USA 102 , 3301 –3306 10.1073/pnas.0500095102 15728366 Doherty D. (2009 ). Joubert syndrome: insights into brain development, cilium biology, and complex disease. Semin. Pediatr. Neurol. 16 , 143 –154 10.1016/j.spen.2009.06.002 19778711 Domire J. S. Green J. A. Lee K. G. Johnson A. D. Askwith C. C. Mykytyn K. (2011 ). Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet–Biedl syndrome proteins. Cell. Mol. Life Sci. 68 , 2951 –2960 10.1007/s00018-010-0603-4 21152952 Follit J. A. Li L. Vucica Y. Pazour G. J. (2010 ). The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence. J. Cell Biol. 188 , 21 –28 10.1083/jcb.200910096 20048263 Foord S. M. Bonner T. I. Neubig R. R. Rosser E. M. Pin J.-P. Davenport A. P. Spedding M. Harmar A. J. (2005 ). International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacol. Rev. 57 , 279 –288 10.1124/pr.57.2.5 15914470 Fredriksson R. Lagerström M. C. Lundin L.-G. Schiöth H. B. (2003 ). The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63 , 1256 –1272 10.1124/mol.63.6.1256 12761335 Garcia-Gonzalo F. R. Reiter J. F. (2012 ). Scoring a backstage pass: mechanisms of ciliogenesis and ciliary access. J. Cell Biol. 197 , 697 –709 10.1083/jcb.201111146 22689651 Geng L. Okuhara D. Yu Z. Tian X. Cai Y. Shibazaki S. Somlo S. (2006 ). Polycystin-2 traffics to cilia independently of polycystin-1 by using an N-terminal RVxP motif. J. Cell Sci. 119 , 1383 –1395 10.1242/jcs.02818 16537653 Green J. A. Mykytyn K. (2010 ). Neuronal ciliary signaling in homeostasis and disease. Cell. Mol. Life Sci. 67 , 3287 –3297 10.1007/s00018-010-0425-4 20544253 Hamon M. Doucet E. Lefèvre K. Miquel M.-C. Lanfumey L. Insausti R. Frechilla D. Del Rio J. Vergé D. (1999 ). Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors. Neuropsychopharmacology 21 Suppl. , 68S –76S 10.1016/S0893-133X(99)00044-5 10432491 Händel M. Schulz S. Stanarius A. Schreff M. Erdtmann-Vourliotis M. Schmidt H. Wolf G. Höllt V. (1999 ). Selective targeting of somatostatin receptor 3 to neuronal cilia. Neuroscience 89 , 909 –926 10.1016/S0306-4522(98)00354-6 10199624 Harmar A. J. Marston H. M. Shen S. Spratt C. West K. M. Sheward W. J. Morrison C. F. Dorin J. R. Piggins H. D. Reubi J.-C. (2002 ). The VPAC(2) receptor is essential for circadian function in the mouse suprachiasmatic nuclei. Cell 109 , 497 –508 10.1016/S0092-8674(02)00736-5 12086606 Imai Y. Ibata I. Ito D. Ohsawa K. Kohsaka S. (1996 ). A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem. Biophys. Res. Commun. 224 , 855 –862 10.1006/bbrc.1996.1112 8713135 Jin H. White S. R. Shida T. Schulz S. Aguiar M. Gygi S. P. Bazan J. F. Nachury M. V. (2010 ). The conserved Bardet–Biedl syndrome proteins assemble a coat that traffics membrane proteins to cilia. Cell 141 , 1208 –1219 10.1016/j.cell.2010.05.015 20603001 Kang S. H. Fukaya M. Yang J. K. Rothstein J. D. Bergles D. E. (2010 ). NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration. Neuron 68 , 668 –681 10.1016/j.neuron.2010.09.009 21092857 Lee J. H. Gleeson J. G. (2010 ). The role of primary cilia in neuronal function. Neurobiol. Dis. 38 , 167 –172 10.1016/j.nbd.2009.12.022 20097287 Lee J. E. Gleeson J. G. (2011 ). Cilia in the nervous system: linking cilia function and neurodevelopmental disorders. Curr. Opin. Neurol. 24 , 98 –105 10.1097/WCO.0b013e3283444d05 21386674 Levinson D. F. Duan J. Oh S. Wang K. Sanders A. R. Shi J. Zhang N. Mowry B. J. Olincy A. Amin F. (2011 ). Copy number variants in schizophrenia: confirmation of five previous findings and new evidence for 3q29 microdeletions and VIPR2 duplications. Am. J. Psychiatry 168 , 302 –316 10.1176/appi.ajp.2010.10060876 21285140 Louvi A. Grove E. A. (2011 ). Cilia in the CNS: the quiet organelle claims center stage. Neuron 69 , 1046 –1060 10.1016/j.neuron.2011.03.002 21435552 Maka M. Stolt C. C. Wegner M. (2005 ). Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect. Dev. Biol. 277 , 155 –169 10.1016/j.ydbio.2004.09.014 15572147 Marley A. von Zastrow M. (2010 ). DISC1 regulates primary cilia that display specific dopamine receptors. PLoS ONE 5 , e10902 10.1371/journal.pone.0010902 20531939 Marley A. von Zastrow M. (2012 ). A simple cell-based assay reveals that diverse neuropsychiatric risk genes converge on primary cilia. PLoS ONE 7 , e46647 10.1371/journal.pone.0046647 23056384 Maywood E. S. Reddy A. B. Wong G. K. Y. O'Neill J. S. O'Brien J. A. McMahon D. G. Harmar A. J. Okamura H. Hastings M. H. (2006 ). Synchronization and maintenance of timekeeping in suprachiasmatic circadian clock cells by neuropeptidergic signaling. Curr. Biol. 16 , 599 –605 10.1016/j.cub.2006.02.023 16546085 Mukhopadhyay S. Wen X. Ratti N. Loktev A. Rangell L. Scales S. J. Jackson P. K. (2013 ). The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell 152 , 210 –223 10.1016/j.cell.2012.12.026 23332756 Nachury M. V. Seeley E. S. Jin H. (2010 ). Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? Annu. Rev. Cell Dev. Biol. 26 , 59 –87 10.1146/annurev.cellbio.042308.113337 19575670 Pazour G. J. Bloodgood R. A. (2008 ). Targeting proteins to the ciliary membrane. Curr. Top. Dev. Biol. 85 , 115 –149 10.1016/S0070-2153(08)00805-3 19147004 Sarkisian M. R. Arellano J. I. Breunig J. J. (2013 ). Primary cilia in cerebral cortex: growth and functions on neuronal and non-neuronal cells. Cilia And Nervous System Development And Function Tucker K L Caspary T J , ed105 –129 Dordrecht : Springer . Sheward W. J. Lutz E. M. Harmar A. J. (1995 ). The distribution of vasoactive intestinal peptide2 receptor messenger RNA in the rat brain and pituitary gland as assessed by in situ hybridization. Neuroscience 67 , 409 –418 10.1016/0306-4522(95)00048-N 7675176 Stallcup W. B. Dahlin K. Healy P. (1990 ). Interaction of the NG2 chondroitin sulfate proteoglycan with type VI collagen. J. Cell Biol. 111 , 3177 –3188 10.1083/jcb.111.6.3177 2269670 Sun X. Haley J. Bulgakov O. V. Cai X. McGinnis J. Li T. (2012 ). Tubby is required for trafficking G protein-coupled receptors to neuronal cilia. Cilia 1 , 21 10.1186/2046-2530-1-21 23351594 Vacic V. McCarthy S. Malhotra D. Murray F. Chou H.-H. Peoples A. Makarov V. Yoon S. Bhandari A. Corominas R. (2011 ). Duplications of the neuropeptide receptor gene VIPR2 confer significant risk for schizophrenia. Nature 471 , 499 –503 10.1038/nature09884 21346763 Yang X. Boehm J. S. Yang X. Salehi-Ashtiani K. Hao T. Shen Y. Lubonja R. Thomas S. R. Alkan O. Bhimdi T. (2011 ). A public genome-scale lentiviral expression library of human ORFs. Nat. Methods 8 , 659 –661 10.1038/nmeth.1638 21706014 Yang Y. Vidensky S. Jin L. Jie C. Lorenzini I. Frankl M. Rothstein J. D. (2011 ). Molecular comparison of GLT1+ and ALDH1L1+ astrocytes in vivo in astroglial reporter mice. Glia 59 , 200 –207 10.1002/glia.21089 21046559 Yoshimura K. Kawate T. Takeda S. (2011 ). Signaling through the primary cilium affects glial cell survival under a stressed environment. Glia 59 , 333 –344 10.1002/glia.21105 21125655	23862016	PMC3711036	CC BY	2021-01-05 02:35:29	yes	Biol Open. 2013 May 23; 2(7):686-694
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23894414PONE-D-13-1209410.1371/journal.pone.0069102Research ArticleBiologyBiotechnologyApplied MicrobiologySmall MoleculesImmunologyMicrobiologyImmunityImmunoregulationApplied MicrobiologyMolecular Cell BiologyCytometryFlow CytometryMedicineGastroenterology and Hepatology Lactobacillus gasseri SF1183 Affects Intestinal Epithelial Cell Survival and Growth Effects of Probiotic L.gasser on Intestinal CellsDi Luccia Blanda 1 Manzo Nicola 1 Baccigalupi Loredana 1 Calabrò Viola 1 Crescenzi Elvira 2 Ricca Ezio 1 Pollice Alessandra 1 * 1 Department of Biology, University of Naples Federico II-MSA-Via Cinthia, Naples, Italy 2 Istituto di Endocrinologia ed Oncologia Sperimentale-CNR-via S. Pansini, Naples, Italy Salmon Henri Editor INRA, UR1282, France * E-mail: apollice@unina.itCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: AP ER BDL NM. Performed the experiments: BDL NM EC. Analyzed the data: BDL NM LB EC VC ER AP. Contributed reagents/materials/analysis tools: LB VC ER AP. Wrote the paper: ER AP. 2013 23 7 2013 8 7 e6910222 3 2013 6 6 2013 © 2013 Di Luccia et al2013Di Luccia et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host’s intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis. This work was supported by the FARO project (terza tornata) of the University of Naples and Istituto San Paolo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Several recent studies have shown that commensal bacteria, forming the human gut microbiota, establish complex symbiotic interactions with cells of the GastroIntestinal Tract (GIT) and that these interactions significantly contribute to human health [1], [2], [3], [4]. Metagenomic experiments have indicated that the vast majority of the intestinal bacteria belong to two phyla, the Gram-negative Bacteroidetes and the Gram-positive Firmicutes, including the large class of Clostridia and the lactic acid bacteria [5], [6]. However, the composition of the gut microbiota is known to change transiently as a consequence of a variety of factors such as age, diet, enteral infections, pharmacological treatments and immunosuppression [7], [8], [9]. Changes in the microbiota composition have also been associated to several diseases, such as chronic inflammation of the GIT, diabetes and obesity [7], [10], [11], [12], [13], [14], and the oral administration of members of the microbiota has been considered as a potential clinical tool to relieve intestinal dysfunctions [15], [16], [17], [18], [19], [20]. Interest in the beneficial functions of the human microbiota has resulted in the selection of specific strains with putative health-promoting capacities that are recognized as probiotics and are generally selected from isolates of the Lactobacillus or Bifidobacterium species. Probiotic bacteria have been shown capable to modulate systemic inflammation, cell proliferation and apoptosis, and such properties proposed as useful for future immunomodulatory and cancer prevention strategies [13], [14], [21], [22]. In vitro studies have reported the anti-proliferative and pro-apoptotic effects of Lactobacillus and Bifidobacterium spp. in various cancer cell lines [23], [24], [25], [26], while in vivo studies have shown the inhibitory activity of probiotics on liver, bladder and colon tumours in animal models [27], [28], [29], [30]. The molecular mechanisms of interaction between intestinal cells and bacteria have been studied in detail only in few cases and often quorum-sensing autoinducers, communication molecules released by bacteria at high densities, have been shown to modulate host responses either directly or through regulation of bacterial genes involved in gut colonization and host signaling [31], [32]. An example in this context is the quorum-sensing pentapeptide CSF (Competence and Sporulation Factor) of Bacillus subtilis that is taken up by Caco-2 cells via the membrane transporter OCTN2 (organic cation transporter 2) and that contributes to eukaryotic cell homeostasis activating survival pathways (p38 MitogenActivatedProteinKinase (MAPK) and protein kinase B) [33]. In other cases the secreted bacterial effectors have not been identified: still unidentified molecules secreted by Lactobacillus rhamnosus GG were shown to prevent cytokine-induced apoptosis on two different intestinal cell model systems (YAMC-young adult mouse colon; HT29-colon carcinoma) [34]; molecules secreted by L. reuteri were shown to potentiate tumour necrosis factor (TNFα)-induced apoptosis in myeloid leukemia derived cells. In the latter example L. reuteri molecules were found to: i) suppress NF-kB activation by inhibiting IkBa degradation; ii) downregulate nuclear factor-kB (NF-kB)-dependent gene products affecting cell proliferation and survival; iii) promote apoptosis by enhancing mitogen-activated protein kinase (MAPK) activities including c-Jun N-terminal kinase and p38 MAPK [35]. Lactobacillus gasseri is a well characterized species of low GC gram-positive bacteria, known to represent one of the major homofermentative Lactobacillus of the human intestine [36]. We have isolated the SF1183 strain of L. gasseri from an ileal biopsy of a human healthy volunteer and, in particular, from the fraction of bacteria tightly associated to the epithelial cells. SF1183 was shown to have antimicrobial activity against a panel of enteropathogens and to form a matrix (biofilm) in standard laboratory as well as in simulated intestinal conditions [36]. This study investigates the effects of molecules produced and secreted by L. gasseri SF1183 on colorectal HCT116 cells, both at the molecular and cellular level. Since HCT116 cells are responsive to TNFα-induced apoptosis [37], [38], we tested their response to the presence of L. gasseri SF1183 supernatant. Moreover, we extended our analysis to the effects of another inducer of apoptosis to evaluate the specificity of the observed effect. Results and Discussion The Conditioned Medium (CM) of L. gasseri SF1183 Protects HCT116 cells from TNFα Induced Apoptosis Among the most common features of chronic intestinal inflammations, such as Crohn and irritable bowel diseases (IBDs), is the increase in the production of inflammatory cytokines, epithelial cell apoptosis and immune cell infiltration, leading to disruption of the intestinal epithelial integrity. TNFα is among the cytokines more largely produced under these conditions. It is known to regulate both anti- and pro-apoptotic signaling pathways and determine the cell fate by controlling the balance between the two pathways [39]. To study the effects of molecules secreted by L. gasseri on TNFα-induced apoptosis we used the TNFα sensitive HCT116 human colon cancer cells as a model of intestinal epithelial cells [37]. As a marker of apoptosis we followed the proteolytic cleavage of PARP-1, a regulator of the DNA base excision repair pathway essential for the maintenance of genomic integrity and for survival in response to genotoxic insults [40]. PARP-1 is known to be specifically proteolysed by the Caspase 3 to a 24 kDa DNA-binding domain (DBD) and a 89 kDa catalytic fragment during the execution of the apoptotic program [41]. To set up the experimental conditions, HCT116 cells were incubated with 1 nM TNFα for various times and cell extracts analyzed by western blotting with anti-PARP-1 antibody. As shown in Figure 1A, the amount of proteolyzed PARP-1 increased with the time of exposure to TNFα. Therefore we decided to use 8 hours of treatment with 1 nM TNFα to detect either induction or inhibition of PARP cleavage, for all therein experiments involving a TNF-α activation. 10.1371/journal.pone.0069102.g001Figure 1 HCT116 cell response to L. gasseri CM with or without TNFα treatment. Western blot with anti-PARP-1 antibody of whole cell extracts from HCT116 cells incubated in (A) complete cell culture medium supplemented or not with TNFα (1 nM) for 2, 8 or 24 hours; (B) complete cell culture medium supplemented or not with CM (20%v/v) for 16 hours before treatment with 1 nM TNFα for 8 hours; (C) complete cell culture medium supplemented or not with TNFα (1 nM) for 8 hours and MDM (20%v/v) or MDM+lactic acid pH4 (20%v/v). After the treatments cells were collected, lysed and protein concentration determined. Equal amount of cell lysates were fractionated on SDS-PAGE and analyzed by western blotting with antibodies against PARP-1. Actin was used as a loading control. A filter-sterilized conditioned medium (CM) of a L. gasseri SF1183 culture was added (20% v/v) to HCT116 cells and incubated for 16 hours. Then, TNFα was added and, after additional 8 hours of incubation, cells were harvested and whole extracts analyzed by western blotting with anti-PARP-1 antibody. As shown in Figure 1B the bacterial CM alone did not have any effect on PARP-1 cleavage while was able to significantly reduce the TNFa-induced proteolytic activation of PARP-1. L. gasseri is a homofermentative bacterium that, therefore, grows producing lactic acid as the only metabolic end-point of carbohydrate metabolism. As a consequence, its growth medium is acidified during growth to reach a final pH value of 4.0. To verify that the reduction in the extent of PARP-1 cleavage was due to secreted molecules and not to the acidification of the growth medium, the same experiment of Figure 1B was performed adding to HCT116 cells the same amount (20% v/v) of the fresh bacterial growth medium (MDM) either at its normal pH (pH 7.0) or acidified to pH 4.0 with lactic acid. As shown in Fig. 1C, both media did not have any effect on TNFα-induced cleavage of PARP-1 suggesting that the CM of L. gasseri SF1183 contains molecules with anti-apoptotic activity. The CM of L. gasseri SF1183 Contains Bioactive Soluble Molecule(s) Secreted During the Stationary Phase of Growth As a first step toward the characterization of molecule(s) involved in the observed effect, we decided to size-separate the CM of L. gasseri by using a 3 kDa molecular mass cut-off filter. As Figure 2A clearly shows we observed bioactivity largely in the filtrate, indicating a small (less than 3 kDa) molecular mass for the effector(s) molecule(s). Further, different enzymatic treatments of the CM indicated that bioactivity is proteinase-K sensitive, suggesting a proteinaceous nature (Figure 2B). After a heat treatment of 30 minutes at 100°C the CM was still able to reduce the TNFa-induced cleavage of PARP-1 at the same extent of untreated CM (Figure 2C), suggesting that the bioactive molecule(s) is not thermolabile. Often bacteria secrete bioactive molecules during their stationary phase of growth. We thus tested the CM of L. gasseri cultures at different stages of growth and observed bioactivity produced only in early and late stationary phase of growth (24 and 48 hours of growth, respectively) (Figure 2D). All experiments therein reported have been performed by using the size-fractionated (<3 kDa) CM of a late stationary culture of L. gasseri SF1183. 10.1371/journal.pone.0069102.g002Figure 2 L. gasseri secretes thermostable, bioactive molecule(s) of proteinaceous nature during the stationary phase of growth. HCT116 cells were incubated in complete cell culture medium supplemented or not with TNFα (1 nM) for 8 hours and with A) CM fractionated with a cut-off of 3 kDa, or B) CM treated with different enzymes [Trypsin, Proteinase K, DNAse I, RNAse A], or C) CM treated at 100°C for 30 minutes, or D) CM of cultures at the indicated phases of growth. After the treatments, cells were collected, lysed and total cell extracts were analyzed by western blotting with antibodies against PARP-1. Actin was used as a loading control. PARP-1 band intensity was evaluated by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition. Values expressed as ratio with the corresponding actin values and normalised to the reference point (PARP-1 cleavage in medium). Percentage of increase (+) or decrease (–) with respect to the intensity of the reference point are indicated. The CM of L. gasseri SF1183 Affects Cell Proliferation of HCT116 Cells To characterize the cellular response to L. gasseri secreted molecules, we analyzed HCT116 cell number and viability after growth in presence of CM. Briefly, cells were incubated for 24 hours with CM of L. gasseri (20% vol/vol) and then analyzed both for the number of cells by counting in a Burker chamber and for cell viability by MTS assay. As Figure 3A shows, the CM caused a 30% reduction in the number of cells. The MTS assay (Figure 3B) showed a reduction in cell viability of the same order of magnitude. 10.1371/journal.pone.0069102.g003Figure 3 The CM of L. gasseri SF1183 affects HCT116 cell number but not cell viability. Proliferating HCT116 cells were incubated in complete cell culture medium supplemented or not with CM (20%v/v). After 24 hours (A) controls (NT) and CM-treated (CM) cells were collected and counted in a Burker chamber; or (B) incubated with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium as a substrate and the absorbance of converted formazan measured at 490 nm. To get more insights into the cellular response, we looked at the cell-cycle distribution profile and at the expression of cell cycle-related molecular markers in HCT116 cells exposed to TNFα and/or to the CM of L. gasseri. The cell cycle distribution was analyzed by flow cytometry and showed that treatment with TNFα causes a drastic increase in the subG1 cell population (from 4 to 28%) while the pre-treatment of cells with the CM of L. gasseri strongly reduced the TNFα induced effect (Figure 4A), thus supporting our previous data indicating a reduction in the extent of PARP-1 cleavage (see Figure 1B, 2B, 2C). Importantly, we found that the CM alone caused a significant increase (up to 18%) in the G1 population of cells with a compensatory decrease in S/G2 cells, indicating that cells were unable to resume the cell cycle at normal phase transit rate (Figure 4A), consistently with previous MTS and proliferation data. This suggests that, indeed, molecules secreted from L. gasseri can drastically interfere with proliferation of HCT116 cells, rendering them less prone to TNFα induced apoptosis. 10.1371/journal.pone.0069102.g004Figure 4 The CM of L. gasseri SF1183 affects cell proliferation of HCT116 cells. Proliferating HCT116 cells were incubated in complete cell culture medium supplemented or not with CM (20%v/v) and/or TNFα (1 nM). After the treatments, cells were collected and treated for flow-cytometric analysis (A and Fig. S3 in File S1) or western blot (B) with the indicated antibodies. Expression of the cell cycle markers p21WAF1 and pERKs was also investigated to explore the effects of the CM of L. gasseri at the molecular level. p21WAF1 (also known as cyclin-dependent kinase inhibitor 1) is a regulator of cell cycle progression at the S phase that acts as an inhibitor of cyclin-dependent kinase, and occupies a central position in the regulation of the cell cycle in many tissues [42], [43]. Levels of p21WAF1 protein are regulated during the cell cycle at the levels of transcription and protein degradation, although many questions remain on the mechanism of p21 proteolysis [44], [45]. Extracellular signal-regulated kinases (ERK1,2) are members of the MAPK super family that can mediate cell proliferation and apoptosis. Activated (phosphorylated) ERKs, are usually associated with active cell proliferation [46], while p21 increase correlates with a G1 cell cycle arrest [47]. Immunoblots with the appropriate antibodies showed that treatment with CM significantly induced p21WAF independently from TNFα (Figure 4B; compare lanes 1–2 with 3–4) while pERKs expression was inhibited in CM treated cells, strongly supporting the antiproliferative effect of molecule(s) present in L. gasseri supernatant. Altogether these experiments clearly indicate that L. gasseri supernatant exerts a cytostatic but not a cytotoxic effect on epithelial colon cells. The CM of L. gasseri SF1183 Protects HCT116 Cells from Cisplatin Induced Apoptosis To test whether bioactive molecules present in L. gasseri supernatant could exert anti-apoptotic effects against other apoptosis-inducers we preincubated HCT116 cells with CM and then treated them with 30 µM cisplatin to induce the intrinsic apoptotic pathway. As shown in Figure 5A cytofluorimetric analysis indicate that a G1 cell cycle arrest is induced by CM addition which causes cells to be more resistant to cisplatin induced apoptosis. These observations are supported, at the molecular level, with an increase in p21WAF1 levels and a decrease of ERKs activation when CM was added to the cells (Figure 5B, lanes 1,2). Consistently, pretreatment of cells with CM determined a reduction in the extent of PARP-1 cleavage when cells were subjected to cisplatin action (Figure 5B, lanes 3,4). 10.1371/journal.pone.0069102.g005Figure 5 The anti-apoptotic effect of L. gasseri is not specific for TNFα-induced apoptosis. Proliferating HCT116 cells were incubated in complete cell culture medium supplemented or not with CM (20%v/v) and/or cisplatin (30 µM). After the treatments, cells were collected and treated for flow-cytometric analysis (A and Fig. S4 in File S1) or western blot (B) with the indicated antibodies. Altogether our results clearly indicate that probiotic L. gasseri protects intestinal epithelial cells from apoptosis induced by inflammatory cytokines or cytotoxic drugs, causing cell cycle arrest. Conclusions The main result of this report is that the conditioned medium of a stationary culture of the human isolate SF1183 of L. gasseri contains molecule(s) able to affect cell proliferation of HCT116 cells, protecting them from intrinsic as well as extrinsic, TNFα-induced, apoptosis. Chronic inflammations cause an increase in inflammatory cytokines (such as TNFα), epithelial cell apoptosis and immune cell infiltration, leading to disruption of the intestinal epithelial integrity. Therefore, a reduction of cell proliferation could protect epithelial barrier integrity and help in reconstituting tissutal homeostasis. The L. gasseri molecule(s) responsible of the observed effects is proteinaceous, has a small (less than 3 kDa) size and its synthesis is growth phase-dependent, occuring only in bacterial cells in stationary phase. Those properties are suggestive of bacterial quorum-sensing autoinducers, communication molecules produced at high cell density and known to act as modulator of bacterial host responses [31], [32], [33]. Unfortunately, the definition of the chemical nature of the molecule(s) secreted by L. gasseri SF1183 and able to affect HCT116 cells has been so far unsuccessful. The size-fractionated (less than 3 kDa) CM of L. gasseri was analyzed by gel filtration chromatography with a Superdex Peptide 10/300 GL (GE Healthcare Life Sciences) column and two main peaks were obtained (Fig. S1 in File S1). Chromatographic fractions containing either one of the two peaks were tested for the ability to reduce the TNFα-induced cleavage of PARP-1 (Fig. S2A in File S1). Only one of the fractions (Fraction 1) was shown to reduce the TNFα-induced cleavage of PARP-1 at the same extent of the unfractionated CM (Fig. S2B in File S1). Unfortunately, attempts to analyze Fraction 1 by mass-spectrometry have been so far unsuccessful, probably because of the minimal concentration of molecules in the fraction. To define the chemical nature of the molecule(s) affecting HCT116 cells and identify its cellular and molecular targets will then be a future and challenging task. Materials and Methods Bacterial Growth and Preparation of Conditioned Medium Lactobacillus gasseri (SF1183) was grown in MRS broth (Difco, Detroit, MI) for 24 hours at 37°C and the culture diluted and used to inoculate MDM (Glucose 10 g/L, Sodium acetate 5 g/L, KH2PO4 3 g/L, K2HPO4 3 g/L, MgSO4 *7H2O 0.2 g/L, L-Alanine 100 mg/L, L-Arginine 100 mg/L, L-Aspartic acid 200 mg/L, L-Cysteine 200 mg/L, L-Glutamic 200 mg/L, L-Histidine 100 mg/L, L-Isoleucine 100 mg/L, L-Leucine 100 mg/L, L-Lysine 100 mg/L, L-Methionine 100 mg/L, L-Phenylalanine 100 mg/L, L-Serine 100 mg/L, L-Tryptophan 100 mg/L, L-Tyrosine 100 mg/L, L-Valine 100 mg/L, Nicotinic acid 1 mg/L, Pantothenic acid 1 mg/L, Pyridoxal 2 mg/L, Riboflavin 1 mg/L, Cyanocobalamin 1 mg/L, Adenine 10 mg/L, Guanine 10 mg/L, Uracil 10 mg/L) minimal medium. Cells of SF1183 were then grown anaerobically for 48 hours at 37°C. The culture was centrifuged (1000 g for 10 min at RT) and the supernatant (conditioned medium, CM) was filtered-sterilized through a 0.22 µm low-protein binding filter (Millipore, Bedford, MA, US). CM treated with proteases and nucleases was prepared as described above and size fractionated (3-kDa cutoff spin column; Centricon, Millipore). Before treatment with trypsin (GIBCO) or proteinase K (Invitrogen), or DNasi I, or RNasi A (Invitrogen, Life Technology, Monza, Italy) at a final 100 mg/ml concentration for 60 min at 37°C the pH of CM was neutralized with concentrated NaOH (10 N). After the enzymatic treatments CM was acidified to pH 4.0 using concentrated HCl and fractionated as described above to remove the enzymes. Cell Culture and Treatment with Bacterial CM HCT116 cells (ATCC CCL 247) derived from a poorly-differentiated colonic adenocarcinoma and were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37°C in humidified atmosphere of 5% CO2. The bacterial CM was employed for the treatment at 20% v/v concentration in complete growth medium. After incubation of 16 hours with CM (20% v/v), TNFα (1 nM) (Millipore, Milan, Italy) or Cisplatin (30 µM) (Sigma Milan, Italy) was added and cells harvested after 8 hours or 24 hours of treatment. Cells were lysed and cell extracts prepared for Western blot and FACS analysis, respectively, as described below. SDS-PAGE and Western Immunoblot Analysis Cells were harvested in lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, and protease inhibitors) and total protein extract prepared as previously described [48]. Briefly, cell lysates were incubated on ice for 40 minutes, and the extracts were centrifuged at 15000 g for 15 minutes to remove cell debris. Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad). After the addition of 2x Laemmli buffer (SIGMA), samples were boiled at 100°C for 5 minutes and resolved by SDS-polyacrylamide gel electrophoresis (10% or 12%). Proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore Milan, Italy) as previously described [49]. The membranes were blocked in 5% w/v milk buffer (5% w/v non-fat dried milk, 50 mM Tris, 200 mM NaCl, 0,2% Tween 20) and incubated with primary antibody diluted in 5% w/v milk or bovine serum albumine buffer for 2 hours at room temperature or overnight at 4°C. Primary antibodies were anti-rabbit PARP-1 (Cell Signaling, EuroClone, Milan, Italy), anti-rabbit pErks 42/44 (Cell Signaling, EuroClone, Milan, Italy), anti-rabbit p21WAF1 (Santa-Cruz Biotechnology, DBA Milan-Italy), anti-goat β-actin (Santa-Cruz Biotechnology DBA Milan, Italy). Data were visualized by enhanced chemiluminescence method (ECL, GE-Healthcare Milan, Italy) using HRP-conjugated secondary antibody (Santa-Cruz Biotechnology DBA Milan, Italy) incubated 1 hour at room temperature, and analysed by Quantity One ®software of ChemiDoc TMXRS system (Bio-Rad Milan, Italy). Cell Growth and Flow Cytometry Analysis HCT116 cells were plated in 35 mm dishes at the cell density of 2,5×105 cells/plate. For cell growth analysis, cells were cultured in complete growth medium supplemented or not with bacterial CM at 20% v/v concentration for 24 hours. After the treatment, cells were collected and counted in a Burker chamber. Flow cytometry analysis was performed as previously described [50]. Briefly, cells were washed twice with PBS and harvested at 1500 g with 0.05% trypsin in 0.15% Na2EDTA. Cells were then centrifuged, washed in PBS, fixed with ice-cold 70% ethanol, and stored overnight at 4°C. Fixed cells were washed in PBS and then incubated with propidium iodide (50 µg/ml) and RNAse A (10 µg/ml) for 30 min at room temperature. Data acquisition was performed using a CyAn ADP Flow Cytometer (Beckman Coulter, Inc., Milano, Italy) and Summit Software. MTS Assay HCT116 cells were cultured at a density of 2,5×105 cells per well in flat bottomed 6-well plates and supplemented or not with CM (20% v/v) for 24 hours. After treatment, CellTiter 96® AQUEOUS One Solution Reagent (Promega, Madison, WI, US) was added to each well according to the manufacturer’s instructions. After 30 minutes cell viability was determined by measuring the absorbance at 490 nm using a Multiscan spectrum (Thermo Electron Corporation). Supporting Information File S1 Supplemental figures. Figure S1, the CM of L. gasseri was size fractionated with a 3 kDa molecular mass cut-off filter and loaded on a gel filtration chromatographic column (Superdex Peptide 10/300 GL, GE Healthecare Life Sciences). The elution buffer was AMAC 0.3 M. Two main peaks were observed at 220 nm. Figure S2, chromatographic fractions from the experiment of Fig. S1 were tested by western blotting with anti-PARP-1 antibody (A). As a control, cells were also treated with the elution buffer (AMAC 0,3 M). (B) Densitometric analysis of the western blot. PARP-1 band intensity was evaluated by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition. Values are expressed as ratio with the corresponding actin values and normalised to the reference point (PARP-1 cleavage in medium). Percentage of increase (+) or decrease (–) with respect to the intensity of the reference point are indicated. Figure S3, enlargment of part of Fig. 4 showing the output of the FACS analysis. Figure S4, enlargment of part of Fig. 5 showing the output of the FACS analysis. (PDF) Click here for additional data file. We thank Elio Pizzo for helping us with the chromatography experiments and Luciano Di Iorio for technical assistance. ==== Refs References 1 Macpherson AJ , Harris NL (2004 ) Interactions between commensal intestinal bacteria and the immune system . Nat Rev Immunol 4 : 478 –485 .15173836 2 Lozupone CA , Stombaugh JI , Gordon JI , Jansson JK , Knight R (2012 ) Diversity, stability and resilience of the human gut microbiota . Nature 13 489 : 220 –230 . 3 Clemente JC , Ursell LK , Parfrey LW , Knight R (2012 ) The impact of the gut microbiota on human health: an integrative view . Cell 148 : 1258 –1270 .22424233 4 Kau AL , Ahern PP , Griffin NW , Goodman AL , Gordon JI (2011 ) Human nutrition, the gut microbiome and the immune system . Nature 474 : 327 –336 .21677749 5 Eckburg PB , Bik EM , Bernstein CN , Purdom E , Dethlefsen L , et al (2005 ) Diversity of the human intestinal microbial flora . Science 308 : 1635 –1638 .15831718 6 Mahowald MA , Rey FE , Seedorf H , Turnbaugh PJ , Fulton RS , et al (2009 ) Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla. Proc. Natl. Acad. Sci. . USA 106 : 5859 –5864 . 7 Turnbaugh PJ , Hamady M , Yatsunenko T , Cantarel BL , Duncan A , et al (2009 ) A core gut microbiome in obese and lean twins . Nature 457 : 480 –484 .19043404 8 De Filippo C , Cavalieri D , Di Paola M , Ramazzotti M , Poullet JB , et al (2010 ) Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa . Proc Natl Acad Sci USA 107 : 14691 –14696 .20679230 9 Biagi E , Candela M , Franceschi C , Brigidi P (2011 ) The aging gut microbiota: new perspectives. Ageing Res Rev. . 10 : 428 –439 . 10 Wen L , Ley RE , Yu P , Volchkov PY , Stranges PB , et al (2008 ) Innate immunity and intestinal microbiota in the development of Type 1 diabetes . Nature 455 : 1109 –1113 .18806780 11 Li M , Wang B , Zhang M , Rantalainen M , Wang S , et al (2008 ) Symbiotic gut microbes modulate human metabolic phenotypes . Proc Natl Acad Sci USA 105 : 2117 –2122 .18252821 12 Zhang H , DiBaise JK , Zuccolo A , Kudrna D , Braidotti M , et al (2009 ) Human gut microbiota in obesity and after gastric bypass . Proc Natl Acad Sci USA 106 : 2365 –2370 .19164560 13 O’Mahony L , McCarthy J , Kelly P , Shanahan F , Quigley EM (2005 ) Lactobacillus and Bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterol. . 128 : 541 –551 . 14 Sokol H , Pigneur B , Watterlot L , Lakhdari O , Bermúdez-Humarán LG , et al (2008 ) Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients . Proc Natl Acad Sci USA 105 : 16731 –16736 .18936492 15 Gilliland SE , Nelson CR , Maxwell C (1985 ) Assimilation of cholesterol by Lactobacillus acidophilus Appl Environ Microbiol . 49 : 377 –381 . 16 Salminen S , Bouley C , Boutron-Ruault MC , Cummings JH , Franck A , et al (1998 ) Functional food science and gastrointestinal physiology and function . Br J Nutr 80 : S147 –S171 .9849357 17 Perdigon G , Fuller R , Raya R (2001 ) Lactic acid bacteria and their effect on the immune system . Curr Issues Intest Microbiol 2 : 27 –42 .11709854 18 Rafter J (2003 ) Probiotics and colon cancer . Best Pract Res Clin Gastroenterol 17 : 849 –859 .14507593 19 Isolauri E , Salminen S , Ouwehand AC (2004 ) Microbial–gut interactions in health and disease . Best Pract Res Clin Gastroenterol 18 : 299 –313 .15123071 20 Guarner F , Bourdet-Sicard R , Brandtzaeg P , Gill HS , McGuirk P , et al (2006 ) Mechanisms of disease: the hygiene hypothesis revisited . Nat Clin Pract Gastroenterol Hepatol 3 : 275 –284 .16673007 21 Kato I , Endo-Tanaka K , Yokokura T (1998 ) Suppressive effects of the oral administration of Lactobacillus casei on type II collagen-induced arthritis in DBA/1 mice . Life Sci 63 : 635 –644 .9718093 22 Sheil B , McCarthy J , O’Mahony L , Bennett MW , Ryan P , et al (2004 ) Is the mucosal route of administration essential for probiotic function ? Subcutaneous administration is associated with attenuation of murine colitis and arthritis . Gut 53 : 694 –700 .15082588 23 Fichera GA , Giese G (1994 ) Non-immunologically mediated cytotoxicity of Lactobacillus casei and its derivative peptidoglycan against tumor cell lines . Cancer Lett 85 : 93 –103 .7923109 24 Biffi A , Coradini D , Larsen R , Riva L , Di Fronzo G (1997 ) Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line . Nutr Cancer 28 : 93 –99 .9200156 25 Kim JY , Woo HJ , Kim YS , Lee JH (2002 ) Screening for antiproliferative effects of cellular components from lactic acid bacteria against human cancer cell lines . Biotechnol Lett 24 : 1431 –1436 . 26 Orlando A , Refolo MG , Messa C , Amati L , Lavermicocca P , et al (2012 ) Antiproliferative and proapoptotic effects of viable or heat-killed Lactobacillus paracasei IMPC2.1 and Lactobacillus rhamnosus GG in HGC-27 gastric and DLD-1 colon cell lines . Nutr Cancer 64 : 1103 –1111 .23061912 27 Reddy BS , Rivenson A (1993 ) Inhibitory effect of Bifidobacterium longum on colon, mammary, and liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline, a food mutagen. Cancer Res. . 53 : 3914 –3918 . 28 Lim BK , Mahendran R , Lee YK , Bay BH (2002 ) Chemopreventive effect of Lactobacillus rhamnosus on growth of a subcutaneously implanted bladder cancer cell line in the mouse . Jpn J Cancer Res 93 : 36 –41 .11802806 29 Candela M , Guidotti M , Fabbri A , Brigidi P , Franceschi C , et al (2011 ) Human intestinal microbiota: cross-talk with the host and its potential role in colorectal cancer. Crit Rev Microbiol. . 37 : 1 –14 . 30 Matsumoto M , Kurihara S , Kibe R , Ashida H , Benno Y (2011 ) Longevity in mice is promoted by probiotic-induced suppression of colonic senescence dependent on upregulation of gut bacterial polyamine production . PLoS One 6 : e23652 .21858192 31 Sperandio V, Torres AG, Kaper JB (2002) Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol. Microbiol. 43: 809, 2002. 32 Merritt J , Qi F , Goodman SD , Anderson MH , Shi W (2003 ) Mutation in luxS affects biofilm formation in Streptococcus mutans . Infect Immun 71 : 1972 –1979 .12654815 33 Fujiya M , Musch MW , Nakagawa Y , Hu S , Alverdy J , et al (2007 ) The Bacillus subtilis Quorum-Sensing Molecule CSF Contributes to Intestinal Homeostasis via OCTN2, a Host Cell Membrane Transporter . Cell Host & Microbe 1 : 299 –308 .18005709 34 Yan F , Polk DB (2002 ) Probiotic Bacterium Prevents Cytokine-induced Apoptosis in Intestinal Epithelial Cells. J Biol Chem. . 277 : 50959 –50965 . 35 Iyer C , Kosters A , Sethi G , Kunnumakkara AB , Aggarwal BB , et al (2008 ) Probiotic Lactobacillus reuteri promotes TNF-induced apoptosis in human myeloid leukemia-derived cells by modulation of NF-kB and MAPK signaling . Cellular Microbiology 10 : 1442 –1452 .18331465 36 Fakhry S , Manzo N , D’Apuzzo E , Pietrini L , Sorrentini I , et al (2009 ) Characterization of intestinal bacteria tightly bound to the human ileal epithelium. Res. Microbiol. . 160 : 817 –823 . 37 Zwacka MR , Stark L , Dunlop MG (2000 ) NF-kB kinetics predetermine TNFalpha sensitivity of colorectal cancer cells . J Gene Med 2 : 334 –343 .11045427 38 Roemer MM , Roemer K (2001 ) p21 Waf1/Cip1 can protect human colon carcinoma cells against p53-dependent and p53-independent apoptosis induced by natural chemopreventive and therapeutic agents . Oncogene 20 : 3387 –3398 .11423989 39 Yan F , John SK , Polk DB (2001 ) Kinase suppressor of Ras determines survival of intestinal epithelial cells exposed to tumor necrosis factor Cancer Res . 61 : 8668 –8675 . 40 D’Amours D , Sallmann FR , Vishva MD , Poiriers GG (2001 ) Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis . J Cell Sci 114 : 3771 –3778 .11707529 41 Kaufmann SH , Desnoyers S , Ottaviano Y , Davidson NE , Poirier GG (1993 ) Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res . 53 : 3976 –3985 . 42 Sherr CJ , Roberts JM (1999 ) CDK inhibitors positive and negative regulators of G1-phase progression. Genes Dev. . 13 : 1501 –1512 . 43 Weinberg WC , Denning MF (2002 ) p21Waf1 control of epithelial cell cycle and cell fate. Crit Rev Oral Biol Med. . 13 : 453 –464 . 44 Bloom J , Amador V , Bartolini F , DeMartino G , Pagano M (2003 ) Proteasome-Mediated Degradation of p21 via N-Terminal Ubiquitinylation Cell . 115 : 71 –82 . 45 Pollice A , Vivo M , La Mantia G (2008 ) The promiscuity of ARF interactions with the proteasome . FEBS Letters 582 : 3257 –3262 .18805416 46 McCubrey JA , Steelman LS , Chappell WH , et al (2007 ) Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim. Biophys. . Acta 1773 : 1263 –1284 . 47 El-Deiry WS , Tokino T , Velculescu VE , Levy DB , Parsons R , et al (1993 ) WAF1, a potential mediator of p53 tumor suppression . Cell 75 : 817 –825 .8242752 48 Vivo M , Di Costanzo A , Fortugno P , Pollice A , Calabrò V , et al (2009 ) Downregulation of ΔNp63α in keratinocytes by p14ARF-mediated SUMo-conjugation and degradation . Cell Cycle 8 : 3545 –3551 .19829080 49 Vivo M , Ranieri M , Sansone F , Santoriello C , Calogero RA , et al (2013 ) Mimicking p14ARF Phosphorylation Influences Its Ability to Restrain Cell Proliferation . PLoS One 8 : e53631 .23308265 50 Di Luccia B, Manzo N, Vivo M, Galano E, Amoresano A, et al. (2013) A Biochemical and Cellular Approach to Explore the Antiproliferative and Prodifferentiative Activity of Aloe Arborescens Leaf Extract. Phytother Res. doi: 10.1002/ptr.4939.	23894414	PMC3720908	CC BY	2021-01-05 17:34:15	yes	PLoS One. 2013 Jul 23; 8(7):e69102
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23894578PONE-D-13-1834110.1371/journal.pone.0070020Research ArticleDexamethasone Rescues Neurovascular Unit Integrity from Cell Damage Caused by Systemic Administration of Shiga Toxin 2 and Lipopolysaccharide in Mice Motor Cortex Dexamethasone Reduces Stx2 NeurotoxicityPinto Alipio 1 Jacobsen Mariana 1 Geoghegan Patricia A. 2 Cangelosi Adriana 2 Cejudo María Laura 1 Tironi-Farinati Carla 1 Goldstein Jorge 1 * 1 Laboratorio de Neurofisiopatología, Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina 2 Centro Nacional de Control de Calidad de Biológicos (CNCCB), – ANLIS “Dr. Carlos G. Malbrán”, Ciudad Autónoma de Buenos Aires, Argentina Neyrolles Olivier Editor Institut de Pharmacologie et de Biologie Structurale, France * E-mail: jogol@fmed.uba.arCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: AP CTF JG PG AC. Performed the experiments: AP MJ PG AC MC CTF JG. Analyzed the data: AP JG PG AC. Contributed reagents/materials/analysis tools: JG AP PG AC. Wrote the manuscript: AP JG. 2013 23 7 2013 8 7 e7002003 5 2013 14 6 2013 © 2013 Pinto et al2013Pinto et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) that can lead to fatal encephalopathies. Neurological abnormalities may occur before or after the onset of systemic pathological symptoms and motor disorders are frequently observed in affected patients and in studies with animal models. As Stx2 succeeds in crossing the blood-brain barrier (BBB) and invading the brain parenchyma, it is highly probable that the observed neurological alterations are based on the possibility that the toxin may trigger the impairment of the neurovascular unit and/or cell damage in the parenchyma. Also, lipopolysaccharide (LPS) produced and secreted by enterohemorrhagic Escherichia coli (EHEC) may aggravate the deleterious effects of Stx2 in the brain. Therefore, this study aimed to determine (i) whether Stx2 affects the neurovascular unit and parenchymal cells, (ii) whether the contribution of LPS aggravates these effects, and (iii) whether an inflammatory event underlies the pathophysiological mechanisms that lead to the observed injury. The administration of a sub-lethal dose of Stx2 was employed to study in detail the motor cortex obtained from a translational murine model of encephalopathy. In the present paper we report that Stx2 damaged microvasculature, caused astrocyte reaction and neuronal degeneration, and that this was aggravated by LPS. Dexamethasone, an anti-inflammatory, reversed the pathologic effects and proved to be an important drug in the treatment of acute encephalopathies. These studies were supported by CONICET (National Research Council, Argentina) Grant PIP114-200801-00497 and PIP 112-201101-00901 (http://www.conicet.gov.ar/web/conicet/) to JG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and Hemolytic Uremic Syndrome (HUS) [1], the triad of thrombocytopenia, microangiopathic hemolytic anemia and acute renal failure [2], and it is the main cause of acute renal failure and the second cause of chronic renal failure and renal transplantation in children in Argentina [3]. Furthermore, central nervous system (CNS) alterations caused by STEC are a leading cause of mortality among children during the period of acute illness [4–6]. In North America and Europe, 0.72 to 1.44 cases of HUS per 100,000 population are reported each year [7]. The largest outbreak of HUS in Europe took place between the months of May and July 2011 and began in northern Germany. HUS was diagnosed in 855 patients out of a total of 3,842 patients infected with STEC O104: H4. The death toll in Germany was 53 [8,9]. Currently Argentina has the highest occurrence of HUS worldwide, with approximately 420 cases reported annually and an incidence of 17/100,000 in children under 5 years of age [10]. It has been reported that the mortality rate derived from HUS ranges between 0–5% of the cases, and 7-40% when the CNS is involved [11–13]. Approximately 35% of the patients with HUS progress to CNS dysfunction [14–22] but 9-15% of patients have CNS dysfunction even before the first symptoms of HUS, suggesting that damage in the CNS may occur before or concomitantly with other symptoms of the systemic disease [15,16]. CNS symptoms in STEC disease range from decerebrate posture, hemiparesis, ataxia and cranial nerve palsy to ophthalmological dysfunctions, hallucinations, seizures and changes in level of consciousness (from lethargy to coma) [23–26]. Symptoms in mice include lethargy, shivering, abnormal gait, hind limb paralysis, spasm-like seizure, reduced spontaneous motor activity, abnormal gait and pelvic elevation [27,28]. Previous reports claim that severe and even fatal encephalopathy is due to damage found in the microvasculature, neurons and/or astrocytes that compromise normal functioning of the neurovascular unit [23,27,28]. In addition to the known deleterious effects of Stx2, the gram-negative EHEC releases the endotoxin lipopolysaccharide (LPS). LPS is a component of the outer membrane that has no direct cytotoxic action but rather induces a variety of inflammatory mediators when secreted in the gut [29]. In the present study, the harmful cytotoxic effects of co-administration of LPS and Stx2 were studied in the neurovascular unit by the use of specific cell markers. Lycopersicon esculentum lectins, NeuN, glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) antibodies were employed to study distribution of endothelium glycocalyx [30], early signs of neuronal degeneration [31], reactive astrocytes [32], and angioplasticity (angiogenic adaptational changes) [33]. Given that neurological alterations are commonly observed in the motor cortex as well as motor disturbances in STEC-infected patients [23–26], the cell alterations produced in this brain region were specifically analyzed. The study of this area could be clinically relevant to determine predictive factors for generalized seizures and/or other disorders produced in this brain area that may lead to death [8,11–14,17,25,27]. Animal models have been established and analyzed to define the nature of a disease in humans. To this end, animal models must resemble the human disease [34]. Therefore, the objective of this paper was (i) to study the contribution of LPS to pathogenicity in the neurovascular unit in mice brain following systemic administration of a sub-lethal dose of Stx2, and (ii) to determine whether these pathogenic changes include an inflammatory component. To this end, the glucocorticoid Dexamethasone, an anti-inflammatory and possible neuroprotectant, was challenged to neutralize the toxic action of LPS with Stx2. Materials and Methods Stx2 Protein Purification Stx2 was purified by affinity chromatography under native conditions as previously described [35]. Briefly, recombinant E. coli DH5a containing pStx2 were cultured overnight. The supernatant obtained was precipitated in 60% SO4 (NH4) 2 1 mM PMSF, and the pellet was dialyzed overnight, resuspended in phosphate buffer solution (PBS) with a cocktail of protease inhibitors, and incubated with Globotriose Fractogel Resin (IsoSep AB, Tullinge, Sweden). The resin was washed and the toxin eluted with MgCl2. Protein concentration was determined in all the eluates. Protein content in all the fractions was monitored by silver/Coomassie blue staining [36], and the presence of Stx2 in the eluates was confirmed by Western Blot analysis. Results showed a 7.7-kDa band corresponding to Stx2B and a 32-kDa band corresponding to Stx2A. The same batch of toxin was used for all the experiments. The cytotoxic capacity of Stx2 was assessed in Vero cells by the neutral red assay and the cytotoxic dose 50 (CD50) found was about 1 pg/ml [37]. This effect was neutralized by means of preincubation with an anti-subunit 2B monoclonal antibody (Sifin, Berlin, Germany), and not neutralized when using an isotype antibody instead [37]. Lipopolysaccharide (LPS) was removed from the Stx2 solution by using Detoxi-gel (Pierce, Rockford, USA). This Stx2 solution contained less than 0.03 endotoxin units/ml. Neurovascular Toxicity Assays Ninety-six female pathogen-free NIH mice weighing 25-30g were housed in an air conditioned and light-controlled (lights on between 06:00 am and 06:00 pm) animal facility. They were then separated into groups and subjected to the following intravenous (i.v.) treatments: LPS (428.6ng); Stx2 (0.5ng); LPS+Stx2 (0.5ng Stx2+ 428.6ng LPS); vehicle infusion (saline solution) (Table 1). Each animal received two i.v. doses in the lateral tail vein (half the total amount and 100 µl per injection for each treatment) at an interval of 24 hours. Food and water were provided ad libitum. Six mice per group were anesthetized with chloral hydrate (350 mg/kg) and perfused transcardially with 0.9% NaCl solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS) [fixative per animal weight (ml/g)] at the following time intervals after the respective treatment: 2, 4, 7 and 20 days. The LPS used was from E. coli O157:H7 (Sigma, Saint Louis, MO, USA). Brains were removed from the skull and post-fixed with the same fixative solution for 2 hours, and cryoprotected through a daily sequenced passage of increasingly concentrated sucrose solutions (10, 20 and 30%). Brain coronal sections (25 µm thick) were cut with a cryostat, maintained in a cryoprotectant solution (50% PBS 0.1 M, 30% Ethylene glycol, 20% Glycerol) at -20 °C, and subsequently processed for immunofluorescence microscopy. Table 1 Diagram of the study. Experiment n Treatment Days of treatment Total number of mice Neurovascular Toxicity Assays 6 Vehicle 2, 4, 7, 20 96 LPS Stx2 Stx2+LPS Neurovascular Protection Assays 4 Vehicle 4 32 LPS + Saline solution Stx2 + Saline solution Stx2+LPS + Saline solution Dexamethasone LPS + Dexamethasone Stx2 + Dexamethasone Stx2+LPS + Dexamethasone The experimental protocols and euthanasia procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the School of Medicine of Universidad de Buenos Aires, Argentina (Resolution No. 2437/2012). All the procedures were performed in accordance with the EEC guidelines for care and use of experimental animals (EEC Council 86/609). Lectin Histofluorescence Six floating sections (the same number of sections was used for all immunofluorescence assays performed) for each treatment were subjected to Lycopersicum esculentum lectin histochemical marker to study the motor cortex endothelial cells. After several rinses with 10 mM PBS, sections were incubated with biotinylated lectin-10µg/ml 0.3% Triton X-100 in the same buffer (4°C for 24 hours), and subsequently incubated with Alexa-488 Streptavidin (1:100) 0.3% Triton X-100 for 1 hour at room temperature (RT), rinsed 3 times with 10 mM PBS and mounted on slides with a solution of glycerol and PBS 3:1 (sections for other immunofluorescence assays were also mounted in the same solution). Controls were performed using the same procedure but without adding the lectin protein. A green fluorescence filter was used to visualize brain cortex microvessels, and Adobe Photoshop software to assemble the images and obtain merged images (the same procedure was used for the other immunofluorescence assays). VEGF Immunofluorescence After several rinses with 10 mM PBS, brain floating sections were incubated to determine the expression of the vascular endothelial growth factor (VEGF), first with the same buffer but with 0.1% Triton X-100 for one hour, followed by normal goat serum 10% with 0.3% Triton X-100 in PBS, also for one hour. The sections were subsequently incubated with anti-VEGF antibody diluted at 1:500 in 10mM PBS with 0.3% Triton X-100 at 4 °C for 48 hours. After several rinses with Triton X-100 0.025% sections were incubated with goat IgG anti-mouse/Texas Red diluted at 1:200 in the same buffer with 0.3% Triton X-100 for 2 hours at RT. Finally, sections were rinsed with 10mM PBS and mounted on slides. Controls were performed using the same procedure but without adding the primary antibody for VEGF. A red fluorescence filter was used for visualization of immunofluorescence to VEGF localization. NeuN Immunofluorescence After several rinses with 10 mM PBS, sections were incubated with 10 mM PBS Triton X-100 0.1% for one hour, followed by normal goat serum 10% with 0.3% Triton X-100 in the same solution, also for one hour. Sections were subsequently incubated with anti-NeuN antibody diluted at 1:500 in 10mM PBS with 0.3% Triton X-100 at4°C for 48 hours. After several rinses with 10mM PBS Triton X-100 0.025% sections were incubated with goat IgG anti-mouse/Texas Red diluted at 1:200 with 0.3% Triton X-100 in the same buffer for 2 hours at RT. Finally, sections were rinsed with 10mM PBS and mounted on slides. Controls were performed using the same procedure but without adding the primary antibody. A red fluorescence filter was used for visualization of the NeuN immunofluorescence. GFAP Immunofluorescence After several rinses with 10 mM PBS, brain floating sections were incubated with 10 mM PBS 0.1% Triton X-100 for one hour, followed by normal goat serum 10% in PBS 0.3% Triton X-100 for another hour. Sections were then incubated with anti-GFAP antibody (dilution 1:500) in 10 mM PBS 0.3% Triton X-100 at 4°C for 48 hours. After several rinses with 10 mM PBS Triton X-100 0.025%, brain sections were incubated with goat IgG anti-Rabbit/Texas Red (dilution 1:200) in 10mMPBS Triton X-100 0.3% for one and a half hours at RT. Finally, sections were rinsed in 10 mM PBS and mounted on slides. Controls were performed using the same procedure but without adding the primary antibody. A red fluorescence filter was used for visualization of GFAP immunofluorescence. Merging Images The procedure described above was employed to obtain merged images of GFAP and lectin immunofluorescence and/or VEGF and lectin immunofluorescence. The lectin histofluorescence protocol was always performed after GFAP or VEGF immunofluorescence. All analyses were carried out in the same comparable areas. Neurovascular Protection Assays Thirty-two female mice divided into eight groups of 4 mice each were used for this experiment (two groups treated with vehicle, two with LPS, two with Stx2 and two with Stx2+LPS as described above, table 1). Four of these groups were treated with 7.5 mg/kg i.p. Dexamethasone (100 µl per dose) twice a day for 3 days, starting when they received their respective i.v. treatment (vehicle, LPS, Stx2 or Stx2+LPS), and perfused on the fourth day as described above; the other half received 100 µl of i.v. saline solution twice a day, also for three days, and were perfused on the fourth day. The perfusion and treatment procedures to obtain the brains were performed as previously described. Analysis of Micrographs A total of 32 brain motor cortex micrographs per treatment were analyzed. For this purpose, eight different sections per treatment were obtained and two micrographs were taken from each hemisphere. Micrographs were taken between cortical layers II (external granular layer) and V (internal pyramidal layer) of M1 and M2 [38] to determine neurodegeneration (NeuN), endothelial damage (lectins), expression of vascular endothelial growth factor (VEGF), and reactive astrocytes (GFAP). A fluorescence Axiophot Zeiss microscope with a 20x objective lens was used. The images obtained were analyzed using the ImageJ software (NIH). Two criteria were used to analyze endothelial damage: changes in glycocalyx expression in microvessels (as the number of glycocalyx particles bound to lectins) and density of microvessels (as the percentage of area occupied by microvessels). The particles analyzed were quantified by conversion into 8-bit and contrast against the background. Moreover, objects with an area less than 10 µm2 were excluded to avoid quantified dots from the background. In addition, VEGF immunopositive particles were quantified as described above. Acquired images were opened using Adobe Photoshop CS software to determine neurodegeneration, and nuclei with normal phenotype were quantified and painted to avoid errors. These data were represented as the percentage of degenerated nuclei in respect of total nuclei per micrograph. The ROI Manager tool on Image-J software was employed to quantify the expression of GFAP and to determine reactive astrocytes. The mean gray option was selected and integral optical density (IOD) was employed to obtain the mean of a grayscale. Statistical Analysis The data are presented as mean ±SEM. In the case of different toxicity treated-groups and their respective controls were challenged with dexamethasone at one time point (4 days of treatment) in the neurovascular protection assays, statistical significance was performed using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post hoc tests. In the case of comparison of different treatment groups at different time points in the neurovascular toxicity assays, two-way analysis of ANOVA was used followed Bonferroni post hoc test (GraphPad Prism 4, GraphPad Software, Inc.). The criterion for significance was p<0.001 for all the experiments. Samples subjected to the neurovascular protection assays were independent from those assayed for neurovascular toxicity. The number of animals and the corresponding brain section samples used in the neurovascular protection assays (dexamethasone assays) yielded error bars with low dispersion and therefore it was not necessary to subject additional animals and/or brain sections to these treatments. Results Intravenous administration of a sublethal dose of Stx2 changes the profile of microvessels and LPS exacerbates these changes in the brain motor cortex Lectin fluorescence binding to glycoconjugates was used to detect the changes in the microvasculature profile until day twenty of treatment. Lectins are non-immune proteins that bind with high affinity to glycoconjugates present in the glycocalyx of endothelial cells. Representative micrographs obtained from saline-treated control mice showed continuous lectin fluorescence binding throughout all microvessels. Microvessels of saline-treated mice were well preserved, with continuous and defined edges in comparison with those treated with the toxins. In addition, microvessels occupied a larger area in the cortex per observed field than microvessels treated with the toxins (Figure 1A, E, I, M). In toxin-treated mice, discontinuous lectin fluorescence binding distributed in patches with poorly defined edges was observed and, consequently, the lectin microvessel density was significantly decreased (Figure 1H). Microvessels from Stx2 plus LPS (Stx2+LPS)-treated mice were maximally damaged after four days of treatment (Figure 1H) compared with Stx2-alone- or LPS-alone-treated mice. However, after twenty days, microvessels from the Stx2+LPS, Stx2 and LPS treatments recovered a normal appearance similar to vehicle-treated microvessels (Figure 1P). All the observed changes in the microvasculature were confirmed by morphometric analysis: the density of microvessels (calculated as the percentage of microvessels that occupy a determined area) and the number of positive glycocalyx-particles bound to fluorescence lectins were determined. A maximal and significant decrease in density of microvessels (p<0.001) was observed in the Stx2+LPS-treated mice compared with the Stx2-treated ones, and in both groups in comparison with the saline-treated control mice between days four and seven of treatment (Figure 1Q), while a maximal and significant increase in the number of positive glycocalyx particles bound to fluorescence lectins (p<0.001) was observed as a result of microvessel fragmentation in Stx2+LPS-treated mice compared with Stx2-treated ones, and in both groups in comparison with saline-treated control mice between days four and seven of treatment (Figure 1R). 10.1371/journal.pone.0070020.g001Figure 1 Changes in the expression of glycocalyx particles bound to fluorescent lectins in the cerebral microvasculature of mice motor cortex. Micrographs A, E, I and M show the microvasculature profile in the control groups; B, F, J and N: LPS treatment; C, G, K and O: Stx2 treatment; D, H, L and P: Stx2+LPS treatment; at two, four, seven and twenty days after the respective treatment. Arrows (H) show microvessels devoid of glycocalyx particles bound to fluorescent lectins. Stx2+LPS decreased microvessel density (Q) and increased the number of positive glycocalyx particles (R). : significant differences between treated and control groups; : significant differences between Stx2 and Stx2+LPS treatments (p <0.001). Dexamethasone recovered the density of microvessels and glycocalyx integrity Treatment with Dexamethasone recovered glycocalyx distribution in microvessels and significantly reduced the number of fragmented glycocalyx particles bound to fluorescent lectins after four days of treatment with LPS, Stx2 and Stx2+LPS (Figure 2J). It also maintained the integrity of microvessel edges in the three experimental groups described (Figure 2B–D, F–H) and the density of microvessels was increased (Figure 2I). 10.1371/journal.pone.0070020.g002Figure 2 Dexamethasone maintains microvessel integrity. Fluorescence micrographs show changes in the profile of microvessels from the motor cortex after four days of treatment with saline (A and E), LPS (B and F), Stx2 (C and G) and Stx2+LPS (D and H) followed by i.p. injection of saline (A through D) or Dexamethasone (E through H). Dexamethasone rescues microvessel density (I) and glycocalyx distribution (J), similar to density and distribution in the control groups. K: negative control in mice motor cortex obtained by not adding Lycopersicum esculentum lectins. : significant differences between Dexamethasone and saline treatments. Intravenous administration of a sublethal dose of Stx2 inhibits the expression of VEGF, and this is exacerbated by LPS Following the observation that the toxins changed the profile of microvessels, it was postulated that they could also change the expression of VEGF, an angiogenic growth factor that may appear under regenerative processes. An anti-VEGF antibody was employed to evaluate whether LPS, Stx2 or both toxins combined changed the expression of VEGF in motor cortex microvessels. VEGF expression was observed in microvessels, and it co-localized with glycocalyx particles bound to lectin fluorescence (Figure 3Q). A significant decrease in the expression of VEGF was observed two days after administration of LPS, Stx2 and Stx2+LPS (Figure 3A–D), although no significant differences were observed between the Stx2 and Stx2+LPS treatments. Maximum VEGF reduction was observed after seven days of treatment with Stx2+LPS (Figure 3H, L). Total reduction of VEGF was also observed after the treatment with Stx2 and/or LPS as from day seven (Figure 3J, K). However, a restoration tendency in VEGF expression was observed after twenty days (Figure 3N–P). 10.1371/journal.pone.0070020.g003Figure 3 Changes in the expression of VEGF. Micrographs A, E, I and M: basal expression of VEGF with saline treatment; B, F, J and N: LPS treatment; C, G, K and O: Stx2 treatment; D, H, L and P: Stx2+LPS treatment; at two, four, seven and twenty days after the respective treatment. Stx2+LPS fully decreased VEGF expression after four days of treatment until day seven (H and L). LPS (J) or Stx2 (K) treatment fully decreased VEGF expression as from day seven. Micrograph Q shows co-localization of VEGF and glycocalyx particles bound to lectins in the endothelium. Stx2+LPS significantly decreased VEGF expression as compared to LPS or Stx2 treatment (R) (p <0.001). : significant differences between the treated and saline groups. :significant differences between Stx2+LPS and Stx2 treatments (p <0.001). Dexamethasone restores the basal expression of VEGF Dexamethasone succeeded in significantly elevating the basal expression of VEGF, which was reduced by the action of LPS and Stx2 (Figure 4), and reestablished the inhibited expression of VEGF caused by the treatment with Stx2+LPS after four days of i.v. administration (Figure 4I). 10.1371/journal.pone.0070020.g004Figure 4 Dexamethasone restores the expression of VEGF. Micrographs show the expression of VEGF by immunofluorescence in mice motor cortex after four days of treatment with saline (A and E), LPS (B and F), Stx2 (C and G) and Stx2+LPS (D and H) followed by i.p. injection of saline (without Dexamethasone, A through D) or Dexamethasone (E through H). VEGF expression is quantified under different treatments (I). J: negative control by not adding the primary antibody. : significant differences between mice treated with Dexamethasone and those treated with saline (p <0.001). Intravenous administration of a sublethal dose of Stx2 produces neurodegeneration, and LPS exacerbates this Anti-NeuN antibody was employed to determine whether systemic administration of LPS, Stx2 or Stx2+LPS caused neurodegeneration. A nuclear dot staining pattern by indirect immunofluorescence and/or negative nuclear immunofluorescence with perinuclear immunofluorescence pattern for Neu-N confirmed a neurodegenerative phenotype, while a conserved and homogeneous nuclear immunofluorescence pattern for Neu-N confirmed healthy neurons (Figure 5A). Neurons in degenerative state were observed two days after administration of LPS, Stx2 or Stx2+LPS (Figure 5Q). It must be noted that a significant increase in the number of degenerated neurons was observed in animals treated with Stx2+LPS as compared to the Stx2 or LPS treatments after four, seven and even twenty days. Accordingly, maximal neurodegeneration was observed four days after administration of Stx2+LPS (Figure 5H) and it decreased at seven and twenty days (Figure 5P). 10.1371/journal.pone.0070020.g005Figure 5 Changes in the expression of NeuN. Micrographs show immunofluorescence staining for NeuN in the nucleus of neurons in the motor cortex labeled with anti-NeuN. A, E, I and M: vehicle; B, F, J and N: LPS; C, G, K and O: Stx2; D, H, L and P: Stx2+LPS; at two, four, seven and twenty days after the respective treatment. Arrows show nuclei of degenerating neurons (H); asterisk shows normal nuclei of neurons (A). Quantification of phenotypic nuclear abnormalities in neurons at different days and for different treatments (Q). : significant differences among all treated and control groups, * significant differences between Stx2 and Stx2+LPS treatments (p <0.001). Dexamethasone protects neurons against Stx2 and LPS A significant decrease in the number of degenerated neurons in all groups treated with toxin (LPS, Stx2 or Stx2+LPS) was observed when challenged with a dose of Dexamethasone (Figure 6I). It was found that Dexamethasone protected about 30% of neurons against the administration of Stx2+LPS after 4 days. 10.1371/journal.pone.0070020.g006Figure 6 Dexamethasone decreases the percentage of degenerative neurons. Micrographs show the expression of NeuN by immunofluorescence in mice motor cortex immunolabeled with anti-NeuN antibody after four days of treatment with saline (A and E), LPS (B and F), Stx2 (C and G), Stx2+LPS (D and H) followed by i.p. injection of saline (A through D) or Dexamethasone (E through H). Dexamethasone protected neurons from the cytotoxic action of LPS, Stx2 and Stx2+LPS (I). J: negative control by not adding the primary antibody. : significant differences between mice treated with Dexamethasone and those treated with saline (p <0.001). Intravenous administration of a sublethal dose of Stx2 produces reactive astrocytes, and the combination with LPS exacerbates them The study of glial fibrillary acidic protein (GFAP) expression by immunofluorescence was carried out to determine whether i.v. administration of LPS, Stx2 and/or Stx2+LPS produced reactive astrocytes. GFAP is a cytoskeletal protein produced in astrocytes and expression thereof increases following a noxious event. It was observed that Stx2 administration increased the expression of GFAP in reactive astrocytes as from two days after the LPS, Stx2 and Stx2+LPS treatment (Figure 7R). Quantification of GFAP levels was performed by integral optical density (IOD) imaging. Maximum expression of GFAP was observed after four days of Stx2+LPS treatment in comparison with the Stx2, LPS or vehicle treatments (Figure 7H), while minimum expression of GFAP was observed after 20 days for all treatments (Figure 7N–P). 10.1371/journal.pone.0070020.g007Figure 7 A sub-lethal dose of the toxins causes reactive astrocytes. Immunofluorescence using an anti-GFAP antibody was employed to show reactive astrocytes in mice motor cortex. A, E, I and M: saline-treated astrocytes; B, F, J and N: LPS-treated astrocytes; C, G, K and O: Stx2-treated astrocytes; D, H, L and P: Stx2+LPS-treated astrocytes; at two, four, seven and twenty days after the respective treatment. Some reactive astrocytes contact microvessels that express VEGF (Q). Quantification of reactive astrocytes (R). Stx2+LPS treatment caused maximum astrocyte reaction (H). : significant differences between toxin-treated and control groups, *: significant differences between Stx2 and Stx2+LPS treatments (p <0.001). Dexamethasone reduces the number of reactive astrocytes The Dexamethasone treatment significantly reduced the expression levels of GFAP in all treated groups, except in the vehicle one (Figure 8J), and it concomitantly also reduced the number of reactive astrocytes. 10.1371/journal.pone.0070020.g008Figure 8 Dexamethasone reduces reactive astrocytes. Micrographs show the expression of GFAP by immunofluorescence with anti-GFAP antibody in astrocytes after four days of treatment with saline (A and E), LPS (B and F), Stx2 (C and G) and Stx2+LPS (D and H) followed by i.p. injection of saline (A through D) or Dexamethasone (E through H). Quantification of reactive astrocytes (I) under all treatments. J: negative control by not adding the primary antibody. : significant differences between mice treated with Dexamethasone and those treated with saline (p <0.001). Discussion Various authors have reported that systemic infection with Stx2 or STEC causes brain damage in different animal models [39–41]. However, the contribution of LPS secreted by EHEC to brain damage by Stx2 has not been considered. The present study has shown strong evidence that LPS enhances the cytotoxic action of Stx2 in microvasculature, astrocytes and neurons of mice motor cortex. This is consistent with previous findings by other authors showing that in vitro administration of Stx2 together with LPS results in an enhanced synergistic cytotoxic effect compared with Stx2 alone on human umbilical vein endothelial cells [42], and also that anti-LPS antibodies belonging to the O157:H7 serotype have been found in the serum of HUS patients along with clinical evidence of endotoxemia [43,44]. Stx2 alone is not enough to obtain a complete murine model of HUS infection but such model should also include LPS [45]. Therefore, the present study aimed to determine for the first time the contribution of secreted LPS to the encephalopathy caused by the systemic administration of a sub-lethal dose of Stx2 in a murine model that emulates a pathological condition observed in patients infected with Stx2 who suffer from acute encephalopathy. The alterations observed in the microvasculature support the fact that Stx2 crosses the blood–brain barrier [34,35]. In line with this observation, in the present study it was observed that co-treatment with Stx2 and LPS led to significant alteration of the endothelium involving discontinuity of the endothelial glycocalyx and compromising the integrity of the blood–brain barrier. It is known that the glycocalyx contributes to vascular protection in vessel walls [46] and to maintenance of vascular permeability [47,48]. Therefore, the intact glycocalyx is necessary for the maintenance of normal vascular function and its discontinuity compromises integrity of the blood–brain barrier [49]. Damage to the endothelium was progressive and it coincided with maximum damage of neurons and astrocytes after the fourth day of treatment. Endothelial cell damage was accompanied by a decrease in the expression of VEGF. This event was previously observed in primary culture of human podocytes treated with Stx [50] and it supports our findings. Recent reports indicate that alteration or loss of VEGF contributes to degeneration of neurons [51] and this may have occurred in our animal model. Conversely, VEGF treatment enhances neuronal survival and neurite outgrowth in explanted brain cortex or substantia nigra [51,52], as well as maturation in primary cortical neurons [53] in response to different stress situations [51,53]. However, whether the neurotrophic effects of VEGF protect neurons or are mediated indirectly by glial cells still remains to be elucidated. Therefore, co-treatment of Stx2 and LPS may reduce VEGF expression that could contribute to neuronal degeneration. In the present model of Stx2+LPS injury, the BBB became more permeable and therefore the toxin reached the brain parenchyma. This is consistent with previous studies showing that co-administration of Stx and LPS results in more severe hemorrhage compared with Stx2 alone [54]. Accordingly, astrocytes, which also constitute the neurovascular unit, become injured [35,55]. They are the largest number of cells in the CNS and react in response to all types of insults [32] such as trauma, ischemia, neurodegenerative [56] or infective diseases [57] through a phenomenon known as astrogliosis. At this stage GFAP is dramatically deregulated [32] and LPS may produce and/or regulate specific aspects of reactive astrocytes during inflammatory processes [58]. Direct damage to neurons by Stx2 has been previously demonstrated [35]. Systemic administration of the toxin in this study also caused neuronal damage. Co-administration of both toxins increased the number of damaged neurons. Possible deleterious actions of pro-inflammatory and/or other elements raised by Stx2 and LPS on neuronal damage should also be taken into account and are currently under consideration for future research. It is known that LPS produces an immune response that causes increased production of TNFα and IL-1β in microglia through the p38α mitogen-activated protein kinase, leading to neurotoxicity [59,60]. In addition, it has been observed that co-cultures of microglia and neurons treated with LPS release TNFα, causing loss of synaptic proteins and eventually neuronal death [60]. In our model mechanisms involving inflammatory responses could indicate the microglia as a central target for the effects of both toxins, as it belongs to the monocyte-macrophage lineage [61]. It has been reported that peripheral blood monocytes, granulocytes and alveolar macrophages are targets of Stx2 binding and toxicity through the Gb3 receptor [62]. In addition, it has been demonstrated that Ricin, a toxin with RNA N-glycosidases of 28S RNA action that presents the same ribotoxicity as Stx2 [63], is responsible for initiating upstream events that lead to inflammatory responses [64] by activation of stress-activated protein kinases (SAPKs) [65]. Therefore it is posited that the microglia may mediate the observed neurotoxicity through the SAPKs pathway, in a way similar to macrophage response to Stx2 or Ricin. As reported by some authors, the use of antibiotics is not recommended in STEC infections as this may release more Stx2 into the digestive tract [66]. Accordingly, administration of the antibiotic ciprofloxacin to mice infected with E. coli O157:H7 resulted in the production of elevated levels of Stx2 [67], which would increase the chances of the condition being aggravated by HUS in children and adults [68–70]. Consequently, it is important to develop a treatment with neuroprotective agents. Dexamethasone has proved to be a good neuroprotective candidate as in previous experiments we found that it increases survival of mice challenged with two lethal doses of Stx2 by 50% (unpublished data). Dexamethasone is a glucocorticoid and one of the most common corticosteroids used in medicine. Its biological response is thirty times more potent than endogenous cortisol [71], it succeeds in reducing plasma IL-1β and it may provide neuroprotective effects [72]. Dexamethasone also increases the expression of occludin, a protein present in tight junctions localized between BBB endothelial cells [71], making the BBB less permeable. In the present study it was observed that Dexamethasone rescued the integrity of the neurovascular unit function following the detrimental action of both toxins by restoring the normal distribution of the endothelial glycocalyx and the basal expression of VEGF. Moreover, it significantly decreased the astrocyte reaction in all treatments and rescued about 60% of neurons from a degenerative phenotype. This drug therefore appears to reduce inflammation and BBB permeability in the CNS. The differences observed in cell damage in the brain cortex between the treatments employing Stx2 free of LPS and Stx2 with LPS were conclusive. In the encephalopathy mediated by Stx2, the contribution of LPS and the inflammatory agents produced are significantly relevant and so cannot be ignored [43,44,73]. In conclusion, co-treatment with Stx2 and LPS increased the cytotoxic effects of Stx2 but Dexamethasone, a potent anti-inflammatory, protected neuronal integrity and decreased cerebral damage. Conversely, Dexamethasone treatment suggests that cytokines such as TNF-α and/or IL-1β may be involved in the encephalopathy. ==== Refs References 1 O’Brien AD , Kaper JB (1998 ) Shiga toxin-producing Escherichia coli: yesterday, today, and tomorrow . In: Kaper JB O’Brien AD Escherichia coli O157:H7 and Other Shiga Toxin-Producing E. coli Strains. Am Soc Microbiology Washington, DC . pp. 1 -11 . 2 Proulx F , Seidman EG , Karpman D (2001 ) Pathogenesis of Shiga toxin-associated hemolytic uremic syndrome . Pediatr Res 50 : 163 -171 . doi:10.1203/00006450-200108000-00002. PubMed: 11477199.11477199 3 Rivas M , Caletti MG , Chinen I , Refi SM , Roldán CD et al. (2003 ) Home-prepared hamburger and sporadic hemolytic uremic syndrome, Argentina . Emerg Infect Dis 9 : 1184 -1186 . doi:10.3201/eid0909.020563. PubMed: 14531383.14531383 4 Exeni RA (2001 ) syndrome urémico hemolítico . Arch Latin Nefr Ped 1 : 35 -56 . 5 Eriksson KJ , Boyd SG , Tasker RC (2001 ) Acute neurology and neurophysiology of haemolytic-uraemic syndrome . Arch Dis Child 84 : 434 -435 . doi:10.1136/adc.84.5.434. PubMed: 11316694.11316694 6 Oakes RS , Siegler RL , McReynolds MA , Pysher T , Pavia AT (2006 ) Predictors of fatality in postdiarrheal hemolytic uremic syndrome . Pediatrics 117 : 1656 -1662 . doi:10.1542/peds.2005-0785. PubMed: 16651320.16651320 7 Grisaru S , Midgley JP , Hamiwka LA , Wade AW , Samuel SM (2011 ) Diarrhea-associated hemolytic uremic syndrome in southern Alberta: A long-term single-centre experience . Paediatr Child Health 16 : 337 -340 . PubMed: 22654544.22654544 8 Weissenborn K , Donnerstag F , Kielstein JT , Heeren M , Worthmann H et al. (2012 ) Hemolytic-uremic syndrome in adults neurologic manifestations of e coli infection-induced hemolytic-uremic syndrome in adults . Neurology 79 : 1466 -1473 . doi:10.1212/WNL.0b013e31826d5f26. PubMed: 22993286.22993286 9 Voyer LE (1998 ) Síndrome urémico hemolítico: aspectos epidemiológicos de clínicas y patogenia . In: Seijo A Larghi O Espinosa M Rivas M Sabatini M Temas de zoonosis y enfermedades emergentes . Buenos Aires : Asociación Argentina de Zoonosis pp. 46 -49 . 10 Rivas M , Padola NL , Luchessi PM , Masana M (2010 ) diarrheogenic Escherichia coli in Argentina . In: Torres AG Pathogenic Escherichia coli in Latin America . Oak Park IL, USA : Bentham Science Publishers Ltd. pp. 142 -161 . 11 Upadhyaya K , Barwick K , Fishaut M , Kashgarian M , Siegel NJ (1980 ) The importance of nonrenal involvement in hemolytic uremic syndrome . Pediatrics 65 : 115 -120 . PubMed: 7355005.7355005 12 Shetb KJ , Swick HM , Haworth N (1986 ) Neurologic involvement in hemolytic uremic syndrome . Ann Neurol 19 : 90 -99 . doi:10.1002/ana.410190120. PubMed: 3947042.3947042 13 Hahn JS , Havens PL , Higgins JJ , O’Rourke PP , Estroff JA et al. (1989 ) Neurologic complications of hemolytic uremic syndrome . J Child Neurol 4 : 108 -113 . doi:10.1177/088307388900400206. PubMed: 2715605.2715605 14 Bale JF Jr ., Brasher C , Siegler RL (1980 ) CNS manifestations of the hemolytic-uremic syndrome. Relationship to metabolic alterations and prognosis . Am J Dis Child 134 : 869 -872 . PubMed: 7416114.7416114 15 Brascher C , Siegler RL (1981 ) The hemolytic-uremic syndrome . West J Med 134 : 193 -197 . PubMed: 7269554.7269554 16 Karmali MA , Petric M , Lim C , Fleming PC , Arbus GS et al. (1985 ) The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli . J Infect Dis 151 : 775 -782 . doi:10.1093/infdis/151.5.775. PubMed: 3886804.3886804 17 Rooney JC , Anderson RM , Hopkins IJ (1971 ) Clinical and pathologic aspects of central nervous system involvement in the haemolytic uraemic syndrome . Proc Aust Assoc Neurol 8 : 67 -75 .5153469 18 Sheth KJ , Swick HM , Haworth N (1996 ) Neurological involvement in hemolytic-uremic syndrome . Ann Neurol 19 : 90 -93 . 19 Siegler RL , Pavia AT , Christofferson RD , Milligan MK (1994 ) A 20-year population-based study of postdiarrheal hemolytic uremic syndrome in Utah . Pediatrics 94 : 35 -40 . PubMed: 8008534.8008534 20 Taylor CM , White RH , Winterborn MH , Rowe B (1986 ) Haemolytic-uraemic syndrome: clinical experience of an outbreak in the West Midlands . Br Med J (Clin Res Ed) 292 : 1513 -1516 . doi:10.1136/bmj.292.6534.1513. 21 Upadhyaya K , Barwick K , Fishaut M , Kashgarian M , Siegel NJ (1980 ) The importance of nonrenal involvement in hemolytic-uremic syndrome . Pediatrics 65 : 115 -120 . PubMed: 7355005.7355005 22 Verweyen HM , Karch H , Allerberger F , Zimmerhackl LB (1999 ) Enterohemorrhagic Escherichia coli (EHEC) in pediatric hemolytic-uremic syndrome: a prospective study in Germany and Austria . Infection 27 : 341 -347 . doi:10.1007/s150100050040. PubMed: 10624594.10624594 23 Cimolai N , Morrison BJ , Carter JE (1992 ) Risk factors for the central nervous system manifestations of gastroenteritis-associated hemolytic-uremic syndrome . Pediatrics 90 : 616 -621 . PubMed: 1408519.1408519 24 Gianantoni CA , Vitacco M , Mendilaharzu F , Gallo GE , Sojo ET (1973 ) The hemolytic-uremic syndrome . Nephron 11 : 174 -192 . doi:10.1159/000180229. PubMed: 4542964.4542964 25 Hamano S , Nakanishi Y , Nara T , Seki T , Ohtani T et al. (1993 ) Neurological manifestations of hemorrhagic colitis in the outbreak of Escherichia coli O157:H7 infection in Japan . Acta Paediatr 82 : 454 -458 . doi:10.1111/j.1651-2227.1993.tb12721.x. PubMed: 8518521.8518521 26 Tapper D , Tarr P , Avner E , Brandt J , Waldhausen J (1995 ) Lessons learned in the management of hemolytic uremic syndrome in children . J Pediatr Surg 30 : 158 -163 . doi:10.1016/0022-3468(95)90554-5. PubMed: 7738732.7738732 27 Obata F , Tohyama K , Bonev AD , Kolling GL , Keepers TR et al. (2008 ) Shiga toxin 2 affects the central nervous system through receptor globotriaosylceramide localized to neurons . J Infect Dis 198 : 1398 -1406 . doi:10.1086/591911. PubMed: 18754742.18754742 28 Tironi-Farinati C , Geoghegan PA , Cangelosi A , Pinto A , Loidl CF et al. (2013 ) A Translational Murine Model of Sub-Lethal Intoxication with Shiga Toxin 2 Reveals Novel Ultrastructural Findings in the Brain Striatum . PLOS ONE 8 (1): e55812 . doi:10.1371/journal.pone.0055812. PubMed: 23383285.23383285 29 Zhang H , Peterson JW , Niesel DW , Klimpel GR (1997 ) Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production . J Immunol 159 : 4868 -4878 . PubMed: 9366412.9366412 30 Mazzetti S , Frigerio S , Gelati M , Salmaggi A , Vitellaro-Zuccarello L (2004 ) Lycopersicon esculentum lectin: an effective and versatile endothelial marker of normal and tumoral blood vessels in the central nervous system . Eur J Histochem 48 : 423 -428 . PubMed: 15718209.15718209 31 Robertson CL , Puskar A , Hoffman GE , Murphy AZ , Saraswati M et al. (2006 ) Physiologic progesterone reduces mitochondrial dysfunction and hippocampal cell loss after traumatic brain injury in female rats . Exp Neurol 197 : 235 -243 . doi:10.1016/j.expneurol.2005.09.014. PubMed: 16259981.16259981 32 Little AR , O’Callaghan JP (2001 ) Astrogliosis in the adult and developing CNS: is there a role for proinflammatory cytokines? Neurotoxicology 22 : 607 -618 . doi:10.1016/S0161-813X(01)00032-8. PubMed: 11770882.11770882 33 Benderro GF , Sun X , Kuang Y , LaManna JC (2012 ) Decreased VEGF expression and microvascular density, but increased HIF-1 and 2a accumulation and EPO expression in chronic moderate hyperoxia in the mouse brain . Brain Res 1471 : 46 -55 . doi:10.1016/j.brainres.2012.06.055. PubMed: 22820296.22820296 34 Obata F (2010 ) Influence of Escherichia coli shiga toxin on the mammalian central nervous system . Adv Appl Microbiol 71 : 1 -19 . doi:10.1016/S0065-2164(10)71001-7. PubMed: 20378049.20378049 35 Goldstein J , Loidl CF , Creydt VP , Boccoli J , Ibarra C (2007 ) Intracerebroventricular administration of Shiga toxin type 2 induces striatal neuronal death and glial alterations: an ultrastructural study . Brain Res 1161 : 106 -115 . doi:10.1016/j.brainres.2007.05.067. PubMed: 17610852.17610852 36 Candiano G , Bruschi M , Musante L , Santucci L , Ghiggeri GM et al. (2004 ) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis . Electrophoresis 25 : 1327 -1333 . doi:10.1002/elps.200305844. PubMed: 15174055.15174055 37 Tironi-Farinati C , Loidl CF , Boccoli J , Parma Y , Fernandez-Miyakawa ME et al. (2010 ) Intracerebroventricular Shiga toxin 2 increases the expression of its receptor globotriaosylceramide and causes dendritic abnormalities . J Neuroimmunol 222 : 48 -61 . doi:10.1016/j.jneuroim.2010.03.001. PubMed: 20347160.20347160 38 Paxinos G , Franklin KBJ (2001 ) The mouse brain in stereotaxic coordinates . San Diego, CA : Academic Press . 39 Fujii J , Kinoshita Y , Kita T , Higure A , Takeda T et al. (1996 ) Magnetic resonance imaging and histopathological study of brain lesions in rabbits given intravenous verotoxin 2 . Infect Immun 64 : 5053 -5060 . PubMed: 8945546.8945546 40 Mizuguchi M , Sugatani J , Maeda T , Momoi T , Arima K et al. (2001 ) Cerebrovascular damage in young rabbits after intravenous administration of Shiga toxin 2 . Acta Neuropathol 102 : 306 -312 . PubMed: 11603804.11603804 41 Kita E , Yunou Y , Kurioka T , Harada H , Yoshikawa S et al. (2000 ) Pathogenic mechanism of mouse brain damage caused by oral infection with Shiga toxin-producing Escherichia coli O157:H7 . Infect Immun 68 : 1207 -1214 . doi:10.1128/IAI.68.3.1207-1214.2000. PubMed: 10678928.10678928 42 Louise CB , Obrig TG (1992 ) Shiga toxin-associated hemolytic uremic syndrome: combined cytotoxic effects of shiga toxin and lipopolysaccharide (endotoxin) on human vascular endothelial cells in vitro . Infect Immun 60 : 1536 -1543 . PubMed: 1548077.1548077 43 Bitzan M , Moebius E , Ludwig K , Muller-Wiefel DE , Heesemann J et al. (1991 ) High incidence of serum antibodies to Escherichia coli O157 lipopolysaccharide in children with hemolytic-uremic syndrome . J Pediatr 119 : 380 -385 . doi:10.1016/S0022-3476(05)82049-9. PubMed: 1880650.1880650 44 Koster F , Levin J , Walker L , Tung K , Gilman R et al. (1978 ) Hemolytic-uremic syndrome after shigellosis. Relation to endotoxemia and circulating immune complexes . N Engl J Med 298 : 927 -933 . doi:10.1056/NEJM197804272981702. PubMed: 642973.642973 45 Keepers TR , Psotka MA , Gross LK , Obrig TG (2006 ) A murine model of HUS: Shiga toxin with lipopolysaccharide mimics the renal damage and physiologic response of human disease . J Am Soc Nephrol 17 : 3404 -3414 . doi:10.1681/ASN.2006050419. PubMed: 17082244.17082244 46 Nieuwdorp M , Meuwese MC , Vink H , Hoekstra JBL , Kastelen JJP et al. (2005 ) The endothelial glycocalyx: a potential barrier between health and vascular disease . Curr Opin Lipidol 16 : 507 -511 . doi:10.1097/01.mol.0000181325.08926.9c. PubMed: 16148534.16148534 47 Henry CB , Duling BR (1999 ) Permeation of the luminal capillary glycocalyx is determined by hyaluronam . Am J Physiol 277 : H508 -H514 . PubMed: 10444475.10444475 48 Vink H , Duling BR (2000 ) Capillary endothelial surface layer selectively reduces plasma solute distribution volume . Am J Physiol Heart Circ Physiol 278 : H285 -H289 . PubMed: 10644610.10644610 49 Ueno M (2009 ) Mechanisms of the Penetration of Blood-Borne Substances into the Brain . Curr Neuropharmacol 7 : 142 -149 . doi:10.2174/157015909788848901. PubMed: 19949573.19949573 50 Psotka MA , Obata F , Kolling GL , Gross LK , Saleem MA et al. (2009 ) Shiga toxin 2 targets the murine renal collecting duct epithelium . Infect Immun 77 : 959 -969 . doi:10.1128/IAI.00679-08. PubMed: 19124603.19124603 51 Rosenstein JM , Mani N , Khaibullina A , Krum JM (2003 ) Neurotrophic effects of vascular endothelial growth factor on organotypic cortical explants and primary cortical neurons . J Neurosci 23 : 11036 -11044 . PubMed: 14657160.14657160 52 Silverman WF , Krum JM , Mani N , Rosenstein JM (1999 ) Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explants cultures . Neuroscience 90 : 1529 -1541 . doi:10.1016/S0306-4522(98)00540-5. PubMed: 10338318.10338318 53 Khaibulina AA , Rosenstein JM , Krum JM (2004 ) Vascular endothelial growth factor promotes neurite maturation in primary CNS neuronal cultures . Brain Res Dev Brain Res 148 : 59 -68 . doi:10.1016/j.devbrainres.2003.09.022. PubMed: 14757519.14757519 54 Sugatani J , Igarashi T , Munakata M , Komiyama Y , Takahashi H et al. (2000 ) Activation of coagulation in C57BL/6 mice given verotoxin 2 (VT2) and the effect of co-administration of LPS with VT2 . Thromb Res 100 : 61 -72 . doi:10.1016/S0049-3848(00)00305-4. PubMed: 11053618.11053618 55 Boccoli J , Loidl CF , Lopez-Costa JJ , Creydt VP , Ibarra C et al. (2008 ) Intracerebroventricular administration of Shiga toxin type 2 altered the expression levels of neuronal nitric oxide synthase and glial fibrillary acidic protein in rat brains . Brain Res 1230 : 320 -333 . doi:10.1016/j.brainres.2008.07.052. PubMed: 18675791.18675791 56 Pekny M , Wilhelmsson U , Bogestål YR , Pekna M (2007 ) The role of astrocytes and complement system in neural plasticity . Int Rev Neurobiol 82 : 95 -111 . doi:10.1016/S0074-7742(07)82005-8. PubMed: 17678957.17678957 57 Jacob A , Hensley LK , Safratowich BD , Quigg RJ , Alexander JJ (2007 ) The role of the complement cascade in endotoxin-induced septic encephalopathy . Lab Invest 87 : 1186 -1194 . doi:10.1038/labinvest.3700686. PubMed: 17922019.17922019 58 Sofroniew MV , Vinters HV (2010 ) Astrocytes: biology and pathology . Acta Neuropathol 119 : 7 -35 . doi:10.1007/s00401-009-0619-8. PubMed: 20012068.20012068 59 Murray CL , Skelly DT , Cunningham C (2001 ) Exacerbation of CNS inflammation and neurodegeneration by systemic LPS treatment is independent of circulating Il-1β and IL-6 . J Neuroinflammation 8 : 50 . 60 Xing B , Bachstetter AD , Van-Eldik LJ (2011 ) Microglial p38α MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFα . Mol Neurodegener 6 : 84 . doi:10.1186/1750-1326-6-84. PubMed: 22185458.22185458 61 Kettenmann H , Hanisch UK , Noda M , Verkhratsky A (2011 ) Physiology of microglia . Physiol Rev 91 : 461 -553 . doi:10.1152/physrev.00011.2010. PubMed: 21527731.21527731 62 Winter KRK , Stoffregen WC , Dean-Nystrom EA (2004 ) Shiga toxin binding to isolated porcine tissues and peripheral blood leukocytes . Infect Immun 72 : 6680 -6684 . doi:10.1128/IAI.72.11.6680-6684.2004. PubMed: 15501802.15501802 63 Saxena SK , O’Brien AD , Ackerman EJ (1989 ) Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site when microinjected into Xenopus oocytes . J Biol Chem 264 : 596 -601 . PubMed: 2642481.2642481 64 Lindauer ML , Wong J , Iwakura Y , Magun BE (2009 ) Pulmonary Inflammation Triggered by Ricin Toxin Requires Macrophages and IL-1 Signaling . J Immunol 183 : 1419 -1426 . doi:10.4049/jimmunol.0901119. PubMed: 19561099.19561099 65 Iordanov MS , Pribnow D , Magun JL , Dinh TH , Pearson JA et al. (1997 ) Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the alpha-sarcin/ricin loop in the 28S rRNA . Mol Cell Biol 17 : 3373 -3381 . PubMed: 9154836.9154836 66 Kimmitt PT , Harwood CR , Barer MR (2000 ) Toxin gene expression by Shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response . Emerg Infect Dis 6 : 458 -465 . doi:10.3201/eid0605.000503. PubMed: 10998375.10998375 67 Zhang X , McDaniel AD , Wolf LE , Keusch GT , Waldor MK et al. (2000 ) Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice . J Infect Dis 181 : 664 -670 . doi:10.1086/315239. PubMed: 10669353.10669353 68 Katz J , Lurie A , Kaplan BS , Krawitz S , Metz J (1971 ) Coagulation findings in the hemolytic-uremic syndrome of infancy: similarity to hyperacute renal allograft rejection J Pediatr 78: 426-434 69 Bell BP , Griffin PM , Lozano P , Christie DL , Kobayashi JM et al. (1997 ) Predictors of hemolytic uremic syndrome in children during a large outbreak of Escherichia coli O157:H7 infections . Pediatrics 100 : E12 . doi:10.1542/peds.100.1.e12. PubMed: 9200386.9200386 70 Dundas S , Todd WT , Stewart AI , Murdoch PS , Chaudhuri AK et al. (2001 ). Cent Scotland Escherichia Coli O157:H7 outbreak: risk factors for the hemolytic uremic syndrome and death among hospitalized patients Clin Infect Dis 33 : 923 -931 .11528561 71 Forster C , Waschke J , Burek M , Leers J , Drenckhahn D (2006 ) Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific . J Physiol 573 : 413 -425 . doi:10.1113/jphysiol.2006.106385. PubMed: 16543270.16543270 72 Liu CC , Chien CH , Lin MT (2000 ) Glucocorticoids reduce interleukin-1β concentration and result in neuroprotective effects in rat heatstroke . J Physiol 527 : 333 -343 . doi:10.1111/j.1469-7793.2000.t01-1-00333.x. PubMed: 10970434.10970434 73 Siegler RL , Pysher TJ , Lou R , Tesh VL , Taylor FB Jr (2001 ) Response to Shiga toxin-1, with and without lipopolysaccharide, in primate model of hemolytic uremic syndrome . Am J Nephrol 21 : 420 -425 . doi:10.1159/000046288. PubMed: 11684808.11684808	23894578	PMC3720947	CC BY	2021-01-05 17:34:17	yes	PLoS One. 2013 Jul 23; 8(7):e70020
==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2013/438243Research ArticleSide-by-Side Comparison of the Biological Characteristics of Human Umbilical Cord and Adipose Tissue-Derived Mesenchymal Stem Cells Hu Li 1 Hu Jingqiong 2 Zhao Jiajia 1 Liu Jiarong 1 Ouyang Weixiang 3 Yang Chao 2 Gong Niya 1 Du Luyang 1 Khanal Abhilasha 1 Chen Lili 1 1Department of Stomatology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan, Hubei 430022, China2Stem Cell Center, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China3Department of Gynaecology and Obstetrics, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, ChinaLili Chen: chenlili@whuh.comAcademic Editor: Susan A. Rotenberg 2013 7 7 2013 2013 43824328 2 2013 3 5 2013 7 5 2013 Copyright © 2013 Li Hu et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Both human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been explored as attractive mesenchymal stem cells (MSCs) sources, but very few parallel comparative studies of these two cell types have been made. We designed a side-by-side comparative study by isolating MSCs from the adipose tissue and umbilical cords from mothers delivering full-term babies and thus compared the various biological aspects of ASCs and UC-MSCs derived from the same individual, in one study. Both types of cells expressed cell surface markers characteristic of MSCs. ASCs and UC-MSCs both could be efficiently induced into adipocytes, osteoblasts, and neuronal phenotypes. While there were no significant differences in their osteogenic differentiation, the adipogenesis of ASCs was more prominent and efficient than UC-MSCs. In the meanwhile, ASCs responded better to neuronal induction methods, exhibiting the higher differentiation rate in a relatively shorter time. In addition, UC-MSCs exhibited a more prominent secretion profile of cytokines than ASCs. These results indicate that although ASCs and UC-MSCs share considerable similarities in their immunological phenotype and pluripotentiality, certain biological differences do exist, which might have different implications for future cell-based therapy. ==== Body 1. Introduction Stem cells are self-renewable and capable of differentiating into at least two distinctive cell types. Mesenchymal stem cells (MSCs) are a population of stem cells, widely present in a large number of tissues including bone marrow, adipose tissue, umbilical cord blood and the cord itself, brain, liver, muscle, dental pulp, skin, and fetal tissues [1–6]. Owing to their multipotentiality, rapid proliferation, and strong capacities for self-renewal, mesenchymal stem cells hold great promise for tissue engineering and are therefore suitable seed cells for future cell therapy. Bone-marrow-derived MSCs (BMSCs) are the most commonly used MSCs for scientific and clinical purposes. Nevertheless, there are some limitations of BMSCs, such as the significant decrease in relative number of MSCs in the marrow and their differentiation potential with age [7]. In addition, the isolation procedure is invasive and may lead to complications and morbidity [8]; therefore it is necessary to find an alternative source of MSCs that have functions similar to the BMSCs but overcome these key limitations and portray a part of successful alternative. In recent years, adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been explored as new MSCs sources with obvious advantages over BMSCs [9, 10]. UC-MSCs are different from human umbilical cord-blood-derived mesenchymal stem cells (UCB-MSCs). Studies have shown that these MSCs, derived from Wharton's jelly tissue of the human umbilical cord, are actually better in many aspects than UCB-MSCs [11]. Both ASCs and UC-MSCs have drawn the attention of researchers due to their convenient harvesting procedures, excellent proliferation and differentiation abilities, less susceptibility to contamination of tumor cells, and no ethical restrictions. Various characteristics of ASCs and UC-MSCs have been studied, and many aspects focusing on their potential application in wound repair, tissue reconstruction, and disease treatment have been investigated [12–14]. But so far very few direct comparative studies focusing on these two types of cells have been made. We therefore designed a side-by-side comparative study. In consideration to keep uniformity of tissue sources and internal factors, we compared ASCs and UC-MSCs derived from the same donor. To this end, MSCs were isolated from the adipose tissue and umbilical cord from mothers delivering full-term babies, and thus side-by-side comparisons among various biological aspects including their in vitro cell culture dynamics, immunological phenotypes, multidifferentiation, proliferation and antiapoptic abilities, and their cytokine expression profiles were made. We found that although ASCs and UC-MSCs share considerable similarities in their immunological phenotype and multipotentiality, certain biological differences do exist, including their adipogenesis, neurogenesis capability, and cytokine secretion profiles, which might have different implications for future cell-based therapy. To our knowledge, very few side-by-side comparisons among various biological aspects were made between ASCs and UC-MSCs derived from the same individual. We believe that our finding will aid in future decision making in choosing the most suitable seed cell for cell-based therapy. 2. Material and Methods 2.1. Isolation and Culture of ASCs and UC-MSCs Human subcutaneous adipose tissues and umbilical cords were obtained from mothers (18–30 years old) planning on cesarean sections after obtaining written informed consent and approval by the Ethics Committee of Wuhan Union Hospital. ASCs and UC-MSCs were, respectively, isolated according to the procedures described by Bunnell et al. [15] and Seshareddy et al. [16]. Briefly, samples were washed with phosphate-buffered saline (PBS, Hyclone) to remove red blood cells. The adipose tissue was chopped into small pieces of about 25–50 mm3 and digested with 0.1% collagenase type I (Gibco) at 37°C for 60 min. The single cell suspension was obtained by filtering the digested material through a 100 μm mesh filter to remove tissue debris. The ASC-containing cell suspension was centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in specific MSC culture medium (Cyagen). The umbilical cord was transected longitudinally along the umbilical cord blood vessels. After careful removal of these blood vessels using forceps, Wharton's jelly tissue was chopped into small pieces of 1 mm3 and treated with 0.1% collagenase type I for 16 hours at 37°C and then washed and treated with 2.5% trypsin (Gibco) for 30 min at 37°C with agitation. Finally, ASCs and UC-MSCs were cultured in proliferation medium and seeded in 25 cm2 flasks (Costar) at a density of 5 × 107 cells/mL. After 48–72 h incubation, nonadherent cells were removed by medium changing. Around 5–7 days after seeding, the cells reached 80% confluency. These cells were passaged using trypsinization. Cells at culture passage P3–P7 were used in the following studies. 2.2. Flow Cytometry Cultured ASCs and UC-MSCs at passage 3 were trypsinized (0.25% trypsin-EDTA), washed twice with PBS (PH = 7.4), and suspended in PBS at a concentration of 5 × 106/mL, and then 1 mL sample was incubated with fluorescein isothiocyanate- (FITC-) conjugated monoclonal rabbit anti-human CD13, CD14, CD44, CD90, CD105, and CD34 (BD Biosciences) or isotype control for 30 min at 4°C according to the recommendation of the manufacturer. Finally, they were washed twice with PBS, centrifuged, and fixed in 4% paraformaldehyde. The stained cells were analyzed using a standard Becton-Dickinson FACSAria instrument and the CellQuest Pro software (BD Biosciences). 2.3. Growth Curves In order to compare the growth curves of ASCs and UC-MSCs, the cells at passage 3 were trypsinized and modulated to the concentration of 2 × 104/mL, and then the cells were seeded in 96-well plates (100 μL/well, Costar). 24 hours later, 5 experimental wells and 1 control well were selected randomly to add CCK8 (10 μL/well), and after 2-hour incubation, their absorbance values were tested by enzyme immunoassay analyzer, and the mean values were calculated. The growth curves were drawn after 9 successive days of continuous detection. 2.4. Determination of Cells Antiapoptotic Ability In order to compare the antiapoptotic ability of ASCs and UC-MSCs, the cells at passage 3 were trypsinized and modulated to the concentration of 3 × 105/mL, and then the cells were seeded in 6-well plates. Until 80% confluent, 1 × 10−6 mol/L dexamethasone was added, and the cells were collected by trypsinization after 48-hour incubation and then suspended in 500 μL binding buffer, followed by adding 5 μL Annexin V-FITC and 5 μL Propidium Iodide. After incubation in the dark at room temperature for 5–15 min, the cells were analyzed by flow cytometry. 2.5. Multidifferentiation Ability Test of ASCs and UC-MSCs For adipogenic differentiation, ASCs and UC-MSCs were cultured in DMEM/F12 supplemented with 10% FBS, 1 mM dexamethasone, 0.5 mM methyl-isobutyl-xanthine, 10 mg/mL insulin, and 100 mM indomethacin (all from Sigma) for 3 weeks. At the end of the incubation, adipogenic differentiation was assayed by Oil-Red-O (Sigma) staining for lipid droplets. For osteogenic differentiation, ASCs and UC-MSCs were cultured in DMEM/F12 supplemented with 10% FBS, 0.1 mM dexamethasone, 10 mM b-glycerolphosphate, and 50 mM ascorbic acid (all from Sigma) for about 3 weeks. At the end of incubation, osteogenic differentiation was assayed by Alizarin red (Sigma) staining for calcium deposition. RNA was isolated from UC-MSCs and ASCs before and after the induction using Trizol reagent (Invitrogen, Carlsbad, CD, USA). cDNA was transcribed using Superscript III First Strand cDNA Synthesis kit following manufacturer's instructions (Invitrogen, Carlsbad, CD, USA). Quantitative real-time-PCR was performed with SYBR Green PCR reagents on an ABI Prism 7300 detection system (Applied Biosystems, Foster City, CA, USA). Beta-actin was used as an internal control. The normalized fold expression was obtained using the 2−ΔΔCT method. Primers used for real-time PCR were summarized in Supplementary Table 1. (See Supplementary Material available online at http://dx.doi.org/10.1155/2013/438243). For neurogenic differentiation, ASCs and UC-MSCs were cultured in either group A, DMEM/F12 supplemented with B27 (1 : 50) (Gibco), N2 (1 : 100) (Gibco), 20 ng/mL basic fibroblast growth factor (bFGF, Invitrogen), and 20 ng/mL epithelial growth factor (EGF, Invitrogen), or group B, DMEM/F12 supplemented with 5 μmol/L retinoic acids (RA) for 6–10 days. The induction medium was refreshed every 3 days. At the end of the induction period, neurogenic differentiation was assayed by immunofluorescence staining for neural and glial-specific protein expression. MSCs were washed with PBS and then incubated for 1 hour at room temperature with the following antibodies: rabbit anti-NSE mAb at final concentrations of 1/250 and mouse anti-GFAP mAb (Millipore, at final concentrations of 1/300), respectively. Primary antibodies were developed with secondary Dylight 488-goat anti-rabbit IgG and Dylight 546-rat anti-mouse IgG, both at final concentrations of 1/500. Secondary antibodies were incubated for 45 minutes at room temperature in the dark. After labeling, the cells were fixed with 0.4% paraformaldehyde and then covered with antifade mounting medium. Slides were immediately examined on a three-color immunofluorescence microscope (Nikon Instruments Inc.). 2.6. Preparation of ASC-CM and UC-MSC-CM and Protein Microarray Analysis of ASC-CM and UC-MSC-CM ASCs and UC-MSCs at passage 3 were cultured in specific mesenchymal stem cell growth medium until cells were approximately 80% confluent; the medium was replaced with serum-free DMEM/F12. Following incubation in serum-free medium for 48 h, the conditioned medium was collected, centrifuged at 1000 rpm for 5 minutes, and filtered through a 0.22 μm syringe filter. ASC-CM and UC-MSC-CM were conserved at −20°C, and 5 mL medium was assayed by RayBio Biotin Label-based Human Antibody Array I (Cat no.: AAH-BLM-1-2, Norcross, GA, USA), which can detect the expression levels of 507 human proteins in cell culture supernatants simultaneously. 2.7. Statistical Analysis All values are expressed as mean ± SD. Comparisons between two groups were analyzed by Students' t-test and comparisons between more than two groups were analyzed by ANOVA. A value of P < 0.05 was considered statistically significant. All analyses were performed with SPSS 16.0. 3. Results 3.1. Morphologies of ASCs and UC-MSCs Most of the primary ASCs adhered within 24 hours after plating and demonstrated polygonal or round morphology, and the cells stretched out pseudopodia and displayed similar fibroblast-like or spindle-shaped morphology around 2 days. ASCs proliferated rapidly within 5–7 days and gradually fused into a single layer, arranged in long spindle and distributed in clusters. The primary UC-MSCs began to adhere within 12 hours and formed scattered spindle morphology in 72 hours. UC-MSCs proliferated rapidly in 6–12 days and gradually fused into a sheet, parallel arrangement, and spiral-shaped distribution (Figure 1). 3.2. Flow Cytometry ASCs and UC-MSCs surface receptor molecules CD13, CD14, CD44, CD90, CD105, and CD34 were detected by flow cytometry. Results showed that ASCs and UC-MSCs both exhibited positive surface antigenicity for CD13, CD44, CD90, and CD105 and exhibited negative surface antigenicity for CD14 and CD34. Flow cytometry results are shown in Figure 2, and the expression of immunological phenotypes of ASCs and UC-MSCs is listed in Table 1. Statistical analysis showed that there was no significant difference in the surface antigenicity profiles of these two types of cells (P > 0.05). 3.3. Growth Curves Growth curves of ASCs and UC-MSCs demonstrated that they had the following characteristics in common: in the first 12–18 hours, cells proliferated slowly and then entered the logarithmic growth phase, which continued for 5-6 days, and reached cell growth plateau in 7-8 days. The notable point was that their proliferation rates were basically the same in the first four days; however, UC-MSCs proliferated significantly faster than ASCs from the fifth day (P < 0.05). The results are shown in Figure 3. 3.4. Antiapoptotic Ability of ASCs and UC-MSCs The flow cytometry results showed good antiapoptotic capacity of ASCs and UC-MSCs when induced by the high concentration of dexamethasone, which was not statistically different (P > 0.05). 3.5. Multidifferentiation Capabilities of ASCs and UC-MSCs ASCs and UC-MSCs were able to efficiently differentiate into adipocytes and osteocytes. The adipogenesis of ASCs is much more prominent and efficient than that of UC-MSCs. ASCs changed to round-shaped adipocytes already around 2 days after induction, and at about 2 weeks, 95% of the ASCs were efficiently induced into adipocytes, whereas for UC-MSCs, clear morphology change began 5 days after induction and complete differentiation occurred three weeks after induction. The oil red staining for adipocytes is also more prominent for ASCs in comparison to UC-MSCs (Figure 4(a)). For osteogenic induction, UC-MSCs and ASCs from the same individual (passage 4) were cultured under osteogenic induction medium. Dramatic morphological changes already began 2 days after induction. At the end of two weeks, most MSCs already transformed from spindle-shaped MSCs to osteoblasts-like cells. Quantitative real-time PCR results were shown in Figure 5. As is shown, there was comparable significant upregulation of alkaline phosphatase, osteocalcin, and Runx2 in both UC-MSCs and ASCs. Osteocalcin is more significantly upregulated in UC-MSCs in comparison to ASCs, whereas alkaline phosphatase and Runx2 are more significantly upregulated in ASCs in comparison to UC-MSCs (Figure 5). Although no upregulation of leptin gene was identified, the equivalent upregulation of osteogenic genes of alkaline phosphatase, osteocalcin, and Runx2, together with equivalent Alizarin red staining (Figure 4(b)), suggests equivalence of osteogenic differentiation capacity for UC-MSCs and ASCs. However, the differences were displayed (P < 0.05) in induction time and differentiation rate when they differentiated into neuron-like cells. We tried two induction methods: retinoid acid induction and neurobasal media induction. ASCs responded better to both methods. ASCs differentiated into neuron-like cells easily in culture medium of DMEM/F12 + B27 + N2 + bFGF + EGF (neurobasal media). Clear morphology changes begin as soon as two days after induction and became more prominent around 4-5 days. ASCs quickly lost their spindle-shaped morphology and displayed a bipolar or multipolar outlook. Neurites growth was most obvious around day 6, and multiple interconnections among cells can be seen. Longer cultivation in neurobasal medium (more than 10 days) leads to more prominent neurites outgrowth. Retinoid acid can induce ASCs into neurogenic differentiation as well. Clear morphology changes begin around 3 days after induction and became much more prominent around 5-6 days. Longer exposure (more than 10 days) and higher concentration (more than 5 uM) of retinoid acid lead to obvious cell death. The morphology of neurons induced by retinoid acid is significantly different from neurobasal media-induced neurons in that they are much smaller and usually display a spiky outlook (Figure 4). Interestingly, alone DMEM/F12 medium can induce ASCs to transform to neuron-like cells, similar to RA induction method (data not shown). The differentiation rate of ASCs is 38.6 ± 11.2% for neurobasal medium induction method and 45.5 ± 8.3% for RA induction method. The results are shown in Figure 4 and Table 2. Both conditions can induce UC-MSCs to differentiate into neuron-like cells as well but with a much lower efficiency (22.3 ± 4.8% for neurobasal medium induction method and 18.4 ± 5.6% for RA induction method (P < 0.01)). These results can be repeated in five individuals (Table 2). 3.6. Protein Microarray Analysis of ASC-CM and UC-MSC-CM ASC-CM and UC-MSC-CM were assayed to determine the cytokines secreted by ASCs and UC-MSCs. Protein microarray analyses are shown in Figure 5; both these conditioned media contained a variety of cytokines. Signal value ratio of content in UC-MSC-CM and ASC-CM was used to analyze these cytokines, and statistical analysis had been carried out for those cytokines whose signal value exceeded 300, and the ratio was more than 1.5 or less than 0.66. The levels of expression of macrophage inflammatory protein 2 (MIP-2), interleukin 6 (IL-6), and growth-regulated oncogene (GRO) in UC-MSC-CM were significantly higher than those in ASC-CM, while the levels of expression of CD27 and neuregulin in ASC-CM were significantly higher than those of UC-MSC-CM. The results are shown in Figure 6 and Table 3. 4. Discussion Tissue engineering technology is one of the most promising means for solving difficult problems of tissues, organs defection, and wound healing [17, 18]. How to select suitable seed cells as well as their large-scale amplification has been an important issue for tissue engineering [19]. Adipose and umbilical cord tissues are good sources for mesenchymal stem cells, and both these cells have potential for multidifferentiation [20, 21]. In this study, we compared the similarities and differences between ASCs and UC-MSCs, aiming to provide a theoretical basis for clinical selection and application of seed cells. In consideration to keep uniformity of tissue sources and internal factors, we compared ASCs and UC-MSCs derived from the same donor. In our study, we compared the cell morphologies and surface markers of mesenchymal stem cells from two different sources, the adipose and umbilical cord tissues. Two types of cells had similar morphologies and typical surface markers of MSCs, such as positive expression for CD13, CD44, CD90, and CD105 and negative expression for hematopoietic stem cells marker phenotypes of CD14, CD34, and there was no significant statistical difference in expression levels. The above results confirmed that, as mesenchymal stem cells, ASCs did not exhibit significant difference with UC-MSCs in the basic phenotypic characteristics of stem cells, which was consistent with the previous reports [22]. The proliferation and antiapoptotic abilities are very important for mesenchymal stem cells to maintain their characteristics of stem cells for long time in vitro. Related articles reported that ASCs and UC-MSCs both had strong self-renewal capacity [23, 24]. In this study, we in vitro cultured ASCs and UC-MSCs derived from the same individual and detected their proliferation ability. Their growth curves demonstrated that, compared with ASCs, UC-MSCs exhibited stronger proliferation ability, and the difference was significant (P < 0.05). The possible reason was that umbilical cord-derived cells might contain a lot of cytokines associated with the growth and development for neonatus, which could promote cell division and proliferation. Although UC-MSCs had stronger proliferation ability, in some of the existing reports, the proliferation ability of ASCs was superior to terminally differentiated cells and some other stem cells, and this ability basically met most of the experimental requirements [25]. Dexamethasone has biphasic effect on MSCs. In appropriate concentration (1 × 10−9 mol/L), dexamethasone can promote the osteogenic differentiation of MSCs [26], and there are also some studies which report that high concentration (higher than 1 × 10−8 mol/L) of dexamethasone could induce the apoptosis of MSCs [27]. So we chose 1 × 10−6 mol/L concentration of dexamethasone to induce apoptosis in this study, but our results showed that ASCs and UC-MSCs both had strong antiapoptotic ability, and there was no significant difference. Multidifferentiation ability also is one of the factors for wide application of stem cells in tissue engineering field. There are many studies indicating that MSCs can differentiate into the three mesodermal cells in the appropriate induction environment [28]. The results of this study showed that both ASCs and UC-MSCs were able to efficiently differentiate into adipocytes and osteocytes. The adipogenesis of ASCs is much more prominent and efficient than that of UC-MSCs. We hypothesized that MSCs derived from different tissues still carry some of the reminiscent features of the original tissue. Some of the tissue-specific genes might already be and still are turned on, which might lead to the results we saw. We are particularly interested in neurogenic differentiation of both types of MSCs. We speculated that there must be some differences in the induction efficiency and in their response to different induction medium. We initially thought that UC-MSCs might respond better to neuronal induction since UC-MSCs had been widely used for treatment of neurological disorders with some noticeable clinical effects. But to our surprise, we found that ASCs responded much better to various induction methods than UC-MSCs. We tried two induction methods: retinoid acid (RA) induction and neurobasal medium induction. RA is a general differentiation and transdifferentiation agent for the generation of neurons [29]. It has been shown to induce the differentiation of cells during embryonic maturation into distinct organs, including the generation of neurons [30], whereas the culture medium of DMEM/F12 + B27 + N2 + bFGF + EGF is the medium generally used to produce neurospheres. These two induction methods had been reported in previous studies [31, 32]. We found that ASCs respond well to both methods. For neurobasal medium induction protocol, clear morphology changes begin as soon as two days after induction and became more prominent around 4-5 days. Neurites growth was most obvious around day 6, and multiple interconnections among cells can be seen. Retinoid acid can induce neuronal induction as well. Clear morphology changes begin around 3 days after induction and became much more prominent around 5-6 days. The neurons induced by retinoid acid are usually much smaller and usually display a spiky outlook. Interestingly, alone DMEM/F12 medium can induce ASCs to transform to a neuron-like cell, similar to RA induction method. Other papers have reported neural induction of UC-MSCs to various neuronal types. Here we failed to observe a prominent response of UC-MSCs to the above-mentioned induction methods. Our results suggested that, in the process of neuronal differentiation, ASCs might be more sensitive to different neuronal induction signals. These are probably due to the expression of multiple neurogenic genes that are already expressed in ASCs which might facilitate the neuronal induction process. However, further exploration of the exact mechanism for this phenomenon is necessary. The mechanisms of actions of stem cells on tissue regeneration and wound healing include two hypotheses [33, 34]: differentiation theory and paracrine theory, and the latter is more recognized, which means that stem cells can secrete a variety of cytokines acting on surrounding cells or migrate to tissue defect sites through cell homing, to participate in the reconstruction of tissues. ASC-CM and UC-MSC-CM contained a variety of cytokines secreted by cells [27]; we found that the types of cytokines were nearly the same in ASC-CM and UC-MSC-CM through protein microarray analysis, but the differences existed in expression levels of cytokines. The content of some cytokines varied greatly; for example, the expressions of MIP-2, IL-6, and GRO in UC-MSC-CM were significantly higher than those in ASC-CM, while the expressions of CD27 and neuregulin in ASC-CM were significantly higher than those in UC-MSC-CM. MIP-2 is the main chemotactic cytokine of neutrophil, and it can specifically promote neutrophil migrating to the inflammatory tissue, to get rid of pathogens and participate in the body's defense reaction [35]; MMP-1 is involved in mediation of a wide range of physiological and pathological processes in the body, such as the formation of embryo, tissue remodeling, wound healing, inflammation, and apoptosis [36]. IL-6 can promote the proliferation of a variety of cells, and this might be one of the reasons for UC-MSCs proliferating faster than ASCs [37]. In addition, IL-6 and IL-3 can synergistically promote cell differentiation, and in our protein microarray analysis results, the levels of expression of IL-3 in ASCs and UC-MSCs supernatants were similar (results not shown); therefore, in most aspects of differentiation, the capacity of two types of stem cells did not exhibit significant difference. As a member of NGFR/TNFR gene superfamily, CD27 is expressed in most of peripheral T cells and can be used as a second messenger with its ligand CD70 to promote T cells proliferation [38]. It is also involved in the process of T cells differentiation and the immune reaction of cells [39]. Neuregulin, a neuromodulation protein, is a kind of nutritional factor containing epidermal growth factor-like domain that plays an important role in nervous system development process [40]. Different contents of these cytokines indicated that suitable stem cells could be selected depending on the different needs in the tissue engineering fields. 5. Conclusion In summary, the basic biological characteristics of mesenchymal stem cells derived from adipose and umbilical cord tissues are similar to each other, and both of them have strong self-renewal capacity, antiapoptotic capacity, and multidifferentiation capacity. The types of cytokines were nearly the same in ASC-CM and UC-MSC-CM, but there were differences in the levels of expression of certain cytokines. Interestingly, we found that ASCs responded much better to various neuronal induction methods in comparison to UC-MSCs. In consideration of the wide use of human umbilical cord-derived MSCs in stem cell therapy for treatment of neurological disorders, we suggest that human adipose mesenchymal stem cells may also be a valuable source for stem cell therapy in the future. Supplementary Material The Primer sequences (5'-3') used for Real-time PCR are listed in Supplementary Table 1. Flow cytometric images of immunophenotyping with SSEA-PE antibody are shown in Supplementary Figure 1. Click here for additional data file. Authors' Contribution Li Hu and Jingqiong Hu contributed equally to this work as first authors. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grants no. 31110103905 and no. 31201100). Figure 1 Morphologies of ASCs and UC-MSCs cultured ex vivo. ((a)–(d)) Morphology of ASCs. (a) P0; (b) P1; (c) P2; (d) P3. ((e)–(h)) Morphology of UC-MSCs. (e) P0; (f) P1; (g) P2; (h) P3. Scale bar = 200 μm. ASCs: adipose mesenchymal stem cell; UC-MSC: human umbilical cord mesenchymal stem cell, P: passage. Figure 2 Immunophenotyping of ASCs and UC-MSCs. (a1)–(a4) Flow cytometry analysis of ASC; (b1)–(b4) flow cytometry analysis of UC-MSCs. Figure 3 Proliferation and antiapoptotic ability of ASCs and UC-MSCs. (a) Growth curves of ASCs and UC-MSCs showed that UC-MSCs proliferated significantly faster than ASCs from the fifth day (P < 0.05). (b) Flow cytometric analysis of antiapoptotic ability of ASCs and UC-MSCs. (c) Statistical analysis of antiapoptotic ability of ASCs and UC-MSCs. Results showed that these two types of cells had good antiapoptotic capacity, and there was no significant difference (P > 0.05). Figure 4 Multilineage differentiation of ASCs ((a1), (b1), (c1), and (d1)) and UC-MSCs ((a2), (b2), (c2), and (d2)). (a1) and (a2) Adipogenesis; (b1) and (b2) osteogenesis; (c1) and (c2) neurogenesis; (d1) and (d2) immunofluorescence staining of NSE for neurogenic differentiation. Scale bar = 100 μm. Figure 5 Relative quantification of osteogenic gene expression using real-time PCR after osteogenic induction in ASC and UC-MSCs. The mRNA levels were normalized using the expression of the reference gene (beta-actin). Results were from three independent experiments. ASC: adipose tissue-derived mesenchymal stem cells; UC-MSC: umbilical cord-derived mesenchymal stem cells. Figure 6 Protein microarray analysis of ASC-CM (a) and UC-MSC (b) and changes of difference proportion of a variety of cytokines. Results showed that there were a lot of cytokines in ASC-CM and UC-MSC, and the contents of these cytokines were not exactly the same in two conditioned mediums, and the cytokines whose content had very obvious difference were MIP-2, IL-6, CRO, and MMP-1. Table 1 Comparison of expression of surface markers of ASCs and UC-MSCs. Antibody ASCs (%) UC-MSCs (%) P CD13 97.46 ± 1.97 96.81 ± 1.66 0.681 CD44 97.67 ± 1.55 97.51 ± 1.14 0.897 CD90 96.56 ± 1.21 98.03 ± 1.22 0.212 CD105 96.45 ± 0.91 97.18 ± 1.33 0.481 CD14 1.12 ± 0.13 0.96 ± 0.14 0.209 CD34 1.32 ± 0.30 1.10 ± 0.17 0.315 Table 2 Comparison of neurogenic differentiation of ASCs and UC-MSCs (n = 5). Cell DMEM/F12 + RA DMEM/F12 + N2 + B27 + EGF + bFGF Time (days) Successful differentiation rate (%) Time (days) Successful differentiation rate (%) ASCs 4.8 ± 1.3 45.5 ± 8.3 3.8 ± 2.2 38.6 ± 11.2 UC-MSCs 7.1 ± 2.1 18.4 ± 5.6 9.5 ± 2.2 22.3 ± 4.8 P 0.001 0.001 0.001 0.001 Results of neurogenic differentiation of ASCs and UC-MSCs are presented. ASCs responded better to both methods, exhibiting the higher differentiation rate and relatively shorter induction time than UC-MSCs in the same induction method. Table 3 Proportion of some cytokines content in ASC-CM and UC-MSC-CM. Cytokines ASC-CM UC-MSC-CM UC-MSC-CM/ASC-CM MIP-2 121.00 1,578.59 13.05 IL-6 194.50 1,329.72 6.84 GRO 303.00 1,433.86 4.73 MMP-1 501.00 1,599.55 3.19 IL-8 160.50 486.48 3.03 Thrombospondin-1 355.50 734.03 2.06 TIMP-2 408.00 778.37 1.91 Pentraxin3/TSG-14 352.00 661.00 1.88 Osteoprotegerin/TNFRSF11B 423.00 792.94 1.87 EG-VEGF/PK1 324.50 583.56 1.80 TRAILR4/TNFRSF10D 332.50 589.96 1.77 ENA-78 175.00 308.44 1.76 Vasorin 355.00 620.63 1.75 ErbB2 199.00 340.87 1.71 Decorin 333.00 570.32 1.71 Angiopoietin-2 267.50 447.65 1.67 IFN-beta 320.00 534.58 1.67 Flt-3 Ligand 245.00 403.09 1.65 FLRG 383.50 576.94 1.50 CXCR6 450.50 296.74 0.66 Activin RIA/ALK-2 393.00 253.94 0.65 D6 385.00 247.32 0.64 NRG3 1,328.50 804.41 0.61 Siglec-9 476.50 281.08 0.59 MMP-20 1,085.00 636.95 0.59 sFRP-3 622.00 359.18 0.58 CCR3 343.00 179.81 0.52 HCR/CRAM-A/B 1,261.00 604.52 0.48 Heregulin/NDF/GGF/Neuregulin 478.00 223.72 0.47 CD27/TNFRSF7 369.50 161.72 0.44 Part of cytokines were listed in table. Signal value of these cytokines was more than 300, and their content proportion in UC-MSC-CM and ASC-CM was more than 1.5 or less than 0.66. Results showed that the content of MIP-2, IL-6, and GRO in UC-MSC-CM was significantly more than that in ASC-CM, while the content of HCR, Heregulin, and CD27 in ASC-CM was significantly more than that in UC-MSC-CM. ==== Refs 1 Prockop DJ Marrow stromal cells as stem cells for nonhematopoietic tissues Science 1997 276 5309 71 74 2-s2.0-0030889354 9082988 2 Zuk PA Zhu M Mizuno H Multilineage cells from human adipose tissue: implications for cell-based therapies Tissue Engineering 2001 7 2 211 228 2-s2.0-0035067539 11304456 3 Wang L Tran I Seshareddy K Weiss ML Detamore MS A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stromal cells for cartilage tissue engineering Tissue Engineering Part A 2009 15 8 2259 2266 2-s2.0-68749116404 19260778 4 Joyce NC Harris DL Markov V Zhang Z Saitta B Potential of human umbilical cord blood mesenchymal stem cells to heal damaged corneal endothelium Molecular Vision 2012 18 547 564 2-s2.0-84859054130 22419848 5 Usas A Mačiulaitis J Mačiulaitis R Jakuboniene N Milašius A Huard J Skeletal muscle-derived stem cells: implications for cell-mediated therapies Medicina 2011 47 9 469 479 2-s2.0-84856628579 22156603 6 Saichanma S Bunyaratvej A Sila-Asna M In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells International Journal of Ophthalmology 2012 5 2 158 163 22762041 7 Mueller SM Glowacki J Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges Journal of Cellular Biochemistry 2001 82 4 583 590 2-s2.0-0034895190 11500936 8 Lee SY Miwa M Sakai Y In vitro multipotentiality and characterization of human unfractured traumatic hemarthrosis-derived progenitor cells: a potential cell source for tissue repair Journal of Cellular Physiology 2007 210 3 561 566 2-s2.0-33846602261 17171634 9 Secco M Zucconi E Vieira NM Multipotent stem cells from umbilical cord: cord is richer than blood! Stem Cells 2008 26 1 146 150 2-s2.0-38349052370 17932423 10 Sterodimas A De Faria J Nicaretta B Pitanguy I Tissue engineering with adipose-derived stem cells (ADSCs): current and future applications Journal of Plastic, Reconstructive and Aesthetic Surgery 2010 63 11 1886 1892 2-s2.0-77958009280 11 Zeddou M Briquet A Relic B The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood Cell Biology International 2010 34 7 693 701 2-s2.0-77955093312 20187873 12 Meruane M Rojas M Marcelain K Chile S The use of adipose tissue-derived stem cells within a dermal substitute improves skin regeneration by increasing neoangiogenesis and collagen synthesis Plastic and Reconstructive Surgery 2012 130 1 53 63 2-s2.0-84857981609 22418720 13 Ra JC Kang SK Shin IS Stem cell treatment for patients with autoimmune disease by systemic infusion of culture-expanded autologous adipose tissue derived mesenchymal stem cells Journal of Translational Medicine 2011 9 1, article 181 2-s2.0-80054716864 14 Wu KH Chan CK Tsai C Effective treatment of severe steroid-resistant acute graft-versus-host disease with umbilical cord-derived mesenchymal stem cells Transplantation 2011 91 12 1412 1416 21494176 15 Bunnell BA Flaat M Gagliardi C Patel B Ripoll C Adipose-derived stem cells: isolation, expansion and differentiation Methods 2008 45 2 115 120 2-s2.0-45849112916 18593609 16 Seshareddy K Troyer D Weiss ML Method to isolate mesenchymal-like cells from Wharton's Jelly of umbilical cord Methods in Cell Biology 2008 86 101 119 2-s2.0-42949144838 18442646 17 Falanga V Stem cells in tissue repair and regeneration Journal of Investigative Dermatology 2012 132 6 1538 1541 22584501 18 Cerqueira MT Marques AP Reis RL Using stem cells in skin regeneration: possibilities and reality Stem Cells and Development 2012 21 8 1201 1214 22188597 19 Pillai RG Stem cells for ocular tissue engineering and regeneration Current Topics in Medicinal Chemistry 2011 11 13 1606 1620 2-s2.0-79959988953 21446913 20 Gaiba S França LP França JP Ferreira LM Characterization of human adipose-derived stem cells Acta Cirúrgica Brasileira 2012 27 7 471 476 22760832 21 Margossian T Reppel L Makdissy N Stoltz JF Bensoussan D Huselstein C Mesenchymal stem cells derived from Wharton's jelly: comparative phenotype analysis between tissue and in vitro expansion Bio-Medical Materials and Engineering 2012 22 4 243 254 22785368 22 Najar M Raicevic G Boufker HI Mesenchymal stromal cells use PGE2 to modulate activation and proliferation of lymphocyte subsets: combined comparison of adipose tissue, Wharton’s Jelly and bone marrow sources Cellular Immunology 2010 264 2 171 179 2-s2.0-77955093536 20619400 23 Camassola M de Macedo Braga LM Chagastelles PC Nardi NB Methodology, biology and clinical applications of human mesenchymal stem cells Methods in Molecular Biology 2012 879 491 504 22610579 24 Xu Y Huang S Ma K Fu X Han W Sheng Z Promising new potential for mesenchymal stem cells derived from human umbilical cord Wharton’s jelly: sweat gland cell-like differentiative capacity Journal of Tissue Engineering and Regenerative Medicine 2012 6 8 645 654 2-s2.0-80053458535 21916019 25 Beeson W Woods E Agha R Tissue engineering, regenerative medicine, and rejuvenation in 2010: the role of adipose-derived stem cells Facial Plastic Surgery 2011 27 4 378 387 2-s2.0-79960801341 21792781 26 Park DG Kim KG Lee T-J Optimal supplementation of dexamethasone for clinical purposed expansion of mesenchymal stem cells for bone repair Journal of Orthopaedic Science 2011 16 5 606 612 2-s2.0-82955212879 21720802 27 Boomsma RA Geenen DL Mesenchymal stem cells secrete multiple cytokines that promote angiogenesis and have contrasting effects on chemotaxis and apoptosis PLoS ONE 2012 7 4 28 Anzalone R Iacono ML Corrao S New emerging potentials for human wharton’s jelly mesenchymal stem cells: immunological features and hepatocyte-like differentiative capacity Stem Cells and Development 2010 19 4 423 438 2-s2.0-77950603767 19958166 29 Maden M Retinoic acid in the development, regeneration and maintenance of the nervous system Nature Reviews Neuroscience 2007 8 10 755 765 2-s2.0-34648816863 17882253 30 Nagashima M Sakurai H Mawatari K Koriyama Y Matsukawa T Kato S Involvement of retinoic acid signaling in goldfish optic nerve regeneration Neurochemistry International 2009 54 3-4 229 236 2-s2.0-60949101813 19114071 31 Huang T He D Kleiner G Kuluz J Neuron-like differentiation of adipose-derived stem cells from infant piglets in vitro Journal of Spinal Cord Medicine 2007 30 1 S35 S40 2-s2.0-34548294717 17874685 32 Kwon EB Lee JY Piao S Kim IG Ra JC Lee JY Comparison of human muscle-derived stem cells and human adipose-derived stem cells in neurogenic trans-differentiation Korean Journal of Urology 2011 52 12 852 857 2-s2.0-84455175987 22216399 33 Kondo T Ishida Y Molecular pathology of wound healing Forensic Science International 2010 203 1–3 93 98 2-s2.0-78649321650 20739128 34 Baiguera S Jungebluth P Mazzanti B MacChiarini P Mesenchymal stromal cells for tissue-engineered tissue and organ replacements Transplant International 2012 25 4 369 382 2-s2.0-84858441307 22248229 35 Heine SJ Olive D Gao J-L Murphy PM Bukrinsky MI Constant SL Cyclophilin a cooperates with MIP-2 to augment neutrophil migration Journal of Inflammation Research 2011 4 1 93 104 2-s2.0-79959487580 22096373 36 Mahajan N Dhawan V Mahmood S Malik S Jain S Extracellular matrix remodeling in Takayasu's arteritis: role of matrix metalloproteinases and adventitial inflammation Archives of Medical Research 2012 43 5 406 410 22868188 37 Tu B Du L Fan QM Tang Z Tang TT STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastasis of osteosarcoma Cancer Letters 2012 325 1 80 88 22743617 38 Deola S Panelli MC Maric D Helper B cells promote cytotoxic T cell survival and proliferation independently of antigen presentation through CD27/CD70 interactions Journal of Immunology 2008 180 3 1362 1372 2-s2.0-40749131713 39 Claus C Riether C Schürch C Matter MS Hilmenyuk T Ochsenbein AF CD27 signaling increases the frequency of regulatory T cells and promotes tumor growth Cancer Research 2012 72 14 3664 3676 22628427 40 Bare DJ Becker-Catania SG DeVries GH Differential localization of neuregulin-1 type III in the central and peripheral nervous system Brain Research 2011 1369 10 20 2-s2.0-78651083963 21044615	23936800	PMC3722850	CC BY	2021-01-05 10:05:37	yes	Biomed Res Int. 2013 Jul 7; 2013:438243
==== Front Pan Afr Med JPan Afr Med JPAMJThe Pan African Medical Journal1937-8688The African Field Epidemiology Network PAMJ-15-1210.11604/pamj.2013.15.12.1831ResearchAssociation between the use of biomass fuels on respiratory health of workers in food catering enterprises in Nairobi Kenya Keraka Margaret 1Ochieng Carolyne 2Engelbrecht Jacobus 3&Hongoro Charles 341 Department of Public Health, Kenyatta University, Kenya2 London School of Hygiene, United Kingdom3 Department of Environmental Health, Tshwane University of Technology, South Africa4 Health Systems Research Unit, South African Medical Research Council, South Africa& Corresponding author: Jacobus Engelbrecht, Department of Environmental Health, Tshwane University of Technology, Private Bag X680, Pretoria, South Africa06 5 2013 2013 15 1223 6 2012 06 11 2012 © Margaret Keraka et al.2013The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction Indoor air pollution from biomass fuel use has been found to be responsible for more than 1.6 million annual deaths and 2.7% of the global burden of disease. This makes it the second biggest environmental contributor to ill health, behind unsafe water and sanitation. Methods The main objective of this study was to investigate if there was any association between use of bio-fuels in food catering enterprises and respiratory health of the workers. A cross-sectional design was employed, and data collected using Qualitative and quantitative techniques. Results The study found significantly higher prevalence of respiratory health outcomes among respondents in enterprises using biomass fuels compared to those using processed fuels. Biomass fuels are thus a major public health threat to workers in this sub-sector, and urgent intervention is required. Conclusion The study recommends a switch from biomass fuels to processed fuels to protect the health of the workers. Indoor air pollutionbiomass fuelshealthrespiratoryworkers ==== Body Introduction Worldwide, approximately 50% of people, almost all in developing countries, rely on biomass fuels in the form of wood, dung and crop residues for domestic energy [1, 2]. Biomass fuels are commonly burned in inefficient simple stoves and in poorly ventilated conditions. Due to the construction of such stoves acceptable principles of combustion is not complied to. According to [3] the three condition required for proper combustion are proper proportioning of fuel and oxygen (air), mixing of fuel and oxygen and ignition temperature of the fuel. In such situations, biomass fuel use generates substantial emissions of many health-damaging pollutants, including respirable particulates and carbon monoxide, and results in indoor air pollution exposures often far exceeding national standards and international guidelines [4]. Where biomass fuels are used, the average daily exposure concentration of children under the age of 5 to PM10 (particulate matter smaller than 10 microns in diameter) is approximately 1500µg/m3 and that of adult women approximately 5000 µg/m3. This is many times higher than the latest United States Environmental Protection Agency (US: EPA) standard, which states that individuals should not to exceed PM10 levels of 150 µg/m3 for a 24-hour period [5]. Several studies firmly associate biomass fuel use with acute lower respiratory tract infections, chronic obstructive pulmonary disease and lung cancer. Each of these three health outcomes is a major disease category in most societies. According to the World Health Organisation (WHO) [6], household biomass fuel use is likely to be a major cause of disease burden in communities where it is prevalent. Globally, WHO attributes 2.7% of all ill-health to indoor smoke from biomass fuels, nearly all in developing countries. Evidence is also emerging that exposure to indoor air pollution from biomass fuels may increase the risk of a number of other important conditions, including tuberculosis, low birth weight, and cataract [4, 7–9]. In developing countries, according to WHO [6], around 4.9% of deaths and 4.4% of Daily Adjusted Life Years (DALYs) are attributed to indoor air pollution from solid biomass fuels. The above mentioned studies indicate that those who are mostly at risk are women, because they are responsible for food preparation and cooking, and infants/young children who are usually with their mothers near the cooking area. However, no study had previously been done to determine if other groups of people who spend a lot of time indoors during cooking using biomass fuels for instance workers in food catering enterprises are also vulnerable. This is the gap the study aimed to fill, focusing on respiratory health given that the pollutants are inhaled. Workers in food catering enterprises were particularly chosen since these enterprises are the largest biomass fuel energy consumer among all small and medium enterprises under the cottage and service sector category [10]. The general objective of this study was therefore to determine the association between use of biomass fuels in food catering enterprises and respiratory health outcomes exhibited by the workers. The specific objectives of the study were: To assess the determinants of exposure to indoor air pollution from biomass fuels; To determine the major fuel types used by food catering enterprises in Nairobi and factors determining their choice; To establish prevalence of respiratory health symptoms exhibited by the workers; and To investigate the influence of exposure factors on prevalence of respiratory health outcomes exhibited. Methods The study employed a cross-sectional design as most of the enterprises were built on illegal land and are constantly demolished. Employment in the sector is also temporary, with workers constantly shifting from one form of activity to another. This is a general characteristic of the informal sector. The study was carried out in the city of Nairobi between June 2008 and January 2009 According to the 1999 population and housing census, Nairobi's population was 2,807,154 with 1,397,963 males and 1,409,191 females. The number of males working for pay was estimated at 454,456 and females 209,514 [11]. Unemployment figures were 95,535 and 73,128 for males and females respectively. A large proportion of the unemployed engage in informal sector activities as a source of income. In 2004, Nairobi province accounted for 24.2% and the highest informal sector employment of 1,343,000 persons [11]. The study population comprised of both male and female workers (employees and employers) in food catering enterprises. Workers at these enterprises were the main respondents, while the food catering enterprises were taken as the sampling unit. A combination of cluster sampling and simple random sampling were used. To select the enterprises, a list of all the estates (clusters) in each of the eight divisions of Nairobi was made. One cluster was then picked at random from each Division. There were 250 enterprises in the eight clusters. Thirty per cent of the enterprises were randomly selected amounting 60 enterprises. Listing of the workers was done in the enterprises which had a total of 5 workers per enterprise hence a total of 400 workers. All the workers were targeted but only 368 were available for the interview. Hence 368 respondents were interviewed. The study employed questionnaires for the workers, interview guides for the proprietors and observation checklists to observe if protective gear was used. Health outcomes were expressed in terms of respiratory symptoms such as cough, phlegm, breathlessness and wheezing as defined by the Medical Research Council (MRC) questionnaire for respiratory symptoms, chronic respiratory illnesses such as Chronic Obstructive Pulmonary Disease (COPD), asthma, and Acute Lower Respiratory Tract Infection (ALRI) which has been used in similar studies [12]. The MRC questionnaire was modified to suit local conditions. Data were analysed using the SPSS software to generate descriptive statistics for independent and intermediate variables, while chi-square statistic was used to test the association between respiratory symptoms such as cough, phlegm, wheezing and breathlessness (dependent variables) and the independent variables or exposure factors. Confounding variables such as smoking, age and fuel used at home were treated as independent factors in separate analyses. Although broader environmental factors such as temperature, humidity, air velocity, ambient air quality and others might have contributed to the observed respiratory health symptoms, the principal focus of the study was on the effects of indoor exposure and type of biomass used across enterprises. Results Respondents’ characteristics The mean age of respondents was 28.6 years. A large proportion of the respondents (58.5%) were aged 21- 30 years of which 56% of the respondents were males and 44% females. Forty six (46%) had primary level of education, 39% had secondary education and 13% had tertiary education. Two thirds (67%) of the respondents earned 3000 Kenya Shillings (Kshs)(US$35) or less per month, and were mostly casual employees. Over fifty percent of the respondents (59.5%) had worked in food catering enterprises for not more than 2 years, with 55% spending less than six hours a day in the cooking area. Most respondents (62.4%) used kerosene for home cooking. Charcoal was the second and only biomass fuel used at home, accounting for 20.3% of the respondents. Liquid Petroleum Gas (LPG) was used by 8.6% of respondents. Some respondents (7.3%) used various fuels of varying quantities hence could not classify any as the main fuel type. Enterprise characteristics Most enterprises’ structures were made of tin, which is generally the dominant construction material of informal enterprises, given their temporary nature. Concrete was mainly used by registered enterprises. A few enterprises were constructed with mud, and accounted for 5.5% of the respondents. Other enterprises were made of brick, while others used a mixture of materials such as wood and tin. Most of the kitchens were very small, measuring 3m < sup2 or less. Only 14.6% of respondents worked in enterprises that covered areas greater than 7m2. Two thirds of the enterprises had windows or chimneys as forms of ventilation. Enterprises that had windows as the main form of ventilation constituted 41.1% while those with chimneys accounted for 12.4%. Others had both chimneys and windows, accounting for 13.2%. Nearly one third of the respondents (33.2%) worked in enterprises that had neither windows nor chimneys, but other forms of ventilation. These ranged from large open spaces on the roof or wall to very tiny holes on the upper part of the walls that would hardly let out smoke from the kitchens. Some enterprises consisted of space with wire-mesh surrounding the entire wall of the kiosk especially in areas where customers were served, not in the kitchens. Only 29% of the enterprises were registered by the Registrar of Companies of Kenya. The remaining 71% were not registered meaning that they were operating illegally. Nearly all the enterprises (95.5%) had 1 to 5 employees, thus falling in the micro-enterprises category. Small enterprises (with 6 to 20 employees) accounted for only 9%. Most enterprises (81%) reported using biomass fuels as the main form of fuel, whereas processed fuels accounted for only 19%. Charcoal was the most utilised fuel type (58.9%), followed by fuelwood (18.6%) and electricity (14.1%). LPG came fourth, accounting for 4.9% of the businesses, followed by sawdust (3.5%). No enterprise reported using kerosene as a main fuel type. Charcoal was the most preferred biomass fuel. Even though many enterprises used fuelwood and sawdust, they were utilised as substitute fuels rather than main forms of fuel. Kerosene was also used as a substitute fuel. Despite the fact that charcoal was preferred to fuel wood, this should not be encouraged given that charcoal production contributes to emissions such as greenhouse gasses for example CO2 as well as other air pollutants for example volatile organic compounds including benzene that is a known carcinogen [13]. Results from the enterprise proprietors indicate a shift to cleaner fuels such as liquid petroleum gas and electricity would not be affected due to cost implications. Prevalence of respiratory health symptoms Nearly half (49%) of the respondents reported having one form of respiratory symptom or another whilst 51% did not experience any of the listed symptoms. Other respondents had more than one symptom (16.5%), while a few (4%) reported experiencing all the symptoms under investigation. Table 1 shows that the most experienced symptom was cough at 42% of the study population. Cough tends to be the first indication of irritation of the respiratory tract and that explains why it had the highest prevalence. The remaining symptoms were, however, reported by less than one fifth of respondents: breathlessness at 18%, wheezing at 15%, and lastly phlegm being the least experienced symptom at 14.5%. Table 1 Frequency distribution of responses by respiratory symptoms Respiratory Symptoms Number of responses (%) TOTAL Yes No Cough 155 (42.0%) 215 (58.0%) 370 (100.0%) Phlegm 54 (14.5%) 316 (85.5%) 370 (100.0%) Breathlessness 67 (18.0%) 303 (82.0%) 370 (100.0%) Wheezing 56 (15.0%) 314 (85.0%) 370 (100.0%) All symptoms 15 (4.0%) 355 (96.0%) 370 (100.0%) Multiple responses allowed Presence of these respiratory symptoms over a long period of time are considered indicative of respiratory health ailments especially in studies where it is impossible to ascertain the presence of respiratory diseases [8]. The study focused on respondents who had experienced these symptoms in the past one year. The study assessed all the symptoms in relation to exposure factors that could have influenced their presence. Factors influencing respiratory health outcomes Influence of age: A higher prevalence of most respiratory health symptoms is observed amongst those aged above 29 years (Table 2). The only exception was cough with equal proportions between those under and above 29 years. The difference in respiratory health outcomes between the two age categories were statistically significant: for phlegm (x2=18.78; df = 1; p= 0.004) and all symptoms (x2=6.16; df = 1; p = 0.047). Table 2 Cross tabulation of respondents by respiratory symptoms and age Respiratory symptoms Number of respondents with symptoms (%) TOTAL 29 years and below Above 29 years Cough 103 (42.0) 52 (41.6) 155 (42.0%) Phlegm 34 (13.9) 20 (16.0) 54 (14.5%) Breathlessness 43 (17.6) 24 (19.2) 67 (18.0%) Wheezing 33 (13.5) 23 (18.4) 56 (15.0%) All symptoms 6 (2.4) 9 (7.2) 15 (4.0%) Multiple responses allowed This finding can be explained by duration of exposure as those who were older had worked for longer periods in food catering enterprises, hence been exposed to the harmful pollutants for a longer period of time. Most of old people had worked in food catering enterprises for longer periods and had more stable business compared to the lower age-groups, of whom the majority were new entrants in the sector hence had not experienced long term exposure. Fuel used at home: Uses of charcoal at home recorded the highest prevalence for most respiratory health symptoms (cough, 48%; phlegm, 17.3% and breathlessness, 26.7%) followed by Kerosene users. A significant association existed between the type of fuel used at home by the respondents and cough (x2=13.04; df = 5; P = 0.023), with prevalence in charcoal users being nearly three times higher (48.1%) than that in LPG users (17.1%). Breathlessness was also found to be significantly higher in charcoal and kerosene users compared to those using other fuel types (x2=11.12; df =2; P = 0.049). It was found that 78.7% of those using kerosene at home were using biomass fuels in the enterprises they worked in, hence leading to the exposure. Given that most respondents (82%) worked for nearly 12 hours a day for six days a week, they spent limited time at home. Hence most of their exposure occurred in the work environment. Ventilation: The prevalence of respiratory health symptoms was higher for those with other forms of ventilation, followed by those with only windows. Those with chimney came in a distance third, and those with chimney and window had the least prevalence (Table 3). Table 3 Cross tabulation of respondents by symptoms and forms of ventilation Respiratory symptoms Number of respondents with symptoms (%) P – Value Chimney Window Chimney & Window Other Cough 6 (13.0%) 71 (46.7%) 3 (6.1%) 75 (60.9%) 0.000 Phlegm 2 (4%) 26 (17.1%) 2 (3.8%) 24 (19.5%) 0.005 Breathlessness 6 (13%) 27 (17.8%) 3 (6.1%) 31 (25.2%) 0.008 Wheezing 2 (4.0%) 28 (18.4%) 3 (5.7%) 23 (18.7%) 0.058 All symptoms 0 6 (3.9%) 0 9 (7.5%) 0.035 * χ2 Test, p <0.05 Those using other forms of ventilation recorded the highest prevalence for nearly all respiratory health outcomes (with the exception of wheezing). The largest differences were observed between those with other forms of ventilation and those with chimney & window. For instance, the prevalence of cough was 60.9% in those with other forms of ventilation, and only 6.1% in those with chimney and window. There was significant association between forms of ventilation and nearly all the health symptoms (Table 3), with enterprises having chimney and window recording significantly lower prevalence of symptoms compared to those that had other forms of ventilation. Influence of fuels used in enterprises Two analyses were done on respiratory health symptoms and fuel types. The first was based on all fuel types while the second involved the binary classification scheme, separating the study population into those using biomass fuels and those using processed fuels. Table 4 shows that fuel wood and sawdust users reported the highest prevalence of most of the respiratory health symptoms, followed by charcoal users. LPG users came in third while electricity users had the least prevalence of symptoms, with none of them reporting experiencing wheezing. Table 4 The relationship between reported health symptoms and fuel types Respiratory symptoms Number of respondents with symptoms (%) TOTAL Charcoal Fuelwood Sawdust Electricity LPG Cough 89 (40.8%) 51 (73.9%) 8 (61.5%) 4 (7.7%) 3 (16.7%) 155 (42.0%) Phlegm 31 (14.2%) 16 (23.2%) 3 (23.1%) 3 (5.8%) 1 (5.6%) 54 (14.5%) Breathlessness 43 (19.7%) 16 (23.2%) 3 (23.1%) 4 (7.7%) 1 (5.6%) 67 (18.0%) Wheezing 40 (18.3%) 13 (18.8%) 3 (23.1%) 0 0 56 (15.0%) All symptoms 9 (4.1%) 5 (7.2%) 1 (7.7%) 0 0 15 (4.0%) Significant association was established between fuel types and most respiratory health outcomes as shown in Table 5, with respondents using charcoal, fuel wood and sawdust recording higher prevalence than those using electricity and LPG. Table 5 Association between fuel type and respiratory health outcomes. Symptoms Fuel Types χ2 df P value* Cough Charcoal versus electricity 68.48 4 0.000 Phlegm Fuel wood versus LPG 11.35 4 0.023 Breathlessness Fuel wood versus LPG 10.06 4 0.039 Wheezing Sawdust vs electricity/LPG 17.4 4 0.020 All symptoms Sawdust vs electricity/LPG 4 0.051 Notes: p < 0.05 Although charcoal has poorer combustion efficiencies than other biomass fuels, the charcoal production process creates a fuel that burns with far less smoke than wood at the point of end-use, leading to lower emissions of PM10. This is because charcoal has undergone some form of processing during which some of its PM content has been released, unlike raw wood. During the study, it was observed that enterprises using fuelwood and sawdust were visibly smoky, and respondents were coughing during the interviews. Enterprises using charcoal were however less smoky, and after the charcoal had caught fire, no smoke was visible. This would therefore explain why charcoal users had lower prevalence of respiratory symptoms compared to fuelwood and sawdust users, and why charcoal was not significantly associated with most symptoms. Charcoal users reported higher prevalence of all the symptoms under investigation compared to electricity and LPG users. For instance, they recorded 75% prevalence of cough compared to only 19% recorded by electricity users. This is because charcoal is a biomass fuel, and biomass fuels have generally been associated with significant emissions of PM10 in relation to processed fuels. Influence of forms of fuels used When classified on the basis of binary fuel types, biomass fuel users reported higher prevalence of all the symptoms compared to those using processed fuels (Figure 1). Prevalence of cough was nearly 5 times higher (49%) in biomass fuel users compared to processed fuel users (10%). For phlegm and breathlessness, the prevalence was up to three times higher in biomass fuel users compared to processed fuel users. Figure 1 Distribution of respondents by symptoms and binary fuel types None of the processed fuel users reported experiencing wheezing or all the symptoms, yet biomass fuel users reported prevalence of 19% and 15% respectively. Prevalence of phlegm and breathlessness were up to three times higher in biomass fuel users compared to processed fuel users. The remaining symptoms were more than four times higher in biomass fuel users. None of the processed fuel users reported experiencing wheezing or all the symptoms, yet biomass fuel users reported prevalence of 19% and 15% respectively. The differences were found to be statistically significant for some symptoms (cough, p = 0.000; phlegm, p = 0,011; breathless, p = 0.004; wheezing, p = 0.000). Discussion These findings are consistent with most studies that have related biomass fuel use to respiratory health outcomes, even though all the previous studies were done in households. In a household study by [14] that used case-control design by reporting fuel type (wood vs. cleaner fuel) as a proxy for exposure, those who were exposed to smoke from biomass fuels reported higher prevalence of respiratory abnormalities as compared with users of cleaner fuels. For instance, prevalence of COPD and ALRI among unprocessed fuel users was three times higher compared to those using processed fuels. Similarly, much higher prevalences of all respiratory symptoms were found for the unprocessed fuel users. Studies in Kenya by [15, 16] in Mpala Ranch also revealed significantly higher prevalence of respiratory ailments among biomass fuel users. However, the high prevalence of symptoms among kerosene users found in this study is inconsistent with these studies, given that kerosene is a processed fuel. The inconsistency could therefore imply that the high prevalence observed did not arise from exposure at home, but from occupational environment. Many studies show that biomass fuels are detrimental in kitchens that are poorly ventilated; therefore implying that ventilation is a crucial parameter in determining respiratory health outcomes. Ventilation is a major factor influencing the concentration of pollutants, hence a key factor in determining exposure and health outcomes. Poor ventilation therefore is associated with more symptoms. A study by [17] found similar results, whereby there was a significant reduction in the risk of contracting ALRI between groups of people using charcoal relative to groups using fuel wood. Households using charcoal stoves had PM10 concentrations of around 500 µg/m3, while households using wood in an open fire had concentrations over 3000 µ/m3. The risk of adult men contracting ALRI was 44% lower in households using charcoal rather than fuel wood, while reduction in risk for adult women was 65%. The study indicates that despite the fuel type used, a key intervention measure would be to ensure adequate ventilation to protect health of the workers. Chimneys would especially be useful, as enterprises fitted with chimneys reported the least prevalence of respiratory health outcomes. This kind of intervention would yield other positive benefits such as becoming an income generating activity for people in the informal sector, as most of the chimneys were jua kali made (Swahili language directly translated as “hot sun” meaning that the enterprises are conducted in the open outdoor environment. Security could have been a factor, but they need to be made aware of the danger they are exposing themselves to by cooking with biomass fuels in poorly ventilated conditions. Registration of the enterprises is also key, as this would enable them to make investments in ventilation knowing they'll be able to operate those facilities for a given period of time. Most of the unregistered enterprises had very small kitchen that could not fit in chimneys even if they had that option. The results confirm what has been established by previous studies, that indoor air pollution from biomass fuels greatly compromises respiratory health of exposed populations. The study has thus demonstrated that workers in food catering enterprises who use biomass fuels are facing a great public health risk that needs attention. The results imply that switching from biomass fuels to processed fuels would lead to significant reductions in the prevalence of respiratory health problems. At the same time, the results indicate that some forms of biomass fuel are more harmful to health compared to others. In this instance, those using charcoal had significantly lower prevalence of respiratory health outcomes compared to those using fuelwood. Therefore a switch from fuelwood to charcoal would significantly reduce the health threat. Though in the long run, if the economic status of the study population is improved, it is recommended that there is a switch to liquid petroleum gas (LPG) and electricity as a cleaner source of energy supply for cooking purposes. Conclusion The study demonstrated that there is an association between use of biomass fuels in food catering enterprises and prevalence of respiratory disease symptoms among the workers. Biomass fuel use is therefore a major health threat, not just to women and children in households but also to workers in food catering enterprises. Improving access to cleaner and more efficient energy to these enterprises will lead to marked improvements in health and well being of the many workers engaged in the sub-sector. Aside from the fuel types, the study demonstrated that other factors including registration status of enterprises and ventilation are significant determinants of health symptoms. Therefore in addition to fuel switch, measures such as registering the enterprises and improving ventilation in them would lead to health benefits. Based on the study findings the following measures to reduce the effects of indoor air pollution from biomass fuels on respiratory health of workers in food catering enterprises are proposed: Switch from biomass fuels to processed fuels, given the significant differences observed in health effects associated with the two forms of fuels. However, given that cost was found to be a major determinant of fuels choice, efforts aimed at reducing the cost of processed fuels should first be put in place. These include reduction in cost of appliances such as cookers and cylinders, fuel subsidies and sale of processed fuels in smaller quantities. This can be achieved through formulation of national level policies that would encourage supply and distribution of improved cleaner fuels. Switch from highly polluting biomass fuels such as fuelwood to less polluting ones such as charcoal, which was associated with lower prevalence of symptoms. This could be a more viable option, given that the cost implication involved is lower compared to switching that of switching to processed fuels. Reduction in charcoal costs can also be achieved through change of current policy that makes charcoal production and transportation illegal yet legalizes its sale. This intervention however needs to be balanced with potential environmental impacts that could result from greenhouse gas emissions associated with charcoal production and use. Other general measures to include: education and awareness creation, ensuring improved ventilation through regular inspections, advise workers to reduce the number of hours spent indoors, registration of the enterprises and significant changes in Nairobi City Council Bylaws. A study by [18] revealed that elevated levels of air pollution during indoor activities can lead to increase in risk of respiratory health problems such as chronic bronchitis and obstructive pulmonary disease (assessed clinically and by spirometry. There is also a direct correlation between time of exposure and presented symptoms. Acknowledgments The authors would like to acknowledge the Kenya Ministry of Health for technical and logistical support, the Kenyatta University for funding the project and the workers at the food catering enterprises for their willingness to participate in the project. Competing interests The authors declare that there is no competing interest (financial or non-financial). Funding for the project was received from the Kenyatta University in Nairobi, Kenya. Authors’ contributions Margaret Keraka: She was responsible for initialising the research and acted as lead researcher of the project. She was also responsible for the formulation of the research problem and objectives. Carolyne Ochieng: She was responsible for the development of the measurement instruments, collection of the data, and literature survey. Jacobus Engelbrecht: He was responsible for the drafting of the manuscript and acted as advisor and collaborator during the project. Charles Hongoro: He was responsible for reviewing the quantitative analysis of the data and the peer review of the final article. ==== Refs References 1 United Nations Development Programme. Department of Economic and Social Affairs & World Energy Council World Energy Assessment: the challenge of sustainability 2000 New York United Development Programme 2 Energy After Rio: Prospects & Challenges 1997 New York United Nations Development Programme 3 Boiler Operator’s Guide 1998 New York McGraw Hill 4 Ezzati M Kammen DM Quantifying the Effects of Exposure to Indoor Air Pollution from Biomass Combustion on Acute Respiratory Infections in Developing Countries Environ Health Perspect 2001 5 109 5 481 8 11401759 5 United States Environmental Protection Agency Greenhouse gases from small-scale combustion devices in developing countries: Phase IIA Household Stoves in India 1997 India 6 World Health Organisation Indoor Air Pollution from Biomass Fuel 2000 Geneva 7 Bruce N Perez Padilla R Albalak R Indoor Air Pollution in developing Countries: A Major Environmental and Public Health Challenge Bull World Health Organ. 2000 78 9 1078 92 11019457 8 Ezzati M Kammen DM Indoor Air Pollution from Biomass combustion as a Risk Factor for Acute Respiratory Infections in Kenya: An Exposure-Response Study Lancet 2001 8 25 358 9282 619 24 11530148 9 Smith KR Samet JM Romieu I Bruce N Indoor Air Pollution in developing Countries and Acute Lower Respiratory Infections in Children Thorax 2000 6 55 6 518 32 10817802 10 Republic of Kenya Economic Survey 2004 2004 Nairobi Central Bureau of Statistics, Office of the President/Ministry of Planning and National Development 11 Republic of Kenya Ministry of Energy. Study on Kenya’s Energy Demand, Supply and Policy Strategy for Households, Small Scale Industries and Service Establishments 2001 Nairobi Government Printers 12 Cotes JE Chinn DJ Medical Research Council questionnaire on respiratory symptoms Occupational Medicine. 2007 57 5 388 13 Air and Waste Association Air Pollution Engineering Manual 2000 New York John Wiley & Sons, Inc 14 Commission on Macroeconomics and Health Addressing the Impact of Household Energy and Indoor Air Pollution on the Health of the Poor: Implications for Policy Action and Intervention Measures 2001 http://www.cmhealth.org/wg5.htm . Accessed 24th April 2010. 15 Ezzati M Saleh H Kammen DM The contributions of emissions and spatial microenvironments to exposure to indoor air pollution from biomass combustion in Kenya Environ Health Perspect 2000 9 108 9 833 9 11017887 16 Indoor Air Pollution: Formaldehyde and Other Carbonyls emitted from various cookstoves The 7th International Conference on Indoor Air Quality and Climate 1996 Nagoya, Japan In the proceedings of indoor air 96 17 Kituyi E Marifu L Jumba IO Wandiga SO Andreae MO Heals G Biofuel consumption rates and patterns in Kenya Biomass and Bio-energy. 2001 20 2 83 99 18 The Health Effects of Indoor Air Exposure in Developing Countries 2002 World Health Organisation	23898361	PMC3725321	CC BY	2021-01-04 23:00:28	yes	Pan Afr Med J. 2013 May 6; 15:12
==== Front BMC Res NotesBMC Res NotesBMC Research Notes1756-0500BioMed Central 1756-0500-6-1822364813710.1186/1756-0500-6-182Case ReportDoes changing from a first generation antipsychotic (perphenazin) to a second generation antipsychotic (risperidone) alter brain activation and motor activity? A case report Berle Jan Øystein 1jaob@helse-bergen.noLøberg Else-Marie 12else.marie.loeberg@psych.uib.noFasmer Ole Bernt 134ole.fasmer@kliniskmedisin.uib.no1 Division of Psychiatry, Haukeland University Hospital, Bergen, Norway2 Department of Biological and Medical Psychology, University of Bergen, Bergen, Norway3 Department of Clinical Medicine, Section for Psychiatry, Faculty of Medicine and Dentistry, University of Bergen, Bergen, Norway4 K way2013 6 5 2013 6 182 182 21 12 2012 26 4 2013 Copyright © 2013 Berle et al.; licensee BioMed Central Ltd.2013Berle et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background In patients with schizophrenia, altered brain activation and motor activity levels are central features, reflecting cognitive impairments and negative symptoms, respectively. Newer studies using nonlinear methods have addressed the severe disturbances in neurocognitive functioning that is regarded as one of the core features of schizophrenia. Our aim was to compare brain activation and motor activity in a patient during pharmacological treatment that was switched from a first- to a second-generation antipsychotic drug. We hypothesised that this change of medication would increase level of responding in both measures. Case presentation We present the case of a 53-year-old male with onset of severe mental illness in adolescence, ICD-10 diagnosed as schizophrenia of paranoid type, chronic form. We compared brain activation and motor activity in this patient during pharmacological treatment with a first-generation (perphenazin), and later switched to a second-generation (risperidone) antipsychotic drug. We used functional magnetic resonance imaging (fMRI) to measure brain activation and wrist worn actigraphy to measure motor activity. Conclusion Our study showed that brain activation decreased in areas critical for cognitive functioning in this patient, when changing from a first to a second generation antipsychotic drug. However the mean motor activity level was unchanged, although risperidone reduced variability, particularly short-term variability from minute to minute. Compared to the results from previous studies, the present findings indicate that changing to a second-generation antipsychotic alters variability measures towards that seen in a control group, but with reduced brain activation, which was an unexpected finding. SchizophreniaAntipsychoticBrain activationfMRINeurocognitiveMotor activity ==== Body Background Altered brain activation and reduced motor activity levels are central features of schizophrenia. Altered brain activation has often been reported in frontal regions during cognitive demanding tasks [1,2]. The term hypofrontality has been used by several authors to define this effect, which also has been seen in first-episode patients with schizophrenia [2] and neuroleptic-naive patients [1]. Recent studies present however a more complex picture, including for instance compensatory increased activation in other brain regions [1], altered brain activation in schizophrenia is seen as a reflection of the cognitive impairments in this patient group [2]. Cognitive impairments with clinical consequences are seen in a majority of patients with schizophrenia [3,4]. Reduced motor activity is one of several negative symptoms in schizophrenia. Motor retardation can be described as a reduction in motor activity as reflected in slowing or retardation of movements and speech, and reduced body tone. In addition, side-effects of the motor type are commonly reported when treating these disorders with antipsychotic medication [5], particularly when using first generation antipsychotics (FGA). Furthermore, a relationship between hypofrontality and physical anhedonia in schizophrenia has been reported [6]. Both cognitive impairments and negative symptoms have been shown to be important prognostic predictors in schizophrenia, predicting functional outcome better than positive symptoms [3,7]. It would be of clinical importance if there was a difference between a first-generation antipsychotic (FGA) and a second-generation antipsychotic (SGA) drug with regard to brain activation and motor activity. SGAs are hypothesized as having a more beneficial influence on the negative symptoms than the older (FGA) antipsychotics. The SGAs also have different pharmacological profiles with regard to brain regions involved in cognition and movement. Thus, although functional neuroimaging traditionally is a research tool, studies of brain activation and motor activity may also have clinical consequences, on an individual level where there is a need for objective measures. The brain imaging method fMRI is a technique that allows detection of the brain areas that are involved in the performance of a cognitive or emotional task. We chose an arithmetic task with working memory load as the stimulus, or task, paradigm to tap cognitive processes attributed to frontal brain areas and brain areas rich in dopaminergic receptors [8], which could reflect effects of medication. The task was similar to the experimental design used by Hugdahl et al. [9]. Assessment of motor activity in psychiatry is seldom done using objective methods, in contrast to for example in neurology. In our study motor activity was monitored by a wrist worn actigraph. Actigraphy is an objective method to register accumulated motor activity over time for later analysis of movement frequency and amplitude. In schizophrenia, actigraphy has been used to investigate activity levels and circadian rest-activity phases and motor activity pattern [10,11]. The aim of our study was to explore putative changes in brain activation and in motor activity in a patient with schizophrenia during antipsychotic treatment with the FGA perphenazine and after switching to the SGA risperidone. We hypothesised that this change of medication would increase brain activation and motor activity. Case presentation The patient The patients was male, Caucasian, and 53 years old. There were numerous incidences of severe mental illness in his family. His intellectual level was within the normal range; basic education, but no training or education as skilled worker or to a higher level. He had no significant somatic illness nor brain damage or other injuries. His alcohol consumption was within acceptable levels in the population and no use of illegal drugs was reported. This patient had symptom onset of severe mental illness in young adulthood; paranoid psychotic symptoms, auditory hallucinations, episodes with aggressive behaviour, dysfunction in activities of daily living. He was diagnosed with schizophrenia, paranoid type, chronic form, chronic phase (F20.0 in the ICD-10), although in periods affective symptoms of depression were present as well. Methods In the first registration period the patient was treated with perphenazine decanoate 216 mg im/14th day, and in addition received chlorprotixene 100 mg and valproate 1200 mg daily. Drug monitoring revealed a trough serum level of perphenazine of 22 nmol/L, well above the recommended reference range of 1 to 6 nmol/L. Serum valproate was 467 nmol/L. In the second registration period the antipsychotic medication was switched into risperidone (Risperdal Consta®), a long-acting intramuscular formulation. Drug monitoring of risperidone (s-risperidone + 9-OH risperidone) revealed a trough serum level of 114 nmol/L, serum level within the recommended reference range. Genotyping revealed that the patient was a CYP2D6 slow metabolizer (CYP2D64/CYP2D64). The fMRI image acquisitions were done both during treatment with FGA, and again at follow-up ~6 months after the patient had been switched to a SGA. Cognitive function was examined using a mental arithmetic and working memory task where the patient had to add two-and-two successive numbers presented visually in LCD goggles, and press a response button placed on the chest whenever the sum of the numbers seen in the goggles was 10 [Figure 1]. fMRI was performed with a 1.5 T Siemens Vision Plus scanner equipped with 25 mT/m gradients. Initial scanning of anatomy was done with a T1W 3D FLASH pulse sequence. Thereafter, serial imaging with 100 BOLD sensitive echo planar (EPI pulse sequence) whole brain measurements were done during the task. Each EPI volume measurement consisted of 40 axial slices which constituted an image volume (FA/TA/TE/FOV/matrix = 500/6 s/84 ms/230 mm/64×64). The in-plane pixel size was 3.44 × 3.44 mm, each slice with a thickness of 3.0 mm, thus creating nearly isotropic voxels. The first 10 image volumes were discarded prior to statistical analyses, to avoid artifacts due to stimulus novelty. Figure 1 Task performed during the fMRI recordings. There were 3 ON and 3 OFF blocks during task performance, presented in a box-car design. The digit stimuli were the numbers “1” through “9”. There were 16 trials with digit stimuli presented during each ON block, thus the total number of trials was 48. Each digit stimulus was presented for 300 ms, with 2200 ms blank interstimulus intervals (ISIs) in between. Thus, the total ON blocks lasted 120 sec, with a total of 60 sec OFF blocks. The ON and OFF blocks were alternated within the run. The digit stimuli were presented with the Micro Electronic Laboratory (MEL2) software (Psychology Software Tools Inc.). The OFF blocks consisted of resting with no stimulus presentations. The patient viewed the digit stimuli in electronic goggles consisting of a LCD-screen (Magnetic Resonance Technology Inc.) that were connected to a PC outside the MR chamber, which contained the MEL software. A response button was placed on the participant’s chest that he was instructed to press according to the specific instructions for each run. The patient was instructed to “add each consecutive number seen in the goggles to the previous one, and press the button whenever the sum was 10”. There were 6 presentations where the sum was “10” among the 16 trials for each ON block. Thus, the total number of target presentations, across the three ON blocks, were 18. The fMRI set-up was repeated twice in two separate sessions, with the different medications, respectively. The fMRI images were statistically analyzed using the SPM5 software (http://www.fil.ion.ucl.ac.uk/spm). The data were first pre-processed, including realigning images, smoothing (8 mm) and normalizing to the standard MNI template and co-ordinate system, and then subjected to significance testing with t-tests. Motor activity was monitored with an actigraph worn at the right wrist (Actiwatch, Cambridge Neurotechnology Ltd, England). The right wrist was chosen for the convenience of the participant. Previous studies have shown small differences between the right and left wrist. Total activity counts were recorded during one minute intervals. Motor activity was monitored on a 24 hour basis during two separate two week periods 6 months apart performed when the patient was on FGA (perphenazine), and later after switching to SGA (risperidone). The fMRI were performed in close proximity to these two periods. Both medications were given as intramuscular depot injections to ensure adherence to treatment, and therapeutic drug monitoring (TDM) were performed on both medications to ensure adequate dosage. All samples were analyzed by a LC/MSD method. Genotyping of CYP2D6 was also performed to evaluate the patient’s metabolic capacity. Average activity per minute, the standard deviation (SD), the root mean square successive differences (RMSSD), the relation between RMSSD and SD (RMSSD/SD), and sample entropy were calculated for one continuous activity period each day, defined as the longest period containing not more than 9 consecutive minutes with zero activity. These are the same mathemathical methods used in our previous studies [10,11]. Results are presented as mean ± SD for 21 periods during perphenazine treatment and 27 periods during risperidone treatment. Sample entropy is a nonlinear measure, indicates the degree of regularity (complexity) of time series, and is the negative natural logarithm of an estimate of the conditional probability that subseries of a certain length (m) that match point-wise, within a tolerance (r), also match at the next point. For the present study m = 2 and r = 0.2 were chosen. SPSS version 15.0 was used for the statistical analyses. Results The results from the fMRI acquisition showed significant activations during the mental arithmetic task when analyzed separately for the two sessions (using an uncorrected significance threshold of p < .001), in the occipital lobe, right parietal lobule, and pre-central / SMA area, and in an extended area in the pre-motor areas in the frontal lobe (BA 4/6), and in the superior parietal lobule (BA7). The parietal activation for the mental arithmetic task would fit previous findings of parietal cortex activations for number calculations, in particular with a right-sided laterality effect. Figure 2 shows the difference activation between session 1 (FGA) and 2 (SGA). Figure 2 fMRI results. We also compared the activation in condition 2 (SGA) and condition 1 (FGA), by subtracting the images for the respective condition, using an uncorrected significance threshold of p < .01 due to loss of degrees of freedom. This showed remaining activations when subtracting images for condition 2 from images for condition 1 in the right inferior frontal/insula region, (BA 13) extending across the premotor cortex, in the superior parietal lobule (BA 7) and in the occipital lobe/lingual gyrus (BA 18). Increasing the significance threshold to p < .001 did not yield any significantly remaining effects for condition 1 compared to condition 2. Results of the actigraph recordings are shown in Table 1. Mean daytime motor activity was not changed after switching of medication. The standard deviation was slightly, but not significantly, lower with risperidone than with perphenazine. The RMSSD however, was significantly reduced after switching to risperidone, and also the RMSSD/SD ratio. Table 1 Results from actigraphic recordings (mean ± SD) Perphenazin Risperidone Mean activity 301 ± 79 298 ± 48 SD 270 ± 67 254 ± 43 RMSSD 250 ± 48 216 ± 19** RMSSD/SD 0.94 ± 10 0.87 ± 12* Sample entropy 1.35 ± 0.18 1.33 ± 0.29 * p <0.05 t-test. ** p <0.01 t-test. Conclusions This study showed that brain activation decreased in areas critical for cognitive functioning, when a patient with schizophrenia was changed from FGA to a SGA medication. Total motor activity, however, was not altered during this change in medication, although our findings interestingly indicate that a change to SGA alters variability measures towards what was seen in a healthy control group shown in one of our prior studies [11]. We do not have test-retest data for fMRI from this particular study. This may represent a limitation of the study, since intra- and inter-individual variability generally is a problem in fMRI studies. However we do not think that our findings are caused by such confounding effects, since other studies, using variants of the same paradigm have shown similar patterns as found after medication in the present study [9,12]. An additional limitation of the fMRI data is the possibility that some of the changes from the first to the second session could be driven by learning effects and by the instruction to press the response button on target trials, which could lead to reduced activation due to facilitation. This is however not likely to explain all the findings that also were outside of areas to be affected. Another limitation is the rather weak statistical effects, not surviving standard correction procedures for multiple significance tests. Such procedures are typically applied to group average data, and may have reduced applicability for a single case study. A case study like the current study has its obvious limitations when it comes to generalizations to the population of schizophrenia patients. However, group averages based on large samples could equally be criticised for lacking in representativeness for a single patient, which is what the clinician is facing. Group averages are by definition not fully representative for any individual subject in the group, both because of the effect of statistical averaging and from the perspective of individual variation in response to antipsychotic medication (which is substantial in schizophrenia). Thus, reporting data for a single patient, measured longitudinally, being his own control, may yield clinically relevant data. The patterns of variability in the motor activity after switching antipsychotics from a FGA to a SGA are not easy to interpret. While the mean motor activity level and sample entropy were unchanged, risperidone reduced variability, particularly the variability from minute to minute, reflected in the RMSSD and also the RMSSD/SD ratio, towards what has been reported in healthy controls [11]. Nonlinear methods have addressed the severe disturbances in neurocognitive dysfunctioning as one of the core features of schizophrenia [13,14]. Wrist worn actigraphy for motor registration and magnetic resonance imaging for brain activation have shown correlations between motor activity and changes in brain structure [15]. Our findings comparing a patient switching from a first to a second generation antipsychotic supports these findings. The high perphenazine level observed in our patient could be explained by the low CYP2D6 metabolic capacity [16]. The decreased activation in the right inferior frontal and insula region is somewhat of a puzzle since one would have expected increased activation. It has been suggested that the inferior frontal gyrus and anterior insula areas are involved in complex attentional and working memory processing. Possibly, these brain areas are involved in cognitive control related to attentional focus on stimuli that are urgent or close in time and space [17], and it is unclear how such functions relate to reduced activation. Moreover, the right fronto-insular cortex has been implicated in a wide range of cognitive control mechanisms involved in a variety of cognitive control processes, including conflict and error monitoring, interference resolution, and response selection [18]. The inferior frontal gyrus and anterior insula has also been associated with psychosis. Insular dysfunction may be important for the development of psychosis due to its influence on the salience network [19]. Furthermore, the right inferior frontal area and posterior insula has been indicated in auditory hallucinations [20]. We conclude that changes in neuronal brain activation decreased in areas critical for cognitive functioning when a patient with schizophrenia was changed from a FGA to a SGA. At the same time although the total motor activity was not altered, variability measures changed towards values seen in healthy controls. Our study underscores the value of casuistic reports of schizophrenic patients examined during different conditions using objective recording methods in pharmacological treatment studies. Although the actigraph data were partly in accordance with the hypothesis, the brain activation results were unexpected, and not hypothesized and would need careful replication in a group study with proper statistical power, before any firm conclusions can be reached. Consent Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interest The authors declare that they have no competing interests. Authors’ contributions JØB introduced the patient to this study. JØB and OBF made the motor activity measures. EML performed the cognitive investigations. All authors participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgement This research has been supported financially by the legacy of Gerda Meyer Nyquist Gulbrandson & Gerdt Meyer Nyquist. We thank Kenneth Hugdahl, and the Bergen fMRI Group, University of Bergen and Haukeland University Hospital, Bergen, Norway for making the acquisition and analysis of the fMRI data, and for making the data available to us. ==== Refs Andreasen NC O’Leary DS Flaum M Nopoulos P Watkins GL Boles Ponto LL Hichwa RD Hypofrontality in schizophrenia: distributed dysfunctional circuits in neuroleptic-naïve patients Lancet 1997 349 1730 1734 10.1016/S0140-6736(96)08258-X 9193383 Molina V Sanz J Reig S Martínez R Sarramea F Luque R Benito C Gispert JD Pascau J Desco M Hypofrontality in men with first-episode psychosis Br J Psychiatry 2005 186 203 208 10.1192/bjp.186.3.203 15738500 Green MF Cognitive impairment and functional outcome in schizophrenia and bipolar disorder J Clin Psychiatry 2006 67 suppl 9 3 8 discussion 36–42 16965182 Palmer BW Dawes SE Heaton RK What do we know about neuropsychological aspects of schizophrenia? Neuropsychol Rev 2009 19 365 384 10.1007/s11065-009-9109-y 19639412 Lund A Thomsen T Kroken R Smievoll AI Landrø NI Barndon R Ersland L Iversen J Sundet K Lundervold A Asbjørnsen A Rund BR Hugdahl K “Normalization” of brain activation in schizophrenia. An fMRI study Schizophr Res 2002 58 333 335 10.1016/S0920-9964(01)00369-3 12409175 Park IH Kim JJ Chun J Jung YC Seok JH Park HJ Lee JD Medial prefrontal default-mode hypoactivity affecting trait physical anhedonia in schizophrenia Psychiatry Res 2009 171 155 165 10.1016/j.pscychresns.2008.03.010 19217758 Milev P Ho BC Arndt S Andreassen NC Predictive values of neurocognition and negative symptoms on functional outcome in schizophrenia: a longitudinal first-episode study with 7-year follow-up Am J Psychiatry 2005 162 495 506 10.1176/appi.ajp.162.3.495 15741466 Chou YH Halldin C Farde L Clozapine binds preferentially to cortical D1-like dopamine receptors in the primate brain: a PET study Psychopharmacology 2006 185 29 35 10.1007/s00213-005-0219-9 16395606 Hugdahl K Rund BR Lund A Asbjørnsen A Egeland J Ersland L Landrø NI Roness A Stordal KI Sundet K Thomsen T Brain activation measured with fMRI during a mental arithmetic task in schizophrenia and major depression Am J Psychiatry 2004 161 286 293 10.1176/appi.ajp.161.2.286 14754778 Berle JØ Hauge ER Oedegaard KJ Holsten F Fasmer OB Actigraphic registration of motor activity reveals a more structured behavioural pattern in schizophrenia than in major depression BMC Res Notes 2010 3 149 10.1186/1756-0500-3-149 20507606 Hauge ER Berle JØ Ødegaard KJ Holsten F Fasmer OB Nonlinear analysis of motor activity shows differences between schizophrenia and depression: a study using Fourier analysis and sample entropy PLoS One 2011 6 1 e16291 10.1371/journal.pone.0016291 Landrø NI Rund BR Lund A Sundet K Mjellem N Asbjørnsen A Thomsen T Ersland L Lundervold A Smievoll AI Egeland J Stordal K Roness A Sundberg H Hugdahl K Honig’s Model of working memory and brain activation : an fMRI study Neuroreport 2001 12 4047 4054 10.1097/00001756-200112210-00038 11742236 Kim D Zemon V Superstein A Butler PD Javitt DC Dysfunction of early-stage visual processing in schizophrenia: harmonic analysis Schizophr Res 2005 76 55 65 10.1016/j.schres.2004.10.011 15927798 Tschacher W Scheier C Hashimoto Y Dynamic analysis of schizophrenia courses Biol Psychiatry 1997 41 428 437 10.1016/S0006-3223(96)00039-X 9034537 Farrow TF Hunter MD Wirlkinson ID Green RD Spence SA Structural brain correlates of unconstrained motor activity in people with schizophrenia Br J Psychiatry 2005 187 481 482 10.1192/bjp.187.5.481 16260826 Dahl ML Cytochrome p450 phenotyping/genotyping in patients receiving antipsychotics: useful aid to prescribing? Clin Pharmacokinet 2002 41 453 470 10.2165/00003088-200241070-00001 12083975 Tops M Boksem MAS A potential role of the inferior frontal gyrus and anterior insula in cognitive control, brain rhythms, and event-related potentials Front Psychol 2011 2 1 14 10.3389/fpsyg.2011.00330 21713130 Sridharan D Levitin DJ Menon V A critical role for the right fronto-insular cortex in switching between central-executive and default-mode network PNAS 2011 105 12569 12574 10.1073/pnas.0800005105 18723676 Palaniyappan L Liddl PF Aberrant cortical gyrification in schizophrenia: a surface-based morphometry study J Psychiatry Neurosci 2012 37 399 406 10.1503/jpn.110119 22640702 Sommer IEC Diederen KMJ Blom JD Willems A Kushan L Slotema K Boks MPM Daalman K Hoek HW Neggers SFW Kahn RS Auditory verbal hallucinations predominantly activate the right inferior frontal area Brain 2008 131 3169 3177 10.1093/brain/awn251 18854323	23648137	PMC3726322	CC BY	2021-01-05 01:28:27	yes	BMC Res Notes. 2013 May 6; 6:182
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23922721PONE-D-13-1132910.1371/journal.pone.0069485Research ArticleBiologyBiochemistryBioenergeticsEnergy-Producing OrganellesMetabolismOxygen MetabolismMedicineClinical GeneticsMitochondrial DiseasesOncologyBasic Cancer ResearchMetastasisTumor PhysiologyCancers and NeoplasmsBreast TumorsInvasive Ductal CarcinomaMitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species Mitochondrial Dysfunction Increases ROS/HIF1αMa Jia 1 Zhang Qing 2 Chen Sulian 1 Fang Binbin 3 Yang Qingling 1 Chen Changjie 1 Miele Lucio 4 Sarkar Fazlul H. 5 Xia Jun 1 * Wang Zhiwei 6 7 * 1 Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu, Anhui, China 2 Department of Orthopedics, The Center Hospital of Bengbu, Anhui, China 3 Research Center of Clinical Laboratory Science, Bengbu Medical College, Bengbu, Anhui, China 4 University of Mississippi Cancer Institute, Jackson, Mississippi, United States of America 5 Department of Pathology and Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan, United States of America 6 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America 7 Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China Ushio-Fukai Masuko Editor University of Illinois at Chicago, United States of America * E-mail: zwang6@bidmc.harvard.edu (ZW); xiajunbbmc@126.com (JX)Competing Interests: Co-author Dr. Sarkar is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: JM ZW. Performed the experiments: JM QZ SC BF QY JX CC. Analyzed the data: JM CC ZW. Contributed reagents/materials/analysis tools: JM SC. Wrote the paper: JM LM FS ZW. 2013 29 7 2013 8 7 e6948515 3 2013 10 6 2013 © 2013 Ma et al2013Ma et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species. This work was supported by funding from the National Natural Science Foundation of China (81172087), Anhui Province College Excellent Young Talents Fund (2011SQRL084), and the Natural Science Research key Project of Education Office of Anhui Province (KJ2012A196). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Cancer cells display mitochondrial dysfunction to make cells adapt glycolysis to generate ATP even in the presence of oxygen, namely Warburg effect [1]. The mitochondrial dysfunction has been found to be associated with the development of human cancers [2], [3]. It has been reported that mitochondrial dysfunction could be caused by inhibitors of mitochondrial electron transport chain [4], pathogenic mutations in mitochondrial DNA (mtDNA) [3], and mutations in nuclear gene coded electron transport chain proteins [2]. Additionally, accumulating evidence suggests that cancer cells exhibit increased intrinsic reactive oxygen species (ROS) stress partly due to mitochondrial malfunction [5], [6]. The increased ROS in cancer cells may in turn affect certain redoxsensitive molecules and further lead to stimulation of cellular proliferation, cell migration and invasion, contributing to carcinogenesis [7], [8]. However, the underlying molecular mechanisms by which mitochondrial dysfunction increases ROS production and subsequently leads to tumorigenesis are not fully understood. Emerging evidence suggests that mitochondrial malfunction and hypoxia in the tumor microenvironment are considered as two major factors contributing to the Warburg effect [9], [10]. In solid tumors, hypoxia, which is an oxygen tension below physiologic levels, develops as abnormal proliferation outstrips the blood supply [11]. This hypoxic region is involved in tumor malignancy and proliferation, resulting in the development of resistance to radiotherapy [12]. Hypoxia-inducible factor-1 (HIF-1), a transcription factor that regulates the cellular response to hypoxia, induces several genes that mediate tumorigenesis [13], [14]. It is known that HIF-1 is a heterodimer that consists of the oxygen-sensitive HIF-1α subunit and the constitutively expressed HIF-1β subunit [15], [16]. Under normoxic conditions, HIF-1a is hydroxylated by prolyl hydroxylases on the proline residues in the oxygen-dependent degradation domain [17], [18]. In hypoxic conditions, low oxygen leads to HIF-1α stabilization due to the inhibition of prolyl-hydroxylation and subsequent reduction in HIF-1α ubiquitination and degradation [18]. In addition to the regulation of HIF-1α by oxygen supply, there are also different HIF activators that include growth factors, hormones, cytokines and viral proteins [19]. Interestingly, related observations of ROS regulating HIF-1α expression appear to be controversial. For example, multiple studies have shown that increased HIF-1α expression contributes to mitochondrial activity and ROS formation during hypoxia [20], [21]. However, other studies have demonstrated a decrease in HIF-1α with increasing ROS [22]. Moreover, some studies have shown no effects on mitochondrial ROS [23]. These controversy results suggest that further study is required to investigate the relationship between HIF-1 α and ROS. In the present study, we treated SKBR3 and 4T1 breast cancer cells by an inhibitor of mitochondrial electron transport complex I, rotenone, for 1–2 weeks to establish mitochondrial dysfunction subclones. Each subclone was confirmed to have mitochondrial dysfunction by measurement of oxygen consumption, glucose uptake, and lactate production. We found that mitochondrial dysfunction subclones had elevated levels of ROS production. We further analyzed (a) whether ROS are required for induction of tumor cell migration and invasion; (b) whether ROS production regulate HIF-1α and vascular endothelial growth factor (VEGF) expression; (c) whether ROS govern tumor cell migration and invasion through the regulation of HIF-1α and VEGF expression. This work provides the molecular insight into the role of ROS in the regulation of breast cancer cell migration and invasion. Materials and Methods Reagents Antibody against HIF-1α was purchased from BD Biosciences. Antibodies against VEGF and β-actin were purchased from Santa Cruz Biotechnology. Rotenone, PEG-catalase, and antioxidant N-acetyl cysteine were obtained from Sigma. 2′,7′-Dichlorofluorescein diacetate (CM2-DCFHDA) was purchased from Invitrogen. mitoTEMPO was purchased from Enzo Life Sciences. 8 µm pore Transwell inserts and Matrigel were bought from BD Biosciences. Cell Culture SKBR3 and 4T1 breast cancer cells were bought from American Type Culture Collection (Rockville, MD). SKBR3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and penicillin (100 units/ml) and streptomycin (100 µg/ml). Murine 4T1 breast cancer cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and penicillin (100 units/ml) and streptomycin (100 µg/ml). The subclone cells used in this study were generated by rotenone treatment as described previously [4]. It is important to note that rotenone was absent after clone generation during the clone studies. Briefly, cells were treated for 24 h with rotenone (100 nmol/L), then cultured in drug-free medium for 48 h. Followed by two more cycles of rotenone treatment, cells were then plated at a density of 200 per dish in drug-free medium to allow the formation of colonies, which caused an increase in superoxide generation measured by flow cytometry. Among these clones, two clones with higher ROS from SKBR3 cells were named as A and B, and two clones with higher ROS from 4T1 cells were marked as C and D for further studies. ROS Measurement Intracellular ROS generation was assessed using 2′,7,-dichlorofluorescein diacetate. Briefly, 1×106 cells were plated on the 6-well plates and incubated with DCFH-DA (10 mmol/L) for 30 min at 37°C. Cells were washed and harvested in Hank’s buffered salt solution (HBSS) and analyzed immediately using a BD FACScan flow cytometer. Data were analyzed as single parameter frequency histogram using cell Quest software (BD Biosciences). Results are presented as mean fluorescence intensity. Hydrogen Peroxide Measurement The cells were lysed in 100 µl lysis buffer supplied by the H2O2 assay kit (Beyotime Institute of Biotechnology, China) to determine the intracellular H2O2 concentration. The supernatants were obtained by centrifuging at 12,000×g for 10 min. The H2O2 concentrations were measured with the assay kit according to the manufacturer’s instructions [24]. Briefly, 50 µl sample solution was incubated with 100 µl reaction solution at room temperature for 30 min, and then the absorption at 560 nm was measured. The H2O2 concentration was calculated by the standard curve made from the standard solutions. Mitochondrial Respiratory Defect Measurement ATP measurements were performed using ATP assay Kit (Beyotime). We also investigated the mitochondrial oxidative phosphorylation contribution on ATP production with the addition of ATP synthase inhibitor, oligomycin, as previously described [25]. Briefly, cells were grown to approximately 80% confluence, and then incubated in DMEM containing the following combinations of substrates and inhibitors: 4.5 mg/ml glucose plus 110 mg/l pyruvate; glucose plus 15 mg/ml of oligomycin. Cells were incubated with 15 mg/ml of oligomycin for 20 min before harvesting. The remaining steps were following the manufacturer’s instructions. Cellular glucose uptake was measured by incubating cells in glucose-free medium with 0.2 Ci/mL [3H] 2-deoxyglucose (specific activity, 40 Ci/mmol) for 60 minutes. After the cells were washed with ice-cold PBS, the radioactivity in the cell pellets was quantified by liquid scintillation counting. Cellular lactate level was measured under normoxia with lactate assay kit (Biovision) following the manufacturer’s instructions. The absorbance was recorded using a microplate reader at a 570-nm wavelength. The data were normalized to the control group. Transwell Migration and Invasion Assays The migration of breast cancer cells and their subclones was performed using a 24-well transwell chamber (Corning) containing gelatin-coated polycarbonate membrane filter (6.5 mm diameter, 8 µm pore size). 1×105 cells suspended in 100 µl culture medium with 1% FBS were seeded into the upper chamber. The lower chamber contained 600 µl culture medium with 10% FBS as a chemoattractant. After 24 h incubation at 37°C in 5% CO2, non-migrated cells were scraped from the upper surface of the membrane with a cotton swab, and migrated cells remaining on the bottom surface were fixed with 4% paraformaldehyde, staining with giemsa and photographed under a microscope at 20 magnification. The numbers of migrated cells were counted under a light microscope in 5 randomly-selected fields for each chamber. The invasion of the cancer cells were performed by the same procedure as in the migration assay except that the chamber filter were coated with matrigel (BD Biosciences) and 5×105 cells were seeded into the upper chamber. Wound Healing Assay 2×105 cells were added to each well of 6-well plate and cultured at 37°C in 5% CO2 until more than 80% confluent. They were then scratched with a standard 200 µl pipette tip, wounded monolayers were washed twice to remove nonadherent cells and images were captured at 0 h, 24 h and 48 h after wounding using a Nikon Eclipse TE300 microscope and a Nikon Plan Fluor 4×0.13 objective. Western Blotting Analysis Western blotting was performed as previously described [26]. Briefly, cells were lysed with a whole-cell extract buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecylsulfate, 5 mM EDTA, 4 mg/ml glycerophosphate) containing freshly protease inhibitors. Total proteins were collected by centrifugation. The proteins were quantified by BCA protein assay. Twenty-five micrograms of lysate proteins were separated by SDS–PAGE and subsequently transferred to a PVDF membrane. The membranes were blocked with a solution of Tris-buffered saline (TBS), 0.1% Tween-20 (TBS-T) containing 5% nonfat milk. The membranes were incubated overnight at 4°C with primary antibodies against human HIF-1α, VEGF, and β-actin. The membranes were washed with TBS-T, and secondary antibodies were added to the membrane for 1 h at 37°C. Membranes were washed with TBS-T, visualized with ECL reagent (Millipore), and exposed to film. RNAi-mediated Inhibition of HIF-1α The target sequence of HIF-1α was selected [27] as followed: 5′-TACGTTGTGAGTGGTATTATT-3′. The 21 nt target sequence served as the basis for the design of the two complementary 55-mer siRNA template oligonucleotides that were synthesized, annealed, and ligated annealed siRNA template were inserted into the pSilencer 4.1-CMV vecter (Ambion). Briefly, shRNA was designed by using the siRNA software according to the sequence target as follows: Forward oligo, 5′-GAT CCC GTT GTG AGT GGT ATT ATT TTC AAG AGA AAT AAT ACC ACT CAC AAC GTA A-3′, Reverse oligo, 5′-AGC TTT ACG TTG TGA GTG GTA TTA TTT CTC TTG AAA TAA TAC CAC TCA CAA CGG-3′. The double stranded DNA sequence was obtained through annealing after chemosynthesis and was inserted into PsilencerTM4.1-CMV vector. The insert production was transformed into E. coli cells. Then the clones were picked and sequenced to verify the inserts. Transient transfection was performed using the Cationic lipid Lipofectamine2000 (Invitrogen) with HIF-1a shRNA vector. Results Generation of Subclones with Higher ROS Rotenone has been accepted as a blocker of the electron flow through inhibition of complex I and subsequently causing an increase in the production of superoxide due to electron flow bifurcation [4]. Therefore, we used rotenone to increase superoxide generation which leads to subsequent induction of ROS stress in SKBR3 and 4T1 cells. After three cycles of rotenone treatment, cells were plated in drug-free medium to allow the formation of colonies. After subclones are generated, each 18 subclones from SKBR3 and 4T1 cells were picked to measure ROS level. Among 18 SKBR3 subclones, 15 subclones showed significantly higher ROS production compared to their parental SKBR3 cells, 3 subclones showed almost the same ROS level as their parental SKBR3 cells (data not shown). Among 18 4T1 subclones, 14 subclones have obviously higher ROS production than that of their parental 4T1 cells, and 4 subclones showed no difference in ROS production with their parental 4T1 cells (data not shown). Four clones with highest ROS were selected for further studies. Two SKBR3 cells’ subclones with higher ROS were named A and B (Figure S1A), while C and D subclones were selected from 4T1cells (Figure S1B). Subclones Exhibiting Mitochondrial Dysfunction To determine whether increased ROS affect mitochondrial dysfunction, we performed the mitochondrial respiratory defect measurement. As illustrated in Figure 1A, both A and B clones showed increased in glucose uptake and lactate production compared to their parental SKBR3 cells (Fig. 1A). Moreover, both A and B clones had similar total ATP content compared with the parental SKBR3 cells: however; ATP synthesis sensitive to oligomycin that assumed to be contributed by mitochondrial ATP synthase further decreased in A and B clones compared with parental SKBR3 cells (Fig. 1B). Similar results were found in 4T1 cells (Fig. 1B). These results suggest that up-regulation of glycolysis was sufficient to compensate the decreased ATP generation in the mitochondria. 10.1371/journal.pone.0069485.g001Figure 1 Biochemical characterization of mitochondrial dysfunction of both clones of breast cancer cells. A, Comparison of ATP synthase (left), glucose uptake (middle), and lactate production (right) in SKBR3 cells. P<0.01 vs SKBR3 cells, P<0.05 vs SKBR3 cells, #P<0.05 vs SKBR3 cells (n = 3). B, Comparison of ATP synthase (left), glucose uptake (middle), and lactate production (right) in 4T1 cells. P<0.05 vs 4T1 cells, *P<0.01 vs 4T1 cells, #P<0.05 vs 4T1 cells (n = 3). ROS Promoted Cell Motility and Invasion As ROS have been reported to be involved in tumor metastasis [8], we tested the motility and invasive potential of each subclones. Furthermore, to evaluate whether increased ROS were essential for this process, cells were pretreated with the antioxidant N-acetyl cysteine (NAC) before the Transwell assay and wound healing assay. As we expected, four subclones exhibited greater motility compared to their parental cells. More importantly, these migration capacities were significantly inhibited by NAC (Fig. 2A, 2B). Consistent with these results, we observed that these subclones with higher ROS showed highly invasion capacity, which can be inhibited by NAC (Figure S2). In agreement with the Transwell assay, the wound healing assay showed significantly accelerated wound closure in these subclone cells compared to their parental cells after scratch assay (Fig. 3A, 3B). NAC effectively inhibited the wound closure in these subclones (Fig. 3A, 3B). These results indicated that the aggressive cellular behaviors of subclones might be regulated by ROS. 10.1371/journal.pone.0069485.g002Figure 2 Effect of ROS on migration and invasion capacity of subclones and their parental cells by transwell assay. A, SKBR3 subclones migrated faster than parental SKBR3 cells. NAC was able to effectively inhibit the migration of SKBR3 subclones. P<0.05 vs SKBR3 cells, *P<0.01 vs A clone, #P<0.01 vs B clone (n = 3). B, 4T1 subclones migrated faster than parental 4T1 cells. NAC was able to effectively inhibit the migration of 4T1 subclones. P<0.05 vs 4T1 cells, *P<0.01 vs C clone, #P<0.01 vs D clones (n = 3). 10.1371/journal.pone.0069485.g003Figure 3 The migration capacity of subclones and their parental cells was measured by wound healing assay. A–B, The subclones migrated faster than the parental cells SKBR3 cells (A) and 4T1 cells (B). NAC was able to effectively inhibit the migration of the subclones. Inhibition of ROS Generation Decreased Cell Motility and Invasion To further confirm whether ROS were increased in subclone cells, we measured the H2O2 concentrations in these subclones using the commercial assay kit [24]. As expected, we found that H2O2 was increased in these subclones (data not shown), and NAC significantly inhibited generation of H2O2 in subclones (Figure 4A). Furthermore, H2O2 promoted cell migration and invasion, while its scavenger PEG-catalase decreased cell migration and invasion in subclone cells (Figure 4B, 4C, 4D, Figure S3), suggesting that ROS play a role in cell motility and invasion. More importantly, mitochondria-target SOD mimetic, mito-TEMPO [28] inhibited migration and invasion in subclone cells (Figure 4B, 4C, 4D, Figure S3). 10.1371/journal.pone.0069485.g004Figure 4 PEG-catalase and mito-TEMPO inhibited migration and invasion in subclone cells. A, The H2O2 concentrations were measured in subclone cells treated with NAC. P<0.01 vs control in A clone, P<0.01 vs control in B clone, #P<0.01 vs control in C clone, ##P<0.05 vs control in D clones (n = 3). B, Wound healing assay was conducted to measure the migration capacity of A subclone cells treated with 100 µM H2O2, 200 units/ml PEG-catalase, 25 nM mito-TEMPO, respectively. Images were captured at 24 h after wounding. C, Migration assay was performed in A subclone cells treated with indicated reagents. D, Invasion capacity of A subclone cells treated with indicated reagents was detected by transwell assay. ROS Induced HIF-1α and VEGF Expression Next, we explore the molecular mechanism of ROS-mediated migration and invasion. Since mitochondrial ROS production has been implicated in the stabilization of HIF-1α during hypoxia [20], we measured HIF-1α expression under both hypoxia and normoxia conditions. As cobalt chloride (CoCl2) has been accepted as a hypoxic mimetic agent, we used 500 µmol/L CoCl2 to treat cells for 30 minutes before the cells were lysed. As shown in Figure 5, the subclones exhibited increased expression of HIF-1α under normoxic and hypoxic conditions. NAC inhibited not only normoxic HIF-1α expression but also hypoxic HIFα expression in both subclones (Figure 5A, 5B). VEGF, a crucial angiogenic factor for controlling angiogenesis and vasculature, is one of the most prominent HIF-1 target genes. Therefore, we further measured VEGF expression in these subclones. We found that these subclones have increased expression of VEGF (Figure 5A, 5B). We also observed that VEGF expression was significantly decreased after NAC treatment in both subclones (Figure 5A, 5B). These results suggest that ROS could induce HIF-1α and consequently leads to increased VEGF expression. To further validate the role of ROS in regulation of HIF-1α, the subclone cells were treated with PEG-calatase and mito-TEMPO, respectively. We observed that H2O2 upregulated HIF-1α expression, whereas both PEG-calatase and mito-TEMPO down-regulated the expression of HIF-1α in subclone cells (Figure 5C, 5D). Taken together, our findings demonstrated that ROS play a critical role in cell migration and invasion partly through induction of HIF-1α. 10.1371/journal.pone.0069485.g005Figure 5 ROS promoted HIF-1α and VEGF expression in subclone cells. A, ROS led to increased expression of HIF-1α and VEGF in SKBR3 subclones. SKBR3 subclones showed higher normoxic and hypoxic HIF-1α level than the parental SKBR3 cells. VEGF expression was more increased in SKBR3 subclones than in the parental SKBR3 cells. HIF-1α and VEGF expression in SKBR3 subclone were significantly inhibited by NAC. B, ROS led to increased expression of HIF-1α and VEGF in 4T1 subcloneswhereas NAC was able to attenuate the ROS induced expression of HIF-1α and VEGF. C–D, H2O2 promoted HIF-1α expression, while PEG-catalase and mito-TEMPO inhibited HIF-1α expression in A clone (C) and C clone (D). ROS Promoted Cell Motility by Upregulation of HIF-1α Expression To determine the role of HIF-1α in ROS-mediated cell motility and invasion, we constructed the expression vector of HIF-1α small interfering RNA to silence HIF 1α expression. The constructs were sequenced to confirm that there are no unwanted mutations (Fig. 6A). The interfering effects were determined by Western blotting analysis. HIF-1α protein expression was significantly inhibited by its shRNA transfection (Fig. 6B). Then we measured cell mobility and invasion ability after depletion of HIF-1α. The results indicate that HIF-1α shRNA transfected cells significantly decreased cells mobility and invasive capacity compared to control cells (Fig. 6C, 6D, 6E). Our results suggest that rotenone initially generate mitochondrial dysfunction and increased ROS production which, in turn, affect tumor cell motility and invasive capacity by upregulation of HIF-1α expression. 10.1371/journal.pone.0069485.g006Figure 6 ROS promoted aggressive cellular behaviors by up-regulating HIF-1α expression. A, The sequence of inserts in the shRNA vector clone. B, Cells with HIF-1α shRNA showed decreased HIF-1α expression compared with the control. C, Cells with HIF-1α shRNA showed decreased migration ability compared with the control as documented by transwell migration assay. P<0.05 vs A clone (n = 3) D, Cells with HIF-1α shRNA showed decreased invasive capacity compared with the control as documented by transwell invasion assay. **P<0.05 vs A clone (n = 3) E, Wound healing assay showed that cells with HIF-1α shRNA migrated slower than the control. Discussion Mitochondria possess many biological functions, including the production of ATP, housing numerous biochemical reactions, generating ROS, and governing apoptosis [29]. Numerous studies have suggested that these mitochondrial processes may play important roles in tumor initiation and progression [30], [31]. For example, mitochondrial ROS have been implicated in malignant cell transformation [32]. Moreover, it has been suggested that mitochondrial ROS may be important in the maintenance of malignant phenotype through regulation of HIF-1α [28]. In this study, we tested the hypothesis that additional mitochondrial alterations acquired after malignant transformation may increase ROS production which will further contribute to cancer development via ROS-dependent HIF-1α and VEGF pathways to promote cancer cell migration and invasion. To achieve our goal, we choose rotenone to induce mitochondrial dysfunction to investigate whether rotenone could induce ROS production. Both subclones of breast cancer cells showed higher ROS consistent with declined mitochondrial respiration function, as suggested by decreased ATP generation through oxidative phosphorylation, increased ATP generation through glycolysis, higher glucose uptake and lactate production. These subclones also exhibited increased cellular motility and ability to invade through Matrigel. These malignant behaviors were inhibited by the antioxidant NAC, PEG-Calatase, and mito-TEMPO, indicating the important role of ROS, which is associated with mitochondrial dysfunction. Although some researchers investigated the role of ROS in carcinogenesis, the exact mechanisms how ROS are involved in tumorigenesis are unclear. Recently, a number of studies have suggested that mitochondrial ROS are involved in the stabilization and activation of HIF under hypoxic conditions [21]. It is known that HIF-1α, which is induced by hypoxia, growth factors, and oncogenes, plays a pivotal role in tumor growth and angiogenesis [13]. ROS affect HIF-1α expression under gastric ischemic conditions, suggesting that ROS can regulate HIF-1α expression in gastric ischemia [33]. Moreover, increased ROS and ROS-dependent stabilization of HIF under conditions of normal oxygen tension have also been reported in cancer cells by suppression of SdhB expression [20]. Furthermore, Xia et al. found that the stabilization of HIF-1α via ROS generation led to the binding of HIF-1α to the FoxM1 promoter, resulting in increased FoxM1 oncoprotein expression in hepatocellular carcinoma [34]. Recently, mitochondria-target antioxidant mito-TEMPO has been demonstrated to inhibit redox-dependent HIF-1α-mediated cancer pro-survival signaling pathways [28]. Consistent with these reports, our results clearly suggest that increased ROS production is required for HIF-1α stabilization in the mitochondrial dysfunction cells induced by rotenone, and these effects were attenuated by antioxidant NAC, PEG-Calatase, and mito-TEMPO. Therefore, our study indicates that ROS promote breast cancer progression, which is in part mediated through up-regulation of HIF-1α expression in breast cancer cells. Multiple studies have demonstrated that ROS regulate VEGF expression in various human cancers [35]. For example, Xia et al. reported that ROS regulate angiogenesis and tumor growth, which is mediated through upregulation of VEGF [36]. Moreover, Liu et al. found that ROS up-regulate VEGF and HIF-1α through the activation of Akt and p70S6K in human cancer cells [37]. Interestingly, HIF-1α has been found to control VEGF in a variety of human cancer cells [38], [39]. Notably, HIF-1α-mediated up-regulation of VEGF is important in the switch to the angiogenic phenotype during early tumorigenesis [40]. Therefore, these findings suggest that ROS govern VEGF production which is in part mediated through up-regulation of HIF-1α. In line with these reports, we revealed that high levels of ROS production caused elevated VEGF expression by regulating HIF-1α. Conversely, the decrease of HIF-1α expression by ROS inhibitors suppressed VEGF transcriptional activation. Taken together, our present study suggests that mitochondrial dysfunction in breast cancer cells with high ROS production promotes cell mobility and invasion which is in part mediated through HIF-1α and VEGF. However, further in-depth investigation is warranted to explore the molecular insight into the role of ROS-mediated tumorigenesis in vivo. Supporting Information Figure S1 ROS were measured in subclones by DCF-DA method. A–B, comparison of ROS generation by parental SKBR3 cells (A) and 4T1 cells (B), and their subclones as assessed by using CM-H2DCF-DA measurement done by flow cytometry. (TIF) Click here for additional data file. Figure S2 Effect of ROS on migration and invasion capacity of subclones and their parental cells by transwell assay. A, The invasive capacity of SKBR3 subclones was higher than the parental SKBR3 cells and was effectively inhibited by NAC. B, The invasive capacity of 4T1 subclones was higher than the parental 4T1 cells and was effectively inhibited by NAC. (TIF) Click here for additional data file. Figure S3 PEG-catalase and mito-TEMPO inhibited migration and invasion in C subclone cells. A, The migration capacity of C subclone cells treated with different reagents was measured by wound healing assay. B, Migration assay was performed in C subclone cells treated with indicated reagents. C, Invasion capacity of C subclone cells treated with indicated reagents was detected by transwell assay. (TIF) Click here for additional data file. ==== Refs References 1 Warburg O (1956 ) On the origin of cancer cells . Science 123 : 309 –314 .13298683 2 Kwong JQ , Henning MS , Starkov AA , Manfredi G (2007 ) The mitochondrial respiratory chain is a modulator of apoptosis . J Cell Biol 179 : 1163 –1177 .18086914 3 Wu YT , Wu SB , Lee WY , Wei YH (2010 ) Mitochondrial respiratory dysfunction-elicited oxidative stress and posttranslational protein modification in mitochondrial diseases . Ann N Y Acad Sci 1201 : 147 –156 .20649551 4 Pelicano H , Lu W , Zhou Y , Zhang W , Chen Z , et al (2009 ) Mitochondrial dysfunction and reactive oxygen species imbalance promote breast cancer cell motility through a CXCL14-mediated mechanism . Cancer Res 69 : 2375 –2383 .19276362 5 Moreno-Sanchez R , Rodriguez-Enriquez S , Marin-Hernandez A , Saavedra E (2007 ) Energy metabolism in tumor cells . FEBS J 274 : 1393 –1418 .17302740 6 Pelicano H , Martin DS , Xu RH , Huang P (2006 ) Glycolysis inhibition for anticancer treatment . Oncogene 25 : 4633 –4646 .16892078 7 Bauer G (2012 ) Tumor cell-protective catalase as a novel target for rational therapeutic approaches based on specific intercellular ROS signaling . Anticancer Res 32 : 2599 –2624 .22753718 8 Ishikawa K , Takenaga K , Akimoto M , Koshikawa N , Yamaguchi A , et al (2008 ) ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis . Science 320 : 661 –664 .18388260 9 Salem AF , Whitaker-Menezes D , Lin Z , Martinez-Outschoorn UE , Tanowitz HB , et al (2012 ) Two-compartment tumor metabolism: autophagy in the tumor microenvironment and oxidative mitochondrial metabolism (OXPHOS) in cancer cells . Cell Cycle 11 : 2545 –2556 .22722266 10 Balliet RM , Capparelli C , Guido C , Pestell TG , Martinez-Outschoorn UE , et al (2011 ) Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection . Cell Cycle 10 : 4065 –4073 .22129993 11 Henze AT , Acker T (2010 ) Feedback regulators of hypoxia-inducible factors and their role in cancer biology . Cell Cycle 9 : 2749 –2763 .20603601 12 Hockel M , Vaupel P (2001 ) Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects . J Natl Cancer Inst 93 : 266 –276 .11181773 13 Kappler M , Taubert H , Schubert J , Vordermark D , Eckert AW (2012 ) The real face of HIF1alpha in the tumor process . Cell Cycle 11 : 3932 –3936 .22987151 14 Amelio I , Melino G (2012 ) The “Sharp” blade against HIF-mediated metastasis . Cell Cycle 11 : 4530 –4535 .23187809 15 Briston T , Yang J , Ashcroft M (2011 ) HIF-1alpha localization with mitochondria: a new role for an old favorite? Cell Cycle 10 : 4170 –4171 .22101263 16 Bertozzi D , Iurlaro R , Sordet O , Marinello J , Zaffaroni N , et al (2011 ) Characterization of novel antisense HIF-1alpha transcripts in human cancers . Cell Cycle 10 : 3189 –3197 .21897117 17 Rapisarda A , Shoemaker RH , Melillo G (2009 ) Antiangiogenic agents and HIF-1 inhibitors meet at the crossroads . Cell Cycle 8 : 4040 –4043 .19923892 18 Maxwell PH (2004 ) HIF-1’s relationship to oxygen: simple yet sophisticated . Cell Cycle 3 : 156 –159 .14712080 19 Denko NC (2008 ) Hypoxia, HIF1 and glucose metabolism in the solid tumour . Nat Rev Cancer 8 : 705 –713 .19143055 20 Guzy RD , Hoyos B , Robin E , Chen H , Liu L , et al (2005 ) Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing . Cell Metab 1 : 401 –408 .16054089 21 Schroedl C , McClintock DS , Budinger GR , Chandel NS (2002 ) Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species . Am J Physiol Lung Cell Mol Physiol 283 : L922 –931 .12376345 22 Callapina M , Zhou J , Schmid T , Kohl R , Brune B (2005 ) NO restores HIF-1alpha hydroxylation during hypoxia: role of reactive oxygen species . Free Radic Biol Med 39 : 925 –936 .16140212 23 Vaux EC , Metzen E , Yeates KM , Ratcliffe PJ (2001 ) Regulation of hypoxia-inducible factor is preserved in the absence of a functioning mitochondrial respiratory chain . Blood 98 : 296 –302 .11435296 24 Sheng R , Gu ZL , Xie ML , Zhou WX , Guo CY (2010 ) Epigallocatechin gallate protects H9c2 cardiomyoblasts against hydrogen dioxides- induced apoptosis and telomere attrition . European journal of pharmacology 641 : 199 –206 .20553906 25 Park JS , Sharma LK , Li H , Xiang R , Holstein D , et al (2009 ) A heteroplasmic, not homoplasmic, mitochondrial DNA mutation promotes tumorigenesis via alteration in reactive oxygen species generation and apoptosis . Hum Mol Genet 18 : 1578 –1589 .19208652 26 Wang Z , Inuzuka H , Zhong J , Fukushima H , Wan L , et al (2012 ) DNA damage-induced activation of ATM promotes beta-TRCP-mediated Mdm2 ubiquitination and destruction . Oncotarget 3 : 1026 –1035 .22976441 27 Martin SE , Jones TL , Thomas CL , Lorenzi PL , Nguyen DA , et al (2007 ) Multiplexing siRNAs to compress RNAi-based screen size in human cells . Nucleic Acids Res 35 : e57 .17392344 28 Nazarewicz RR, Dikalova A, Bikineyeva A, Ivanov S, Kirilyuk IA, et al.. (2013) Does Scavenging of Mitochondrial Superoxide Attenuate Cancer Prosurvival Signaling Pathways? Antioxidants & redox signaling. 29 Dohi T , Altieri DC (2005 ) Mitochondrial dynamics of survivin and “four dimensional” control of tumor cell apoptosis . Cell Cycle 4 : 21 –23 .15611629 30 Li Y , Rempe DA (2010 ) During hypoxia, HUMMR joins the mitochondrial dance . Cell Cycle 9 : 50 –57 .20016276 31 Pavlides S , Tsirigos A , Vera I , Flomenberg N , Frank PG , et al (2010 ) Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the “reverse Warburg effect”: a transcriptional informatics analysis with validation . Cell Cycle 9 : 2201 –2219 .20519932 32 Haigis MC , Deng CX , Finley LW , Kim HS , Gius D (2012 ) SIRT3 is a mitochondrial tumor suppressor: a scientific tale that connects aberrant cellular ROS, the Warburg effect, and carcinogenesis . Cancer research 72 : 2468 –2472 .22589271 33 Wang T , Leng YF , Zhang Y , Xue X , Kang YQ (2011 ) Oxidative stress and hypoxia-induced factor 1alpha expression in gastric ischemia . World J Gastroenterol 17 : 1915 –1922 .21528068 34 Xia L , Mo P , Huang W , Zhang L , Wang Y , et al (2012 ) The TNF-alpha/ROS/HIF-1-induced upregulation of FoxMI expression promotes HCC proliferation and resistance to apoptosis . Carcinogenesis 33 : 2250 –2259 .22831955 35 Forsythe JA , Jiang BH , Iyer NV , Agani F , Leung SW , et al (1996 ) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1 . Mol Cell Biol 16 : 4604 –4613 .8756616 36 Xia C , Meng Q , Liu LZ , Rojanasakul Y , Wang XR , et al (2007 ) Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor . Cancer Res 67 : 10823 –10830 .18006827 37 Liu LZ , Hu XW , Xia C , He J , Zhou Q , et al (2006 ) Reactive oxygen species regulate epidermal growth factor-induced vascular endothelial growth factor and hypoxia-inducible factor-1alpha expression through activation of AKT and P70S6K1 in human ovarian cancer cells . Free Radic Biol Med 41 : 1521 –1533 .17045920 38 Buchler P , Reber HA , Buchler M , Shrinkante S , Buchler MW , et al (2003 ) Hypoxia-inducible factor 1 regulates vascular endothelial growth factor expression in human pancreatic cancer . Pancreas 26 : 56 –64 .12499918 39 Fukuda R , Kelly B , Semenza GL (2003 ) Vascular endothelial growth factor gene expression in colon cancer cells exposed to prostaglandin E2 is mediated by hypoxia-inducible factor 1 . Cancer Res 63 : 2330 –2334 .12727858 40 Fang J , Yan L , Shing Y , Moses MA (2001 ) HIF-1alpha-mediated up-regulation of vascular endothelial growth factor, independent of basic fibroblast growth factor, is important in the switch to the angiogenic phenotype during early tumorigenesis . Cancer Res 61 : 5731 –5735 .11479208	23922721	PMC3726697	CC BY	2021-01-05 17:35:04	yes	PLoS One. 2013 Jul 29; 8(7):e69485
==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2013/434561Research ArticleDownregulation of ADAM10 Expression Inhibits Metastasis and Invasiveness of Human Hepatocellular Carcinoma HepG2 Cells Yue Yuan 1 Shao Yuan 2 Luo Qing 3 Shi Lei 4 Wang Zuoren 4 1Department of Pharmacy, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, China2Department of the Otorhinolaryngology, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, China3Department of General Surgery, The 417 Hospital of Nuclear Industry, Xi'an, Shaanxi 710600, China4Department of Hepatobiliary Surgery, First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, ChinaLei Shi: s81145558@gmail.comAcademic Editor: Paul Higgins 2013 14 7 2013 2013 43456117 4 2013 19 6 2013 21 6 2013 Copyright © 2013 Yuan Yue et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. This study aims to investigate the effects of ADAM10 expression on metastasis and invasiveness of human hepatocellular carcinoma HepG2 cells. Methods. The HepG2 cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10, respectively. Cell migration assay and Transwell invasiveness assay were performed to detect the effects of ADAM10 knockdown on migration and invasiveness of HepG2 cells. Western blotting and real-time RT PCR were performed to investigate the effects of knock-down of ADAM10 on protein and mRNA levels of E-cadherin gene. Results. Cell migration and invasiveness of HepG2 cells transfected with ADAM10 siRNA were significantly decreased, when compared with the cells transfected with the control siRNA, suggesting that the downregulation of ADAM10 expression inhibits cell migration and invasiveness. The Western blotting results suggest that the down-regulation of ADAM10 expression increases E-cadherin protein levels. The real-time RT-PCR results indicated that the mRNA level of E-cadherin is not detectably affected by the knock-down of ADAM10 gene. Conclusions. Expression of ADAM10 may be related to cell migration and invasiveness of human hepatocellular carcinoma HepG2 cells via a mechanism related to E-cadherin. ==== Body 1. Introduction The migration and invasiveness-related characteristics of hepatoma cells are usually influenced by the expression levels of a variety of cell membrane molecules [1–3]. The invasive growth and metastasis of tumor tissues can be achieved by the escape from the immunologic surveillance [4, 5]. Currently, there is no effective therapy for liver cancer patients, who are diagnosed with distant metastasis or unsuitable for surgery. Therefore, prevention of metastasis is one of the major aims of treatment for primary carcinoma of the liver. And therefore, the molecular targets and novel drugs are necessary to be studied. Metastasis generally involves shedding of tumor cells from the primary sites and their infiltration into peripheral tissues, after which the cells can enter vasculature and form embolus [6–9]. Tumor infiltration is caused by various factors, where intercellular adhesion is reduced and thus tumor cells adhere to the basal membrane and then move out of the primary site as the extracellular matrix is degraded [10–12]. Cellular adhesion molecules play an important role in tumor metastasis. The cell adhesion protein E-cadherin is a member of the superfamily of calcium-dependent cell adhesion proteins [13]. Decreased expression level of E-cadherin reduces intercellular adhesion, changes cellular polarity and morphology, and thus dissociates tumor cells, which then makes cells infiltrate into peripheral tissues [14]. It was demonstrated in vitro that when E-cadherin is added into tumor cell culture, the aggregated cells are dissociated with increased invasiveness and enhanced mobility [15]. The mobility of tumor cells is also influenced by extracellular matrix, the dissolution of which may change the three-dimensional cell structure and result in dedifferentiated morphology and enhanced invasiveness and migration capability of the tumor cells [16]. The metalloproteinase region of the ADAMs family can degrade extracellular matrix and thus influence tumor progression [17, 18]. In this study, the effects of ADAM10-specific siRNAs on the migration capability and invasiveness of HepG2 cells are investigated. It is also determined whether and how the expression of E-cadherin is correlated with the migration and invasiveness of HepG2 cells. 2. Materials and Methods 2.1. Cells and Reagents Human hepatocellular carcinoma HepG2 cells were provided by the Experimental Center of Biomedical Research, Medical College of Xi'an Jiaotong University. The cells were maintained in RPMI-1640 medium (Sigma-Aldrich Co., Ltd., Irvine, CA, USA) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin at 37°C with 5% CO2 and 100% humidity. Lipofectamine 2000 was purchased from Invitrogen Corporation, USA. 2.2. Cell Migration Assay The Transwell chamber was put into a 24-well culture plate. Single-cell suspension (200 μL) containing HepG2 cells were added into the chamber to incubate for 24 h. The membrane was washed for three times, immersed in methanol, and left for 15 min at room temperature to fix the cells. The membrane was then immersed in Giemsa for 20–30 min at room temperature and washed with water. The cells that had migrated through the pores to the lower surface of the membrane were counted under microscope. The total number of cells in five vision fields, including the center and the four corner squares, was recorded for each sample. 2.3. Transwell Invasiveness Assay Matrigel including laminin, heparin sulfate proteoglycan, TGF-β, entactin, and fibroblast growth factor was diluted with FBS-free DMEM with a ratio of 1 : 50. A volume of 50 μL of the diluted Matrigel was added to the Transwell bottom chamber. A volume of 50 μL of DMEM was also added to the chamber. After 30 min, HepG2 cells at exponential growth stage were treated with 0.25% trypsase and added with DMEM to prepare 1 × 106/mL single-cell suspension. A Transwell chamber was put into a 24-well plate, and 600 μL of DMEM containing 10% FBS was added. A volume of 200 μL of the prepared single-cell suspension was also added into the Transwell chamber. The cells were cultured at 37°C with 5% CO2 for 24 h. All of the liquid was removed from the Transwell chamber and the bottom chamber. The cells on the upper surface of the Transwell membrane were also removed. The membrane was washed with PBS for three times, immersed in methanol and kept for 15 min at room temperature. The membrane was then immersed in Giemsa for 30 min at room temperature and washed with water. The cells that have migrated through the pores to the lower surface of the membrane were counted under microscope. 2.4. siRNA Interference The human HepG2 cells were transfected with medium, the empty cDNA3.1 vector, or 80 pmol of siRNA against the human ADAM10 message (5′-AGACAUUAUGAAGGAUUAUTT-3′) or a negative control siRNA (5′AGGUAGUGUAAUCGCCUUGTT3′) using X-tremeGENE (Roche, USA). Two days agter-delivery of the siRNA, the cells were transfected with 20 pmol ADAM10 siRNA (or control siRNA) using Lipofectamine 2000 (Invitrogen, USA). After 2 days, total protein and total RNAs were harvested and subjected to immunoblot or real-time RT-PCR analyses. The control siRNA and the ADAM10 siRNA were provided by Shanghai Allcare Biomedical Development Co., Ltd. (Shanghai, China). 2.5. Real-Time Reverse Transcription-PCR (RT-PCR) Total RNAs were harvested from cells using the Trizol RNA extraction kit (Costar, USA). The RT-PCR reaction kit was purchased from Takara Biological Engineering Company (Japan). RNA (1 μL) was reverse-transcribed into cDNA using random primers in a Reverse Transcription system according to the manufacturer's instructions. Expression of E-cadherin mRNAs was quantified by quantitative PCR using an ABI Prism Sequence Detection System (Applied Biosystems, USA). An assay reagent containing premixed primers and a VIC-labeled probe (Applied Biosystems; cat# 4310884E) was used to detect expression of endogenous β-actin mRNA. Template-negative and RT-negative conditions were used as controls. Amplification of E-cadherin cDNAs and the endogenous β-actin cDNA were monitored by levels in FAM and VIC fluorescent intensities, respectively, using the ABI 7900 software. The relative amounts of E-cadherin transcript were normalized to the amount of β-actin mRNA in the same sample. The levels (mean value) of E-cadherin transcripts in cells were calculated (mean ± SD). E-cadherin primers (forward primer, 5′ GCTCATCAATAGGCGGTA; backward primer, 5′ GTTTATGGCCGATCTTAT) were synthesized by Shanghai Allcare Biomedical Development Co., Ltd. The RT-PCR experiments were repeated at least 3 times. 2.6. Western Blot Analysis Total proteins were harvested from HepG2 cells, separated on 10% SDS/PAGE gels, and then subjected to immunoblot analyses. The primary antibodies against E-cadherin and β-actin were purchased from Santa Cruz, CA, USA (anti-E-cadherin, cat# sc-21791, 1 : 200; anti-ADAM10, cat# sc-28358, 1 : 200; anti-β-actin, cat# sc-130301, 1 : 10,000). Secondary antibody used in this study was goat anti-mouse IgG-HRP (cat# sc-2005, 1 : 10,000, Santa Cruz, CA, USA). Bound antibodies were detected using the ECL system (Pierce Biotechnology). The experiments were performed for at least 3 times. 2.7. Statistical Analyses The results were analyzed with the statistical analysis software SPSS 13.0. The values were given as mean ± SD. One-way ANOVA was used for multigroup comparison with Student's t-test. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Downregulation of ADAM10 Expression Inhibits Migration of HepG2 Cells To determine if downregulation of ADAM10 protein affects migration of human hepatocellular carcinoma HepG2 cells, the cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10. As shown in Figure 1, the results of in vitro migration assay showed that there was no significant difference in the number of cells moving through the bottom pores among the blank group (transfected with medium only), the empty vector group, and the negative control siRNA group. However, the number of cells moving through the bottom pores in the ADAM10 siRNA group was reduced by 70.2%, 68.2%, and 71.3%, when compared with the blank group, the empty vector group, and the negative control group, respectively. The differences were statistically significant (P < 0.05). These results suggest that down-regulation of ADAM10 expression inhibits migration of HepG2 cells. 3.2. Down-Regulation of ADAM10 Expression Inhibits Invasiveness of HepG2 Cells To determine if down-regulation of ADAM10 protein affects invasiveness of human hepatocellular carcinoma HepG2 cells, the cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10. As shown in Figure 2, the result of the cell invasiveness assay using Transwell basement membrane kit showed that there was no significant difference in the number of cells that had passed through the simulated basement membrane among the blank group, the empty vector group, and the negative control siRNA group. However, the number of cells that had passed through the simulated basement membrane in the ADAM10 siRNA group was reduced by 74.4%, 72.9%, and 69.3%, when compared with the blank group, the empty vector group, and the negative control group, respectively. The differences were statistically significant (P < 0.05). These results suggest that down-regulation of ADAM10 expression inhibits invasiveness of HepG2 cells. 3.3. Down-Regulation of ADAM10 Expression Results in Increased Level of E-Cadherin Protein To determine if the knock-down of ADAM10 by siRNA affects expression of E-cadherin, the cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10. The total proteins were extracted at 72 h after transfection, and Western blot analysis was performed. As shown in Figure 3, the levels of ADAM10 were decreased by about 30% upon transfection of siRNA against ADAM10 when compared with the medium control condition. Transfection of empty vector or control siRNA did not alter the ADAM10 levels significantly. There were no significant differences in E-cadherin protein expression level at 72 h after transfection among the cells transfected with medium, the empty vector, or the control siRNA. However, E-cadherin protein expression level in the cells transfected with the ADAM10 siRNA was significantly increased when compared with the levels in the cells transfected with medium, the empty vector, or the control siRNA. These results suggest that the down-regulation of ADAM10 expression increases E-cadherin protein levels. 3.4. Knockdown of ADAM10 by siRNA Does Not Affect mRNA Level of E-Cadherin To determine if the knock-down of ADAM10 by siRNA affects expression of E-cadherin, the cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10. The total RNAs were extracted at 24, 48, 72, and 96 h after transfection and real-time RT-PCR was performed. In RT-PCR detection, relative quantification method was used for the determination of E-cadherin mRNA changes with β-actin serving as an internal reference. As given in Table 1, there were no significant differences of E-cadherin mRNA expression levels among cells transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10 at 24, 48, 72, and 96 h after transfection (P > 0.05). These results suggest that knock-down of ADAM10 by siRNA does not affect mRNA level of E-cadherin. 4. Discussion The present study has investigated changes of the invasiveness and migration capability of HepG2 cells and the changes in the expression levels of the cell adhesion molecule of E-cadherin when ADAM10 expression was down-regulated. Cell migration assays were performed to investigate the effect of the inhibited ADAM10 expression on the mobility of HepG2 cells. The results suggested that migration of HepG2 cells was decreased upon knock-down of ADAM10. The reduced expression level of ADAM10 may decrease the interaction between its disintegrin region and integrins, thus decreasing the migration of tumor cells. Decreased expression level of ADAM10 may decrease the degradation of extracellular matrix, and thus restoring the three-dimensional structure of the matrix. It is therefore indicated that ADAM10 may play a role in the migration of HepG2 cells. This study also indicated possible correlation between the migration of HepG2 cells and the expression level of E-cadherin. The protein expression level of E-cadherin in cells transfected with siRNA against ADAM10 was significantly higher (P < 0.05) than that in the other 3 groups (cells transfected with medium, the empty vector, or the control siRNA). The result suggested a possible correlation between the decrease of ADAM10 protein expression and the increase of E-cadherin protein expression, which may be possibly explained by the decreased degradation of the cell adhesion molecule E-cadherin by the metalloproteinase region of ADAM10. However, the expression of E-cadherin was unchanged at the level of mRNAs, which indicated that the regulation of E-cadherin by ADAM10 should be performed at the posttranscriptional level. Schirrmeister et al. [19] discovered that the dedifferentiation of gastric cancer cells and the decrease of intercellular adhesion were significantly correlated to hydrolysis of E-cadherin, where ADAM10 played an important role. Dittmer et al. [20] also found that down-regulation of ADAM10 expression could decrease the migration capability of and adhesion between breast cancer cells, which was accompanied with decrease of E-cadherin level. The increased ADAM10 expression was also demonstrated to regulation the release of other intercellular adhesion molecules and thus to influence the behavior of the cells, and was significantly correlated to changes of tumor cell mobility in multiple tumor types including cervical carcinoma, lymphoma, lung cancer and melanomata [21, 22]. However, the association of ADSAM10 expression with the liver cells was not clear. In this study, we found that inhibition of ADAM10 expression decreases the migration capability of HepG2 cells and is correlated to the upregulation of E-cadherin expression. Transwell simulated basement membrane assays were performed to investigate the effect of the inhibited ADAM10 expression on the invasiveness of HepG2 cells. The result showed that down-regulation of ADAM10 expression inhibits invasiveness of HepG2 cells. The change in invasiveness of tumor cells is subject to the effects of a variety of factors in human body. In this study, the decrease of cell invasiveness in the ADAM10 siRNA group may be related to less dissolution of the basement membrane and less destructuring of extracellular matrix caused by decreased level of ADAM10 expression. Meanwhile, the increased expression level of E-cadherin protein may also play a role in the alteration of invasiveness of HepG2 cells. It is reported [23] that E-cadherin expression levels in low-invasive tumor cells are significantly higher than in the high-invasive tumor cells. Conflict of Interests All authors declare no financial competing interests. All authors declare no nonfinancial competing interests. All authors declare no possible conflict of interests with any trademark mentioned in this paper. Authors' Contribution Yuan Yue and Yuan Shao contributed equally to this work. Acknowledgment This work was supported by Research Projects of Shaanxi Province (no. 2012SF2-09-04). Figure 1 Downregulation of ADAM10 expression affects migration of HepG2 cells. The HepG2 cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10. The in vitro migration assay was performed. (a) The effect of ADAM10 knockdown on the migration capability of HepG2 cells. (b) Diagram of the effects of ADAM10 knock-down on the migration capability of HepG2 cells. The membrane was then immersed in Giemsa for 20–30 min at room temperature and washed with water. The cells that had migrated through the pores to the lower surface of the membrane were counted under microscope. The total number of cells in five vision fields including the center and the four corner squares was recorded for each sample. Migration ratios of HepG2 cells were calculated relative to the medium control. P < 0.05 when compared with the medium control. Figure 2 Down-regulation of ADAM10 expression inhibits invasiveness of HepG2 cells. The HepG2 cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10. The Transwell invasiveness assay was performed. (a) The effect of ADAM10 knock-down on the invasiveness of HepG2 cells. (b) Diagram of the effect of ADAM10 knock-down on the invasiveness of HepG2 cells. A volume of 50 μL of the diluted Matrigel was added to the Transwell bottom chamber. A volume of 50 μL of DMEM was also added to the chamber. The cells that have migrated through the pores to the lower surface of the membrane were counted under microscope. Invasion ratios of HepG2 cells were calculated relative to the medium control. P < 0.05 when compared with the medium control. Figure 3 Down-regulation of ADAM10 expression results in increased level of E-cadherin protein. HepG2 cells were transfected with medium only, the empty vector, the control siRNA, or siRNA against ADAM10. The total proteins were prepared and immunoblot analysis was performed to analyze the expression of E-cadherin, ADAM10, and β-actin. The cellular β-actin served as a loading control. (a) Representative blots were shown. (b) The relative E-cadherin protein levels were given. The Experiments were repeated for more than 3 times. *P < 0.05 when compared with the level of E-cadherin in the medium control. # P < 0.05 when compared with the level of ADAM10 in the medium control. Table 1 E-cadherin mRNA levels (mean ± SD) in different groups of HepG2 cells transfected with ADAM10 siRNA or the control siRNA. Cells harvested at different time points No transfection Empty vector Negative control siRNA ADAM10 siRNA HepG2 (24 h) 1.0 ± 0.201 0.998 ± 0.205 0.973 ± 0.255 0.938 ± 0.281 HepG2 (48 h) 1.0 ± 0.304 0.976 ± 0.234 0.948 ± 0.321 0.929 ± 0.264 HepG2 (72 h) 1.0 ± 0.219 0.969 ± 0.294 0.957 ± 0.285 1.012 ± 0.279 HepG2 (96 h) 1.0 ± 0.229 0.958 ± 0.302 0.968 ± 0.315 0.976 ± 0.322 ==== Refs 1 Zheng X Gai X Wu Z Liu Q Yao Y Metastasin leads to poor prognosis of hepatocellular carcinoma through partly inducing EMT Oncology Reports 2013 29 5 1811 1818 23483190 2 Wu D Ding J Wang L microRNA-125b inhibits cell migration and invasion by targeting matrix metallopeptidase 13 in bladder cancer Oncology Letters 2013 5 3 829 834 23425975 3 Wang ZG Jia MK Cao H Bian P Fang XD Knockdown of Coronin-1C disrupts Rac1 activation and impairs tumorigenic potential in hepatocellular carcinoma cells Oncology Reports 2013 29 3 1066 1072 23292607 4 Hoenicke L Zender L Immune surveillance of senescent cells—biological significance in cancer- and non-cancer pathologies Carcinogenesis 2012 33 6 1123 1126 22470164 5 Canning C O’Brien M Hegarty J O’Farrelly C Liver immunity and tumour surveillance Immunology Letters 2006 107 2 83 88 2-s2.0-33751180964 17101177 6 Smith HA Kang Y The metastasis-promoting roles of tumor-associated immune cells Journal of Molecular Medicine 2013 91 4 411 429 23515621 7 Gil M Seshadri M Komorowski MP Abrams SI Kozbor D Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases Proceedings of the National Academy of Sciences of the United States of America 2013 110 14 E1291 E1300 23509246 8 Zhao G Huang ZM Kong YL Cortactin is a sensitive biomarker relative to the poor prognosis of human hepatocellular carcinoma World Journal of Surgical Oncology 2013 11 1 p. 74 23518204 9 Schneider M Hadaschik B Hallscheidt P Manual repositioning of intra-atrial kidney cancer tumor thrombus: a technique reducing the need for cardiopulmonary bypass Urology 2013 81 4 909 914 23414691 10 Man YG Stojadinovic A Mason J Tumor-infiltrating immune cells promoting tumor invasion and metastasis: existing theories Journal of Cancer 2013 4 1 84 95 23386907 11 Ramasubba C Cohen SP Cooled sacroiliac radiofrequency denervation for the treatment of pain secondary to tumor infiltration: a case-based focused literature review Pain Physician 2013 16 1 1 8 23340529 12 Umansky V Sevko A Tumor microenvironment and myeloid-derived Suppressor cells Cancer Microenviron 2012 13 Canel M Serrels A Frame MC Brunton VG E-cadherin-integrin crosstalk in cancer invasion and metastasis Journal of Cell Science 2013 126 2 393 401 23525005 14 Griffin JR Wriston CC Peters MS Lehman JS Decreased expression of intercellular adhesion molecules in acantholytic squamous cell carcinoma compared with invasive well-differentiated squamous cell carcinoma of the skin American Journal of Clinical Pathology 2013 139 4 442 447 23525614 15 Boterberg T Vennekens KM Thienpont M Mareel MM Bracke ME Internalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16 Cell Adhesion and Communication 2000 7 4 299 310 2-s2.0-0033980552 10714391 16 Luca AC Mersch S Deenen R Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines PLoS One 2013 8 3 e59689 17 Przemyslaw L Boguslaw HA Elzbieta S Malgorzata SM ADAM and ADAMTS family proteins and their role in the colorectal cancer etiopathogenesis BMB Reports 2013 46 3 139 150 23527857 18 Cochet M Donneger R Cassier E 5-HT4 receptors constitutively promote the non-amyloidogenic pathway of APP cleavage and interact with ADAM10 ACS Chemical Neuroscience 2013 4 1 130 140 23336052 19 Schirrmeister W Gnad T Wex T Ectodomain shedding of E-cadherin and c-Met is induced by Helicobacter pylori infection Experimental Cell Research 2009 315 20 3500 3508 2-s2.0-70449532978 19665015 20 Dittmer A Hohlfeld K Lützkendorf J Müller LP Dittmer J Human mesenchymal stem cells induce E-cadherin degradation in breast carcinoma spheroids by activating ADAM10 Cellular and Molecular Life Sciences 2009 66 18 3053 3065 2-s2.0-69949141694 19603142 21 Soundararajan R Sayat R Robertson GS Marignani PA Triptolide: an inhibitor of a disintegrin and metalloproteinase 10 (ADAM10) in cancer cells Cancer Biology and Therapy 2009 8 21 2054 2062 2-s2.0-73549100626 19783906 22 Erin N Zhao W Bylander J Chase G Clawson G Capsaicin-induced inactivation of sensory neurons promotes a more aggressive gene expression phenotype in breast cancer cells Breast Cancer Research and Treatment 2006 99 3 351 364 2-s2.0-33748337099 16583263 23 Cheng GZ Chan J Wang Q Zhang W Sun CD Wang L-H Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel Cancer Research 2007 67 5 1979 1987 2-s2.0-33947192053 17332325	23936798	PMC3727112	CC BY	2021-01-05 10:05:37	yes	Biomed Res Int. 2013 Jul 14; 2013:434561
==== Front Diagn PatholDiagn PatholDiagnostic Pathology1746-1596BioMed Central 1746-1596-8-962377343610.1186/1746-1596-8-96ResearchCollecting duct carcinoma of the kidney: a clinicopathological study of five cases Wang Xiangyang 1wangxydr@163.comHao Jianwei 1haojianwei45@hotmail.comZhou Ruijin 1zhouruijinzz@163.comZhang Xiangsheng 1xiangshengz@yeah.netYan Tianzhong 1tianzhongy@163.comDing Degang 1dingdgang@163.comShan Lei 1shanleil@126.comLiu Zhonghua 1liuzhh45@163.com1 Department of Urology, Henan Provincial People’s Hospital, Zhengzhou 450003, China2013 17 6 2013 8 96 96 8 3 2013 6 5 2013 Copyright © 2013 Wang et al.; licensee BioMed Central Ltd.2013Wang et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Objective To investigate the clinicopathological features of collecting duct carcinoma (CDC) and improve its diagnosis and treatment. Methods A retrospective analysis was performed with clinical data including follow-up results of five patients with CDC. Results A total of 5 cases, including 4 males and 1 female, were included in this analysis with the average age 54 years (range 42 to 65). Patients mainly suffered from lumbar pain, hematuria, abdominal mass and low grade fever. Four patients underwent radical nephrectomy while another received palliative nephrectomy. Lymph node metastasis occurred in 3 cases and renal hilum fat metastasis happened to 2 other cases. Tumors was located in the renal medulla and presented invasive growth. They had a tubulopapillary architecture with the hobnail-shaped cells protruding into the glandular lumen, and were accompanied by interstitial fibrosis and dysplasia of epithelial cells in collecting ducts adjacent to the tumors. One tumor was staged at AJCC II, two at AJCC III and two at AJCC IV. Postoperative interferon immunotherapy was applied in 2 cases. Patients were followed up for 5 to 18 months and the average survival time was 10 months. Conclusion The CDC exhibits special clinicopathological features, high degree of malignancy and poor prognosis. The diagnosis depends on the histopathological examination. Early detection and early surgical treatment are still the main methods to improve the prognosis of patients with CDC. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3702794279387989 Renal tumorCollecting duct carcinomaDiagnosisPathology ==== Body Background Collecting duct carcinoma (CDC) is a type of kidney cancer that originates in the duct of Bellini of the kidney and also known by several synonyms like Bellini duct carcinoma, medullary renal carcinoma, distal renal tubular carcinoma and distal nephron carcinoma [1,2]. CDC is an unusual variant of renal cell carcinoma and accounts for about 1% of all renal cell carcinomas. CDC is differentiated from other renal cell carcinomas by its characteristic location, typical histological appearance and poor prognosis [1]. CDC metastasizes to regional lymph nodes in approximately 80% of cases, to the lung or adrenal gland in 25% and to the liver in 20% [3]. Average survival time has been reported to be 22 months [4]. Several treatments have been proposed but with limited efficacy, including radiation therapy, immunotherapy, chemotherapy, as well as combination therapy [4-6]. Although many relevant studies have been reported [7-9], it is necessary to gather more clinicopathological features of CDC to better diagnose and treat it. Therefore, we reported our experience with 5 CDC patients from August 2001 to September 2010 and explored their clinicopathological features and treatments in combination with literature review. Materials and methods Clinical data Five patients (4 males and 1 female) with pathologically diagnosed CDC were included. The average age was 54 years (range 42 to 65). The tumors were found in the right kidneys in 3 cases and in left kidneys in 2 cases. Clinical manifestations included waist and abdomen pain in 4 cases, hematuria in 3 cases, low grade fever in 1 case, and local mass in 1 case. Extracorporeal shock wave lithotripsy was historically applied for 1 patient with ureteral stones. Accessory examinations revealed an increase in the levels of red blood cells and urine protein in 3 cases, elevated erythrocyte sedimentation rate in 3 cases, and negative results of urine cytology in all of 5 cases. Analyses by Color Doppler ultrasound and computerized tomography (CT) demonstrated that tumors were located in the center of kidney and near the pelvis; that invasion of renal hilum was observed in 2 cases, containing surrounding of tumor tissue on the renal artery in 1 case; that the diameter of tumors was between 4.6 and 10.5 cm with average 6.8 cm; and that hydrocalycosis was observed in 3 cases. Type-B ultrasonic examination found hypoechoic masses with ill-defined border in the central region around renal sinus as well as pelvis invasion of the mass in some cases (Figure 1). Magnetic resonance imaging (MRI) indicated isointensity on Tl-weighted images (T1WI) and hypointensity on T2WI in the renal sinus and confirmed caliectasis (Figure 2). Masses near the renal sinus were also detected by CT, which were further determined as slight or moderate, progressive, delayed enhancement by enhanced dynamic scanning (Figure 3). Figure 1 Ultrasound showing left renal tumor. A: Ultrasound revealed hypoechoic masses (arrow) with ill-defined border in the central region around renal sinus. B: Color doppler flow imaging of mass (arrow) indicated decreased blood flow signals. The tumor invaded renal pelvis inward and infiltrated vessel pedicle outward, and caused caliectasis. AO: abdominal aorta; LRA: left renal artery. Figure 2 MRI showing the left renal tumor. The mass in the left renal sinus showed homogeneous isointensity on T1WI (A, arrow) and hypointensity on T2WI (B, arrow). Figure 3 CT showing left renal tumor. Plain CT scan revealed a heterogeneous density in the left renal tumor (A, arrow). The borders between the tumor and abdominal aorta, kidney and renal vein were not clear. Enhanced CT scan suggested a slight enhancement in the arterial phase (B, arrow) and a moderate enhancement in the venous stage (C, arrow). Clinical stages WHO classification of renal carcinoma (2004) was chosen as the pathological diagnostic criteria, and TNM and clinical staging were conducted based upon the clinicopathological information of the cases and the criteria developed by American Joint Committee on Cancer (AJCC) in 2002. Treatment methods Radical nephrectomy was applied to 4 cases. Palliative nephrectomy was applied to another case as the tumor tissues in this patient surrounded the renal artery and vein and integrated with multiple lymph nodes. Primary lesion tissues and Para-aortic lymph nodes were removed as much as possible. Postoperative immunotherapy was administered for 3 cases by subcutaneous administration of 9 million units of interferon-α each time (3 times a week and 12 weeks a cycle). Chemoradiotherapy was not given to any of the five cases. Results Pathological results Visual examination indicated that the tumors were mainly found in the renal medulla, gray or yellow, showing necrosis or hemorrhage; they exhibited invasive growth with ill-defined border and no pseudocapsule (Figure 4A). Microscopical inspection suggested that tumor cells had a tubulopapillary architecture and formed hobnail pattern along the glandular tube. They also showed eosinophilic or basophils properties, ill-defined border, large nuclei, and prominent nucleoli, and were accompanied by interstitial connective tissue reaction. Primary CDC or dysplasia was seen in tissues around the tumors (Figure 4B, C, D, E and F). Renal hilar lymph node metastasis appeared in 3 cases. Immunohistochemical tests showed cytokeratin (CK) (+), peanut agglutinin (PNA) (+) and high-molecular-weight cytokeratin (CK34BE12) (+) in all of 5 cases, Vimentin (+) and epithelial membrane antigen (EMA) (+) in 4 cases, low molecular weight cytokeratins (CAM5.2) (+) and Ki67 (+) in 2 cases, and alpha-fetoprotein (AFP) (−), chromogranin A (CgA) (−), synaptopodin (SYN) (−), Villin (−) and Wilms' tumor gene 1 (WT1) (−) for all cases. TNM staging and clinical staging are shown in Table 1. Figure 4 Pathological examination. A: The gray renal tumor without capsule. B: The tumor cells exhibiting a tubulopapillary architecture. C: Hobnail-shaped cells protruding into the glandular lumen (arrow). D: The tumor cells exhibiting eosinophilic property with ill-defined bound, large and atypical nuclei, and prominent nucleoli. E: The tumor interstitial connective tissue reaction. F: Collecting duct carcinoma in situ or dysplasia (indicated by an arrow). Table 1 Staging and treatment outcomes of five collecting duct carcinoma patients 1 2 3 4 5 Venous tumor thrombus No Yes No No No Lymphatic metastasis No No 1/8 5/6 3/4 Metastasis No No No Fat tissue in renal hilus Fat tissue in renal hilus TNM staging T2N0M0 T3bNOMO T2N1M0 T4N2M0 T4N2MO AJCC staging II III III IV IV Operation Radical nephrectomy Radical nephrectomy Radical nephrectomy Palliative nephrectomy Radical nephrectomy Immunotherapy (IFN -α) No 2 cycles No No 1 cycle Survival time (month) 18 12 9 6 5 Treatment outcomes Multiple metastases to liver, vertebrae, lung and retroperitoneal happened to all 5 cases at 5–18 months after operation, and the average survival time was 10 months (Table 1). Discussions CDC is located in the renal medulla and originates from the epithelial cells of Bellini collecting ducts [1]. Because of its unique biological and pathologic characteristics that are different from the other renal cell carcinomas, it’s considered to be an independent histological type. Currently, WHO names it as Bellini duct carcinoma [10]. CDC can occur at any age, and is more common in young adults. Men are more susceptible than women with the ratio of about 2:1. Tokuda et al. [11] report that the average onset age is 58 years and male patients account for 71.6% of the cases. In present study, the average age was 54 years. Common clinical symptoms of CDC include painless gross hematuria, lumbar abdominal pain, waist and abdominal mass, fatigue, fever, and weight loss. It has a short and fast course. Normally, metastasis occurs in most of patients before treatment, including bone metastasis and lymph node metastasis [11]. Three cases of lymph node metastasis and two cases of renal hilum fat metastasis were reported in the present study. Researchers have reported that CDC shows similar biological properties to those of urothelial cell carcinoma. Thus it is considered that they both originate from renal tubular and can occur simultaneously [12]. Imaging examinations are the main methods for CDC diagnosis. The tumors are hypo-vascular with ill-defined border, and pose invasions to the renal cortex and renal sinus [13,14]. Hydrocalycosis often occurs because of the extruding from the tumors. Color ultrasound can reflect a hypoechoic, homogeneous or heterogeneous mass with irregular morphology and ill-defined bounder, as well as reduced blood flow signal. CT is able to detect the invasions of tumors into pelvis and renal cortex. Calcification and hemorrhage can also be seen in some cases. Mild to moderate uneven delayed enhancement can be detected in dynamic contrast-enhanced scan [15]. MRI gives iso-intensity or hyper-intensity on T1WI and hypo-intensity on T2WI. The 5 cases in this study showed similar symptoms. CDC doesn’t have specific imaging features that distinguish it from other types of renal cell carcinoma such as renal medullary carcinoma, sarcomatoid renal cell carcinoma, and renal pelvis carcinoma, so its diagnosis requires pathological examination. The pathological examination is the gold standard for diagnosis of CDC. As the tumors grow, they usually infiltrate into renal pelvis, renal cortex, and even renal hilum. CDC usually presents a tubulopapillary architecture, and tumor cells form hobnail pattern along the glandular tube. Poorly differentiated tumor cells show nest-shaped, rope-like, sarcomatoid or adenoid cystic morphology, with or without interstitial connective tissue reaction [15-17]. Kidney cancer can be classified as multiple types based on the origin of tumor cell types, including renal cell carcinoma (RCC, the most common type), tubulocystic renal carcinoma [18], renal solitary fibrous tumor [19], renal pigmented paraganglioma [20], and renal endocrine tumors [21]. In addition to histological analyses, genetical and biochemical approaches are becoming more and more important for the differentiation and diagnosis of renal carcinomas. Genetical analyses include gene copy numbers, chromosomal imbalances, gene mutations and single nucleotide polymorphism (SNP) analysis [18]. Many biomolecules, including epithelial-mesenchymal transition (EMT) markers such as N-cadherin [22] and vimentin [19] and human leucocyte molecules such as HLA-G and HLA-E [23], are reported to be biomarkers for renal cancer. Immunohistochemical examination of these biomarkers is important for the determination of the origin and the diagnosis of CDC. Cancer cell have positive expressions of CK (AE1/AE3), CK7, CK19, EMA, vimentin, CK34BE12, PNA and ulex europeus agglutinin (UEA), and negative expression of CD10 and CK20. Combination of CK34BE12 and PNA is able to detect 90% of CDC [24]. The results from pathological and immunohistochemical examinations are the important basis for the diagnosis of CDC and for differentiating it from other types of kidney cancer. Radical nephrectomy is the major method to treat CDC. As the tumor cells spread in cortical collecting tubule, which results in poor prognosis, tumor enucleation and partial nephrectomy are not favorable. However, radiotherapy, chemotherapy and immunotherapy have limited efficacy on CDC [12]. The postoperative survival time for the 2 cases of stage IV was 5 to 6 months, 18 months for the case of stage II, and 9 to 12 months for the 2 patients of stage III. Therefore, early detection and early surgery are the best way to prolong the renal collecting duct carcinoma survival time. Recently, there are a few reports on the effectiveness of targeted therapy with Sunitinib and sorafenib in treatment of CDC [25-27]. However, there’s a study indicating no response of targeted therapy [17]. Therefore, the efficacy of targeted therapy on CDC remains to be demonstrated. Overall, our reports are beneficial supplements for better understanding the clinicopathological features of CDC. At the same time, the treatments and corresponding outcomes are valuable information for guiding future clinical practice. Consent Written informed consent was obtained from the patient for publication of this report and any accompanying images. Competing interests The authors declare that they have no competing interests. Authors’ contribution XYW and JWH participated in the design, analyses and data interpretation and drafted the manuscript. RJZ, XSZ, TZY, DGD, LS, and ZHL helped to retrieve pathologic and clinical information and provide valuable insight during manuscript preparation. All authors reviewed and approved the final manuscript. ==== Refs Singh I Nabi G Bellini duct carcinoma: review of diagnosis and management Int Urol Nephrol 2002 1 91 95 12549647 Verdorfer I Culig Z Hobisch A Bartsch G Hittmair A Duba HC Erdel M Characterisation of a collecting duct carcinoma by cytogenetic analysis and comparative genomic hybridisation Int J Oncol 1998 13 3 461 464 9683779 Davis CJ JrMostofi FK Sesterhenn IA Renal medullary carcinoma. The seventh sickle cell nephropathy Am J Surg Pathol 1995 19 1 11 10.1097/00000478-199501000-00001 7528470 Dimopoulos MA Logothetis CJ Markowitz A Sella A Amato R Ro J Collecting duct carcinoma of the kidney Br J Urol 1993 71 388 391 10.1111/j.1464-410X.1993.tb15978.x 8499979 Coogan CL McKiel CF JrFlanagan MJ Bormes TP Matkov TG Renal medullary carcinoma in patients with sickle cell trait Urology 1998 51 1049 1050 10.1016/S0090-4295(98)00104-6 9609653 Peyromaure M Thiounn N Scotte F Vieillefond A Debre B Oudard S Collecting duct carcinoma of the kidney: a clinicopathological study of 9 cases J Urol 2003 170 1138 1140 10.1097/01.ju.0000086616.40603.ad 14501710 Del Sordo R Giansanti M Bellezza G Sidoni A Thyroid transcription factor 1 expression in a case of renal collecting duct carcinoma Hum Pathol 2012 43 1153 1154 author reply 1154 10.1016/j.humpath.2012.03.004 22703591 Li B Ding Q A case report of collecting duct carcinoma of the kidney coexistent with giant adrenal myelolipoma Can Urol Assoc J 2012 6 E97 E100 22511446 Yang GE Seo JW Park JH Distal ureteral seeding metastasis of collecting duct carcinoma manifesting as deep vein thrombosis Clin Radiol 2012 67 936 939 10.1016/j.crad.2012.02.012 22513238 Lopez-Beltran A Scarpelli M Montironi R Kirkali Z WHO classification of the renal tumors of the adults Eur Urol 2004 2006 49 798 805 Tokuda N Naito S Matsuzaki O Nagashima Y Ozono S Igarashi T Collecting duct (bellini duct) renal cell carcinoma: a nationwide survey in japan J Urol 2006 176 40 43 discussion 43 10.1016/S0022-5347(06)00502-7 16753362 Orsola A Trias I Raventos CX Espanol I Cecchini L Orsola I Renal collecting (bellini) duct carcinoma displays similar characteristics to upper tract urothelial cell carcinoma Urology 2005 65 49 54 10.1016/j.urology.2004.08.012 15667862 Yoon SK Nam KJ Rha SH Kim JK Cho KS Kim B Kim KH Kim KA Collecting duct carcinoma of the kidney: CT and pathologic correlation Eur J Radiol 2006 57 453 460 10.1016/j.ejrad.2005.09.009 16266796 Bansal P Kumar S Mittal N Kundu AK Collecting duct carcinoma: a rare renal tumor Saudi J Kidney Dis Transpl 2012 23 810 812 10.4103/1319-2442.98166 22805397 Kim JK Kim TK Ahn HJ Kim CS Kim KR Cho KS Differentiation of subtypes of renal cell carcinoma on helical CT scans Am J Roentgenol 2002 178 1499 1506 10.2214/ajr.178.6.1781499 12034628 Chao D Zisman A Pantuck AJ Gitlitz BJ Freedland SJ Said JW Figlin RA Belldegrun AS Collecting duct renal cell carcinoma: clinical study of a rare tumor J Urol 2002 167 71 74 10.1016/S0022-5347(05)65385-2 11743278 Husillos A Herranz-Amo F Subira D Lledo E Molina-Escudero R Hernandez-Fernandez C Collecting duct renal cell carcinoma Actas Urol Esp 2011 35 368 371 10.1016/j.acuroe.2011.01.003 21450372 Gabriela Q-G Sergio P-O Karime C-O Richard G Schwartz MR Ayala AG Monzon FA Synchronous clear cell renal cell carcinoma and tubulocystic carcinoma: genetic evidence of independent ontogenesis and implications of chromosomal imbalances in tumor progression Diagn Pathol 2012 7 21 10.1186/1746-1596-7-21 22369180 Tsan-Yu H Yi-Che CC Wen-Hsiang C Siu-Chung C Liang-Che C Cheng-Cheng H Hui-Ping C Jim-Ray C De novo malignant solitary fibrous tumor of the kidney Diagn Pathol 2011 6 96 10.1186/1746-1596-6-96 21970525 Ling Z Jie L Honglei Z Jiping D Pigmented paraganglioma of the kidney: a case report Diagn Pathol 2012 7 77 10.1186/1746-1596-7-77 22741527 Qingfu Z Jian M Siyang Z Xueshan Q Primary micro neuroendocrine tumor arising in a horseshoe kidney with cyst: report of a case and review of literature Diagn Pathol 2012 7 126 10.1186/1746-1596-7-126 22999194 Carl Ludwig B Bernhard H Arne S Heinz-Joachim R Felix B N-cadherin is differentially expressed in histological subtypes of papillary renal cell carcinoma Diagn Pathol 2012 7 95 10.1186/1746-1596-7-95 22888908 Leos K Ivo V Jan D Ivo C Dalibor P Alexandr P Radek L Martina R Pavel F Zdenka K Ondrej S HLA-G and HLA-E specific mRNAs connote opposite prognostic significance in renal cell carcinoma Diagn Pathol 2012 7 58 10.1186/1746-1596-7-58 22640987 Li M Vuolo MA Weidenheim KM Minsky LS Collecting-duct carcinoma of the kidney with prominent signet ring cell features Mod Pathol 2001 14 623 628 10.1038/modpathol.3880361 11406666 Miyake H Haraguchi T Takenaka A Fujisawa M Metastatic collecting duct carcinoma of the kidney responded to sunitinib Int J Clin Oncol 2011 16 153 155 10.1007/s10147-010-0116-z 20686910 el Tazi M Essadi I Tazi MF Ahellal Y M’Rabti H Errihani H Retraction: metastatic collecting duct carcinoma of the kidney treated with sunitinib World J Surg Oncol 2011 9 136 10.1186/1477-7819-9-136 22024397 el Tazi M Essadi I Tazi MF Ahellal Y M’Rabti H Errihani H Metastatic collecting duct carcinoma of the kidney treated with sunitinib World J Surg Oncol 2011 9 73 10.1186/1477-7819-9-73 21752265	23773436	PMC3728027	CC BY	2021-01-05 01:30:49	yes	Diagn Pathol. 2013 Jun 17; 8:96
==== Front J Ovarian ResJ Ovarian ResJournal of Ovarian Research1757-2215BioMed Central 1757-2215-6-522387033210.1186/1757-2215-6-52ResearchFollicle stimulating hormone modulates ovarian stem cells through alternately spliced receptor variant FSH-R3 Patel Hiren 1hiren_p85@rediffmail.comBhartiya Deepa 1bhartiyad@nirrh.res.inParte Seema 1seema.parte@gmail.comGunjal Pranesh 1praneshgunjal@gmail.comYedurkar Snehal 1snehaly8@gmail.comBhatt Mithun 1mithun_bhatt2003@yahoo.co.in1 Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai 400012, INDIA2013 20 7 2013 6 52 52 5 6 2013 12 7 2013 Copyright © 2013 Patel et al.; licensee BioMed Central Ltd.2013Patel et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background We have earlier reported that follicle stimulating hormone (FSH) modulates ovarian stem cells which include pluripotent, very small embryonic-like stem cells (VSELs) and their immediate descendants ‘progenitors’ termed ovarian germ stem cells (OGSCs), lodged in adult mammalian ovarian surface epithelium (OSE). FSH may exert pleiotropic actions through its alternatively spliced receptor isoforms. Four isoforms of FSH receptors (FSHR) are reported in literature of which FSH-R1 and FSH-R3 have biological activity. Present study was undertaken to identify FSHR isoforms mediating FSH action on ovarian stem cells, using sheep OSE cells culture as the study model. Methods Cultures of sheep OSE cells (a mix of epithelial cells, VSELs, OGSCs and few contaminating red blood cells) were established with and without FSH 5IU/ml treatment. Effect of FSH treatment on self-renewal of VSELs and their differentiation into OGSCs was studied after 15 hrs by qRT-PCR using markers specific for VSELs (Oct-4A, Sox-2) and OGSCs (Oct-4). FSH receptors and its specific transcripts (R1 and R3) were studied after 3 and 15 hrs of FSH treatment by immunolocalization, in situ hybridization and qRT-PCR. FSHR and OCT-4 were also immuno-localized on sheep ovarian sections, in vitro matured follicles and early embryos. Results FSH treatment resulted in increased stem cells self-renewal and clonal expansion evident by the appearance of stem cell clusters. FSH receptors were expressed on ovarian stem cells whereas the epithelial cells were distinctly negative. An increase in R3 mRNA transcripts was noted after 3 hrs of FSH treatment and was reduced to basal levels by 15 hrs, whereas R1 transcript expression remained unaffected. Both FSHR and OCT-4 were immuno-localized in nuclei of stem cells, showed nuclear or ooplasmic localization in oocytes of primordial follicles and in cytoplasm of granulosa cells in growing follicles. Conclusions FSH modulates ovarian stem cells via FSH-R3 to undergo potential self-renewal, clonal expansion as ‘cysts’ and differentiation into oocytes. OCT-4 and FSHR proteins (required initially to maintain pluripotent state of VSELs and for FSH action respectively) gradually shift from nuclei to cytoplasm of developing oocytes and are later possibly removed by surrounding granulosa cells as the oocyte prepares itself for fertilization. FSHFSHROvaryStem cellsOCT-4VSELs ==== Body Introduction Follicle stimulating hormone (FSH) is a pleiotropic hormone produced by the pituitary that exerts diverse actions on the gonads like growth, proliferation, differentiation, facilitates steroidogenesis and also acts as an anti-apoptotic survival factor in vitro, besides being associated with post-menopausal bone loss [1] and is also implicated in various kind of tumors [2-4]. As per current understanding, FSH receptors are localized on the granulosa cells in the ovary and Sertoli cells in the testis, which constitute the somatic niche and provide physical and biochemical support (source of growth factors and cytokines) to germ cells during their differentiation and development recently reviewed [5]. However, several published reports using ovarian tissue or immortalized ovarian epithelial cell lines suggest that besides granulosa cells, FSH receptors are also localized on the normal ovary surface epithelium (OSE) [6-8], ovarian tumors [9-15], oocytes and cleavage stage mouse embryos [16,17]. Evidence is also available that blocking FSH action results in azoospermia in non-human primates [18] and a significant loss of primordial follicles in hamster ovaries [19]. Thus it remains rather ambiguous at present whether FSH regulates germ cells function indirectly through the granulosa or Sertoli cells or does FSH exert direct action on both the somatic and germ cell compartment in the gonads. Results from our group suggest that FSH may be exerting direct action on ovarian stem cells in the ovary, besides the well-studied action on the granulosa cells. We have reported that besides the granulosa cells of antral follicles, FSH receptors are also expressed in adult mouse OSE, which houses the pluripotent very small ES-like stem cells (VSELs) and ovarian germ stem cells (OGSCs). Treatment with pregnant mare serum gonadotropin (PMSG) activates VSELs and OGSCs in the OSE, and results in augmented neo-oogenesis and primordial follicle assembly [20]. Stimulatory effect of FSH on the VSELs residing in the OSE was also demonstrated by us in cortical tissue culture of human and marmoset ovarian cortical tissue. The OSE underwent extensive proliferation in response to FSH treatment and both pluripotent stem cells and germ cells were increased in number along with certain degree of transition of primordial follicles [21]. Pioneering work done by Sairam’s group has shown that FSH may exert multiple effects on the gonads through alternatively spliced FSH receptors (FSHR) and four different alternatively spliced isoforms of FSHR are reported [22,23]. The canonical FSH-R1 is a 75 kDa member of the G-protein coupled receptor superfamily, expressed on the granulosa cells of growing follicles responsible for steroidogenesis via the cAMP signal transduction pathway. Whereas FSH-R3 is a 39 kDa protein expressed by both surface epithelial and granulosa cells and has topology of a growth factor receptor and promotes DNA synthesis leading to proliferation via mitogen-activated protein kinase (MAPK) pathway, specifically the extracellular-regulated kinase (ERK) signaling cascade and voltage-dependent calcium channels [14,24,25]. R1 and R3 transcripts differ from each other in the exons 9 to 11; R1 has exons 9 and 10 and lacks exon 11 whereas R3 lacks exons 9 & 10 and has a putative exon 11 [25,26]. Published literature suggests that R3 transcript is probably the more pre-dominant transcript in ovaries. Quantitative RT-PCR studies on sheep granulosa cells collected from follicles in different development stages show that R3 transcript is highly regulated and is 20–50 fold more abundant than R1 in small to medium sized follicles, whereas in pre-ovulatory follicles R3 is 5-fold more expressed than R1 [27]. Interestingly R3 is also more regulated during follicular development after PMSG treatment compared to R1 in mice [26,28]. Li et al. [14] have shown that R3 signaling promotes proliferation of ovarian cancer cells. Differential roles played by the two transcripts R1 and R3 in various biological processes, can be easily dissected by a careful designing of primer and probe sequences [14,26]. It becomes vital, at this juncture to delineate the differential regulation of R1 and R3 in response to PMSG/FSH treatment that results in augmented stem cell activity and primordial follicle assembly in adult mammalian (mouse, monkey and human) ovary reported recently by our group [20,21]. Thus the present study was undertaken to examine the effect of FSH treatment on R1 and R3 receptor isoforms and stem cell specific markers for VSELs (Oct-4A, Sox-2) and OGSCs (Oct-4) on sheep OSE cells cultured in vitro. In addition, immunolocalization studies were carried out for FSHR and OCT-4 on sheep ovarian sections to study how OCT-4 (one of the 27 crucial maternally inherited genes), transitions and is differentially expressed during oogenesis (in vitro matured MI and MII oocytes) and early embryogenesis. Materials and methods The study was approved by the Institute Animal Ethics Committee and sheep ovaries obtained from local abattoir were transported in 0.9% normal saline containing antibiotics (Penicillin 100 U/mL, Streptomycin 100 μg/mL; Invitrogen, USA) at ambient temperature adjusted to 22 ± 3°C within an hour of slaughter. Few ovaries were fixed in 10% neutral buffered formalin (NBF) at 4°C, some were immediately frozen for RNA studies and remaining was used for establishing cultures. Granulosa cells from immature and mature sheep ovarian follicles (collected and pooled during routine in vitro maturation of sheep eggs in the lab as reported earlier [29] as well as immature and mature oocytes and embryos were also studied for expression of both FSHR and OCT-4 proteins and their mRNA transcripts. Sheep ovary surface epithelial cells (OSE) culture Ovaries were rinsed gently several times in calcium-and magnesium-free Dulbecco’s phosphate-buffered saline (DPBS; Invitrogen) containing antibiotics. Any extraneous tissue was dissected out carefully without disturbing the OSE layer. The ovaries were subsequently placed in plain high-glucose DMEM/F12 (Sigma Aldrich, USA) containing antibiotics and their surface was gently scraped with the help of a sterile blunt cell scraper to release the cells as described earlier [30]. These cells were spun at 1000 g for 10 mins at room temperature (RT) and finally re-suspended in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) with antibiotics and were cultured in 5% CO2 incubator at 38.5°C with or without FSH (5 IU/ml, human urinary FSH, Kuanart Pharmaceuticals, India) for 3 and 15 hrs. Preparation of sheep OSE cell smears The initial scraped OSE cells and the whole cell suspension (attached as well as floating) after culture was used to make smears on poly L-lysine (Sigma Aldrich) coated slides for H&E and other studies. For in situ hybridization (ISH) utmost precautions were taken during various steps to prevent RNA degradation and the slides were rinsed in 0.1% diethyl pyrocarbonate (DEPC, Sigma Aldrich) treated water to remove any traces of RNases prior to use. Smears were stored at 4°C till further use. Immuno-localization studies Immuno-localization for FSHR and OCT-4 were carried out on both surface epithelial cell smears and on paraffin sections of sheep ovaries. For FSHR immunolocalization, an antipeptide antibody raised in rabbits against 285–309 region of rat FSHR (with no homology with LHR and TSHR) [31] was used since it showed cross-reactivity with sheep ovarian tissue. OCT-4 polyclonal antibody (Abcam, UK) localized differentially to nuclei or cytoplasm of stem cells depending on whether the stem cells are pluripotent (VSELs) or initiated differentiation into progenitors (OGSCs), as reported earlier by our group [30,32]. SSEA-4 is a cell surface marker for pluripotent stem cells (Millipore, USA) and is expressed by both VSELs and OGSCs are reported earlier by our group [30]. Briefly the paraffin embedded ovarian sections were de-paraffinized and incubated with 3% hydrogen peroxide (Qualigens, India) in methanol for 1 hr and then gradually hydrated in descending series of methanol. This was followed by antigen retrieval by immersing the slides in boiling sodium citrate (SSC) buffer at pH 6 for 5 mins (FSHR) and 20 mins (OCT-4). After cooling, the slides were washed with 1X Tris buffered saline (TBS) buffer for 5 mins and then permeabilized (for OCT-4) with 0.3% Triton X-100 in TBS buffer for 5–7 mins. Then after three washes with TBS buffer (5 mins each), the slides were blocked with 10% normal goat serum (NGS) and 1% bovine serum albumin (BSA) in TBS overnight at 4°C to prevent non-specific staining. Next day, after removing excess blocking reagent, the slides were incubated with primary antibody against FSHR (1 in 200 dilution) and OCT-4 (1 in 50 dilution) for 2 hrs at RT. This was followed by 4–5 washes and then detection was carried out according to manufacturer’s instructions using anti Rabbit Vecta ABC kit (Vector Laboratories, USA). Color reaction was performed using diaminobenzidine (Biogenex, USA) and after obtaining appropriate staining, the slides were dipped in water and counterstained with Haematoxylin. Slides were later viewed under bright-field 90i microscope (Nikon, Japan) and representative fields were photographed. For OSE smears, similar procedure was used as mentioned above with the omission of de-paraffinization and antigen retrieval steps. For immunofluorescence studies, the OSE smears were hydrated in phosphate buffer saline (PBS), permeabilized (for OCT-4) with 0.3% Triton X for 5 mins, followed by 2 hrs blocking in 10% NGS and 1% BSA in PBS. After removing excess blocking, the smears were incubated overnight with primary antibody against FSH receptor (1:100), OCT-4 (1:50) and SSEA-4 (1:50) at 4°C. Next day the slides were brought to RT and then washed 3–4 times with PBS (5 mins each) to remove excess unbound antibody. Then the smears were incubated with anti-rabbit secondary antibody Alexaflour 488 (1:1000) for 2 hrs at RT. After washes with PBS, the smears were then counterstained with propidium iodide (PI, Sigma Aldrich; 5 mg/ml) and mounted using Vectashield and stored at 4°C till viewing. The slides were scanned under laser scanning confocal fluorescent microscope (LSM 510-META, ZEISS, Germany) and representative fields were photographed. RNA extraction and cDNA synthesis RNA was extracted from scraped OSE cells using TRIZOL (Invitrogen) reagent by standard protocol followed by DNase I (Amersham Biosciences, USA) treatment at 37°C for 30 mins to remove any genomic DNA contamination. Reverse transcription of cDNA was performed using iScript cDNA synthesis kit (Bio-Rad, USA) according to the manufacturer’s instructions. Briefly, RNA was incubated with 5× iScript reaction mix and iScript reverse transcriptase mix. The reaction was carried out in G-STORM thermocycler (Gene Technologies, UK). The reaction mix was first incubated at 25°C for 5 mins, then at 42°C for 30 mins and finally at 85°C for 5 mins. Selection of primers for qRT-PCR studies Primers for FSHR isoforms (Table 1) were taken from an earlier publication [26] and were basically designed from exon 10 (FSH receptor transcript R1) and exon 11 (FSH receptor transcript R3) respectively of the FSHR gene. Markers specific for pluripotency (Oct-4A and Sox-2) were selected for studying VSELs. Oct-4A is a marker for pluripotent state and once the pluripotent stem cell starts differentiating, nuclear OCT-4 is no longer required and shifts to the cytoplasm and major associated transcript is Oct-4B. We have earlier reported that VSELs express Oct-4A (amplified by Oct-4A primer) and OGSCs (immediate descendants of VSELs) express Oct-4B (amplified by Oct-4 primer which amplifies all isoforms). These primers (Table 1) have been used to detect VSELs and OGSCs [30,32] and have been designed based on earlier publication [33]. Table 1 Primer details and cycling conditions used in the study Sequence Annealing temperature Amplicon size FSH Receptor Transcripts R1 CATTCACTGCCCACAACTTTCATC 60°C 84 bp TGAGTGTGTAATTGGAACCATTGGT R3 TCTCCACTGCTGCACTGTTGGGCT 55°C 382 bp ATTCAAATACAGGAAATAGAGAAA Pluripotent Stem Cell Marker (VSELs) Oct-4A CAATTTGCCAAGCTCCTAAA 53°C 290 bp TTGCCTCTCACTTGGTTCTC Sox-2 TGATACGGTAGGAGCTTTGC 56°C 362 bp CTTTTGCCCCTTTAGAGACC Differentiation Marker (OGSCs) Oct- 4 (all isoforms) GAGCCGAACCCTGAGGAGTCCC 66°C 225 bp CAGCAGGGGCCGCAGCTTAC Housekeeping Gene Gapdh GCC CAG AAC ATC ATC CCT G 60°C 232 bp GGT CCT CAG TGT AGC CTA G Oct-4A is a true marker for pluripotent stem cells[33]. Oct-4 primers amplify both Oct-4A and other isoforms including Oct-4B. Oct-4A reflects VSELs whereas several fold increase in Oct-4 reflects increase in the number of OGSCs suggesting differentiation. qRT-PCR studies The expression levels of Fsh-r1, Fsh-r3, VSELs (Oct-4A, Sox-2) and OGSCs (Oct-4) specific markers and housekeeping transcript Gapdh were estimated using CFX96 Real-Time PCR system (Bio-Rad Laboratories, USA) using SYBR Green chemistry (Bio-Rad). The amplification conditions were: initial denaturation at 94°C for 3 mins followed by 40 cycles comprising of denaturation at 94°C for 30 seconds, primer annealing at specific temperature for 30 seconds, and extension at 72°C for 30 seconds. The final extension was carried out for 5 mins at 72°C. The fluorescence emitted at each cycle was captured during the extension step of each cycle. The homogeneity of the PCR amplicons was verified by running the products on 2% agarose gels. All PCR amplifications were carried out in duplicate. Mean Ct values generated in each experiment using the CFX Manager software (Bio-Rad) were used to calculate the mRNA expression levels. Since delta Ct is inversely proportional to relative mRNA expression levels, the values were calculated manually by the delta Ct method. The relative expression levels of each transcript from three different experiments are represented individually due to considerable variation in the initial population of cells. In Situ Hybridization (ISH) on OSE smears Expression of FSH receptor transcripts were studied using specific oligo probes by non-radioactive method and all the reagents were purchased from Roche (Roche Diagnostics; Germany) and Sigma. The OSE cells after 15 hrs of FSH treatment were fixed in paraforma aldehyde in PBS prepared using DEPC treated water for 20 mins, smears were prepared on 3-aminopropyltriethoxysilane-coated glass slides then air dried and stored at 4°C until use. The probes used for in situ hybridization for R1 was from exon 10 (5’-TCTTT CCCATCTTTG GCATC −3’) and for R3 was from exon 11 (5’-ATATATT CAAAGATAAA CATACACCAA GAGAA-3’) commercially synthesized (Sigma) and labeled with Digoxigenin using the 3’ tailing kit according to the manufacturer’s instructions (Roche). The specificity of the probe sequence was established by examining its homology with other sequences in the database. All database searches were carried out using the BLAST search engine at http://www.ncbi.nlm.nih.gov. For ISH, the smears were hydrated and refixed in 2% PFA for 10 mins. After washing in 0.1 M PBS (pH 7.0), the slides were incubated in 2X SSC (1X SSC comprised 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7) for 15 mins at RT. Pre-hybridization was carried out at 42°C for 1 hr in a pre-hybridization cocktail containing 50% formamide, 4X sodium saline citrate (SSC), 5X Denhardt’s solution, 0.25% yeast tRNA, 0.5% sheared Salmon sperm DNA, and 10% dextran sulphate. After pre-hybridization, the smears were hybridized overnight at 42°C with the labeled probe diluted in the pre-hybridization mix at a concentration of 5 pmol/μl. The sections were stringently washed in varying concentrations of SSC containing 0.1% Tween 20 (4X SSC, 20 mins twice; 2X SSC, 20 min twice; 1X SSC, 10 min once) followed by blocking for 2 hrs at RT in blocking solution containing 2% NGS, 0.1% Triton X-100 in 0.1 M Tris–HCl buffer (pH 7.5). After blocking, the sections were incubated overnight at 4°C in alkaline phosphatase-conjugated anti-Dig antibody diluted (1:500) in the above blocking solution. The slides were then extensively washed in 0.1 M Tris–HCl (pH 7.5) and equilibrated in 0.1 M Tris–HCl (pH 9.5) for 10 mins. Detection was carried out at pH 9.5 at RT in a solution of nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-2-indoyl phosphate (BCIP) containing 0.2% levamisole and mounted using aquamount. The sections were viewed and representative fields were photographed using 90i bright-field microscope (Nikon, Japan). The smears incubated using a sense probe served as negative controls. Results Studies carried out on sheep ovary surface epithelium FSH effect on OSE smears Cells visualized after H & E staining of OSE smears (Figure 1) included the epithelial cells and putative stem cells interspersed with occasional red blood cells. Epithelial cells were easily identified by their cuboidal to spindle shape with oval, pale stained nucleus and abundant cytoplasm, whereas the putative stem cells were spherical in shape with a typical dark stained nucleus, minimal cytoplasm and high nucleo-cytoplasmic ratio. The stem cells comprised of two distinct populations based on their size. These included the very small embryonic-like stem cells (VSELs) and their immediate descendants ‘progenitors’ which were slightly bigger termed the ovarian germ stem cells (OGSCs) and small clusters of OGSCs, representing rapid proliferation with incomplete cytokinesis. Germ cell clusters are also termed ‘cysts’ or ‘nests’, are well defined structures in fetal ovaries and their presence in adult ovary is a crucial evidence in support of postnatal oogenesis and primordial follicle assembly. We have described and characterized these stem cells in our earlier publications [30,34]. The VSELs were always present singly whereas the OGSCs were observed singly and also as small clusters (Figure 1A). 15 hrs of FSH treatment induced stem cell proliferation and led to a marked increase in number of ‘cysts’ reflecting clonal expansion (Figure 1B). The epithelial cells also appeared bigger in size. 15 hrs cultures without FSH treatment showed altered morphology of the epithelial cells however, the stem cells did not show clonal expansion (Figure 1C). Figure 1 Effect of FSH treatment on sheep ovary surface epithelium (OSE) smears. (A, a) Freshly prepared sheep OSE smear after H & E staining. Epithelial cells (spindle shaped cells with pale nuclei and abundant cytoplasm) and distinct populations of putative stem cells including the VSELs (arrow) and OGSCs (asterisk) are evident along with red blood cells (RBCs) (B, b) Note the increase in number of stem cells and germ cell ‘cysts’ after 15 hrs of FSH treatment. The nests represent rapid stem cells clonal expansion with incomplete cytokinesis and characteristic of the OGSCs (C, c) OSE smear after 15 hrs culture without FSH. These observations were made in more than three different experiments. Note absence of stem cells activity. Epithelial cells after 15 hrs culture (B, b, C, c) appear similar and relatively bigger in size compared to the epithelial cells in fresh OSE (A, a). Scale bar represents 20 μm. FSH effect on ovarian stem cells The VSELs expressed nuclear OCT-4 (Figure 2A&B) confirming their pluripotent state whereas the slightly bigger OGSCs expressed cytoplasmic OCT-4. The cytoplasmic continuity due to incomplete cytokinesis amongst the rapidly dividing cells in the cell clusters (formed as a result of clonal expansion of stems cells which undergo rapid divisions and as a result the daughter cells remain connected) was demonstrated by using a cell surface marker SSEA-4 (Figure 2C). Figure 2 Characterization of stem cells in sheep OSE smears. (A) OCT-4 immunolocalization in OSE smear after 15 hrs of culture with FSH. Note that only stem cells express OCT-4 whereas the somatic epithelial cells are devoid of OCT-4. Scale bar represents 20 μm (B) Confocal imaging of ovarian stem cells stained for OCT-4 with PI as the nuclear counterstain. VSELs express nuclear OCT-4 whereas the slightly bigger OGSCs and also the ‘cysts’ express cytoplasmic OCT-4. First column shows OCT-4 staining, middle panel shows PI staining whereas the third column is the merged image. (C) Confocal image of a cell surface marker SSEA-4 to show cytoplasmic continuity in a germ cell nest or a ‘cyst’. Z stack imaging of a cell cluster collected after FSH treatment shows cytoplasmic continuity between cells due to incomplete cytokinesis and is a distinct feature of clonal expansion of a stem cell. Magnification 63X with 3X zoom (D) qRT-PCR results showing increased relative mRNA expression of pluripotent VSELs specific (Oct-4A and Sox-2) and OGSCs specific (Oct-4) transcripts after 15 hrs of FSH treatment (black bars) compared to without FSH (grey bars). Results are representative of three different experiments. qRT-PCR analysis of pluripotent stem cell markers (Oct-4A and Sox-2) and those suggestive of initiation of differentiation of VSELs (Oct-4) into OGSCs revealed that all the transcripts were up-regulated in response to FSH treatment by 15 hrs of culture. Increase in Oct-4A and Sox-2 suggested that VSELs were undergoing potential self-renewal and increased Oct-4 suggested increased differentiation and proliferation of OGSCs to form ‘cysts’ (Figure 2D). FSH effect on FSH receptor expression in OSE After 15 hrs of FSH treatment, FSHR was immunolocalized in the VSELs, OGSCs and cell clusters whereas the epithelial cells were distinctly negative (Figure 3A). Confocal imaging further confirmed the presence of FSHR (Figure 3B) and OCT-4 in the stem cells (Figure 2B). As evident the small sized VSELs express nuclear FSHR and OCT-4. The slightly bigger OGSCs and the germ cell nests expressed cell surface and cytoplasmic staining for FSHR and OCT-4 respectively. Figure 3 FSH receptor studies on sheep OSE smears. (A) FSH receptors are immuno-localized on the stem cells and germ cell nests ‘cysts’ whereas the epithelial cells remain distinctly negative. (B) Confocal imaging of FSHR localization on the stem cells. VSELs show nuclear staining whereas the OGSCs have cell surface expression of FSHR. In situ hybridization results using specific oligo probes for (C) Fsh-r1 and (D) Fsh-r3 transcripts on FSH treated sheep OSE smears. As evident Fsh-r1 transcript is observed in the nuclei of both VSELs (arrow) and OGSCs (asterix). Fsh-r3 transcript is localized both in the nuclei and cytoplasm even in the germ cell nests ‘cysts’. Presence of Fsh-r3 in both cytoplasm and nuclei suggests active involvement of this transcript during FSH action on the stem cells. Since we did not have specific antibodies for the isoforms, they were studied using specific oligoprobes at the transcript level by in situ hybridization as well as by qRT-PCR. In agreement with immunolocalization data, FSHR transcripts also localized in the stem cells and epithelial cells were negative (Figure 3C&D). Fifteen hours after FSH treatment, FSH receptor transcript R1 mRNA showed nuclear localization whereas R3 transcript mRNA was both nuclear and cytoplasmic compartments suggesting active translation. The small clusters of stem cells invariably showed cytoplasmic Fsh-r3. Hybridization with sense probe gave no staining. qRT-PCR was carried out on three different biological experiments (Figure 4). FSH appeared to differentially regulate R3 transcript compared to R1. As evident, R1 transcript was detected in two of the three samples prior to culture (4A) and there was not much change in expression pattern of R1 transcript mRNA after 3 hrs (4B) and 15 hrs (4C) of FSH treatment. The second sample, which showed no R1 transcript mRNA initially, did express the same after culture. Only one of the three samples expressed R3 transcript prior to culture (4D) but the levels increased several times after culture. There was marginal increase in R3 transcript with and without FSH treatment at 3 hrs in the first experiment but other two experiments showed 5–10 times increased expression in untreated group whereas after 3 hrs of FSH treatment FSH receptor R3 transcript was 30–40 times more compared to expression pattern prior to culture (4E). By 15 hrs, R3 similar to R1 transcripts were expressed at basal levels similar to those observed prior to culture (4F). Figure 4 Relative mRNA expression (Y-axis) by qRT-PCR for Fsh-r1 and Fsh-r3 transcripts. Upper panel shows R1 transcript of FSH receptor mRNA expression in (A) initial scraped OSE and after (B) 3 hrs and (C) 15 hrs with (dark blue bars) and without (light blue bars) FSH treatment. Please note that R1 transcript levels are not affected much by FSH treatment. Lower panel shows R3 transcript of FSH receptor mRNA expression in (D) initial scraped OSE and after (E) 3 hrs (F) and 15 hrs with (red bars) and without (pink bars) FSH treatment. Note the increased expression (more than ten-fold) of R3 transcript after FSH treatment at 3 hrs followed by a reduction to basal levels at 15 hrs (please note a change in Y-axis scale to appreciate a difference between D-F). The relative expression levels of each transcript from three different experiments are represented individually (see Results section for more details). It was not possible for us to combine all the three samples and represent consolidated data because of variability in the expression pattern of FSH receptor transcripts prior to culture. We have no control over sheep ovaries brought from slaughter house, state of cycle they are in and thus OSE scrapings may have variable number of stem cells (which express FSHR). Since we are studying a biological response and receptor mRNA transcripts, only a minimal change in expression was expected in response to FSH treatment (compared to when abundantly expressed transcripts are studied). Thus the results are not even expressed as fold change after FSH treatment over untreated control. Studies done on sheep ovarian sections Immuno-localization on sheep ovarian sections (Figure 5) revealed a similar staining pattern for both FSH-R and OCT-4. FSHR and OCT-4 staining was observed in the OSE. The oocytes in the primordial follicles also stained positive for both FSHR and OCT-4 however, the surrounding granulosa cells remained distinctly negative. In primary to secondary follicles, FSHR and OCT-4 staining was observed in both the oocyte nucleus and the ooplasm whereas the granulosa cells remained negative. Interestingly in the mature follicles, both FSHR and OCT-4 were detected in the cytoplasm of the granulosa cells whereas the surrounding thecal layer remained negative. Cytoplasmic FSHR and OCT-4 in the granulosa cells was confirmed by confocal microscopy. In certain fields a distinct spatial gradient of staining pattern was clearly apparent with stronger staining in the granulosa cells surrounding the antral cavity. Figure 5 Immunolocalization of FSH receptors on sheep ovarian sections (i) FSH receptors. (A) Ovary surface epithelial cells and primordial follicles showing positive staining for FSHR (B) Primordial follicles (asterix) exhibit positive FSHR staining in the oocyte nucleus as well as in the ooplasm whereas the surrounding granulosa cells are negative for FSHR (arrow) (C) Secondary follicle showing positive staining for FSHR in oocyte nucleus and ooplasm and negative for surrounding granulosa cells (D &E) Developing antral follicles show FSHR positive in both ooplasm and surrounding granulosa cells. (ii) OCT-4 (A &B) certain regions of ovary surface epithelium stain positive for OCT-4. Primordial follicles exhibit positive OCT-4 staining in the ooplasm (arrow) as well as in the nuclei (asterix) of the oocytes. The secondary follicles show distinct staining in the ooplasm whereas the surrounding granulosa cells in both primordial and secondary follicles are negative for OCT-4. (C &D) Large antral follicles have distinct staining for OCT-4 in the granulosa cells whereas the theca cells are negative and note a gradient in the staining intensity at higher magnification. Scale bar is 20 μm. Images of representative area were photographed using 90i Nikon microscope. Studies done on sheep granulosa cells, oocytes and embryos obtained in vitro Confocal microscopy after immunostaining (Figure 6A) was carried out on the MI and MII oocytes and surrounding granulosa cells. Both (a) FSHR and (b) OCT-4 were observed in the cytoplasm of granulosa cells. (c-f) OCT-4 was consistently expressed in the oocytes, early stage embryo and in the blastocyst stage in both the trophoectoderm as well as in the inner cell mass. qRT-PCR analysis on pooled granulosa cells collected from the surface of MI and MII oocytes showed a reduction in relative mRNA expression for FSH receptor transcripts R1 and R3, Oct-4A and Oct-4 (Figure 6B). R3 mRNA transcript was significantly higher compared to R1 transcript in MI granulosa cells in agreement with published literature [26-28]. Figure 6 Immunolocalization and PCR of FSHR and OCT-4 in the granulosa cells, oocytes and embryo. (A) Confocal microscopy shows the presence of (a) FSH receptors and (b) OCT-4 in the cytoplasm of the granulosa cells (c-f) OCT-4 localization in MI and MII oocytes and early embryo. Note the MI oocyte shows both nuclear and cytoplasmic OCT-4 whereas in the MII oocyte OCT-4 was observed in the ooplasm in the 4–8 cells embryo and blastocyst OCT-4 was observed in the blastomeres surrounding trophoectodermal cells as well as in the inner cell mass. (B) qRT-PCR analysis of FSH receptor transcripts R1 and R3, Oct-4A and total Oct-4 in the granulosa cells surrounding MI and MII oocytes. R3 transcript mRNA is significantly highly expressed compared to the canonical R1 transcript in the granulosa cells. Note a reduction in all the transcripts in the granulosa cells collected from MII oocytes. Magnification is 40X and granulosa cells are image at 4X optical zoom. The relative expression of transcripts is a representation of pooled samples analyzed once. Discussion Ovarian stem cells including pluripotent, very small embryonic-like stem cells (VSELs), and slightly larger ‘progenitors’ termed ovarian germ stem cells (OGSCs) along with small cell clusters termed the ‘cysts’ or ‘nests’ interspersed with the epithelial cells and occasional RBCs were easily visualized in scraped sheep ovary surface epithelium (OSE). The present study for the first time demonstrates that the expression of FSH receptors in the OSE is restricted to the stem cells. FSH interaction through R3 transcript with the stem cells resulted in the potential self-renewal of VSELs (increased expression of Oct-4A and Sox-2) and their differentiation into ‘cysts’ comprising OGSCs (increased expression of Oct-4) representing initial steps during oogenesis (Figure 7). These results are in contradiction to the existing paradigm that initial primordial follicle growth is independent of FSH action. Rather we show that FSH directly acts on the stem cells via FSH-R3, besides the well-studied action of FSH via canonical FSH-R1 on the granulosa cells. These results possibly provide a novel explanation for the significant reduction in primordial follicle numbers when FSH action was blocked using polyclonal antibody during perinatal development of hamster ovaries [19]. Figure 7 Schematic representation of the study results towards better understanding of postnatal oogenesis and follicle assembly. Small insert represents a model proposed earlier by us [34]. Blue line represents sheep ovarian surface epithelium (OSE). Gentle scraping of OSE shows the presence of epithelial cells (ECs), red blood cells (RBCs) and stem cells (VSELs, OGSCs and cysts). A crucial pluripotent marker OCT-4 (represented in brown) shows nuclear expression in VSELs and cytoplasmic in OGSCs and cyst. FSH acts via FSH receptor isoform R3 (rather than the canonical R1 isoform) on the stem cells and regulates self-renewal of VSELs increased (Oct-4A and Sox-2) and clonal expansion of the OGSCs (increased Oct-4) and formation of cysts (rapid proliferation of OGSCs with incomplete cytokinesis). The OGSCs get surrounded by somatic granulosa cells (formed by epithelial-mesenchymal transition of OSE cells) resulting in primordial follicle (PF) assembly. PF undergo transition into primary follicle and OCT-4 is observed in the ooplasm in the developing oocytes whereas the surrounding granulosa cells remain distinctly negative. In pre-antral to antral follicles, OCT-4 expression gradually decreases in the ooplasm whereas cytoplasmic OCT-4 is observed in the surrounding granulosa cells and interestingly the staining is relatively dark in cumulus granulosa cells compared to distal granulosa cells (please refer to Figure 5). In vitro matured sheep oocytes show cytoplasmic OCT-4 staining in surrounding granulosa cells which is reduced in MII compared to MI oocytes. Nuclear OCT-4 reappears in the developing embryo post-fertilization providing it a pluripotent state. In addition to OCT-4, FSHR which is also required for initial stem cell function during oogenesis exhibited a similar staining pattern. The present study also demonstrated an interesting shift in the staining pattern of FSHR and OCT-4 from the nuclei of VSELs to the nuclei and/or ooplasm of the developing oocytes in the primordial to primary follicles (surrounding granulosa cells being distinctly negative) and in the cytoplasm of the granulosa cells of the growing follicles along with a gradual loss in the developing oocytes. The results raise an interesting question whether later on during development of the follicles, cytoplasmic FSHR and OCT-4 in the granulosa cells are newly synthesized proteins with specific functions or are removed from the developing oocyte (which has minimal machinery of its own to degrade proteins) as it prepares itself for the next journey post-fertilization. FSH-R1 is known to be expressed on the granulosa cells of growing follicles however a reduction in R1 and R3 and Oct-4 mRNA was noted in the granulosa cells surrounding the MII oocytes compared to MI oocytes. We were indeed intrigued as to why two such un-related proteins show similar staining pattern? OCT-4 (a crucial maternally inherited protein) reappeared in the nucleus of the developing oocyte. By studying OCT-4 expression in the ovarian stem cells, developing follicles and early embryo, we demonstrate how embryogenesis indeed begins during oogenesis as suggested earlier by [35]. We have split further discussion into two parts. Role of FSH and its receptor transcripts in modulating stem cell function in vitro Existence of stem cells in the adult ovary is a debatable issue and recently Woods and Tilly [36] summarized the work carried out over the last decade in Professor Tilly’s lab and equated the ovarian stem cells to the spermatogonial stem cells. Whereas Lie and Spradling [37] deny the presence of stem cells in mouse ovary as they neither detected rapidly dividing germ stem cells nor ‘cysts’ although adult testis and fetal ovary (positive controls) gave them the expected staining pattern. Our study provides support in favor of postnatal oogenesis and also shows the formation of cysts after FSH treatment in vitro (Figure 1B). The relatively subtle nature of postnatal oogenesis in adult mouse ovary compared to fetal ovary and adult testis, may explain the negative results of Lie and Spradling [37]. We have earlier reported presence of ‘cysts’ in adult human ovaries [34]. Results of the present study clearly show that these ‘cysts’ become prominent after FSH treatment suggesting that ovarian stem cells are directly modulated by FSH. Further, the stem cells termed OGSCs by our group are indeed similar to the OSCs reported by Tilly’s group. However, besides the SSCs in testis and OSCs in ovary proposed by Tilly’s group [36], we have also reported the presence of more primitive stem cells termed VSELs in both testis and ovary [38]. FSH receptors were immuno-localized in the nuclei of the VSELs (Figure 3A&B) and comprised of both the transcripts FSH-R1 and FSH-R3. This was easily demonstrated using specific oligo-probes and primers in the nuclei of the VSELs by in situ hybridization (Figure 3C&D) and qRT-PCR (Figure 5A-F). An increased expression of Fsh-r3, from undetectable expression in the initial culture by qRT-PCR analysis, was noted as early as 3 hrs in FSH treated group and subtle differences persisted even at 15 hrs compared to untreated control. Stimulatory effect of FSH on the stem cells via FSH-R3 was clearly evident on H & E stained OSE smears (Figure 1A&B) and by the up-regulation of its mRNA transcripts (Figure 4). There was a dramatic increase in the ‘cysts’ which stained positive for FSHR (Figure 3A&B) and OCT-4 (Figure 2A&B) suggesting that the stem cells undergo rapid clonal expansion with incomplete cytokinesis (Figure 2C) in response to the FSH treatment. This was further confirmed by increased mRNA expression of pluripotent markers (Oct-4A and Sox-2) and germ cells specific marker (Oct-4) by qRT-PCR analysis (Figure 2D). This early response of FSH- FSH-R3 possibly occurs through the MAPK pathway and is well documented in literature [26] however; our data shows for the first time that this FSH action is restricted to the stem cells located in the OSE. FSH-FSH-R3 interaction on the VSELs resulted in their proliferation (increased expression of Oct-4A, Sox-2) and differentiation (increased expression of Oct-4B) representing initial steps involved in oogenesis. It is an early effect since Fsh-r3 levels increase within 3 hrs of FSH treatment whereas Fsh-r1 levels were not altered (Figure 4). We have earlier reported similar clonal expansion of the stem cells in situ after PMSG treatment in adult mice ovary [20]. Thus a concomitant up-regulation of both Fsh-r3 and Oct-4 mRNA in response to the treatment supports a potential role of FSH in modulating ovarian stem cells in the present study, in agreement to the earlier reports [20,21]. If indeed FSH-R3 (which lacks exon 10) is the key player to mediate FSH action on stem cells resulting in neo-oogenesis during postnatal life, one could easily explain why the extensive studies undertaken to search for mutations in the exon 10 of FSH receptor in cases of amenorrhea [39] and ovarian tumors [40] have failed to yield any results. The number of mutations reported so far in FSHR gene remains low compared to almost 30 reported for LH receptor [41]. On similar grounds, Oktay et al. [42] failed to detect FSH receptors on primordial follicles resulting in the existing paradigm that initial PF growth is gonadotropin independent [43]. However, a closer scrutiny of the primers used for doing RT-PCR by Oktay’s group showed that they were selected from exon 10 whereas the present study shows that the primordial follicles express FSH-R3 that lacks exon 10 [25,26]. The standard action of FSH-FSH-R1 on the granulosa cells via cAMP pathway has remained the focus of the studies published over decades in the available literature. But FSH-FSH-R3 interaction evidently plays a crucial role during neo-oogenesis and primordial follicle assembly. Sullvian et al. [27] have also reported that alternatively spliced Fsh-r3 is the pre-dominant transcript of FSHR in sheep. Babu et al. [26] have earlier reported a similar two-fold up-regulation of Fsh-r3 compared to Fsh-r1 after PMSG treatment in mice. Thus initial lack of knowledge that FSH may exert its pleiotropic actions through alternatively spliced FSH-R isoforms has resulted in the existing confusion in the field of ovarian biology that PF growth is gonadotropin independent and needs to be revised. An association of increased FSH with ovarian cancers, ‘the gonadotropin theory of ovarian tumorigenesis’ exists [15,44-46]. More than 90% of ovarian cancers arise from the OSE [47]. The development of ovarian tumors is related to excessive gonadotropin production associated with the onset of menopause or premature ovarian failure [48] and almost 80-90% of them occur with advanced age [49]. Schiffenbauer and colleagues [50] have reported that human epithelial ovarian cancers progress faster in ovariectomized mice due to elevated FSH and LH levels. Furthermore, Li et al. [14] have earlier shown that FSH-R3 signaling promotes proliferation of ovarian cancer cells. We propose that certain yet not well understood changes occur with age in the ovarian microenvironment, which are unable to support VSELs/OGSCs differentiation into oocytes and primordial follicle assembly in the OSE. This altered interaction of the stem cells with the microenvironment ‘niche’ results in menopause. Due to altered cell signaling in certain cases, the increased FSH levels with advanced age possibly push the VSELs via FSH-R3 to undergo uncontrolled proliferation resulting in cancer [30,34,51]. VSELs are the embryonic remnants in adult body tissues that may possibly give rise to tumors in the body [52]. It is possible that the VSELs lodged in the OSE result in ovarian cancers by responding through FSH-R3 to high levels of FSH. Thus a greater understanding of FSH-FSH-R3 action during neo-oogenesis from the VSELs lodged in the OSE provided by the present study and a possible association of these VSELs with ovarian cancers opens up newer avenues for further research. But if FSH-FSH-R3 interaction with ovarian stem cells is indeed crucial for neo-oogenesis and PF assembly in postnatal ovary, FSHRKO mice ovaries should have been devoid of follicles altogether. However, the small follicles up to pre-antral stage exist. Also the FSHRKO mice exhibit increased incidence of ovarian tumors in complete absence of ovulation. These mutants show various tumor cell types including those related to ovarian surface epithelium around 12–15 months of age [53]. At this juncture it is quite intriguing to find out whether certain compensatory mechanisms or altered cell signaling pathways may result in PF assembly and increased cancer incidence in these animals. However, a well-defined block in further maturation of the PF is undisputable and exists in the FSHRKO mice. Balla et al. [54] have reported a significant reduction in primordial follicle numbers in 2 day old ovaries of FORKO mice, suggesting a direct and important role of FSH-FSHR interaction during ovarian development. Ghadami et al. [55] reported that Fshr mRNA is readily expressed in the ovaries of FSHRKO mice after bone marrow transplantation. Similarly testosterone secretion has been reported in LHRKO mice [56] on transplanting testicular side population cells into the interstitial spaces. Thus FSHR biology in FSHRKO mice with stem cells perspective warrants further investigation. Similarly streak gonads are reported in women who are homozygous to an inactivating mutation of the FSHR [57] and those with an aberrant FSH beta gene that causes premature protein subunit termination [58]. Aittomaki’s group studied cAMP levels and concluded that the mutation resulted in decreased FSHR activity that may have led to ovarian failure. However, FSH-FSH-R3 action via MAPK pathway remains poorly studied in these reports. Our results are in agreement with recent review [59] that Gs/cAMP/PKA pathways may not be the sole mechanism for FSH action (existing paradigm for more than several decades). We conclude that FSH acts through transcript variants by alternate splicing resulting in protein diversity to overcome limited number of genes in the genome and to perform multiple biological functions. FSHR and OCT-4 immunolocalization on sheep ovaries Immunolocalization studies on OSE smears show that both FSHR and OCT-4 have nuclear expression in the VSELs whereas the epithelial cells are negative for both. As the stem cells differentiate into oocytes, OCT-4 expression shifts to the cytoplasm i.e. ooplasm of oocytes whereas surrounding granulosa cells were distinctly negative. More mature follicles show positive cytoplasmic staining in the granulosa cells and more intense in the cells in close association with the developing oocyte. Based on the results we propose that both OCT-4 and FSHR gradually shift from the developing oocyte to the granulosa cells as the follicles mature. As the oocytes mature from MI to MII stage, a dramatic reduction of total Oct-4 and R1 and R3 mRNA transcripts is observed in the surrounding granulosa cells Figure 7. During intraovarian growth, the oocyte diameter increases 8–10 times with a simultaneous increase in 500 fold increase in volume and fully grown oocyte is the largest cell in the body with minimal cytoplasmic organelles including mitochondria, Golgi and endoplasmic reticulum [60] and lacks lysosomes. It outsources many of its functions to the surrounding cumulus cells [61] while preparing itself for early embryonic development. Degradation of oocyte proteins is an essential component of egg-to-embryo transition so that the oogenic program gets erased and makes way for somatic development [62,63]. Several transcripts crucial for early embryonic development accumulate whereas a large number of maternal RNA and proteins get degraded during meiotic maturation of the oocyte. Thus it is likely that proteins like FSH and OCT-4 (in the present study) which were required during early oogenesis shift from the nuclei of the oocyte to the ooplasm and then to the granulosa cells possibly through the transzonal projections (granulosa cell extensions that traverse the zona pellucida onto the oocyte cell surface) [64,65] and also explains why we observe a gradient in staining pattern (Figure 5). Conclusions Several important insights into FSH role in modulating ovarian stem cells activity through FSH-R3 resulting in primordial follicle assembly in the adult mammalian ovaries may be garnered from the present study. Various proteins which play an important role during early oogenesis are eventually removed from the developing oocyte by the surrounding granulosa cells through the transzonal projections. Maternally inherited protein e.g. OCT-4 exhibited nuclear expression in the VSELs and once they initiate differentiation, OCT-4 protein was detected in the cytoplasm of the developing oocyte and later in the cytoplasm of the granulosa cells. Once the oocyte matured from MI to MII state, OCT-4 possibly is de novo synthesized in the oocyte and persists in the early embryo giving it a pluripotent status. More studies need to be undertaken to substantiate this hypothesis. Present study opens up several directions for further research. Better understanding of postnatal oogenesis and follicular assembly may lead to better management of ovarian pathologies, cancer and infertility. Suppressing FSH-R3 may also provide a newer alternative for fertility control and the putative exon 11 of FSH should also be screened for mutations in cases of ovarian pathologies including pre-mature ovarian failure and ovarian cancers. It also reveals an interesting link between OSE, stem cells and FSH. This may help explain why majority of ovarian cancers arise in OSE (from stem cells) and also provide an explanation for gonadotropin theory of ovarian cancers. Competing interests The authors declare that they have no competing interests. Authors’ contributions HP carried out all the experiments, data analysis, interpretation and manuscript preparation. DB was responsible for conceptualizing the study, planning experiments, providing scientific inputs, data interpretation and manuscript preparation. SP helped perform few experiments and reviewed the manuscript. PG, SY and MB provided technical help. All authors read and approved the final manuscript. Acknowledgements This work is supported by Institute core support (Indian Council for Medical Research, Government of India, New Delhi). We thank Dr Smita Mahale, NIRRH for FSH receptor antibody. ==== Refs Sun L Peng Y Sharrow AC Iqbal J Zhang Z FSH directly regulates bone mass Cell 2006 125 2) 247 260 16630814 Ben-Josef E Yang SY Ji TH Bidart JM Garde SV Chopra DP Porter AT Tang DG Hormone-refractory prostate cancer cells express functional follicle-stimulating hormone receptor (FSHR) J Urol 1999 161 3 970 976 10.1016/S0022-5347(01)61831-7 10022736 Mariani S Salvatori L Basciani S Arizzi M Franco G Petrangeli E Spera G Gnessi L Expression and cellular localization of follicle-stimulating hormone receptor in normal human prostate, benign prostatic hyperplasia and prostate cancer J Urol 2006 175 6 2072 2077 10.1016/S0022-5347(06)00273-4 16697805 Radu A Pichon C Camparo P Antoine M Allory Y Couvelard A Fromont G Hai MT Ghinea N Expression of follicle-stimulating hormone receptor in tumor blood vessels N Engl J Med 2010 363 17) 1621 1630 20961245 Siegel ET Kim HG Nishimoto HK Layman LC The molecular basis of impaired follicle-stimulating hormone action: evidence from human mutations and mouse models Reprod Sci 2013 20 3 211 33 10.1177/1933719112461184 23184658 Zheng W Magid MS Kramer EE Chen YT Follicle-stimulating hormone receptor is expressed in human ovarian surface epithelium and fallopian tube Am J Pathol 1996 148 1 47 53 8546225 Parrott JA Doraiswamy V Kim G Mosher R Skinner MK Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer Mol Cell Endocrinol 2001 172 1–2 213 222 11165055 Ji Q Liu PI Chen PK Aoyama C Follicle stimulating hormone-induced growth promotion and gene expression profiles on ovarian surface epithelial cells Int J Cancer 2004 112 5 803 814 10.1002/ijc.20478 15386376 Minegishi T Kameda T Hirakawa T Abe K Tano M Ibuki Y Expression of gonadotropin and activin receptor messenger ribonucleic acid in human ovarian epithelial neoplasms Clin Cancer Res 2000 6 7 2764 2770 10914722 Syed V Ulinski G Mok SC Yiu GK Ho SM Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells Cancer Res 2001 61 18 6768 6776 11559549 Parrott JA Nilsson E Mosher R Magrane G Albertson D Pinkel D Gray JW Skinner MK Stromal-epithelial interactions in the progression of ovarian cancer: influence and source of tumor stromal cells Mol Cell Endocrinol 2001 175 1–2 29 39 11325514 Abd-Elaziz M Moriya T Akahira J Nakamura Y Suzuki T Sasano H Immunolocalization of nuclear transcription factors, DAX-1 and Ad4BP/SF-1, in human common epithelial ovarian tumors: correlations with StAR and steroidogenic enzymes in epithelial ovarian carcinoma Int J Gynecol Pathol 2005 24 2 153 163 10.1097/01.pgp.0000155075.75209.42 15782072 Choi JH Wong AS Huang HF Leung PC Gonadotropins and ovarian cancer Endocr Rev 2007 28 4 440 461 10.1210/er.2006-0036 17463396 Li Y Ganta S Cheng C Craig R Ganta RR Freeman LC FSH stimulates ovarian cancer cell growth by action on growth factor variant receptor Mol Cell Endocrinol 2007 267 1–2 26 37 17234334 Bose CK Follicle stimulating hormone receptor in ovarian surface epithelium and epithelial ovarian cancer Oncol Res 2008 17 5 231 238 10.3727/096504008786111383 18980020 Patsoula E Loutradis D Drakakis P Kallianidis K Bletsa R Michalas S Expression of mRNA for the LH and FSH receptors in mouse oocytes and preimplantation embryos Reproduction 2001 121 3 455 461 10.1530/rep.0.1210455 11226072 Méduri G Charnaux N Driancourt MA Combettes L Granet P Vannier B Loosfelt H Milgrom E Follicle-stimulating hormone receptors in oocytes? J Clin Endocrinol Metab 2002 87 5 2266 2276 10.1210/jc.87.5.2266 11994374 Rao AJ Ramachandra SG Ramesh V Couture L Abdennebi L Salesse R Remy JJ Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor Reprod Biomed Online 2004 8 4 385 391 10.1016/S1472-6483(10)60921-2 15149560 Roy SK Albee L Requirement for follicle-stimulating hormone action in the formation of primordial follicles during perinatal ovarian development in the hamster Endocrinology 2000 141 12 4449 4456 10.1210/en.141.12.4449 11108254 Bhartiya D Sriraman K Gunjal P Modak H Gonadotropin treatment augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries? J Ovarian Res 2012 5 1 32 10.1186/1757-2215-5-32 23134576 Parte S Bhartiya D Manjramkar DD Chauhan A Joshi A Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture J Ovarian Res 2013 6 1 20 10.1186/1757-2215-6-20 23547966 Khan H Yarney TA Sairam MR Cloning of alternately spliced mRNA transcripts coding for variants of ovine testicular follitropin receptor lacking the G protein coupling domains Biochem Biophys Res Commun 1993 190 3 888 894 10.1006/bbrc.1993.1132 8439338 Sairam MR Babu PS The tale of follitropin receptor diversity: a recipe for fine tuning gonadal responses? Mol Cell Endocrinol 2007 260–262 163 171 23160140 Babu PS Krishnamurthy H Chedrese PJ Sairam MR Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor. Role in hormone signaling and cell proliferation J Biol Chem 2000 275 36 27615 27626 10869352 Touyz RM Jiang L Sairam MR Follicle-stimulating hormone mediated calcium signaling by the alternatively spliced growth factor type I receptor BiolReprod 2000 62 4 1067 1074 Babu PS Danilovich N Sairam MR Hormone-induced receptor gene splicing: enhanced expression of the growth factor type I follicle-stimulating hormone receptor motif in the developing mouse ovary as a new paradigm in growth regulation Endocrinology 2001 142 1 381 389 10.1210/en.142.1.381 11145601 Sullivan R Eborn DR Faris BR Grieger DM Rozell TG Expression of follicle-stimulating hormone receptor variants during the estrous cycle of the ewe Biol Reprod 2009 81 346 (Poster) Babu PS Jiang L Sairam AM Touyz RM Sairam MR Structural features and expression of an alternatively spliced growth factor type I receptor for follitropin signaling in the developing ovary Mol Cell Biol Res Commun 1999 2 1 21 27 10.1006/mcbr.1999.0139 10527886 Nandedkar P Chohan P Patwardhan A Gaikwad S Bhartiya D Parthenogenesis and somatic cell nuclear transfer in sheep oocytes using polscope Indian J Exp Biol 2009 47 7 550 558 19761038 Parte S Bhartiya D Telang J Daithankar V Salvi V Zaveri K Hinduja I Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary Stem Cells Dev 2011 20 8 1451 1464 10.1089/scd.2010.0461 21291304 Ghosalkar JD Dharma SJ Nandedkar TD Mahale SD Identification of the region 285–309 of follicle stimulating hormone receptor as a bioneutralizing epitope J Reprod Immunol 2007 74 1–2 24 33 17280718 Bhartiya D Kasiviswanathan S Unni SK Pethe P Dhabalia JV Patwardhan S Tongaonkar HB Newer insights into premeiotic development of germ cells in adult human testis using Oct-4 as a stem cell marker J Histochem Cytochem 2010 58 12 1093 1106 10.1369/jhc.2010.956870 20805580 Liedtke S Stephan M Kögler G Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research Biol Chem 2008 389 7 845 850 18627312 Bhartiya D Sriraman K Parte S Stem cell interaction with somatic niche may hold the key to fertility restoration in cancer patients Obstet Gynecol Int 2012 921082 PMID: 22548074 10.1155/2012/921082 Gosden RG Oogenesis as a foundation for embryogenesis Mol Cell Endocrinol 2002 186 2 149 153 10.1016/S0303-7207(01)00683-9 11900888 Woods DC Tilly JL An evolutionary perspective on adult female germline stem cell function from flies to humans Semin Reprod Med 2013 31 1 24 32 10.1055/s-0032-1331794 23329633 Lei L Spradling AC Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles Proc Natl Acad Sci 2013 110 21 8585 8590 10.1073/pnas.1306189110 23630252 Bhartiya D Unni S Parte S Anand S Very small embryonic-like stem cells: implications in reproductive biology Biomed Res Int 2013 2013 682326 PMID: 23509758 10.1155/2013/682326 23509758 Achrekar SK Modi DN Meherji PK Patel ZM Mahale SD Follicle stimulating hormone receptor gene variants in women with primary and secondary amenorrhea J Assist Reprod Genet 2010 27 6 317 326 10.1007/s10815-010-9404-9 20237833 Bas F Pescovitz OH Steinmetz R No activating mutations of FSH receptor in four children with ovarian juvenile granulosa cell tumors and the association of these tumors with central precocious puberty J Pediatr Adolesc Gynecol 2009 22 3 173 179 10.1016/j.jpag.2008.10.003 19539204 Doherty E Pakarinen P Tiitinen A Kiilavuori A Huhtaniemi I Forrest S Aittomäki K A novel mutation in the FSH receptor inhibiting signal transduction and causing primary ovarian failure J ClinEndocrinol Metab 2002 87 3 1151 1155 10.1210/jc.87.3.1151 Oktay K Briggs D Gosden RG Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles J Clin Endocrinol Metab 1997 82 11 3748 3751 10.1210/jc.82.11.3748 9360535 Edson MA Nagaraja AK Matzuk MM The mammalian ovary from genesis to revelation Endocr Rev 2009 30 6 624 712 10.1210/er.2009-0012 19776209 Konishi I Kuroda H Mandai M Review: gonadotropins and development of ovarian cancer Oncology 1999 57 Suppl 2 45 48 10545802 Konishi I Gonadotropins and ovarian carcinogenesis: A new era of basic research and its clinical implications Int J Gynecol Cancer 2006 16 1 16 22 10.1111/j.1525-1438.2006.00425.x 16445604 Mertens-Walker I Baxter RC Marsh DJ Gonadotropin signalling in epithelial ovarian cancer Cancer Lett 2012 324 2 152 159 10.1016/j.canlet.2012.05.017 22634496 Auersperg N Wong AS Choi KC Kang SK Leung PC Ovarian surface epithelium: biology, endocrinology, and pathology Endocr Rev 2001 22 2 255 288 10.1210/er.22.2.255 11294827 Cramer DW Welch WR Determinants of ovarian cancer risk. II. Inferences regarding pathogenesis. J Natl Cancer Inst 1983 71 4 717 721 6578367 Sell A Bertelsen K Andersen JE Strøyer I Panduro J Randomized study of whole-abdomen irradiation versus pelvic irradiation plus cyclophosphamide in treatment of early ovarian cancer Gynecol Oncol 1990 37 3 367 373 10.1016/0090-8258(90)90369-V 2351321 Schiffenbauer YS Abramovitch R Meir G Nevo N Holzinger M Itin A Keshet E Neeman M Loss of ovarian function promotes angiogenesis in human ovarian carcinoma Proc Natl Acad Sci USA 1997 94 24 13203 13208 10.1073/pnas.94.24.13203 9371824 Massasa E Costa XS Taylor HS Failure of the stem cell niche rather than loss of oocyte stem cells in the aging ovary Aging (Albany NY) 2010 2 1 1 2 20228937 Ratajczak MZ Shin DM Liu R Marlicz W Tarnowski M Ratajczak J Kucia M Epiblast/germ line hypothesis of cancer development revisited: lesson from the presence of Oct-4+ cells in adult tissues Stem Cell Rev 2010 6 2 307 316 10.1007/s12015-010-9143-4 20309650 Aravindakshan J Chen XL Sairam MR Chronology and complexities of ovarian tumorigenesis in FORKO mice: age-dependent gene alterations and progressive dysregulation of Major Histocompatibility Complex (MHC) Class I and II profiles Mol Cell Endocrinol 2010 329 1–2 37 46 20615452 Balla A Danilovich N Yang Y Sairam MR Dynamics of ovarian development in the FORKO immature mouse: structural and functional implications for ovarian reserve Biol Reprod 2003 69 4 1281 1293 10.1095/biolreprod.103.015552 12801993 Ghadami M El-Demerdash E Zhang D Salama SA Binhazim AA Archibong AE Chen X Ballard BR Sairam MR Al-Hendy A Bone marrow transplantation restores follicular maturation and steroid hormones production in a mouse model for primary ovarian failure PLoS One 2012 7 3 e32462 10.1371/journal.pone.0032462 22412875 Lo KC Lei Z Rao CV Beck J Lamb DJ De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of leydig stem cells Endocrinology 2004 145 9 4011 5 10.1210/en.2003-1729 15123536 Aittomäki K Lucena JL Pakarinen P Sistonen P Tapanainen J Gromoll J Kaskikari R Sankila EM Lehväslaiho H Engel AR Nieschlag E Huhtaniemi I de la Chapelle A Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure Cell 1995 82 6 959 968 10.1016/0092-8674(95)90275-9 7553856 Layman LC McDonough PG Mutations of follicle stimulating hormone-beta and its receptor in human and mouse: genotype/phenotype Mol Cell Endocrinol 2000 161 1–2 9 17 10773385 Gloaguen P Crépieux P Heitzler D Poupon A Reiter E Mapping the follicle stimulating hormone-induced signaling networks Front Endocrinol (Lausanne) 2011 2 45 10.3389/fendo.2011.00045 22666216 Zamboni L Mishell DR JrBell JH Baca M Fine structure of the human ovum in the pronuclear stage J Cell Biol 1966 30 3 579 600 10.1083/jcb.30.3.579 6008199 Su YQ Sugiura K Eppig JJ Mouse oocyte control of granulosa cell development and function: paracrine regulation of cumulus cell metabolism Semin Reprod Med 2009 27 1 32 42 10.1055/s-0028-1108008 19197803 Mehlmann LM Losing mom’s message: requirement for DCP1A and DCP2 in the degradation of maternal transcripts during oocyte maturation Biol Reprod 2013 88 1 10 10.1095/biolreprod.112.106591 23221396 Stitzel ML Seydoux G Regulation of the oocyte-to-zygote transition Science 2007 316 5823 407 408 10.1126/science.1138236 17446393 Albertini DF Combelles CM Benecchi E Carabatsos MJ Cellular basis for paracrine regulation of ovarian follicle development Reproduction 2001 121 5 647 653 10.1530/rep.0.1210647 11427152 Thomas FH Vanderhyden BC Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth Reprod Biol Endocrinol 2006 4 19 10.1186/1477-7827-4-19 16611364	23870332	PMC3728228	CC BY	2021-01-05 01:57:27	yes	J Ovarian Res. 2013 Jul 20; 6:52
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23936228PONE-D-12-2754710.1371/journal.pone.0070558Research ArticleBiologyAnatomy and PhysiologyEndocrine SystemEndocrine PhysiologyHormonesBiochemistryHormonesMolecular Cell BiologySignal TransductionSignaling in Selected DisciplinesOncogenic SignalingMedicineAnatomy and PhysiologyEndocrine SystemEndocrine PhysiologyHormonesEndocrinologyEndocrine PhysiologyHormonesOncologyCancers and NeoplasmsGenitourinary Tract TumorsProstate CancerUrologyProstate DiseasesProstate CancerSHBG Is an Important Factor in Stemness Induction of Cells by DHT In Vitro and Associated with Poor Clinical Features of Prostate Carcinomas SHBG in Prostate CarcinomaMa Yuanyuan 1 2 Liang Dongming 1 Liu Jian 1 Wen Jian-Guo 3 Servoll Einar 4 Waaler Gudmund 4 Sæter Thorstein 4 Axcrona Karol 5 Vlatkovic Ljiljana 1 Axcrona Ulrika 1 Paus Elisabeth 6 Yang Yue 2 Zhang Zhiqian 7 Kvalheim Gunnar 8 Nesland Jahn M. 1 Suo Zhenhe 1 9 * 1 Department of Pathology, The Norwegian Radium Hospital, Institute of Clinical Medicine, Oslo University Hospital, Faculty of Medicine, University of Oslo, Oslo, Norway 2 Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Thoracic Surgery II, Peking University Cancer Hospital & Institute, Beijing, China 3 Department of Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Henan, China 4 Department of Surgery, Soerlandet Hospital, Arendal, Norway 5 Departments of Urology, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Oslo, Norway 6 Department of Medical Biochemistry, Oslo University Hospital, University of Oslo, Oslo, Norway 7 Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Cell Biology, Peking University Cancer Hospital & Institute, Beijing, China 8 Departments of Cell Therapy, The Norwegian Radium Hospital, Oslo University Hospital, University of Oslo, Oslo, Norway 9 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Henan, China Aldabe Rafael Editor Centro de Investigación en Medicina Aplicada (CIMA), Spain * E-mail: zhenhes@medisin.uio.noCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: YM DL KA GK JMN ZS. Performed the experiments: YM DL JL EP. Analyzed the data: YM JW ES GW TS KA LV UA EP YY ZZ GK JMN ZS. Contributed reagents/materials/analysis tools: ZS. Wrote the paper: YM DL JMN ZS. 2013 30 7 2013 8 7 e705589 9 2012 24 6 2013 © 2013 Ma et al2013Ma et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Androgen plays a vital role in prostate cancer development. However, it is not clear whether androgens influence stem-like properties of prostate cancer, a feature important for prostate cancer progression. In this study, we show that upon DHT treatment in vitro, prostate cancer cell lines LNCaP and PC-3 were revealed with higher clonogenic potential and higher expression levels of stemness related factors CD44, CD90, Oct3/4 and Nanog. Moreover, sex hormone binding globulin (SHBG) was also simultaneously upregulated in these cells. When the SHBG gene was blocked by SHBG siRNA knock-down, the induction of Oct3/4, Nanog, CD44 and CD90 by DHT was also correspondingly blocked in these cells. Immunohistochemical evaluation of clinical samples disclosed weakly positive, and areas negative for SHBG expression in the benign prostate tissues, while most of the prostate carcinomas were strongly positive for SHBG. In addition, higher levels of SHBG expression were significantly associated with higher Gleason score, more seminal vesicle invasions and lymph node metastases. Collectively, our results show a role of SHBG in upregulating stemness of prostate cancer cells upon DHT exposure in vitro, and SHBG expression in prostate cancer samples is significantly associated with poor clinicopathological features, indicating a role of SHBG in prostate cancer progression. This work is supported by the The Norwegian Radium Hospital Research Foundation and National Natural Science Foundation of China, grant no 81272824. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Prostate cancer is a common malignancy in Western countries [1]. Prostate cancer cell growth is initially dependent on androgens. The standard treatment for patients with primary metastatic hormone-dependent prostate cancer is androgen deprivation, and this treatment modality can originally inhibit tumor growth [2], [3]. However, the androgen ablation therapy eventually fails and androgen independent castration resistant prostate cancer (CRPC) develops [4], [5]. The treatment option for metastatic prostate cancer is limited. Metastatic cancer cells are believed to include rare cells that are phenotypically undifferentiated, also called cancer stem cells (CSCs). CSCs are hypothesized to have similar stem cell capacity such as self-renewal, differentiation and initiation of new tumors and are associated with resistance to chemotherapy and radiotherapy [6], [7]. Potent androgens such as testosterone and 5α-dihydrotestosterone (DHT) play an important role in the development of normal prostate and prostate cancer [8], [9]. Testosterone derived in testes is converted to its active form-DHT in the prostate [10]. Androgen receptor (AR) is an androgen-activated transcription factor and a member of the superfamily of nuclear hormone receptors. Sex hormone-binding globulin (SHBG) has also shown a pivotal effect on development of prostate cancers by regulating androgen. SHBG is a 90-kd glycoprotein which is able to bind to sex hormones like testosterone and estradiol, and especially with higher affinity for DHT. In human, SHBG is most expressed in hepatocytes and secreted into plasma [11]. It is also expressed in several other tissues such as testis, breast and prostate which are classic target tissues for androgens and estrogens [12]–[14]. Importantly, SHBG has been demonstrated in tissue sections of human prostate cancers as well as prostate cancer cell lines PC-3, DU145 and LNCaP by immunohistochemistry for protein examination and in situ hybridization for SHBG mRNA, suggesting that SHBG is locally regulated and produced [15]. The initial step of androgen and estrogen signaling though SHBG requires binding to its specific receptor (RSHBG) on selected cell membranes. Thereafter, subsequent binding of an appropriate androgen or estrogen to the SHBG-RSHBG complex is activated which results in accumulation of cAMP in prostate cancer [16], [17] and breast cancer [18], [19]. Reported downstream effects of SHBG include protein kinase A (PKA) activation [20], induced prostate specific antigen (PSA) expression [21], increased apoptosis [22], and seemingly disparate findings of reduced MCF-7 breast cancer cell growth [23] and increased ALVA-41 prostate cancer cell growth [24]. In this study, we intended to study whether addition of DHT to prostate cancer cell lines LNCaP and PC-3 could influence their stem-like properties. We did observe that upon DHT treatment in vitro, prostate cancer cells were revealed with higher clonogenic potential and higher expression levels of stem cell markers CD44, CD90, Oct3/4 and Nanog. In parallel with these findings, the expression of SHBG in these cells was also upregulated after DHT stimulation, and the induction of Oct3/4 and Nanog by DHT was associated with SHBG expression verified by SHBG siRNA knock-down experiments, indicating an important role of SHBG in maintaining cell stemness which may have clinical consequence. Immunohistochemical evaluation of SHBG in clinical samples was then conducted. Weakly positive and areas negative for SHBG expression in the benign prostate tissues was revealed, while most of the prostate carcinomas were strongly positive for SHBG. In addition, the expression of SHBG in the prostate carcinomas was significantly associated with higher Gleason grade score, seminal vesicle invasions and lymph node metastasis. Materials and Methods The ethical committee of the Health Region South-East of Norway has approved this study (REK 2.2007.219). All individuals involved in this project have given written informed consent for the original human work that produced the tissue samples and written informed consent to publish these case details. Cell Lines and Cell Treatment Human prostate cancer cell lines PC-3 and LNCaP were obtained from the American Type Culture Collection (ATCC). All cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified 5% CO2 incubator at 37°C. After allowing cells to attach onto the flasks, the cells were transferred into phenol red-free RPMI 1640 supplemented with 10% charcoal-stripped FBS (androgen-free medium) for overnight. DHT (1 nM or 10 nM; Sigma-Aldrich) was dissolved in ethanol and added in androgen-free medium for cell culture and the corresponding concentration of ethanol was used as blank control [25]. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) Assays PC-3 (1000/well) and LNCaP (2000/well) cells were planted in 96-well plates. After the cells attached to plate, 1 nM or 10 nM DHT was added into the androgen-free medium for variable times. At each time point as indicated, the cells were added with MTT (Sigma-Aldrich) and cultivated at 37°C for 4 hours. Then 200 µL of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added to each well and mixed thoroughly. The plates were shaken for 15 min and absorbance was determined using spectrophotometer at a wavelength of 570 nm. Colony Formation Assay 500/well single cells of PC-3 cells and 1000/well single cells of LNCaP cells were seeded in six-well plates with/without DHT (1 nM or 10 nM) in androgen-free medium as mentioned above for 14 days before the cells were gently washed with PBS and fixed by 4% buffered formalin for 15 min. Subsequently, 1% crystal violet was used to stain the colonies for 30 min. The plates were gently washed with PBS and dried before colony evaluation under microscope. Colony number which contained more than 30 cells was counted and colony formation efficiency was determined as colony formation efficiency = colonies/input cells × 100%. Sphere Formation Assay The sphere assay was performed as described in our previous study [26]. Single PC-3 (500 cells/well) and LNCaP (1000 cells/well) cells were planted in ultralow attachment six-well plates (Ultra low cluster plates, Life sciences). The cells were cultivated in androgen-free medium as mentioned above added with/without DHT (1 nM or 10 nM) for 14 days. More than 30 cells within a sphere was regarded as a full sphere and counted under inverse microscopy. Sphere formation efficiency was determined as following: sphere formation efficiency = sphere/input cells × 100%. Flow Cytometry Based on our previous study [26], the expressions of the surface markers CD44, CD24 and CD90 in the cells treated with/without DHT (10 nM) for 48 hours were analyzed by flow cytometry using a CD44 antibody conjugated with allophycoyanin (APC), a CD24 antibody conjugated with fluorescein isothiocyanate (FITC), and a CD90 antibody conjugated with phycoerythrin (PE). The corresponding APC Mouse IgG2b, FITC Mouse IgG2a, and PE Mouse IgG1 isotype controls were used as negative control and the viable and single cells were gated for analyses on a flow cytometer (Becton Dickinson, San Jose, CA, USA). All the antibodies and isotype controls were obtained from BD Pharmingen. Immunoblotting Cells were washed with ice-cold PBS twice and homogenized in lyses buffer (25 mM Tris HCl pH 7.6, 100 mM NaCl, 1% NP40, 1% Sodium deoxycholate, 0.1% SDS, Thermo Scientific Pierce, Germany) added with protease inhibitors (0.1 uM Aprotinin, 1.0 mM PMSF, 1 uM Leupeptin, 1 uM Pepstatin) immediately before use. The total protein content in samples was measured by the Bio-Rad protein assay (Hercules, CA, USA) according to the manufacturer’s instruction. Equal amounts of protein were resolved by 10% SDS-PAGE and electro-transferred to polyvinylidene difluoride transfer membrane in a Trans-Blot apparatus (Bio-Rad, Hercules, CA). 5% non-fat milk was used to block the membranes and the primary antibodies with optimized concentrations including AR (1 µg/ml), Oct3/4 (2 µg/ml), Nanog (1 µg/ml), SHBG (1 µg/ml) and GAPDH (0.2 µg/m) from R&D system were added to incubate for overnight at 4 °C. The corresponding secondary antibodies conjugated with HRP were then used to incubate the membranes. Immuno-complexes were visualized by enhanced chemiluminescence detection system (GE Healthcare, UK). PSA and SHBG Measurements The PC-3 and LNCaP cells were cultivated in the androgen-free medium added with 1 nM/10 nM DHT for variable periods. Cell culture media at different culture periods were then collected, centrifuged to remove the cellular debris and stored at −70°C for further PSA and SHBG measurement. Total PSA was measured by the time-resolved fluoroimmunometric dual-label assay performed with the AutoDELFIA instrument (AutoDelfia Prostatus PSA Total/Free, Turku, Finland),. The assay was calibrated against WHO standards, with an assay sensitivity of 0.05 ng/ml and interassay coefficient of variation below 5% over the standard range from 0 to 260 ng/ml. The human SHBG ELISA kit (Alpha Diagnostic International, USA) was used for quantitative determination of SHBG in the medium according to the manufacturer’s instruction. A calibrator curve was established and serum positive control was also included for every run. Transient Transfection SHBG siRNA (sc-44847) and siRNA control (sc-33007) for transient transfection were obtained from Santa Cruz Biotechnology. 2×105 cells per well were planted at six-well plates for 24 hours. SHBG siRNA (50 pmols) and siRNA control (50 pmols) were dissolved within OPTI-MEM Reduced Serum Medium and cultivated for 30 min at room temperature separately. Then the transfection mixtures were added onto the 60–80% confluent cells in six-well plates for 5 hours’ cultivation. The medium was aspirated and replaced with fresh normal growth medium for 24 hours. Then the medium was changed to androgen-free medium added with/without DHT (10 nM) for 48 hours. The cells were washed with PBS and harvested for further experiments. Clinical Samples Radical prostatectomy specimens from 117 patients with clinically localized prostate cancer were included in this study. All the patients underwent radical retropubic prostatectomy (RRP) in the period from 1985 until 2006 at Soerlandet County Hospital, Arendal, Norway. The clinical samples were routinely processed for histological diagnosis. Final histological classification in this study was done according to the International Society of Urological Pathology (ISUP, 2005) consensus on Gleason grading of prostate cancer in this study. Gleason grade score, surgical margin (SM) status, seminal vesicle invasion (SVI) and extraprostatic extension (EPE) for the malignant tumors were recorded. In total, five of these patients were found to have lymph node metastasis upon histological re-examination. In addition, formalin-fixed and paraffin-embedded samples from 10 prostate hyperplasia samples were retrieved from the archives of The Norwegian Radium Hospital and included in this study as well. Immunocytochemistry and Immunohistochemistry 4µm paraffin sections from paraffin blocks with either cell lines or clinical samples were prepared. After deparaffinization, the sections were treated with hydrogen peroxide (H2O2) for 5 min to block the endogenous peroxidase. The antibodies for Oct3/4 (AF1759, R&D), Nanog (AF1997, R&D), AR (ABIN165648, DAKO) and two different antibodies for SHBG (CAB-20000TRH, Creative BioMart; AF2656, R&D) were used. The sections were incubated with the primary antibody for 30 min at room temperature. The liver tissue was used as positive control according to the instructions and the non-immune corresponding IgG was used as negative control. After rinsed with DAKO wash buffer, corresponding EnVision FLEX+Linker reagent was added and incubated for 15 min at room temperature before the slides were incubated with the EnVision FLEX+HRP for 30 min at room temperature. The sections were rinsed, colour reaction developed with DAB reagent, counterstained with hematoxylin for 20 seconds, dehydrated and mounted with cover slips before microscopy evaluation. The results were evaluated by two pathologists without knowledge about the patient data. The immunohistochemical results for SHBG for the 117 patients with prostate cancers were assessed according to a previous study [27] as following: 0, no staining like the negative control staining; 1, if a weak positivity was revealed; 2 if a moderate positive staining was observed; and 3 if a strong staining intensity was found. Since only a few carcinomas were either weakly positive or moderately positive, the scores of all the samples were defined as two groups: scores with 1 or 2 were defined as weak expression group, and score 3 was defined as strong expression group. Statistical Analyses All the experiments were performed at least three times. Data are shown as mean ± S.D, Student’s t-test was used to analyze the surface markers expressions and one-way ANOVA was used to assess the cell growth and clonogenicity between different treatments. Comparisons between SHBG expression and clinicopathology variables were analyzed by Pearson chi-square test and Mann-Whitney U test. SPSS software (version 18.0) was used for data analysis and statistical significance was considered as P<0.05. Results Proliferation and Clonogenicity are Stimulated by DHT Cell growth of prostate cancer cell lines LNCaP and PC-3 treated with two different concentrations (1 nM or 10 nM) of DHT for variable periods of time were examined by MTT assays. Both low and high concentrations of DHT could stimulate the proliferation of androgen-responsive LNCaP cells in both time-dependent and concentration-dependent manners with significantly statistical difference after 48 hours (Figure 1A left panel). The PC-3 cells cultivated with DHT also grew relatively faster than the cell without DHT, although no significant difference was observed in these cells (Figure 1A right panel). We further investigated whether DHT treatment could influence clonogenicity of these two prostate cancer cell lines. As shown in Figure 1B, more clones were demonstrated in cells cultivated with 1 nM DHT and even more clones were observed in both cell lines with 10 nM DHT treatment compared to the control cells without DHT. As also shown in Figure 1C, dose-dependent higher colony formation efficiency was observed in both cell lines. Consistently, DHT treatment could also improve sphere growth for both cell lines, and result in higher sphere formation efficiency for both PC-3 and LNCaP cells in dose -dependent manner as well (Figure 1D and E). 10.1371/journal.pone.0070558.g001Figure 1 DHT induces cell growth and clonogenicity in prostate cancer cell lines. (A) Cell growth curves show statistically significant difference in LNCaP cells with/without DHT treatment, but not in PC-3 cells (* means P<0.05). (B) Representative photographs of colony formation in both cell lines demonstrate that more colonies were formed by 1 nM DHT treatment and even more colonies were obtained by 10 nM DHT treatment (bar scale: 50 mm). (C) Histograms of colony formation efficiency show statistically higher efficiencies in the cells treated with low concentration of DHT (P<0.01), and even higher efficiencies in the cells by high concentration of DHT (P<0.001). (D) Representative photographs for sphere formation for both cell lines in the cells with/without DHT treatment (bar scale: 50 µm). (E) Histograms for sphere formation efficiency show higher efficiencies in the cells added with 1 nM DHT (P<0.05), and even higher efficiencies in the cells stimulated with 10 nM DHT (P<0.01). Stem Cell Surface Markers are Upregulated by DHT CD44, a candidate stem cell marker for prostate cancer, was upregulated in both prostate cancer cell lines treated with 10 nM DHT for 48 hours, although relatively less prominent in PC-3 cells. As shown in Figure 2A, there was a 1.68-fold increase in CD44 expression in LNCaP cells and 1.22-fold increase in PC-3 cells after DHT treatment. The influence of DHT on the expression of CD24 in these two cell lines was not apparent, with a slightly higher CD24 expression in both cell lines after addition of DHT, with about 1.06-fold increase in LNCaP cells and 1.09-fold increase in PC-3 cells (Figure 2B). Since CD90 has been implicated as a stem cell marker in different studies, we also examined its expression in these cells with/without DHT treatment. As shown in Figure 2C, significantly higher levels of CD90 expression were displayed in both cell lines under DHT treatment (1.81-fold and 1.60-fold increases in LNCaP and PC-3 cells, respectively). 10.1371/journal.pone.0070558.g002Figure 2 Stem-like phenotype analyses by flow cytometry. (A–C) CD44, CD24 and CD90 expressions were analyzed by flow cytometry in LNCaP and PC-3 cells cultivated with/without DHT in 10 nM for 48 hours. CD44 was induced by DHT treatment in both cell lines (A). No statistically significant difference of CD24 expression level was shown in both cell lines (B). CD90 expression was significantly increased by DHT stimulation in both cell lines (C). (* means P<0.05). The Stemness Factors Oct3/4 and Nanog are Induced upon DHT Treatment In an attempt to further explore whether stem-like properties of prostate cancer cells were altered in response to DHT, the expressions of stemness factors Oct3/4 and Nanog were investigated in LNCaP and PC-3 cells cultivated with/without DHT for variable periods of time. Both Oct3/4 and Nanog expressions began to be upregulated after 24 hours’ DHT treatment in both cell lines in a concentration-dependent manner (Figure 3A), although Nanog expression was relatively low in comparison to the expression of Oct3/4. The expressions of these two factors were also examined by immunocytochemistry in the cells treated with or without DHT for 48 hours (Figure 3B). In response to DHT treatments, higher levels of Oct3/4 and Nanog expressions were observed in both cell lines. These results were in good agreement with the findings obtained by Western blotting. 10.1371/journal.pone.0070558.g003Figure 3 DHT increases Oct3/4 and Nanog expressions in prostate cancer cell lines. (A) The higher expressions of Oct3/4 and Nanog by immunoblotting assay are shown in LNCaP and PC-3 cells treated with 1 nM and 10 nM DHT treatments, respectively, for variable periods of times. (B) The immunocytochemical staining shows higher levels of these two factors in both cell lines treated with different concentrations of DHT. Human seminoma tissue sections were used as positive controls for these two antibodies (bar scale: 50 µm). AR Expression and PSA Secretion are Stimulated by DHT in LNCaP Cells In consistent with a previous study [28], we also found that AR was positive in LNCaP cells by the methods of Western blotting (Figure 4A left panel) and immnocytochemistry (Figure 4B). The LNCaP cells cultivated in the androgen-free medium showed relatively low level of AR expression and its expression was increased by DHT treatment in both time-dependent and concentration-dependent manners (Figure 4A). Low level of PSA (<5 ng/ml) in LNCaP cells was detected in the androgen-free media. Moreover, there were 4.7-fold and 20.1-fold PSA increases in 24 hours and 48 hours in 1 nM DHT cultivations, respectively, while there were 5.3-fold and 28.4-fold PSA increases in the same time periods in 10 nM DHT cultication in the LNCaP cells (Figure 4A right panel). Immunocytochemistry demonstrated weak nuclear immunostaining for AR in LNCaP cells and stronger expression of this receptor was observed in the cells treated with 1 nM DHT, but strongest AR expression was seen in the cells cultivated with 10 nM DHT (Figure 4B). However, AR was undetectable in PC-3 cells with/without DHT treatment by immunocytochemistry (Figure 4B). In consistent with the negative expression of AR, no PSA was identified by the ELISA assay in the PC-3 cultured media. 10.1371/journal.pone.0070558.g004Figure 4 DHT upregulates AR expression and increases PSA secretion in LNCaP cells. (A) DHT upregulates AR expression in LNCaP cells in both time-dependent and concentration-dependent manners (left panel); there are also higher levels of PSA in the DHT treated cells revealed by the ELISA method (right panel). (B) Immunocytochemistry shows nuclear positivity of AR in LNCaP cells and its expression is upregulated by DHT treatment; but AR expression is negative in the PC-3 cells with/without DHT treatment (bar scale: 50 µm). SHBG is Upregulated by DHT Since the expressions of Oct3/4 and Nanog could be upregulated in both AR positive LNCaP cells and AR negative PC-3 cells, induction of Oct3/4 and Nanog by DHT through AR was ruled out, at least in the PC-3 cells. Considering the high SHBG affinity to DHT [16], [21], [24], SHBG expression was further examined in LNCaP and PC-3 cells in response to DHT treatment. As shown in Figure 5A, membranous and cytoplasmic SHBG expression was seen in both LNCaP and PC-3 cell lines by immunocytochemistry. Furthermore, 48 hours 1 nM DHT treatment could result in higher levels of SHBG expression, and the highest levels of this protein expression were observed in the cells treated with 10 nM DHT. Western blotting analyses confirmed increased expression of SHBG by DHT treatments in both cell lines (Figure 5A). In parallel with the DHT-induced SHBG expression, Oct3/4 and Nanog expressions were also correspondingly increased by 10 nM DHT treatments in these cells demonstrated by Western blotting. 10.1371/journal.pone.0070558.g005Figure 5 DHT upregulates the expression of stemess factors through SHBG. (A) Stronger immunocytochemical staining of SHBG is shown in LNCaP and PC-3 cells treated with DHT (left panel, bar scale: 50 µm). Immunoblotting demonstrates higher levels of SHBG, Oct3/4 and Nanog expressions in the cells treated with DHT (right panel). (B) SHBG specific siRNA results. SHBG knockdown in both LNCaP and PC-3 cells was verified by immunoblotting (left panel); 10 nM DHT induces expressions of Oct3/4 and Nanog in the cells transfected with the siRNA control, but such an induction disappears in the cells transfected with the specific SHBG siRNA (right panel). (C) LNCaP and PC-3 cells treated with specific SHBG siRNA grow relatively slower compared to the cells cultivated with control siRNA. (* means P<0.05). (D) Flowcytometry of CD44 and CD90 (left and right panel, respectively). While 10 nM DHT treatment for 48 hrs induces its expression in both cell lines for both genes (left parts of both panels), there is no CD44 and CD90 expression difference in these cells after specific SHBG siRNA knockdown compared to the siRNA control cells (right parts of both panels). Induction of the Stemness Factors is Associated with SHBG Expression To further investigate whether SHBG was involved in the upregulation of Oct3/4, Nanog, CD44 and CD90 in response to DHT, RNA interference assay was used to suppress endogenous SHBG expression in LNCaP and PC-3 cells. The transient transfection effect was obtained using the siRNA SHBG compared to the siRNA control and the immunoblotting analyses demonstrated that the SHBG-specific siRNA successfully inhibited SHBG expression after transfection for 24 and 48 hours in both cell lines (Figure 5B left panel). Thus, the cells were treated with/without DHT for 48 hours after the cells transfected with siRNA-control or siRNA-SHBG for 24 hours. While higher expressions of Oct3/4 and Nanog was still observed in both LNCaP and PC-3 cells transfected with the siRNA controls and treated with DHT in the cells, the DHT induction of these two factors was blocked in the LNCaP and PC-3 cells treated with the SHBG specific siRNA (Figure 5B right panel), suggesting the induction of these two factors by DHT through SHBG. Relatively slower growth of the LNCaP and PC-3 cells after SHBG knockdown by the specific siRNA was observed, although there was no significant growth difference in the LNCaP cells (Figure 5C). We further examined the effect of SHBG siRNA on the expression of CD44 and CD90 by flowcytometry since their induction was repeatedly observed after DHT treatment. As shown in Figure 5D, no expression induction of CD44 and CD90 could be observed in both cell lines after DHT treatments. SHBG is Highly Expressed in Human Prostate Carcinoma Taking the above results together, greater stem-like properties were displayed by DHT treatment in prostate cancer cell lines and SHBG might play an important role in the induction of stemness features in these cells upon DHT treatment, indicating a potential role of SHBG in prostate carcinomas. To address this question, SHBG expression was investigated in clinical samples including benign and malignant prostate tissues by immunohistochemistry. SHBG expression in liver tissue was always kept as positive control in each running (Figure 6A). Weak and areas negative for SHBG expression were seen in the benign tumor samples. Strong SHBG expression was observed in the prostate carcinomas, especially in the high Gleason grade tumors with highly infiltrating tumor cells (Figure 6B). In the malignant tumors, there were 21 (18%) samples weakly positive, 39 samples (33%) moderately positive, and 57 samples (49%) strong positive for SHBG expression. Typical scores of the prostate carcinomas are shown in Figure 6C. 10.1371/journal.pone.0070558.g006Figure 6 Immunohistochemistry of SHBG expression in prostate cancer tissues. (A) Positive and negative control of SHBG in liver tissues. (B) Weak SHBG positivity is shown in a benign prostate tumor and strong SHBG immunoreactivity is revealed in a malignant Gleason score 8 cancer tissue. (C) Representative immunohistochemical images of different scores of prostate cancer samples are shown. Bar scale in all of the images is 150 µm. SHBG Expression is Significantly Associated with Poor Clinicopathological Characteristics The clinical and pathological characteristics of the 117 patients with prostate cancer are summarized in Table 1. The associations between clinical/pathological features and SHBG expression levels were further analyzed by Pearson chi-square and Mann-Whitney U methods (Table 2). High Gleason grade score for patients with prostate cancer showed a correlation with higher level SHBG expression (p = 0.013). It was also found that tumors with higher levels of SHBG expression were more likely to have seminal vesicle invasion compared to the patients with lower level SHBG expression (p = 0.017). Importantly, all the tumors with lymph node involvement were shown strong SHBG immunostaining (p = 0.009). All the results show that SHBG expression in prostate carcinomas is significantly associated with poor clinicopathological features. 10.1371/journal.pone.0070558.t001Table 1 Clinical and pathologic characteristics for 117 patients with malignant prostate cancer. Variable: Median (range) or Frequency (%) Age: 61 years (44–72) Preoperative PSA: 7.8 ng/ml (3.4–36.0) Follow-up: 108 months (14–296) Gleason score: <7 34 (29) 7a 48 (41) 7b 18 (15) >7 17 (15) TNM staging: pT2 62 (53) pT3 55 (47) PSA, prostatic specific antigen. 10.1371/journal.pone.0070558.t002Table 2 Comparison of clinical and pathologic characteristics by tumor SHBG intensity. SHBG ICH score p-value <3 (weak and moderate) 3 (strong) N 60 57 Age (years) ≤61 33 26 0.310 >61 27 31 Preop PSA (ng/ml) ≤7.8 25 27 0.535 >7.8 35 30 Gleason score <8 56 44 0.013* 8–10 4 13 UICC staging pT2 36 26 0.119 pT3 24 31 EPE − 33 25 0.194 + 26 32 SVI − 57 46 0.017* + 3 11 N − 48 32 0.009* § + 0 5 EPE, extraprostatic extension; SVI, seminal vesicle invasion; N+: lymph node metastases; * indicates significant difference with p value minus 0.05. § means 85 patients underwent pelvic lymphadenectomy. Discussion Androgens are essential for prostate cancer development and prolonged administration of testosterone could induce prostate cancer in rodents [29], [30]. Human prostate cancer disease mostly develops into advanced stage-CRPC and even forms metastatic tumors in other organ eventually, although the androgen deprivation treatment is initially effective. Development of CRPC is most probably attributed to cells with stemness features, which endorse the cells with a unique capability in invasion, metastasis and resistance against most of the conventional cancer therapies. The LNCaP cell line is a human prostate adenocarcinoma cell line derived from lymph node metastasis. This cell line is AR positive. PC-3 is a classical prostatic cell line originally derived from advanced androgen independent bone metastatic prostate cancer. In consistent with the previous study [31], DHT significantly induced proliferation of LNCaP cells. However, we could also see growth stimulation in PC-3 cells, although in a significantly less prominent manner. Due to the notorious association of androgen in prostate carcinogenesis, we attempted to assess whether DHT treatment could influence the stemness features of cells in vitro. Indeed, by colony formation and sphere formation assays, both cell lines exhibited a significantly higher colony and sphere formation efficiency, in a dose-dependent manner within the range of 1 nM to 10 nM of DHT. These observations highly indicated an involvement of stemness related molecules. We further examined the DHT influence on the expressions of CD44, CD24 and CD90 by flow cytometry, since all these molecules have been linked to cells’ stemness. After 48 hours treatment of DHT, significantly higher level of CD44 expression could be repeatedly seen in the LNCaP cells, while the CD44 induction in the PC-3 cells was not the same prominent as in the LNCaP cells, although higher levels of CD44 expression were also observed. This difference may be explained with the fact that PC-3 cells already expressed higher level of CD44 (Figure 2A), and therefore DHT induction of CD44 expression in these cells might be limited. In a previous study, we found that the isolated CD44bright cells of prostate cancer cell lines displayed higher clonogenicity with significantly higher expression levels of stemness-related factors than the corresponding CD44dim cells [26]. The induction of CD44 expression in the current study by DHT is in line with the colony and sphere formation assays. However, CD24 expression in prostate cancer stem cell has been controversial [32], although lack or low expression levels of CD24 were suggested to identify tumor stem cells [33]. We did not find significant difference in CD24 expression in these two cell lines with or without DHT application, indicating that CD24 expression is not associated with cells’ colony formation and sphere formation capability under our experimental condition. CD90, a cell surface molecule, has been identified in a variety of cells including stem and progenitor cells with a function of stemness maintenance [34]–[37]. In good agreement with these findings and our clonogenicity assays, significant increase of CD90 expression was observed in both cell lines upon DHT treatment. Oct3/4 and Nanog play an important role in maintenance of self-renewal of embryonic stem cell and primordial germ cells. These stemness factors are frequently overexpressed in histologically poorly differentiated tumors than in those well-differentiated tumors [38], an indication that their expressions may be upregulated in the cells with higher stem-like features. Hence we asked whether the expressions of these factors were influenced by DHT. As we expected, significantly higher levels of Oct3/4 and Nanog expressions were found in the DHT treated LNCaP and PC-3 cells in both time and concentration dependent manners, verified by both Western blotting and immunocytochemistry. All the above results pinpoint to a possibility that DHT indeed upregulates cell stemness of prostate cancer cells in vitro. The fundamental biological role of DHT has been characterized via AR. Thus, we further determined to examine the AR status of these two cell lines, in case any AR positivity in the PC-3 cells due to any undiscovered reason or mechanism. However, we verified that the PC-3 cells were still AR negative and not inducible with DHT addition while the LNCaP cells were AR positive and DHT could upregulate the expression of AR in these cells as well, a finding well in line with the previous report (Bonaccorsi et al., 2000). Therefore, we reasoned that it is unlikely the upregulation of the stemness factors Oct3/4, Nanog, CD44 and CD90 in these prostate cancer cell lines was through AR, since similar results were also observed in the AR-negative PC-3 cells. In addition to the genomic action like the classic androgen-AR signaling, a growing body of evidence has suggested that androgen could exert rapid non-genomic effects [39]–[42] such as activated ion channel to increases in free intracellular calcium [43]or sodium [44]. Androgen can stimulate the conventional second messenger signal transduction cascades including PKA, protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in prostate cancer cells [45]–[47] or in skeletal muscle cells through MAPK pathway [48], [49]. Moreover, androgen binding by SHBG can also stimulate cAMP and PKA in the prostate cancer cell line LNCaP [16], [20]and the SHBG receptor (SHBG-R) could connect to the G protein complex which may conversely bind androgens or influence the activity of a membrane androgen-binding protein indirectly [40]. The fact that the AR negative PC-3 cells responded to DHT treatment with the same stemness upregulation as LNCaP cells rules out the possibility of DHT-AR interaction in stemness maintenance, at least in the PC-3 cells. This encouraged us to explore additional non-genomic molecular association. Since it is known that except AR, DHT has highest binding affinity to SHBG, SHBG should be a molecular candidate to examine in this context. Our results show that SHBG is expressed in both the AR positive LNCaP cells and the AR negative PC-3 cells, verified with both immunocytochemistry and western blotting methods, which are in line with the former report of Hryb et al [15]. Importantly, we found that SHBG was significantly upregulated in both cell lines with DHT stimulation, in parallel with the increasing expressions of Oct3/4, Nanog, CD44 and CD90 upon DHT treatment. However, SHBG secretion was not detectable in the media of LNCaP and PC-3 cells with or without DHT application. This may be due to the sensitivity of the ELISA kit which is usually used for the serum examination of clinical samples with relatively high concentration of SHBG level, or the SHBG expressed in these cells is not secretory. In line with our findings, Loukovaara et al. already reported that 10 nM testosterone and 100 nmol/L to 1 µmol/L cortisol resulted in higher levels of SHBG expression in HepG2 cells, but these treatments did not increase its release into the culture medium, using solid phase two-site fluoroimmunometric assay [50]. The result from Janne’s group also revealed that in the kidney of mice model shbg transgene expression is androgen dependent and exogenous androgen increases human SHBG mRNA levels in the kidneys of female mice [51]. Collectively, these results have linked the SHBG expression with cells’ stemness. To further verify this possibility, specific SHBG siRNA was used to suppress endogenous SHBG expression in both cell lines. We have repeatedly demonstrated that when SHBG was blocked down by specific siRNA in these cells, DHT could not upregulate the expressions of the stemness factors Oct3/4, Nanog, CD44 and CD90 in these cells longer, indicating a direct interaction between the induction of these factors by DHT and SHBG. Therefore our results suggest that DHT-SHBG pathway may play an important role in induction of stem-like properties in prostate cancer cells and SHBG expression in prostate cancer samples may be of clinical consequence. In clinical sample study, all the ten benign prostate tissues were weak and areas negative for SHBG expression. Generally higher levels of SHBG expression were identified in prostate cancer samples compared to the benign prostate tissues. In the prostate carcinomas, infiltrating tumor cells or tumor cells located in the poorly differentiated areas were always strongly positive, while the well-differentiated glandular structures of cancer were always weakly positive for SHBG. Further statistical analyses showed significant association of SHBG with poor pathological characteristics, including high Gleason grade score, involvement of seminal vesicle invasion and lymph node metastasis. In a serum SHBG study, Andrea et al. have also found a similar correlation and suggested that SHBG may be identified as a multivariate predictor of lymph node invasion in prostate cancer patients undergoing extended pelvic lymph node dissection [52]. SHBG in prostate cancer cells is not secretory as we disclosed in our current study. However, it merits further analysis of the mechanism of high levels SHBG in prostate cancer patients as reported by Salonia et al [52]. In conclusion, we have demonstrated that DHT could upregulate prostate cancer cell stemness in vitro via SHBG and SHBG expression in prostate carcinomas is significantly associated with higher Gleason grade score, seminal vesicle invasion and lymph node metastasis. All our in vitro and clinical sample results indicate an important role of SHBG in prostate cancer progression. We are grateful to Ellen Hellesylt, Mette Synnøve Førsund, and Leni Tøndevold Moripen for their assistance with immunocytochemistry; to Idun Dale Rein and Kirsti Solberg Landsverk for assistance with flow cytometry. ==== Refs References 1 Gronberg H (2003 ) Prostate cancer epidemiology . Lancet 361 : 859 –864 .12642065 2 Huggins C, Hodges CV (2002) Studies on prostatic cancer. I. The effect of castration, of estrogen and of androgen injection on serum phosphatases in metastatic carcinoma of the prostate. 1941. J Urol 167: 948–951; discussion 952. 3 Labrie F , Dupont A , Belanger A , Cusan L , Lacourciere Y , et al (1982 ) New hormonal therapy in prostatic carcinoma: combined treatment with an LHRH agonist and an antiandrogen . Clin Invest Med 5 : 267 –275 .6819101 4 Kong D , Heath E , Chen W , Cher ML , Powell I , et al (2012 ) Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM . PLoS One 7 : e33729 .22442719 5 Litvinov IV , De Marzo AM , Isaacs JT (2003 ) Is the Achilles’ heel for prostate cancer therapy a gain of function in androgen receptor signaling ? J Clin Endocrinol Metab 88 : 2972 –2982 .12843129 6 Houghton J , Morozov A , Smirnova I , Wang TC (2007 ) Stem cells and cancer . Semin Cancer Biol 17 : 191 –203 .16762563 7 Tysnes BB (2010 ) Tumor-initiating and -propagating cells: cells that we would like to identify and control . Neoplasia 12 : 506 –515 .20651980 8 Berger R , Febbo PG , Majumder PK , Zhao JJ , Mukherjee S , et al (2004 ) Androgen-induced differentiation and tumorigenicity of human prostate epithelial cells . Cancer Res 64 : 8867 –8875 .15604246 9 Voigt KD , Bartsch W (1986 ) Intratissular androgens in benign prostatic hyperplasia and prostatic cancer . J Steroid Biochem 25 : 749 –757 .2433505 10 Chokkalingam AP , Stanczyk FZ , Reichardt JK , Hsing AW (2007 ) Molecular epidemiology of prostate cancer: hormone-related genetic loci . Front Biosci 12 : 3436 –3460 .17485312 11 Joseph DR (1994 ) Structure, function, and regulation of androgen-binding protein/sex hormone-binding globulin . Vitam Horm 49 : 197 –280 .7810071 12 Fortunati N , Catalano MG , Boccuzzi G , Frairia R (2010 ) Sex Hormone-Binding Globulin (SHBG), estradiol and breast cancer . Mol Cell Endocrinol 316 : 86 –92 .19770023 13 Kahn SM , Hryb DJ , Nakhla AM , Romas NA , Rosner W (2002 ) Sex hormone-binding globulin is synthesized in target cells . J Endocrinol 175 : 113 –120 .12379495 14 Kahn SM , Li YH , Hryb DJ , Nakhla AM , Romas NA , et al (2008 ) Sex hormone-binding globulin influences gene expression of LNCaP and MCF-7 cells in response to androgen and estrogen treatment . Adv Exp Med Biol 617 : 557 –564 .18497082 15 Hryb DJ , Nakhla AM , Kahn SM , St George J , Levy NC , et al (2002 ) Sex hormone-binding globulin in the human prostate is locally synthesized and may act as an autocrine/paracrine effector . J Biol Chem 277 : 26618 –26622 .12015315 16 Nakhla AM , Khan MS , Rosner W (1990 ) Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 3′,5′-monophosphate . J Clin Endocrinol Metab 71 : 398 –404 .2166070 17 Rosner W , Hryb DJ , Khan MS , Nakhla AM , Romas NA (1992 ) Sex hormone-binding globulin. Binding to cell membranes and generation of a second messenger . J Androl 13 : 101 –106 .1597393 18 Fissore F , Fortunati N , Comba A , Fazzari A , Gaidano G , et al (1994 ) The receptor-mediated action of sex steroid binding protein (SBP, SHBG): accumulation of cAMP in MCF-7 cells under SBP and estradiol treatment . Steroids 59 : 661 –667 .7701543 19 Fortunati N , Fissore F , Fazzari A , Piovano F , Catalano MG , et al (1999 ) Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG) . J Steroid Biochem Mol Biol 70 : 73 –80 .10529004 20 Fortunati N , Fissore F , Fazzari A , Becchis M , Comba A , et al (1996 ) Sex steroid binding protein exerts a negative control on estradiol action in MCF-7 cells (human breast cancer) through cyclic adenosine 3′,5′-monophosphate and protein kinase A. Endocrinology . 137 : 686 –692 . 21 Nakhla AM , Romas NA , Rosner W (1997 ) Estradiol activates the prostate androgen receptor and prostate-specific antigen secretion through the intermediacy of sex hormone-binding globulin . J Biol Chem 272 : 6838 –6841 .9054366 22 Catalano MG , Frairia R , Boccuzzi G , Fortunati N (2005 ) Sex hormone-binding globulin antagonizes the anti-apoptotic effect of estradiol in breast cancer cells . Mol Cell Endocrinol 230 : 31 –37 .15664449 23 Fortunati N , Fissore F , Fazzari A , Berta L , Benedusi-Pagliano E , et al (1993 ) Biological relevance of the interaction between sex steroid binding protein and its specific receptor of MCF-7 cells: effect on the estradiol-induced cell proliferation . J Steroid Biochem Mol Biol 45 : 435 –444 .8388711 24 Nakhla AM , Rosner W (1996 ) Stimulation of prostate cancer growth by androgens and estrogens through the intermediacy of sex hormone-binding globulin . Endocrinology 137 : 4126 –4129 .8828467 25 Mabjeesh NJ , Willard MT , Frederickson CE , Zhong H , Simons JW (2003 ) Androgens stimulate hypoxia-inducible factor 1 activation via autocrine loop of tyrosine kinase receptor/phosphatidylinositol 3′-kinase/protein kinase B in prostate cancer cells . Clin Cancer Res 9 : 2416 –2425 .12855613 26 Ma Y , Liang D , Liu J , Axcrona K , Kvalheim G , et al (2011 ) Prostate Cancer Cell Lines under Hypoxia Exhibit Greater Stem-Like Properties . PLoS One 6 : e29170 .22216200 27 Ishiguro H , Akimoto K , Nagashima Y , Kagawa E , Sasaki T , et al (2011 ) Coexpression of aPKClambda/iota and IL-6 in prostate cancer tissue correlates with biochemical recurrence . Cancer Sci 102 : 1576 –1581 .21535317 28 Bonaccorsi L , Carloni V , Muratori M , Salvadori A , Giannini A , et al (2000 ) Androgen receptor expression in prostate carcinoma cells suppresses alpha6beta4 integrin-mediated invasive phenotype . Endocrinology 141 : 3172 –3182 .10965888 29 Noble RL (1977 ) The development of prostatic adenocarcinoma in Nb rats following prolonged sex hormone administration . Cancer Res 37 : 1929 –1933 .858144 30 Pollard M , Luckert PH , Schmidt MA (1982 ) Induction of prostate adenocarcinomas in Lobund Wistar rats by testosterone . Prostate 3 : 563 –568 .7155989 31 Sirab N, Terry S, Giton F, Caradec J, Chimingqi M, et al. (2011) Androgens regulate Hedgehog signalling and proliferation in androgen-dependent prostate cells. Int J Cancer. 32 Kristiansen G , Machado E , Bretz N , Rupp C , Winzer KJ , et al (2010 ) Molecular and clinical dissection of CD24 antibody specificity by a comprehensive comparative analysis . Lab Invest 90 : 1102 –1116 .20351695 33 Hurt EM , Kawasaki BT , Klarmann GJ , Thomas SB , Farrar WL (2008 ) CD44+ CD24(-) prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis . Br J Cancer 98 : 756 –765 .18268494 34 He J, Liu Y, Zhu T, Zhu J, Dimeco F, et al. (2011) CD90 is identified as a marker for cancer stem cells in primary high-grade gliomas using tissue microarrays. Mol Cell Proteomics. 35 Kobayashi S , Takebe T , Inui M , Iwai S , Kan H , et al (2011 ) Reconstruction of human elastic cartilage by a CD44+ CD90+ stem cell in the ear perichondrium . Proc Natl Acad Sci U S A 108 : 14479 –14484 .21836053 36 Masson NM , Currie IS , Terrace JD , Garden OJ , Parks RW , et al (2006 ) Hepatic progenitor cells in human fetal liver express the oval cell marker Thy-1 . Am J Physiol Gastrointest Liver Physiol 291 : G45 –54 .16769813 37 Petersen BE , Goff JP , Greenberger JS , Michalopoulos GK (1998 ) Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat . Hepatology 27 : 433 –445 .9462642 38 Ben-Porath I , Thomson MW , Carey VJ , Ge R , Bell GW , et al (2008 ) An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors . Nat Genet 40 : 499 –507 .18443585 39 Falkenstein E , Tillmann HC , Christ M , Feuring M , Wehling M (2000 ) Multiple actions of steroid hormones–a focus on rapid, nongenomic effects . Pharmacol Rev 52 : 513 –556 .11121509 40 Heinlein CA , Chang C (2002 ) The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions . Mol Endocrinol 16 : 2181 –2187 .12351684 41 Revelli A , Massobrio M , Tesarik J (1998 ) Nongenomic actions of steroid hormones in reproductive tissues . Endocr Rev 19 : 3 –17 .9494778 42 Wehling M (1997 ) Specific, nongenomic actions of steroid hormones . Annu Rev Physiol 59 : 365 –393 .9074769 43 Perusquia M , Villalon CM (1999 ) Possible role of Ca2+ channels in the vasodilating effect of 5beta-dihydrotestosterone in rat aorta . Eur J Pharmacol 371 : 169 –178 .10357254 44 Farnsworth WE (2004 ) The androgen receptor of the prostate plasma membrane - an hypothesis . Med Hypotheses 62 : 954 –957 .15142656 45 Gonzalez-Guerrico AM , Meshki J , Xiao L , Benavides F , Conti CJ , et al (2005 ) Molecular mechanisms of protein kinase C-induced apoptosis in prostate cancer cells . J Biochem Mol Biol 38 : 639 –645 .16336777 46 Rodriguez-Berriguete G , Fraile B , Martinez-Onsurbe P , Olmedilla G , Paniagua R , et al (2012 ) MAP Kinases and Prostate Cancer . J Signal Transduct 2012 : 169170 .22046506 47 Sadar MD , Hussain M , Bruchovsky N (1999 ) Prostate cancer: molecular biology of early progression to androgen independence . Endocr Relat Cancer 6 : 487 –502 .10730903 48 Hamdi MM , Mutungi G (2010 ) Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres . J Physiol 588 : 511 –525 .20008468 49 Hamdi MM , Mutungi G (2011 ) Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2-dependent mechanism . J Physiol 589 : 3623 –3640 .21606113 50 Loukovaara M , Carson M , Adlercreutz H (1995 ) Regulation of production and secretion of sex hormone-binding globulin in HepG2 cell cultures by hormones and growth factors . J Clin Endocrinol Metab 80 : 160 –164 .7829605 51 Janne M , Hogeveen KN , Deol HK , Hammond GL (1999 ) Expression and regulation of human sex hormone-binding globulin transgenes in mice during development . Endocrinology 140 : 4166 –4174 .10465289 52 Salonia A , Briganti A , Gallina A , Karakiewicz P , Shariat S , et al (2009 ) Sex hormone-binding globulin: a novel marker for nodal metastases prediction in prostate cancer patients undergoing extended pelvic lymph node dissection . Urology 73 : 850 –855 .19038425	23936228	PMC3728318	CC BY	2021-01-05 17:35:12	yes	PLoS One. 2013 Jul 30; 8(7):e70558
==== Front Transl Med UniSaTransl Med UniSaTranslational Medicine @ UniSa2239-97472239-9747Università di Salerno tm-03-57ReviewThe novel therapeuthic targets in the treatment of chronic pain Palomba Rosa 1Bonaccia Paola 1Graffi Marco 1Costa Francesca 11 Department of Surgery, Anaesthesiology and Emergency-Resuscitation, “Giuseppe Zannini”, University of Naples “Federico II”, Napoly, Italy, (giovannini47@libero.it)30 4 2012 May-Aug 2012 3 57 61 2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Effective treatment for neuropathic pain is still lacking, because of poor understanding of pathological mechanisms at the molecular level. Chronic pain (inflammatory and neuropathic pain) is believed to be caused by aberrant neuronal responses along the pain transmission pathways. Both peripheral and central origins are likely to be involved in chronic pain, although their contribution may be different depending on the various forms of chronic pain. Glial cells have recently been implicated in neuropathic pain. These cells form close interactions with neurons and thus may modulate nociceptive transmission under pathological conditions. We will first examine the recent progress in the role of glia in neuropathic and inflammatory pain, with particular emphasis on microglia. Finally, we will discuss how the study of the interaction between neuronal and microglial mechanisms can open the door to new therapeutic opportunities, designed to act on the mechanisms underlying the disease (“disease-oriented”) using natural endogenous substances. Neuropathic painGliaPalmitoylethanolamide ==== Body I. INTRODUCTION To develop a better treatment for neuropathic pain, a comprehensive understanding of its pathogenesis is required. Chronic pain (such as inflammatory and neuropathic pain) is believed to be caused by aberrant neuronal responses along the pain transmission pathway from dorsal root ganglion (DRG) to spinal cord, thalamus and cortex. Both peripheral and central origins are likely to be involved in chronic pain, although their contribution may be different depending on the various forms of chronic pain. It has been recently reported that neurons are not the only cell type involved in chronic pain states. Glial cells, including astrocytes and microglia, are emerging as possible additional players in the initiation and maintenance of neuropathic and inflammatory pain. These glial cells have close interactions with neurons and thus modulate pain transmission particularly under pathological conditions [1–2]. II. THE NOVEL TARGETS Current therapeutic strategies for neuropathic pain aim to reduce the excitability of neurons by modulating ion channel activity (such as gabapentin and lidocaine) or by enhancing endogenous inhibitory mechanisms (such as tricyclic antidepressants and opioids). As we know, NMDA receptors play a critical role in synaptic plasticity within pain transmission pathways and are thus likely to be important in neuropathic pain. NR2B subunit-containing NMDA receptors are localized in pain-relevant structures, such as in superficial layers of the dorsal spinal horn, thalamus, hippocampus and cortex. The restricted distribution of NR2Bmakes it promising as a candidate target of side effect free analgesic drugs. Indeed, NR2B antagonists, such as ifenprodil and related compounds, are effective in neuropathic pain in animals, and show better separation between efficacy and side effects in human patients than non-selective NMDA receptor blockers. Our understanding of pathological pain has evolved from solely neuronal mechanisms to neuronglial interactions. In particular, astrocytes and microglia act as possible modulators of neuropathic pain by releasing a number of cytokines and chemokines. Astrocytes and microglia play different roles in relation to neuronal activity; however, they do have some overlapping functions in mediating CNS innate immune response [3]. Both astrocytes and microglia are activated in neuropathic pain, and their activation leads to pro-inflammatory responses with pathological effects, such as neuronal hyperexcitability, neurotoxcity and chronic inflammation. Astrocytes are the most abundant glial cell type in the CNS. In addition to their neuron-supportive functions, astrocytes also directly alter neuronal communication because they completely encapsulate synapses and are in close contact with neuronal somas [3,4]. The close astrocyte–neuron contact allows for astrocyte activation by neurotransmission as astrocytes express various functional neurotransmitter receptors. These include ionotropic non-NMDA and NMDA receptors as well as metabotropic glutamate (mGluR3 and mGluR5), purinergic and substance P receptors. After astrocyte activation, the extracellular signal-regulated kinase (ERK; also known as mitogenactivated protein kinase 1 (MAPK1)) and c-Jun N-terminal kinase (JNK; also known as MAPK8) signalling pathways are activated [5]. These lead to an increase in the synthesis of inflammatory factors (interleukin 1β (IL-1 β), IL-6, tumour-necrosis factor-α (TNF α), prostaglandin E2 (PGE2) and nitric oxide(NO), which finally alter glial glutamate transporter function and gap-junction proteins, which are known to facilitate astrocyte–astrocyte activation through Ca2+ cascades. Although similar pathways are activated in microglia after nerve injury, the temporal patterns of enzyme activation and pro-inflammatory cytokine release are distinct for microglia and astrocytes.. Furthermore, chronic astrocyte activation after nerve injury has been shown to involve ERK activation and subsequent down-regulation of excitatory amino acid transporters (glutamate transporter 1 (GLT1) and glutamate–aspartate transporter (GLAST) leading to a decrease in glutamate uptake and an increase in excitatory synaptic transmission. During chronic neuropathic conditions, astrocytes remain activated in response to the initial microglia-derived inflammatory factors; this is likely to account for their ongoing responses during these conditions. Microglia are the resident macrophages and main immune-response cells in the CNS [6]. They comprise 5–10% of the glial cell population and are quite evenly distributed in the brain. Under pathological conditions, these cells are activated and exhibit chemotactic, phagocytoxic and secretory responses to various stimuli. Resting ramified microglia rapidly transform into an activated state in most pathological conditions, including host defense against infectious organisms, autoimmune inflammation, ischemia, trauma, neurodegeneration and neuropathic pain [6,7]. Activation of microglia is accompanied by: changes in morphology, characterized by hypertrophy with retracted processes and associated with proliferation or microgliosis; upregulation of immune surface antigens included CD11b, P2X4 receptors, toll-like receptor 4, CD44, and MHC II. Finally, the activation of spinal microglia in neuropathic pain is characterized by phosphorylation of MAP kinases, including the p38 and Src-family kinases. Interestingly, spinal microglia activation occurs during the early phase of neuropathic pain and precedes astrogliosis, supporting the current hypothesis that microglia may be important for initiation, while astrocytes are important for the maintenance of neuropathic pain [8,10]. It is important to point out that none of the spinal glial cells project to the brain. Thus, the influence of glial changes may act through ascending neuronal transmission. Using transgenic mice in which microglia are selectively labeled with GFP, recent studies[ 9] performed systematic mapping of microglia in major pain-related brain areas following nerve injury. They have confirmed the microglial activation occurs only in spinal cord but not in supraspinal structures; Zhang et all showed the possibility that microglia are altered at the biochemical and molecular levels in neuropathic pain. Indeed, upregulation of microglial and astrocytic markers such as OX-42 and GFAP was observed in rat brain after peripheral administration of complete Freunds adjuvant (CFA) to produce inflammation [11]. Interestingly, astrogliosis was observed in the ACC after sciatic nerve ligation. In the setting of neuropathic pain, peripheral neurons transmit signals to spinal dorsal horn neurons, releasing neurotransmitters such as calcitonin gene-related protein (CGRP), substance P, glutamate, and ATP. Locally in the dorsal horn, there are also other neurotransmitters involved, such as GABA, glycine, serotonin. Spinal microglia activation happens via several signaling pathways, which include ATP and its receptors (P2X and P2Y receptor), fractalkine and CX3CR1, monocyte chemotactic protein (MCP-1) and CCR2. Similar to microglia in the brain, spinal microglia show fast chemotaxis in response to local application of ATP [12]. Microglia are known to express both ionotropic receptors, such as P2X4 and P2X7, and metabotropic receptors, such as P2Y6 and P2Y12 [13,14]. Activation of P2X4 in microglia facilitates BDNF release [15], while activation of P2X7 in microglia induces IL1β release and CXCL2 production [16,17]. Interestingly, The P2Y12 receptor in microglia is reported to mediate ATP-induced microglial chemotaxis [18,19] while P2Y6 may mediate microglial phagocytosis. Moreover, ATP induced both inward and outward current in resting microglia, which may be mediated by P2X and P2Y receptors, respectively [14]. In models of neuropathic pain, both P2X4 and P2Y12 receptors are upregulated in microglia, but not in neurons or astrocytes in the dorsal horn. Fractalkine and its receptor CX3CR1 are also involved in microglial activation associated with neuropathic pain. Fractalkine is a neuronal transmembrane glycoprotein that can be released after being cleaved by proteolysis; fractalkine activates p38 in spinal microglia and produces mechanical allodynia and thermal hyperalgesia [20]. The cleavage of fractalkine may involve cathepsin S, a cysteine protease that is expressed in spinal microglia. It has been shown that noxious stimulation of primary afferent fibers induces release of cathepsin S from microglia; the process may require P2X7 activation [21]. Microglial cells constitutively express CX3CR1, and its expression is markedly upregulated in models of neuropathic pain. In addition, MCP-1 and its receptor CCR2 are involved in microglial activation and neuropathic pain. Intrathecal injection of MCP-1 produced tactile allodynia, Microglial cells in the dorsal horn express CCR2, which may mediate MCP-1's effect: microglial morphology transitions and p38 activation in microglia. How might activated microglia contribute to neuropathic pain? One intriguing pathway involving ATP and the P2X4 receptor has been proposed. Peripheral nerve injury leads to an upregulation of P2X4 receptors in activated microglia. ATP acts on the P2X4 receptor in microglia and induces intracellular Ca2+ elevation and phosphorylation of p38, which subsequently increase BDNF synthesis and release. BDNF released from microglia may produce a shift in neuronal anion gradient with potassium-chloride exporter KCC2 reduced in dorsal horn neurons. This prompts a disinhibition and thus facilitates mechanical allodynia after nerve injury. It has been shown that p38 activation turns on the transcription factor NF-κB, which leads to the expression of IL-1β, IL6, and COX-2 [22]. These and other cytokines and chemokines released by activated microglia, such as tumor necrosis factor-α(TNFα), PGE2, and nitric oxide, will amplify microglial activation in an autocrine manner and may act directly on dorsal horn neurons to cause behavioral sensitization. Among the receptors we know to be upregulated on immune response cells under neuropathic pain conditions, there are also the cannabinoids receptors (CB1 and CB2). Cannabinoids suppress behavioural responses to noxious stimulation and suppress nociceptive transmission through activation of CB1 and CB2 receptor subtypes. CB1 receptors are mainly expressed at high levels in the central nervous system (CNS), whereas CB2 receptors are found predominantly, but not exclusively, on immune cells outside the CNS. Activation of CB1 and CB2 receptors inhibits adenylyl cyclase [23,24] and activates mitogen-activated protein kinase [25] through binding of the α-subunit of the Gi/o protein. Only CB1 receptors suppress calcium currents and activate inward-rectifying potassium channels: these effects are associated with depression of neuronal excitability and transmitter release. Thus, differences in receptor distribution and signal transduction mechanisms are likely to account for the relative absence of the CNS side effects induced by CB2 agonists. Microglia or macrophages exspress CB2 receptors in a different way in vivo or in vitro: in vitro its levels depend on the local environment and the combination of inflammatory molecules [26]; in vivo CB2 is not expressed equally in all microglial populations, but rather it is predominantly present in perivascular or activated microglia [27]. Treatment of activated microglia cultures with anandamide, the main endogenous CB1-ligand, decreases the expression of the inducible NOS-2 and the production of nitrites in a CB1- and CB2-dependent manner [28]. In addition, through CB2 receptors, cannabinoids inhibit the expression of TNF-a, IL-1b and the p40 subunit of IL-12 and IL-23 by microglia/macrophages [29]. Astrocytes express CB1 and CB2 receptors and they respond to cytokines secreted by the immune cells by regulating the production of molecules involved both in the bystander injury and in the protection of CNS tissue. Among these molecules, astrocytes express NOS-2 and they produce NO in response to several inflammatory signals. Cannabinoids inhibit the release of nitrites by astrocytes through a mechanism involving the CB2 receptor. In these cells, cannabinoids also inhibit the inflammation-induced expression of TNF-a, IL-1b and IL-6 through CB1 and CB2 receptors [28]. III. THE NOVEL THERAPEUTHIC STRATEGIES All these considerations together suggest that novel pharmacotherapies targeting CB2 receptors may have considerable therapeutic potential for suppressing inflammatory and neuropathic pain states. In animal models of tissue and nerve injury-induced nociception, CB2-selective agonists suppress hyperalgesia and allodynia and normalize nociceptive thresholds without inducing analgesia [30]. CB2 selective agonists may also be more efficacious in suppressing hypersensitivity to mechanical as opposed to thermal stimulation for reasons that remain incompletely understood. As mentioned above, a particularly beneficial aspect of the pharmacological profile of CB2 agonists is the failure of these compounds to induce adverse CNS side effects associated with activation of CB1 receptors. The available literature supports the efficacy of CB2 agonists in suppressing persistent pain states following acute administration. However, the impact of long-term treatment with CB2 agonists on antihyperalgesic efficacy and immune system function remains largely unknown [31]. More work is needed to identify the limitations associated with therapeutic strategies targeting CB2 receptors and to explore the therapeutic potential of multimodal analgesic strategies that combine CB2-mediated pharmacotherapies for pain with other agents directed at different analgesic targets. Such strategies offer the potential to produce synergistic antihyperalgesic effects with a more beneficial therapeutic ratio compared to conventional analgesics (for example, by combining a CB2-selective agonist with lower doses of opiates, CB1 agonists or nonsteroidal anti-inflammatory drugs that are below the threshold for inducing undesirable side effects). Taking into account that the endocannabinoids are synthesized only “on demand” and that tissue accumulation of N-acylated glycerophospholipids, and free N-acylamides, such as anandamide (AEA) and palmitoylethanolamide(PEA), occurs in some pathological conditions known to be associated with inflammatory and pain reactions, several studies have been carried out to identify the beneficial effects associated with therapeutic use of PEA [32]. PEA is an endogenous fatty acid and with properties comparable to anandaminde, as well as other endogenous cannabinoids. PEA may behave as local autacoids capable of negatively modulating mast cell activation (ALIA mechanism) [33]. In keeping with this hypothesis, the direct anti-inflammatory effect of PEA is parly linked to its modulation of mast cells degranulation, thus to inhibition of the release of several pro-inflammatory enzymes such as iNOS, chynase and metalloproteinase MMP-9, as well as mediators such as nitric oxide and TNF-α. PEA pharmacological effects could be mediated by its interactions with CB2 receptors, or CB2 like-receptors, highly exspressed on mast cells and neuronal cells [32]. Lipids like N-palmitoylethanolamine can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions. The signaling lipid PEA is known to activate and membrane-associated, intracellular and nuclear receptors (PPAR-alpha) and regulate many physiological functions related to the inflammatory cascade, and thus is of high interest in the treatment of neuropathic, or gliopathic pain. Meanwhile it is an established fact that anandamide (AEA) and PEA regulate directly or indirectly many of the same pathophysiological processes, including pain perception, inflammation, convulsions and neuroprotection. There are a number of biological effects of endocannabinoids which can be enhanced by related endogenous fatty acid derivatives which are devoid of some of these primary effects. The last mechanism of action is called the entourage effect [34]. The fatty acids as PEA seem to be co-synthesized and co-released with the endocannabinoids such as anandaminde. Anandamide and PEA both are present in the spinal cord, but the concentration of PEA is most probably 10 times higher. The entourage effect of PEA most probably is due to its function of amplifier activity anti-inflammatory and antinociceptive of other endogenous compounds such as endocannabinoid anandamide. It has been suggested that PEA enhances the effects of AEA by acting as a competitive inhibitor of the enzymatic degradation of endocannabinoids, and increaseing the affinity of the AEA for the CB1 and vanilloid receptors (TRPV1), highly exspressed on neuronal and mast cells [34]. Transient Receptor Potential Vanilloid 1 (TRPV1) is a Ca2+ permeant non-selective cation channel and they are exspressed highly on sensorial fiber nerve endings (FNE) and they can act as a transducers of noxious temperature and chemical stimuli [35]. TRPV1 expression is increased in inflammation and neuropathic pain because of the retrograde transport of nerve growth factor (NGF) released at the site of peripheral tissue injury to the DRG soma. The possibility of using TRPV1 receptor agonists, such as capsaicin, to alleviate pain is an interesting concept. Capsaicin acts first exciting FNE, than leading to TRPV1 down-regulation. As a result of this FNE show a loss of pain sensibility known as analgesia. Moreover capsaicin inhibits the retrograde transport of NGF. Activation of TRPV1 by capsaicin can induce persistent depolarization of the nerve terminals causing a decrease in their ability to generate and propagate action potentials [36]. On the other hand, TRPV1 activation causes a massive influx of Ca2+ that in the long-term can cause nerve terminal degeneration. The use of transdermal capsaicin has been shown to reduce FNE density in epidermis in a reversible manner and to cause a long-lasting loss of thermal sensitivity without affecting mechanical sensitivity. This effect is dose dependent and it suggests the use of a novel generation of analgesics suche as the patch to high concentrations of capasaicin in the treatment of neuropathic or gliopathic pain. ==== Refs REFERENCES [1] Inoue K Tsuda M Microglia and neuropathic pain Glia 2009 57 1469 1479 19306358 [2] McMahon SB Malcangio M Current challenges in glia-pain biology Neuron 2009 64 46 54 19840548 [3] Milligan ED Watkins LR Pathological and protective roles of glia in chronic pain Nat Rev Neurosci 2009 10 23 36 19096368 [4] Zhuo M Neuronal mechanism for neuropathic pain Mol Pain 2007 3 14 17553143 [5] Matricon J Gelot A Etienne M Lazdunski M Muller E Ardid D Spinal cord plasticity and acid-sensing ion channels involvement in a rodent model of irritable bowel syndrome Eur J Pain 2011 15 335 343 20888277 [6] Streit WJ Mrak RE Griffin WS Microglia and neuroinflammation: a pathological perspective J Neuroinflammation 2004 1 14 15285801 [7] Tsuda M Inoue K Salter MW Neuropathic pain and spinal microglia: a big problem from molecules in “small” glia Trends Neurosci 2005 28 101 107 15667933 [8] Ji RR Suter MR p38 MAPK, microglial signaling, and neuropathic pain Mol Pain 2007 3 33 17974036 [9] Boucsein C Kettenmann H Nolte C Electrophysiological properties of microglial cells in normal and pathologic rat brain slices Eur J Neurosci 2000 12 2049 2058 10886344 [10] Gosselin RD Suter MR Ji RR Decosterd I Glial Cells and Chronic Pain Neuroscientist 2010 [11] Raghavendra V Tanga FY DeLeo JA Complete Freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflammatory cytokine expression in the CNS Eur J Neurosci 2004 20 467 473 15233755 [12] Chen T Koga K Li XY Zhuo M Spinal microglial motility is independent of neuronal activity and plasticity in adult mice Mol Pain 2010 6 19 20380706 [13] Inoue K Microglial activation by purines and pyrimidines Glia 2002 40 156 163 12379903 [14] James G Butt AM P2Y and P2X purinoceptor mediated Ca2+ signaling in glial cell pathology in the central nervous system Eur J Pharmacol 2002 447 247 260 12151016 [15] Trang T Beggs S Wan X Salter MW P2X4-receptor-mediated synthesis and release of brain-derived neurotrophic factor in microglia is dependent on calcium and p38-mitogen-activated protein kinase activation J Neurosci 2009 29 3518 3528 19295157 [16] Clark AK Staniland AA Marchand F Kaan TK McMahon SB Malcangio M P2X7-dependent release of interleukin-1beta and nociception in the spinal cord following lipopolysaccharide J Neurosci 2010 30 573 582 20071520 [17] Shiratori M Tozaki-Saitoh H Yoshitake M Tsuda M Inoue K P2X7 receptor activation induces CXCL2 production in microglia through NFAT and PKC/MAPK pathways J Neurochem 2010 114 810 819 20477948 [18] Haynes SE Hollopeter G Yang G Kurpius D Dailey ME Gan WB Julius D The P2Y12 receptor regulates microglial activation by extracellularnucleotides Nat Neurosci 2006 9 1512 1519 17115040 [19] Koizumi S Shigemoto-Mogami Y Nasu-Tada K Shinozaki Y Ohsawa K Tsuda M Joshi BV Jacobson KA Kohsaka S Inoue K UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis Nature 2007 446 1091 1095 17410128 [20] Zhuang ZY Kawasaki Y Tan PH Wen YR Huang J Ji RR Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine Brain Behav Immun 2007 21 642 651 17174525 [21] Clark AK Wodarski R Guida F Sasso O Malcangio M Cathepsin S release from primary cultured microglia is regulated by the P2X7 receptor Glia 2010 58 1710 1726 20629190 [22] Krakauer T Molecular therapeutic targets in inflammation: cyclooxygenase and NF-kappaB Curr Drug Targets Inflamm Allergy 2004 3 317 324 15379601 [23] Slipetz DM O’Neill GP Favreau L Dufresne C Gallant M Gareau Y Activation of the human peripheral cannabinoid receptor results in inhibtion of adenylyl cyclase Mol Pharmacol 1995 48 352 361 7651369 [24] De Petrocellis L Bisogno T Ligresti A Bifulco M Melck D Di Marzo V Effect on cancer cell proliferation of palmitoylethanolamide, a fatty acid amide interacting with both the cannabinoid and vanilloid signalling systems Fundam Clin Pharmacol 2002 16 297 302 12570018 [25] Bouaboula M Poinot-Chazel C Marchand J Canat X Bourrié B Rinaldi-Carmona M Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor. Involvement of both mitogen-activated protein kinase and induction of Krox-24 expression Eur J Biochem 1996 237 704 711 8647116 [26] Maresz K Carrier EJ Ponomarev ED Hillard CJ Dittel BN Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli J Neurochem 2005 95 437 445 16086683 [27] Clayton N Marshall FH Bountra C O’Shaughnessy CT CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain Pain 2002 96 253 260 11972997 [28] Gutierrez T Farthing JN Zvonok AM Makriyannis A Hohmann AG Activation of peripheral cannabinoid CB1 and CB2 receptors suppresses the maintenance of inflammatory nociception: a comparative analysis Br J Pharmacol 2007 150 153 163 17160008 [29] DeLeo JA Yezierski RP The role of neuroinflammation and neuroimmune activation in persistent pain Pain 2001 90 1 6 11166964 [30] Clayton N Marshall FH Bountra C O’Shaughnessy CT CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain Pain 2002 96 253 260 11972997 [31] Fox A Kesingland A Gentry C McNair K Patel S Urban L 2001 The role of central and peripheral Cannabinoid1 receptors in the antihyperalgesic activity of cannabinoids in a model of neuropathic pain Pain 92 91 100 11323130 [32] Re G Barbero R Miolo A Di Marzo V Palmitoylethanolamide, endocannabinoids and related cannabimimetic compounds in protection against tissue inflammation and pain: potential use in companion animals Vet J. 2007 1 173 1 21 30 Epub 2005 Dec 1. 16324856 [33] Skaper SD Buriani A Dal Toso R Petrelli L Romanello S Facci L Leon A The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons Proc Natl Acad Sci U S A 1996 4 30 93 9 3984 9 8633002 [34] Bachur NR Masek K Melmon KL Udenfriend S Fatty acid amides of ethanolamine in mammalian tissues J Biol Chem 1965 3 240 1019 24 14284696 [35] Avelino A Cruz C Nagy I Cruz F 2002 Vanilloid receptor 1 expression in the rat urinary tract Neuroscience 109 787 798 11927161 [36] Holzer P 1991 Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons Pharmacol Rev 43 143 201 1852779	23905053	PMC3728790	CC BY	2021-01-04 23:01:17	yes	Transl Med UniSa. 2012 Apr 30; 3:57-61
==== Front Transl Med UniSaTransl Med UniSaTranslational Medicine @ UniSa2239-97472239-9747Università di Salerno tm-05-07ArticlesVasopressin vs Terlipressin in Treatment of Refractory Shock Scarpati G Piazza O University of Salerno, Salerno, Italy(giulianascarpati@me.com)4 1 2013 Jan-Apr 2013 5 22 27 2013This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Arginine vasopressin (AVP) and its synthetic, long-acting analog terlipressin (TP) are potent alternative vasoconstrictors in the treatment of septic patients with catecholamine-refractive vasodilatatory shock. Recent clinical data suggest that early administration of AVP analogues may be advantageous compared to a last resort therapy. However, it is still unknown whether vasopressin and terlipressin are equally effective for hemodynamic support in shock. Despite important pharmacological differences between the two drugs the use of either substance is determined mainly by local availability and institutional inventory. The current literature suggests that neither AVP nor TP should be administered in high doses in shock. Furthermore, increasing evidence indicates that early administration of terlipressin may improve outcome as compared to a last-resort treatment. Low-dose infusion of AVP has been demonstrated to be a safe adjunct in the management of refractory shock. Evidence from experimental studies and initial clinical reports suggests that continuous low-dose infusion of TP may stabilize hemodynamics in shock. In this review we briefly described differences in pharmacokinetics and pharmacodynamics between AVP and Terlipressin (TP) in treatment of refractory shock. VasopressinTerlipressin ==== Body I. VASOPRESSIN IN SHOCK Vasopressin (AVP) is a polypeptide with a disulphide bond between the two cysteine amino acids [1]. In humans AVP is encoded by the mRNA for preproneurophysin II. After cleavage of the signal peptide, the resulting prohormone contains AVP (nine amino acids), neurophysin II (95 amino acids) and a glycopeptide (39 amino acids). The prohormone is synthesized of the supraoptic and paraventricular nuclei of the hypothalamus. The final hormone is transported by the neurones of the hypothalamo-neurohypophyseal bundle of the pituitary gland to the secretion site, namely the posterior hypophysis. It is then stored in granule form. Of the total stock of vasopressin, 10–20% can be rapidly released into the bloodstream [2]. Secretion diminishes if the stimulus continues. This kinetic action explains the biphasic course of vasopressin plasma concentrations during septic shock, with an early elevation followed by subsequent diminution [3]. Vasopressin secretion is complex and depends upon plasma osmolarity and blood volume. The central osmoreceptors that regulate vasopressin secretion are located near to the supraoptic nucleus in the anterolateral hypothalamus in a region with no blood-brain barrier.[4]. There are also peropheralosmoreceptors at he level of the hepatic portal vein that detect early the osmotic impact of ingestion of foods and fluids. The afferent pathways reach the magnocellularneurones of the hypothalamus via the vagal nerve. These neurones are depolarized by hypertonic conditions and hyperpolarized by hypotonic conditions [5]. In contrast to osmotic stimulation, arterial hypotension and hypovolaemia stimulate vasopressin exponentially. Arterial hypotension is the principal stimulus for vasopressin secretion via arterial baroreceptors located in the aortic arch and the carotid sinus [6]. It is transported by the vagal and glossopharyngeal nerves toward the nucleus tractussolitarus and then toward the supraoptic and paraventricular nuclei. Inhibition of this secretion is principally linked to volume receptors located in the cardiac cavities [7]. In a physiological situation, inhibition is constant because of diminishes then vasopressin secretion increases [8]. If central venous pressure diminishes, then these receptors first stimulate secretion of natriuretic factor, the sympathetic system, and renin secretion. Vasopressin is secreted when arterial pressure falls to the point that it can no longer be compensated for by the predominant action of the vascular baroreceptors [9–11]. Other stimuli can favour secretion of vasopressin. These include hypercapnia, hypoxia, hyperthermia, pain, nausea, morphine and nicotine [12]. At the hormone level, numerous molecules are direct stimulators, including acetylcholine, histamine, nicotine, angiotensin II, prostaglandins, dopamine and, especially, the adrenergic system [13]. Noradrenaline (norepinephrine) has a complex effect on vasopressin secretion [12]. At low concentrations it increases activity. At high concentrations it inhibits the production of vasopressin [14]. Nitric oxide (NO), through cGMP, is a powerful neurohormonal inhibitor of vasopressin. This pathway is of fundamental importance in the case of septic shock [15,16]. Opiates, alcohol, γ-aminobutyric acid, and auricular natriuretic factor are also inhibitors. Vasopressin acts through several receptors, These receptors are different from those of catecholamines. Vasopressin has a direct vasoconstrictor effect on systemic vascular smooth muscle via V1receptors. The same type of receptor was found on platelets, which are another storage location for vasopressin [17, 18]. The V2 receptors in the renal collecting tubule are responsible for regulating osmolarity and blood volume. At certain concentrations, vasopressin provokes vasodilatation in some vascular regions. Vasopressin also acts as a neurotransmitter. The vasoconstrictor activity of vasopressin, which is mediated by the receptors, is intense in vitro. There is also a V1 probable indirect action on vascular smooth muscle cells by local inhibition of NO production [19]. However, under physiological conditions, vasopressin has only a minor effect on arterial pressure [20]. One experimental hypothesis is that the vasopressor effect of vasopressin is secondary to its capacity to inhibit smooth muscle cell K+-ATP channels [21]. This moderate effect observed in vivo can be explained by the indirect bradycardic effect resulting from vasopressin’s action on baroreflexes [22]. This effect on baroreflexes is mediated by the cerebral V1receptors [23]. It requires integrity of the cardiac baroreflexes because it disappears after administration of a ganglioplegic agent. Vasopressin concentrations of approximately 50 pg/ml are required before any significant modification becomes apparent [24, 25]. In shock the haemodynamic response to vasopressin becomes important in maintaining arterial pressure and tissue perfusion. Administration of V1 receptor antagonists in animals in haemorrhagic shock increases hypotension [26]. Vasopressin concentrations increase during the initial phase of shock. Thus, contrary to what is observed under physiological conditions, when the autonomous nervous system is deficient and baroreflexes altered the vasopressor effect becomes predominant and prevents severe hypotension [27]. However, its trigger differs from that of catechol-amines on several levels. Vasopressin provokes a reduction in cardiac output and its vasoconstrictor activity is heterogeneous on a topographical level [28]. Its administration provokes vasoconstriction in skin, skeletal muscle, adipose tissue, pancreas and thyroid [26]. This vasoconstriction is less apparent in the mesenteric, coronary and cerebral territories under physiological conditions [29]. Its impact on digestive perfusion is under debate. Two studies conducted in patients with septic shock [30, 31] demonstrated absence of impact of vasopressin on splanchnic circulation. In contrast, in a recent study conducted in animals in a state of endotoxaemic shock [32], a reduction in digestive perfusion with vasopressinadministration was observed. Finally, contrary to catechol-amines, whose effect can only be additive, vasopressin potentiatesthe contractile effect of other vasopressor agents [33]. The vasodilatation of certain vascular regions with vasopressin is an further major difference from catecholamines. This effect occurs at very low concentrations [34]. The literature is limited on this subject. Animal studies have been reported, butthey were not conducted in the context of sepsis. Some authors reported vasodilatation at a cerebral level in response to vasopressin, with more marked sensitivity to vasopressin in the circle of Willis [32]. The mechanism of this vasodilatation can be explained by production of NO at the level of the endothelial cells. The receptors involved have not been clearly identified. It has been shown that vasopressin provokes vasodilatation of the pulmonary artery both under physiological and hypoxic conditions [35]. The V1 receptors are involved and cause endothelial liberation of NO [36]. The renal effect of vasopressin is complex. In response to blood hyperosmolarity it reduces urine output through its action on the V2receptors, which induce reabsorption of water. Inversely, it has diuretic properties in case of septic shock and congestive heart failure [37]. The mechanisms involved in the reestablishment of diuresis are poorly understood. The principal hypothetical mechanisms are a counter-regulation of the V2 receptors and selective vasodilatation of the afferent arteriole (under the action of NO) in contrast to vasoconstriction of the efferent arteriole [38]. Patel and coworkers [31] recently reported a randomized study in which there were significant improvements in diuresis and creatinine clearance in patients with septic shock under vasopressin treatment as compared with patients treated with noradrenaline. It has been shown in nonseptic rats that elevated concentrations of this hormone provoked a dose-dependent fall in renal blood output, glomerular filtration, and natriuresis [39]. All of the investigators who found a beneficial effect following treatment with vasopressin for septic shock used minimal doses, allowing for readjustment to achieve physiological concentrations. Vasopressin acts on the corticotrophic axis by potentiating the effect of the corticotrophin-releasing hormone on the hypophyseal production of adrenocorticotrophic hormone [40]. The ultimate effect is an elevation in cortisolaemia [41], which is of interest in the case of septic shock because cortisol levels can be lowered. At a supraphysiological dose, vasopressin acts as a platelet-aggregating agent [42]. The coagulation problems in septic shock make this effect undesirable. However, the doses used are unlikely to provoke a significant aggregation effect. II. TERLIPRESSIN IN SHOCK Terlipressin (TP) is a synthetic analogue of AVP characterized by greater selectivity for the V1 receptor than AVP [43]. The vasopressor (V1 receptor-mediated) to antidiuretic (V2 receptor-mediated) ratios of AVP and TP are 1 and 2.2, respectively (Fig.1).[44]. The elimination half-life of TP is longer than that of AVP (50 vs. 6 min) [45]. As a prodrug, TP is cleaved by endopeptidases, resulting in retarded release of the active metabolite lysine vasopressin (LVP). Data on TP plasma concentrations after bolus injection of the drug are limited, and unfortunately no data on plasma levels after continuous TP infusion are currently available. Following bolus injection of 10 μg/kg TP in 14 healthy volunteers (equivalent to 0.7mg in a 70-kg subject), Nilsson et al. reported a peak plasma level of approx. 52,000 pg/ml within 5 min and a decline to approx. 2,750 pg/ml within 1 hour after administration. Notably, the very high TP peak plasma levels measured at this time point may have occurred because levels were determined during the distribution half-life of the drug. With respect to the different preparations (prodrug vs. active agent) the pharmacokinetics of TP and AVP are difficult to compare. Following enzyme kinetics the organism degrades TP into diglycyl-, monoglycyl-lysine vasopressin, and LVP. In the same study Nilsson et al. determined LVP plasma levels after bolus injection of 5 μg/kg TP (equivalent to 0.35 mg in a 70-kg subject). Peak LVP plasma concentrations were detected 60 min after TP bolus and averaged 106 pg/ml. Thus plasma concentrations of the main bioactive component LVP after 5 μg/kg TP bolus injection (i.e., 106 pg/ml) are comparable to plasma levels reached by continuous infusion of 1.8–2.4U/h AVP (i.e., 100–300 pg/ml). Forsling et al. [46] reported that LVP plasma levels in healthy humans increased 40–60 min after intravenous bolus administration of 7.5 μg/kg TP and reached its peak after 60–120 min. Whereas the onset of antidiuretic effects after TP infusion were observed after approx. 120 min, the onset of vasopressor effects was detected after only 3 min. These observations indicate that the renal V2 receptor-mediated effects after TP injection are dependent on the release of LVP, while TP exerts intrinsic effects on V1 receptors. The fact that the effective half-life of TP is markedly longer than that of AVP (4–6 h vs. 6–20 min) provides a theoretical rationale for administering TP as intermittent bolus infusion of 0.5–1 mg (–2) in patients with septicshock [47– 50]. The first clinical trial of the efficacy of terlipressin in septic shock was performed in a small case series of eight patients [50]. Terlipressin was administered as a single bolus of 1 mg (the dosage used in gastroenterological practice) in patients with septic shock refractory to catecholamine–hydrocortisone–methylene blue. A significant improvement in blood pressure was obtained in these patients during the first 5 hours. Cardiac output was reduced, which might have impaired oxygendelivery. Partial or total weaning from catecholamines was possible. No other side effect was observed. Another study was conducted in 15 patients with catecholamine-dependent septic shock (noradrenaline ≥ 0.6 μg/kg per min). An intravenous bolus of 1 mg terlipressin was followed by an increase in MAP and a significant decrease in cardiac index. Oxygen delivery and consumption were significantly decreased [48]. Gastric mucosal perfusion was evaluated by laser Doppler flowmetry and was increased after terlipressin injection. The ratio between gastric mucosal perfusion and systematic oxygen delivery was also significantly improved after terlipressin injection. These findings could be related to a positive redistribution effect of cardiac output on hepatosplanchnic circulation, with an increase in blood flow to the mucosa. The adverse effects of terlipressin on oxygen metabolism were also emphazised in an experimental study conducted in sheep [51]. Terlipressin was given by continuous infusion (10–40 mg/kg per hour) and was responsible for a significant decrease in cardiac index and oxygen delivery. Oxygen consumption decreased whereas oxygen extraction increased. These modifications may carry a risk for tissue hypoxia, expecially in septic states in which oxygen demand is typically incrased. Terlipressin was also used in children [52] in a short case series of four patients with catecholamine-resistant shock. MAP increased, allowing reduction or withdrawal of noradrenaline. Two children died. III. SUMMARY AND CONCLUSIONS Both AVP and TP have been proven effective in restoring sepsis-related arterial hypotension and reducing catecholamine requirements in the experimental [53–55] and clinical [56,48,49,30] setting. In high doses, however, administration of either compound may be associated with adverse effects, basically related to excessive systemic and/or regional vasoconstriction, leading to a reduction in cardiac output and systemic oxygen delivery [30,49], impairment of intestinal microcirculation [48,57], increase in pulmonary vascular resistance [58, 59], ischemic skin lesions [60], or elevated surrogate parameters of liver injury [61,62]. The higher V1 receptor selectivity of TP towards AVP may be more potent in restoring refractory hypotension related to septic shock. The longer effective half-life of TP than AVP may help avoiding rebound hypotension after discontinuation of the drug [63] but, on the other hand, carries the risk of excessive vasoconstriction after bolus injection [64]. Results from experimental studies and clinical case reports suggest that continuous infusion of low-dose TP [65] may stabilize hemodynamics in shock with reduced side effects towards injections of 1 mg (–2) boli [60]. However, future randomized controlled clinical trials are warranted to elucidate the significance of continuous low-dose and bolus TP infusion vs. infusion of AVP in patients with shock. Due to the lack of evidence from comparative studies, bolus or continuous administration of TP should currently be limited to controlled clinical trials. Fig. 1. Signal transduction of vasopressin analogues on V1 receptor in vascular smooth muscle cells. Stimulation of V1 receptors by vasopressin analogues such as arginine vasopressin (AVP) and terlipressin (TP) mediates the hydrolysis of phosphatidylinositol bisphosphate to inositol triphosphate (IP3) and diacylglycerol (DAG) via phospholipase C (PLC). Those second messengers facilitate actin-myosin interactions by increasing intracellular calcium (Ca2+) concentrations through various mechanisms including activation of receptor-operated Ca2+ channels, voltage-gated Ca2+ channels via protein kinase C (PKC), and emptying of intracellular Ca2+ stores. ==== Refs REFERENCES [1] Barberis C Mouillac B Durroux T Structural Bases of Vasopressin/Oxytocin Receptor Function J Endocrinol 1998 156 223 229 9518866 [2] Holmes CL Patel BM Russell JA Physiology of Vasopressin Relevant to Management of Septic Shock Chest 2001 120 989 999 11555538 [3] Sklar AH Schrier RW Central Nervous System Mediators of Vasopressin Release Physiol Rev 1983 63 1243 1280 6197715 [4] Nicolet-Barousse L Sharshar T Paillard M Raphael JC Annane D Vasopressin: a Hormone with Multiple Functions MèdThèrEndocrinol 2001 7 757 764 [5] Bourque CW Oliet SH Richard Osmoreceptors, Osmoreception, and Osmoregulation Front Neuroendocrinol 1994 15 231 274 7859914 [6] Forrest P Vasopressin and shock Anaesth Intensive Care 2001 29 463 472 11669425 [7] Leng G Dyball RE Russell JA Neurophysiology of Body Fluid Homeostasis Comp BiochemPhysiol A 1988 90 781 788 [8] Bisset GW Chowdrey HS Control of Release of Vasopressin by Neuroendocrine Reflexes Q J ExpPhysiol 1988 73 811 872 [9] Goldsmith SR Francis GS Cowley AW Cohn JN Response of Vasopressin and Norepinephrine to Lower Body Negative Pressure in Humans Am J Physiol 1993 243 H970 H973 7149050 [10] Trasher TN Baroreceptor Regulation of Vasopressin and Renin Secretion: Low Pressure Versus High Pressure Receptors Front Neuroendocrinol 1994 15 157 187 7813742 [11] O’Donnell CP Thompson CJ Keil LC Thrasher TN Renin and Vasopressin Responses to Graded Reductions in Atrial Pressure in Conscious Dogs Am J Physiol 1994 266 R714 R721 8160864 [12] Leng G Brown CH Russell JA Physiological Pathways Regulating the Activity of Magnocellular Neurosecretory Cells Prog Neurobiol 1999 57 625 655 10221785 [13] Sklar AH Schrier RW Central Nervous System Mediators of Vasopressin Release Physiol Rev 1983 63 1243 1280 6197715 [14] Day TA Randle JC Renaud LP Opposing α- and β-Adrenergic Mechanisms Mediate Dose-Dependent Actions of Norepinephrine on Supraoptic Vasopressin Neuronesin Vivo Brain Res 1985 358 171 179 3000512 [15] Forrest P Vasopressin and Shock Anaesth Intensive Care 2001 29 463 472 11669425 [16] Nicolet-Barousse L Sharshar T Paillard M Raphael JC Annane D Vasopressin. a Hormone with Multiple Functions MédThérEndocrinol 2001 7 757 764 [17] Thibonnier M Roberts JM Characterization of Human Platelet Vasopressin Receptors J Clin Invest 1985 76 1857 1864 2997293 [18] Bichet DG Razi M Lonergan M Arthus MF Papukna V Kortas C Barjon JN Human Platelet Fraction Arginine vasopressin. Potential Physiological Role J Clin Invest 1987 79 881 887 2950136 [19] Abboud FM Floras JS Aylward PE Guo GB Gupta BN Schmid PG Role of Vasopressin in Cardiovacular and Blood Pressure Regulation Blood Vessels 1990 27 106 115 2242439 [20] Abboud FM Floras JS Aylward PE Guo GB Gupta BN Schmid PG Role of Vasopressin in Cardiovacular and Blood Pressure Regulation Blood Vessels 1990 27 106 115 2242439 [21] Wakatsuki T Nakaya Y Inoue I Vasopressin Modulates K+-Channel Activities of Cultured Smooth Muscle Cells from Porcine Coronary Artery Am J Physiol 1992 263 H491 H496 1387293 [22] Reid I Role of Vasopressin Deficiency in the Vasodilation of Septic Shock Circulation 1997 95 1108 1110 9054835 [23] Luk J Role of V1 Receptors in the Action of Vasopressin on the Baroreflex Control of Heart Rate Am J Physiol 1993 265 R524 R529 8214142 [24] Cowley AW Jr Switzer SJ Guinn MM Evidence and Quantification of the Vasopressin ArterialPressure Control System in the Dog Circ Res 1980 46 58 67 7349918 [25] Mohring J Glanzer K Maciel JA Jr Dusing R Kramer HJ Arbogast R Koch-Weser J Greatly Enhanced Pressor Response to Antidiuretic Hormone in Patients with Impaired Cardiovascular Reflexes Due to Idiopathic Orthostatic Hypotension J CardiovascPharmacol 1980 2 367 376 [26] Laszlo FA Laszlo F Jr De Wield D Pharmacology and Clinical Perspectives of Vasopressin Antagonists Pharmacol Rev 1991 43 73 108 1852013 [27] Jordan J Tank J Dietrich A Clarck JP Benton MD Vasopressin and Blood Pressure in Humans Hypertension 2000 36 E3 E4 11116134 [28] Liard JF Deriaz O Schelling P Thibonnier M Cardiac Output Distribution During Vasopressin Infusion or Dehydration in Conscious Dogs Am J Physiol 1982 243 H663 H669 7137358 [29] Altura BM Dose–Response Relationships for Arginine Vasopressin and Synthetic Analogs on Three Types of Rat Blood Vessels: Possible Evidence for Regional Differences in Vasopressin Receptor Sites Within a Mammal J PharmacolExpTher 1975 193 413 423 [30] Kerr JC Jain KM Swan KG Rocko JM Effects of Vasopressin on Cardiac Output and its Distribution in the Subhuman Primate J VascSurg 1985 2 443 449 [31] Dunser MW Mayr AJ Ulmer H Knotzer H Sumann G Pajk W Arginine Vasopressin in Advanced Vasodilatory Shock. A Prospective, Randomized, Controlled Study Circulation 2003 107 2313 2319 12732600 [32] Patel BM Chittock DR Russell JA Walley KR Beneficial Effects of Short-Term Vasopressin Infusion During Severe Septic Shock Anesthesiology 2002 96 576 582 11873030 [33] Suzuki Y Satoh S Oyama H Takayasu M Shibuya M Regional Differences in the Vasodilator Response to Vasopressin in Canine Cerebral Arteries in Vivo Stroke 1993 24 1049 1053 7686696 [34] Karmazyn M Manku MS Horrobin DF Changes of Vascular Reactivity Induced by Low Vasopressin Concentrations: Interactions with Cortisol and Lithium and Possible Involvement of Prostaglandins Endocrinology 1978 102 1230 1236 744021 [35] Okamura T Toda M Ayajiki K Toda N Receptor Subtypes Involved in Relaxation and Contraction by Arginine Vasopressin in Canine Isolated Short Posterior Ciliary Arteries J Vasc Res 1997 34 464 472 9425999 [36] Eichinger MR Walker BR Enhanced Pulmonary Arterial Dilation to Arginine Vasopressin in Chronically Hypoxic Rats Am J Physiol 1994 267 H2413 H2419 7528996 [37] Evora PR Pearson PJ Schaff HV Arginine Vasopressin Induces Endothelium Dependant Vasodilatation of the Pulmonary Artery: V1-Receptor- Mediated Production of Nitric Oxide Chest 1993 103 1241 1245 8131474 [38] Eisenman A Armali Z Enat R Bankir L Baruch Y Low Dose Vasopressin Restores Diuresis both in Patients with Hepatorenal Syndrome and in Anuric Patients with End-Stage Heart Failure J Intern Med 1999 246 183 190 10447787 [39] Edwards RM Trizna W Kinter LB Renal Microvascular Effects of Vasopressin and Vasopressin Antagonists Am J Physiol 1989 256 F274 F278 2916660 [40] Harrison-Bernard LM Carmines PK Juxtamedullary Micro-Vascular Responses to Arginine Vasopressin in Rat Kidney Am J Physiol 1994 267 F249 F256 8067385 [41] Chrousos G The Hypothalamic-Pituitary-Adrenal Axis and Immune-Mediated Inflammation N Engl J Med 1995 32 1351 1360 7715646 [42] Antoni FA Vasopressinergic Control of Pituitary Adrenocorticotropin Secretion Comes on Age Front Neuroendocrinol 1993 14 76 122 8387436 [43] Leung FW Jensen DM Guth PH Endoscopic Demonstration That Vasopressin but Not Propranolol Produces Gastric Mucosal Ischaemia in Dogs with Portal Hypertension GastrointestEndosc 1988 34 310 313 [44] Pesaturo AB Jennings HR Voils SA Terlipressin: Vasopressin Analog and Novel Drug for Septic Shock Ann Pharmacother 2006 40 2170 2177 17148649 [45] Bernadich C Bandi JC Melin P Bosch J Effects of F-180, a New Selective Vasoconstrictor Peptide, Compared with Terlipressin and Vasopressin on Systemic and Splanchnic Hemodynamics in a Rat Model of Portal Hypertension Hepatology 1998 27 351 356 9462630 [46] Nilsson G Lindblom P Ohlin M Berling R Vernersson E Pharmacokinetics of Terlipressin After Single i.v. Doses to Healthy Volunteers Drugs Exp Clin Res 1990 16 307 314 2086166 [47] Forsling ML Aziz LA Miller M Davies R Donovan B Conversion of Triglycyl vasopressin to Lysine-Vasopressin in Man J Endocrinol 1980 85 237 244 7400711 [48] Leone M Albanese J Delmas A Chaabane W Garnier F Martin C Terlipressin in Catecholamine Resistant Septic Shock Patients Shock 2004 22 314 319 15377885 [49] Morelli A Rocco M Conti G Orecchioni A De Gaetano A Cortese G Effects of Terlipressin on Systemic and Regional Haemodynamics in Catecholamine Treated Hyperkinetic Septic Shock Intensive Care Med 2004 30 597 604 14673520 [50] Albanese J Leone M Delmas A Martin C Terlipressin or Norepinephrine in Hyperdynamic Septic Shock: a Prospective, Randomized Study Crit Care Med 2005 33 1897 1902 16148457 [51] O’Brien A Clapp L Singer M Terlipressin for Norepinephrine-Resistant Septic Shock Lancet 2002 359 1209 1210 11955542 [52] Westphal M Stubbe H Sielenkamper AW Borgulya R Van Aken H Ball C Terlipressin Response in Healthy and Endotoxinemic Sheep: Impact on Cardiopulonary Performance and Global Oxygen Intensive Care Med 2003 29 301 308 12594590 [53] Rodriguez-Nunez A Fernandez Sanmartin M Martinon-Torres F Gonzalez-Alonso N Martinon- Sanchez JM Terlipressin for Catecholamine-Resistant Septic Shock in Children Intensive Care Med 2004 30 477 480 14767584 [54] Westphal M Stubbe H Sielenkamper AW Ball C Van Aken H Borgulya R Effects of Titrated Arginine Vasopressin on Hemodynamic Variables and Oxygen Transport in Healthy and Endotoxemic Sheep Crit Care Med 2003 31 1502 1508 12771625 [55] Scharte M Meyer J Van Aken H Bone HG Hemodynamic Effects of Terlipressin (a Synthetic Analog of Vasopressin) in Healthy and Endotoxemic Sheep Crit Care Med 2001 29 1756 1760 11546979 [56] Martikainen TJ Tenhunen JJ Uusaro A Ruokonen E The Effects of Vasopressin on Systemic and Splanchnic Hemodynamics and Metabolism in Endotoxin Shock AnesthAnalg 2003 97 1756 1763 [57] Landry DW Levin HR Gallant EM Ashton RC Jr Seo S D’Alessandro D Vasopressin Deficiency Contributes to the Vasodilationof Septic Shock Circulation 1997 95 1122 1125 9054839 [58] Klinzing S Simon M Reinhart K Bredle DL Meier-Hellmann A High-Dose Vasopressin Is Not Superior to Norepinephrine in Septic Shock CritCare Med 2003 31 2646 2650 [59] Scharte M Meyer J Van Aken H Bone HG Hemodynamic Effects of Terlipressin (a Synthetic Analog of Vasopressin) in Healthy and Endotoxemic Sheep Crit Care Med 2001 29 1756 1760 11546979 [60] Lange M Morelli A Ertmer C Koehler G Broking K Hucklenbruch C Continuous Versus Bolus Infusion of Terlipressin in Ovine Endotoxemia Shock 2007 28 623 629 17589382 [61] Piazza O Scarpati G Rispoli F Iannuzzi M Tufano R De Robertis E Terlipressin in Brain-Death Donors Clin Transplant 2012 11 2 10.1111/ctr.12038 [62] Dunser MW Mayr AJ Ulmer H Knotzer H Sumann G Pajk W Arginine Vasopressin in Advanced Vasodilatory Shock: a Prospective, Randomized, Controlled Study Circulation 2003 107 2313 2319 12732600 [63] Luckner G Dunser MW Jochberger S Mayr VD Wenzel V Ulmer H Schmid S Arginine Vasopressin in 316 Patients with Advanced Vasodilatory Shock Crit Care Med 2005 33 2659 2666 16276194 [64] Pesaturo AB Jennings HR Voils SA Terlipressin: Vasopressin Analog and Novel Drug for Septic Shock Ann Pharmacother 2006 40 2170 2177 17148649 [65] Westphal M Traber DL Low Dose Terlipressin for Emodynamic Support in Sepsis and Systemic Inflammatory Response Syndrome: Art for (he) Art’s Sake or State of the Art? Crit Care Med 2005 33 455 457 15699859 [66] Morelli A Ertmer C Lange M Westphal M Continuous Terlipressin Infusion in Patients with Septic Shock: Less May Be Best, and the Earlier the Better? Intensive Care Med 2007 33 1669 1670 17530219	23905079	PMC3728816	CC BY	2021-01-04 23:01:18	yes	Transl Med UniSa. 2013 Jan 4; 5:22-27
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23936343PONE-D-13-1519610.1371/journal.pone.0069669Research ArticleBiologyImmunologyImmunityInflammationInnate ImmunityMolecular Cell BiologyCellular Stress ResponsesSignal TransductionMedicineCardiovascularAtherosclerosisCardiovascular PharmacologyVascular BiologyDrugs and DevicesDrug Research and DevelopmentDrug DiscoveryCardiovascular PharmacologyThe Effect of Soluble RAGE on Inhibition of Angiotensin II-Mediated Atherosclerosis in Apolipoprotein E Deficient Mice The Effect of Soluble RAGE on AtherosclerosisLee Dajeong 1 3 Lee Kyung Hye 2 Park Hyelim 3 6 Kim Soo Hyuk 1 3 Jin Taewon 1 3 Cho Soyoung 3 5 Chung Ji Hyung 1 3 Lim Soyeon 3 5 * Park Sungha 3 4 6 * 1 Graduate Program in Science for Aging and Yonsei Research Institute of Aging Science, Yonsei University, Seoul, Republic of Korea 2 Medical Science Research Institute, Kyung Hee University Medical Center, Seoul, Republic of Korea 3 Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea 4 Division of Cardiology, Yonsei University College of Medicine, Seoul, Republic of Korea 5 Severance Integrative Research Institute for Cerebral and Cardiovascular Diseases, Yonsei University College of Medicine, Seoul, Republic of Korea 6 Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Republic of Korea Kocher Olivier Editor Harvard Medical School, United States of America * E-mail: shpark0530@yuhs.ac (SP); slim724@yuhs.ac (SL)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SL SP. Performed the experiments: DL. Analyzed the data: DL SL SP. Contributed reagents/materials/analysis tools: KHL HP SHK TJ SC JHC. Wrote the paper: DL SL SP. 2013 1 8 2013 8 8 e6966912 4 2013 11 6 2013 © 2013 Lee et al2013Lee et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background The cross talk between RAGE and angiotensin II (AngII) activation may be important in the development of atherosclerosis. Soluble RAGE (sRAGE), a truncated soluble form of the receptor, acts as a decoy and prevents the inflammatory response mediated by RAGE activation. In this study, we sought to determine the effect of sRAGE in inhibiting AngII-induced atherosclerosis in apolipoprotein E knockout mice (Apo E KO). Methods and Results 9 week old Apo E KO mice were infused subcutaneously with AngII (1 µg/min/kg) and saline for 4 weeks using osmotic mini-pumps. The mice were divided into 4 groups 1. saline infusion and saline injection; 2. saline infusion and sRAGE injection; 3. AngII infusion and saline injection; 4. AngII infusion and sRAGE injection. Saline or 0.5 µg, 1 µg, to 2 µg/day/mouse of sRAGE were injected intraperitoneally daily for 28 days. We showed that atherosclerotic plaque areas in the AngII-infused Apo E KO mice and markers of inflammation such as RAGE, ICAM-1, VCAM-1, and MCP-1 were increased in aorta compared to that of the Apo E KO mice. However, the treatment of 0.5 µg, 1 µg, and 2 µg of sRAGE in AngII group resulted in the dose-dependent decrease in atherosclerotic plaque area. We also demonstrated that sRAGE decreased RAGE expression level as well as inflammatory cytokines and cell adhesion molecules in AngII or HMGB1 treated-rat aorta vascular smooth muscle cells. Conclusion The results demonstrated that partical blockade of RAGE activation by sRAGE prevent AngII -induced atherosclerosis. Therefore these results suggested that first, RAGE activation may be important in mediating AngII-induced atherogenesis, and second, AngII activation is a major pathway in the development of atherosclerosis. Taken together, results from this study may provide the basis for future anti- atherosclerotic drug development mediated through RAGE activation. This work was supported by grants from the National Research Foundation Grant (NRF-2010-0003855) and the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (A085136). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Activation of the renin-angiotensin system (RAS)-signaling pathway, through the activation of the NADPH oxidase system, results in increased oxidative stress and vascular inflammation and has a significant role in the pathogenesis of atherosclerosis. Previous studies have shown that activation of RAS signaling is associated with increased expression of the Receptor for Advanced Glycation Endproducts (RAGE) at the site of vascular inflammation [1], [2]. RAGE is an important endogenous pattern recognition receptor important for initiation of innate immune responses, and is a member of the immunoglobulin superfamily of cell surface molecules specific for diverse endogenous ligands [3], [4]. Activation of RAGE by ligands such as HMGB1, S100A/calgranulin, and advanced glycation end products is associated with induction of oxidative stress, increased inflammatory cytokines, and recruitment of proinflmmatory cells [5], [6]. RAGE signaling has been linked to various chronic inflammatory diseases such as diabetes, atherosclerosis, and inflammatory renal disease [7], [8]. Thus, the increase in damaged self-proteins during the initiation of vascular inflammation and atherogenesis may result in RAGE activation-mediated inflammation. Angiotensin II (AngII), through activation of NADPH oxidase, increases oxidative stress and vascular inflammation and is known to accelerate the development of atherosclerosis [9], [10]. However, there have been no reports regarding the role of RAGE during the progression of AngII-mediated atherosclerosis. Prior studies have shown that infusion of angiotensin II for 4 weeks in Apo E knockout mice is associated with acceleration of atherosclerosis beyond that observed in untreated Apo E knockout mice [11]. Therefore, in this study, we sought to determine the inhibitory effect of soluble RAGE (sRAGE), a decoy receptor that blocks RAGE activation and inhibits inflammatory responses mediated by RAGE activation, in an AngII-induced atherosclerosis model using Apolipoprotein E knockout (Apo E KO) mice. Materials and Methods Expression and Purification of sRAGE-Fc Fusion Protein The purified mouse sRAGE-Fc fusion protein was purchased from A&R Therapeutics (Daejeon, Republic of Korea) for the experiment. Briefly, For the sRAGE-Fc construction, a leader sequence (gene ID: K02149; protein: AAA51633); mouse IgG H chain (primer set: 5′-ATAGGCTAGCGCCACCATGGGATGG-3′, 5′-TGTGTGAGTTTTGTCCGAGTGGACA TCTGT-3′), a.a 23–341 of mouse sRAGE (primer set: 5′-GGTCAGAACATCA CAGCCCGG ATTG-3′, 5′-TGTGTGAGTTTTGTCCCCCATGGTGCAAGA-3′), and the human IgG1 Fc region (primer set: 5′-GGCTAGCGTACCCAGCCCAGACTC-3′, 5′-CCAGCTCGAGCTATTTACCCGGAGACAG-3′) were amplified, and the overlap extension PCR was performed. To express the desired domain, PCR product was treated with SfiI and ligated into pYK 602-His vector (vector constructed by KRIBB). To express the mRAGE-Fc, Mouse sRAGE-Fc was transfected into HEK293E cells and collected supernatants every other day. To purify, a protein A-Sepharose column (Amersham Biosciences, Piscataway, NJ, USA) was used according to the manufacturer’s instructions. The purified recombinant sRAGE was dialyzed with PBS, analyzed by SDS-PAGE. After quantification, mRAGE was aliquoted and stored at −70°C for experiment. And analysis with the Limulus amebocyte lysate test kit (Cape Cod, East Falmouth, MA, USA) was performed to examine the endotoxin level. Animal Studies Apo E KO male mice on a C57BL/6J background were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and all animal experiments conformed to the Guide for the Care and Use of Laboratory Animals that was published by the US National Institute of Health (NIH Publication No. 8523, revised 1985). 9-week-old Apo E KO mice were anaesthetized by intraperitoneally injection of zoletil (30 mg/kg) and xylazine (10 mg/kg), and then were infused subcutaneously with AngII (Sigma, St. Louis, USA) at a concentration of 1 µg/min/kg and saline for 4 weeks using osmotic mini-pumps (Alzet, model 2004; flow rate = 0.25 µl/hour). Mice were divided into 4 groups: 1. saline infusion and saline IP injection; 2. saline infusion and sRAGE IP injection (A&R Therapeutics, Daejeon, Republic of Korea); 3. AngII infusion and saline IP injection; 4. AngII infusion and sRAGE IP injection. sRAGE was injected daily for 28 days and the concentration of sRAGE varied from 0.5 µg, 1 µg, to 2 µg/day/mouse for each group to determine dose responsiveness (N = 10 for each groups). After waiting for 28 days after the initial injection period, the animals were sacrificed for the necessary analysis. The animals were fed a standard diet ad libitum, had free access to water, and were housed in a room with 12 hours of light/dark cycle with a maintained temperature of 25°C for 9 weeks. All animal studies and post-mortem procedures were approved by the Institutional Animal Care and Use Committee of Yonsei University (Approval reference number: 2010-0310, 2011-0008). Determination of Blood Pressure For the blood pressure determination, the mice that were used from for the blood pressure measurement were exposed to the same treatment protocol but were a separate group of mice from the animals sacrificed for pathologic assessment and immunohistochemical assessment. Briefly, 9-week-old Apo E KO mice were anaesthetized by intraperitoneally injection of zoletil (30 mg/kg) and xylazine (10 mg/kg), and then were infused subcutaneously with AngII (Sigma, St. Louis, USA) at a concentration of 1 µg/min/kg and saline for 4 weeks using osmotic mini-pumps (Alzet, model 2004; flow rate = 0.25 µl/hour). Mice were divided into 3 groups: 1. saline infusion and saline IP injection (N = 7); 2. AngII infusion and saline IP injection (N = 7); 3. AngII infusion and 2 µg/day of sRAGE IP injection for 28 days (N = 7). Systolic and diastolic blood pressure was measured in live mice by a non-invasive approach using a validated tail-cuff system that relies on volume pressure recording technology (BP-2000, Visitech Systems, Apex, NC, USA). Blood pressure was monitored on the 55th, 56th day of the 8weeks experimental period. The chamber was kept at 35–36°C and the equipment was set for a maximum inflation pressure of 180 mmHg. Mice were placed in the restrainers several times before the measurements for acclimatization to the environment. All mice were first acclimated to the blood pressure measurements for 3 days (these data were discarded) and then the blood pressure was determined as the average measurement of the subsequent 2 days. For the blood pressure measurement protocol on each day, after the initial acclimatization of the mice for five cycles, the blood pressure was measured for 10 cycles and the average measurement was derived. Analysis of Atherosclerotic Lesion Area Eight weeks after the AngII infusion, mice were anaesthetized by intraperitoneal injection of zoletil (30 mg/kg) and xylazine (10 mg/kg), and then hearts and aortas were perfused with phosphate-buffered saline (PBS) by cardiac puncture. Hearts were removed by cutting the ascending aorta and fixing in 4% (v/v) formalin for at least 24 hours. Aortas were dissected from the proximal ascending aorta and cut at the branch point. Adventitial fat was removed from aorta. Fixed aortas were opened longitudinally and stained with Oil Red-O solution for 12 hours, washed gently with distilled water, and photographed after pinning onto silicone plates. Formalin-fixed hearts were embedded in OCT compound (Leica biosystems, NUSSLOCH, Germany) and stored at −80°C. Frozen heart tissue was cut into 10 µm sections beginning with the lower portion of the heart until the aortic sinus was visible, then placed on tissue section polysine-coated slides (Thermo scientific, Hudson, NH, USA), and stained with Oil Red-O for 12 hours, washed with distilled water briefly, and photographed by microscopy. Lesional areas of whole aortas and plaques of aortic sinuses were calculated using the Image J program (National institutes of Health, USA; http://rsb.info.nih.gov/ij). Oil Red-O stained plaques were imaged from seven different animals, then data were averaged. Immunohistochemistry Mouse aortas were removed from the heart and placed into PBS; adipose tissue was removed in situ. Segments of aortas were fixed in 10% (v/v) formalin solution and immersed in OCT compound, and stored at −80°C. Aorta cross sections (5 µm) were cut at the ascending aorta and the infrarenal abdominal aorta (2–3 mm below the renal artery) and incubated at 45°C for 12 hours to allow tissue to adhere to the slide. To stain with anti-RAGE, anti-MCP-1 (abcam®, Cambridge, UK), anti-ICAM-1, or anti-VCAM-1(Santa Cruz Biotechnology Inc., USA), sections were incubated with primary antibodies at 4°C for overnight, followed by fixation in acetone at −20°C for 10 minutes. Primary antibodies were used at1∶100. Stained slides were washed in PBS then incubated with biotinylated polyclonal secondary antibodies, and the slides were visualized using 3,3′-diaminobenzidine before glycerol gelatin mounting (Sigma, St. Louis, USA). Stained slides imaged from seven different animals were photographed by microscopy and were analyzed using the Image J program (National institutes of Health, USA; http://rsb.info.nih.gov/ij). RNA Extraction from Mouse Aorta Tissues and Reverse Transcription PCR The expression levels of various genes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Mouse aortas were removed from the heart and were stored in RNAlater Tissue Protect solution (QIAGEN, Mainz, Germany). Total RNA was isolated from aorta tissue using RNA isolation from the fibrous tissue kit (QIAGEN, Mainz, Germany). Complementary DNA (cDNA) was generated with the Reverse Transcription System (Bioneer, Seoul, Republic of Korea) according to the manufacturer’s instructions. 1 µg of total RNA was reverse-transcribed in a 20 µl reaction, with cDNA pre-mixed and 0.5 µg oligo-(dT) 15 primer at 42°C for 5 minutes. The reaction was terminated by heating at 90°C for 1 hour. PCR was performed with the AccuPower PCR Premix (Bioneer, Seoul, Republic of Korea) and amplification conditions were as follows: reaction volume, 20 µL; primer, 10 pM; and template genomic DNA 1 µg. PCR was carried out in a thermal cycler using the following conditions: 95°C for 3 minutes, 95°C for 1 minute and then individual conditions for each gene. All PCR products were separated by electrophoresis on 1% agarose gels and visualized using staining with Gel-red (Biotium, Hayward, CA, USA) by Gel-Doc (Bio-Rad, Hercules, CA, USA). The mouse gene primer sequences used for PCR were: mRAGE, forward 5′-CCTGGGTGCTGGTTCTTGCTCT-3′ and reverse 5′-GATCTGGGTGCTCTTACGGTCC-3′ (nucleotides 31–52 and 12091-230 in GenBank accession no. L33412); mICAM-1, forward 5′- GAGAGTGGACCCAACTGGAA-3′ and reverse 5′- CTTTGGGATGGTAGCTGGAA-3′; mVCAM-1, forward 5′- CCCAAGGATCCAGAGATTCA-3′ and reverse 5′- ACGTCAGAACAACCGAATCC-3′ [12]; mMCP-1, forward 5′- GGCTCA GCCAGATGCAGTTAA-3′ and reverse 5′- GTGAATGAGTAGCAGCAGGTGAGT-3′ (nucleotides 154–174 and 181–204 in GenBank Ref NM011333); and mGAPDH, forward 5′- AATGCATCCTGCACCACCAACTGC-3′ and reverse 5′- GGAGGCCATGTAGGCCATGAGGTC-3′ [13]. All samples were measured in triplicates for statistical analysis. Quantitative Real-Time PCR PCRs were performed on LightCycler®480II System using the LightCycler® 480 SYBR green I master mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The reaction mixtures conditions were as follows: final volume, 20 µL; cDNA, 500 ng;, 1 and forward and reverse primer, 10 pM. After pre-incubation at 95°C for 10 minutes, we performed 45 PCR cycles, each consisting of a denaturation step (95°C for 10 seconds) and an annealing step (58°C for 25 seconds). The intensity of the expression of each gene quantitated using LightCycler®480 Software 1.5.0 (Roche Diagnostics, Mannheim, Germany). Values were expr essed in arbitrary units. Relative values of mRNA levels were determined by that of GAPDH gene, and expressed as fold change over the control. The mouse gene primer sequences used for PCR were: mRAGE, forward 5′-AAC ACA GCC CCC ATC CAA-3′, and reverse, 5′-GCT CAA CCA ACA GCT GAA TGC-3′ [14]; mICAM-1, forward 5′-CGC TGT GCT TTG AGA ACT GTG-3′ and reverse 5′-ATA CAC GGT GAT GGT AGC GGA-3′ [15]; mMCP-1, forward 5′-CTG AAG CCA GCT CTC TCT TCC T-3′ and reverse 5′-CAG GCC CAG AAG CAT GAC A-3′ [16] and mGAPDH, forward 5′-CAT GGC CTT CCG TGT TCC TA-3′and reverse 5′-GCG GCA CGT CAG ATC CA-3′ [17]. All samples were measured in triplicates for statistical analysis. Cell Culture and Treatment Rat aorta vascular smooth muscle cells (VSMCs) were purchased from Biobud (Seongnam-si, Gyeonggi-do, Republic of Korea). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Thermo Fisher scientific Inc., USA) containing 10% fetal bovine serum (FBS), Penicillin (10,000 Units/mL) and Streptomycin (10,000 µg/mL) in a humidified incubator at 37°C under an atmosphere of 5% CO2 and 95% air. All the experiments were performed with cells at the 6th passage and incubated for 24 hours in serum-free medium before treatment. The cells were incubated with Ang II (100 nM) or sRAGE (0.5, 1, 2 µg/ml) for 24 hours and followed by HMGB1 (1 µg/ml) for 15 minutes before the end of the incubation period. Human recombinant HMGB1 was purchased from A&R Therapeutics (Daejeon, Republic of Korea). Western Blot Analysis Mouse aortas were removed from the heart and homogenized immediately and lysed with RIPA buffer (Biosesang, Seongnam-si, Gyeonggi-do, Republic of Korea) containing a protease inhibitor cocktail (Calbiochem, Darmstadt, Germany). Cells were washed twice with cold PBS and lysed using the same buffer as aorta tissues. The protein concentration was measured by bicinchoninic acid protein assay. Equal amounts of total protein samples were loaded and separated by 10%–12.5% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and electro-blotted to a polyvinylidene difluoride membrane. The membranes were blocked by Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween 20) containing 10% non-fat dried milk at room temperature for 1 hour and membranes were washed twice with TBS-T and anti-RAGE, anti-ICAM-1, anti-MCP-1, or anti-TNF-α(abcam®, Cambridge, UK); primary antibodies were incubated overnight at 4°C. Membranes were then washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. Signals were visualized using enhanced chemiluminescent detection (Millipore Corporation, Billerica, MA, U.S.A.). All samples were measured in triplicates for statistical analysis. Statistical Analysis Data were expressed as the mean ± SD. ANOVA with Bonferroni’s correction were used to compare the means of two numeric values. Data analyses were done with commercially available GraphPad Prism 5 software. p-values<0.05 were considered statistically significant. Results Infusion of AngII Accelerates Atherosclerosis To assess the activation of AngII with or without sRAGE, we generated an atherosclerosis model in Apo E KO mice over a period of 8 weeks. Mortality in the AngII-infused Apo E KO mice significantly higher compared to Apo E KO mice injected with saline (p-value = 0.013). However, there was no significant difference in mortality between Apo E KO mice injected with saline and AngII-infused Apo E KO mice that received sRAGE injection (p-value = 0.103). Although statistically non-significant, there was a tendency for the sRAGE injection group to have better survival compared to the angiotensin II infusion group (p-value = 0.220) (Figure 1A). 10.1371/journal.pone.0069669.g001Figure 1 Survival and Development of Atherosclerotic Lesions in Apo E KO mice with AngII-induced atherosclerosis. A. Survival graph of Angiotensin II-induced atherosclerosis in Apo E KO mice with saline injection, AngII-infused, and AngII with sRAGE-treated groups. B. Immunohistochemistry sections of aorta (left = ascending aorta, right = infrarenal abdominal aorta) staining for inflammation markers (RAGE, ICAM-1, VCAM-1, and MCP-1) from Apo E KO mice saline injection and AngII-induced atherosclerotic Apo E KO mice (magnification: x10 and below RAGE data: x20). C. Decreased intensity in atherosclerotic areas of sRAGE-treated AngII-induced Apo E KO mice compared to AngII-induced Apo E KO mice (n = 7). The positive stain is expressed in arbitrary units normalized against a control. p<0.01, Scale bar = 200 µm. To check whether activation of NF-κB associated cell adhesion molecules and cytokines are associated with atherosclerosis formation with AngII treatment, cell adhesion molecules and cytokines were examined by immunohistochemistry in Apo E KO mice under several conditions at both the ascending aorta and the infrarenal abdominal aorta (Figure 1B). As activation of RAGE is known to increase inflammation markers and adhesion molecules [6], we chose representative cytokines and adhesion molecules for assessment. Aortic arches for experiments were collected and Cryo-sections were made for immunohistochemistry. Serial sections were stained and analyzed to detect the following markers of inflammation; RAGE, ICAM-1, VCAM-1, and MCP-1. Inflammatory markers were markedly increased in AngII-infused Apo E KO mice, whereas 2 µg of sRAGE injection was associated with significant reduction in the expression of inflammatory markers. Use of 0.5 µg or 1 µg of sRAGE also produced a concentration-dependent reduction in these inflammatory markers (Figure 1C). sRAGE Prevents AngII-induced Atherosclerotic Plaque Formation As plaque formation is an important phenomenon in the atherosclerotic progress, we examined lesion progression in the aorta. Atherosclerotic lesion areas were markedly increased in AngII-infused mice compared to untreated controls, but sRAGE injection showed a marked reduction in atherosclerotic plaque areas, which were detected by Oil Red-O staining of en face and aortic sinuses (Figure 2 and 3). Oil Red-O staining of whole aortas showed more than a 2.5-fold increase in the atherosclerotic plaque area in AngII-infused Apo E KO mice compared to age-matched, untreated Apo E KO animals. In contrast, treatment with 2 µg of sRAGE resulted in a 70% decrease in the atherosclerotic plaque area as compared to mice that received only AngII-infusion (Figure 2). Quantitation of staining results is shown in Figure 2B. We injected lower doses of sRAGE, and found that injection of either 0.5 µg or 1 µg of sRAGE also resulted in a significant dose-dependent reduction in atherosclerotic plaque area to approximately 50%and 60%of that seen in mice that received AngII, respectively. Increased atherosclerosis was also distinct at the aortic sinus in the AngII-infused group, in which the lesion area was increased by over 12-fold compared to untreated Apo E KO mice. The administration of sRAGE decreased the average lesion area by 80% compared to the AngII-infused Apo E KO mice (Figure 3A and 3B). These findings suggest that sRAGE inhibits the development of AngII-mediated atherosclerosis. 10.1371/journal.pone.0069669.g002Figure 2 Quantification of Atherosclerotic Lesions. A. Development of atherosclerotic plaque areas in en face aortas (n = 7, representative picture) stained with Oil Red-O stain from Apo E KO mice. B. The percent of atherosclerotic plaque area in the Apo E KO saline injection and in mice that received infusions of AngII with or without various doses of sRAGE. Relative quantification (% plaque area) was performed. p<0.05 and *p<0.01. 10.1371/journal.pone.0069669.g003Figure 3 Quantification of Atherosclerosis. A. Photomicrographs of atherosclerotic lesions from aortic sinuses of Apo E KO saline injection mice, AngII infused, and AngII infused with various doses of sRAGE (n = 7); scale bar, 200 µm. B. Quantitative data, p<0.01. sRAGE Decreases Expression of Inflammation Markers and Adhesion Molecules We next used whole aorta tissue lysates to measure the representative protein expression and mRNA levels of proteins as downstream targets of RAGE activation, such as the inflammatory marker MCP-1 and adhesion molecule ICAM-1 (Figure 4 and 5). Western blotting showed increased expression of RAGE, ICAM-1, and MCP-1 in AngII-infused Apo E KO mice. Treatment with sRAGE significantly decreased inflammatory markers in a concentration-dependent manner. In mice treated with 2 µg/ml of sRAGE, there was a significant reduction in the expression of RAGE, ICAM-1, and MCP-1 (Figure 4A and 4B). Similar findings were also observed when RT-PCR (Figure 5A) and quantitative Real-time PCR (Figure 5B) were performed for the expression of RAGE, ICAM-1, and MCP-1in AngII-infused Apo E KO mice with or without sRAGE. Protein and mRNA expression data demonstrate that increased levels of inflammatory markers induced by AngII can be significantly inhibited by RAGE blockade. 10.1371/journal.pone.0069669.g004Figure 4 Effect of sRAGE in Apo E KO mice on Expression of Cytokines and Adhesion Molecules. A-B. Apo E KO mice infused with AngII were treated with various concentrations of sRAGE. Expressions of RAGE, ICAM-1 and MCP-1 were detected by western blot. The optical density is expressed in arbitrary units normalized against β-actin control. Data in histograms represent mean ± SD from individual 3 experiments. Lane 1, saline injection; lane 2, infusion of AngII; lane 3, infusion of AngII with 0.5 µg/day of sRAGE; lane 4, infusion of AngII with 1 µg/day of sRAGE; lane 5, infusion of AngII with 2 µg/day of sRAGE, p<0.01. 10.1371/journal.pone.0069669.g005Figure 5 Effects of sRAGE on mRNA Levels in Aorta of AngII-induced Apo E KO mice. A. Reverse transcription PCR analysis of RAGE, ICAM-1 and MCP-1 gene expression. Data in histograms represent mean ± SD from 3 experiments. Lane 1, saline injection; lane 2, infusion of AngII; lane 3, infusion of AngII with 0.5 µg/day of sRAGE; lane 4, infusion of AngII with 1 µg/day of sRAGE; lane 5, infusion of AngII with 2 µg/day of sRAGE. B. RAGE, ICAM-1 and MCP-1 mRNA expression level were determined by Real-Time PCR. Results are means ± SD from 3 experiments each performed in duplicate. p<0.05, p<0.01, * p<0.001 and ns = non-significant. sRAGE Decreases Inflammation in Rat Aorta Vascular Smooth Muscle Cells We first investigated whether sRAGE could inhibit expression of RAGE protein levels in VSMCs treated with AngII or HMGB1. Quantitative western blot analysis revealed that HMGB1 or AngII upregulated RAGE expression respectively compared to control VSMCs and HMGB1 with AngII showed a synergistic effect on RAGE expression. However, 0.5–2 µg/ml of sRAGE significantly decreased the AngII/HMGB1-induced RAGE expression by approximately 1.3-fold. We next investigated the effects of sRAGE on ICAM-1, MCP-1, and TNF-α protein levels. In agreement with our previous results, 0.5–2 µg/ml of sRAGE blocked AngII-induced activation of each of the above-mentioned markers by approximately 1.5-fold (Figure 6A and 6B). 10.1371/journal.pone.0069669.g006Figure 6 Effect of sRAGE in Rat Aorta Vascular Smooth Muscle Cells. Western blot analysis showing expression of A, RAGE inflammation receptor protein, ICAM-1, MCP-1, and TNF-α; and B, Quantitative data from panel A. Expression of RAGE, ICAM-1, MCP-1, TNF-α, and β-actin were detected by western blot. Lane 1, control; lane 2, AngII-treated for 24 hours (100 nM); lane 3, HMGB1 treated for 15 minutes (1 µg/ml); lane 4, AngII plus HMGB1; lane 5, AngII plus HMGB1 and sRAGE (0.5 µg/ml); lane 6, AngII plus HMGB1 and sRAGE (1 µg/ml); lane 7, AngII plus HMGB1 and sRAGE (2 µg/ml); lane 8, treatment with sRAGE alone at 2 µg/ml. The optical density is expressed in arbitrary units normalized against a control. Data in histograms represent mean ± SD from 3 experiments. p<0.01 vs control, † p<0.01 vs lane 4, # p<0.01 vs lane 4, ns = non-significant. Association AngII and Blood Pressure The results from the mice tail blood pressure at the 55,56th day of experiment showed that compared to Apo E knockout mice injected with saline (group 1), mice chronically infused with Ang II(group 2) were associated with significant increase in the systolic and diastolic blood pressure (p<0.05). However, the systolic and diastolic blood pressure between group 2 and mice infused with AngII and injected with 2 µg/day of SRAGE (group 3) was not significant. Discussion Activation of the renin-angiotensin-aldosterone (RAS) system is important in the pathogenesis of cardiovascular disease. Evidence from numerous basic and clinical studies has unequivocally demonstrated the clinical benefit of blocking RAS activation on improvement of the prognosis of cardiovascular disease [18], [19]. Angiotensin II, through the activation of the NADPH oxidase, may increase the degree of oxidative stress and vascular inflammation, which contributes to the pro-atherogenic effect of angiotensin II [20], [21]. Studies have shown that infusion of angiotensin II for 4 weeks in Apo E knockout mice is associated with acceleration of atherosclerosis beyond that of the control Apo E knockout mice [11]. Infusion of angiotensin II was also associated with neointimal proliferation of the vessel wall (Figure 1B) which was attenuated by sRAGE administration. There are several studies to support this result, in which NADPH oxidase inhibitor have been shown to suppress the angiotensin II induced neointimal formation [22], [23]. RAGE is a multi-ligand signal transduction receptor for AGEs, HMGB1, and S100/calgranulin. This receptor is an important mediator of innate immune responses to endogenous ligands, and is thus, known as an alarmin [5], [24]. RAGE activates inflammatory cascades through a combination of NF-κB activation, increasing reactive oxygen species, and promoting leukocyte recruitment as an adhesion receptor [24]. Enhanced expression of RAGE has been demonstrated in various chronic inflammatory diseases such as diabetes, atherosclerosis, rheumatoid arthritis, and inflammatory kidney disease [25]. Recently, a study by Ihara et al. demonstrated that in diabetic Apo E knockout mice, there was an increased expression of RAGE that was significantly suppressed in diabetic Apo E knockout/Angiotensin II type 1 receptor knockout mice [26]. Since RAGE expression is downregulated after knockout of the angiotensin II type 1 receptor, it is conceivable that angiotensin II activation will result in increased RAGE expression and activity. This is the first study, to our knowledge, to demonstrate that inhibition of RAGE activation by administration of the RAGE decoy receptor, sRAGE, results in a significant, dose-dependent decrease in angiotensin II induced atherosclerosis formation and improvement in 60 day survival. In this experiment, we used a recombinant sRAGE-Fc fusion protein cloned from a mammalian cell system. As previous studies have shown that soluble protein-Fc fusion proteins dramatic increase in half-life of soluble protein [27] we were able to demonstrate attenuation of atherosclerosis using a much lower dose than previously described [28]. In another published experiment, we have demonstrated the efficacy of 2 µg of the same sRAGE-Fc fusion protein in attenuating lupus nephritis in NZB-WF1 lupus nephritis mice model [29]. The novelty of this finding is the fact that angiotensin II infusion was associated with markedly increased expression of RAGE in the aortas of non-diabetic Apo E knockout mice. RAGE activation is commonly associated with diabetes, a condition that is connected with increased oxidative stress and inflammation. The dose-dependent effects of sRAGE on inhibiting atherosclerosis indicate that RAGE might be important in mediating angiotensin II-induced progression of atherosclerosis. However, the mechanism linking angiotensin II activation with RAGE activation is not defined. We speculate that the increased oxidative stress and inflammation associated with angiotensin II activation results in leukocyte infiltration and cellular necrosis, which will result in increased extracellular release of S100/calgranulin and HMGB1. We showed that RAGE activation acts to increase the degree of inflammation and upregulation of RAGE expression. Although there is a report that S100B stimulates proliferation and migration in vascular smooth muscle through RAGE-mDia1 interaction [30], a mechanism to link angiotensin II activation with RAGE expression is unclear at the present time. The results from our immunohistochemical staining, western blot and RT-PCR analyses demonstrated increased expression of RAGE, ICAM-1, and MCP-1 in the aortas of angiotensin II-administered Apo E knockout mice, all of which were significantly attenuated with sRAGE administration. We also checked systolic and diastolic blood pressure to demonstrate whether or not the effect of sRAGE in AngII is related to regulation of blood pressure as blood pressure could be a factor that could accelerate atherosclerosis independent from the effect of angiotensin II [31]. Although AngII significantly increased blood pressure compared to control, the administration of sRAGE failed to decrease blood pressure when compared to the AngII-infused mouse, suggesting that effects of sRAGE for AngII is independent from blood pressure regulation (Figure 7). Further, it is interesting to note the decreased expression of RAGE was observed in the aortas of animals treated with sRAGE. RAGE is a unique receptor in that it is positively upregulated following ligand activation. As such, the inhibition of RAGE activation by sRAGE may act to attenuate the expression of RAGE as well as the expression of various chemokine and adhesion molecules that are mediated by RAGE activation. 10.1371/journal.pone.0069669.g007Figure 7 Measurement of Mice Blood Pressure. A. The results from the mice tail systolic and B, diastolic blood pressure. Compared to group 1 and group 2 were associated with significant increase in the blood pressure but, between group 2 and group 3 was not significant. N = 7 for each groups. *p<0.05, ns = non-significant. One of the limitations of this study was the fact that as sRAGE is an indirect inhibitor of RAGE activation acting through binding of RAGE ligands, we cannot rule out the possibility that inhibition of Toll-Like Receptor activation may have contributed to the results. As TLR 2/4 activation can be mediated by RAGE ligands such as HMGB1 [32], sRAGE is not a specific blocker of RAGE activation as the administration of sRAGE may partially inhibit TLR4 activation. This has been demonstrated in a previous experiment demonstrating the efficacy of sRAGE in attenuating atherosclerosis in RAGE KO mice [32]. However, we still believe that sRAGE has a significant effect in blocking RAGE activation because firstly, Liliensiek’s results are limited to adaptive immune responses such as EAE or DTH models [33] and secondly, increased expression of RAGE and inflammatory markers by AngII were significantly reduced under sRAGE treatment in our experiment (Fig. 1C). Further studies using RAGE blocking antibodies or RAGE/Apo E double knockout mouse models will be needed. In conclusion, the results from this study suggest that RAGE activation may be important in mediating AngII-induced atherosclerosis which was shown to be independent from blood pressure elevation. In addition, as AngII activation is a major pathway in the development of atherosclerosis, the results from this study may provide the basis for future anti-atherosclerotic drug development focused on targeted RAGE activation. The authors are grateful to Dr. Jong-gil Park (Division of Life and pharmaceutical Science, Ewha Womans University) and Se-Hoon Kim (Division of Pathology, Yonsei University College of Medicine) for critical comments, and we thank Kun-Bae Bang (Division of Pathology, Yonsei University College of Medicine) for excellent technical assistance. ==== Refs References 1 Yusuf S , Sleight P , Pogue J , Bosch J , Davies R , et al (2000 ) Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators . N Engl J Med 342 : 145 –153 .10639539 2 Mehta PK , Griendling KK (2007 ) Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system . Am J Physiol Cell Physiol 292 : C82 –97 .16870827 3 Schmidt AM , Vianna M , Gerlach M , Brett J , Ryan J , et al (1992 ) Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface . J Biol Chem 267 : 14987 –14997 .1321822 4 Neeper M , Schmidt AM , Brett J , Yan SD , Wang F , et al (1992 ) Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins . J Biol Chem 267 : 14998 –15004 .1378843 5 Chavakis T , Bierhaus A , Al-Fakhri N , Schneider D , Witte S , et al (2003 ) The pattern recognition receptor (RAGE) is a counterreceptor for leukocyte integrins: a novel pathway for inflammatory cell recruitment . J Exp Med 198 : 1507 –1515 .14623906 6 Vazzana N , Santilli F , Cuccurullo C , Davi G (2009 ) Soluble forms of RAGE in internal medicine . Intern Emerg Med 4 : 389 –401 .19727582 7 Bierhaus A , Humpert PM , Morcos M , Wendt T , Chavakis T , et al (2005 ) Understanding RAGE, the receptor for advanced glycation end products . J Mol Med (Berl) 83 : 876 –886 .16133426 8 Alexiou P , Chatzopoulou M , Pegklidou K , Demopoulos VJ (2010 ) RAGE: a multi-ligand receptor unveiling novel insights in health and disease . Curr Med Chem 17 : 2232 –2252 .20459381 9 Weiss D , Kools JJ , Taylor WR (2001 ) Angiotensin II-induced hypertension accelerates the development of atherosclerosis in Apo E-deficient mice . Circulation 103 : 448 –454 .11157699 10 Lin L , Park S , Lakatta EG (2009 ) RAGE signaling in inflammation and arterial aging . Front Biosci 14 : 1403 –1413 . 11 Saraff K , Babamusta F , Cassis LA , Daugherty A (2003 ) Aortic dissection precedes formation of aneurysms and atherosclerosis in angiotensin II-infused, apolipoprotein E-deficient mice . Arterioscler Thromb Vasc Biol 23 : 1621 –1626 .12855482 12 Lee HW , Na YJ , Jung PK , Kim MN , Kim SM , et al (2008 ) Nerve growth factor stimulates proliferation, adhesion and thymopoietic cytokine expression in mouse thymic epithelial cells in vitro . Regul Pept 147 : 72 –81 .18276023 13 Choi JW , Lee KH , Kim SH , Jin T , Lee BS , et al (2011 ) C-reactive protein induces p53-mediated cell cycle arrest in H9c2 cardiac myocytes . Biochem Biophys Res Commun 410 : 525 –530 .21679689 14 Chen CY , Abell AM , Moon YS , Kim KH (2012 ) An advanced glycation end product (age)-receptor for ages (rage) axis restores adipogenic potential of senescent preadipocytes through modulation of p53 protein function . J Biol Chem 287 : 44498 –44507 .23150674 15 Ling HP , Li W , Zhou ML , Tang Y , Chen ZR , et al (2013 ) Expression of intestinal myeloid differentiation primary response protein 88 (myd88) following experimental traumatic brain injury in a mouse model . J Surg Res 179 : e227 –234 .22498027 16 Radeke HH , Janssen-Graalfs I , Sowa EN , Chouchakova N , Skokowa J , et al (2002 ) Opposite regulation of type II and III receptors for immunoglobulin G in mouse glomerular mesangial cells and in the induction of anti-glomerular basement membrane (GBM) nephritis . J Biol Chem 277 : 27535 –27544 .11983693 17 Patole PS , Schubert S , Hildinger K , Khandoga S , Khandoga A , et al (2005 ) Toll-like receptor-4: renal cells and bone marrow cells signal for neutrophil recruitment during pyelonephritis . Kidney Int 68 : 2582 –2587 .16316333 18 Brewster UC , Setaro JF , Perazella MA (2003 ) The renin-angiotensin-aldosterone system: cardiorenal effects and implications for renal and cardiovascular disease states . Am J Med Sci 326 : 15 –24 .12861121 19 Dzau V (2005) The cardiovascular continuum and renin-angiotensin-aldosterone system blockade. J Hypertens Suppl 23: S9–17. 20 Cheng ZJ , Vapaatalo H , Mervaala E (2005 ) Angiotensin II and vascular inflammation . Med Sci Monit 11 : RA194 –205 .15917731 21 Pueyo ME , Gonzalez W , Nicoletti A , Savoie F , Arnal JF , et al (2000 ) Angiotensin II stimulates endothelial vascular cell adhesion molecule-1 via nuclear factor-kappaB activation induced by intracellular oxidative stress . Arterioscler Thromb Vasc Biol 20 : 645 –651 .10712386 22 Weaver M , Liu J , Pimentel D , Reddy DJ , Harding P , et al (2006 ) Adventitial delivery of dominant-negative p67phox attenuates neointimal hyperplasia of the rat carotid artery . Am J Physiol Heart Circ Physiol 290 : H1933 –1941 .16603705 23 Dourron HM , Jacobson GM , Park JL , Liu J , Reddy DJ , et al (2005 ) Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery . Am J Physiol Heart Circ Physiol 288 : H946 –953 .15388496 24 Jandeleit-Dahm K , Watson A , Soro-Paavonen A (2008 ) The AGE/RAGE axis in diabetes-accelerated atherosclerosis . Clin Exp Pharmacol Physiol 35 : 329 –334 .18290873 25 Bierhaus A , Stern DM , Nawroth PP (2006 ) RAGE in inflammation: a new therapeutic target? Curr Opin Investig Drugs 7 : 985 –991 . 26 Ihara Y , Egashira K , Nakano K , Ohtani K , Kubo M , et al (2007 ) Upregulation of the ligand-RAGE pathway via the angiotensin II type I receptor is essential in the pathogenesis of diabetic atherosclerosis . J Mol Cell Cardiol 43 : 455 –464 .17761193 27 Wooley PH , Dutcher J , Widmer MB , Gillis S (1993 ) Influence of a recombinant human soluble tumor necrosis factor receptor FC fusion protein on type II collagen-induced arthritis in mice . J Immunol 151 : 6602 –6607 .8245488 28 Bucciarelli LG , Wendt T , Qu W , Lu Y , Lalla E , et al (2002 ) RAGE blockade stabilizes established atherosclerosis in diabetic apolipoprotein E-null mice . Circulation 106 : 2827 –2835 .12451010 29 Lee SW, Park KH, Park S, Kim JH, Hong SY, et al.. (2013) Soluble receptor for advanced glycation end products alleviates nephritis in NZB/WF1 mice. Arthritis Rheum. 30 Rai V , Maldonado AY , Burz DS , Reverdatto S , Yan SF , et al (2012 ) Signal transduction in receptor for advanced glycation end products (RAGE): solution structure of C-terminal rage (ctRAGE) and its binding to mDia1 . J Biol Chem 287 : 5133 –5144 .22194616 31 Chae CU , Lee RT , Rifai N , Ridker PM (2001 ) Blood pressure and inflammation in apparently healthy men . Hypertension 38 : 399 –403 .11566912 32 Yu M , Wang H , Ding A , Golenbock DT , Latz E , et al (2006 ) HMGB1 signals through toll-like receptor (TLR) 4 and TLR2 . Shock 26 : 174 –179 .16878026 33 Liliensiek B , Weigand MA , Bierhaus A , Nicklas W , Kasper M , et al (2004 ) Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response . J Clin Invest 113 : 1641 –1650 .15173891	23936343	PMC3731311	CC BY	2021-01-05 17:47:15	yes	PLoS One. 2013 Aug 1; 8(8):e69669
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23940730PONE-D-13-0908910.1371/journal.pone.0071251Research ArticleBiologyImmunologyImmunologic SubspecialtiesTransplantationMolecular Cell BiologySignal TransductionSignaling CascadesCalcineurin Signaling CascadeMedicineClinical ImmunologyImmunologic SubspecialtiesTransplantationClinical Research DesignRetrospective StudiesGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis BOncologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaComparison of Steroid-Free Immunosuppression and Standard Immunosuppression for Liver Transplant Patients with Hepatocellular Carcinoma Immunosuppression after Liver TransplantXing Tonghai 1 Huang Li 1 Yu Zhenhai 2 Zhong Lin 1 Wang Shuyun 1 Peng Zhihai 1 * 1 Department of General Surgery, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Organ Transplantation Center of Shanghai, Shanghai, China 2 Department of General Surgery, Shandong Qianfoshan Hospital, Ji’nan, China Starkel Peter Editor St. Luc University Hospital, Belgium * E-mail: zhihai-peng@hotmail.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: TX ZP. Performed the experiments: LZ SW. Analyzed the data: LH. Contributed reagents/materials/analysis tools: ZY. Wrote the paper: TX. 2013 6 8 2013 8 8 e7125128 2 2013 28 6 2013 © 2013 Xing et al2013Xing et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Immunosuppression therapy following liver transplantation often includes steroids. However, extended corticosteroid therapy is associated with numerous complications. This study evaluated the efficacy and safety of using basiliximab in place of a corticosteroid for immunosuppression following liver transplantation for hepatocellular carcinoma (HCC) in Chinese patients. The records of 178 patients with HCC who underwent orthotopic liver transplantation from January 2003 to December 2009 were retrospectively reviewed. All patients received immunosuppression therapy that contained either basiliximab (n = 78) or steroids (n = 100) in addition to tacrolimus and mycophenolate mofetil. Assessments included complications related to liver transplantation, occurrence of steroid side effects, recurrence of HCC, and patient and graft survival. A smaller proportion of patients receiving basiliximab compared with steroids experienced de novo diabetes (38.7% vs. 91.0%, respectively) or long-term de novo diabetes mellitus (7.7% vs. 38.0%, respectively) (both, P<0.0001). The median overall and disease free survival was similar between basiliximab (50.8 months and 19.6 months, respectively) and steroid treated patients (64.2 months and 23.8 months, respectively). The 5-year overall survival and disease free survival rates was also similar between the basiliximab (42.5% and 38.9%, respectively) and steroid (50.5% and 39.2%) groups (all, P>0.730). However, in patients who met the Milan criteria basiliximab was associated with greater 5-year overall survival rate as compared with steroid therapy (88.9% vs. 57.4%, respectively, P = 0.022). These findings provide further evidence of the negative impact of steroids as a part of immunosuppression therapy following liver transplantation for HCC. This work was sponsored by a grant from Shanghai Nature Science Fund project (No. 10ZR1423900), a grant from Science and Technology Department of Shanghai (No. 09411952400) and a grant from the National Key Technology R&D Program in the 11th Five year Plan of China (No. 2008BAI60B03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer death worldwide [1]. In 2008, it was estimated that about 700,000 cancer deaths were due to HCC, and half of these were in China [1]. Major risk factors for HCC include chronic infections with hepatitis B (HBV) or C (HCV) viruses, foodstuff contamination with aflatoxins, and increased alcohol consumption [2]–[5]. Approximately 7% of the population in China are positive for HBV, and the elevated presence of HBV infection is reflected in the higher rate of HCC in China [6], [7]. Orthotopic liver transplantation (OLT) is the treatment of choice for patients with hepatic cirrhosis related to HCC [8]. Standard immunosuppressive treatment following liver transplantation includes corticosteroids and calcineurin inhibitors (CNIs). This treatment strategy has improved survival of patients with liver transplants; however, long-term steroid-based immunosuppression is associated with serious complications and increased morbidity and mortality [9]. Existing evidence suggests that extended corticosteroid therapy is associated with complications including diabetes, hypercholesterolemia, and atrial hypertension [9], [10]. Recent treatment strategies have been aimed at limiting corticosteroid use to improve the quality of life in transplant recipients [11]. Promising clinical results with the CNI tacrolimus in liver transplant patients have resulted in the development of tacrolimus-based protocols with minimal use of corticosteroids [11]–[14]. Basiliximab is a monoclonal antibody to the α chain of the interleukin (IL)-2 receptor of T-cells, and is used to prevent organ rejection following transplantation [15]. Several studies have investigated the use of basiliximab as part of immunosuppression therapy in liver transplant recipients [16]–[20], but only a few have examined its use the absence of steroids [16], [19]. Prior studies have found that the choice of immunosuppressive therapy can affect outcomes, including recurrence of HCC [21], [22]. The objective of this study was to compare the clinical outcomes of Chinese HCC patients who received basiliximab as part of their immunosuppressive therapy with those who received steroid-based therapy following liver transplantation. Methods This study was a retrospective chart review of patients who underwent OLT at the Department of General Surgery, Shanghai First People’s Hospital between January 2003 and December 2009. The study was approved by the ethics committee of the hospital, and followed the principles of the Declaration of Helsinki. Written consent was given by the patients for their information to be stored in the hospital database and used for research. Study Population Eligible patients were ≥ 18 years or age and had undergone cadaveric OLT for histologically proven HCC. The criteria for a patient being eligible for liver transplantation in China differ from those used in Western countries. In China, there are 2 criteria, the Hangzhou and Shanghai criteria. The Hangzhou criterion specify a total tumor diameter ≤8 cm, or a total tumor diameter >8 cm with a histopathological grade I or II, and preoperative alpha-fetoprotein level ≤400 ng/mL. The Shanghai criterion requires a solitary lesion ≤9 cm without macrovascular or lymph node invasion, or extrahepatic metastasis [23], [24]. Patients were not included in this study if they received a liver transplant for acute liver failure, received multiple organ transplantation, had received more than one liver transplant, had prior transplants of other organs, had autoimmune hepatitis, biliary cirrhosis, ABO-ABO-incompatible liver transplantation, or were infected with human immunodeficiency virus. Study Design Medical records were reviewed for demographic and pre-, intra- and post-operative clinical information. Patients were retrospectively classified by the traditional Milan criteria (single tumor ≤5 cm, or 2–3 tumors with none exceeding 3 cm, and no vascular invasion and/or extrahepatic spread) [21] and the University of California San Francisco (UCSF) criteria (single tumor ≤6.5 cm, or 2–3 tumors with none exceeding 4.5 cm with a total tumor diameter ≤8 cm, and no vascular invasion and/or extrahepatic spread) [22]. All patients received methylprednisolone 500 mg as a single intravenous dose before reperfusion during the transplantation procedure. Patients who received steroid-based immunosuppressive induction therapy also received intravenous methylprednisolone on days 1 through 7 after transplantation, beginning at 300 mg/day on day 1 and tapering down to 40 mg/day on day 7. These patients subsequently received oral prednisone from day 8 through day 90, beginning at 20 mg/day on day 8, and followed a gradual tapering schedule to discontinuation. Patients not receiving steroid-based therapy post-operatively received 2 doses of intravenous basiliximab 20 mg, with the first dose at 6 hours after reperfusion and a second dose on postoperative day 4. Postoperatively, all patients received the CNI inhibitor tacrolimus in combination with mycophenolate mofetil (MMF). Tacrolimus was initiated at an oral dose of 0.05 mg/kg/day in two divided doses. The dose was subsequently adjusted to achieve a whole blood trough concentration (measured just prior to the next dose) of 5–10 ng/mL. Tacrolimus treatment was withheld when patients showed insufficient renal function (creatinine >120 µmol/L or creatinine clearance <40 mL/min). MMF, 750 mg twice daily, was initiated after confirmation of the absence of pancytopenia (hematocrit >26% and platelet count >50,000 cells/mm3). At 90 days post-surgery, immunosuppressive therapy was steroid-free for all patients. If there was mild to moderate rejection of the transplanted liver, the dose of tacrolimus or MMF was increased. If there was severe rejection, patients were given methylprednisolone (500 mg) for 3 days with or without an increase in tacrolimus or MMF. All acute rejection episodes were verified by liver biopsy, and if confirmed using the criteria of the fifth Banff Consensus conference [23], patients received an intravenous bolus of 500 mg methylprednisolone per day for 3 consecutive days. If liver function tests showed improvement, steroid therapy with oral methylprednisolone or prednisone was continued. If liver function did not improve, the rejection episode was considered to be steroid-resistant and the tacrolimus dose was increased (0.1 mg/kg/day in two divided doses to achieve 8–10 ng/mL whole blood trough concentration levels) and no steroids were given. The dose of MMF could be increased as determined by the treating physician. Anti-infective prophylaxis was administered according to local practice. The most common protocol was the administration of antibiotics (amoxicillin/clavulanate or aztreonam) for 5 to 7 days without antiviral prophylaxis. Antiviral prophylaxis with valganciclovir was administered only to cytomegalovirus (CMV) mismatch patients (CMV IgG-positive donor/CMV IgG-negative recipient). The postoperative anti-HBV protocol included administration of lamivudine plus low-dose intramuscular HBV immunoglobulin [24]. All patients were followed-up in outpatient clinics until of the end of 2012. Study Assessments The primary endpoints were patient overall survival (OS) and disease-free survival (DFS). Secondary endpoints included the incidence of biopsy-proven acute rejection, the incidence and severity of HCC recurrence, graft survival, recurrence of HBV infection, incidence of adverse events related to immunosuppressive therapy, incidence of infection, and incidence of metabolic complications (diabetes mellitus, hypertension, and hyperlipidemia). Diabetes mellitus, hypertension, and hyperlipidemia were diagnosed according to the guidelines of the World Health Organization. De novo diabetes was defined as diabetes mellitus diagnosed within 30 days postoperatively in patients who did not have diabetes before transplantation. Long-term de novo diabetes was defined as diabetes mellitus newly diagnosed within 30 days postoperatively with active disease continuing beyond 30 days postoperatively. De novo hypertension and de novo hyperlipidemia were defined as hypertension and hyperlipidemia newly diagnosed within one year postoperatively in patients who did not have these conditions preoperatively. Recurrence of HCC was monitored by ultrasonography performed monthly for 6 months, and then every 3 months for the first year, every 6 months for the second year, then annually thereafter. Computed tomography (CT) scans were performed if the results of the ultrasonography were not conclusive. HCC recurrence was also monitored by measurement of alpha fetoprotein serum levels every month for 6 months, followed by every 2 months for the next 6 months, then biannually. Recurrence of HBV was monitored by evaluating presence of HBV surface antigen and HBV DNA in serum. These tests were performed at each follow up visit. Biopsies were performed when clinically required. Patients were evaluated for these outcomes during their postoperative hospital stay, and their follow-up examinations at 1, 2, 3, 6, 9, and 12 months during the first year post-surgery, and every 3–6 months in subsequent years. Statistical Analysis Continuous variables were summarized by mean ± standard deviation or median with inter-quartile range (IQR, the range between the 25th and 75th percentile) depending on normality of the distribution of the data. Categorical variables were expressed by frequencies and percentages. The differences in the distribution of the demographic and clinical characteristics between the steroid and basiliximab groups were detected by independent t-test or Wilcoxon rank sum test for continuous variables, and by Chi-square test or Fisher’s exact test for categorical variables, as appropriate. Overall survival (OS) time was defined as the length of time from the date of liver transplantation to the date of death or last follow up visit. Patients were censored in the DFS analysis if they were disease free (without HCC recurrence) at the last visit, but either HCC recurrence or death was counted as an event in the DFS analysis. The survival curves were constructed by the Kaplan-Meier method with log-rank test to detect the difference between the basiliximab and steroid groups, for OS and DFS, respectively. Kaplan-Meier survival curves for DFS were also constructed for patients based on the Milan and UCSF criteria. Cox’s proportional hazard regressions were performed to calculate crude and adjusted hazard ratios (HRs), with 95% confidence interval (CIs), for effects of immunosuppression therapy group (basiliximab vs. steroid group) and other potential prognostic factors of OS and DFS. The multivariate Cox’s proportional hazard regression model was constructed using the backward selection procedure, wherein variables that did not improve the model fit at P<0.05 were discarded. Treatment group, age, gender, and transplant year were always forced in the model for adjustment. The statistical analyses were all performed with SAS software version 9.2 (SAS Institute Inc., Cary, North Carolina). A two-tailed P<0.05 indicated statistical significance. Results Recipient Demographic and Baseline Characteristics Of the 543 patients who received a liver transplant at our hospital, 178 were eligible for the study (Figure 1). There were more males (n = 156 [87.6%]) than females (n = 22 [12.4%], and the mean age of the patients was 49.2±8.7 years (range, 21.3 to 72.9 years). The distribution of age, gender, alpha-fetoprotein level, Child-Pugh score, type of HCC, meeting of the Milan and UCSF criteria, presence of diabetes mellitus, hypertension, hyperlipidemia, HBV infection, HCV infection, cirrhosis, and treatment with preoperative antiviral therapy were similar between the basiliximab and steroid group (all P>0.05; Table 1). Tumor number, size, and stage were similar between groups (Table 1). The distribution of transplant year was significantly different between the two groups (P<0.0001); all patients who received a liver transplant prior to 2006 were given steroid therapy, and most patients from 2007 to 2009 received basiliximab therapy (Table 1). 10.1371/journal.pone.0071251.g001Figure 1 Consort diagram. 10.1371/journal.pone.0071251.t001Table 1 Recipient demographic and pre-operative characteristics. Basiliximab (n = 78) Steroid (n = 100) P-value Gender Female 7 (9.0) 15 (15.0) 0.226¶ Male 71 (91.0) 85 (85.0) Age 48.7±8.4 49.6±8.9 0.485† AFP1 207.0 (6.3, 897.3) 200.0 (24.5, 1000.0) 0.497‡ Transplant year 2003–2005 0 (0.0) 79 (79.0) <0.0001¶ 2006–2009 78 (1.00) 21 (21.0) Child–Pugh score2 5–6 40 (52.0) 46 (46.0) 0.211¶ 7–9 33 (42.9) 41 (41.0) 10–15 4 (5.2) 13 (13.0) HCC Primary cancer 69 (88.5) 93 (93.0) 0.294¶ Recurrent cancer 9 (11.5) 7 (7.0) HBV positive 72 (92.3) 92 (92.0) 0.940¶ HCV positive 1 (1.3) 2 (2.0) 1.000§ Cirrhosis 68 (87.2) 93 (93.0) 0.190¶ Number of tumors 1.0 (1.0, 4.0) 1.0 (1.0, 3.0) 0.247† Diameter of largest tumor (cm) 3.8 (2.0, 7.0) 4.0 (2.2, 8.5) 0.479† TNM tumor stage Stage I 15 (19.2) 26 (26.0) 0.135¶ Stage II 23 (29.5) 38 (38.0) Stage III 39 (50.0) 36 (36.0) Stage IV 1 (1.3) 0 (0.0) Milan Criteria2 Within Milan 28 (36.4) 36 (36.0) 0.960¶ Beyond Milan 49 (63.6) 64 (64.0) UCSF Criteria1 Within UCSF 31 (40.8) 40 (41.2) 0.953¶ Beyond UCSF 45 (59.2) 57 (58.8) Diabetes mellitus 3 (3.9) 11 (11.0) 0.079¶ Hypertension 6 (7.7) 6 (6.0) 0.655¶ Hyperlipidemia 3 (3.8) 0 (0.0) 0.082§ Preoperative antiviral therapy 16 (20.5) 30 (30.0) 0.151¶ Data are presented as number (percentage), median (IRQ), or mean ± standard deviation. † Independent t-test; ‡ Wilcoxon rank sum test; ¶ Chi-square test; § Fisher’s exact test. 1 Two subjects in basiliximab group and three in the steroid group were missing data. 2 One subject in basiliximab group was missing data. AFP = alpha-fetoprotein; HBV = hepatitis B virus; HCC = hepatocellular carcinoma; HCV = hepatitis C virus; IQR = interquartile range; SD, standard deviation; UCSF, University of California San Francisco. Recipient Postoperative Status, Complications, Steroid Side Effects, and Postoperative Immunosuppression Following liver transplantation, the median follow-up time in the basiliximab and steroid groups was 37.2 (8.7, 52.6) months and 19.5 (4.1, 83.3) months, respectively (P = 0.819), and the mean follow-up time in the two groups was 33.4±23.8 months and 39.9±40.1 months, respectively (P = 0.180) (Table 2). In both treatment groups, most patients did not experience recurrence of HBV (>88%) (P = 0.099) or de novo HBV infection (>66%) (P = 1.0). 10.1371/journal.pone.0071251.t002Table 2 Recipient postoperative status, complications, and immunosuppressive therapya. Basiliximab (n = 78) Steroid (n = 100) P-value Follow-up time (month) Median (IQR) 37.2 (8.7, 52.6) 19.5 (4.1, 83.3) 0.819† Mean ± SD 33.4±23.8 39.9±40.1 0.180§ Mortality, perioperative period 4 (5.1) 11 (11.0) 0.162‡ Mortality 34 (43.6) 42 (42.0) 0.832‡ Cause of death Graft failure 1 (2.9) 1 (2.4) 0.591‡ Hemorrhage 3 (8.8) 1 (2.4) Multi-organ failure 22 (64.7) 30 (71.4) Respiratory complication 1 (2.9) 1 (2.4) Died after re-transplantation 1 (2.9) 4 (9.5) Recurrent disease 6 (17.7) 4 (9.5) Other 0 (0.0) 1 (2.4) HBV recurrence n = 72 8 (11.1) n = 92 4 (4.4) 0.099‡ De novo HBV infection n = 6 2 (33.3) n = 8 2 (25.0) 1.000¶ De novo diabetes n = 75 29 (38.7) n = 89 81 (91.0) <0.0001‡ Long-term de novo diabetes n = 75 3 (4.0) n = 89 27 (30.3) <0.0001‡ De novo hypertension n = 72 4 (5.6) n = 94 5 (5.3) 1.000¶ De novo hyperlipidemia n = 75 3 (4.0) 1 (1.0) 0.315¶ Pleural effusion 63 (80.8) 54 (54.0) 0.0002‡ Postoperative infection 33 (42.3) 23 (23.0) 0.006‡ Biliary complication 6 (7.7) 5 (5.0) 0.538¶ Renal failure 1 (1.3) 7 (7.0) 0.081¶ Pulmonary edema 2 (2.6) 5 (5.0) 0.469 Intra-abdominal bleeding 7 (9.0) 5 (5.0) 0.294‡ Intra-abdominal collection/abscess 6 (7.7) 1 (1.0) 0.045¶ Vascular complication 2 (2.6) 3 (3.0) 1.000¶ CMVpp65 antigenemia 0 (0.0) 1 (1.0) 1.000¶ Primary graft nonfunction 0 (0.0) 1 (1.0) 1.000¶ Chronic rejection 0 (0.0) 0 (0.0) NA GVHD 0 (0.0) 0 (0.0) NA PTLD 0 (0.0) 0 (0.0) NA Postoperative immunosuppressive therapy b Recipient alive at end of study 44 (56.4) 58 (58.0) Maintenance immunosuppressant Tacrolimus 42 (95.5) 54 (93.1) 0.697¶ MMF 40 (90.9) 58 (100.0) 0.032¶ Sirolimus 0 (0.0) 5 (8.6) 0.068¶ Immunosuppression protocol, n (%) Tacrolimus+MMF+sirolimus 0 (0.0) 5 (8.6) 0.017¶ Tacrolimus+MMF 38 (86.4) 49 (84.5) Tacrolimus only 4 (9.1) 0 (0.0) MMF only 2 (4.6) 4 (6.9) Data are presented as number (percentage), median (IRQ), or mean ± standard deviation. a The number of patients for the basiliximab and steroid groups are 78 and 100, respectively unless indicated otherwise. b The number of patients for the basiliximab and steroid groups are 44 and 58, respectively. † Wilcoxon rank sum test; ‡ Chi-square test; ¶ Fisher’s exact test; § independent t-test. CMV = cytomegalovirus; GVHD = graft versus host disease; MMF = mycophenolate mofetil; NA = not available; PTLD = post-transplant lymphoproliferative disorder. Table 2 summarizes the postoperative complications and steroid side effects. A lower proportion of patients treated with basiliximab compared with steroids had de novo diabetes and long-term de novo diabetes (both, P<0.0001). A greater percentage of basiliximab patients experienced pleural effusion, postoperative infection, and intra-abdominal collection/abscess compared with those treated with steroids (all, P<0.05). Other complications were comparable between the two groups. No patients experienced chronic rejection, graft versus host disease (GVHD), or post-transplant lymphoproliferative disorder (PTLD). Of the surviving patients at the end of the study who received immunosuppression therapy, more patients in the steroid group required MMF as compared to the basiliximab group (100.0% vs. 90.9%, respectively; P = 0.032) (Table 2). The distribution of immunosuppression protocols between two groups were significantly different (P = 0.017), with the most common treatment for both groups being tacrolimus plus MMF (>80%) (Table 2). Of the surviving patients receiving immunosuppression therapy, one patient in the steroid group and three in the basiliximab group were switched from a CNI to sirolimus due to the occurrence of renal dysfunction as evidenced by elevated creatinine and proteinuria. MMF was discontinued in one patient in the steroid group and three patients in the basiliximab group due to leukopenia (white blood cell count <2000/mm3). Recipients with Acute Rejection A total of 22 patients experienced biopsy-proven acute rejection, and the rejection rate was similar between the groups (P = 0.869) (Table 3). The rejection time was significantly different between basiliximab and steroid treated patients (P = 0.013). In the basiliximab group, rejection most often occurred within the first 2 weeks, and in the steroid group between 2 and 6 months (Table 3). All patients had mild to moderate rejection severity based on Banff’s schema for grading liver allograft rejection; no cases of severe rejection occurred. All episodes of rejection were successfully treated. The revised treatments protocols used to treat acute rejection did not differ between the groups (all, P = 0.67), and basiliximab and steroid treated patients had similar mortality per revised treatment protocol (Table 3). For both treatment groups, the most common revised protocol was tacrolimus and glucocorticoids. 10.1371/journal.pone.0071251.t003Table 3 Recipients with acute rejection. Basiliximab (n = 78) Steroid (n = 100) P-value Acute rejection 10 (12.8) 12 (12.0) 0.869† Rejection time after transplantation 0–14 days 8 (80.0) 2 (16.7) 0.013‡ 15–30 days 2 (20.0) 6 (50.0) 2–6 months 0 (0.0) 3 (25.0) 7–12 months 0 (0.0) 1 (8.3) Revised treatment protocol Tacrolimus 4 (40.0) 4 (33.3) 0.607‡ Glucocorticoida 3 (30.0) 6 (50.0) MMF 1 (10.0) 0 (0.0) Tacrolimus+glucocorticoida 1 (10.0) 0 (0.0) Tacrolimus+MMF 1 (10.0) 2 (16.7) Mortality, by revised treatment protocol Tacrolimus 1 (25.0) 2 (50.0) 1.000‡ Glucocorticoida 2 (66.7) 4 (66.7) 1.000‡ MMF 0 (0.0) – NA Tacrolimus+glucocorticoida 1 (100.0) – NA Tacrolimus+MMF 0 (0.0) 0 (0.0) NA Data are presented as number (percentage). a Glucocorticoid treatment consisted of oral methylprednisolone or oral prednisone. † Chi-square test; ‡ Fisher’s exact test. MMF = mycophenolate mofetil, NA = non-available. Recipients with HCC Recurrence HCC recurred at a similar frequency in patients in the basiliximab and steroid groups (44.9% vs. 38.0%, respectively; P = 0.355) (Table 4). Approximately one quarter of patients in each group experienced intrahepatic recurrence and extrahepatic recurrence with no statistically significant differences in rates between groups (Table 4). For extrahepatic recurrence, the most common site was the lung (77.3% and 56.5% for basiliximab and steroid patients, respectively). About 40% of patients in both groups experienced HCC recurrence within 1 year, with 28.4% having intrahepatic recurrence and 28.4% have extrahepatic recurrence. None of the three patients positive for HCV experienced HCV recurrence. 10.1371/journal.pone.0071251.t004Table 4 Recipients with HCC Recurrencea. Basiliximab(n = 78) Steroid(n = 100) P-value Overall recurrence of HCC 35 (44.9) 38 (38.0) 0.355† Intrahepatic recurrenceb n = 74 25 (33.8) n = 94 29 (30.9) 0.686† Extrahepatic recurrence/transferb n = 74 23 (31.1) n = 94 23 (24.5) 0.340† Transferred locationc Lung n = 22 17 (77.3) 13 (56.5) 0.458‡ Bone n = 22 2 (9.1) 4 (17.4) Lung+bone n = 22 2 (9.1) 2 (8.7) Bone+brain n = 22 0 (0.0) 1 (4.4) Lung+bone+brain n = 22 1 (4.6) 0 (0.0) Lung+brain n = 22 0 (0.0) 1 (4.4) Abdomen n = 22 0 (0.0) 2 (8.7) Recurrence of HCC within 1 year 29 (37.2) 28 (28.0) 0.193† Intrahepatic recurrence within 1 yearb n = 74 21 (28.4) n = 94 21 (22.3) 0.370† Extrahepatic recurrence/transfer within 1 yearb n = 74 21 (28.4) n = 94 18 (19.2) 0.160† Transferred location within 1 yearc Lung n = 20 16 (80.0) 9 (50.0) 0.199‡ Bone n = 20 2 (10.0) 4 (22.2) Lung+bone n = 20 1 (5.0) 2 (11.1) Lung+bone+brain n = 20 1 (5.0) 0 (0.0) Lung+brain n = 20 0 (0.0) 1 (5.6) Abdomen n = 20 0 (0.0) 2 (11.1) Data are presented as number (percentage). a The number of patients for basiliximab and steroid groups are 78 and 100, respectively unless indicated otherwise. † Chi-square test; ‡ Fisher’s exact test. NA: non-available. b Four subjects in the basiliximab group and 6 subjects in the steroid group had missing data. c One subject in the basiliximab group was missing data. HCC = hepatocellular carcinoma. Overall Survival and Disease-free Survival In the two groups, 5.1% of the patients receiving basiliximab and 11.0% of the patients receiving steroids died within 1 month postoperatively. During the course of the study, a similar number of patients died in the basiliximab (43.6%) and steroid (42.0%) groups (P = 0.832). The most common cause of death for the basiliximab and steroid groups was multiple organ failure (64.7% and 71.4%, respectively), and the second most common cause of death was recurrent disease (17.7% and 9.5%, respectively). The median OS and DFS for the basiliximab group were 50.8 months and 19.6 months, respectively, and for the steroid group were 64.2 months and 23.8 months, respectively. The 5-year OS rate was similar between the basiliximab and steroid groups (42.5% vs. 50.5%; P = 0.734) (Figure 2), as was the 5-years DFS rate (38.9% vs. 39.2%; P = 0.913) (Figure 3a). 10.1371/journal.pone.0071251.g002Figure 2 Overall survival of recipients between basiliximab and steroid groups (log-rank test, P = 0.734). 10.1371/journal.pone.0071251.g003Figure 3 Disease-free survival between basiliximab and steroid groups for (A) all recipients (log-rank test, P = 0.913); (B) recipients within Milan criteria (log-rank test, P = 0.022); (C) recipients within UCSF criteria (log-rank test, P = 0.079). In the group of patients with HCC exceeding the Milan criteria, there were 64 patients in the steroid group and 50 patients in the basiliximab, and based on follow-up to date there are 12/64 and 11/50 patients who survived in the steroid and basiliximab groups, respectively. Stratifying patients by the Milan and UCSF criteria indicated that the 5-year OS rate was significantly different between the basiliximab and steroid groups for patients who met the Milan criteria (5-year OS: 88.9% vs. 57.4%; log-rank test, P = 0.022) (Figure 3b). The 5-year OS was similar for patients treated with basiliximab or steroids who met UCSF criteria (5-year OS: 83.5% vs. 58.5%; log-rank test, P = 0.079) (Figure 3c). The Cox proportional hazard regression multivariate model that included therapy group, gender, age, transplant year, TNM tumor staging, and Milan criteria, after controlling for the other variables, found higher TNM staging was associated with higher mortality (Stage III+ vs. Stage I, adjusted HR = 3.08, 95% CI: 1.28–7.42; P = 0.012) (Table 5). This analysis also found that patients meeting the Milan criteria had a lower mortality (meeting vs. not meeting, adjusted HR = 0.35, 95% CI: 0.17–0.73; P = 0.005). 10.1371/journal.pone.0071251.t005Table 5 Cox proportional hazard regression model for overall survival. Univariate Multivariate† crude HR (95% CI) P-value adjusted HR (95% CI) P-value Group Basiliximab 1.08 (0.68–1.72) 0.733 0.58 (0.27–1.21) 0.146 Steroid 1.00 (reference) – 1.00 (reference) – Gender Female 1.00 (reference) – 1.00 (reference) – Male 0.98 (0.52–1.87) 0.961 0.89 (0.46–1.72) 0.723 Age (y) <50 1.00 (reference) – 1.00 (reference) – ≥50 0.71 (0.45–1.12) 0.140 0.64 (0.40–1.02) 0.059 AFP1 <200 1.00 (reference) – ≥200 1.54 (0.97–2.45) 0.067 Transplant year 2003–2005 0.86 (0.54–1.37) 0.516 0.57 (0.27–1.18) 0.130 2006–2009 1.00 (reference) – 1.00 (reference) – Child–Pugh score2 5–6 1.00 (reference) – 7–9 0.72 (0.44–1.17) 0.180 10–15 0.91 (0.43–1.89) 0.792 Diabetes mellitus No 1.00 (reference) – Yes 0.94 (0.41–2.16) 0.879 HBV No 1.00 (reference) – Yes 0.47 (0.25–0.88) 0.019 Cirrhosis No 1.00 (reference) – Yes 0.44 (0.24–0.82) 0.010 HCC Primary liver cancer 1.00 (reference) – Recurrent hepatocellular carcinoma 1.38 (0.63–3.03) 0.420 No. of tumor <2 1.00 (reference) – ≥2 1.29 (0.82–2.02) 0.278 Diameter of largest tumor (cm) <5 1.00 (reference) – ≥5 2.35 (1.49–3.71) 0.0002 TNM tumor staging for HCC, n (%) Stage I 1.00 (reference) – 1.00 (reference) – Stage II 1.84 (0.85–4.01) 0.122 1.50 (0.67–3.38) 0.327 Stage III+ 5.54 (2.69–11.42) <0.0001 3.08 (1.28–7.42) 0.012 Milan Criteria2, n (%) Within Milan 0.21 (0.11–0.38) <0.0001 0.35 (0.17–0.73) 0.005 Beyond Milan 1.00 (reference) – 1.00 (reference) – UCSF Criteria1, n (%) Within UCSF 0.22 (0.13–0.38) <0.0001 Beyond UCSF 1.00 (reference) – Preoperative antiviral therapy, n (%) No 1.00 (reference) – Yes 1.14 (0.69–1.88) 0.616 1 n = 173; 2 n = 177. † In the multivariate model, data of 177 subjects were included. AFP = alpha-fetoprotein; CI = confidence interval; HBV = hepatitis B virus; HCC = hepatocellular carcinoma; HCV = hepatitis C virus; HR = hazard ratio; UCSF = University of California San Francisco. The Cox proportional hazard regression multivariate model for DFS that included therapy group, gender, age, transplant year, TNM tumor staging, and UCSF criteria, after controlling for the other variables, also found higher TNM staging was associated with higher rate of HCC recurrence (Stage III+ vs. Stage I, adjusted HR = 3.02, 95% CI: 1.35–6.78; P = 0.007) (Table 6). This analysis also found that patients meeting the UCSF criteria had lower recurrence of HCC (within vs. beyond, adjusted HR = 0.37, 95% CI: 0.20–0.71; P = 0.003). 10.1371/journal.pone.0071251.t006Table 6 Cox proportional hazard regression model for disease-free survival. Univariate Multivariate crude HR (95% CI) P-value adjusted HR (95% CI) P-value Group Basiliximab 0.98 (0.65–1.47) 0.913 0.53 (0.27–1.06) 0.073 Steroid 1.00 (reference) – 1.00 (reference) – Gender Female 1.00 (reference) – 1.00 (reference) – Male 1.05 (0.58–1.89) 0.83 (0.46–1.51) 0.541 Age (y) <50 1.00 (reference) – 1.00 (reference) – ≥50 0.76 (0.51–1.13) 0.72 (0.48–1.09) 0.118 AFP1 <200 1.00 (reference) – ≥200 1.64 (1.08–2.49) Transplant year 2003–2005 0.98 (0.65–1.47) 0.57 (0.29–1.11) 0.098 2006–2009 1.00 (reference) – 1.00 (reference) – Child–Pugh score2 5–6 1.00 (reference) – 7–9 0.65 (0.42–1.01) 10–15 0.90 (0.47–1.73) Diabetes mellitus No 1.00 (reference) – Yes 1.33 (0.67–2.64) HBV No 1.00 (reference) – Yes 0.50 (0.27–0.92) 0.025 Cirrhosis No 1.00 (reference) – Yes 0.47 (0.26–0.85) 0.012 HCC Primary liver cancer 1.00 (reference) – Recurrent hepatocellular carcinoma 1.07 (0.52–2.22) 0.855 No. of tumor <2 1.00 (reference) – ≥2 1.30 (0.87–1.95) 0.201 Diameter of largest tumor (cm) <5 1.00 (reference) – ≥5 2.57 (1.71–3.86) <0.0001 TNM tumor staging for HCC, n (%) Stage I 1.00 (reference) – 1.00 (reference) – Stage II 1.72 (0.87–3.40) 0.118 1.56 (0.77–3.15) 0.219 Stage III+ 5.47 (2.90–10.29) <0.0001 3.02 (1.35–6.78) 0.007 Milan Criteria2, n (%) Within Milan 0.22 (0.13–0.38) <0.0001 Beyond Milan 1.00 (reference) – UCSF Criteria1, n (%) Within UCSF 0.22 (0.13–0.36) <0.0001 0.37 (0.20–0.71) 0.003 Beyond UCSF 1.00 (reference) – 1.00 (reference) – Preoperative antiviral therapy, n (%) No 1.00 (reference) – Yes 1.36 (0.88–2.10) 0.172 1 n = 173; 2 n = 177. † In the multivariate model, data of 173 subjects were included. AFP = alpha-fetoprotein; HBV = hepatitis B virus; HCC = hepatocellular carcinoma; HCV = hepatitis C virus; UCSF, University of California San Francisco. Discussion This study compared the efficacy and safety of immunosuppressive therapy based on either basiliximab or corticosteroids in Chinese HCC patients following liver transplantation. Although all patients received 1 dose of methylprednisone during the operation, the patients treated with basiliximab did not receive steroids during the post-operative period. Patients who received basiliximab had a significantly lower incidence of postoperative de novo diabetes and long-term de novo diabetes than patients who received steroids. The rates of de novo hypertension, de novo hyperlipidemia, acute rejection, and HCC recurrence were similar between the groups. The median OS and DFS, and the 5-year OS and DFS were comparable between the two groups. However for patients who met the Milan criteria, 5-year OS of patients treated with basiliximab was longer compared with those who received steroids. The immune system plays a direct role in controlling the tumor growth, and evidence is accumulating that the choice of immunosuppressive therapy following HCC-related liver transplantation may affect treatment outcomes such as survival and HCC recurrence [21], [22]. For example, sirolimus (an inhibitor mTOR) based therapy is associated with longer recurrence-free survival, OS, and lower recurrence-related mortality than tacrolimus-based therapies [22], [25]–[27]. Several studies have indicated that steroid therapy may impact HCC recurrence following transplantation. One study found that a risk factor for HCC recurrence following transplantation was the dose of steroids given within 180 days of transplantation [28]. Another study found that basiliximab plus tacrolimus resulted in lower HCC recurrence than a tacrolimus-based treatment regimen that reduced steroid use over 3–6 months [29]. This same study found that removal of steroid therapy 3 months after transplantation was associated with a lower 1-year survival rate than steroid-maintenance therapy (39% vs. 69%; P<0.05) [29]. Our study did not find a difference in the HCC recurrence rate between treatments. This may be due to, at least in part, to differences in the patient criteria used for transplant eligibility, or different samples sizes. How certain immunosuppressive regimens influence HCC recurrence it not clear. Some findings suggest that steroids may result in protection of tumor cells from apoptosis [30]–[32]. Further studies are required to understand the underlying molecular mechanisms that influence how certain immunosuppressive regimens influence HCC recurrence in the transplant recipients. Similar to our findings, prior studies have not found a difference in the OS rate, or HCV of HBV infection rates, between basiliximab and steroid containing treatments [16], [19]. In our study, there was difference in 5-year OS for patients meeting the Milan criteria who were treated with steroids compared with those receiving basiliximab (88.9% vs. 57.4%, respectively; log-rank test, P = 0.022). These findings suggest that long-term steroid treatment can have a negative outcome for the subclass of patients that meet the Milan criteria. The reasons for the decrease in OS associated with steroid use in some patients are not clear, but may reflect the impact of steroid-associated adverse effects such a diabetes mellitus. Ours and prior studies [11], [12], [14] found the proportion of patients developing postoperative diabetes was lower (although not always significantly lower) in steroid-free compared with steroid-containing immunosuppressive regimens. Although multiple studies indicate the negative effects associated with steroid use in liver transplant patients, steroid-avoidance protocols are not generally implemented in most clinical settings due to the concern of acute rejection. The effect of steroids on the rate of acute rejection is not clear; some studies have found that the presence of steroids in the immunosuppressive protocol was associated with higher acute graft rejection [12], [33], [34], while others, like this study, have not found this association [11], [13], [14]. Two prior studies have compared the efficacy and safety of basiliximab versus steroid-based immunosuppressive therapy [16], [19]. Similar to our findings, in one study basiliximab was associated with lower rates of diabetes than the steroid-containing therapy [19]. However, in the other study basiliximab and steroid therapies were similar in regards to the proportion of patients developing diabetes [16]. The differences in the results may reflect differences in the study populations, as the prior studies included other patients in addition to those with HCC. It may also result from differences in immunosuppressive regimens [16], [19]. The observation in our study that patients who met the Milan criteria and were treated with basiliximab had longer 5-year OS than those receiving steroids is consistent with patients meeting this criteria having a better prognosis [8]. In this study, a higher proportion of patients treated with basiliximab had postoperative infections, including intra-abdominal abscesses. This may reflect that basiliximab therapy requires the therapeutic target concentration of tacrolimus to be rapidly achieved, hence the overall initial dose of tacrolimus was higher in the basiliximab than the steroid group. The increased immunosuppression resulting from the high levels of tacrolimus may have promoted infections in patients prone to infections. There are a number of limitations of this study that should be taken into consideration, which include the small number of patients in each group, relatively short follow-up length and the retrospective nature of the study. In addition, all patients who received a liver transplant prior to 2006 were given steroid therapy, and most patients from 2007 to 2009 received basiliximab. This non-random distribution may have possibly confounded some of the findings. However, there were no differences in operative or other treatment protocols between those two time periods. The high mortality rate may blind the real effect of the immunosuppression protocol on survival. Lastly, many Chinese patients carry HBV infection and more than 90% of the patients in this study had a history of HBV infection. In contrast, only about 1% had a history of HCV infection. Thus, a comparison of underlying disease (HBV negative and HCV positive) with respect to the protocols cannot be performed. However, a recent study indicated that survival outcomes after liver transplantation were significantly better in HBV-HCC patients than in HCV-HCC patients [35]. Conclusion This study found that the use of basiliximab instead of steroids as part of the immunosuppression therapy following liver transplantation in HCC patients was associated with a longer 5-year overall survival in patients that met the Milan criteria and a lower occurrence of diabetes. There was no difference between treatments in regards to HCC recurrence. These findings are consistent with the negative impact of steroids on morbidity and mortality and suggest that basiliximab is an effective immunosuppression therapy following liver transplantation. The authors thank Long Jianyan, Li Wen and Zhang Yuzheng for their assistance with statistical analysis. ==== Refs References 1 Ferlay J , Shin HR , Bray F , Forman D , Mathers C , et al (2010 ) Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008 . Int J Cancer 127 : 2893 –2917 .21351269 2 Walter SR , Thein HH , Gidding HF , Amin J , Law MG , et al (2011 ) Risk factors for hepatocellular carcinoma in a cohort infected with hepatitis B or C. J Gastroenterol Hepatol . 26 : 1757 –1764 . 3 Yang JD , Roberts LR (2010 ) Hepatocellular carcinoma: A global view . Nat Rev Gastroenterol Hepatol 7 : 448 –458 .20628345 4 Song IH , Kim KS (2009 ) Current status of liver diseases in Korea: hepatocellular carcinoma . Korean J Hepatol 15 Suppl 6S50 –S59 .20037280 5 Chuang SC , La Vecchia C , Boffetta P (2009 ) Liver cancer: descriptive epidemiology and risk factors other than HBV and HCV infection . Cancer Lett 286 : 9 –14 .19091458 6 Wang Y , Jia J (2011 ) Control of hepatitis B in China: prevention and treatment . Expert Rev Anti Infect Ther 9 : 21 –25 .21171874 7 Zhang S , Li RT , Wang Y , Liu Q , Zhou YH , Hu Y (2010 ) Seroprevalence of hepatitis B surface antigen among pregnant women in Jiangsu, China, 17 years after introduction of hepatitis B vaccine . Int J Gynaecol Obstet 109 : 194 –197 .20152977 8 Waly Raphael S , Yangde Z , Yuxiang C (2012 ) Hepatocellular carcinoma: focus on different aspects of management . ISRN Oncol 2012 : 421673 .22655206 9 Pageaux GP , Calmus Y , Boillot O , Ducerf C , Vanlemmens C , et al (2004 ) Steroid withdrawal at day 14 after liver transplantation: a double-blind, placebo-controlled study. Liver Transpl . 10 : 1454 –1460 . 10 Stegall MD , Everson G , Schroter G , Bilir B , Karrer F , et al (1995 ) Metabolic complications after liver transplantation. Diabetes, hypercholesterolemia, hypertension, and obesity. Transplantation . 60 : 1057 –1060 . 11 Foroncewicz B , Mucha K , Ryszkowska E , Ciszek M , Ziółkowski J , et al (2009 ) Safety and efficacy of steroid-free immunosuppression with tacrolimus and daclizumab in liver transplant recipients: 6-year follow-up in a single center. Transplant Proc . 41 : 3103 –3106 . 12 Boillot O , Mayer DA , Boudjema K , Salizzoni M , Gridelli B , et al (2005 ) Corticosteroid-free immunosuppression with tacrolimus following induction with daclizumab: a large randomized clinical study. Liver Transpl . 11 : 61 –67 . 13 Becker T , Foltys D , Bilbao I , D’Amico D , Colledan M , et al (2008 ) Patient outcomes in two steroid-free regimens using tacrolimus monotherapy after daclizumab induction and tacrolimus with mycophenolate mofetil in liver transplantation. Transplantation . 86 : 1689 –1694 . 14 Klintmalm GB , Davis GL , Teperman L , Netto GJ , Washburn K , et al (2011 ) A randomized, multicenter study comparing steroid-free immunosuppression and standard immunosuppression for liver transplant recipients with chronic hepatitis C. Liver Transpl . 17 : 1394 –403 . 15 Novartis. Simulect prescribing information (basilliximab for injection). 2012. 16 Lupo L , Panzera P , Tandoi F , Carbotta G , Giannelli G , et al (2008 ) Basiliximab versus steroids in double therapy immunosuppression in liver transplantation: a prospective randomized clinical trial . Transplantation 86 : 925 –931 .18852657 17 Gruttadauria S , Vasta F , Mandalà L , Cintorino D , Piazza T , et al (2005 ) Basiliximab in a triple-drug regimen with tacrolimus and steroids in liver transplantation . Transplant Proc 37 : 2611 –2613 .16182762 18 Ramirez CB , Marino IR (2007 ) The role of basiliximab induction therapy in organ transplantation . Expert Opin Biol Ther 7 : 137 –148 .17150025 19 Liu CL , Fan ST , Lo CM , Chan SC , Ng IO , et al (2004 ) Interleukin-2 receptor antibody (basiliximab) for immunosuppressive induction therapy after liver transplantation: a protocol with early elimination of steroids and reduction of tacrolimus dosage . Liver Transpl 10 : 728 –733 .15162466 20 Neuhaus P , Clavien PA , Kittur D , Salizzoni M , Rimola A , et al (2002 ) Improved treatment response with basiliximab immunoprophylaxis after liver transplantation: results from a double-blind randomized placebo-controlled trial . Liver Transpl 8 : 132 –142 .11862589 21 Welker MW , Bechstein WO , Zeuzem S , Trojan J (2013 ) Recurrent hepatocellular carcinoma after liver transplantation - an emerging clinical challenge . Transpl Int 26 : 109 –118 .22994652 22 Menon KV , Hakeem AR , Heaton ND (2013 ) Meta-analysis: recurrence and survival following the use of sirolimus in liver transplantation for hepatocellular carcinoma . Aliment Pharmacol Ther 37 : 411 –419 .23278125 23 Fan J , Yang GS , Fu ZR , Peng ZH , Xia Q , et al (2009 ) Liver transplantation outcomes in 1,078 hepatocellular carcinoma patients: a multi-center experience in Shanghai, China . J Cancer Res Clin Oncol 135 : 1403 –1412 .19381688 24 Zheng SS , Xu X , Wu J , Chen J , Wang WL , et al (2008 ) Liver transplantation for hepatocellular carcinoma: Hangzhou experiences . Transplantation 85 : 1726 –1732 .18580463 25 Zhou J , Fan J , Wang Z , Wu ZQ , Qiu SJ , et al (2006 ) Conversion to sirolimus immunosuppression in liver transplantation recipients with hepatocellular carcinoma: Report of an initial experience . World J Gastroenterol 12 : 3114 –3118 .16718799 26 Vivarelli M , Dazzi A , Zanello M , Cucchetti A , Cescon M , et al (2010 ) Effect of different immunosuppressive schedules on recurrence-free survival after liver transplantation for hepatocellular carcinoma . Transplantation 89 : 227 –231 .20098287 27 Zhou J , Wang Z , Wu ZQ , Qiu SJ , Yu Y , et al (2008 ) Sirolimus-based immunosuppression therapy in liver transplantation for patients with hepatocellular carcinoma exceeding the Milan criteria . Transplant Proc 40 : 3548 –3553 .19100435 28 Miyagi S , Kawagishi N , Sekiguchi S , Akamatsu Y , Sato K , et al (2012 ) The relationship between recurrences and immunosuppression on living donor liver transplantation for hepatocellular carcinoma . Transplant Proc 44 : 797 –801 .22483499 29 Chen ZS , He F , Zeng FJ , Jiang JP , Du DF , et al (2007 ) Early steroid withdrawal after liver transplantation for hepatocellular carcinoma . World J Gastroenterol 13 : 5273 –5276 .17876900 30 Uen YH , Ko PH , Yin PH , Liu TY , Chi CW , et al (2008 ) Glucocorticoid protects hepatoma cells against metabolic stress-induced cell death . Int J Oncol 33 : 1263 –1270 .19020760 31 Zhang C , Kolb A , Mattern J , Gassler N , Wenger T , et al (2006 ) Dexamethasone desensitizes hepatocellular and colorectal tumours toward cytotoxic therapy . Cancer Lett 242 : 104 –111 .16338063 32 Wanke I , Schwarz M , Buchmann A (2004 ) Insulin and dexamethasone inhibit TGF-beta-induced apoptosis of hepatoma cells upstream of the caspase activation cascade . Toxicology 204 : 141 –154 .15388240 33 Sgourakis G , Radtke A , Fouzas I , Mylona S , Goumas K , et al (2009 ) Corticosteroid-free immunosuppression in liver transplantation: a meta-analysis and meta-regression of outcomes . Transplant Int 22 : 892 –905 . 34 Martín-Mateos RM , Graus J , Albillos A , Arocena C , Rodríguez Gandía MA , et al (2012 ) Initial immunosuppression with or without basiliximab: a comparative study. Transplant Proc . 44 : 2570 –2572 . 35 Hu Z , Zhou J , Wang H , Zhang M , Li S , et al (2013 ) Survival in liver transplant recipients with hepatitis B- or hepatitis C-associated hepatocellular carcinoma: the Chinese experience from 1999 to 2010 . PLoS One 8 : e61620 .23613886	23940730	PMC3735494	CC BY	2021-01-05 17:36:07	yes	PLoS One. 2013 Aug 6; 8(8):e71251
==== Front Chem Cent JChem Cent JChemistry Central Journal1752-153XBioMed Central 1752-153X-6-692280908310.1186/1752-153X-6-69Research ArticleRetracted: Luciferase-transfected colon adenocarcinoma cell line (DLD-1) for use in Orthotopic Xenotransplantation studies Siddique Muhammad Rashid 1rstahiri@googlemail.comShynder Steve 1steveshynder@yahoo.comAshraf Muhammad Aqeel 2chemaqeel@gmail.comYusoff Ismail 3ismaily70@um.edu.myWajid Abdul 4wajidkambo@yahoo.com1 Guy Hilton Research Centre, Keele University, Staffordshire, UK2 Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia3 Department of Geology, University of Malaya, 50603 Kuala Lumpur, Malaysia4 Department of Chemistry, The Islamia University of Bahawlapur, Bahawlapur 63100, Pakistan2012 18 7 2012 6 69 69 3 5 2012 2 7 2012 Copyright © 2012 Siddique et al.; licensee Chemistry Central Ltd.2012Siddique et al.; licensee Chemistry Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Renilla Luciferase reporter gene (rLuc) GL4.82 and GL4.13 promoter are key player in transfection, but precise knowledge of its targets in colon cancer remains limited. The aim of this study was to characterize the best transfection technique to produce a stable transfected colon DLD1 (colorectal adenocarcinoma cell line), therefore imaging based approaches were employed. Results DLD1 cells were transfected with a Plasmid (SV40-RLuc) carrying Renilla luciferase under the control of the SV-40 promoter, by using two different transfection techniques. Cells expressing the required DNA were isolated after antibiotic (Puramycin) selection. Clones of DLD-1/SV40-RLuc were produced using two different techniques (96 well plates and Petri dish) and their florescence intensity was recorded using IVIS machine (Calliper Life Sciences, Hopkinton, USA). Both techniques were characterized with the help of serial dilution technique. Results from this study substantiated that electroporation is the best. As expected, clones varied in their specific luciferase activity along with the dilutions. With the increase in cell concentration increase in intensity of florescence was recorded. Conclusions Based on the results we are confident that this transfected cell line DLD-1/SV40-RLuc (colorectal adenocarcinoma cell line) is the best for further Orthotopic Xenotransplantation Studies and in-vivo experiments as well. Investigation shows that DLD1/SV-rLuc cells have gained little bit resistance against both drugs therefore further study is suggested to know the reasons. DLD1Reporter geneElectroporationExGen transfectionMTT assayA retraction article was published for this article. It is available from the following link; http:// http://journal.chemistrycentral.com/content/6/1/69 . ==== Body Background Human body is a complex of billions of cells, which grow, divide, and die in programmed fashion, and when any of these designed fashion undergoes uncontrolled or abnormal causes cancer. Cancer is a lethal disease; in 2007, 1554845 cancer related deaths were recorded in UK [1]. Good News is that despite increase in incidence, cancer mortality is decreasing [2] yet there is great need of new drug discovery and development. Drug discovery and development procedure involves; acquisition, preclinical screening, production and formulation, toxicology, clinical trials, and general medical practice [3]. Screening drugs for their intracellular effects is a crucial part of the drug discovery and development process. To support any investigational new drug, there is an essential need of carefully designed preclinical drug in vitro and in vivo studies. Most of the researchers in drug development agree that the demonstration of anti-tumor efficacy in preclinical, typically animal based model of cancer is a key determinant in both, and compounds for testing should have shown target biomarker modulation in vitro and in vivo[4]. From 1950s to 1970s syngeneic murine models were used. Subcutaneous transplantation of human tumor xenograft in nude mice was used in next decades and now with the advancement of transgenic knock out models, interest in using genetically engineered mouse models (GEMMS) has sparked [5]. While syngeneic murine tumor models remain valuable for studying immunotherapeutic or targeting metastasis, the human tumor xenograft models established by inoculation of human cancer cell lines into the mice, which is immunodeficient, have been used for test of cytotoxic anticancer agents and new antineoplastic treatment modalities [6]. Since early 1990 new drug developments have moved from general cytotoxic agents to molecular target directed therapeutic, eventually, there was a need to identify tumor types and individual patient tumors that express the target and could benefit from therapies in clinical trials, therefore in vivo models used in preclinical development should be oriented, disease-to-disease or target directed [7]. This is still less clear and point of debate that which is the best model to use with respect to predict for clinical trails? Currently, nine out of ten experimental drugs fail in clinical studies because we cannot accurately predict how they will behave in people based on laboratory and animal studies. In Biomedical sciences use of animals as models to help understand and predict responses in humans, in toxicology and pharmacology in particular remains both the major tool for biomedical advances and a source of significant controversy [8]. During 19th century rise in use of animals rose up critically, which induced pressure on British government to amend Cruelty to Animal Act of 1876 in 1986 [9]. Recent developments of new techniques have permitted researchers to minimize the use of animals at a certain level. It has been very important element in the current success of biomedical research. Very first example of this was in the development of polio vaccine [10]. Since then human and animal cells culture techniques have improved greatly. Anyhow alternatives are nothing but new techniques that may lead to reduce the use of animal in research e.g. paper chromatography, radioimmunoassay, non-invasive imaging and use of Orthotopic models etc. has been main factor behind rapid development of biomedical research [11]. Currently research directions are towards specific cancer target, due to advanced development in cancer biology scientists are also focusing towards translational medicine and diagnosis. In use mouse tumor models have been improved in reliability of imitating human disease. Orthotopic implantation (Organ-specific) reproduces human tumor and metastasis, which has replaced ectopic subcutaneous implantation in experiments [12]. In Orthotopic models strong fluorescent are labeled with green fluorescent protein along with video detectors, allows the monitoring of details of growth, angiogenesis, and metastasis. External imaging is limited by light scattering in deep tissues, especially inside the skin. Signal reduction is markedly eradicated by opening a reversible skin-flap in the light path, which increases the detection sensitivity at much higher extent [13]. The problem of this technique depth of tissue is thereby minimized and many tumors that were previously hidden are now clearly observable [14]. In recent years, a significant development has been made in the choice of imaging cancer non-invasively in murine tumor models. This includes new materials that are imaging probes, which selectively accumulate in tumors, or that become activated by tumor-specific molecules in animal experimentation [15]. Other tumor imaging’s techniques that rely upon the detection of gene expression (reporter) in the body of the animals have been developed [16], which made a significant impact on both the versatility and the specificity of tumor imaging of animals. Optical imaging is rapidly developing field of research aimed at non-invasively interrogates animals for disease progression, evaluating the effects of a drug, assessing the pharmacokinetics behavior of a drug, or identifying molecular biomarker of disease. Optical imaging is mainly based on quantifying the qualitative changes in the emission of light by bioluminescence and fluorescence, which requires incorporation of a vector reporter gene into the cells [17]. In fluorescence imaging, fluorescently tagged agent is injected in mice which carry cells or tissue expressing a florescent transgene, imaged using different light tight highly sensitive CCD camera [18]. Bioluminescence imaging is based upon the sensitive detection of visible light that is produced during enzyme mediated oxidation of a molecular substrate when that enzyme is expressed in animals as molecular reporter [19]. There are several other optical techniques being developed, as FTIR Spectroscopy, Raman Spectroscopy and Multiphoton Imaging but, very low cost and high throughput capability of Bioluminescence imaging and Fluorescence imaging make these important to the drug discovery and development process [20,21]. Key to non-invasive imaging is to incorporation of these reporter genes (talked above) in to the cells “Transfection”. Transfection is the introduction of foreign DNA into eukaryotic or prokaryotic cells [22]. It is the delivery of DNA, mRNA, proteins, and macromolecules into the cells. Goal for the transfection is to study the regulation of gene as well as protein expression and function [23]. Frederick Griffith, 1928 first transformed nonpathogenic pneumococcus bacteria into virulent variety by mixing nonpathogenic pneumococcus with heat-killed pathogenic bacteria. Avery, et al., 1944 reported that the pure DNA is originally the transforming factor [24]. There are different methods of transfection commonly used such as DEAE-dextran for the delivery of nucleic acids into cells for transient transfection [25], calcium phosphate for transient and stable both type of transfection of a variety of cell types [26], lipofection is much reproducible and efficient than other transfection modalities [27], microinjection for the introduction of functional proteins, genes, inhibitors of enzyme activities and antibodies into living cells [28], protoplast fusion is very efficient with high level of transfection about 10% [29], electroporation is proven to be efficient method for transfection of many cell types [30] though it requires very precise and accurate setting for duration and strength of the current for each type of the cells used and ExGen 500 used both in-vitro and in- vivo, and wide range of cell types can be transfected with this reagent [31]. It has shown very higher transfection efficiencies as compared to other cationic lipids and polymers [32,33]. The objective of this study was to develop a stable transfected cell line DLD-1 colon adenocarcinoma and at the same time characterize two different techniques of transfection on the basis of their efficacy. Characterize these transfected cells on the basis of their growth and chemo sensitivity to two standard drugs (Doxorubicin, MB2) for further use in orthotopic xenotransplantation studies. Results Characterization of the techniques DLD1 cells were transfected with a SV40-RLuc carrying Renilla luciferase under the control of the SV-40 promoter, using two different techniques. Cells expressing the required DNA vector (Figure 1) were isolated using antibiotic selection method [3]. To characterize the best technique among both techniques used for transfection (ExGen500 and Electroporation) in this study, substrate coelenterazine 50 μg/ml was added to each well and output was recorded, using IVIS machine with in 10 second of the substrate addition. Electroporation and ExGen500 technique, Electroporation was found the best [3] shows, as ELec1’s output was recorded 5.77 × 105 p/sec/cm2/sr while ExGen2’s output was recorded 3.46× 105 p/sec/cm2/sr. While from ExGen1, 2, and 3 ExGen 2 was the best and from ELec1 and 2, ELec1 was found the best. As expected, clones varied in their specific luciferase activity (measured by standard luminometer -data not shown-). Figure 2 represents light emission in live cells corresponding to five different clones after the treatment of cells with the substrate 50 μg/ml Coelenterazine. Figure 1 Cloning of SV40 region from GL4.13 into Nhe1 & Hind III site of GL4.82 produces SV40-RLuc. Figure 2 All transfected cells (ExGen1, 2,3 and Elec1, 2) added substrate 5 μl of coelenterazine (50 μg/ml), Output (p/sec/cm2/sr) was recorded using IVIS machine with in 10 second of the substrate addition. Bioluminescence intensity is directly proportional to cell concentration Following selection of the best clones serial dilutions were prepared to show correlation between cell number and photon output. Bioluminescence intensity of FLuc serial dilutions was recorded using IVIS machine. It was found that bioluminescence intensity of FLuc cells increases in a gradual manner, with the increase in cell concentration. At the concentration of 1 × 105 cells/well it shows an output of 1.52 × 104 (p/sec/cm2/sr). While a slight decrease has been noticed in the intensity of light at 5 × 105cells/ml where it shows 1.02 × 104 (p/sec/cm2/sr) (Figure 3). Results were measured (mean ± s.d). All types of rLuc cells were serial diluted and treated with substrate. Results showed that bioluminescence intensity of the ELec1 increases in a gradual manner with the increase in cell concentrations, while ExGen1 and 2 did not show gradual increase in the light emission. On the other hand ExGen3 showed very higheroutput of 4.70 × 105 (p/sec/cm2/sr) (Figure 4) at the concentration of 5 × 105 cells/ml while at 1 × 106 cells/ml, it showed a decrease in the output 3.67 × 105 (p/sec/cm2/sr) (Figure 4). Anyhow ExGen2 showed consistent increase in output with higher concentrations of cells, 2.86 × 105 (p/sec/cm2/sr) at 1 × 106 cells/ml. ELec1 was found producing highest intensity than any other one, showing 6.12 × 105 (p/sec/cm2/sr) at 1 × 106 cells/ml. Figure 3 FLuc cells plated at range of concentration from 5 × 10 cells/well to from 1 × 106cells/well, treated with 30 mg/ml D-Luciferin at 1:200 dilution and Imaged using IVIS machine. Shows highest intensity of light at the concentration of 1 × 105cells/well. Results were measured (mean ± sd). Figure 4 Three independent DLD1/SV-rLuc/ExGen and Two independent DLD1/SV-rLuc/ELec clones were seeded in 96-well plates at different cell densities, ranging from 5 × 10 cells/well to 1 × 106 cells/well and treated with the Renilla luciferase substrate Coelenterazine. 50 μg/ml, output (p/sec/cm2/sr) was recorded using IVIS machine with in 10 second of the substrate addition. Characterization on the basis of cloning 96 well cloning After the selection of best clones, we also evaluated the cloning techniques between 96 well plate cloning and ring cloning, single cell clones of ELec1 (Best transfected cells) from 96 well plates were imaged directly. As expected, clones varied in their specific luciferase activity. We selected 7 best clones (Figure 5) on the bases of their light emission ranging from 1.384 × 105 to 2.612 × 105 (p/sec/cm2/sr). In (Figure 5) it represents light emission in living cells corresponding to 7 clones after the treatment of cells with 25 μg/ml Coelenterazine. Figure 5 Seven independent DLD1/SV-rLuc/ELec clones were selected after seeding of 10 cells/ml in 96-well plates and treated with coelenterazine 25 μg/ml after overnight incubation. Output (p/sec/cm2/sr) was immediately quantified using IVIS machine. Ring cloning In case of ring cloning we selected ELec1 and ExGen2 to image. Results shown in (Figure 6) light intensity of ELec1 ranging from 4 × 105 to 1.57 × 106 (p/sec/cm2/sr), and of ExGen2 it ranges from 1 × 105 to 1.275 × 106 (p/sec/cm2/sr). Three best resulting clones from ring cloning were selected for further process. Figure 6 Nine independent clones of DLD1/SV-rLuc/ExGen2 and of DLD1/SV-rLuc/ELec1 were transferred from patri dishes to the 24-well plates at and treated with coelenterazine 25 μg/ml after overnight incubation. Output (p/sec/cm2/sr) was immediately quantified using IVIS machine. Growth curve results To ensure that transfected cell shows no difference in their proliferation activity after transfection, we determined growth curve for Normal DLD1, FLuc, rLuc cells, results shown in (Figure 7). Following subculture, the control cells progressed through a characteristic growth cycle, nearing the peak phase at Day4. Normal DLD1 cells along with the other transfected cells, Fluc and rLuc, were cultivated at the concentration of 5 × 104 cell/ml. Growth of all cells appears to be very similar with Fluc having the highest rate of growth as compare to others. All the cells have shown nearly same trend of growth. Figure 7 Above showing growth curve of Normal DLD1, DLD1/SV-FLuc, rLuc cells over the period of seven days. Cells were maintained in 10 ml RPMI supplemented with 1% L-glutamine, 1% Sodium Pyruvate, and 5% Fetal bovine syrup (FBS), incubated at 37°C 5% CO2. Counted manually using haemocytometer. Results were measured (mean ± s.d). MTT ASSAY In vitro growth inhibition by MB2 (ICT in-house drug) and Doxorubicin was tested against all the cell types wild type DLD1 and transfected DLD1/SV-FLuc, DLD1/SV-rLuc cells, to ensure that transfected cells have no change in their response to drug as compared to normal DLD-1 cells. Cells were tested after 96 hours of drug exposure. Data was recorded by using Teacan reader set at the 540 nm filter. Results (Figures 8 and 9) described that DLD1/SV-rLuc transfected cells have gained little bit resistant against both of the drugs as ic50 for normal DLD-1 cells for MB2 is (30 nM +/− 10 nM), for DLD1/SV-FLuc = (30 nM +/− 10 nM) and DLD1/SV-rLuc = (100 nM +/− 30 nM) (Figure 4). Ic50 of DLD1 cells for doxorubicin is = (100 nM +/− 30 nM) (Figure 6) and of DLD1/SV-FLuc = (100 nM +/− 30 nM) and DLD1/SV-rLuc = (300 nM +/− 100 nM) was observed. Anyhow all cell types have shown more or less same behavior to the drug exposure. With increase in drug concentration, all cells have shown gradual decay in percentage viability. Results were measured as (mean ± s.d). Figure 8 MTT assay result; The effects of MB2 on DLD-1, DLD1/SV-rLuc, and DLD1/SV-FLuc cells exposed to serial dilution of MB2 ranging from .1 ηM to 1 μM for 96 hours showing gradual decrease in % viability with the increasing dose of MB2. Absorbance values were blanked against DMSO and the absorbance of cells exposed to medium only (i.e. no drug added) was taken as 100% cell viability. Figure 9 MTT assay result; The effects of Doxorubicin on DLD-1, DLD1/SV-rLuc, and DLD1/SV-FLuc cells exposed to serial dilution of Doxorubicin ranging from 10 ηM to 100 μM for 96 hours showing gradual decrease in % viability with the increasing dose of Doxorubicin. Absorbance values were blanked against DMSO and the absorbance of cells exposed to medium only (i.e. no Drug added) was taken as 100% cell viability. Discussion Imaging techniques are emerging as important research tools in the scientific industry, very useful especially in diagnostics which are the main problem of the time. Even though our understanding of functional roles of these reporter genes in cancers is steadily increasing, knowledge about Renilla Luciferase reporter gene GL4.82 and GL4.13 promoter used in colon adenocarcinoma is still largely missing. Hence focused on the characterization of the best transfection technique to get the stable cell line after transfection, which could result in a fine biomarker in targeting colon adenocarcinoma cells. Supporting previous findings that imaging techniques are largely used in tumor studies, cell growth profile of DLD1, DLD1/SV-rLuc Cells and DLD1/SV-FLuc cells were established. Cell lines demonstrated that proliferation levels of DLD1, DLD1/SV-rLuc Cells and DLD1/SV-FLuc cells were significantly same relative to non-tumorigenic cell lines [34,35]. Growth reduction of DLD-1 cells upon transient miR-145 transfection, which implies that miR-145 possesses a tumor-suppressor function in vitro has already been reported [36]. Here, immunocytochemistry has been used as a method of identification of potential targets using two different drugs. It has the advantage over strictly computational target prediction that it is not only based on the presence of a seed site and sequence features of the potential target, but takes into account whether the target is expressed in the considered cell line and whether the target is regulated on the transcript level [37-39]. Since this approach is solely based on changes observed on transcript level, targets exclusively regulated by translational repression will not be identified. Many of the targets identification techniques used here have previously been implicated in cancer [40,41]. A number of florescence genes with florescence function, many of which have also been associated with colon cancer, were identified as potential biomarkers based on the microarray analysis [42-44]. The cell membrane associated fraction of VANGL1 increases with differentiation and was demonstrated to co-localize with E-cadherin in human colon cells [45,46]. Previously reported miR-145 targets including OCT4, SOX2 and KLF4 involved in the promotion of stem cell proliferation were not expressed in DLD-1 cells [47,48]. Experimental validation of these biomarkers demonstrated a significant success in in-vitro studies as well as in in-vivo[49]. The regulation in luciferase assays further validates that this is the result of direct interactions [50]. The reason of the different effects of luminesce in the different assays is likely due to a lack of direct comparability between these assays [51]. This difference could also be due to other binding factors involved in regulation of the endogenous transcript, as these binding sites are not present in the 3′UTR or cDNA fragments used in the cloned luciferase reporter constructs [52-54]. A number of studies have linked increased use of these biomarkers in cancer studies with increased cell motility and tumor invasion [55]. In conclusion, using a microarray based approach we have identified additional targets for the cancer-associated miRNA miR-145 in colon cancer cells [56]. These transfection techniques could be a milestone in identifying different targets in colon cancers as well as other cancer types [57-59]. As the problem for the early diagnose stays the same in this era we need to have more studies following these lines of the studies which could save time and get very early diagnose of the cancer at the same time. On the same time we strongly suggest the further use of the same cell lines been transfected in this studies in Orthotopic Xenotransplantation Studies, as we best stable cells were frozen down already. These studies can elaborate the use of such biomarkers in In-Vivo studies. Conclusions Renilla Luciferase reporter gene GL4.82 and GL4.13 promoter Renilla luciferases (rLuc) GL4.82 have emerged as important gene regulators and are recognized as key players in transfection (rLuc) GL4.82 is reported to be down regulated in several cancers, but knowledge of its targets in colon cancer remains limited. To investigate the role of (rLuc) GL4.82 in colon cancer, we have employed imaging based approach to characterize the best transfection technique to get a stable transfected colon DLD1 cell line. Based on the best of its imaging results in-vitro we hereby urge the use of this cell line in Orthotopic Xenotransplantation Studies and in-vivo experiments. DLD1/SV-rLuc cells gained little bit resistance against both drugs; more studies designed for this particular plasmid need to be carrying out to know the reason why they gained that resistance while other transfected line did not. Methods Materials DLD1 (colon adenocarcinoma cells) obtained from European Collection of Cell Cultures Salisbury UK (ECACC™). DLD1/Sv-Fluc transfected cells obtained from IN HOUSE CELL CULTURE COLLECTION of ICT. Roswell Park Memorial Institute 1640 (RPMI) medium supplemented with, 1% L-glutamine, 1% Sodium Pyruvate, 10% fetal bovine serum (FBS), all purchased from Sigma Aldrich, St. Louis, USA. Hanks Balanced Salt Solution (HBSS), T/E .25% Trypsin EDTA Solution, Serum free media (SFM), Doxorubicin Hydrochloride, MTT (Thiazolyl Blue Tetrazolium Bromide, Approx 98% TLC), DMSO (Dimethyl Sulfoxide, ≥ 99.9% ACS Spectrophotometric grade) and Puramycin dihydrochloride solution 10 mg/ml also obtained from Sigma Aldrich, St. Louis, USA. Gene Pulser Xcell Machine, Renilla Luciferase reporter gene GL4.82, and GL4.13 promoter obtained from Promega, Madison, USA. Fermentas, Maryland, USA provided 150 mM NaCl, and ExGen 500. Coelenterazine (native coelenterazine), a substrate for Renilla Luciferase from Biotium Hayward, CA (the compound (1 mg/ml) was dissolved in methanol). D-luciferin Firefly potassium salt, a substrate for Fire Fly Luciferase from Xenogen Alameda, CA (A 30-mg/ml stock in PBS was filtered through 0.22 μm filters before use). IVIS 50 imaging machine from Calliper Life Sciences, Hopkinton, USA. MB2 (Vascular Disrupting Agent) was taken from in house production of ICT. Cell lines, culture conditions DLD1 cells, DLD1/Sv-Flu previously cultured and DLD1/SV40-rLuc transfected with Renilla luciferase (rLuc) GL4.82 reporter gene under the control of Simian Virus 40 GL4.18 promoter, cells were maintained in complete RPMI 1640 medium and incubated in humidified incubator at 37°C and 5% CO2 level. Passage of all types of cells was always done by seeding in complete RPMI 1640, incubating at 37°C and 5% CO2 level, washing twice by 10 ml/wash HBSS and counting manually with the help of haemocytometer after trypsinization. Transfection of the cells Renilla Luciferase reporter gene GL4.82 (Figure 2) and SV40 GL4.13 (Figure 1) promoter been used to make the reporter gene by cloning SV40 region from GL4.13 into the NheI & Hind III site of GL4.82 made SV40-RLuc as a vector reporter to transfect the DLD1 cells [60]. Transfection methods Two different techniques of transfection; I. Electroporation II. ExGen500 Electroporation 1× 107 cells/ml were used for Electroporation Transfection, so resuspended the required concentration of the cells in 400 μl of serum free media (SFM) after trypsinization of the cells. Transferred the cell suspension to the 2 mm vial. 0.88 μg/ml SV40-RLuc were added to each sample (needed 20 μg of Plasmid in 400 μl of cell suspension). Gene Pulser Xcell Machine was used for electroporation. Two methods of electroporation were used; Exponential electroporation; the machine was set at voltage 250volts, Capacitance 1000volts, and resistance ∞. Sample 1 was electroporated and these cells were named DLD1/SV- rLuc/ELec1. Square Wave Method; the machine was set at voltage 250volts, Pulse Length 10 nm, and Number of Pulse 1. Sample 2 was electroporated and these cells were named DLD1/SV-rLuc/ELec2. Following the transfection, Cells were transferred in to T25 flasks and incubated in humidified incubator at 37°C 5% CO2. ExGen500 Transfection Initially, we recommend the use of 1ug of DNA and 3.3 ul (6 equivalents) of ExGen 500 per well of 6-well plate. Cells were plated in 6 well plates at 5 × 104 cells/well and incubated overnight at 37°C 5% CO2 level. 150 mM NaCl solution was made up. The ExGen 500 and DNA complex was made by adding 1 μg of SV40-RLuc in to 100 μl of 150 mM NaCl, vortaxing gently and spinning down and then adding ExGen 500 at the concentrations shown in (Table 1). Following this, the solution was mixed immediately for 10 second then incubated for 10 minutes at room temperature. Then 100 μl of each sample was added to each well containing 3 ml complete RPMI 1640 respectively. Then plate was incubated at 37°C and 5% CO2 level for 48 hours. Colonies were visible after 2 to 3 weeks. Colonies were picked using sterile cloning rings and expanded to form sub lines. Table 1 ExGen500’s Volume used for transfection Sample Amount of DNA Volume Of ExGen500 μl at equivalents DLD1/SV-rLuc/ExGen1 1 μg 8.23 μl DLD1/SV-rLuc/ExGen2 1 μg 9.87 μl DLD1/SV-rLuc/ExGen3 1 μg 11.52 μl Puramycin selection/cloning To select the cells expressing the required DNA vector (Figure 1), all types of cell were treated with 50 μg/ml of Puramycin because SV40-RLuc is Puramycin resistant that works as selection marker. Optimal concentration for puramycin was tested earlier at different concentrations with normal DLD1 cells data not included here. So, purely transfected cells expressing the required DNA vector (Figure 1), sustained within the puramycin were harvested and plated in Petri dishes and 96 well plates at 10 cells/ml following the procedure described earlier. Plates were incubated at 37°C 5% CO2 level for 2–3 days. Single cell clones were selected in both, Petri dishes and 96 well plates for imaging. Single cell best observed clones in 96 well plates were imaged directly. While best clones from Petri dishes were passaged to the 24 well plates using method descried earlier. Serial dilutions To check the ratio of bioluminescence and cell numbers both DLD1/SV-FLuc and DLD1/SV-rLuc cells were serially diluted. Each type of cells were serially diluted at the concentrations ranging from 1 × 104 cells/well to 1 × 106 cells/well and DLD1/SV-FLuc at the concentrations ranging from 5 × 10 cells/well to 1 × 106cells/well was plated in 96 well plates. Incubated overnight at 37°C 5% CO2 level for imaging. Imaging and quantification of bioluminescence data The in vivo Imaging System (IVIS), consists of a cooled camera (CCD) mounted on a light- tight chamber (dark box), a camera controller, a camera cooling system, and a Windows based computer system, were used for data acquisition and analysis. Each 96/24-well plate sample was placed in the specimen chamber mounted with the CCD camera cooled to −90°C, with a field of view set at 25 cm above the sample shelf. The photon emission, transmitted from cell samples was measured in p/sec/cm2/sr. The bioluminescence color images were viewed using the living image software overlay (Xenogen). Bioluminescence was measured for the plates set up for imaging using substrate 50 μg/ml Coelenterazine for RLuc cells and 30 mg/ml stock solution of D-Luciferin at 1: 200 dilution for FLuc cells. To check the effect of lower concentration of Coelenterazine, 25 μg/ml of Coelenterazine was used to image ring cloning and 96 well plate cloning. Images were taken within the 10 sec of the Coelenterazine addition to the cells, due to the quick nature of the reaction. Coelenterazine was tested alone1st in blank 2nd in only media. Growth curve Growth curves were determined to ensure that transfected cells show no difference to normal cells in their proliferation activity. Normal DLD1 cells, DLD1/SV-rLuc Cells and DLD1/SV-FLuc cells were seeded at 5 × 104cells/ml. Fourteen flasks of each line were seeded. To count, cells were washed with Hanks, trypsinized, centrifuged, and resuspended in 5 ml of media and then counted manually using Haemocytometer every day for 7 days. Two flask of each type of cells were used every day to have precise average count of the cell. MTT assay The MTT assay was done to check that transfected and normal cells had same Chemosensitivity (transfection has not changed their nature to react with the chemicals). In brief, normal DLD1, DLD1/SV-rLuc Cells and DLD1/SV-FLuc cells were seeded at 1x105cells/ml into 96 well plates. Column 1 containing culture medium 200 μl/well, only to blank the Spectrophotometer. Some drugs affect the optical density (OD) of the medium and require a separate blank. Colum 2 containing 200 μl/well only cell suspension but no drug exposure serves as control wells to calculate the control cell survival percentage rest of the plate cultivated with 180 μl/well of cell suspension. The cells then left to adhere for 24 h in incubator at 37°C 5% CO2 level. Next day, serial dilutions of both drugs were prepared (Table 2). Table 2 Drug concentrations of MB2 and Doxorubicin used in MTT assay Blank Control MB2 drug concentration Blank Control .1 nM .3 nM 1 nM 3 nM 10 nM 30 nM 100 nM 300 nM 1 μM Blank Control Doxorubicin Drug Concentration Blank Control 10 nM 30 nM 100 nM 300 nM 1 μM 3 μM 10 μM 30 μM 100 μM MB2 ranges from 1 μM to .1 ηM and Doxorubicin ranging from 100 μM to 10 ηM, and added 20 μl of each dilution to the plates respectively leaving Blank and control with no drug. The plates were incubated at 37°C with 5% CO2 level for 96 hours. MTT stock solution 5 mg/ml was prepared in distilled water. Stock solution of MTT was then diluted in complete RPMI 1640 at 1/10 ratio. All the media from the plates were removed and MTT solution 200 μl/well was added in each well leaving blank. The plates were incubated for 4 hours at 37°C with 5% CO2. MTT was removed from the plates very carefully and 150 μl of DMSO was added. Finally the absorbance at 540 nm of the plates was read with the Tecan plate reader. MTT assay were done in triplicate. Absorbance values were blanked against DMSO and the absorbance of cells exposed to medium only i.e. no Drug added was taken as 100% cell viability i.e. the control. Freezing down the cells The best clones resulting high intensity of bioluminescence were washed with hanks, trypsinized, centrifuged and resuspended in 1 ml of complete RPMI 1640. Cells suspension was transferred to the 1.5 ml vials with 1:10 of DMSO and stored in freezer at −80°C for future use. Competing interests The authors declare that they have no competing interests. Authors’ contributions MRS and SS carried out the reporter gene cloning SV40 to make SV40-RLuc as a vector reporter to transfect the DLD1 cells and biochemical characterizations, MAA performed transfection studies. IY and AW carried out imaging and quantification of bioluminescence data. All authors have read and approved the final manuscript. Acknowledgments This research work is collaborative research work conducted by Cell Culture Lab, Institute of Cancer Therapeutics, Bradford University, UK, Department of Chemistry, University of Malaya and Department of Chemistry, The Islamia University of Bahawalpur, Pakistan. The research was supported by grant from the Cell Culture Lab, Institute of Cancer therapeutics, Bradford University, UK, and by UM research grants PV039-2011. ==== Refs Kalabis J Rosenberg I Podolsky DK Vangl1 protein acts as a downstream effector of intestinal trefoil factor (ITF)/TFF3 signaling and regulates wound healing of intestinal epithelium J Biol Chem 2006 281 6434 6441 10.1074/jbc.M512905200 16410243 Horvath CM STAT proteins and transcriptional responses to extracellular signals Trends Biochem Sci 2000 25 496 502 10.1016/S0968-0004(00)01624-8 11050435 Johnson M Sharma M Henderson BR IQGAP1 regulation and roles in cancer Cell Signal 2009 10 1471 1478 19269319 Sandelin A Alkema W Engstrom P Wasserman WW Lenhard B JASPAR: an open- access database for eukaryotic transcription factor binding profiles Nucleic Acids Res 2004 32 D91 D94 10.1093/nar/gkh012 14681366 Chen HC Chen GH Chen YH Liao WL Liu CY MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma Br J Cancer 2009 100 1002 1011 10.1038/sj.bjc.6604948 19293812 Gramantieri L Ferracin M Fornari F Veronese A Sabbioni S Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma Cancer Res 2007 67 6092 6099 10.1158/0008-5472.CAN-06-4607 17616664 Nicolau B Catherine C Anna LB Francois BLE Stefan Z Rainer K Vincet D Review Article in vivo mouse imaging and spectroscopy in drug discovery Nanomed Nanobiotech 2007 20 154 185 Meng Y Eugene B Jin-Wei W Ping J Xiaoen W Fang-Xian S Michael B Moossa AR Sheldon P Robert MH Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model PNAS 2002 99 3824 3829 10.1073/pnas.052029099 11891294 Lin Y Weissleder R Tung CH Synthesis and properties of sulfhydryl-reactive near infrared cyanine fluorochromes for fluorescence imaging Mol Imaging 2003 2 87 92 10.1162/153535003322331975 12964306 Nathan CS George HP Michael WD Advances in fluorescent protein technology J Cells Sc 2007 120 4247 4260 10.1242/jcs.005801 Contage PR Whole animal cellular and molecular imaging to accelerate drug development Drug Discov Today 2002 7 555 562 10.1016/S1359-6446(02)02268-7 12047855 Contag CH Living mammals using a Bioluminesce reporter Photochem Photobiol 1997 66 523 531 10.1111/j.1751-1097.1997.tb03184.x 9337626 Ruxana TS Timothy SB Bioluminescence imaging Proc Am Thorac Soc 2005 2 537 540 10.1513/pats.200507-067DS 16352761 Wilson T Hastings JW Bioluminescence Annu Rev Cell Dev Biol 1998 14 197 230 10.1146/annurev.cellbio.14.1.197 9891783 de Wet JR Wood KV Helinski DR DeLuca M Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli Proc Natl Acad Sci USA 1985 82 7870 7873 10.1073/pnas.82.23.7870 3906652 Selden RF Transfection using DEAE-dextran 2001 Chap 10. 10.14 Joseph S David WR Calcium-phosphate-mediated transfection of eukaryotic cells with plasmid DNAs: protocols 2006 10.1101/pdb.prot3871 Felgner P L: A highly efficient, lipid-mediated DNA-transfection procedure Proc Natl Acad Sci USA 1987 21 7413 7417 2823261 Straubinger RM Duzgunes N Papahadjopoulos D pH-sensitive liposomes: introduction of foreign substances into cells FEBS Lett 1991 179 713 730 Graessmann A Wolf H Bornkamm GW Expression of Epstein-Barr virus genes in different cell types after microinjection of viral DNA Proc Natl Acad Sci USA 1980 77 433 436 10.1073/pnas.77.1.433 6244558 Mello CC Kramer JM Stinchcomb D Ambros V Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences EMBO J 1991 10 3959 3970 1935914 Kleber S Sancho-Martinez I Wiestler B Beisel A Gieffers C Yes and PI3K bind CD95 to signal invasion of glioblastoma Cancer Cell 2008 13 235 248 10.1016/j.ccr.2008.02.003 18328427 Thomas SM Brugge JS Cellular functions regulated by Src family kinases Annu Rev Cell Dev Biol 1997 13 513 609 10.1146/annurev.cellbio.13.1.513 9442882 Pena SV Melhem MF Meisler AI Cartwright CA Elevated c-yes tyrosine kinase activity in premalignant lesions of the colon Gastroenterology 1995 108 117 124 10.1016/0016-5085(95)90015-2 7806032 Christoffersen NR Shalgi R Frankel LB Leucci E Lees M p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC Cell Death Differ 2010 17 236 245 10.1038/cdd.2009.109 19696787 Arvidsson S Kwasniewski M Riano-Pachon DM Mueller-Roeber B QuantPrime–a flexible tool for reliable high-throughput primer design for quantitative PCR BMC Bioinforma 2008 9 460 465 10.1186/1471-2105-9-460 Gentleman R Carey V Huber W Hu T Irizarry R Bioinformatics and computational biology solutions using R and bioconductor 2005 New York: Springer 473 Huber W von Heydebreck A Sultmann H Poustka A Vingron M Huber W von Heydebreck A Sultmann H Poustka A Vingron M Bioinformatics 2002 18 Suppl 1S 96 104 Schaffner W Direct transfer of cloned genes from bacteria to mammalian cells Proc Natl Acad Sci USA 1980 77 2163 2167 10.1073/pnas.77.4.2163 6246525 Purves WK Life: the science of biology 2001 Sinauer Associates 316 317 Nickoloff JA Nickoloff JA Preface Electroporation protocols for microorganisms 1995 Totowa, New Jersey: Humana Press v vi Withers HL Nickoloff JA Direct plasmid transfer between bacterial species and electrocuring Electroporation protocols for microorganisms 1995 New Jersey: Humana Press, Totowa 47 54 Weaver JC Nickoloff JA Electroporation theory: concepts and mechanisms Electroporation protocols for microorganisms 1995 Totowa, New Jersey: Humana Press 1 26 Kelley RL Meller VH Gordadze PR Roman G Davis RL Kuroda MI Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin Cell 1999 98 513 522 10.1016/S0092-8674(00)81979-0 10481915 Niall S Ray G Jean G Are animal models predictive for humans? Philos Ethics Humanit Med 2009 4 2 10.1186/1747-5341-4-2 19146696 Weber M Davies JJ Wittig D Oakeley EJ Haase M Lam WL Schubeler D Chromosome-wide and promoterspecific analyses identify sites of differential DNA methylation in normal and transformed human cells Nat Genet 2005 37 853 862 10.1038/ng1598 16007088 Rakyan VK Down TA Thorne NP Flicek P Kulesha E Gra FS Tomazou EM Backdahl L Johnson N Herberth M Howe KL Jackson DK Miretti MM Fiegler H Marioni JC Birney E Hubbard TJ Carter NP Tavare S Beck S An integrated resource for genome-wide identification and analysis of human tissue-specific differentially methylated regions (tDMRs) Genome Res 2008 18 1518 1529 10.1101/gr.077479.108 18577705 Lister R Pelizzola M Dowen RH Hawkins RD Hon G Tonti-Filippini J Nery JR Lee L Ye Z Ngo QM Edsall L Antosiewicz-Bourget J Stewart R Ruotti V Millar AH Thomson JA Ren B Ecker JR Human DNA methylomes at base resolution show widespread epigenomic differences Nature 2009 462 315 322 10.1038/nature08514 19829295 Margueron R Reinberg D Chromatin structure and the inheritance of epigenetic information Nat Rev Genet 2010 11 285 296 10.1038/nrg2752 20300089 Russell WMS Burch RL The principles of humane experimental technique 1959 London: Methuen Van den Heuvel MJ Dyan AD Shillaker RO Evaluation of the BTS approach to the testing of substances and preparations for their acute toxicity Hum Toxicol 1987 5 151 157 Scott KL Advances in imaging mouse tumour models in vivo J Pathol 2005 205 194 205 10.1002/path.1697 15641018 Santoro MM Samuel T Mitchell T Reed JC Stainier DY Birc2 (cIap1) regulates endothelial cell integrity and blood vessel homeostasis Nat Genet 2007 39 1397 1402 10.1038/ng.2007.8 17934460 Smyth G Gentleman RCV, Dudoit S, Irizarry R, Huber W, Gentleman RCV, Dudoit S, Irizarry R, Huber W Limma: linear models for microarray data Bioinformatics and computational biology 2005 Springer, New York: Solutions using R and Bioconductor 397 420 Xu N Papagiannakopoulos T Pan G Thomson JA Kosik KS MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells Cell 2009 137 647 658 10.1016/j.cell.2009.02.038 19409607 Cordes KR Sheehy NT White MP Berry EC Morton SU miR-145 and miR-143 regulate smooth muscle cell fate and plasticity Nature 2009 460 705 710 19578358 Xin M Small EM Sutherland LB Qi X McAnally J MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury Genes Dev 2009 23 2166 2178 10.1101/gad.1842409 19720868 Liu X Sempere LF Galimberti F Freemantle SJ Black C Uncovering growth- suppressive MicroRNAs in lung cancer Clin Cancer Res 2009 15 1177 1183 10.1158/1078-0432.CCR-08-1355 19228723 Akao Y Nakagawa Y Naoe T MicroRNAs 143 and 145 are possible common onco- microRNAs in human cancers Oncol Rep 2006 16 845 850 16969504 Wieder J Soloway MS Incidence, etiology, location, prevention, and treatment of positive surgical margins after radical prostatectomy for prostate cancer J Urol 1998 160 299 315 10.1016/S0022-5347(01)62881-7 9679867 Barraclough J Hodgkinson C Hogg A Dive C Welman A Increases in c-Yes expression level and activity promote motility but not proliferation of human colorectal carcinoma cells Neoplasia 2007 9 745 754 10.1593/neo.07442 17898870 Joseph S Preclinical development of molecularly targeted agents in oncology 2007 707 772 Michael RB Kenneth DP Some practical considerations and applications of the national cancer Institute in vitro anticancer drug discovery screen Drug Develop Res 1995 34 91 109 10.1002/ddr.430340203 Angelike MB Heinz HF Screening using animal system Drug Develop Res 2002 16 285 289 Wang X Tang S Le SY Lu R Rader JS Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth PLoS One 2008 3 e2557 10.1371/journal.pone.0002557 18596939 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP MicroRNA targeting specificity in mammals: determinants beyond seed pairing Mol Cell 2007 27 91 105 10.1016/j.molcel.2007.06.017 17612493 Lim LP Lau NC Garrett-Engele P Grimson A Schelter JM Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs Nature 2005 433 769 773 10.1038/nature03315 15685193 Nielsen CB Shomron N Sandberg R Hornstein E Kitzman J Determinants of targeting by endogenous and exogenous microRNAs and siRNAs RNA 2007 13 1894 1910 10.1261/rna.768207 17872505 Sahin U Blobel CP Ectodomain shedding of the EGF-receptor ligand epigen is mediated by ADAM17 FEBS Lett 2007 581 41 44 10.1016/j.febslet.2006.11.074 17169360 Francois P Tumor and Cancers in Graeno-Roman Times 2005 91 4	22809083	PMC3737038	CC BY	2021-01-05 01:28:35	yes	Chem Cent J. 2012 Jul 18; 6:69
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23950881PONE-D-12-0499110.1371/journal.pone.0069001Research ArticleOverexpression of the Insulin Receptor Isoform A Promotes Endometrial Carcinoma Cell Growth IR-A Overexpression in Endometrial CarcinomasWang Chun-Fang Zhang Guo Zhao Li-Jun Qi Wen-Juan Li Xiao-Ping Wang Jian-Liu Wei Li-Hui * Department of Obstetrics and Gynecology, Peking University People’s Hospital, Peking University, Beijing, China Lobaccaro Jean-Marc A Editor Clermont Université, France * E-mail: weilh@bjmu.edu.cnCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: XL JW LW. Performed the experiments: CW GZ WQ. Analyzed the data: CW GZ LZ. Contributed reagents/materials/analysis tools: XL. Wrote the manuscript: CW GZ. 2013 7 8 2013 8 8 e6900115 2 2012 10 6 2013 © 2013 Wang et al2013Wang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Epidemiological studies have demonstrated that type 2 diabetes mellitus (T2DM) and hyperinsulinemia are associated closely with endometrial carcinoma risk, although the molecular mechanism remains unclear. Insulin receptor isoformA expression is upregulated in many cancer cells and tissues, which suggests that IR-A-mediated signaling pathways may have important implications for cancer pathogenesis. We measured the expression of insulin receptor isoforms (IR-A and IR–B in the normal endometrium tissues, the endometrial carcinoma tissues and the endometrial carcinoma cell lines. We found that the total insulin receptor (IR) and IR-A expression mRNA levels and the ratio of IR-A to total IR in endometrial carcinoma specimens were significantly higher than them in control endometrial tissue specimens(P<0.05). Further analysis indicated that the tendency was more prominently in patients with T2DM. IR-A mRNA was differentially expressed in four endometrial carcinoma cell lines (Ishikawa, KLE, RL95-2 and HEC-1-A. RL95-2 cells have a low endogenous IR-A expression, and these were used to construct a stable cell line overexpressing IR-A. We found that IR-A overexpression significantly increased cell proliferation, the proportion of cells in S phase, activation of the Akt pathway and tumorigenicity of xenografts in nude mice. In contrast, there was no significant difference in the the percentage of apoptotic cells between cells overexpressing IR-A and control cells. Moreover, levels of phosphorylated ERK1/2 protein were significantly decreased in cells overexpressing IR-A relative to controls. These findings reveal the pivotal role of IR-A in endometrial cancer carcinogenesis, and suggest that the association of elevated IR-A levels with cell proliferation and tumorigenicity may be causally linked to its effect on the proportion of cells in S phase and the activation of the Akt pathway. This work was supported by grants from the Specialized Research Fund for the Doctoral Program of Higher Education (No.200800010095) and the National Natural Science Foundation of China (No. 30973181). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Endometrial cancer is the third most common malignancy of the female genital tract reported in China. The incidence and mortality rate of endometrial carcinoma has increased in recent years [1]. An improved understanding of the factors regulating endometrial cancer cell growth should lead to better treatment options. The insulin receptor (IR) belongs to a subfamily of receptor tyrosine kinases that includes the insulin-like growth factor (IGF) 1receptor(IGF-1R) and the insulin-receptor-related receptor. Members of this family of receptors are tetrameric proteins consisting of two extracellular α-subunits and two transmembrane β-subunits linked by disulfide bonds [2]. The human IR is encoded by a large single insulin receptor gene comprising 22 exons and the protein is expressed as two different isoforms that differ at the carboxyl terminus of the α-subunits by 12 amino acids [3]. The presence (IR–B, or IR exon 11+) or absence (IR-A, or exon 11-) of these residues alters the functional properties of the isoform. IR–B is a classical IR that regulates glucose uptake. In contrast, IR-A has potent mitogenic and anti-apoptotic functions and plays a key role in cell proliferation [4]. Expression of the insulin receptor isoform A (IR-A) has been predominantly detected in cancers of the breast, lung, colon [5], thyroid [6], ovaries [7], and smooth and striated muscle [8]. IR-A expression is upregulated in many cancer cells and tissues, which suggests that IR-A-mediated signaling pathways may have important implications for cancer pathogenesis [9]. Understanding the IR-A expression and function in the endometrial cancer tissues and cells is critical for advancing our knowledge of endometrial cancer biology, and could lead to the development of novel tumor-specific therapies. The present study aims to determine the expression of IR-A in the normal endometrium tissues, the endometrial carcinoma tissues and the cell lines. endometrial carcinoma tissues, and endometrial carcinoma cell lines (Ishikawa, KLE, HEC-1-A and RL95-2, which was named RL95-2-CON in this paper). Thenwe explored the role of IR-A in human endometrial cancer development. Results Expression of insulin receptor isoforms and IGF-2 in endometrial cancer cell lines and tissues To assess expression of IR and IGF-2 in human endometrial cancer cell lines, reverse transcription polymerase chain reaction (RT-PCR) and Real time RT-PCR were performed using RNA from the HEC-1-A, Ishikawa, KLE, RL95-2-CON endometrial cancer cell lines, positive control Liver cancer cell line Hep-G2 and breast cancer cell line MCF-7. RT-PCR shown that IR-A, IR–B and IGF-2 are differentially expressed in HEC-1-A, Ishikawa, KLE and RL95-2-CON endometrial cancer cells(Figure 1A). The real time RT-PCR results shown the relative expression levels of IR-A and total IR in four endometrial cancer cells (Figure 1B). The ratio of IR-A to total IR which got from the real time RT-PCR results was highest in Ishikawa cells and reduced in HEC-1-A, KLE and RL95-2-CON cells(Figure 1C). 10.1371/journal.pone.0069001.g001Figure 1 IR-A expression in endometrial carcinoma cell lines. A. Expression of IR-A (444 bp), IR–B (480 bp), IGF-2 (214 bp) and GAPDH (226 bp) mRNA in endometrial carcinoma cell lines was measured by RT-PCR. The results indicates that two transcript isoforms of insulin receptors (IR-A and IR–B) and IGF-2 were co-expressed in HEC-1-A, Ishikawa, KLE and RL95-2–CON cells. RL95-2–CON has a much higher ratio of IR–B expressed than IR-A. B. Real-time RT-PCR indicates the relative levels of IR-A and total IR mRNA in endometrial carcinoma cell lines, normalized to GAPDH. C.The ratio of IR-A/ IR in endometrial cancer cell lines from real-time RT-PCR results. D. ELISA quantitation of IGF-2 protein secreted by RL95-2–CON, RL95-2–IR-A, KLE, HEC-1-A and Ishikawa cells. Previous studies have reported that IR-A is a high affinity receptor for IGF-2 [10], To further confirm whether the endometrial cancer cell lines can secrete IGF-2, enzyme-linked immunosorbent assay (ELISA) was performed using cell culture supernatants from the HEC-1-A, Ishikawa, KLE, RL95-2-con and RL95-2-IR-A cell lines. We observed that IGF-2 secretion was highest in RL95-2–CON cells and reduced in RL95-2–IR-A, KLE, HEC-1-A and Ishikawa cells. There was no difference in IGF-2 secretion between RL95-2–CON and RL95-2–IR-A cells(Figure 1D). RT-PCR results of tissue specimens shown that mRNA encoding IR-A was expressed in 78 of the 103 endometrial carcinoma samples(75.7%) and in 21 of the 60 normal endometrial tissue samples(35%) and the electrophoretic assay of representative examples was shown in Figure 2. 10.1371/journal.pone.0069001.g002Figure 2 Expression of IR-A/ IR–B in endometrial carcinoma tissues and control normal endometrium tissues. A. Representative examples of the endometrial carcinoma tissues (total 103cases). Lane M shown the DNA marker and Lanes T1–T19 correspond to 19 separate endometrial carcinoma patients B. Representative examples of control normal endometrium tissues(total 60 cases). Lanes N1–N25 correspond to 25 separate patients with normal endometrium. Real time RT-PCR results of tissue specimens was shown in table 1, The total IR and IR-A expression levels and the ratio of IR-A to total IR in endometrial carcinoma specimens were significantly higher than them in control endometrial tissue specimens(P<0.05). Further analysis indicated that the tendency was more prominently in patients with type 2 diabetes mellitus (T2DM) (table 2). Table 1 Relative expression levels of IR and IR-A in endometrial cancer and control endometrial tissues. IR / GAPDH IR-A / GAPDH IR-A / IR endometrial cancer 0.018821±0.013805 0.012744±0.011868 0.62964±0.196977 Control endometrial tissues 0.013779±0.012518 0.00827±0.009825 0.502148±0.17704 P value <0.05 <0.05 <0.05 Table 2 Relative expression levels of IR and IR-A in endometrial cancer and control endometrial tissues from patients with type 2 diabetes mellitus. IR / GAPDH IR-A / GAPDH IR-A / IR endometrial cancer 0.02224±0.011295 0.014868±0.007912 0.68192±0.193396 Control endometrial tissues 0.016076±0.014604 0.009154±0.012137 0.5391±0.18775 P value <0.05 <0.01 <0.01 Overexpression of IR-A in RL95-2-CON cells To investigate the role of IR-A in endometrial carcinogenesis and the potential interaction with T2DM signalling pathway, we synthesized stable RL95-2-CON cells that overexpressed IR-A because IR-A mRNA expression in the parental RL95-2-CON cell line is low. Full-length IR-A cDNA was cloned into the pcDNA3.1 eukaryotic expression plasmid (IR-A–pcDNA3.1) and used to construct the RL95-2-CON cell line stably overexpressing IR-A (RL95-2–IR-A). The IR-A mRNA expression in one cloning of the RL95-2–IR-A stable cell line was determined to be 111.41 times higher than in parental cells by real-time RT-PCR and this cloning was used in the follow-up experiments. IR-A overexpression enhances cell proliferation To investigate the effect of IR-A overexpression on cell proliferation, we plotted 7-day growth curves for RL95-2–IR-A, RL95-2–CON and RL95-2–NC using OD490 nm values, The growth curves for RL95-2–CON and RL95-2–NC were similar, but IR-A overexpression significantly enhanced the proliferation of RL95-2-CON cells when plated 2000 cells/well starting cells(Figure 3A). According to OD490 nm values at 72 hours when plated different number of starting cells, we observed that IR-A overexpression can’t significantly enhanced the proliferation of RL95-2-CON cells when plated 10000 cells/well or more starting cells(Figure 3B). 10.1371/journal.pone.0069001.g003Figure 3 IR-A overexpression increases cell proliferation rate. A. Proliferation of RL95-2–IR-A, RL95-2–CON and RL95-2–NC cells was measured over a 7-day period (plate 2000 cells/well as starting cells) B. Proliferation of the three cells was measured after 72 hours (plate 2000, 5000, 10000, 20000 cells/well as starting cells). 6 replicate wells were included per sample and the data points were present as means±SD. , P < 0.05 vs. control. IR-A overexpression leads to increased cell division To determine whether the IR-A overexpression could influence the cell cycle progression of RL95-2-CON cells, flow cytometry was used to quantitate the proportion of RL95-2–IR-A cells containing in S phase DNA content relative to controls. We found that 25.65% and 26.39% of RL95-2–CON and RL95-2–NC cells, respectively, were in contained S phase cells, but that this was increased to 35.04% in RL95-2–IR-A cells (Figure 4A, C). The proportion of RL95-2–IR-A cells containing S phase DNA content was significantly higher than controls (P<0.05). 10.1371/journal.pone.0069001.g004Figure 4 IR-A overexpression affects cell cycle parameters. A. Flow cytometry analysis of the S phase DNA content in RL95-2-IR-A, RL95-2–CON and RL95-2–NC samples. B The percentage of apoptotic cells in three cells. C,D. The proportion of cells containing S phase DNA and the percentage of apoptotic cells are presented as means±SD. , P < 0.05 vs. control. To further investigate the potential anti-apoptotic role of IR-A in endometrial carcinoma, we used a double labeling technique using Annexin-V and PI to distinguish between apoptotic and necrotic cells. The percentage of apoptotic cells was 5.47% and 5.32% in the RL95-2–CON and RL95-2–NC control cells, but was reduced to 4.59% in RL95-2–IR-A cells (Figure 4B, D). There was no significant difference of the percentage of apoptotic cells between RL95-2–IR-A and control cells(P>0.05). PI3K-Akt and ERK signaling are differentially regulated in cells overexpressing IR-A As no IR-A specific antibodies are available, we conﬁrmed that expression of IR-A protein was increased in RL95-2–IR-A cells by quantitating the total insulin receptor expression using an insulin receptor (IR) pan-specific antibody. As shown in Figure 5A, B. IR protein expression in RL95-2–IR-A cells was approximately three-fold higher than in either RL95-2–CON or RL95-2–NC cells. 10.1371/journal.pone.0069001.g005Figure 5 Effect of IR-A overexpression on downstream signaling pathways. A. Western blot analysis of IR protein expression and expression of downstream signaling proteins in RL95-2-CON, RL95-2–NC and RL95-2–IR-A cells. B. IR protein expression in RL95-2–IR-A cells is significantly higher than that in RL95-2–CON and RL95-2–NC cells. C. The relative expression of phospho-Akt is signiﬁcantly increased and the relative expression of phosphorylated ERK1/2 reduced in RL95-2–IR-A cells than controls. , #, P < 0.05 vs. control. The PI3K-Akt and ERK signaling plays a role in the regulation of various cellular processes such as proliferation, differentiation, development, survival and apoptosis [11]. So we measured phospho-ERK1/2, phospho-Akt and total ERK1/2 and Akt to detect the activation of these two signaling pathways. Total ERK1/2 and Akt protein expression were not affected by IR-A overexpression. However, levels of phospho-Akt were signiﬁcantly increased in RL95-2–IR-A cells relative to controls, while levels of phospho-ERK1/2 were reduced (Figure 5C). Inhibition of the PI3K–Akt pathway reverses the effects of IR-A overexpression In order to confirm the results that IR-A -induced cell proliferation through PI3K/Akt pathway, we treat the RL95-2–IR-A cells with PI3K inhibitor (LY294002) [12]. Levels of phosphorylated Akt were reduced 48 h after treatment with 5, 10, 20 or 40 µM LY294002. Further, the proportion of RL95-2–IR-A cells in S phase was reduced and the proliferation rate of RL95-2–IR-A cells was reduced 48 h after treatment with 20 µM LY294002(Figure 6). 10.1371/journal.pone.0069001.g006Figure 6 PI3K inhibition reverses the effects of IR-A overexpression. A. Western blot analysis of phospho-Akt and total Akt levels 48 h after treatment with 5, 10, 20 and 40 µM of LY294002. B. Growth curves of RL95-2–IR-A cells following treatment with 20 µM LY294002 and without. C. Flow cytometry analysis indicating the proportion of RL95-2–IR-A cells containing S phase DNA at 48 h after treatment with 20 µM LY294002. D. Flow cytometry analysis indicating the percentage of apoptotic cells in RL95-2–IR-A cells at 48 h after treatment with 20 µM LY294002. , P < 0.05 vs. control. IR-A overexpression increases tumorigenicity of endometrial carcinoma cells To explore whether IR-A overexpression in RL95-2-CON cells affects their tumorigenicity of nude mice xenografts, we injected RL95-2-IR-A, RL95-2–CON or RL95-2–NC cells into BALB/c nude mice and monitored tumor volumes every week for 5 weeks. As shown in Figure 7, there are subcutaneous tumor in all three group mice and the histology of tumours was endometrial carcinoma. RL95-2–IR-A xenografts developed larger tumors than those of RL95-2–CON and RL95-2–NC control cells, indicating that 1R-A overexpression exerts a strong tumor-promoting effect on endometrial carcinoma cells in vivo. 10.1371/journal.pone.0069001.g007Figure 7 Tumorigenicity of the RL95-2–IR-A, RL95-2–CON and RL95-2–NC cells in a xenograft model. A. Photographs of the inoculated BALB/c nude mice five weeks after inoculation, showing the tumor size. B. RL95-2–IR-A, RL95-2–CON and RL95-2–NC cells were injected groups of five mice and tumor volumes were measured using calipers every week after the inoculation. C. Five weeks after inoculation, tumors were excised, fixed, and stained by hematoxylin and eosin (H&E; 400× magnification). *, P < 0.05 vs. control. Discussion In this study, we investigated the role of IR-A in endometrial carcinogenesis in vitro and in vivo. We found that IR-A is expressed at similar levels in normal endometrial tissues, endometrial carcinoma tissues and endometrial carcinoma cells. However, further analysis showed that IR-A expression was significantly higher in endometrial carcinomas from patients with T2DM than that in control patients. A number of epidemiological studies have demonstrated that type 2 diabetes mellitus (T2DM) is an important risk factor for many cancers, including endometrial carcinoma, although the molecular mechanism remains unclear [13]. the insulin receptor (IR) have been found to be overexpressed in cancer cells [14,15] and signaling through IR is increased in hyperinsulinemia [16]. Several studies have firmly established that IGF-2 elicits its biological effects through IR-A. For instance, IGF-2 is a more potent mitogen than insulin in mouse fibroblasts expressing only IR-A and not IGF-IR (R-/IR-A cells) [4]. Previous studies have also shown that endometrial carcinomas can express IGF-2 [17]. Interestingly, we confirmed that endometrial carcinoma cells, and particularly the RL95-2-CON cell line, secrete IGF-2 by RT-PCR and Elisa. Taken together, these data support that IR-A overexpression drives endometrial carcinogenesis, and we additionally propose that increased IGF-2 secretion in cells overexpressing IR-A may further stimulate the IR pathway. Our study also shown that cell lines with overexpression of IR-A(RL95-2-IR-A) was successfully constructed and overexpression of IR-A showed a significant proliferation-promoting effect in vitro on RL95-2-CON cell lines which originally has a low expression level of IR-A. Flow cytometry analysis shown that one possible cause of cell growth faster in RL95-2–IR-A may be due to the DNA content in S phase increased. The xenotransplant nude mice model datas shown that the average tumor volume in RL95-2–IR-A xenotransplant mice group were signiﬁcantly bigger than those in the control group indicating that the IR-A could increased the growth of RL95-2-CON cells in vivo. We discovered that the PI3K/Akt signaling pathway was activated and and the MAP kinase pathway was inactivated in cells overexpressing IR-A level, indicating that IR-A-mediated proliferation occurs through the PI3K/Akt signaling pathway and not the MAP kinase pathway. The PI3K/AKT axis regulates essential cellular functions including cell survival, proliferation, migration, and angiogenesis [18]. the PI3K-Akt signaling pathway is implicated in human diseases including diabetes and cancer. Dysregulation and activation of the pathway is common in a large fraction of most human tumor types [19]. Further, PI3K-Akt pathway inhibition led to a reduction in Akt phosphorylation, a reduction in the proportion of cells in S phase and a reduction in growth rate of cells overexpressing IR-A. Our hypothesis may be perfect for that IR-A overexpression drives endometrial carcinogenesis through the PI3K/Akt signaling pathway. In conlusion, IR-A expression in endometrial carcinoma patients with T2DM was significantly higher than that in patients without T2DM, and it is likely that IR-A overexpression can promote endometrial carcinoma cell proliferation. It is possible that the level of IR-A expression could make a significant contribution to cell growth and survival in endometrial carcinomas. However, the speciﬁc pathogenesis and molecular mechanism requires further investigation. Materials and Methods Reagents CellTiter 96® AQueous One Solution Cell Proliferation Assay was purchased from Promega (USA). The Annexin V-FITC & PI Apoptosis Detection Kit was purchased from Beijing Puli Lai Gene Technology Co. Ltd (China). The anti-insulin receptor antibody was purchased from Abcam (Cambridge, UK); anti-phospho-p44/42 MAP Kinase (Thr202/Tyr204), anti-p44/42 MAPK, anti-phospho-Akt (Ser473), anti-Akt, anti β-actin and anti-rabbit IgG horseradish peroxidase (HRP)- conjugated antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). LY294002 was purchased from Invitrogen (Carlsbad, CA, USA). Tissue samples and cell lines With approval from the ethics committee for research involving Human Subjects of Peking University People’s Hospital and patients’ written informed consent, Samples of 103 endometrial carcinomas were obtained from patients treated with surgery in the Peking University People’s Hospital from November 2007 to July 2011. 18 of them are patients with type 2 diabetes mellitus (T2DM). 60 normal endometrial tissues were obtained from patients who received hysterectomy for early stage ovarian cancer or early stage Cervical Cancer during the same period. 9 of them are patients with T2DM. The tissue samples were immediately snap frozen and stored at liquid nitrogen. All endometrial carcinomas and normal endometrial tissues were review confirmed by two pathologists. The mean ages of patients with endometrial carcinomas and controls were 61 ± 11 years and 56± 14 separately. The difference was not statistically significant. Ishikawa, HEC-1a, KLE and RL95-2-CON endometrial cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in our laboratory. Breast cancer cell line MCF-7 was donated by Professor Mao Zebin in department of Biochemistry, Peking University Basic Medical School and Liver cancer cell line Hep-G2 was donated by Hepatobiliary Surgery Center of Peking University People’s Hospital, both of them was obtained from American Type Culture Collection initially. All cell lines were maintained under 5% CO2 at 37° C in appropriate culture medium containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). Ishikawa, HEC-1a, MCF-7, and Hep-G2 cells were cultured in DMEM and KLE and RL95-2-CON cells were cultured in DMEM/F12 (Gibco-BRL, Gaithersburg, MD). Reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR Total RNA was prepared from endometrial cancer cells and frozen tissue samples using Trizol reagent (Invitrogen, USA) and cDNA was synthesized from 2 µg total RNA using a QuantScript RT Kit (Tiangen, China), and used for PCR ampliﬁcation. Two pairs of primers were used to amplify the insulin receptor isoforms. F1,5’-AGG CAG GCG GAA GAC AGT-3’ and R1,5’-GAT GCG ATA GCC CGT GAA-3’ ampliﬁed IR-A and IR–B mRNA fragments of 444 bp and 480 bp, respectively; F2,5’-AAC CAG AGT GAG TAT GAG GAT-3’ and R2,5’-CCG TTC CAG AGC GAA GTG CTT-3’ ampliﬁed IR-A and IR–B mRNA fragments of 600 bp and 636 bp, respectively. Primers used to amplify IGF-2 were F,5’-CTG TGC TAC CCC CGC CAA GT-3’ and R,5ʹ-ACG TTT GGC CTC CCT GAA CG-3’, producing a 214 bp fragment. As a control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was ampliﬁed using the primers F,5’-GAA GGT GAA GGT CGG AGT C-3’ and R,5’-GAAGATGGTGATGGGATTTC-3’, producing a 226 bp fragment. To accurately quantify insulin receptor mRNA expression, IR-A, IR and GAPDH cDNAs were ampliﬁed using real-time PCR. Primers, probe and PCR Master Mix (GAPDH, IR-A, IR) were purchased from Applied Biosystems (USA). All reagents were kept on ice and the probe mix was kept in the dark. PCR conditions were: 50° C for 2 min, 95° C for 10 min, then 40 cycles of 92° C for 15 s and 60° C for 60 s. The comparative Ct method was used to calculate the relative differences in mRNA expression. Enzyme-linked immunosorbent assay IGF-2 protein secretion was measured in culture medium using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R and D Systems). The detection limit of the kit is 15 pg/mL. Construction of RL95-2-CON cell lines overexpressing IR-A Full-length IR-A cDNA was amplified from the INSR-TOPO-XL (BC117172) plasmid using high-fidelity DNA polymerase, cloned into the pcDNA3.1 eukaryotic expression vector (Invitrogen, USA) and verified by restriction digest (two bands of the expected sizes, 5.5 kb and 4 kb) and sequencing. The IR-A–pcDNA3.1 plasmid was transformed into E. coli MAX DH10B and amplified, then transfected into RL95-2-CON cells using Lipofectamine 2000 (Invitrogen, USA). Stably transfected cell clone were done using G418 scanning. Western blot and RT PCR analysis for detecting IR-A protein and mRNA in stably transfected cell clone. Stable cells clone containing IR-A–pcDNA3.1 were named RL95-2–IR-A; those containing empty pcDNA3.1 plasmid were named RL95-2–NC; and the parental cell line was named RL95-2-CON. Cell proliferation The MTS assay was used to generate 7-day growth curves and OD 490nm at 96h for RL95-2–CON, RL95-2–NC and RL95-2–IR-A cell lines, according to the protocol for the CellTiter 96® AQueous One Solution Cell Proliferation Assay. The proliferation rate of RL95-2–IR-A cells treated with 0 or 20 µM of the PI3K inhibitor LY294002 for 48 h was also established. Each treatment was administered to cells at the same time in six individual wells per experiment, and the experiments were repeated three times. Flow cytometry Cells (5 x 105) were seeded into 6-well plates and incubated overnight in DMEM/F12 containing 10% FBS, and then harvested for flow cytometry analysis as described previously [20]. Chilled ethanol (4° C) was added to the cell suspension to a final concentration of 70% and incubated at 4° C overnight. After washing, cells were resuspended in PBS and strained through a 400-µm mesh sieve (Wako, Osaka, Japan) to exclude cell aggregates. RNase (100 µg/ml final concentration) was added to the cell suspension and incubated at 37° C for 30 min in the dark. Cells were then stained with propidium iodide (PI; 100 µg/ml) in PBS for 30 min at room temperature. The DNA content of cell samples was measured using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). Apoptotic cells were detected according to the flow cytometry protocol of the Annexin V-FITC & PI Apoptosis Detection Kit (Beijing Biosea Biotechnology Co., Ltd, China) using a FACSCalibur flow cytometer. Cell cycle progression was determined for RL95-2–IR-A cells treated with 0 or 20 µM of LY294002 for 48 h. Western blot analysis RL95-2–IR-A,RL95-2–NC and RL95-2–CON cells were harvested and protein extraction was performed as described previously [21]. Protein concentration was determined using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. Soluble protein (30 µg) was separated on a 10% SDS-PAGE gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Membranes were blocked by incubation with 5% skimmed milk in Tris-buffered saline containing 0.1% (v/v) Tween-20 (Sigma-Aldrich, St. Louis, MO, USA; TBS-T) at room temperature for 2 h. Phosphorylated ERK1/2, total ERK1/2, phosphorylated Akt, total Akt and β-actin proteins were detected by incubating membranes overnight at 4° C with the relevant antibodies (1:1000 dilution) in TBS-T containing 5% BSA. After washing three times for 5 min with TBS-T, membranes were incubated for 2 h at room temperature with HRP-conjugated secondary antibody (1:5000 dilution) in TBS-T containing 5% skimmed milk. Proteins were detected using the ECL Plus Western blotting detection system (GE Healthcare, Chalfont St. Giles, UK). Protein band intensity was measured using the MicroChemi chemiluminescence gel imaging system (Israel DNR). RL95-2–IR-A were treated with 0, 5, 10, 20 or 40 µM LY294002 PI-3K inhibitor for 48 h, then phospho-Akt expression was measured by Western blotting Xenograft assay In vivo studies were performed in athymic nude mice to examine the tumorigenicity of control RL95-2-CON and IR-A-overexpressing cells. Female four-week-old BALB/c nude mice were obtained from the Beijing Vital River Laboratory Animal Technology Co Ltd (Certificate No: SCXK 2006-0008) and maintained in speciﬁc pathogen-free facilities approved by the Animal Care and Use Committee of Peking University People’s Hospital. Nude mice were randomly divided into three groups (five mice per group). RL95-2-IR-A, RL95-2-NC or RL95-2-CON cells (5×106) were inoculated subcutaneously into the right ﬂank of each mouse. Tumor growth was measured weekly using vernier calipers and tumor volume was calculated using the formula [4]: length (mm) × width2 (mm2)/2. Animals were sacriﬁced five weeks post- inoculation. Tissue samples were harvested for histological analysis, ﬁxed and embedded in parafﬁn wax, and 3 µm sections were cut. Statistical analysis All statistical analyses were performed using SPSS13.0 software (SPSS, Chicago, IL, USA). Comparisons among all cell groups were performed using one-way analysis of variance (ANOVA) and P-Values less than 0.05 was considered to be statistically signiﬁcant. The Tukey’ post hoc test was used for samples where differences were statistically signiﬁcant. ==== Refs References 1 Amant F , Moerman P , Neven P , Timmerman D , Van Limbergen E et al. (2005 ) Endometrial cancer . Lancet . 366 : 491 -505 . doi:10.1016/S0140-6736(05)67063-8. PubMed: 16084259.16084259 2 Giudice J , Leskow FC , Arndt-Jovin DJ , Jovin TM , Jares-Erijman EA (2011 ) Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B . J Cell Sci 124 : 801 -811 . doi:10.1242/jcs.076869. PubMed: 21303927.21303927 3 Moller DE , Yokota A , Caro JF , Flier JS (1989 ) Tissue-speciﬁc expression of two alternatively spliced insulin receptor mRNAs in man . Mol Endocrinol 3 : 1263 –1269 . doi:10.1210/mend-3-8-1263. PubMed: 2779582.2779582 4 Denley A , Bonython ER , Booker GW , Cosgrove LJ , Forbes BE et al. (2004 ) Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR . Mol Endocrinol 18 : 2502 –2512 . doi:10.1210/me.2004-0183. PubMed: 15205474.15205474 5 Frasca F , Pandini G , Scalia P , Sciacca L , Mineo R et al. (1999 ) Insulin receptor isoformA,a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells . Mol Cell Biol 19 : 3278 –3288 . PubMed: 10207053.10207053 6 Vella V , Pandini G , Sciacca L , Mineo R , Vigneri R et al. (2002 ) A novel autocrine loop involving IGF-II and the insulin receptor isoform-A stimulates growth of thyroid cancer . J Clin Endocrinol Metab 87 : 245 –254 . doi:10.1210/jc.87.1.245. PubMed: 11788654.11788654 7 Kalli KR , Falowo OI , Bale LK , Zschunke MA , Roche PC et al. (2002 ) Functional insulin receptors on human epithelial ovarian carcinoma cells: implications for IGF-II mitogenic signaling. Endo-crinology 143: 3259–3267 8 Sciacca L , Mineo R , Pandini G , Murabito A , Vigneri R et al. (2002 ) In IGF-I receptor-deficient leiomyosarcoma cells autocrine IGF-II induces cell invasion and protection from apoptosis via the insulin receptor isoform A . Oncogene 21 : 8240 –8250 . doi:10.1038/sj.onc.1206058. PubMed: 12447687.12447687 9 Denley A , Wallace JC , Cosgrove LJ , Forbes BE (2003 ) The insulin receptor isoform exon 11- (IR-A) in cancer and other diseases: a review . Horm Metab Res 35 : 778 –785 . doi:10.1055/s-2004-814157. PubMed: 14710358.14710358 10 Ulanet DB , Ludwig DL , Kahn CR , Hanahan D (2010 ) Insulin receptor functionally enhances multistage tumor progression and conveys intrinsic resistance to IGF-1R targeted therapy . Proceedings of the National Academyof Sciences of the United States of America 107 : 10791 –10798 . PubMed: 20457905. 11 Alvino CL , Ong SC , McNeil KA , Delaine C , Booker GW et al. (2011 ) Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II . PLOS ONE 6 : e27488 . doi:10.1371/journal.pone.0027488. PubMed: 22140443.22140443 12 Shaul YD , Seger R (2007 ) The MEK/ERK cascade: from signaling specificity to diverse functions . Biochim Biophys Acta 1773 : 1213 –1226 . doi:10.1016/j.bbamcr.2006.10.005. PubMed: 17112607.17112607 13 Lai JP , Sandhu DS , Yu C , Moser CD , Hu C et al. (2010 ) Sulfatase 2 protects hepatocellular carcinoma cells against apoptosis induced by the PI3K inhibitor LY294002 and ERK and JNK kinase inhibitors . Liver Int 30 : 1522 -1528 . doi:10.1111/j.1478-3231.2010.02336.x. PubMed: 21040406.21040406 14 Hemminki K , Li X , Sundquist J , Sundquist K (2010 ) Risk of cancer following hospitalization for type 2 diabetes . Oncologist 15 : 548 -555 . doi:10.1634/theoncologist.2009-0300. PubMed: 20479278.20479278 15 Papa V , Pezzino V , Costantino A , Belfiore A , Giuffrida D et al. (1990 ) Elevated insulin receptor content in human breast cancer . J Clin Invest 86 : 1503 –1510 . doi:10.1172/JCI114868. PubMed: 2243127.2243127 16 Gallagher EJ , LeRoith D (2010 ) The proliferating role of insulin and insulin-like growth factors in cancer . Trends Endocrinol Metab 21 : 610 –618 . doi:10.1016/j.tem.2010.06.007. PubMed: 20663687.20663687 17 McGrath M , Lee IM , Buring J , De Vivo I (2011 ) Common genetic variation within IGFI, IGFII, IGFBP-1, and IGFBP-3 and endometrial cancer risk . Gynecol Oncol 120 (2): 174 -178 . doi:10.1016/j.ygyno.2010.10.012. PubMed: 21078522.21078522 18 Garrett JT , Chakrabarty A , Arteaga CL (2011 ) Will PI3K pathway inhibitors be effective as single agents in patients with cancer? Oncotarget 2 : 1314 -1321 . PubMed: 22248929.22248929 19 Cantley LC (2002 ) The phosphoinositide 3-kinase pathway . Science 296 : 1655 –1657 . doi:10.1126/science.296.5573.1655. PubMed: 12040186.12040186 20 Zhang G , Li X , Zhang L , Zhao L , Jiang J , Wang J et al. (2010 ) The expression and role of hybrid insulin/insulin-like growth factor receptor type 1 in endometrial carcinoma cells . Cancer Genet Cytogenet 200 : 140 –148 . doi:10.1016/j.cancergencyto.2010.04.007. PubMed: 20620597.20620597 21 Zhang L , Li X , Zhao L , Zhang L , Zhang G et al. (2009 ) Nongenomic effect of estrogen on the MAPK signaling pathway and calcium influx in endometrial carcinoma cells . J Cell Biochem 106 (4): 553 -562 . doi:10.1002/jcb.22017. PubMed: 19160418.19160418	23950881	PMC3737217	CC BY	2021-01-05 17:36:50	yes	PLoS One. 2013 Aug 7; 8(8):e69001
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23951180PONE-D-13-1271810.1371/journal.pone.0071518Research ArticleBiologyGeneticsGene expressionDNA transcriptionMolecular cell biologyCellular StructuresCell NucleusGene expressionDNA transcriptionSignal TransductionSignaling CascadesTyrosine Kinase Signaling CascadeSignaling in Cellular ProcessesNuclear SignalingTranscriptional SignalingNuclear Receptor SignalingMapping C-Terminal Transactivation Domains of the Nuclear HER Family Receptor Tyrosine Kinase HER3 Mapping Minimal Transactivation Domains of HER3Brand Toni M. Iida Mari Luthar Neha Wleklinski Matthew J. Starr Megan M. Wheeler Deric L. * Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America Picard Didier Editor University of Geneva, Switzerland * E-mail: dlwheeler@wisc.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: TB DW. Performed the experiments: TB MI NL MW MS. Analyzed the data: TB NL DLW. Contributed reagents/materials/analysis tools: TB MI NL MW MS. Wrote the paper: TB MI DW. 2013 8 8 2013 8 8 e7151826 3 2013 2 7 2013 © 2013 Brand et al2013Brand et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B1) and 27 (B2) amino acids in length that conferred the majority of HER3’s transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B1 and B2 regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B1 and B2 regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs. The project described was supported, in part, by grant P30CA014520 from the National Cancer Institute, by the Clinical and Translational Science Award (CTSA) program, previously through the National Center for Research Resources (NCRR) grant 1UL1RR025011, and now by the National Center for Advancing Translational Sciences (NCATS), grant 9U54TR000021, grant RSG-10-193-01-TBG from the American Cancer Society (DLW), and by NIH grant T32 GM08.1061-01A2 from Graduate Training in Cellular and Molecular Pathogenesis of Human Diseases (TMB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The ErbB/HER family of receptor tyrosine kinases (RTKs) consists of four family members: the epidermal growth factor receptor (EGFR/ErbB1), HER2 (ErbB2/Neu), HER3 (ErbB3), and HER4 (ErbB4). This family of RTKs has been highly implicated in the formation and progression of various cancers via aberrant overexpression, kinase activation, and mutation [1], [2]. Classically, HER family members function from the cell surface, where binding to cognate ligands can induce receptor homo- or hetero-dimerization with other HER family receptors [3]. The HER2 receptor does not bind to any known ligands, however, its dimerization arm is innately positioned in an open conformation. This process leads to the activation of each receptors’ tyrosine kinase and the subsequent phosphorylation of tyrosine residues located on their C-terminal tails. Phosphorylated tyrosine residues recruit various intracellular adaptor and effector molecules that result in the propagation of growth promoting signal transduction cascades [1], [2]. Membrane-bound HER receptors activate numerous tumor promoting signaling cascades via this mechanism, including the PI3K/AKT, Ras/Raf/Mek/Erk, PLCγ/PKC, and signal transducer and activator of transcription (STAT) pathways [1], [2]. While the classical membrane-bound functions of HER family RTKs have been extensively studied, accumulating data suggest that these receptors can be found in the cell’s nucleus where they can function as co-transcriptional activators [4], [5]. To date, EGFR, HER2, and a nuclear variant of HER3 have been shown to function as co-transcriptional activators for cyclin D1 [6]–[8]. Clinically, nuclear EGFR has been correlated with poor overall survival in breast [9], [10], ovarian [11], oropharyngeal [9], [12], and gallbladder [13] cancers. Nuclear EGFR has also been shown to play a role in resistance to numerous cancer therapies, including radiation [14]–[18], cisplatin [17]–[19], and the anti-EGFR therapies cetuximab [20] and gefitinib [21]. Collectively, these pivotal studies suggest that nuclear HER family receptors may enhance the tumorigenic phenotype of cancer cells, and therefore their nuclear roles must be further elucidated. The HER3 receptor has recently come to the forefront as playing a key role in HER family driven cancers [22], [23]. HER3 is a unique HER family member in that it has diminished tyrosine kinase activity due to the lack of specific amino acids within its kinase domain [24], [25]. However, HER3 plays a crucial role as an allosteric activator of other HER family members in addition to functioning as a signaling substrate through the direct recruitment and activation of PI3K [26], [27]. With the discovery of the various functions of nuclear EGFR and HER2, recent interest has prompted the investigation of nuclear HER3. HER3 was first identified to be nuclear localized in normal and malignant mammary epithelial cells in 2002 [28]. HER3 was also shown to be prominently nuclear localized in malignant prostate cancer tissues, where it was correlated with risk of disease progression [29]. Most recently, two nuclear C-terminal splice variants of HER3 were identified and shown to function as co-transcriptional activators [8], [30]. Thus, the functions of nuclear HER3 are just beginning to unfold. In the current study we focused on identifying the amino acid regions on the C-terminal tail of HER3 that function as transactivation domains (TADs). First, numerous cancer cell lines were characterized for the expression of HER3, which was prominently nuclear localized in its full-length form. Next, various HER3 intracellular cytoplasmic domain (ICD) regions fused to the Gal4 DNA binding domain (Gal4DBD) were analyzed, and the C-terminal domain (CTD, amino acids distal to the tyrosine kinase domain) of HER3 was shown to activate the Gal4 upstream activation sequence (UAS) fused to luciferase. To identify specific C-terminal TADs, we created nine HER3-CTD truncation mutants and identified a bipartite region of 34 and 27 amino acids in length (denoted as B1 and B2) that contained the majority of HER3’s transactivation potential. To determine if the identified B1 and B2 TADs could augment nuclear HER3’s transcriptional function in cells, we first identified that full-length nuclear HER3 could associate and activate a 122 bp region of the cyclin D1 promoter when overexpressed. Importantly, the overexpression of HER3 lacking the B1 and B2 regions was severely hindered both in activating the 122 bp region of the cyclin D1 promoter and in enhancing transcription from the endogenous cyclin D1 gene. Collectively, these data suggest that this bipartite region of HER3 functions as a prominent TAD to mediate HER3’s nuclear functions. Materials and Methods Cell Lines The human breast cancer cell line MCF-7, human colorectal cancer (CRC) cell lines LoVo, CaCO2, and CHOK1 cells were purchased from ATCC (Manassas, VA, USA). The human NSCLC line NCI-H226R was developed as a cetuximab resistant cell line previously described [31], [32]. The human breast cancer cell lines SKBr3, BT474, HCC1954, and BT549 were kindly supplied by Dr. J. Boerner (Wayne State University School of Medicine, Karmanos Cancer Institute, MI, USA) [33], the human NSCLC cell line H522 was kindly supplied by Dr. R. Salgia (The University of Chicago Medical Center, IL, USA) [34], and human HNSCC cell lines SCC-1 and SCC-6 were kindly supplied by Dr. T. Carey (University of Michigan, MI, USA) [35]. All cell lines were maintained in their respective media with 10% fetal bovine serum and 1% penicillin and streptomycin; SKBr3, BT549, SCC-1, and SCC-6 were maintained in Dulbecco’s modified Eagle’s medium (Mediatech Inc., Manassas, VA, USA); BT474, HCC1954, and H226R were maintained in RPMI 1640 (Mediatech Inc.); CaCO2 was maintained in minimum essential medium eagle (Mediatech Inc.); LoVo and CHOK1 cells were maintained in F12K medium (Mediatech Inc.); MCF-7 cells were maintained in DMEM/F12K medium (Mediatech Inc.). Antibodies and Compounds All antibodies were obtained from the following sources: HER3-C- TERM (SC-285), HER3-N-TERM (SC-292557), Histone H3 (SC-28654), EGFR (SC-03), HER2 (SC-284), Gal4DBD (SC-510), HRP-conjugated goat-anti-rabbit IgG, and HRP-conjugated goat-anti mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). EGFR (1902-1, EP38Y) was purchased from Epitomics/Abcam (Cambridge, MA, USA). pHER3-Y1222 (#4784), pHER3-Y1289 (#4791), and GAPDH (#2118) were purchased from Cell Signaling Technology (Beverly, MA, USA). α-tubulin was purchased from Calbiochem (San Diego, CA, USA). Neuregulin-1 (NRG) was purchased from R&D Systems (Minneapolis, MN, USA). Cellular Fractionation and Immunoblotting Analysis Cellular fractionation was performed as previously described [6], [36]. Cells were plated in either 10 cm or 15 cm dishes. At ∼80–90% confluency, cells were scraped in PBS and swelled in cytoplasmic lysis buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 2 mM MgCl2, 0.5% NP40, 1 mM Na3VO4, 1 mM PMSF, 1 mM β-glycerophosphate (BGP), 10 ug/ml of leupeptin and aprotinin) for 15 min on ice. Cells were then homogenized by 30–40 strokes in a tightly fitting Dounce homogenizer and checked under microscope for intact nuclei. The homogenate was centrifuged at 1,500 g for 5 min at 4°C to sediment the nuclei. The nuclear pellet was washed 5 times in cytoplasmic lysis buffer to ensure complete removal of cytosolic membranes. After washes, the nuclear pellet was lysed in the same buffer with the addition of 0.5 M NaCl. Nuclear pellets were sonicated for 10 sec, and vortexed for 30 sec 3 times. The extracted nuclear lysate was centrifuged at 15,000 g for 10 min at 4°C, and the supernatants were collected as nuclear lysate. Whole cell protein lysate was obtained through lysis with RIPA buffer (50 mM HEPES, pH 7.4, 150 mM NaCl,.1% Tween-20, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM Na3VO4, 1 mM PMSF, 1 mM BGP, and 10 ug/ml of leupeptin and aprotinin). Samples were sonicated for 10 sec, and then centrifuged at 15,000 g for 10 min at 4°C. All protein lysates were quantified by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (∼20 ug) were fractionated by SDA-PAGE, transferred to a PVDF membrane (Millipore, Billerica, MA, USA), and analyzed by incubation with the appropriate primary antibody overnight at 4°C. Membranes were then subjected to incubation with HRP-conjugated secondary antibodies for 1 hr at room temperature. ECL chemiluminescence detection system was used to visual proteins with either of the following reagents: ECL Western Blotting Substrate (Promega Cooperation, Madison, WI, USA) or SuperSignal West Dura Extended Duration Chemiluminescent Substrate (Thermo Fisher Scientific, Walham, MA, USA). Immunoprecipitation of HER3 Cells were lysed with NP-40 lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Deoxycholic acid, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 mM BGP, 1 mM Na3VO4, and 10 ug/ml of leupeptin and aprotinin). Lysates were sheered via syringe 3 times, and then centrifuged at 15,000 g for 10 min at 4°C. 250 ug of protein was incubated overnight at 4°C with 2 ug of primary C-TERM or N-TERM HER3 antibody. Normal human IgG (Sigma, St. Louis, MO) was used as a negative control. Next day, 25 ul of protein A/G agarose beads (Santa Cruz Biotechnology) were added for 3 hr at 4°C. The immunoprecipitates were pelleted by centrifugation and washed 5 times with NP-40 lysis buffer. The captured immunocomplexes were eluted by boiling in 2×SDS sample buffer for 5 min and subjected to immunoblot analysis as described above. Immunofluorescent Staining of HER3 Approximately 3×103 cells/well were seeded on a four-well glass chamber slide (Millipore). 24 hr later, cells were washed briefly with PBS, and fixed with 4% methanol-free formaldehyde for 15 min at room temperature. Cells were rinsed with PBS again, and permeabilized with PBS containing 0.2% TritonX-100 for 15 min. Cells were blocked in 5% normal goal serum diluted in PBS containing 0.3% Triton X-100 for 1 hr at room temperature, and primary C-TERM or N-TERM HER3 antibody was incubated overnight at 4°C. The next day, cells were rinsed with PBS 3 times, and incubated with Alexa Fluor-546 rabbit secondary antibody for 30 min at room temperature (Life Technologies, Carlsbad, CA, USA). Cells were rinsed with PBS again and mounted with ProLong gold with DAPI antifade mounting solution (Life Technologies). Fluorescence microscopy and photography were performed using a Nikon 80 i upright confocal microscope. Plasmid Construction and Transfection Wild-type human HER3-pSPORT6 vector was kindly provided to us by Dr. P. Bertics (University of Wisconsin-Madison, WI, USA). HER3 was subsequently amplified via polymerase chain reaction and subcloned into KpnI/NotI restriction sites of the pcDNA6/V5-HisA vector (Life Technologies). PCDNA3.0-EGFRWT was kindly supplied by Dr. J. Boerner (Wayne State University School of Medicine, Karmanos Cancer Institute, MI, USA). EGFR-ICD and CTD, and HER3-ICD, JKD, and CTD were cloned into the pM-Gal4DBD expression vector using the SmaI/XbaI restriction sites (Clontech, Mountain View, CA, USA). Gal4DBD-EGFR, Gal4DBD-HER3, pcDNA6-HER3, pcDNA6-HER3ΔB1B2, pcDNA6-HER3WT-ICD, and pcDNA6-HER3ΔB1B2-ICD constructs were generated using the Phusion Site-Directed Mutagenesis Kit (Finnzymes, Keilaranta, Finland). pSPORT6-HER3-Y1222F/Y1289F construct was created via QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions. All mutations were verified for correct orientation and integrity via sequencing. The pGL4.35[luc2P/9XGal4UAS/Hygro], pGL3-Basic-Luciferase, and pRL-Tk-Renilla vectors were purchased from Promega. The 122 bp region of the cyclin D1 promoter (−33− +89) was subcloned into the pGL3-Basic promoterless vector using the KpnI/HindIII restriction sites. The sequences for this primer set are the following: CyD1 FWD 5′-CGGGGTACCCCGGGCTT GATCTTTGCT-3′, CyD1 REV 5′-CCCAAGCTTGACTCTGCTGCTCGCTGCTA-3′. All plasmid transfections were performed using Lipofectamine LTX and Opti-MEM I (Life Technologies) according to the manufacturer’s instructions. Cells were analyzed 24–72 hr post transfection for protein, luciferase activity, and mRNA. For siRNAs, cells were transfected with siHER3 (ON-TARGETplus SMARTpool HER3, L-003127 Dharmacon, Lafayette, CO, USA) or siNon-targeting (NT) (ON-TARGETplus Non-targeting Pool, D-001810, Dharmacon) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Vehicle (V) treated cells were treated with RNAiMAX only. For luciferase studies post siRNA, cells were transfected 24 hr later with cyclin D1-luciferase and Renilla plasmids, and analyzed for luciferase activity 48 hr later. For protein analysis, cells were lysed 72 hr post transfection and analyzed via western blot for HER3 knockdown. Luciferase Assay Cells were plated in 6-well plates at approximate 70% confluence. For the Gal4 UAS-luciferase assay, cells were transiently transfected with 100 ng plasmid DNA, 1 ug of pGL4.35 Gal4 UAS-luciferase vector (Promega), and 100 ng of pRL-Tk-Renilla vector (Promega). For the cyclin D1 promoter luciferase assay, 2 ug of HER3 expression vectors, 1 ug of the 122 bp cyclin D1 promoter-pGL3 luciferase construct, and 10–100 ng of pRL-Tk-Renilla vector (Promega) were used. 48 hr post transfection, cells were collected for analysis via the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Both Firefly and Renilla luciferase reporters were detected in a 96 well format using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Transfection efficiencies were normalized against DNA content, protein content, and the expression of Renilla Luciferase. Chromatin Immunoprecipitation (ChIP) Cells were plated in 15 cm plates at ∼80% confluency in quadruplicate. Each plate was fixed with formaldehyde at a final concentration of 1% for 15 min at room temperature. Fixation was terminated via 1.25 M glycine for 5 min, and cells were subsequently scraped in ice-cold PBS with 1 mM of PMSF. The cells were pelleted by centrifugation at 1500 rpm for 5 min at 4°C and then lysed in cell lysis buffer (5 mM HEPES, pH 8.0, 85 mM KCl, 0.5% NP-40 and 10 mM sodium pyrophosphate). 10 min later cells were further homogenized via Dounce homogenizer (30 strokes), and centrifuged for 1500 g for 5 min at 4°C. The supernatant was removed and nuclei pellets were lysed in nuclei lysis buffer (Tris-HCl 50 mM, pH 8.1, 10 mM EDTA, 1% SDS and 10 mM sodium pyrophosphate). The lysate was sonicated on ice for varied time points to achieve ∼500 bp fragments of DNA (sonication was optimized via running sheared DNA on 1% agarose gels). The supernatant was pre-cleared with protein A/G agarose beads (Santa Cruz Biotechnology) in dilution buffer (16.7 mM Tris-HCl, pH 8.1, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS and 10 mM sodium pyrophosphate) for 1 hr at 4°C. The pre-cleared lysates were immunoprecipated by incubating with protein A/G beads containing 5 ug of the N-TERM anti-HER3 antibody or human IgG (Sigma) rotating overnight at 4°C. The next day immunocomplexes were centrifuged at 1500 rpm for 5 min, and the beads were washed via light vortex for 15 min time intervals with the following wash buffers: wash buffer I (25 mM Tris-HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS and 10 mM sodium pyrophosphate), wash buffer II (25 mM Tris-HCl, pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS and 10 mM sodium pyrophosphate), wash buffer III (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% deoxycholic acid and 10 mM sodium pyrophosphate), and TE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA, 10 mM sodium pyrophosphate). The bound DNA was eluted twice with elution buffer (10 mM NaHCO3 and 1% SDS). 5 mM NaCl was then added to the pooled DNA and incubated at 68°C overnight. The DNA was recovered and purified using a DNA purification kit (Qiagen, Valencia, CA, USA). The purified chromatin-immunoprecipitated DNA was used as template for qPCR with primers flanking the 122 bp region (−33− +89) of the cyclin D1 promoter: Cyclin D1 FWD 5′-CCGGGCTTGATCTTTGCT-3′, Cyclin D1 REV 5′-GACTCTGCTGCTCGCTGCTA-3′. The qPCR program was: 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec and 55.5°C for 30 sec. The qPCR was performed using the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories). DNA Affinity Pull Down Assay (DAPA) DAPA was performed as previously described [37]. The nucleotide sequences of the 5′ biotinylated oligonucleotides corresponding to the 122 bp cyclin D1 promoter (−33− +89) are the following: Sense 5′- CCGGGCTTGATCTTTGCTTAACAACAGTAACGTCACACGGACTACAGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGGAGCCTCCAGAGGGCTGTCGGCGCAGTAGCAGCGAGCAGCAGAGTC-3′, and Antisense 5′-GACTCTGCTGCTCGCTGCTACTGCGCCGACAGCCCTCTGGAGGCTCCAGGACTTTGCAACTTCAACAAAACTCCCCTGTAGTCCGTGTGACGTTACTGTTGTTAAGCAAAGATCAAGCCCGG-3′. The probes were purchased from Integrated DNA Technologies (Coralville, IA, USA). Following annealing of Sense and Anti-sense oligonucleotdies for 1 hr at 95°C, 4 ug of annealed biotinylated probe was incubated with 500 ug of nuclear cell lysate, and 40 ul of streptavidin-agarose bead suspension (Sigma) in PBS diluted with 1 mM Na3VO4, 1 mM PMSF, 1 mM BGP, 10 ug/ml of leupeptin and aprotinin for 2–3 hr rocking at room temperature. The streptavidin-agarose beads were pelleted by centrifugation and washed 5 times with PBS. The captured probe binding proteins were eluted from the streptavidin-agarose beads by boiling in 2×SDS sample buffer for 5 min and subjected to immunoblot analysis for HER3 binding. cDNA Synthesis and Quantitative PCR Total RNA from cells was prepared using an RNeasy Mini kit (Qiagen, Inc., Valencia, CA). cDNA from total RNA of cells was synthesized using qScript cDNA SuperMix (Quanta BioSciences, Inc., Gaithersburg, MD, USA) according to manufacturer’s protocol. qPCR analysis was performed using a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories). The primer sets used for this analysis were purchased from Life Technologies TaqMan® Gene Expression Assay: Cyclin D1 (Hs00765553_m1) and β-actin (Hs99999903_m1). For amplification cDNA was combined with primers and TaqMan universal PCR Master Mix (Life Technologies) according to the manufacturer’s instructions. All reactions were performed in triplicate. To determine the normalized value, 2−ΔΔCt values were compared between cyclin D1 and endogenous control (β-actin) samples, where the change in crossing threshold (ΔCt) = CtCyclin D1-Ctβ-actin and ΔΔCt = ΔCt(HER3WT or HER3ΔB1ΔB2)-ΔCt(Vector). Statistical Analysis Student t-tests were used to evaluate the significance of changes in all reporter and expression assays as compared to vector only or non-targeting controls. Statistical analysis comparing antibody pull downs in all ChIP assays were also evaluated via Student t-test. Differences were considered statistically significant if P≤0.05. Results The HER3 Receptor is Localized to the Nucleus in its Full-length Form The overexpression of the HER3 receptor has been observed in cancers of the breast [38], non-small cell lung (NSCLC) [39], head and neck (HNSCC) [40], and colon (CRC) [41]. Therefore various cell lines from each cancer type were probed for HER3 expression, including the breast cancer cell lines SKBr3, MCF-7, BT474, HCC1954, and BT549, NSCLC cell lines H522 and H226R (a cell line previously developed to be resistant to the anti-EGFR monoclonal antibody cetuximab [31], [32]), HNSCC lines SCC6 and SCC1, and the CRC lines LoVo and CaCO2 ( Figure 1A ). Results from this experiment indicated that HER3 was expressed broadly across a variety of tumor types. Further, isolation of nuclei from H226R, SKBr3 and MCF-7 cells indicated that full-length HER3 (∼185 kDa) could be detected in the nucleus, whereas nuclear HER3 was undetectable in the HER3 low-expressing breast cancer cell line BT549 ( Figure 1B ). The plasma membrane associated proteins clathrin and dynamin, as well as the endoplasmic reticulum (ER) associated protein calnexin, were used to demonstrate nuclear fraction (Nuc) purity from other membrane-bound proteins and ER proteins. Additionally, the cytoplasmic protein α-tubulin and the nuclear protein Histone H3 were used as non-nuclear fraction (NN) and Nuc purity controls. 10.1371/journal.pone.0071518.g001Figure 1 The HER3 receptor is localized to the nucleus in its full-length form. A. HER3 is expressed in numerous cancer cell lines. Whole cell protein lysates were isolated from various breast, lung, HNSCC, and colon cancer cell lines. Lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin was used as a loading control. B. HER3 is localized to the nucleus in cancer cell lines. H226R, SKBr3, MCF-7, and BT549 cells were harvested for whole cell (WC), non-nuclear (NN) and nuclear (Nuc) protein, fractionated on SDS-PAGE followed by immunoblotting for HER3. Clathrin, dynamin, calnexin, α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. C. Specificity of nuclear HER3 by siRNA. H226R and SKBr3 cells were harvested for NN and Nuc protein 48 hr post treatment with siHER3 or non-targeting (NT) siRNA. Experimental procedure as in 1B. α-tubulin and Histone H3 were used as loading and purity controls for the NN and Nuc fractions, respectively. D. Full-length HER3 is localized to the nucleus. H226R and SKBr3 cells were harvested for WC, NN, and Nuc lysate. WC lysates were harvested 48 hr post treatment with siHER3. 250 ug of cell lysate was immunoprecipitated with an N-TERM HER3 antibody or human IgG control. The immunoprecipitates were fractionated on SDS-PAGE followed by immunoblotting for HER3 with a C-TERM antibody. To validate the specificity of the nuclear HER3 signal detected in Figure 1B , we used siRNA to knockdown HER3 expression and subsequently performed nuclear fractionation in H226R and SKBr3 cell lines. The knockdown of HER3 led to a loss of the 185 kDa band in both the NN and Nuc fractions, whereas vehicle (V) and non-targeting (NT) siRNA did not affect HER3 expression ( Figure 1C ); this demonstrates that the nuclear 185 kDa band detected is specific for HER3. To further show that nuclear HER3 is full-length within the nucleus, we performed IP analysis using an antibody that recognizes the extracellular N-terminal region of HER3 (N-TERM) followed by immunoblot analysis with an antibody that recognizes the C-terminal region of HER3 (C-TERM) in the presence of HER3 siRNA. HER3 could be effectively immunoprecipitated with an N-TERM antibody from whole cell lysate (WC) in both H226R and SKBr3 cells, which was prevented upon knockdown of HER3 with siRNA ( Figure 1D ). IP analysis was further performed from NN and Nuc lysate harvested from H226R and SKBr3 cells using the same N-TERM HER3 antibody ( Figure 1D ), demonstrating that full-length HER3 can be extracted from the Nuc fraction. Confocal immunofluorescent (IF) microscopy was used to visualize HER3 localization in H226R and SKBr3 cells using both C-TERM and N-TERM HER3 antibodies ( Figure 2 ). HER3 was detected using an Alexa Fluor-546 labeled secondary antibody (visualized in red). Merging DAPI and 546 labeled HER3 yielded distinct nuclear HER3 signals in both cell lines with little bleed through from the 405 laser (used to visualize DAPI). Additionally, we detect minimal HER3 staining in the HER3 low expressing cell line BT549, demonstrating specificity for the HER3 primary antibodies. These data demonstrate that full-length HER3 is localized to the nucleus in various cancer cell lines. 10.1371/journal.pone.0071518.g002Figure 2 Confocal Immunofluorescent staining of nuclear HER3. C-TERM and N-TERM HER3 antibodies were used to visualize HER3 localization in H226R, SKBr3, and BT549 cells. Alexa Fluor-546 secondary antibody was used to visualize HER3 (RED) and DAPI was used to visualize the nucleus (BLUE). Merged images were magnified to depict the nuclear localization of HER3 (see white arrows). Magnification 600X. The C-terminus of HER3 Contains a Strong Transactivation Domain Recent studies have demonstrated that EGFR, HER2, and small splice variants of HER3 can function as co-transcriptional activators for various gene targets [4], [5], [8], [30]. The weak tyrosine kinase activity of HER3 and its role in resistance to numerous therapeutic agents promoted our investigation into the non-kinase functions of HER3, including its transactivation potential. To investigate this, we utilized the Gal4/luciferase assay system, where the Gal4 DNA binding domain (Gal4DBD) of the yeast transcription factor Gal4 was fused to the intracellular domain (ICD), juxtamembrane and tyrosine kinase domain (JKD), and the C-terminal domain (CTD) of HER3. As a control for this assay, the EGFR-ICD and CTD were fused to the Gal4DBD as performed by Lin et al [6] ( Figure 3A ). While the EGFR-CTD strongly activated Gal4 UAS-luciferase, the EGFR-ICD was less capable of transactivating UAS-luciferase ( Figure 3B ) recapitulating previous findings [6]. The overexpression of the HER3-CTD led to strong transactivation of Gal4 UAS-luciferase (∼8.7 fold) in CHOK1 cells, while the HER3-ICD and HER3-JKD constructs resulted in minimal luciferase activity that were not statistically different from luciferase detected from the Gal4DBD vector control ( Figure 3C ). To broaden the scope of this finding we transfected two other cell lines and observed similar results ( Figure 3D ). Overall, these data demonstrate that the HER3-CTD contains strong transactivation potential. 10.1371/journal.pone.0071518.g003Figure 3 The C-terminus of HER3 contains a strong transactivation domain. A. EGFR intracellular domain (ICD) map and plasmid validation. The intracellular domain (ICD) and C-terminal domain (CTD) of EGFR were fused to the Gal4 DNA binding domain (Gal4DBD). CHOK1 cells were transfected with each construct for 48 hr prior to harvesting WC lysate and fractionation on SDS-PAGE followed by immunoblotting for EGFR and Gal4DBD. EGFRWT vector was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control. B. EGFR-CTD contains strong transactivation potential. CHOK1 cells were transfected with EGFR-ICD or EGFR-CTD constructs, UAS-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. C. HER3-ICD map and plasmid validation. The HER3- ICD, juxtamembrane and tyrosine kinase domain (JKD), and CTD were fused to the Gal4DBD. Transfection was performed the same as in 1A and immunoblot analysis was performed for HER3 and Gal4DBD. HER3WT was transfected into CHOK1 cells as a positive control. α-tubulin was used as a loading control. D. HER3-CTD contains strong transactivation potential. CHOK1, H226R, and SKBr3 cells were transfected with HER3-ICD, HER3-JKD, and HER3-CTD constructs, UAS-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graphs are representative of four independent experiments. Luciferase activity was normalized to DNA content, protein content, and the expression of Renilla luciferase in all assays. Luciferase activity detected for each construct was normalized to the Gal4DBD vector control. Data points are represented as mean+/−s.e.m. p<0.05. TM (transmembrane); JM (juxtamembrane); KD (kinase domain). Minimal Mapping of the HER3 C-terminus Demonstrates the Existence of a Bipartite Transactivation Domain To further understand the capability of HER3 to function as a transcription factor, we sought to map the minimal C-terminal TADs. Previous reports suggest that TADs often contain proline-rich regions [42], [43]; therefore, we analyzed the CTD of HER3 for proline-rich sequences. Two C-terminal proline-rich regions were identified, one between amino acids 1,142–1,150, and a second between amino acids 1,208–1,214. To investigate if these proline-rich sequences function as TADs both regions were deleted from the HER3-CTD ( Figure 4A ). The ability for HER3-CTD deleted of proline-rich region 1 (CTDΔP1), proline-rich region 2 (CTDΔP2), or both proline-rich regions (CTDΔP1ΔP2) to transactivate Gal4 UAS-luciferase was not statistically different from that of the HER3-CTD ( Figure 4B ). These data suggest that HER3’s C-terminal proline-rich regions do not confer strong transactivation potential and therefore may not function as prominent TADs. 10.1371/journal.pone.0071518.g004Figure 4 Minimal mapping of the HER3 C-terminus demonstrates the existence of a bipartite transactivation domain. A. HER3-CTD proline deletion mapping and validation. The HER3-CTD deleted of proline-rich region 1 (CTDΔP1), proline-rich region 2 (CTDΔP2), and both regions (CTDΔP1ΔP2) were fused to the Gal4DBD. CHOK1 cells were transfected with each construct for 48 hr prior to harvesting WC lysate and fractionation on SDS-PAGE followed by immunoblotting for Gal4DBD. B. The HER3-CTD proline-rich regions do not function as prominent TADs. CHOK1 cells were transfected with the HER3-CTD, CTDΔP1, CTDΔP2, or CTDΔP1ΔP2, UAS-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 6). The graph is representative of three independent experiments. C. HER3-CTD minimal transactivation domain mapping and validation. Nine consecutive regions of the HER3-CTD of increasing length were fused to the Gal4DBD (CTD1-CTD9). CHOK1 cells were transfected with each construct for 48 hr prior to harvesting WC lysate and fractionation on SDS-PAGE followed by immunoblotting for Gal4DBD. D. HER3-CTD contains two regions with strong transactivation potential. CHOK1 cells were transfected with CTD1-CTD9, UAS-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 3). The graph is representative of three independent experiments. E. HER3-CTD bipartite deletion mapping and validation. The regions with strong transactivation potential were deleted from the HER3-CTD, denoted as CTDΔB1 and CTDΔB2. The double deletion mutant CTDΔB1ΔB2 was also constructed. As a control, a region of similar size to B1 and B2 was deleted, denoted as CTDΔR1. CHOK1 cells were transfected with each construct for 48 hr prior to harvesting WC lysate and fractionation on SDS-PAGE followed by immunoblotting for Gal4DBD. F. HER3 contains a strong bipartite C-terminal TAD. CHOK1 cells were transfected with CTDΔB1, CTDΔB2, CTDΔB1ΔB2, or CTDΔR1, UAS-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 6). The graph is representative of four independent experiments. Luciferase activity was normalized to DNA content, protein content, and the expression of Renilla luciferase in all assays. Luciferase activity detected for each construct was normalized to the pM Gal4DBD vector control. Data points are represented as mean +/− s.e.m. P<0.05. To identify HER3 TADs we next constructed a series of nine Gal4DBD HER3-CTD mapping mutants, where small regions of the HER3-CTD were sequentially added back to each construct until the entire HER3-CTD was restored ( Figure 4C ). The transactivation capability of each mapping mutant (CTD1–CTD9) was then tested via Gal4 UAS-luciferase assays in CHOK1 cells ( Figure 4D ). The CTD1 and CTD2 constructs had minimal transactivation activity (similar activity to the Gal4DBD vector control), while CTD3–5 expression yielded slightly more transactivation activity. However, CTD6 demonstrated a 5.0 fold increase in Gal4 UAS-luciferase activation (∼3.0 fold higher than CTD5, p<0.001). The CTD7 construct demonstrated similar transactivation activity to CTD6, while CTD8 displayed a 7.5 fold increase in Gal4 UAS-luciferase activation (∼2.5 fold higher than CTD7, p<0.01), which recapitulated the luciferase activity detected from the full-length HER3-CTD. The CTD9 construct exhibited similar transactivation activity to CTD8 and the full-length HER3-CTD. These data suggest that the HER3-CTD contains two prominent TADs, one between CTD5 and CTD6, and a second between CTD7 and CTD8. To validate the bipartite TAD identified in Figure 4D , both regions were deleted from the HER3-CTD ( Figure 4E ). The first deletion mutant consisted of the region located between CTD5 and CTD6 (amino acids 1,215–1,248) denoted as CTDΔB1 and the second deletion mutant consisted of the region located between CTD7 and CTD8 (amino acids 1,281–1,302) denoted as CTDΔB2. The double bipartite deletion mutant was also constructed, denoted as CTDΔB1ΔB2. To ensure that the B1 and B2 regions are specific TADs, we also deleted one region in the HER3-CTD of approximately the same size as B1 and B2 denoted as CTDΔR1 ( Figure 4E ). While the HER3-CTD transactivated Gal4 UAS-luciferase 7.4 fold higher than the Gal4DBD vector control, CTDΔB1 and CTDΔB2 led to statistically significant reductions in UAS-luciferase activity (3.2 and 2.0 fold decreases), while the CTDΔB1ΔB2 demonstrated the greatest reduction (4.9 fold decrease) ( Figure 4F ). The CTDΔR1 was not hindered in its ability to transactivate UAS-luciferase, demonstrating specificity of the B1 and B2 regions. Collectively, these data demonstrate that the B1 and B2 regions contain the majority of HER3’s transactivation potential. Nuclear HER3 can Associate with a 122 bp Region of the Cyclin D1 Promoter To understand how the identified bipartite TAD influences nuclear HER3’s transcriptional functions we sought to identify a nuclear HER3 gene target. Since full-length nuclear EGFR and HER2 and a nuclear variant of HER3 have been shown to associate with the cyclin D1 promoter [6]–[8], we hypothesized that full-length nuclear HER3 would be similarly associated. As a control, we first validated via chromatin immunoprecipitation (ChIP) analysis that EGFR and HER2 can associate with a 122 bp region of the cyclin D1 promoter (-33 - +89) approximately 6.8 fold (EGFR) and 9.4 fold (HER2) higher than an IgG control ( Figure 5A ). Additionally, DNA affinity precipitation assays (DAPA) were performed using a biotinylated 122 bp cyclin D1 promoter probe. Both EGFR and HER2 associated with the cyclin D1 promoter probe while lysate incubated with beads only lacked association. 10.1371/journal.pone.0071518.g005Figure 5 HER3 can associate with the cyclin D1 promoter. A. Nuclear EGFR and HER2 associate with the cyclin D1 promoter. ChIP using an anti-EGFR, anti-HER2, or normal rabbit IgG antibody was performed with SKBr3 cells and isolated DNA was subsequently used for qPCR with primers flanking the 122 bp cyclin D1 promoter region (n = 3). qPCR specificity for the 122 bp cyclin D1 promoter was confirmed by agarose gel electrophoresis of semi-qPCR products. DAPA analysis was performed using a biotinylated 122 bp cyclin D1 promoter probe incubated with 400 ug of nuclear lysate harvested from SKBr3 cells. Bound proteins were isolated with streptavidin agarose beads and subsequently fractionated on SDS-PAGE followed by immunoblotting for EGFR or HER2. Nuclear lysate incubated with beads only lacked association. B. Nuclear HER3 can associate with the cyclin D1 promoter. ChIP using a N-TERM anti-HER3 or a human IgG antibody was performed with H226R, SKBr3, and BT549 cells. qPCR was performed as in 5A. C. HER3 can associate with a cyclin D1 promoter probe. DAPA analysis was performed using nuclear lysate harvested from H226R, SKBr3, MCF-7, and BT549 cells as in 5A. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. D. HER3 association with the cyclin D1 promoter probe is specific. DAPA analysis was performed as in 5A using nuclear lysate harvested from H226R, SKBr3, MCF-7, and BT549 cells transfected with either non-targeting (NT) or HER3 siRNA for 48 hr. Proteins isolated from the probe were subsequently fractionated on SDS-PAGE followed by immunoblotting for HER3. All data points for ChIP are represented as mean +/− s.e.m and normalized to the IgG control. P<0.05. Next, ChIP was performed using an N-TERM HER3 antibody in H226R, SKBr3, and BT549 cells. Full-length HER3 associated with the 122 bp region of the cyclin D1 promoter in both H226R (3.2 fold) and SKBr3 (5.6 fold) cells while HER3 association with cyclin D1 could not be detected in the HER3 low expressing cell line BT549 ( Figure 5B ). Additionally, HER3 associated with the cyclin D1 promoter probe in a DAPA assay ( Figure 5C ). Detection of HER3 in this assay was specific because siHER3 knockdown studies demonstrated loss of probe binding ( Figure 5D ). Collectively, these data demonstrate that nuclear HER3 can associate with a 122 bp region of the cyclin D1 promoter. Nuclear HER3 can Regulate the Transcription of a 122 bp Region of the Cyclin D1 Promoter via its Bipartite Transactivation Domain Since full-length nuclear HER3 can associate with a 122 bp region of the cyclin D1 promoter, we sought to understand its ability to regulate this region. To do this, the 122 bp region of the cyclin D1 promoter was cloned into a luciferase reporter construct. In Figure 6A , the knockdown of HER3 expression using siRNA decreased cyclin D1-promoter luciferase 24% in H226R, 35% in SKBr3, and 27% in MCF-7 cells compared to cells transfected with NT siRNA. Lysate from the luciferase assay was used to confirm the knockdown of HER3 expression. In Figure 6B , HER3 was overexpressed in SCC6, HCC1954, SKBr3, and BT474 cells, where cyclin D1 promoter-luciferase activity was significantly increased (90%, 50%, 35%, and 65% respectively). Nuclear lysate was obtained to validate the presence of nuclear HER3 upon overexpression. Overall, these experiments demonstrate that HER3 can influence the activation of a 122 bp region of the cyclin D1 promoter in numerous cell lines. 10.1371/journal.pone.0071518.g006Figure 6 Nuclear HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain. A. HER3 knockdown decreases the activation of the cyclin D1 promoter. H226R, SKBr3, and MCF-7 cells were incubated with non-targeting (NT) or HER3 siRNA for 24 hr followed by transfection with the 122 bp cyclin D1 promoter-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n = 6). Percent decreases in cyclin D1 promoter-luciferase activity were normalized to NT transfected cells. The graph is representative of three independent experiments. Luciferase lysate was fractionated on SDS-PAGE followed by immunoblotting for HER3. B. HER3 overexpression activates the cyclin D1 promoter. SCC6, HCC1954, SKBr3, and BT474 cells were transfected with HER3WT or control vector, 122 bp cyclin-D1-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1-promoter luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Nuclear lysate was harvested from each cell line and fractionated on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for the Nuc fraction. C. HER3ΔB1ΔB2 overexpression can prevent the activation of the cyclin D1 promoter while HER3DM remains functional. SCC6, BT474 and HCC1954 cells were transfected with HER3WT, HER3DM, HER3ΔB1ΔB2 or control vector, the 122 bp cyclin-D1-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of seven independent experiments. Inset 1: CHOK1 cells were transfected with HER3WT, HER3ΔB1ΔB2 or control vector for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. Inset 2: CHOK1 cells were transfected with HER3WT, HER3DM, HER3ΔB1ΔB2 or control vector and stimulated for 40 min with 5 nM neuregulin-1. Whole cell lysate was fractionated on SDS-PAGE followed by immunoblotting for indicated proteins. D. HER3 knockdown decreases cyclin D1 expression. SCC6 and BT474 cells were incubated with NT or HER3 siRNA for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to NT transfected cells. The graph is representative of two independent experiments. E. The overexpression of HER3WT enhances cyclin D1 expression while HER3ΔB1ΔB2 is hindered. SCC6 and BT474 cells were transfected with HER3WT, HER3ΔB1ΔB2 or control vector for 72 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of four independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of Renilla luciferase. All data points are represented as mean +/− s.e.m. P<0.05. Next, we measured the ability for full-length HER3 deleted of both the B1 and B2 regions (denoted as HER3ΔB1ΔB2) to transactivate cyclin D1 promoter-luciferase. While the overexpression of wild-type HER3 (HER3WT) led to increases in cyclin D1 promoter-luciferase activity in SCC6, BT474 and HCC1954 cells (97%, 63% and 30% respectively), the overexpression of HER3ΔB1ΔB2 was hindered in its ability to transactivate cyclin D1 in all cell lines (10%, 14% and 0% respectively) ( Figure 6C ). Analysis of the B1 and B2 regions yielded the presence of two tyrosine residues: tyrosine 1222 located in B1, and tyrosine 1289 located in B2. To verify that the loss of luciferase activity by HER3ΔB1ΔB2 was not due to altered signaling dependent on these tyrosines, we tested the ability for a double tyrosine mutant (HER3 Y1222F/Y1289F denoted as HER3DM) to transactivate cyclin D1 promoter-luciferase ( Figure 6C ). The overexpression of HER3DM in all cell lines demonstrated a minor reduction in functionality as compared to HER3WT in this assay (85%, 55%, and 27% respectively); however, the luciferase values between HER3WT and HER3DM were not statistically different from one another in each cell line ( Figure 6C ). Additionally, both HER3WT and HER3ΔB1ΔB2 were effectively nuclear localized (Inset 1, Figure 6C ) and the activation of the downstream signaling molecule AKT was not affected in all mutants (Inset 2, Figure 6C ). These data suggest that the alterations in cyclin D1 luciferase observed upon overexpression of HER3ΔB1ΔB2 were not due to a loss of nuclear HER3 localization or changes in AKT activity. To further understand if the B1 and B2 TADs are required for HER3 induced cyclin D1 expression, we performed quantitative PCR (qPCR) to measure cyclin D1 transcripts after treatment with siHER3 ( Figure 6D ) or overexpression of HER3WT and HER3ΔB1ΔB2 ( Figure 6E ). siRNA knockdown of HER3 in H226R and MCF-7 cells led to significant decreases in cyclin D1 expression (39% and 50% downregulation respectively) as compared to NT treated cells ( Figure 6D ). Importantly, the overexpression of HER3WT vs. HER3ΔB1ΔB2 in SCC6 and BT474 cells demonstrated that HER3WT could significantly enhance cyclin D1 expression (92% and 35% upregulation respectively), while the HER3ΔB1ΔB2 mutant was severely hindered at doing so (8% and 1% upregulation respectively) ( Figure 6E ). The minor increases in cyclin D1 transcripts detected upon HER3ΔB1ΔB2 overexpression were not statistically different from vector transfected cells. Collectively, these data suggest that nuclear HER3 can regulate cyclin D1 expression, in part, through its B1 and B2 TADs. An Intracellular Domain Mutant of HER3 can Regulate the Transcription of Cyclin D1 via its Bipartite Transactivation Domain To further ensure that nuclear HER3, rather than classical signaling emanating from membrane-bound HER3, functions in the regulation of cyclin D1 we created intracellular domain (ICD) truncation mutants of HER3 that lack both the N-terminus and the transmembrane domain (Inset 1, Figure 7A for illustration). HER3WT-ICD (denoted as WT-ICD) and HER3ΔB1ΔB2-ICD (denoted as ΔB1ΔB2-ICD) were overexpressed in SCC6, BT474, and HCC1954 cells alongside the 122 bp cyclin D1 promoter-luciferase construct. While the overexpression of WT-ICD led to increases in cyclin D1 promoter-luciferase activity (60%, 25% and 94% respectively), the overexpression of ΔB1ΔB2-ICD was hindered in its ability to transactivate cyclin D1 in all cell lines (12%, 8% and 11% respectively) ( Figure 7A ). Additionally, both WT-ICD and ΔB1ΔB2-ICD were effectively nuclear localized (Inset 2, Figure 7A ). Next, qPCR was performed to assess the ability for each ICD mutant to augment cyclin D1 expression ( Figure 7B ). The overexpression of WT-ICD in SCC6 and BT474 cells significantly enhanced cyclin D1 expression (200% and 40% upregulation respectively), while ΔB1ΔB2–ICD was hindered at doing so (5% and 12% upregulation respectively). The minor increases in cyclin D1 promoter-luciferase and transcripts detected upon HER3ΔB1ΔB2 overexpression were not statistically different from vector transfected cells. Collectively, these data indicate that HER3 depleted of its membrane-bound functions can effectively regulate the cyclin D1 promoter through its bipartite TAD. 10.1371/journal.pone.0071518.g007Figure 7 An intracellular domain (ICD) mutant of HER3 can regulate a minimal region of the cyclin D1 promoter via its bipartite transactivation domain. A. HER3WT ICD (WT-ICD) can activate the cyclin D1 promoter while HER3ΔB1ΔB2-ICD (ΔB1ΔB2-ICD) is hindered. SCC6, BT474, and HCC1954 cells were transfected with WT-ICD, ΔB1ΔB2-ICD or control vector, the 122 bp cyclin D1-luciferase and Tk-Renilla reporter plasmids for 48 hr prior to quantification by dual luciferase assay (n≥3). Percent increases in cyclin D1 promoter-luciferase were normalized to vector transfected cells. The graph is representative of three independent experiments. Inset 1: Illustration of both the WT-ICD and ΔB1ΔB2-ICD constructs. Inset 2: CHOK1 cells were transfected with WT-ICD and ΔB1ΔB2-ICD for 48 hr prior to harvesting NN and Nuc protein, fractionation on SDS-PAGE followed by immunoblotting for HER3. α-tubulin and Histone H3 were used as loading and purity controls for Nuc fraction. B. The overexpression of WT-ICD can enhance cyclin D1 expression while ΔB1ΔB2-ICD is hindered. SCC6 and BT474 cells were transfected with WT-ICD, ΔB1ΔB2-ICD or control vector for 48 hr prior to harvesting RNA. The mRNA expression of cyclin D1 was determined by qPCR (n = 3) and normalized to vector transfected cells. The graph is representative of three independent experiments. All luciferase values were normalized to DNA content, protein content, and the expression of Renilla luciferase. All data points are represented as mean +/− s.e.m. P<0.05. ICD (intracellular domain). Discussion To date, all HER family RTKs have been shown to be nuclear localized in primary tumor tissues and cancer cell lines where they can function as co-transcription factors [4], [5]. The nuclear expression of both EGFR and HER3 have been correlated with worse disease prognosis in specific cancers [9]–[11], [13], [29], and nuclear EGFR has been shown to enhance resistance to various therapeutic agents [4], [5]. These findings highlight the need to better understand nuclear HER family function. In the current study, we sought to identify the regions on the HER3 receptor that function as TADs to enhance the understanding of HER3’s transcriptional functions. To identify HER3 C-terminal TADs, various regions of the HER3 CTD were fused to the Gal4DBD and tested for their ability transactivate Gal4 UAS-luciferase. Two regions of prominent transactivation potential were identified (B1 and B2) via this method and were further shown to impact nuclear HER3’s ability to regulate both the cyclin D1 promoter and mRNA expression. To date, several investigators have identified full-length nuclear EGFR and HER2 in different tumor types [5], [44]. In the current report, we detected nuclear HER3 in its full-length form with both N- and C- terminal HER3 antibodies ( Figure 1 and 2 ). Additionally ChIP analysis was performed using an N-terminal HER3 antibody ( Figure 5 ). In 2002, Offterdinger et al. observed full-length nuclear HER3 in human breast cancer MTSV1-7 cells [28], which was validated by Koumakpayi et al. in multiple prostate cancer cell lines [29]. Additionally, using both fluorescently labeled HER3 and cell fractionation techniques, Tao et al. demonstrated that full-length HER3 homodimers were highly localized to the nucleus in various cell models [45]. Recently, alternative splice variants of HER3 have been discovered to be nuclear localized, including an 80-kDa fragment in H358 lung cancer cells [8], and a 50-kDa fragment in rat Schwann cells [30]. In the present study, an 80-kDa fragment of HER3 was detected in whole cell lysate from some cell lines, while a 50-kDa fragment was undetectable (data not shown). Thus, in the current study the predominant form of nuclear HER3 detected was that of the full-length receptor. In 2001, a pioneering study by Lin et al. demonstrated that the C-terminus of EGFR (EGFR-CTD) contained strong transactivation potential through the use of a Gal4 UAS-chloramphenicol transferase reporter assay [6], while the EGFR-ICD and EGFR-JKD constructs elicited minimal activation. In the current study we identified that the HER3-CTD also contained strong transactivation potential, similar to that of the EGFR-CTD, while the HER3-ICD and HER3–JKD constructs had minimal activity ( Figure 3 ). Lin et al. hypothesized that the EGFR-JKD may contain negative regulatory sites that hinder the EGFR-CTD from functioning as a transcriptional co-activator [6]. The HER3-JKD may also contain negative regulatory sites, and therefore may inhibit the HER3-CTD from functioning in a Gal4 UAS-luciferase assay. Further experimentation is needed to identify these putative negative regulatory sites and their influence on HER3-CTD function. TADs of transcription factors often contain proline and acidic amino acid rich regions that help recruit proteins necessary for transcription [38], [43]. To define HER3’s TADs, we first identified two prominent C-terminal proline-rich sequences ( Figure 4A ). When the proline-rich sequences were deleted from the HER3-CTD a significant change in Gal4 UAS-luciferase activity was not observed, suggesting that these regions do not function as prominent TADs ( Figure 4B ). Through sequential mapping studies we found two independent regions (B1 and B2) on the HER3-CTD that most prominently activated Gal4 UAS-luciferase ( Figure 4C–4F ). These regions demonstrated specificity because deletion of another region (R1) of similar size did not affect Gal4 UAS-luciferase activity ( Figure 4F ). Motif analysis of this bipartite region did not yield the presence of any known TAD sequence, however various transcription factors contain bipartite TADs, examples including p53 and CREB [46], [47]. Research over the last two decades has demonstrated that TADs are not necessarily rich in any particular sequence, and that TADs have high diversity of sequence and domain structure, unlike the uniform sequence and structure of DBDs [43], [48], [49]. While the bipartite region discovered accounted for ∼66% of HER3’s transactivation potential in a Gal4 UAS-luciferase assay, it did not recapitulate the full activity detected from the entire HER3-CTD, and therefore other CTD regions may function as less prominent TADs. To identify how B1 and B2 influence nuclear HER3 function, we first demonstrated that HER3 can bind to a 122 bp region of the cyclin D1 promoter, a region that was originally found to associate with nuclear EGFR [6]. Interestingly, EGFR, HER2, and HER3 all associated with this relatively small promoter region in SKBr3 cells ( Figure 5 ). Whether HER family dimers exist in the nucleus and function together as co-transcription factors has yet to be determined. We further demonstrate that HER3 lacking the B1 and B2 regions (HER3ΔB1B2) had either reduced ability or an inability to transactivate cyclin D1 promoter-luciferase and cyclin D1 expression in multiple cell lines ( Figure 6 ), suggesting that these regions function as prominent TADs. SCC6 and BT474 cells express endogenous HER3 (See Figure 1A ) and therefore the slight increases in reporter activity detected upon overexpression of HER3ΔB1ΔB2 may have been due to activation by endogenous nuclear HER3. Additionally, both HER3WT and HER3ΔB1ΔB2 were effectively localized to the nucleus (Inset 1, Figure 6C ). Therefore, the lack of cyclin D1 promoter-luciferase detected upon HER3ΔB1ΔB2 overexpression was likely due to its inability to function in the nucleus rather than impairment in nuclear translocation. The specific transcription factors that associate with nuclear HER3 is under current investigation, but we speculate that HER3ΔB1ΔB2 is deficient in the proper association and/or recruitment of these factors. Collectively, these data suggest that the B1 and B2 regions of HER3 function as TADs. One of the major hurdles in the study of nuclear RTKs is to experimentally isolate their nuclear functions from plasma membrane-bound functions. To ensure that the loss of cyclin D1 promoter- luciferase activity detected upon overexpression of HER3ΔB1ΔB2 was due to deficiency in nuclear HER3 functions the tyrosine residues located in the B1 and B2 regions known to play a role in activating signaling cascades were mutated. HER3 mutated at both tyrosine 1222 and 1289 (HER3DM) was only slightly hindered in the activation of the cyclin D1 promoter, unlike HER3ΔB1ΔB2, and both HER3DM and HER3ΔB1ΔB2 were still capable of activating HER3’s downstream effector kinase AKT (Inset 2, Figure 6C ). To further validate that the regulation of cyclin D1 was not a result of classical membrane-bound functions of HER3, ICD mutants of HER3 were created in which HER3WT and HER3ΔB1ΔB2 were deleted of both the N-terminus and transmembrane domain ( Figure 7 ). The WT-ICD, which cannot be localized at the plasma membrane or serve as a dimerization partner to activate classical signaling pathways, was still capable of regulating cyclin D1 luciferase activity and mRNA expression. This finding falls in line with the identified HER3 C-terminal splice variants that have been shown to function as co-transcription factors [8], [30]. Importantly, ΔB1ΔB2-ICD was hindered in cyclin D1 regulation, further supporting the role of these TADs in influencing nuclear HER3 transcriptional function. We speculate that the minor increases in luciferase and mRNA expression observed upon ΔB1ΔB2-ICD overexpression may emanate from endogenous HER3 in SCC6 and BT474 cells and/or the residual transcriptional activity remaining on the C-terminus of the ΔB1ΔB2-ICD. Collectively, these data suggest that the loss of cyclin D1 promoter-luciferase activity and mRNA expression was not due to modulation of signaling pathways emanating from membrane-bound HER3, but likely due to an inability for HER3ΔB1ΔB2 to function as a co-transcription factor. To date, various functions of nuclear localized HER family receptors have been identified. The present study is the first to map specific TADs on a nuclear HER family member, and further TAD mapping studies of both HER2 and EGFR are underway. These studies are important because it is becoming increasingly apparent that nuclear HER family transcriptional functions play a role in cancer biology and that these functions may be independent of their kinase activity [4], [5]. The current study supports these findings because 1) HER3 lacks high levels of kinase activity, and 2) nuclear HER3 regulation of cyclin D1 was independent of plasma membrane-bound HER3 functions and AKT signaling. Since the kinase functions of HER family members may not be solely responsible for the tumorigenic properties of these receptors, the identification of HER family TADs may serve as a map to better target nuclear HER functions in the future. ==== Refs References 1 Yarden Y , Sliwkowski MX (2001 ) Untangling the ErbB signalling network . Nature reviews Molecular cell biology 2 : 127 –137 .11252954 2 Yarden Y , Pines G (2012 ) The ERBB network: at last, cancer therapy meets systems biology . Nature reviews Cancer 12 : 553 –563 .22785351 3 Olayioye MA , Neve RM , Lane HA , Hynes NE (2000 ) The ErbB signaling network: receptor heterodimerization in development and cancer . The EMBO journal 19 : 3159 –3167 .10880430 4 Brand TM , Iida M , Li C , Wheeler DL (2011 ) The nuclear epidermal growth factor receptor signaling network and its role in cancer . Discov Med 12 : 419 –432 .22127113 5 Han W , Lo HW (2012 ) Landscape of EGFR signaling network in human cancers: biology and therapeutic response in relation to receptor subcellular locations . Cancer Letters 318 : 124 –134 .22261334 6 Lin SY , Makino K , Xia WY , Matin A , Wen Y , et al (2001 ) Nuclear localization of EGF receptor and its potential new role as a transcription factor . Nature Cell Biology 3 : 802 –808 .11533659 7 Beguelin W , Diaz Flaque MC , Proietti CJ , Cayrol F , Rivas MA , et al (2010 ) Progesterone receptor induces ErbB-2 nuclear translocation to promote breast cancer growth via a novel transcriptional effect: ErbB-2 function as a coactivator of Stat3 . Molecular and Cellular Biology 30 : 5456 –5472 .20876300 8 Andrique L , Fauvin D , El Maassarani M , Colasson H , Vannier B , et al (2012 ) ErbB3(80 kDa), a nuclear variant of the ErbB3 receptor, binds to the Cyclin D1 promoter to activate cell proliferation but is negatively controlled by p14ARF . Cellular Signalling 24 : 1074 –1085 .22261253 9 Lo HW , Xia W , Wei Y , Ali-Seyed M , Huang SF , et al (2005 ) Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer . Cancer Res 65 : 338 –348 .15665312 10 Hadzisejdic I , Mustac E , Jonjic N , Petkovic M , Grahovac B (2010 ) Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis . Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 23 : 392 –403 . 11 Xia WY , Wei YK , Du Y , Liu JS , Chang B , et al (2009 ) Nuclear Expression of Epidermal Growth Factor Receptor is a Novel Prognostic Value in Patients With Ovarian Cancer . Molecular Carcinogenesis 48 : 610 –617 .19058255 12 Psyrri A , Yu ZW , Weinberger PM , Sasaki C , Haffty B , et al (2005 ) Quantitative determination of nuclear and cytoplasmic epidermal growth factor receptor expression in oropharyngeal squamous cell cancer by using automated quantitative analysis . Clinical Cancer Research 11 : 5856 –5862 .16115926 13 Li CF, Fang FM, Wang JM, Tzeng CC, Tai HC, et al.. (2011) EGFR Nuclear Import in Gallbladder Carcinoma: Nuclear Phosphorylated EGFR Upregulates iNOS Expression and Confers Independent Prognostic Impact. Annals of Surgical Oncology. 14 Dittmann K , Mayer C , Fehrenbacher B , Schaller M , Kehlbach R , et al (2010 ) Nuclear EGFR shuttling induced by ionizing radiation is regulated by phosphorylation at residue Thr654 . FEBS letters 584 : 3878 –3884 .20692258 15 Dittmann K , Mayer C , Fehrenbacher B , Schaller M , Kehlbach R , et al (2011 ) Nuclear epidermal growth factor receptor modulates cellular radio-sensitivity by regulation of chromatin access . Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 99 : 317 –322 .21704408 16 Dittmann K , Mayer C , Rodemann HP (2005 ) Inhibition of radiation-induced EGFR nuclear import by C225 (Cetuximab) suppresses DNA-PK activity . Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 76 : 157 –161 .16024112 17 Dittmann K , Mayer C , Fehrenbacher B , Schaller M , Raju U , et al (2005 ) Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase . The Journal of biological chemistry 280 : 31182 –31189 .16000298 18 Liccardi G , Hartley JA , Hochhauser D (2011 ) EGFR nuclear translocation modulates DNA repair following cisplatin and ionizing radiation treatment . Cancer research 71 : 1103 –1114 .21266349 19 Hsu SC , Miller SA , Wang Y , Hung MC (2009 ) Nuclear EGFR is required for cisplatin resistance and DNA repair . American journal of translational research 1 : 249 –258 .19956435 20 Li C , Iida M , Dunn EF , Ghia AJ , Wheeler DL (2009 ) Nuclear EGFR contributes to acquired resistance to cetuximab . Oncogene 28 : 3801 –3813 .19684613 21 Huang WC , Chen YJ , Li LY , Wei YL , Hsu SC , et al (2011 ) Nuclear Translocation of Epidermal Growth Factor Receptor by Akt-dependent Phosphorylation Enhances Breast Cancer-resistant Protein Expression in Gefitinib-resistant Cells . The Journal of biological chemistry 286 : 20558 –20568 .21487020 22 Baselga J , Swain SM (2009 ) Novel anticancer targets: revisiting ERBB2 and discovering ERBB3 . Nature reviews Cancer 9 : 463 –475 .19536107 23 Campbell MR , Amin D , Moasser MM (2010 ) HER3 comes of age: new insights into its functions and role in signaling, tumor biology, and cancer therapy . Clinical cancer research : an official journal of the American Association for Cancer Research 16 : 1373 –1383 .20179223 24 Sierke SL , Cheng K , Kim HH , Koland JG (1997 ) Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein . The Biochemical journal 322 (Pt 3) : 757 –763 . 25 Jura N , Shan Y , Cao X , Shaw DE , Kuriyan J (2009 ) Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3 . Proceedings of the National Academy of Sciences of the United States of America 106 : 21608 –21613 .20007378 26 Zhang X , Gureasko J , Shen K , Cole PA , Kuriyan J (2006 ) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor . Cell 125 : 1137 –1149 .16777603 27 Prigent SA , Gullick WJ (1994 ) Identification of c-erbB-3 binding sites for phosphatidylinositol 3′-kinase and SHC using an EGF receptor/c-erbB-3 chimera . The EMBO journal 13 : 2831 –2841 .8026468 28 Offterdinger M , Schofer C , Weipoltshammer K , Grunt TW (2002 ) c-erbB-3: a nuclear protein in mammary epithelial cells . The Journal of cell biology 157 : 929 –939 .12045181 29 Koumakpayi IH , Diallo JS , Le Page C , Lessard L , Gleave M , et al (2006 ) Expression and nuclear localization of ErbB3 in prostate cancer . Clinical cancer research : an official journal of the American Association for Cancer Research 12 : 2730 –2737 .16675564 30 Adilakshmi T , Ness-Myers J , Madrid-Aliste C , Fiser A , Tapinos N (2011 ) A nuclear variant of ErbB3 receptor tyrosine kinase regulates ezrin distribution and Schwann cell myelination . The Journal of neuroscience : the official journal of the Society for Neuroscience 31 : 5106 –5119 .21451047 31 Wheeler DL , Huang S , Kruser TJ , Nechrebecki MM , Armstrong EA , et al (2008 ) Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members . Oncogene 27 : 3944 –3956 .18297114 32 Wheeler DL , Iida M , Kruser TJ , Nechrebecki MM , Dunn EF , et al (2009 ) Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab . Cancer Biol Ther 8 : 696 –703 .19276677 33 Irwin ME , Mueller KL , Bohin N , Ge Y , Boerner JL (2011 ) Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib . Journal of cellular physiology 226 : 2316 –2328 .21660955 34 Kanteti R , Nallasura V , Loganathan S , Tretiakova M , Kroll T , et al (2009 ) PAX5 is expressed in small-cell lung cancer and positively regulates c-Met transcription . Laboratory investigation; a journal of technical methods and pathology 89 : 301 –314 .19139719 35 Brenner JC , Graham MP , Kumar B , Saunders LM , Kupfer R , et al (2010 ) Genotyping of 73 UM-SCC head and neck squamous cell carcinoma cell lines . Head & neck 32 : 417 –426 .19760794 36 Lo HW , Hsu SC , Ali-Seyed M , Gunduz M , Xia WY , et al (2005 ) Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway . Cancer Cell 7 : 575 –589 .15950906 37 Wu KK (2006 ) Analysis of protein-DNA binding by streptavidin-agarose pulldown . Methods in molecular biology 338 : 281 –290 .16888365 38 Chiu CG , Masoudi H , Leung S , Voduc DK , Gilks B , et al (2010 ) HER-3 Overexpression Is Prognostic of Reduced Breast Cancer Survival A Study of 4046 Patients . Annals of Surgery 251 : 1107 –1116 .20485140 39 Chen HY , Yu SL , Chen CH , Chang GC , Chen CY , et al (2007 ) A five-gene signature and clinical outcome in non-small-cell lung cancer . The New England journal of medicine 356 : 11 –20 .17202451 40 Erjala K , Sundvall M , Junttila TT , Zhang N , Savisalo M , et al (2006 ) Signaling via ErbB2 and ErbB3 associates with resistance and Epidermal Growth Factor Receptor (EGFR) amplification with sensitivity to EGFR inhibitor gefitinib in head and neck squamous cell carcinoma cells . Clinical Cancer Research 12 : 4103 –4111 .16818711 41 Beji A , Horst D , Engel J , Kirchner T , Ullrich A (2012 ) Toward the Prognostic Significance and Therapeutic Potential of HER3 Receptor Tyrosine Kinase in Human Colon Cancer . Clinical Cancer Research 18 : 956 –968 .22142822 42 Hirai H , Tani T , Kikyo N (2010 ) Structure and functions of powerful transactivators: VP16, MyoD and FoxA . The International journal of developmental biology 54 : 1589 –1596 .21404180 43 Mapp AK , Ansari AZ (2007 ) A TAD further: Exogenous control of gene activation . Acs Chemical Biology 2 : 62 –75 .17243784 44 Wang SC , Lien HC , Xia W , Chen IF , Lo HW , et al (2004 ) Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2 . Cancer Cell 6 : 251 –261 .15380516 45 Tao RH , Maruyama IN (2008 ) All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells . Journal of Cell Science 121 : 3207 –3217 .18782861 46 Harms KL , Chen X (2006 ) The functional domains in p53 family proteins exhibit both common and distinct properties . Cell Death and Differentiation 13 : 890 –897 .16543939 47 Yamamoto KK , Gonzalez GA , Menzel P , Rivier J , Montminy MR (1990 ) Characterization of a Bipartite Activator Domain in Transcription Factor Creb . Cell 60 : 611 –617 .2137373 48 Altmann H , Wendler W , Winnacker EL (1994 ) Transcriptional Activation by Ctf Proteins Is Mediated by a Bipartite Low-Proline Domain . Proceedings of the National Academy of Sciences of the United States of America 91 : 3901 –3905 .8171010 49 Pei DQ , Shih CH (1991 ) An “attenuator domain” is sandwiched by two distinct transactivation domains in the transcription factor C/EBP . Molecular and Cellular Biology 11 : 1480 –1487 .1996105	23951180	PMC3738522	CC BY	2021-01-05 17:36:52	yes	PLoS One. 2013 Aug 8; 8(8):e71518
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23951005PONE-D-13-1134910.1371/journal.pone.0070773Research ArticleBiologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologySignal TransductionMechanisms of Signal TransductionCrosstalkCellular Stress ResponsesGene ExpressionNeuroscienceCellular NeuroscienceMolecular NeuroscienceNeurobiology of Disease and RegenerationNeurochemistryMathematicsStatisticsBiostatisticsMedicineClinical Research DesignAnimal Models of DiseaseNeurologyDementiaAlzheimer DiseaseGadd153 and NF-κB Crosstalk Regulates 27-Hydroxycholesterol-Induced Increase in BACE1 and β-Amyloid Production in Human Neuroblastoma SH-SY5Y Cells Gadd153 and NF-κB Regulate BACE1 ExpressionMarwarha Gurdeep Raza Shaneabbas Prasanthi Jaya R. P. Ghribi Othman * Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, United States of America Cai Huaibin Editor National Institute of Health, United States of America * E-mail: othman.ghribi@med.und.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: GM OG. Performed the experiments: GM SR JP. Analyzed the data: GM OG. Wrote the paper: GM OG. 2013 9 8 2013 8 8 e7077315 3 2013 21 6 2013 © 2013 Marwarha et al2013Marwarha et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.β-amyloid (Aβ) peptide, accumulation of which is a culprit for Alzheimer’s disease (AD), is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC) increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP). We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD. This work was supported by a grant from the National Institutes of Health (NIH) (RO1ES014826). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Alzheimer Disease (AD) is the most common neurodegenerative disorder and the fifth leading cause of death in the elderly. Extracellular deposition of aggregated Amyloid-β (Aβ) peptide in senile plaques and intraneuronal accumulation of aggregated hyperphosphorylated microtubule-associated protein tau (τ) in neurofibrillary tangles (NFT) are the two major pathological hallmarks of AD. The etiology of AD is unknown, but it is widely accepted that increased production and accumulation of Aβ is an instigator of the neurodegenerative processes observed in AD [1]. Reduction in Aβ production and accumulation is an appealing strategy to reduce the progression of AD. Aβ is derived from the amyloid precursor protein (APP) through an initial cleavage by aspartyl protease BACE1 and subsequent cleavage by the γ-secretase enzyme complex [2], [3]. The initial cleavage of APP by BACE1 is the rate-limiting step in Aβ production [2], [3]. Several studies have shown that BACE1 protein, mRNA, and activity are upregulated in AD brains [4]–[7]. Stress in endoplasmic reticulum (ER) may play a role in the pathophysiology of many diseases including AD [8]–[10]. Sustained ER stress upregulates the gene expression of several deleterious transcription factors including that of the growth arrest and DNA damage-induced gene153 (gadd153; also known as C/EBP homologous protein, CHOP). Interestingly, gadd153 has been shown to enhance NF-κB signaling [11], suggesting that gadd153 can crosstalk with NF-κB, and that NF-κB activation can be a downstream event to activated gadd153. Multiple lines of evidence suggest that, in addition to being an important regulator of inflammation, NF-κB also regulates the transcription of BACE1, as evidenced by the presence of κB sites in the BACE1 promoter region [12]–[14]. More evidence of the tight link of NF-κB to the pathophysiology of AD is the observation that this transcription factor is activated in AD patients [12], [13], [15]. Our published studies demonstrated that the cholesterol oxidized metabolite (oxysterol) 27-hydroxycholesterol (27-OHC) increases BACE1 levels in hippocampal organotypic slices from adult rabbits [16] and in human SH-SY5Y neuroblastoma cells [17]. The oxysterol 27-OHC has been shown to accumulate in AD brains [18]. We also showed that 27-OHC induced ER-mediated gadd153 and NF-κB activation in ARPE cells [19] and SH-SY5Y cells [20]. We propose that, because gadd153 and NF-κB may work in concert to upregulate BACE1, a crosstalk between gadd153 and NF-κB would enhance Aβ production and accumulation, and may thus increase the risk for AD. Inhibition of gadd153 would therefore reduce NF-κB and BACE1 expression, prevent Aβ accumulation, and may have a translation potential for reducing the propgression of AD. In the present study, we not only demonstrate the involvement of NF-κB in 27-OHC-induced increase in BACE1 expression levels, but also establish the dynamic interplay between gadd153 and NF-κB in the regulation of BACE1 expression in response to treatment with the oxysterol 27-OHC. Methods Reagents 27-OHC was purchased from Medical Isotopes (Pelham, NH), the NF-κB inhibitor sc514 from Tocris Bioscience (Ellisville, MO), the reporter constructs encoding NF-κB response elements conjugated to the firefly luciferase gene from SA Biosciences (Frederick, MD), and the human BACE1 promoter construct conjugated to the firefly luciferase gene was purchased from SwitchGear Genomics (Menlo Park, CA). All cell culture reagents, with the exception of fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and antibiotic/antimycotic mix (Sigma Aldrich, Saint Louis, MO) were from Invitrogen (Carlsbad, CA). Human SH-SY5Y neuroblastoma cells were purchased from ATCC (Manassas, VA). Cell Culture and Treatments Human neuroblastoma SH-SY5Y cells were grown in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic mix. Cells were maintained at 37°C in a saturated humidity atmosphere containing 95% air and 5% CO2. After reaching 80% confluence, cells were incubated for 24 hours at 37°C in cell medium with vehicle, 5 µM 27-OHC, 5 µM NF-κB inhibitor sc514, or 5 µM 27-OHC +5 µM sc514. siRNA for gadd153 and the respective scrambled non-silencing control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The following human gadd153 double-stranded siRNA sequences (5′ → 3′ orientation) were used (A): Sense GAAGGCUUGGAGUAGACAAtt, Antisense UUGUCUACUCCAAGCCUUCtt; (B): Sense GGAAAGGUCUCAGCUUGUAtt, Antisense UACAAGCUGAGACCUUUCCtt; (C): Sense GUCUCAGCUUGUAUAUAGAtt, Antisense UCUAUAUACAAGCUGAGACtt. The transfection of siRNA was performed in the cells with siRNA transfection reagent (Santa Cruz Biotechnology) and siRNA transfection medium (Santa Cruz Biotechnology) according to the manufacturer’s recommendation. The siRNAs stock solution (10 µM) was prepared by dissolving 3 nmol of siRNAs in 330 µL of RNAse free water and further diluted 1∶50 using transfection reagent and transfection medium following manufacturer’s protocol to yield a final concentration of 200 nM. The cells were transfected for 16 hours followed by 24 hour incubation in normal media before being subjected to respective treatments. Animal Study The precise role of gadd153 in AD and the extent to which gadd153 is activated in AD are ill-defined. In order to determine whether gadd153 and NF-κB are activated in transgenic mice model for AD, we used 3, 6 and 12 month-old male triple transgenic (3xTg-AD) mice (n = 6 per group). The 3xTg-AD mouse model harbors 3 mutant genes, namely amyloid- β protein precursor (AβPPswe), presenilin-1 (PS1M146 V) and tau (P301 L). The 3xTg-AD mice used in the present study are from a colony maintained for more than 7 generations in our facility. Our mice colony was developed from 3xTg-AD mice obtained from Dr. Frederic Calon, Laval University [21], [22]. Dr. Calon’s colony, maintained on a C57BL/6J background for more than 10 generations in his facility, was originally derived from a colony generated from homozygous founders obtained from Dr. Frank LaFerla on a C57BL/6J x129SVJ background [23]. Mice were deeply anesthetized and perfused trans-cardially with phosphate-buffered saline (PBS). Brains were promptly removed and processed for Western blot analyses. Ethics Statement All animal procedures were carried out in accordance with the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of North Dakota (Protocol 110.3-3). Western Blot Analysis Treated SH-SY5Y cells were washed with PBS and trypsinized to collect the cells and centrifuged at 5000 g. The pellet was washed again with PBS and homogenized in NE-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Hippocampi isolated from 3XTg-AD and non-transgenic control mice brains were homogenized in T-PER tissue protein extraction reagent (Thermo Scientific) also containing protease and phosphatase inhibitors. Total, cytosolic, or nuclear proteins (10 µg) were separated on SDS-PAGE gels, transferred to a polyvinylidene difluoride membrane (BioRad, Hercules, CA), and incubated with the following monoclonal antibodies to : gadd153 (1∶100, Abcam, Cambridge, MA), BACE1 (1∶1000; Millipore, Bedford, MA), p65 mouse antibody (1∶200; Cell Signaling, Boston, MA), p50 (1∶200; Cell Signaling, Boston, MA), IKKα (1∶500; Cell Signaling, Boston, MA), anti-IKKβ (1∶500; Cell Signaling, Boston, MA), p-Ser176/180 IKKα/β (1∶500; Cell Signaling, Boston, MA), anti-IKBα (1∶2000, Abcam), and anti p- Ser32 IKBα (1∶200, Santa Cruz). β-actin and Lamin A/C were used as a gel loading control for cytosolic homogenates and nuclear homogenates respectively. The blots were developed with enhanced chemiluminescence (Immun-star HRP chemiluminescent kit, Bio-Rad, Hercules, CA). Bands were visualized on a polyvinylidene difluoride membrane and analyzed by LabWorks 4.5 software on a UVP Bioimaging System (Upland, CA). Quantification of results was performed by densitometry and the results analyzed as total integrated densitometric values (arbitrary units). Quantitative Real Time RT-PCR Analysis Total RNA was isolated and extracted from treated cells using the 5 prime “PerfectPure RNA tissue kit” (5 Prime, Inc., Gaithersburg, MD). RNA estimation was performed using “Quant-iT RNA Assay Kit” and a Qubit fluorometer according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). cDNA was synthesized by reverse transcribing 1 µg of extracted RNA using an iScript cDNA synthesis kit” (BioRad, Hercules, CA). The oligomeric primers (Sigma, St Louis, MO) used to amplify BACE1 mRNA are enumerated in Table 1. The cDNA amplification was performed using an iQ SYBR Green Supermix kit following the manufacturer’s instructions (BioRad, Hercules, CA). The amplification was performed using an iCycler iQ Multicolor Real Time PCR Detection System (BioRad, Hercules, CA). The expressions of specific BACE1 transcripts amplified were normalized to the expression of glyceraldehyde -3-phosphate dehydrogenase (GAPDH). 10.1371/journal.pone.0070773.t001Table 1 Primers designed and used for RT-PCR, EMSA, and ChIP analyses. Gene Primer Gene Bank Accession Number Sequence Assay BACE1 Forward NM 012104 5′-aggttaccttggcgtgtgtc-3′ RT-PCR BACE1 Reverse NM 012104 5′-gaggcaatctttgcaccaat-3′ RT-PCR BACE1 NT 033899 5′-ggctaacatggtgaattcccgtctccacta-3′ EMSA BACE1 Forward NT 033899 5′-tgaggcaggcagataacttg-3′ ChIP BACE1 Reverse NT 033899 5′-gcctcctcaagcgattctc-3′ ChIP ELISA Immunoassay Aβ40 and Aβ42 levels were quantified in the media (secreted) and cellular homogenates (intracellular) using an ELISA immunoassay kit (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol. Following treatments, the culture medium was collected, supplemented with protease and phosphatase inhibitors cocktail, and centrifuged at 16,000 g for 5 min at 4°C. 100 µl of supernatant was used for the quantification of secreted Aβ40 and Aβ42 levels by colorimetric sandwich ELISA according to the manufacturer’s protocol. To measure the levels of intracellular Aβ40 and Aβ42 in the cellular homogenates, cells were trypsinized and collected by centrifugation at 5000 g and the cell pellet (∼100 mg) was homogenized thoroughly with 8x mass of cold 5 M guanidine Hcl/50 mM Tris–HCl. The homogenates were mixed for 3–4 h at room temperature. The samples were diluted with cold reaction buffer (Dulbecco’s phosphate-buffered saline with 5% BSA and 0.03% Tween-20 supplemented with 1x protease inhibitor cocktail) and centrifuged at 16,000 g for 20 min at 4°C. The supernatant was decanted, diluted at 1∶1 with standard diluent buffer, and quantified by colorimetric sandwich ELISA kits. Intracellular Aβ levels in the cellular homogenates were normalized to total protein content in the samples. Treatments were performed in quadruplet, and the quantity of Aβ in each sample was measured in duplicate. The secreted Aβ40 and Aβ42 levels measured in the culture medium are expressed in pg/mL of media. Electrophoretic Mobility Shift Assay (EMSA) The Electrophoretic Mobility Shift Assay (EMSA) was performed using a kit from Active Motif (Carlsbad, CA) following manufacturer’s protocol. Nuclear extract was prepared using NE-PER protein extraction reagent following the manufacturer’s instructions (Thermo Scientific, Rockford, IL). The human BACE1 promoter region spanning 5000 nucleotides upstream of the transcription initiation site in BACE1 gene was scanned for NF-κB binding consensus sequences using the “TFsearch” online program that searches highly correlated sequence fragments against TFMATRIX transcription factor binding site profile database in ‘TRANSFAC’ databases. The human BACE1 promoter contains a NF-κB consensus motif 2549 nucleotides upstream of the transcription start site. The 5′-biotin labeled and unlabeled oligonucleotide probes that correspond to the NF-κB binding site in the human BACE1 promoter region (−2559 to −2530 of the BACE1 promoter) (Table 1) were purchased from Sigma Aldrich (St Louis, MO). 10 µg of nuclear proteins were incubated with either 20 femto moles of biotin labeled oligonucleotide probe or 4 pico moles of unlabelled oligonucleotide. To exhibit specificity of the oligonucleotide probes, unlabelled oligonucleotide probe was used as a specific competitor for binding reactions at a concentration of 200 fold of the concentration of the biotin labeled probe. 1 µg of Poly d(I–C) was used as a non-specific competitor for binding reactions. The resulting binding reaction mix was loaded and resolved on a 5% TBE gel (BioRad, Hercules, CA) followed by transfer onto a nylon membrane. The bands were visualized using the HRP-Streptavidin – Chemiluminescent reaction mix provided with the kit on a UVP Bioimaging System (Upland, CA). Chromatin Immunoprecipitation (ChIP) Analysis ChIP analysis was performed to evaluate the extent of NF-κB binding to the DNA elements in the BACE1 promoter regions respectively using “SimpleChIP™ Enzymatic Chromatic IP kit” from Cell Signaling (Boston, MA). Briefly, cells from each treatment group (3×106 cells) were washed with PBS, trypsinized, centrifuged at 5000 g. The pellet containing the cells was further washed with PBS and cross-linked using 37% formaldehyde for 15 min followed by the addition of glycine solution to cease the cross-linking reaction. The cells were washed with 4x volumes of 1x PBS and centrifuged at ∼220 g for 5 min. The pellet was resuspended and incubated for 10 min in 5 ml of tissue lysis buffer containing DTT and protease and phosphatase inhibitors. The subsequent steps to isolate the cross-linked chromatin were performed according to the manufacturer’s protocol. The cross-linked chromatin from each sample was apportioned into three equal parts. One third of the cross-linked chromatin was set aside as “input”. One third of the cross-linked chromatin from each sample was incubated with 5 µg of p65 mouse antibody (Abcam, Cambridge, MA), while the remaining one third of the cross-linked chromatin from each sample was incubated with 5 µg of normal Rabbit IgG to serve as negative control. The cross-linked chromatin samples were incubated overnight at 4°C with their respective antibodies. The DNA-protein complexes were collected with Protein G agarose beads and washed to remove non-specific antibody binding. The DNA from the DNA-protein complexes from all the samples including the input and negative control was reverse cross-linked by incubation with 2 µL of Proteinase K for 2 hours at 65°C. The crude DNA extract was eluted and then washed several times with wash buffer containing ethanol (provided with the kit) followed by purification with the use of DNA spin columns provided by the manufacturer. The pure DNA was eluted out of the DNA spin columns using 50 µL of the DNA elution buffer provided in the kit. 1 µL of the purified DNA was used for DNA concentration analysis using the “Quant-iT™ dsDNA Assay kit from Invitrogen (Carlsbad, CA) The DNA fragment size was determined by electrophoresis on a 1.2% agarose FlashGelR system (Lonza, Rockland, ME). The relative abundance of the p65 antibody precipitated chromatin containing the NF-κB binding site in the BACE1 promoter region was determined by qPCR using an iQ SYBR Green Supermix kit following the manufacturer’s instructions (BioRad, Hercules, CA) and sequence specific primers (Table 1). The amplification was performed using an iCycler iQ Multicolor Real Time PCR Detection System (BioRad, Hercules, CA).The fold enrichment was calculated using the ΔΔCt method (Livak & Schmittgen, 2001) which normalizes ChIP Ct values of each sample to the % input and background. Luciferase Reporter Assays Constructs encoding NF-κB response element and human BACE1 promoter conjugated to the firefly luciferase gene were used in the study. SH-SY5Y cells were plated in 96-well plates at a density of 2×104cells/well. The cells were transfected when 80% confluent with 0.25 µg of reporter constructs. Respective non-inducible reporter constructs containing constitutively expressing Renilla luciferase were used as negative internal controls. Constitutively expressing GFP constructs were used as positive control to determine transfection efficiency. Cells were incubated for 24 hours with Opti-MEM serum free medium (Invitrogen, Carlsbad, CA) containing the reporter constructs dissolved in transfection reagent. After 24 hours the medium was changed and the cells were incubated in normal DMEM/F12 medium containing 10% FBS and cells were treated with the different treatment regimens. The cells were treated in triplicate and harvested 24 hours later and subjected to dual-luciferases assay. The dual-luciferase assay was performed using a “Dual-Luciferase Reporter Assay System” (Promega, Madison, WI). The luminescence recorded is expressed as Relative Luminescence Units (RLU) and normalized to per mg protein. Unit value was assigned to control and the magnitude of differences among the samples is expressed relative to the unit value of control cells. Statistical Analysis The significance of differences among the samples was assessed by One Way Analysis of Variance (One Way ANOVA) followed by Tukey’s post-hoc test. Statistical analysis was performed with GraphPad Prism software 4.01. Quantitative data for Western blotting analysis are presented as mean values ± S.E.M with unit value assigned to control and the magnitude of differences among the samples being expressed relative to the unit value of control. Quantitative data for RT-PCR analysis are presented as mean values ± S.E.M, with reported values being the product of absolute value of the ratio of BACE1 to GAPDH mRNA multiplied by 1000000. Results 27-OHC Increases BACE1 Expression and Subsequent Aβ Production by Activating NF-κB Signaling NF-κB family of proteins exist as homodimers or heterodimers of five different proteins or subunits, namely – p65 (RelA), RelB, c-Rel, p50, and p52. Among the 15 known heterodimers and homodimers that exist, the p65-p50 heterodimeric NF-κB complex is the most common and well-studied [24]. In resting cells, NF-κB heterodimers are sequestered in the cytosol through interaction with IκB proteins which results in inhibition of NF-κB nuclear translocation and subsequent DNA binding and transcriptional activation [25]. Stimulus specific activation of IκB kinases (IKKs) leads to phosphorylation, ubiquitination and proteasomal degradation of IκB proteins thereby releasing and allowing the nuclear translocation of the free NF-κB dimer [24], [26]. We therefore determined the p65 and p50 nuclear levels in cells treated with 27-OHC as a surrogate marker of NF-κB activation. 27-OHC treatment elicited a 2.2-fold increase in p65 and a 2-fold increase in p50 translocation to the nucleus (Fig. 1a,b). We also determined the involvement of NF-κB in 27-OHC-induced increase in BACE1 expression. To this end, we treated SH-SY5Y cells with 27-OHC in the presence and absence of sc514, a selective IKKβ (IκB kinase β) inhibitor that inhibits IκB phosphorylation, subsequent ubiquitination and degradation thereby keeping NF-κB sequestered in the cytosol and inhibiting NF-κB signaling [25]. sc514 inhibits IKKβ with an IC50 of 3–12 µM [27]. We found that, while 27-OHC induces ∼2-fold increase in BACE1 protein levels (Fig. 1c,d) and 3.5-fold increase in BACE1 mRNA expression (Fig. 1e), treatment with the NF-κB inhibitor substantially reduced the 27-OHC-induced increase in BACE1 expression levels. Additionally, the NF-κB inhibitor did not elicit any significant effects on the basal expression levels of BACE1 (Fig. 1c–e). We next determined, the binding of NF-κB to the BACE1 promoter region using ChIP analysis and BACE1 promoter activity assay using a BACE1 promoter construct. We found that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter (Fig. 1f). To further demonstrate that the changes in NF-κB binding to the BACE1 promoter result in changes in transcription of BACE1, we performed NF-κB reporter activity assay and BACE1 promoter analysis using a dual-luciferase assay system. Dual-luciferase assay shows that 27-OHC elicited a 4-fold increase in NF-κB reporter activity (Fig. 1g). Furthermore, 27-OHC also increased the BACE1 promoter activity by ∼4.5-fold as determined by a dual-luciferase assay analysis (Fig. 1h). Importantly, the NF-κB inhibitor significantly attenuated the NF-κB binding to the BACE1 promoter region by ∼50% in absence or presence of 27-OHC as demonstrated by ChIP analysis (Fig. 1f). Treatment with sc514 also reduced the 27-OHC-induced increase in BACE1 promoter activity by ∼60% as demonstrated by dual luciferase assay (Fig. 1h). This suggests that activation of the transcription factor NF-κB predominantly mediates the 27-OHC-induced increase in BACE1 expression. However, the NF-κB inhibitor did not affect the basal BACE1 promoter activity (Fig. 1h) despite evoking a substantial reduction in the basal binding of NF-κB to the BACE1 promoter (Fig. 1f). 10.1371/journal.pone.0070773.g001Figure 1 27-OHC induces NF-κB activation and subsequently increases NF-κB-mediated BACE1 expression. (a,b) 27-OHC significantly increases the levels of the p65 and the p50 subunits of the NF-κB in the nuclear homogenates, and (c–e) increases the protein and mRNA levels of BACE1; treatment with the NF-κB inhibitor sc514 attenuates the 27-OHC-induced increase in protein and mRNA expression of BACE1. (f) ChIP analysis shows that 27-OHC increases the binding of NF-κB in the BACE1 promoter region. (g) Dual luciferase assay demonstrates that 27-OHC increases the NF-κB transcriptional activity as measured by a significant increase in NF-κB reporter activity. (h) Dual luciferase assay demonstrates that 27-OHC significantly increases the BACE1 promoter activity, while the NF-κB inhibitor sc514 decreases the 27-OHC-induced increase in BACE1 promoter activity. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). p<0.05, p<0.01, and *p<0.001 versus control, † p<0.05, †† p<0.01, and ††† p<0.001 versus 27-OHC. The cleavage of Amyloid-β Precursor Protein (APP) by BACE1 is the rate limiting step in Aβ production. We determined the impact of inhibition of NF-κB activation and signaling on basal as well as 27-OHC-induced increase in Aβ levels. Consistent with our previously published studies [16], [17], 27-OHC evoked ∼2-fold increase in intracellular Aβ42 and a ∼1.7-fold increase in levels intracellular Aβ40 levels as determined by ELISA immunoassay (Fig. 2a,b). Treatment with 27-OHC also elicited a ∼2.2-fold increase in Aβ42 and a ∼1.7-fold increase in Aβ40 secreted in the media (Fig. 2c,d). However in the presence of the NF-κB inhibitor sc514, 27-OHC elicited only a ∼1.4-fold increase in intracellular Aβ42 and no significant increase in intracellular Aβ40 levels compared to basal levels (Fig. 2a,b). Furthermore, the NF-κB inhibitor sc514 also significantly attenuated the 27-OHC-induced increase in secreted Aβ42 by ∼33% and completely reversed the 27-OHC-induced increase in secreted Aβ40. This further suggests that NF-κB plays an indispensable role in 27-OHC-induced increase in BACE1 expression levels and subsequent increase in Aβ levels. However, consistent with the lack of effect of the NF-κB inhibitor on the basal expression levels of BACE1 (Fig. 1d,e) and BACE1 promoter activity (Fig. 1h), the NF-κB inhibitor sc514 did not elicit any effect on the basal levels of Aβ42 (intracellular and secreted) and Aβ42 (intracellular and secreted) (Fig. 2a–d). 10.1371/journal.pone.0070773.g002Figure 2 27-OHC-induced increase in the levels of intracellular as well as secreted forms of Aβ42 and Aβ40 is contingent on NF-κB activation. (a–d) ELISA immunoassay demonstrates that 27-OHC increases the levels of intracellular and secreted Aβ42 and Aβ40, while the NF-κB inhibitor sc514 mitigates the 27-OHC-induced increase in both intracellular and secreted Aβ42 and Aβ40. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). p<0.05, p<0.01, and p<0.001 versus control, †† p<0.01 and ††† p<0.001 versus 27-OHC. 27-OHC Increases NF-κB Activation by Evoking ER Stress and Inducing gadd153 Expression Western blot analyses shows that siRNA to gadd153 efficiently reduces gadd153 levels in cells treated with 27-OHC (Fig. 3a). We determined whether gadd153 expression is required for NF-κB activation. To this end, we determined NF-κB activation and nuclear translocation by determining the nuclear levels of p65 and p50 subunits of the NF-κB heterodimeric complex in response to 27-OHC treatment in both the native and gadd153-silenced SH-SY5Y cells. 27-OHC evoked a 2-fold increase in p65 and a 2.5-fold increase in p50 translocation to the nucleus in cells untreated with siRNA to gadd153 (3b–d). However, 27-OHC treatment s did not elicit significant increase in p65 and p50 levels in the nucleus of gadd153-silenced SH-SY5Y cells (Fig. 3b–d). This suggests that 27-OHC induces NF-κB activation and its subsequent nuclear translocation by evoking ER stress-induced increase in gadd153 expression. 10.1371/journal.pone.0070773.g003Figure 3 27-OHC-induced nuclear translocation of NF-κB is contingent on gadd153 activation. (a) siRNA to gadd153 suppresses the 27-OHC-induced increase in gadd153 protein levels. (b–d) siRNA to gadd153 attenuates the 27-OHC-induced increase in p65 and p50 subunit translocation to the nucleus. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). *p<0.001 versus control, †† p<0.01 and ††† p<0.001 versus 27-OHC. 27-OHC Fails to Increase NF-κB-mediated BACE1 Expression and Aβ Production in gadd153 Silenced Cells As we found that gadd153 is required for 27-OHC-induced NF-κB activation, and given that NF-κB increases BACE1 expression levels, we determined the involvement of gadd153 in 27-OHC-induced increase in BACE1 expression. To this end, we silenced gadd153 gene expression with siRNA in SH-SY5Y cells prior to treatment with 27-OHC. Treatment with 27-OHC elicited only a 1.3-fold increase in BACE1 protein levels and a 1.5-fold increase in BACE1 mRNA in gadd153 silenced cells compared to a ∼2.1-fold and ∼3-fold increase in BACE1 protein and mRNA levels respectively in native SH-SY5Y cells (Fig. 4a–c). However, the silencing of gadd153 expression did not elicit any effect on the basal expression levels of BACE1 (Fig. 4a–c). To correlate the decreased nuclear translocation of NF-κB in gadd153-silenced cells with the decreased expression levels of BACE1, we performed EMSA and ChIP analysis to determine the binding of NF-κB to the BACE1 promoter region in gadd153-silenced cells. Silencing gadd153 expression resulted in a significant reduction in the 27-OHC-induced increase in NF-κB binding to the exogenous DNA sequence that corresponds to the NF-κB-binding site in the BACE1 promoter as determined by EMSA (Fig. 4d). ChIP analysis also corroborated the EMSA data and revealed an ∼47% reduction in the NF-κB binding to the BACE1 promoter in gadd153-silenced cells treated with the 27-OHC compared 27-OHC-treated native cells. Gadd153-silenced cells treated with 27-OHC elicited a ∼4.5-fold increase in NF-κB binding to the BACE1 promoter compared to a ∼8.5-fold increase in NF-κB binding to the BACE1 promoter in 27-OHC-treated native cells (Fig. 4e). 10.1371/journal.pone.0070773.g004Figure 4 27-OHC-induced NF-κB-mediated increase in BACE1 transcription and expression is contingent on gadd153 expression. (a–c) siRNA to gadd153 mitigates the 27-OHC-induced increase in BACE1 protein levels and mRNA expression. (d) EMSA shows that 27-OHC increases the binding of NF-κB to the exogenous DNA sequence that corresponds to the NF-κB binding site in the BACE1 promoter; silencing gadd153 expression reduces the increase in the binding of NF-κB. (e) ChIP analysis shows that siRNA to gadd153 decreases the 27-OHC-induced increase in binding of NF-κB to the BACE1 promoter. (f,g) Dual luciferase assay demonstrates that siRNA to gadd153 decreases the 27-OHC-induced increase in NF-κB transcriptional activity and BACE1 promoter activity. (h) Levels of gadd153 are detectable in hippocampus of 3, 6, and 12 month-old 3xTg-AD, while NF-κB levels are detectable in hippocampus of 6 and 12month-old, and BACE1 levels increasing only in 12 month-old 3xTg-AD. Data in a-g is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). p<0.05, p<0.01, and p<0.001 versus control, †† p<0.01 and ††† p<0.001 versus 27-OHC. To implicate gadd153 as the mediator of 27-OHC-induced increase in NF-κB-targeted BACE1 expression, we next performed dual-luciferase analysis to determine the extent of NF-κB transcriptional activity and BACE1 promoter activity in gadd153-silenced cells. We found that, silencing gadd153 expression results in a ∼50% reduction in the 27-OHC-induced increase in NF-κB transcriptional activity (Fig. 4f). Gadd153-silenced cells treated with 27-OHC exhibited a ∼1.7-fold increase in NF-κB transcriptional activity compared to a ∼3.5-fold increase in NF-κB transcriptional activity in native cells treated with 27-OHC (Fig. 4f). Dual-luciferase assay also demonstrated that knocking-down gadd153 expression in 27-OHC treated SH-SY5Y cells decreased the 27-OHC-induced increase in BACE1 promoter activity by ∼56% (Fig. 4g). Gadd153-silenced cells treated with 27-OHC exhibited a ∼2-fold increase in BACE1 promoter activity compared to a ∼4.2-fold increase in BACE1 promoter activity in native cells treated with 27-OHC (Fig. 4g). These data suggest that gadd153 expression is both necessary and mediates the 27-OHC-induced increase in BACE1 expression via NF-κB activation. However, the silencing of gadd153 expression did not alter the basal NF-κB transcription activity (Fig. 4f) and BACE1 promoter activity (Fig. 4g) as well as did not produce any effect on the extent of basal NF-κB binding to the BACE1 promoter region (Fig. 4e). Western blots for gadd153 show a strong band in hippocampus from the 3xTg-AD mice of 3, 6 and 12 months of age (Fig. 4h). NF-κB (p50) levels are highly detected in 6 and 12 month-old 3xTg-AD mice, but barely detected in the 3 month-old mice (Fig. h). BACE1 levels were substantially increased in the 12 month-old mice compared to 3 or 6 month-old mice. These results indicate that gadd153 protein levels are detected earlier than translocation of NFkB (p50) to the nucleus, which in turn precedes the increase in BACE1 levels. It is noteworthy to mention that Aβ levels increase the 3xTg-AD of more than 6 month of age [28]. As 27-OHC-induced increased expression of gadd153 increases BACE1 expression via NF-κB activation in human neuroblastoma SH-SY5Y cells, we investigated the involvement of gadd153 in 27-OHC-induced increase in Aβ production. Silencing of gadd153 expression effectuated a ∼33% reduction in the 27-OHC-induced increase in intracellular Aβ42 and a ∼22% decrease in 27-OHC-induced increase in intracellular Aβ40 (Fig. 5a,b). Intracellular Aβ42 levels were ∼1.3-fold the basal levels in gadd153-silenced cells treated with 27-OHC compared to ∼2-fold the basal levels in native cells treated with 27-OHC (Fig. 5a). Intracellular Aβ40 levels were not significantly different than the basal levels in gadd153-silenced cells treated with 27-OHC compared to ∼1.7-fold the basal levels in native cells treated with 27-OHC (Fig. 5b). Silencing of gadd153 expression also elicited ∼30% reduction in the 27-OHC-induced increase in secreted Aβ42 and ∼31% decrease in 27-OHC-induced increase in secreted Aβ40 (Fig. 5c,d). Secreted Aβ42 levels were ∼1.3-fold the basal levels in gadd153-silenced cells treated with 27-OHC compared to ∼1.9-fold the basal levels in native cells treated with 27-OHC (Fig. 5c). Similarly, secreted Aβ40 levels were ∼1.2-fold the basal levels in gadd153-silenced cells treated with 27-OHC compared to ∼1.8-fold the basal levels in native cells treated with 27-OHC (Fig. 5d). These data suggest the involvement of gadd153 in the 27-OHC-induced increase in Aβ. However, consistent with the lack of effect of the gadd153 siRNA on the basal expression levels of BACE1 and BACE1 promoter activity, the silencing of gadd153 expression did not elicit significant effects on the basal levels of Aβ42 (intracellular and secreted) and Aβ42 (intracellular and secreted) (Fig. 5a-d). 10.1371/journal.pone.0070773.g005Figure 5 Gadd153 expression is necessary for 27-OHC-induced increase in the levels of Aβ40 and Aβ42. (a,b) ELISA immunoassay demonstrates that siRNA to gadd153 expression attenuates the 27-OHC-induced increase in levels of intracellular and secreted Aβ42 and Aβ40. Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). p<0.05, p<0.01, and p<0.001 versus control, †† p<0.01 and ††† p<0.001 versus 27-OHC. 27-OHC Increases the Activation of NF-κB via the Phosphorylation of IKK in gadd153-Dependent Manner Next, we investigated the mechanism involved in the 27-OHC-induced gadd153-mediated increase in NF-κB activation. NF-κB activation is regulated by the IκBα (inhibitor of kappa B α) protein which masks the nuclear localization signal of the NF-κB subunits and sequesters the NF-κB complex sequestered in the cytosol [25], [26]. NF-κB activation entails the phosphorylation-evoked and proteasome-mediated degradation of IκB proteins resulting in the unmasking of the nuclear localization sequence of NF-κB heterodimeric [29], [30]. The IKK (IκB kinase) triad complex (composed of the catalytic subunits IKKα and IKKβ and the regulatory subunit IKKγ) activates NF-κB signaling pathway and allows the nuclear translocation of NF-κB heterodimeric complex by phosphorylation of the IκBα protein [30]–[32]. IKK complex is itself is activated by phosphorylation of Ser176 and Ser180 in the catalytic loop of IKKα and Ser177 and Ser181 in the catalytic loop of IKKβ [33]. Therefore, we determined the effects of 27-OHC on the phosphorylation of IKKα/β in the native and gadd153-deficient cells. We found that 27-OHC-evokes a 4.5-fold increase in phosphorylation of IKKα at Ser176/Ser180 and a 3-fold increase in the phosphorylation of IKKβ at Ser177/Ser181, thereby accounting for NF-κB activation (Fig. 6a,b). Interestingly, gadd153-silenced cells did not exhibit any increase in IKKα/β phosphorylation in response to 27-OHC, an effect consistent with the lack of NF-κB activation in gadd153-deficient cells treated with 27-OHC. We also found that while 27-OHC induces a significant decrease in IKBα levels and a significant increase in the phosphorylation of IKBα and β, siRNA to gadd153 reverses the 27-OHC-induced alterations in IKBα and p- IKBα levels (c,d). This data indicate that 27-OHC increases the degradation of IKBα. 10.1371/journal.pone.0070773.g006Figure 6 27-OHC increases the activation of NF-κB via the phosphorylation of IKKα/β which is dependent on gadd153 expression. 27-OHC induces a significant increase in the phosphorylation of IKKα/β, and siRNA to gadd153 reverses the 27-OHC-induced increase in phosphorylation of IKKα/β (a,b). 27-OHC induces a significant decrease in IKBα levels and a significant increase in the phosphorylation of IKBα; siRNA to gadd153 reverses the 27-OHC-induced alterations in IKBα and p- IKBα levels (c,d). Data is expressed as Mean+S.E.M and includes determinations made in four separate cell culture experiments (n = 4). **p<0.001 versus control; †† p<0.01 versus 27-OHC and ††† p<0.001 versus 27-OHC. Discussion This study was conceived to elucidate signaling intermediates and transcription factors involved in the regulation of BACE1 expression. We determined the role of the ER stress-induced transcription factor gadd153 and the ubiquitous transcription factor NF-κB in the regulation of BACE1 expression levels in human neuroblastoma cells treated with the oxysterol 27-OHC. We demonstrate that 27-OHC induces BACE1 expression by increasing the activation and nuclear translocation of the transcription factor NF-κB in a gadd153-dependent manner. We found that silencing gadd153 expression significantly attenuated the 27-OHC-induced increase in NF-κB activation and NF-κB-mediated BACE1 expression. We further demonstrate that 27-OHC-induced increase in gadd153 expression results in the phosphorylation of IKKα/β and consequently activation in NF-κB and NF-κB mediated increase in BACE1 expression. Our data also shows gadd153 and NF-ΚB activation in the 3xTg-AD mice, suggesting that these transcription factors may contribute to the increase in BACE1 and the generation of Aβ peptide accumulation in these mice. AD is a multifactorial disease with several factors likely contributing to its pathogenesis. Altered levels of sphingolipidome, ceramides, saturated fatty acids and several phospholipids may be involved in AD [34]–[39]. Previous studies from our laboratory have demonstrated that the cholesterol metabolite 27-OHC increases the expression levels of BACE1 and Aβ, and may also increase the risk for AD [16], [17], [40]. The aspartyl protease BACE1 catalyzes the rate-limiting step in the amyloidogenic processing of APP to generate the Aβ peptide [2], [3]. Some studies have found BACE1 expression levels to be increased in AD [6], [7], while other studies found no changes in BACE1 expression [41]–[43]. Preponderance of contemporary literature now overwhelmingly implicates increased BACE1 protein levels and activity in AD [4]; [44]–[46]. AD brains exhibit increased BACE1 protein levels and activity [4]; [6]; [44]. There is growing consensus that this increase in BACE1 plays a role in the neurodegenerative cascade that leads to AD. Interestingly, there is also suggestion that a positive feedback loop exists between amyloid plaques and BACE1 expression levels. Vassar and colleagues have recently demonstrated that BACE1 is up-regulated in neurons in the immediate vicinity juxtaposing the amyloid plaques [47]. Furthermore, the same study also demonstrated that BACE1 elevation in the neurons that surround the amyloid plaques is an early event preceding the subsequent neuronal loss that eventually occurs, thereby dispelling the speculation that the elevation in BACE1 levels and activity is an epiphenomenon that merely accompanies neuronal loss. Moreover, neuritic plaques containing a dense core of Aβ42 are able to elevate BACE1 expression levels in dystrophic neurites, but not diffuse plaques in transgenic mouse models [47]; [48]. The BACE1 transcription is modulated by a plethora of transcription factors and multitude of transcription factor binding sites have been found in the BACE1 promoter region including those for NF-κB, YY1, Sp1, and PPARγ among others [49]. In this study we determined the role of NF-κB in the 27-OHC-induced increase in BACE1 expression. To this end, we used sc514 to inhibit the activation of NF-κB in SH-SY5Y cells concomitantly treated with 27-OHC. We found that the NF-κB inhibitor significantly reduces the 27-OHC-induced increase in BACE1 expression levels. 27-OHC failed to evoke a similar magnitude of increase in BACE1 expression levels in cells concomitantly treated with sc514 compared to cells treated with 27-OHC alone. 27-OHC also induced the nuclear translocation of the p65 and the p50 NF-κB subunits. To further implicate the increased nuclear translocation of the p65 and the p50 NF-κB subunits with the increased expression of BACE1, we performed a ChIP assay to determine the binding of NF-κB to the BACE1 promoter region. Multiple studies have reported the modulation of BACE1 expression by NF-κB binding to the κB sites in the BACE1 promoter [12], [13]. Using ChIP analysis, we found that 27-OHC induced a marked increase in NF-κB binding to the BACE1 promoter region. To further demonstrate that the increase in the binding of NF-κB in the BACE1 promoter region does indeed result in the modulation of NF-κB-mediated BACE1 gene expression, we performed an NF-κB reporter activity assay to assess NF-κB transcriptional activity and a BACE1 promoter assay to assess changes in BACE1 gene transcription. We found that 27-OHC substantially increases NF-κB reporter activity and BACE1 promoter activity. Furthermore, the NF-κB inhibitor attenuated the 27-OHC-induced increase in BACE1 promoter activity thereby further implicating NF-κB in the 27-OHC-induced upregulation of BACE1 expression. Indeed, NF-κB activity has been found to be increased in autopsied AD-brain tissue as well as increased NF-κB immunoreactivity has been observed in proximity to amyloid plaques [12], [13], [50], [51]. As BACE1 catalyzes the rate-limiting step in the genesis of Aβ peptide via the amyloidogenic processing of APP and given that BACE1 expression levels are tightly associated with Aβ levels, we determined the effects of the NF-κB inhibitor sc514 on the 27-OHC-induced increase in Aβ levels. The NF-κB inhibitor significantly mitigated the 27-OHC-induced increase in both intracellular as well as secreted forms Aβ42 and Aβ40. The ER is an organelle of fundamental importance to cell functions as it is the Ca2+ storage site and where surface and secreted proteins are synthesized, folded and assembled before being transported. Mounting evidence indicates a role of ER stress in the pathophysiology of many diseases including AD [8]–[10]. ER dyshomeostasis triggers stress signaling by activating the unfolded protein response (UPR) [52]. The UPR signaling is intended to protect cells from the consequences of changes in Ca2+ levels and the buildup of misfolded proteins. However, intense or sustained ER stress activates deleterious cascades including the activation of the transcription factor gadd153. It is significant how gadd153 activation is linked to AD as this transcription factor regulates genes and proteins related to the pathophysiology of this disease (see for review [10]). It is also significant that PS1 mutations, a risk factor for familial AD, increases gadd153 protein translation [53]. Previous studies from our laboratory have demonstrated that the ER stress-induced transcription factor gadd153 mediates the 27-OHC-induced increase in BACE1 expression in hippocampal organotypic slices from adult rabbits [40]. However, the molecular mechanisms and signal transductional intermediates utilized by gadd153 to elicit an increase in BACE1 expression had not been determined prior to this study. BACE1 expression is markedly induced by cellular stressors such as oxidative stress [54], hypoxia [55], [56] and glucose deprivation [57] as well as aging [58]. Interestingly, gadd153 expression is also induced by the same cellular stressors and glucose and nutrient deprivation [52]. It is therefore conceivable that gadd153 could be a key factor instigating this stress-mediated induction of BACE1 expression. Recent emerging evidence has implicated ER stress-induced gadd153 expression in NF-κB activation [11]. Therefore, we determined the role of NF-κB in 27-OHC-induced gadd153-mediated upregulation of BACE1. To this end, we knocked-down the expression of gadd153 via siRNA-mediated gene silencing and determined the effects on basal NF-κB levels as well as 27-OHC-induced increased NF-κB levels in the nuclear homogenates. Silencing gadd153 expression did not affect the basal levels of p65 and p50 NF-κB subunits in the nucleus. However, gadd153 silencing reversed the 27-OHC-induced increase in p65 and p50 NF-κB levels in the nucleus. This suggests that increased expression of gadd153 mediates the 27-OHC-induced activation and nuclear translocation of p65 and p50 NF-κB. Consistent with our previous study in hippocampal organotypic slices from adult rabbits [40], silencing of gadd153 expression significantly mitigated the 27-OHC-induced increase in BACE1 expression without affecting the basal expression of BACE1. To further implicate NF-κB as the intermediate in 27-OHC-induced gadd153-mediate increase in BACE1 expression, we subsequently performed EMSA and ChIP analyses to study the binding of NF-κB to the BACE1 promoter region in response to treatment with 27-OHC in a gadd153-deficient paradigm. Consistent with the observation of lack of commensurate increase in BACE1 expression in gadd153-silenced cells treated with 27-OHC, silencing gadd153 expression significantly attenuated the binding of NF-κB in the BACE1 promoter region as revealed by EMSA and ChIP analysis. However, changes in NF-κB binding to the BACE1 promoter may not necessarily evoke changes in transcription of BACE1. Therefore, we next performed NF-κB reporter activity assay to assess NF-κB transcriptional activity and also BACE1 promoter analysis to assess BACE1 transcriptional changes in gadd153-silenced cells treated with 27-OHC. Silencing gadd153 expression significantly reduced the 27-OHC-induced increase in NF-κB reporter activity and BACE1 promoter activity, thereby suggesting that gadd153 mediates the 27-OHC-induced increase in NF-κB transcriptional activity and BACE1 transcription. Our data is consistent with the observation that many signal transduction pathways that are actuated by cellular stressors that evoke ER stress do also indeed increase BACE1 expression levels. However, no gadd153 binding sites have been identified yet in the BACE1 promoter suggesting that gadd153 may not directly regulate BACE1 expression. Recently, it was demonstrated that ER stress-induced activation of gadd153 increases NF-κB signaling [11]. We explored the possibility of this gadd153-NF-κB crosstalk as 27-OHC induces the expression of gadd153 as well as NF-κB activation. Indeed, we found that increased gadd153 expression by 27-OHC positively regulates NF-κB activation and expression of NF-κB target genes. Specifically, we found that 27-OHC induced phosphorylation of IKKα/β that results in the phosphorylation and proteasomal degradation of IκB proteins culminating in the activation of NF-κB is regulated positively by gadd153. However, further studies are warranted to examine the signaling intermediates involved in the gadd153- NF-κB crosstalk that regulates BACE1 expression and therefore could represent a potential therapeutic target in AD. In summary, we demonstrate that the ER stress-activated transcription factor gadd153 positively regulates BACE1 expression via the activation of NF-κB signaling cascade (Fig. 7). Our study provides a valuable insight into the putative molecular mechanisms and signal transduction cascades that regulate BACE1 expression and is therefore of high relevance to the search and design of therapeutic intervention that can reduce BACE1 as well as Aβ overproduction, and AD progression. 10.1371/journal.pone.0070773.g007Figure 7 Schematic representation of the molecular mechanisms involved in the 27-OHC-induced increase in BACE1 expression. 27-OHC activates gadd153 (1) which evokes the phosphorylation of the IKK-complex (2). Phosphorylation IKK complex (3) results in the phosphorylation and subsequent proteasomal degradation of IκB (4), thereby releasing the p65-p50 NF-κB dimer from inhibitory sequestration in the cytosol and allowing the p65-p50 NF-κB dimer to translocate to the nucleus (5). The translocated p65-p50 NF-κB dimer binds to distinct κB sites in the BACE1 promoter region and upregulates BACE1 expression (5). Increased gadd153 expression by 27-OHC may also induce NF-κB activation, nuclear translocation, and subsequently increase BACE1 expression by other mechanism that are yet to be elucidated (6). siRNA to gadd153 reduces the 27-OHC-induced nuclear translocation of NF-κB and thereby attenuates the increase in BACE1(7). The NF-κB inhibitor sc514 also decreases the 27-OHC-induced increase in BACE1 expression by inhibiting the nuclear translocation of NF-κB and subsequent increase in NF-κB-mediated transcription of BACE1 (8). ==== Refs References 1 Hardy J , Allsop D (1991 ) Amyloid deposition as the central event in the aetiology of Alzheimer’s disease . Trends Pharmacol Sci 12 : 383 –388 .1763432 2 Haass C , Hung AY , Schlossmacher MG , Teplow DB , Selkoe DJ (1993 ) beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms . J Biol Chem 268 : 3021 –3024 .8428976 3 Vassar R , Bennett BD , Babu-Khan S , Kahn S , Mendiaz EA , et al (1999 ) Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE . Science 286 : 735 –741 .10531052 4 Fukumoto H , Cheung BS , Hyman BT , Irizarry MC (2002 ) Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease . Arch Neurol 59 : 1381 –1389 .12223024 5 Tyler SJ , Dawbarn D , Wilcock GK , Allen S J (2002 ) alpha- and beta-secretase: profound changes in Alzheimer’s disease . Biochem Biophys Res Commun 299 : 373 –376 .12445809 6 Holsinger RM , McLean CA , Beyreuther K , Masters CL , Evin G (2002 ) Increased expression of the amyloid precursor beta-secretase in Alzheimer’s disease . Ann Neurol 51 : 783 –786 .12112088 7 Hebert SS , Horre K , Nicolai L , Papadopoulou AS , Mandemakers W , et al (2008 ) Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/beta-secretase expression . Proc Natl Acad Sci U S A 105 : 6415 –6420 .18434550 8 Chadwick W , Mitchell N , Martin B , Maudsley S (2012 ) Therapeutic targeting of the endoplasmic reticulum in Alzheimer’s disease . Curr Alzheimer Res 9 : 110 –9 .22329655 9 Stutzmann GE , Mattson MP (2011 ) Endoplasmic reticulum Ca(2+) handling in excitable cells in health and disease . Pharmacol Rev 63 : 700 –27 .21737534 10 Ghribi O (2006 ) The role of the endoplasmic reticulum in the accumulation of beta-amyloid peptide in Alzheimer’s disease . Curr Mol Med 6 : 119 –133 .16472119 11 Park SH , Choi HJ , Yang H , Do KH , Kim J , et al (2010 ) Endoplasmic reticulum stress-activated C/EBP homologous protein enhances nuclear factor-kappaB signals via repression of peroxisome proliferator-activated receptor gamma . J Biol Chem 285 : 35330 –35339 .20829347 12 Kaltschmidt B , Uherek M , Volk B , Baeuerle PA , Kaltschmidt C (1997 ) Transcription factor NF-kappaB is activated in primary neurons by amyloid beta peptides and in neurons surrounding early plaques from patients with Alzheimer disease . Proc Natl Acad Sci U S A 94 : 2642 –2647 .9122249 13 Chen CH, Zhou W, Liu S, Deng Y, Cai F, et al.. (2011) Increased NF-kappaB signalling up-regulates BACE1 expression and its therapeutic potential in Alzheimer’s disease. Int J Neuropsy Pharmacol 1–14. 14 Boissiere F , Hunot S , Faucheux B , Duyckaerts C , Hauw JJ , et al (1997 ) Nuclear translocation of NF-kappaB in cholinergic neurons of patients with Alzheimer’s disease . Neuroreport 8 : 2849 –2852 .9376517 15 Lukiw WJ , Bazan NG (1998 ) Strong nuclear factor-kappaB-DNA binding parallels cyclooxygenase-2 gene transcription in aging and in sporadic Alzheimer’s disease superior temporal lobe neocortex . J Neurosci Res 53 : 583 –592 .9726429 16 Marwarha G , Dasari B , Prasanthi JR , Schommer J , Ghribi O (2010 ) Leptin reduces the accumulation of Abeta and phosphorylated tau induced by 27-hydroxycholesterol in rabbit organotypic slices . J Alzheimers Dis 19 : 1007 –1019 .20157255 17 Prasanthi JRP, Huls A, Thomasson S, Thompson A, Schommer E, et al.. (2009) Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on beta-amyloid precursor protein levels and processing in human neuroblastoma SH-SY5Y cells. Mol Neurodegener 4, 1. 18 Shafaati M, Marutle A, Pettersson H, Lovgren-Sandblom A, Olin M, et al.. (2011) Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer’s patients with the Swedish APP 670/671 mutation. J Lipid Res 52, 1004–1010. 19 Dasari B , Prasanthi JRP , Marwarha G , Singh BB , Ghribi O (2011 ) Cholesterol-enriched diet causes age-related macular degeneration-like pathology in rabbit retina . BMC Ophthalmol 11 : 65 –77 . 20 Marwarha G , Dasari B , Ghribi O (2012 ) Endoplasmic reticulum stress-induced CHOP activation mediates the down-regulation of leptin in human neuroblastoma SH-SY5Y cells treated with the oxysterol 27-hydroxycholesterol . Cell Signal 24 : 484 –92 .21983012 21 Bories C , Guitton MJ , Julien C , Tremblay C , Vandal M , et al (2012 ) Sex-dependent alterations in social behaviour and cortical synaptic activity coincide at different ages in a model of Alzheimer’s disease . PLoS One 7 : e46111 .23029404 22 Hamilton LK , Aumont A , Julien C , Vadnais A , Calon F , et al (2010 ) Widespread deficits in adult neurogenesis precede plaque and tangle formation in the 3xTg mouse model of Alzheimer’s disease . Eur J Neurosci 32 : 905 –20 .20726889 23 Oddo S , Caccamo A , Shepherd JD , Murphy MP , Golde TE , et al (2003 ) Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction . Neuron 39 : 409 –421 .12895417 24 Shih VF , Tsui R , Caldwell A , Hoffmann A (2011 ) A single NFkappaB system for both canonical and non-canonical signaling . Cell Res 21 : 86 –102 .21102550 25 Baeuerle PA , Baltimore D (1988 ) I kappa B: a specific inhibitor of the NF-kappa B transcription factor . Science 242 : 540 –546 .3140380 26 Beg AA , Baldwin AS Jr (1993 ) The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors . Genes Dev 7 : 2064 –2070 .8224838 27 Baxter A , Brough S , Cooper A , Floettmann E , Foster S , et al (2004 ) Hit-to-lead studies: the discovery of potent, orally active, thiophenecarboxamide IKK-2 inhibitors . Bioorg Med Chem Lett 14 : 2817 –2822 .15125939 28 Winton MJ , Lee EB , Sun E , Wong MM , Leight S , et al (2011 ) Intraneuronal APP, not free Aβ peptides in 3xTg-AD mice: implications for tau versus Aβ-mediated Alzheimer neurodegeneration . J Neurosci 31 : 7691 –9 .21613482 29 Brown K , Gerstberger S , Carlson L , Franzoso G , Siebenlist U (1995 ) Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation . Science 267 : 1485 –1488 .7878466 30 Chen Z J , Parent L , Maniatis T (1996 ) Site-specific phosphorylation of IkBα by a novel ubiquitination-dependent protein kinase activity . Cell 84 : 853 –862 .8601309 31 Zandi E , Rothwarf DM , Delhase M , Hayakawa M , Karin M (1997 ) The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation . Cell 91 : 243 –252 .9346241 32 Karin M (1999) How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex. Oncogene 18, 6867–6874. 33 Johnson LN , Noble ME , Owen DJ (1996 ) Active and inactive protein kinases: structural basis for regulation . Cell 85 : 149 –158 .8612268 34 Mielke MM , Haughey NJ , Ratnam Bandaru VV , Schech S , Carrick R , et al (2010 ) Plasma ceramides are altered in mild cognitive impairment and predict cognitive decline and hippocampal volume loss . Alzheimers Dement 6 : 378 –85 .20813340 35 Mielke MM , Haughey NJ , Bandaru VV , Weinberg DD , Darby E , et al (2011 ) Plasma Sphingomyelins are Associated with Cognitive Progression in Alzheimer’s Disease . J Alzheimers Dis 27 : 259 –69 .21841258 36 Han X , Rozen S , Boyle SH , Hellegers C , Cheng H , et al (2011 ) Metabolomics in early Alzheimer’s disease: identification of altered plasma sphingolipidome using shotgun lipidomics . PLoS One 6 : e21643 .21779331 37 Haughey NJ , Bandaru VV , Bae M , Mattson MP (2010 ) Roles for dysfunctional sphingolipid metabolism in Alzheimer’s disease neuropathogenesis . Biochim Biophys Acta1801 : 878 –86 . 38 Bazan NG , Molina MF , Gordon WC (2011 ) Docosahexaenoic Acid signalolipidomics in nutrition: significance in aging, neuroinflammation, macular degeneration, Alzheimer’s, and other neurodegenerative diseases . Annu Rev Nutr 31 : 321 –51 .21756134 39 Kumar A , Bullard RL , Patel P , Paslay LC , Singh D , et al (2011 ) Non-esterified fatty acids generate distinct low-molecular weight amyloid-β (Aβ42) oligomers along pathway different from fibril formation . PLoS One 6 : e18759 .21526230 40 Prasanthi JRP , Larson T , Schommer J , Ghribi O (2011 ) Silencing GADD153/CHOP Gene Expression Protects Against Alzheimer’s Disease-Like Pathology Induced by 27-Hydroxycholesterol in Rabbit Hippocampus . PLoS ONE 6 : e26420 .22046282 41 Sun A , Koelsch G , Tang J , Bing G (2002 ) Localization of beta-secretase memapsin 2 in the brain of Alzheimer’s patients and normal aged controls . Exp Neurol 175 : 10 –22 .12009756 42 Preece P , Virley DJ , Costandi M , Coombes R , Moss SJ , et al (2003 ) Beta-secretase (BACE) and GSK-3 mRNA levels in Alzheimer’s disease. Brain Res . Mol Brain Res 116 : 155 –158 .12941471 43 Leuba G , Wernli G , Vernay A , Kraftsik R , Mohajeri MH , et al (2005 ) Neuronal and nonneuronal quantitative BACE immunocytochemical expression in the entorhinohippocampal and frontal regions in Alzheimer’s disease. Dement . Geriatr Cogn Disord 19 : 171 –183 . 44 Yang LB , Lindholm K , Yan R , Citron M , Xia W , et al (2003 ) Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease . Nat Med 9 : 3 –4 .12514700 45 Li Q , Sudhof TC (2004 ) Cleavage of amyloid-beta precursor protein and amyloid-beta precursor-like protein by BACE 1 . J Biol Chem 279 : 10542 –10550 .14699153 46 Harada H , Tamaoka A , Ishii K , Shoji S , Kametaka S , et al (2006 ) Beta-site APP cleaving enzyme 1 (BACE1) is increased in remaining neurons in Alzheimer’s disease brains . Neurosci Res 54 : 24 –29 .16290302 47 Zhao J , Fu Y , Yasvoina M , Shao P , Hitt B , et al (2007 ) Beta-site amyloid precursor protein cleaving enzyme 1 levels become elevated in neurons around amyloid plaques: implications for Alzheimer’s disease pathogenesis . J Neurosci 27 : 3639 –3649 .17409228 48 Zhang XM , Cai Y , Xiong K , Cai H , Luo XG , et al (2009 ) Beta-secretase-1 elevation in transgenic mouse models of Alzheimer’s disease is associated with synaptic/axonal pathology and amyloidogenesis: implications for neuritic plaque development . Eur J Neurosci 30 : 2271 –2283 .20092570 49 Rossner S , Sastre M , Bourne K , Lichtenthaler SF (2006 ) Transcriptional and translational regulation of BACE1 expression–implications for Alzheimer’s disease . Prog Neurobiol 79 : 95 –111 .16904810 50 Terai K , Matsuo A , McGeer PL (1996 ) Enhancement of immunoreactivity for NF-kappa B in the hippocampal formation and cerebral cortex of Alzheimer’s disease . Brain Res 735 : 159 –168 .8905182 51 Ferrer I , Marti E , Lopez E , Tortosa A (1998 ) NF-kB immunoreactivity is observed in association with beta A4 diffuse plaques in patients with Alzheimer’s disease . Neuropathol Appl Neurobiol 24 : 271 –277 .9775392 52 Oyadomari S , Mori M (2004 ) Roles of CHOP/GADD153 in endoplasmic reticulum stress . Cell Death Differ 11 : 381 –9 .14685163 53 Milhavet O , Martindale JL , Camandola S , Chan SL , Gary DS , et al (2002 ) Involvement of Gadd153 in the pathogenic action of presenilin-1 mutations . J Neurochem 83 : 673 –681 .12390529 54 Tamagno E , Bardini P , Obbili A , Vitali A , Borghi R , et al (2002 ) Oxidative stress increases expression and activity of BACE in NT2 neurons . Neurobiol Dis 10 : 279 –288 .12270690 55 Sun X , He G , Qing H , Zhou W , Dobie F , et al (2006 ) Hypoxia facilitates Alzheimer’s disease pathogenesis by up-regulating BACE1 gene expression . Proc Natl Acad Sci U S A 103 : 18727 –18732 .17121991 56 Zhang X , Zhou K , Wang R , Cui J , Lipton SA , et al (2007 ) Hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation . J Biol Chem 282 : 10873 –10880 .17303576 57 O’Connor T , Sadleir KR , Maus E , Velliquette RA , Zhao J , et al (2008 ) Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis . Neuron 60 : 988 –1009 .19109907 58 Cole SL , Vassar R (2008 ) The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology . J Biol Chem 283 : 29621 –29625 .18650431	23951005	PMC3739769	CC BY	2021-01-05 17:36:57	yes	PLoS One. 2013 Aug 9; 8(8):e70773
==== Front J Biol ChemJ. Biol. ChemjbcjbcJBCThe Journal of Biological Chemistry0021-92581083-351XAmerican Society for Biochemistry and Molecular Biology 9650 Rockville Pike, Bethesda, MD 20814, U.S.A. M113.48425310.1074/jbc.M113.484253Signal TransductionTwo Pore Channel 2 (TPC2) Inhibits Autophagosomal-Lysosomal Fusion by Alkalinizing Lysosomal pH* Inhibition of Autophagy Progression by TPC2Lu Yingying ‡Hao Bai-Xia ‡Graeff Richard ‡Wong Connie W. M. §Wu Wu-Tian §¶Yue Jianbo ‡1From the ‡ Department of Physiology, University of Hong Kong, Hong Kong, China, § Department of Anatomy and State Key Laboratory of Brain and Cognitive Sciences, University of Hong Kong, Hong Kong, China, and ¶ GHM Institute of CNS Regeneration, Jinan University, Guangzhou, China1 To whom correspondence should be addressed. E-mail: jyue@hku.hk.16 8 2013 8 7 2013 8 7 2013 288 33 24247 24263 8 5 2013 1 7 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.2013Author's Choice—Final version full access.Creative Commons Attribution Unported License applies to Author Choice ArticlesBackground: The role and mechanism of NAADP, an endogenous Ca2+ mobilizing nucleotide, in autophagic regulation remain to be determined. Results: Activation of NAADP/TPC2 signaling induced the accumulation of autophagosomes. Conclusion: The NAADP/TPC2 signaling inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH. Significance: Development of agonists or antagonists of NAADP should provide a novel approach to specifically manipulate autophagy. Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca2+ mobilizing messengers, elicits Ca2+ release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca2+ signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression. AutophagyCalciumCalcium ChannelsCalcium Intracellular ReleaseCalcium SignalingLysosomesNAADPTPC2pH ==== Body Introduction Macroautophagy (hereafter referred as autophagy) is an evolutionarily conserved catabolic degradation cellular process. Autophagy starts with sequestering of cytoplasmic material, such as misfolded proteins and/or damaged organelles, inside the phagophore followed by the elongation and closure of the autophagosome, a double-membrane vesicle. The matured autophagosome then fuses with the lysosome to create an autolysosome inside which the autophagosome inner membrane and its luminal contents are degraded by lysosomal enzymes for subsequent recycling of macromolecules. Autophagy can also be markedly induced by a wide variety of stresses, such as nutrient starvation, infection, and aging, for cell survival. Dysfunctional autophagy has been associated with wide-ranging human diseases, e.g. cancer, neurodegenerative diseases, heart disease, diabetes, and infections (1). Autophagy induction is controlled by the ULK1 and ULK2 complexes, and autophagosome formation requires class III phosphatidylinositol 3 kinase (PI3K) complexes. The key autophagy regulator is mTOR Ser/Thr kinase, which negatively regulates autophagy by inhibiting the ULK1/2 complex. Starvation, on the other hand, activates the AMP-activated protein kinase (AMPK) to inactivate mTOR, thereby inducing autophagy. Another key step for autophagy induction is the activation of mammalian Vps34, a class III PI3K. Vps34 is activated by forming a multiprotein complex with beclin-1, UVRAG, and Vps15, to generate phosphatidylinositol 3-phosphate. Two ubiquitin-like conjugation systems, the LC3-II and Atg12-Atg5-Atg16L complex, are essential for the autophagosomal elongation process. The covalent conjugation of Atg12 to Atg5 is catalyzed by the E1-like enzyme Atg7 and the E2-like enzyme Atg10. The conjugation of phosphatidylethanolamine to LC3 is sequentially controlled by the protease Atg4, Atg7, and the E2-like enzyme Atg3. Lipidation of LC3 converts the cytosolic LC3 (LC3-I) to the autophagic vesicle-associated form (LC3-II). Notably, LC3-II is commonly used as a marker of autophagy because lipidated LC3-II exhibits a punctate staining pattern and has faster electrophoretic mobility compared with diffused LC3-I (1, 2). Besides mTOR, other signaling pathways, e.g. JNK, Ras, and Ca2+, can modulate autophagy as well (3, 4). Even after extensive research, the regulation and mechanisms of autophagy induction, autophagosome formation and maturation, and especially autophagosomal-lysosomal fusion, remain elusive in mammalian cells. Although autophagosomal-lysosomal fusion is poorly understood, many factors and proteins, including lysosomal pH (5), Rab7 (6), SNAREs (7), the HOPS complex (8), TECPR1 (9), histone deacetylase-6 (10), ubiquilins (11), Hrs (12), OATL1 (13), COP9 signalosome (14), presenilin 1 (15), the ESCRT III complex (16), LAMP1/2 (17), UVRAG (18), p97VCP (19), LRRK2 (20), and HSP70 (21) have been implicated in regulating this process. We have been studying the effects of lysosomal specific ion channels on autophagosomal-lysosomal fusion. In vertebrates, three TPC2 genes, TPC1, -2, and -3, have been cloned. TPCs contain two putative pore-forming repeats, with each of the repeats containing six transmembrane domains and an intervening pore-forming loop. The transmembrane domain of TPCs is similar to that of voltage-gated Ca2+/Na+ channels. Interestingly, human and rodent only encode TPC1 and TPC2. TPC2 predominantly localizes on lysosomes, whereas TPC1 is mainly on late endosomes, and TPC3 is thought to be in the recycling endosomes. Ever since their identification, TPCs have become the prime candidates for NAADP-mediated Ca2+ release from lysosome-related stores (22–24). NAADP is a metabolite of nicotinamide adenine dinucleotide phosphate (NADP) and is formed by a base-exchange reaction catalyzed by ADP-ribosyl cyclases, which replaces the nicotinamide moiety of NADP with nicotinic acid. NAADP mobilizes Ca2+ from acidic lysosome-related stores in a wide variety of cells, from plant to animal, including human (25). Ample evidence indicates that TPC2 forms NAADP-sensitive Ca2+-permeable channels in many cell types. TPC2 overexpression promotes NAADP-induced Ca2+ release from lysosome-related stores, whereas ablating or knocking-down TPC2 expression blocks NAADP-induced Ca2+ release. Moreover, TPC2 knock-out abolishes NAADP-mediated smooth muscle contraction, a well established function of NAADP (26–33). Besides TPC2, other NAADP receptor candidates have been reported in different cell types, including TPC1 in SKBR3 human breast carcinoma (34, 35), TRP mucolipin 1(TRPML1) in coronary arterial myocytes (36, 37), and TRPM2 or ryanodine receptors in T lymphocytes (38, 39). Interestingly, several recent papers found that NAADP does not directly bind to TPC2, suggesting that NAADP first binds to accessory proteins, which subsequently activate TPC2 or other ion channels for Ca2+ mobilization depending upon the cell type (40–42). Previously, two groups found that activation of NAADP/TPC2 signaling increased LC3-II levels (43, 44), and another report found that down-regulation of TPC2 by presenilin decreased LC3-II (45), thus concluding that TPC2 signaling mainly induces the initiation of autophagy. However, here we found that the increased LC3-II levels by TPC2 signaling are not due to the enhanced autophagy activity but result from the inhibition of basal autophagy progression, which leads to the accumulation of autophagosomes. Inhibition of TPC2 signaling either by TPC2 knockdown or by treatment with a NAADP antagonist, on the other hand, facilitates autophagosomal-lysosomal fusion, thereby decreasing LC3-II levels. EXPERIMENTAL PROCEDURES Antibodies and Reagents LC3 antibody (antibody from MBL was used in study in the embryonic stem (ES) cells, and the one from Novus was used in the study in HeLa cells; 1:500 for the immunofluorescence analysis and 1:1000 for the Western blotting analysis (WB)), p62 antibody (Novus; 1:1000 WB), cathepsin-L antibody (BD Bioscience; 1:250 WB), Lamp-1 and EGFR antibodies (Santa Cruz; 1:250 WB), Lamp-1 antibody (Cell Signaling; 1:500 immunofluorescence analysis), phospho-mTOR and mTOR antibodies (Cell Signaling; 1:500 WB), GAPDH antibody (Sigma; 1:5000 WB), and TPC2 antibody (a custom rabbit polyclonal antibody against rat TPC2; 1:500 immunofluorescence analysis and 1:1000 WB). Bafilomycin A1, rapamycin, and BAPTA-acetoxymethyl (AM) were all purchased from Sigma. NH4Cl was purchased from Merck. Fura-2 AM, dye-quenched (DQ)-BSA-green, lysosensor Green DND-189, and LysoSensor Yellow/Blue DND-160 were purchased from Invitrogen. Cell Culture HeLa cells (ATCC) were maintained in DMEM (Invitrogen) plus 10% fetal bovine serum (Invitrogen) and 100 units/ml penicillin/streptomycin (Invitrogen) at 5% CO2 and 37 °C. ES cells, D3 from Prof. S. Y. Tsang (Chinese University of Hong Kong), and 46C Sox1-GFP from Prof. Austin Smith (University of Cambridge), were normally maintained with feeders (mouse embryonic fibroblasts) in Dulbecco's modified Eagle's medium plus 15% FBS (ES qualified, Invitrogen), 1% nonessential amino acids, 1% penicillin-streptomycin, 0.2% 2-mercaptoethanol, 1000 units/ml leukemia inhibitory factor. Cells are passaged every 2 days. Before any experimental procedures, ES cells are cultured in feeder-free ES medium containing leukemia inhibitory factor on gelatin-coated plates for two passages. The pluripotency of ES cells are periodically assessed by alkaline phosphatase assay and Oct4 immunostaining. TPC2 shRNA, TPC2, GFP-LC3, RFP-GFP-LC3 Lentivirus Production and Infection Three optimal 21-mers were selected in the mouse TPC2 genes (supplemental Table S1). One 21-mer was selected in the GFP gene as a control. These sequences were then cloned into the pLKO.1 vector for expressing shRNA. Likewise, a rat TPC2, GFP-LC3, or RFP-GFP-LC3 (tfLC3) cDNA was cloned into pLenti-CMV-Puro-DEST (Addgene). The shRNA, TPC2, GFP-LC3, or tfLC3 lentivirus production was performed in 293T cells. For infection, cells were plated at a density of 3 × 105 cells/well in 6-well plates. On the next day, 100-μl pools of shRNAs lentivirus were added to the cells in fresh medium containing 8 μg/ml Polybrene. Two days later cells infected with shRNA lentiviruses were selected in fresh medium containing puromycin (3 μg/ml) for 3–5 days. The puromycin-resistant cells were pooled, and the knockdown efficiency was verified by both quantitative real-time RT-PCR and/or Western blot analyses. Intracellular Ca2+ Measurement Intracellular Ca2+ in HeLa cells were measured as described previously (46, 47). Briefly, cells were cultured in 24-well plates at the density of 5 × 104 cells/well in regular DMEM medium overnight. Before Ca2+ measurement, cells were incubated for 30 min in Hanks' balanced salt solution containing 4 μm Fura-2 AM and 0.04% F127 in the dark at room temperature. The cells were then washed with Hanks' balanced salt solution three times and incubated at room temperature for another 10 min. Cells were put on the stage of an Olympus epifluorescence microscope and visualized using a 20× objective. Fluorescence images were obtained by alternate excitation at 340 and 380 nm with emission set at 510 nm. Images were collected by a CCD camera every 3 s. Finally, the images were saved and analyzed by Cell R imaging software. Western Blot Analysis Western blot analysis was performed as described previously (48, 49). Briefly, cells were lysed in an ice-cold EBC lysis buffer (50 mm HEPES at pH 7.5, 0.15 m NaCl, 1 mm EDTA, 1% Nonidet P-40, 150 μm PMSF, 10 mm NaF, 10 ng/ml leupeptin, 1 mm DTT, and 1 mm sodium vanadate) and passed through a 21-gauge needle several times to disperse any large aggregates. Protein concentrations of the cell lysates were determined by Bradford protein assay. 30–100 μg of protein per lane was diluted in the standard SDS sample buffer and subjected to electrophoresis on SDS-PAGE gels. Proteins were then transferred to an Immobilon PVDF membrane (Millipore, Billerica, MA), blocked with 5% milk in TBST (20 mm Tris, 150 mm NaCl, pH 7.6), and incubated with the primary antibody overnight. After washing with TBST, the blots were probed with a secondary antibody (1:5000 dilution) for detection by chemiluminescence. RNA Isolation and Quantitative Real-time RT-PCR Total RNA was extracted from HeLa cells or mouse ES cells at various stages of neural differentiation using an RNA extraction kit (Invitrogen). The quantitative real-time RT-PCR using the SuperScript® III Platinum® One-Step Q-RT PCR Kit (Invitrogen) was performed in a MiniOpticonTM Real-time PCR Detection System (Bio-Rad) according to the manufacturer's instructions. The primers for detecting tpc2 and GAPDH mRNAs are listed in supplemental Table S1. Relative gene expression was normalized to GAPDH expression. Immunofluorescence Staining Analysis Immunofluorescence staining analysis was performed as described previously (50). Briefly, infected or transfected cells grown on coverslips were fixed for 20 min with 4% paraformaldehyde at room temperature and washed twice with PBS, and nuclei were labeled with DAPI to identify each individual cell. For immunostaining analysis, fixed cells were further blocked with 1% goat serum, 1% BSA, 0.1% Triton X-100 in PBS for 1 h. Thereafter, the cover glasses were incubated with primary antibodies for 2 h, washed, and incubated with a secondary antibody (Alexa Fluor® 488 or 568 goat anti-mouse or rabbit IgG, Invitrogen; 1:1000 dilution) for 1 h. Images were captured under a Zeiss LSM710 confocal microscope with a Plan-Apochromat ×63/1.40 oil differential interference contrast objective. Images were processed and analyzed with Zeiss ZEN software. Transmission Electron Microscopy Cell preparation for transmission electron microscopy was performed as described previously (51). Briefly, cells were washed in PBS and fixed in 2% formaldehyde and 2.5% glutaraldehyde for 2 h. After fixation, cells were pelleted, transferred to glass slides, and allowed to dry on a hotplate at 37 °C before embedding in 2% agarose solution. Agarose-embedded blocks were then fixed in the same fixative solution at 4 °C for 4 h. Thereafter, the blocks were washed three times with PBS and post-fixed with 1% osmium tetroxide solution in PBS at 4 °C overnight. Blocks were subsequently dehydrated in graded ethanol, embedded in pure Epon, and kept at 60 °C for 3 days. Ultrathin sections (∼90 nm) were cut and stained with 8% uranyl acetate and lead citrate before observation under electron microscope. For immunoelectron microscopy, cells were incubated with primary antibody (rabbit anti-LC3 antibody, 1:100) at 4 °C overnight. After incubation in the primary antibody, the cells were incubated in biotinylated secondary antibody (biotinylated goat anti-rabbit IgG antibody, 1:200) for 40 min at room temperature. Cells were then incubated in ABC reagents (Vectastain ABC kit) for 60 min at room temperature, sequentially, followed by incubating in 0.05% diaminobenzidine and 0.03% H2O2 for 3–5 min until a brown reaction product was observed. The stained cells were then processed to transmission electron microscopy study as described above. Neural Differentiation The in vitro neural differentiation of mouse ES cells was performed as described previously (52). Briefly, feeder-free ES cells were plated onto 0.1% gelatin-coated plates at a density of 0.8∼1 × 104 cells/cm2 in N2B27 medium (1:1 mixture of DMEM/F12 and neurobasal medium supplemented with N2 and B27 plus 50 μg/ml bovine serum albumin (BSA) fraction V and 20 μm β-mercaptoethanol). The medium was changed every other day until day 8. Lysosomal pH Measurement LysoSensor Green DND-189 is commonly used to qualitatively measure the pH of acidic organelles, such as lysosomes, which become more fluorescent in acidic environments and less fluorescent in alkaline environments (53). Briefly, cells were loaded with 1 μm LysoSensor Green DND-189 in prewarmed regular medium for 20 min at 37 °C. Then the cells were washed twice with PBS and immediately analyzed by flow cytometry (collecting FL1 fluorescence, and 10,000 cells were collected for each sample) or in a microplate reader (excitation/emission = 485/530 nm) in triplicate. Quantification of lysosomal pH was performed using a ratiometric lysosomal pH dye LysoSensor Yellow/Blue DND-160. The pH calibration curve was generated as described previously (54). Briefly, cells were trypsinized and labeled with 2 μm LysoSensor Yellow/Blue DND-160 for 30 min at 37 °C in regular medium, and excessive dye was washed out using PBS. The labeled cells were treated for 10 min with 10 μm monensin and 10 μm nigericin in 25 mm MES calibration buffer, pH 3.5–6.0, containing 5 mm NaCl, 115 mm KCl, and 1.2 mm MgSO4. Quantitative comparisons were performed in a 96-well plate, and the fluorescence was measured with a microplate reader at 37 °C. Light emitted at 440 and 535 nm in response to excitation at 340 and 380 nm was measured, respectively. The ratio of light emitted with 340- and 380-nm excitation was plotted against the pH values in MES buffer, and the pH calibration curve for the fluorescence probe was generated from the plot using Microsoft Excel. NH4Cl Chase Assay HeLa cells were seeded in regular DMEM medium overnight and then incubated with 100 μm NH4Cl in regular DMEM medium without sodium bicarbonate for 20 min at 37 °C. Thereafter, NH4Cl was removed, and the cells were washed with regular medium without sodium bicarbonate continuously and thoroughly. Samples were collected at the indicated time and finally analyzed by Western blotting analyses or for pH measurement in a microplate reader. Statistical Analysis All data were presented as the means ± S.E. Student's t test was performed to determine the differences among grouped data. * denotes statistically significant with p < 0.05. RESULTS Reconstitution of NAADP Competence in HeLa Cells by TPC2 Overexpression HeLa cell line is an autophagy-competent cell line (supplemental Fig. S1, A and B). Yet HeLa cells are not responsive to NAADP-induced Ca2+ changes (Fig. 1A), which might be due to the low expression level of endogenous TPC2 (supplemental Fig. S1C). We thus first constructed a stable rat TPC2-overexpressing HeLa cell line by lentiviral infection. As shown in Fig. 1B, TPC2 was overexpressed in the lysosome as indicated by its co-localization with the lysosomal marker Lamp1 in a subset of cellular puncta (the TPC2-positive vesicles were actually enlarged in some cells as compared with the control). Likewise, the HeLa cells expressing a rat TPC2 249L/P mutant, in which a highly conserved leucine residue within a putative pore region of rat TPC2 was mutated to proline (34), were constructed by lentiviral infection (Fig. 1B). A cell permeant NAADP analog, NAADP-AM (55), induced Ca2+ release only in wild type TPC2-overexpressing HeLa cells and not in TPC2 249L/P mutant-expressing cells (Fig. 1A) and displayed a characteristic bell-shaped dose-response curve due to the activation of its receptor by low concentrations of NAADP and its desensitization by high concentrations of NAADP. The induced Ca2+ release was markedly inhibited by preincubation with Ned-19 (56), an NAADP antagonist (Fig. 1C). These data indicated that HeLa cells can be reconstituted to be NAADP competent via TPC2 overexpression. FIGURE 1. Reconstitution of NAADP-competent HeLa cells by overexpressing TPC2. A, NAADP-AM (100 nm) only induced Ca2+ release in wild type, but not in an inactive mutant, TPC2-overexpressing HeLa cells. B, shown is localization of Lamp1-RFP, TPC2, and TPC2 249L/G in HeLa cells. A rabbit polyclonal TPC2 antibody was used to detect overexpressing TPC2. Scale bar = 5 μm. C, NAADP-AM induced Ca2+ release in TPC2-overexpressing HeLa cells, which was markedly inhibited by Ned-19 (10 μm). Note the bell-shaped concentration dependence curve for NAADP-AM. Quantification of the [Ca2+]i peak induced by drug treatment in A and C is expressed as the mean ± S.E., n = 30–40 cells. The asterisk indicates the results of t test analysis; p < 0.05. Inhibition of Autophagic Flux by TPC2 in HeLa Cells Strikingly, under the electron microscope, large numbers of autophagic vacuoles were observed in TPC2-overexpressing HeLa cells maintained in normal (nutrient-rich) culture conditions but not in control cells (Fig. 2A). Similar to previous reports (43), lipidated LC3-II, a reliable autophagosome marker, was markedly increased in TPC2-overexpressing cells compared with that in TPC2 mutant expressing or control HeLa cells (Fig. 2B). Interestingly, p62, an autophagic substrate (57), was also accumulated in TPC2-overexpressing cells compared with that in control HeLa cells, suggesting that TPC2 overexpression results in a defect of autophagic degradation instead of simply promoting autophagy (Fig. 2B). Consistently, endogenous LC3-II puncta (Fig. 2C) or GFP-LC3 puncta (Fig. 2D and supplemental Fig. S1A) were greatly increased in TPC2-overexpressing cells and did not co-localize with Lamp1, which was similar to the cells treated with bafilomycin (BAF), an inhibitor of the vacuolar proton pump that blocks the fusion of autophagosomes with lysosomes (58). These data suggest that these LC3-II puncta present in TPC2-overexpressing cells are autophagosomes, not autolysosomes. FIGURE 2. TPC2 overexpression induced a defect in autophagic degradation in HeLa cells. A, large numbers of autophagic vacuoles were observed in TPC2-overexpressing cells, but not in control cells, under electron microscope. B, wild type, not an inactive mutant, TPC2 overexpression induced the accumulation of both LC3-II and p62. C, wild type, not an inactive mutant, TPC2 overexpression markedly induced endogenous LC3-II puncta (green) in HeLa cells, which were not colocalized with RFP-Lamp1. Scale bar = 5 μm. Quantification of LC3 puncta is expressed as the mean ± S.E., n = ∼80 cells. The asterisks indicate the results of t test analysis; p < 0.05. D, starvation induced LC3 puncta, some of which were colocalized with LAMP1 puncta, whereas the LC3 puncta induced by bafilomycin or TPC2 expression failed to co-localize with LAMP1 in RFP-LAMP1/GFP-LC3 HeLa cells. Scale bar = 10 μm. Because it is possible that the accumulation of LC3-II in TPC2-overexpressing cells is partly due to the increased early autophagic activity, we briefly (1 h) treated control or TPC2-overexpressing HeLa cells with BAF in normal or serum-free medium. We reasoned that BAF treatment could further induce the accumulation of LC3-II and p62 if TPC2, like starvation, promotes early autophagic progression. However, BAF treatment (1 h) only induced the accumulation of LC3-II and p62 in control cells, not in TPC2-overexpressing cells, regardless of whether cells were in normal or starvation states (Fig. 3A and supplemental Fig. S1B). This suggests that TPC2 is not involved in early autophagy induction. Also notably, starvation not only induced the accumulation of LC3-II but also decreased p62 in both control and TPC2-overexpressing cells (comparing lanes 5 and 6 in Fig. 3A), suggesting that TPC2 overexpression only inhibits but does not abolish the fusion of autophagosomes with lysosomes. FIGURE 3. TPC2 overexpression did not affect the early autophagic progression in HeLa cells. A, bafilomycin (10 nm, 1 h) only induced the accumulation of LC3-II and p62 in control, not TPC2-overexpressing, HeLa cells, whereas starvation induced autophagic progression in both cells. The quantification of the four boxed lanes is shown. B, starvation (60 min) induced RFP-ATG5 puncta, most of which were not colocalized with GFP-LC3 puncta, in both control and TPC2-overexpressing HeLa cells. C, TPC2 failed to induce LC3-II and p62 in ATG5 knockdown cells. D, ATG5 knockdown markedly inhibited TPC2-induced accumulation of LC3-II and p62 in HeLa cells. E, TPC2 overexpression did not affect p62 incorporation into autophagosome in HeLa cells. Scale bar = 10 μm. After the initiation of the phagophores, autophagosomes undergo a stepwise maturation process from early to late autophagosomes, which ultimately fuse with lysosomes to form autolysosomes. Any missteps during the maturation process could lead to the accumulation of autophagosomes (1). Therefore, we next assessed whether TPC2 signaling compromises early autophagosome formation. To do so, GFP-LC3 or GFP-LC3/TPC2-expressing HeLa cells were transfected with RFP-ATG5. Under normal conditions (nutrient-rich), ATG5 was diffuse throughout the cytoplasm in both control and TPC2-overexpressing HeLa cells, again suggesting that TPC2 does not induce autophagy. Starvation (60 min), on the other hand, markedly induced puncta formation of ATG5, some of which were not colocalized with LC3 in both cell types (Fig. 3B). These data suggest that TPC2 signaling does not inhibit the early autophagosome(s) maturation. Subsequently, we tested whether the accumulated autophagosomes in TPC2-overexpressing HeLa cells are the result of basal cellular autophagy activity. To do so, we knocked-down ATG5, which is required for the elongation and closure of autophagosomes in both control and TPC2-overexpressing HeLa cells (supplemental Fig. S2A). As expected, not only did TPC2 overexpression in ATG5-knockdown cells fail to induce autophagosome(s) accumulation (Fig. 3C and supplemental Fig. S2B), but also knockdown of ATG5 in TPC2-overexpressing cells markedly decreased the accumulated LC3-II and p62 (Fig. 3D). These results place TPC2 downstream of ATG5 in autophagy regulation and again suggest that TPC2 overexpression does not completely block the fusion of autophagosomes with lysosomes. p62, as a cargo receptor for some ubiquitinated proteins, is first incorporated into autophagosomes before being targeted to lysosomes for degradation (59). Therefore, it is also necessary to clarify whether accumulated p62 in TPC2-overexpressing cells is due to the defects in cargo incorporation into autophagosomes. As shown in Fig. 3E, under normal culture conditions, p62 puncta were already markedly accumulated in TPC2-overexpressing cells compared with control cells, and most of them were co-localized with LC3-II. Moreover, similar to control cells, starvation further induced LC3-II puncta and p62 puncta in TPC2-overexpressing cells (Fig. 3E). This indicates that TPC2 does not prevent p62 incorporation into autophagosomes and suggests that accumulated p62 in TPC2-overexpressing cells is due to the defects in later stages of autophagy. Inhibition of the Fusion between Autophagosomes and Autolysosomes by NAADP/TPC2 Signaling in HeLa Cells To further differentiate autophagosomes and autolysosomes during autophagy, we infected the control or TPC2-overexpressing HeLa cells with lentiviruses carrying expression cassettes that encode RFP-GFP-LC3 (tfLC3), in which RFP and GFP are tandem-tagged to the N terminus of LC3 (60). Before fusion with lysosomes, the LC3-II-positive autophagosomes are shown by both GFP and RFP signals as yellow puncta, and after fusion, autolysosomes are shown by only RFP signals as red only puncta because GFP loses its fluorescence in acidic pH. As expected, yellow puncta (autophagosomes) were markedly increased with few red puncta signals (autolysosomes) in wild type, not mutant, TPC2-overexpressing cells, which was similar to the effects of BAF in control cells. In contrast, starvation dramatically increased both yellow and red puncta in control cells (Fig. 4, A and B). We further performed immunoelectron microscopy analyses to distinguish autophagosomes from autolysosomes in control and TPC2-overexpressing HeLa cells (Fig. 4C). The cells were incubated with anti-LC3 antibody followed by diaminobenzidine staining and were subsequently subjected to EM analyses. Two types of autophagic structures were quantified: autophagosomes whose surfaces are LC3-positive (Fig. 4C1) and autolysosomes whose inside are LC3-positive (Fig. 4C2). Consistently, the numbers of autophagosomes in TPC2-overexpressing cells were significantly more than those in control cells, whereas autolysosomes were hard to find in both control and TPC2-overexpressing cells (the bottom panel of Fig. 4C). Thus, these data clearly demonstrate that TPC2 overexpression inhibits the fusion between autophagosomes and autolysosomes, thereby resulting in a marked accumulation of autophagosomes. FIGURE 4. TPC2 signaling inhibited the progression of autophagy in HeLa cells. A, HeLa cells were transfected with a RFP-GFP tandem fluorescent-tagged LC3 (tfLC3) plasmid. TPC2 overexpression induced yellow puncta with few red-only puncta signals in cells. In contrast, starvation dramatically increased both yellow and red puncta in control cells. Scale bar = 10 μm. B, quantitation of % of yellow puncta or red only puncta/per cell of A are expressed as the mean ± S.E., n = ∼ 80 cells. The asterisks indicate the results of t test analysis; p < 0.05. C, control and TPC2-overexpressing cells were processed for immunoelectron microscopy analyses. C1, shown are representative autophagosomal structures, whose surfaces are LC3-positive, in TPC2-overexpressing HeLa cells; C2, shown are representative autolysosomal structures, whose inside are LC3-positive, in TPC2-overexpressing HeLa cells. Quantitation of autophagosomes and autolysosomes per cell are expressed as the mean ± S.E., n = ∼ 40 cells (bottom panel). ns, not significant. D, NAADP-AM (100 nm) increased the accumulation of both LC3-II and p62 only in TPC2-overexpressing, not the control, HeLa cells. E, Ned-19 (10 μm) decreased the levels of both LC3-II and p62 in TPC2-overexpressing, but not the control, HeLa cells. Because TPC2 is a NAADP-sensitive Ca2+-permeable channel in lysosomes, we examined the effects of NAADP-AM on the fate of autophagosomes in TPC2-overexpressing cells. As shown in Fig. 4D and supplemental Fig. S3A, NAADP-AM indeed further induced the accumulation of both LC3-II and p62 only in TPC2-overexpressing HeLa cells. On the other hand, treatment with Ned-19, a NAADP antagonist, decreased the levels of both LC3-II and p62 only in TPC2-overexpressing cells (Fig. 4E, supplemental Fig. S3, B and C). Moreover, Ned-19 increased the red LC3 puncta (autolysosomes), whereas it decreased the yellow LC3 puncta (autophagosomes) in TPC2/RFP-GFP-LC3 co-expressing HeLa cells (Fig. 4, A and B). Taken together, our results further demonstrate that the activation of NAADP/TPC2 signaling compromises autophagy progression, resulting in autophagosome accumulation. Inhibition of Autophagic Flux by Endogenous TPC2 Signaling in Mouse Embryonic Stem Cells Upon withdrawal of self-renewal stimuli, mouse ES cells can spontaneously and robustly differentiate into neural progenitors in adherent monoculture (61). Interestingly, autophagy was markedly induced during neural differentiation of mouse ES cells (Fig. 5A and supplemental Fig. S4A). TPC2 is detectable in mouse ES cells (Fig. 5B), and quantitative RT-PCR analyses showed that the expression of TPC2 mRNA was dynamically regulated during neural differentiation of mouse ES cells (supplemental Fig. S4B). Thus, we used lentivirus-mediated short hairpin RNA (shRNA) or cDNA to stably knockdown the expression of TPC2 (Fig. 5B) or to overexpress TPC2 (Fig. 5C), respectively, in mouse ES cells. FIGURE 5. TPC2 signaling inhibited the progression of autophagy during neural differentiation of mouse ES cells. A, in vitro neural differentiation of mouse ES cells initiated by monolayer adherent culture markedly induced LC3-II in D3 mouse ES cells. B, TPC2 knockdown by shRNA in ES cells was determined by TPC2 immunoblot analyses. C, TPC2 is expressed in lysosome-related organelles, as it is co-localized with LysoTracker (red). Scale bar = 50 μm. D, TPC2 overexpression induced accumulation of LC3-II in ES cells during neural differentiation. Treatment with bafilomycin (100 nm) only induced the accumulation of LC3-II in control cells but not in TPC2-overexpressing cells. E, TPC2 knockdown decreased the accumulation of LC3-II in ES cells during neural differentiation. Treatment with bafilomycin (100 nm) induced the accumulation of LC3-II in both control and TPC2 knockdown cells. F, mouse D3 ES cells were infected with RFP-GFP tandem fluorescent-tagged LC3 (tfLC3) lentiviruses. TPC2 overexpression induced yellow puncta with few red only puncta signals, whereas TPC2 knockdown increased the red-only puncta in ES cells after 4 days of neural differentiation. Quantification of at least 50 cells from three independent experiments is expressed as the mean ± S.E. and shown in the right columns. The asterisks indicate the results of t test analysis; p < 0.05. Scale bar = 10 μm. The control scramble-shRNA-infected, TPC2-knockdown, or TPC2-overexpressing cells were then induced to differentiate into neural lineages after the monolayer culture protocol. Similar to HeLa cells, the LC3-II level was markedly accumulated in differentiated TPC2-overexpressing ES cells as compared with that in differentiated control ES cells, and BAF treatment only increased LC3-II levels in control but not in TPC2-overexpressing ES cells (Fig. 5D and supplemental Fig. S4H). On the other hand, LC3-II levels (Fig. 5E, supplemental Fig. S4, C and F) or LC3 puncta (supplemental Fig. S4, D and E) were significantly decreased in differentiated TPC2 knockdown ES cells compared with those in control ES cells. Two opposite mechanisms can lead to less LC3-II level or LC3 puncta in TPC2 knockdown cells; a decrease in autophagic induction causes less LC3-II lipidation, or an enhanced fusion between autophagosome and lysosome leads to a faster LC3-II degradation. Interestingly, BAF treatment further increased LC3-II in TPC2 knockdown cells to a level similar to that in control cells (Fig. 5E and supplemental Fig. S4G), suggesting that the decreased LC3-II level in TPC2 knockdown cells during differentiation is not due to inhibition of autophagic induction but because of the enhanced autophagy progression leads to faster LC3-II degradation. To further assess the possibility that TPC2 knockdown facilitates the fusion between autophagosome and lysosome in order to result in decreased LC3-II levels during differentiation, the tfLC3 was again utilized to distinguish autophagosomes from autolysosomes in differentiated ES cells. Not surprisingly, in tfLC3-expressing ES cells during neural differentiation, more yellow LC3 puncta (autophagosomes) existed in TPC2-overexpressing cells, whereas more red-only LC3 puncta (autolysosomes) appeared in TPC2 knockdown cells (Fig. 5F), indicating that autophagosomal-lysosomal fusion is indeed facilitated by TPC2 knockdown but inhibited by TPC2 overexpression. Clearly, these data further demonstrate that the endogenous TPC2 signaling inhibits the fusion between autophagosomes and autolysosomes as well. In addition, we found that TPC2 knockdown facilitated mouse ES cells to differentiate into neural progenitors, whereas TPC2 overexpression inhibited mouse ES cells differentiated into neural progenitors (61). The Effects of mTOR on TPC2-induced Autophagy Inhibition The mTOR kinase is one of the key regulators for autophagy, and the activation of mTOR complex 1 occurs at the surface of lysosomes and requires v-ATPase activity (62–64). Moreover, increased mTOR activity is found to be essential for termination of autophagy and reformation of lysosomes (65), and it has recently been shown that ATP activates mTOR to inhibit TPC2 gating (66). We, therefore, assessed whether TPC2 inhibits the fusion between autophagosomes and autolysosomes via mTOR. Unexpectedly, TPC2 overexpression in HeLa cells not only failed to affect mTOR activity but also had no effect on the ability of starvation to inhibit mTOR (Fig. 6A and supplemental Fig. S5B). Notably, inhibition of mTOR induced by starvation in TPC2-overexpressing cells was accompanied by not only the increase of LC3-II but also the decrease of p62, suggesting that mTOR inhibition does not increase the ability of TPC2 to inhibit autophagy (Fig. 6A). Similarly, treatment of cells with rapamycin (50 μm), an mTOR inhibitor, for 3 h increased LC3-II levels and decreased p62 levels in both control and TPC2-overexpressing cells (Fig. 6B). Therefore, these data suggest that TPC2 signaling suppresses autophagy maturation independent of mTOR. FIGURE 6. Ca2+, not mTOR, was involved in TPC2-induced autophagosome accumulation in HeLa cells. A, starvation induced LC3-II levels and promoted the degradation of p62 in both control and TPC2-overexpressing HeLa cells accompanied with the inactivation of mTOR. TPC2-induced accumulation of LC3-II and p62 was decreased by Ned-19 (10 μm). B, treatment of cells with rapamycin (50 μm) for 3 h promoted autophagy in both control and TPC2-overexpressing cells. C and D, BAPTA-AM (20 μm) decreased the levels of LC3-II and p62 (C) and LC3-II puncta (D) in TPC2-overexpressing HeLa cells. Quantification of LC3 puncta is expressed as the mean ± S.E., n = ∼ 50 cells. The asterisk indicates the results of t test analysis; p < 0.05. Scale bar = 10 μm. The Effects of Ca2+ on TPC2-induced Autophagy Inhibition NAADP can induce Ca2+ release from lysosomes (25), and we found that NAADP treatment actually inhibited autophagosomal-lysosomal fusion (Fig. 4C and supplemental Fig. S3A). Thus we next examined whether NAADP/TPC2 signaling suppresses autophagy progression via Ca2+ release from lysosomes. As shown in Fig. 6C, after 1 or 2 h of treatment of cells with BAPTA-AM, a Ca2+ chelator, both LC3-II and p62 were markedly decreased in TPC2-overexpressing HeLa cells but not in control cells. Similarly, BAPTA-AM treatment for 1 h significantly decreased LC3-II puncta in TPC2-overexpressing HeLa cells (Fig. 6D). Collectively, these data suggest that the inhibition of autophagy progression by NAADP/TPC2 is dependent on Ca2+. TPC2 on Lysosomal pH in HeLa and Mouse ES Cells Interestingly, brief treatment of cells with BAF (∼1 h) failed to further induce the accumulation of LC3-II and p62 in TPC2-overexpressing cells (Fig. 3A and supplemental Fig. S5A), yet longer treatment with BAF (>3 h) actually further induced the accumulation of these two proteins (supplemental Fig. S5, A and B). Considering that BAF blocks autophagosomal-lysosomal fusion by raising lysosomal pH via inhibiting the V-proton ATPase and that it has been previously shown that NAADP increases the pH of acidic Ca2+ stores in sea urchin egg (67, 68), we speculated that TPC2 overexpression might increase lysosomal pH as well. We first applied LysoSensor Green DND-189 (pKa = ∼ 5.2) to qualitatively measure lysosomal pH (53). LysoSensor Green DND-189 permeates cell membranes and accumulates in acidic intracellular organelles, and its fluorescence increases or decreases in acidic or alkaline environments, respectively. As shown in Fig. 7A, supplemental Fig. S5, C and D, TPC2 overexpression indeed raised lysosomal pH in HeLa cells, which was similar to that by 1 h of BAF treatment but was less than that by 3 h of BAF treatment. This also explains why only 3 h, not 1 h, of BAF treatment further induced the accumulation of LC3-II and p62 in TPC2-overexpressing HeLa cells (supplemental Fig. S5, A and B). We further quantified lysosomal pH by dual-emission ratio imaging of LysoSensor Yellow/Blue DND-160-stained cells (supplemental Fig. S5E) and found that lysosomal pH in TPC2-overexpressing HeLa cells was increased from pH 4.9 in control cells to pH 5.2 (Fig. 7B). Similar results were observed in mouse ES cells (Fig. 7, C and D). In addition, NAADP-AM transiently raised lysosomal pH only in TPC2-overexpressing HeLa cells (supplemental Fig. S5F), which correlated with its effects on LC3-II accumulation (Fig. 3D). Next, we assessed whether reacidifying lysosomes in TPC2-overexpressing HeLa cells could relieve the fusion blockage of autophagosomes and lysosomes. An established ammonium chloride (NH4Cl) pulse-wash technique was adopted to artificially acidify lysosomal pH (69). To do so, cells were initially incubated with NH4Cl. The accumulation of ammonia in lysosomes resulted in an increase of lysosomal pH, which was indicated by a drop of LysoSensor Green DND-189 fluorescence in NH4Cl pulsing cells compared with that in cells without NH4Cl pulsing (time 0 in Fig. 7E). When NH4Cl was removed from the medium by thorough washing, the efflux of ammonia caused the pH in the lysosomes to drop rapidly and transiently reach a value lower than that before the ammonium chloride was added, which was indicated by the increases of LysoSensor Green DND-189 fluorescence in NH4Cl-pulsed cells after washing (Fig. 7E). The transient reacidified lysosomal pH, produced after washing out ammonium chloride, mitigated the fusion blockage in TPC2-overexpressing cells (Fig. 7F). Taken together, these data clearly demonstrate that TPC2 signaling suppresses autophagosomal-lysosomal fusion by alkalizing the lysosomal pH. FIGURE 7. TPC2 signaling increased lysosomal pH to suppress autophagy progression. A, TPC2 overexpression or BAF treatment induced an increase of lysosomal pH in HeLa cells as determined by FACS analyses of Lysosensor DND-189-stained cells. B, quantification of lysosomal pH in control and TPC2-overexpressing HeLa cells as determined by dual-emission ratio imaging of LysoSensor Yellow/Blue DND-160-stained cells. C and D, TPC2 overexpression or BAF treatment induced an increase of lysosomal pH in D3 ES cells as determined by FACS analyses (C) and microplate reader measurement (D) of Lysosensor DND-189-stained cells. E, washing out NH4Cl in both control and TPC2-overexpressing HeLa cells after pulsing with NH4Cl (100 μm) for 20 min transiently reacidified lysosomal pH. The data are expressed as the mean ± S.D.; n = 4. F, washing out NH4Cl decreased the accumulation of LC3-II and p62 in TPC2-overexpressing cells after been pulsing with NH4Cl (100 μm) for 20 min. The first lanes show samples without NH4Cl pulsing. The graph in B and D represents data from three independent experiments, and the data are expressed as the mean ± S.D.; n = 3. The asterisks indicate the results of t test analysis; p < 0.05. TPC2 on Rab7 Recruitment to Autophagosomes Rab7, a small GTPase, is required for autophagosome-lysosome fusion (6). We, thus, assessed the effects of TPC2 overexpression on the localization of Rab7 by transfecting RFP-Rab7 into GFP-LC3 or GFP-LC3/TPC2-overexpressing HeLa cells. We found that starvation induced the co-localization of Rab7 with both GFP-LC3 and Lamp1 in control cells (Fig. 8A). On the other hand, RFP-Rab7 puncta were colocalized with Lamp1, but few of them were colocalized with LC3-GFP in TPC2-overexpressing cells (Fig. 8B). Next, we assessed whether transient reacidified lysosomal pH by NH4Cl pulse-wash in TPC2-overexpressing HeLa cells restores the association of Rab7 with autophagosomes. As shown in Fig. 8C, in the majority (⅔) of TPC2-overexpressing HeLa cells, reacidified lysosomal pH (10 min after NH4Cl washing) markedly decreased LC3 puncta, which is consistent with the fact that re-acidification decreased LC3-II levels in TPC2-overexpressing cells as shown in Fig. 7F. Interestingly, Rab7 failed to co-localize with the remaining LC3 puncta in these cells. We speculate that lysosomal reacidification in the majority of TPC2-overexpressing cells quickly relieves the fusion blockage of autophagosomes and lysosomes to form autolysosomes, thereby leading to the degradation of LC3-II and the quenching of GFP fluorescence. The remaining LC3 puncta in these cells likely represent the immature autophagosomes induced by the repetitive post NH4Cl washing. However, around ⅓ of TPC2-overexpressing cells still contained large numbers of LC3 puncta after removing NH4Cl from the medium, and Rab7 puncta were indeed co-localized with LC3-II in these cells (Fig. 8D). We reason that lysosomal reacidification in these cells might be slow and just start to recruit Rab7 to the autophagosomes but not yet induce the fusion of autophagosomes with lysosomes. Taken together, these results suggest that TPC2 signaling prevents the recruitment of Rab-7 to autophagosomes by alkalizing lysosomal pH to inhibit autophagosomal-lysosomal fusion. FIGURE 8. TPC2 prevents the recruitment of Rab-7 to autophagosomes in HeLa cells. A, starvation (60 min) induced RFP-Rab7 puncta, which were colocalized with GFP-LC3 puncta (left panel) or Lamp1 puncta (right panel) in control HeLa cells. B, RFP-Rab7 puncta were colocalized with Lamp1 (right panel) but not with GFP-LC3 puncta in TPC2-overexpressing HeLa cells. C, removing NH4Cl (10 min) from medium decreased LC3 puncta in the majority (⅔) of TPC2-overexpressing cells pulsed with NH4Cl and RFP-Rab7 failed to colocalize to the remaining LC3 puncta. D, after washing out NH4Cl for 10 min, ⅓ of TPC2-overexpressing cells still contained large number of LC3 puncta, which were co-localized with RFP-Rab7. Scale bar = 10 μm. TPC2 on General Endosomal-Lysosomal Degradation Because the increase of lysosomal pH normally compromises the lysosomal activity, we assessed whether TPC2 signaling simply disrupts the general lysosomal functions to inhibit autophagy maturation. The processing of cathepsin L from the precursor form to its mature form has been commonly used as a marker for lysosomal activity, yet we found that the processing of cathepsin L was only slightly compromised in TPC2-overexpressing HeLa cells compared with that in control cells (Fig. 9A), which might be due to the fact that TPC2 overexpression just marginally increased lysosomal pH (Fig. 7B). Next, an epidermal growth factor receptor (EGFR) degradation assay was performed to examine whether the TPC2 signaling also affects the general endosomal-lysosomal pathway. In this assay both control and TPC2-overexpressing HeLa cells were treated with EGF. In principle, after EGF binds to its receptors (EGFR), the receptor complex undergoes endocytosis and is targeted to lysosomes where it is ultimately degraded. As shown in Fig. 9B and supplemental Fig. S6A, BAF inhibited EGF-triggered EGFR degradation in a dose-dependent manner, whereas TPC2 overexpression had little effect on it, which was similar to that in control cells. Finally, a DQ-BSA-green degradation assay was applied to measure the general endosomal-lysosomal degradation. DQ-BSA-green is a BSA labeled with a self-quenching fluorescent dye. After DQ-BSA-green is delivered to lysosomes via endocytosis, it is hydrolyzed into single dye-labeled peptides by lysosomal proteases, thereby relieving self-quenching, and the fluorescence can subsequently be monitored by flow cytometry. As shown in Fig. 9C and supplemental Fig. S6B, TPC2 overexpression also had little effect on BSA degradation, whereas BAF markedly inhibited it. Collectively, these results document that TPC2 signaling does not inhibit the general endosomal-lysosomal degradation. FIGURE 9. TPC2 did not inhibit general endosomal-lysosomal degradation in HeLa cells. A, shown is processing of cathepsin L from the precursor form to its mature form in control or TPC2-overexpressing HeLa cells treated with or without bafilomycin for 1 or 3 h. B, TPC2 overexpression failed to change EGF-induced EGFR degradation, whereas BAF (100 nm) completely abolished it in HeLa cells. C, TPC2 overexpression did not inhibit the degradation of DQ-BSA-green in HeLa cells, whereas BAF (100 nm) markedly inhibited it. DISCUSSION Here we provide definitive evidence that NAADP/TPC2 signaling induced the accumulation of matured autophagosomes by inhibiting autophagosomal-lysosomal fusion (Figs. 2–5). Perturbing TPC2/NAADP signaling by either TPC2 knockdown or the addition of an NAADP antagonist, on the other hand, facilitated the progression of autophagy by promoting the formation of autolysosomes (Figs. 3–5). Instead of affecting or functioning downstream of mTOR (Fig. 6, A and B), TPC2 signaling actually alkalinized lysosomal pH to suppress autophagosomal-lysosomal fusion (Fig. 7), likely by preventing the recruitment of Rab-7 to autophagosomes (Fig. 8). Yet TPC2 signaling had little effect on general endosomal-lysosomal degradation (Fig. 9). The acidic milieu in lysosomes is generated by the V-ATPase, a multisubunit protein complex that pumps protons into the lysosomal lumen against an electrochemical gradient at the expense of ATP hydrolysis (70). The influx of protons into the lysosomal lumen also creates a lumen-positive membrane potential that, reciprocally, mitigates the ability of the V-ATPase to continue pumping protons. Thus, transporters for cation efflux or anion influx or both must be activated to maintain the balance of acidic pH and membrane potential inside lysosomes (71). Although Cl− influx, possible via ClC-7 or CFTR (cystic fibrosis transmembrane conductance regulator), has been shown to dissipate the restrictive electrical gradient for lysosomal acidification (72, 73), whether Cl− is the main counterion is debatable, and the identity of Cl− transporters in lysosomes also remains controversial (71). Likewise, Ca2+, whose concentration in lysosomes is high and is partially dependent on H+ gradient (74), has been proposed to constitute another counterion pathway. TPC2, TRPM2, and TPML1 have all been shown to mediate Ca2+ release from lysosomes triggered by NAADP or ADPR (adenosine diphosphate ribose) (24, 36). Yet here we found that NAADP treatment or TPC2 overexpression increased lysosomal pH to inhibit autophagosomal-lysosomal fusion (Fig. 7), and chelating Ca2+ by BAPTA-AM (≥ 1 h treatment) relieved TPC2-induced accumulation of both LC3-II and p62 (Fig. 6, C and D), indicating that Ca2+ is required for NAADP/TPC2-induced lysosomal alkalinization and subsequent fusion blockage. These data further suggest that Ca2+ is not the counterion for maintaining high H+ in lysosomes, and the search for the real counterion pathway must continue. Regarding how NAADP/TPC2-triggered Ca2+ release raises lysosomal pH, we speculate that either a putative Ca2+/H+ exchanger (75) or an unidentified lysosomal Ca2+ ATPase (76), similar to SERCA, or the coupling of several lysosomal proton-cation counter-transporters, such as sequential action of Ca2+/Na+ exchangers and Na+/H+ exchangers, can be activated to refill the lysosomal Ca2+ pools at the expense of proton efflux upon Ca2+ release from lysosomes via TPC2. Obviously, deciphering how Ca2+ enters lysosomes is the key to resolving this mystery. However, other than the V-ATPase, ion channels or transporters responsible for the formation of the unique ionic environment, e.g. Na+, Cl−, K+, and Ca2+, inside lysosomes remain unknown (75). A future lysosomal proteomics study should help in identifying the respective transporters. Here we found that the HeLa cell line, an NAADP-incompetent line, can be reconstituted to be NAADP-competent by overexpressing a wild type, but not a pore-mutant, TPC2 (Fig. 1, A and C), and treatment of TPC2-overexpressing cells with Ned-19, a NAADP antagonist, markedly inhibited NAADP-induced Ca2+ release (Fig. 1C). Thus, these data provide unambiguous evidence that NAADP can activate TPC2 for Ca2+ release and echo the previous finding that TPC2 is an NAADP effector (26–31). Yet we also found that TPC2 overexpression in HeLa cells in the absence of exogenous NAADP treatment, already alkalinized lysosomal pH (Fig. 7, A–D) and inhibited autophagy maturation (Figs. 2–5). Although the effects of TPC2 overexpression might be due to endogenous NAADP, the effects of NAADP-AM on Ca2+ release (Fig. 1, A and C) and autophagy inhibition (Fig. 4D and supplemental Fig. S3A) in TPC2-overexpressing cells were transient. It has recently been shown that NAADP does not directly bind to TPC2, arguing that NAADP might first bind to accessory proteins within the TPC2 complex and subsequently activate TPC2 (40–42). Therefore, the effects of TPC2 overexpression in HeLa or mouse ES cells might be caused by accessory proteins in the TPC2 complex, and NAADP, induced by extracellular stimuli or cellular stress (25, 77), could bind to these accessory proteins to further activate TPC2. However, at this stage we still could not exclude the possibility that the effects of TPC2 overexpression are due to endogenous NAADP, as the transient effects of NAADP-AM might be due to its instability in aqueous solution (55). Two recent papers found that TPC2 forms a Na+ channel in lysosomes that can be potentiated by phosphatidylinositol 3,5-disphosphate, an endolysosome-specific phosphoinositide, and inhibited by ATP (66, 78). It was proposed that high intracellular ATP levels enable mTOR to phosphorylate TPC2, thereby keeping it at a closed state. On the other hand, cell starvation results in lower ATP levels and dissociation of mTOR from TPC2, thus activating TPC2 to allow Na+ release from lysosomes (66). However, here we found that overexpression of a wild type, not a pore-mutant, TPC2 in cells maintained in nutrient-rich medium already alkalinized lysosomal pH and suppressed autophagosome-lysosome fusion (Figs. 2–5), suggesting that TPC2 in high ATP levels is already open. Starvation promoted autophagy, e.g. LC3-II induction and p62 degradation, in both control and TPC2-overexpressing cells (Figs. 3A and 6A), arguing that nutrient deprivation does not further activate TPC2 to inhibit autophagy progression. Thus these data suggested that TPC2 suppresses autophagosome-lysosome fusion independent of mTOR or ATP levels. However, the role of intracellular phosphatidylinositol 3,5-disphosphate or Na+ release from lysosome on TPC2-mediated autophagy inhibition remains to be determined. Also notably, these two papers found that NAADP cannot activate TPC2 for Ca2+ release from lysosomes mainly by performing a patch clamp study on enlarged endolysosomes treated with vacuolin-1 (66, 78), which is contrary to some of results presented in this study and a body of work published previously (26–31). Further investigations are needed to resolve the controversy, and a recent study suggests that a GFP tagged on the N terminus of TPC1 disrupts NAADP action (79). Interestingly, mutation of TPC2, M484L, or G734E has been associated with blond versus brown hair by genome-wide association studies for pigmentation (80). The melanosome, a lysosome-related organelle, is the site of synthesis and transport of melanin pigments, thereby providing tissues with color and photoprotection. In addition, autophagy and autophagy regulators have been shown to play roles in both the biogenesis of melanosomes and melanosome destruction (81). Therefore, it is of interest to assess whether and how the two identified TPC2 mutants affect melanosome biogenesis or destruction via autophagy related processes. Without doubt, TPC2 signaling could play roles in other autophagic-related cellular processes. Given that autophagy plays important roles in a wide variety of cellular processes and dysfunctional autophagy has been associated with many human diseases, it is important to further dissect the TPC2 signaling to identify novel regulators in this pathway. Development of novel reagents targeting TPC2 signaling, e.g. NAADP analogues, to manipulate autophagy should provide alternative pharmaceutical intervention in the treatment of autophagy-related human disorders. Along this line, Niemann-Pick disease, a human neurodegenerative lysosomal storage disorder, has been associated with abnormal lysosomal Ca2+ homeostasis, and its cellular phenotype is similar to that in TPC2-overexpressing cells (Fig. 2A) (29, 82). Whether TPC2-mediated autophagic regulation contributes to this disorder is, therefore, of great interest to pursue. Supplementary Material Supplemental Data * This work was supported by Research Grant Council Grants HKU 782709M, HKU 785911M, and HKU 785213M and a Special Fellow Award from the Leukemia and Lymphoma Society of America (to J. Y.). This article contains supplemental Table 1 and Figs. S1–S6. 2 The abbreviations used are: TPC2two pore channel 2 NAADPnicotinic acid adenine dinucleotide phosphate tfLC3RFP-GFP tandem-tagged LC3 BAFbafilomycin-A1 EGFREGF receptor ESembryonic stem WBWestern blot RFPred fluorescent protein AMacetoxymethyl DQdye-quenched. Acknowledgments We thank Grant Churchill for providing several batches of NAADP-AM and Ned-19, Connie Lam and Yong-Juan Zhao in Prof. Hon-Cheung Lee's laboratory for sharing reagents, and King-Ho Cheung, Hon-Cheung Lee, and members of the Yue laboratory for advice on the manuscript. Confocal imaging and FACS analyses were performed in Faculty of Medicine Core Facility at the University of Hong Kong. ==== Refs REFERENCES 1. Yang Z. Klionsky D. J. (2010 ) Eaten alive. A history of macroautophagy . Nat. Cell Biol . 12 , 814 –822 20811353 2. Maiuri M. C. Zalckvar E. Kimchi A. Kroemer G. (2007 ) Self-eating and self-killing. Cross-talk between autophagy and apoptosis . Nat. Rev. Mol. Cell Biol . 8 , 741 –752 17717517 3. Cárdenas C. Foskett J. K. (2012 ) Mitochondrial Ca2+ signals in autophagy . Cell Calcium 52 , 44 –51 22459281 4. Lorin S. Pierron G. Ryan K. M. Codogno P. Djavaheri-Mergny M. (2010 ) Evidence for the interplay between JNK and p53-DRAM signalling pathways in the regulation of autophagy . Autophagy 6 , 153 –154 19949306 5. Klionsky D. J. Elazar Z. Seglen P. O. Rubinsztein D. C. (2008 ) Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy 4 , 849 –950 18758232 6. Jäger S. Bucci C. Tanida I. Ueno T. Kominami E. Saftig P. Eskelinen E. L. (2004 ) Role for Rab7 in maturation of late autophagic vacuoles . J. Cell Sci . 117 , 4837 –4848 15340014 7. Nair U. Jotwani A. Geng J. Gammoh N. Richerson D. Yen W. L. Griffith J. Nag S. Wang K. Moss T. Baba M. McNew J. A. Jiang X. Reggiori F. Melia T. J. Klionsky D. J. (2011 ) SNARE proteins are required for macroautophagy . Cell 146 , 290 –302 21784249 8. Nickerson D. P. Brett C. L. Merz A. J. (2009 ) Vps-C complexes. Gatekeepers of endolysosomal traffic . Curr. Opin. Cell Biol . 21 , 543 –551 19577915 9. Chen D. Fan W. Lu Y. Ding X. Chen S. Zhong Q. (2012 ) A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate . Mol. Cell 45 , 629 –641 22342342 10. Su M. Shi J. J. Yang Y. P. Li J. Zhang Y. L. Chen J. Hu L. F. Liu C. F. (2011 ) HDAC6 regulates aggresome-autophagy degradation pathway of α-synuclein in response to MPP+-induced stress . J. Neurochem . 117 , 112 –120 21235576 11. N'Diaye E. N. Debnath J. Brown E. J. (2009 ) Ubiquilins accelerate autophagosome maturation and promote cell survival during nutrient starvation . Autophagy 5 , 573 –575 19398896 12. Tamai K. Tanaka N. Nara A. Yamamoto A. Nakagawa I. Yoshimori T. Ueno Y. Shimosegawa T. Sugamura K. (2007 ) Role of Hrs in maturation of autophagosomes in mammalian cells . Biochem. Biophys. Res. Commun . 360 , 721 –727 17624298 13. Itoh T. Kanno E. Uemura T. Waguri S. Fukuda M. (2011 ) OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation . J. Cell Biol . 192 , 839 –853 21383079 14. Su H. Li F. Ranek M. J. Wei N. Wang X. (2011 ) COP9 signalosome regulates autophagosome maturation . Circulation 124 , 2117 –2128 21986281 15. Neely K. M. Green K. N. (2011 ) Presenilins mediate efficient proteolysis via the autophagosome-lysosome system . Autophagy 7 , 664 –665 21460614 16. Lee J. A. Beigneux A. Ahmad S. T. Young S. G. Gao F. B. (2007 ) ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration . Curr. Biol . 17 , 1561 –1567 17683935 17. Eskelinen E. L. (2006 ) Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy . Mol. Aspects Med . 27 , 495 –502 16973206 18. Liang C. Lee J. S. Inn K. S. Gack M. U. Li Q. Roberts E. A. Vergne I. Deretic V. Feng P. Akazawa C. Jung J. U. (2008 ) Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking . Nat. Cell Biol . 10 , 776 –787 18552835 19. Ju J. S. Weihl C. C. (2010 ) p97/VCP at the intersection of the autophagy and the ubiquitin proteasome system . Autophagy 6 , 283 –285 20083896 20. Gómez-Suaga P. Churchill G. C. Patel S. Hilfiker S. (2012 ) A link between LRRK2, autophagy and NAADP-mediated endolysosomal calcium signalling . Biochem. Soc Trans . 40 , 1140 –1146 22988879 21. Leu J. I. Pimkina J. Frank A. Murphy M. E. George D. L. (2009 ) A small molecule inhibitor of inducible heat shock protein 70 . Mol. Cell 36 , 15 –27 19818706 22. Galione A. (2011 ) NAADP receptors . Cold Spring Harb. Perspect Biol . 3 , a004036 21047915 23. Hooper R. Patel S. (2012 ) NAADP on target . Adv. Exp. Med. Biol . 740 , 325 –347 22453949 24. Zhu M. X. Ma J. Parrington J. Calcraft P. J. Galione A. Evans A. M. (2010 ) Calcium signaling via two-pore channels. Local or global, that is the question . Am. J. Physiol. Cell Physiol . 298 , C430 –C441 20018950 25. Lee H. C. (2012 ) Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) as messengers for calcium mobilization . J. Biol. Chem . 287 , 31633 –31640 22822066 26. Calcraft P. J. Ruas M. Pan Z. Cheng X. Arredouani A. Hao X. Tang J. Rietdorf K. Teboul L. Chuang K. T. Lin P. Xiao R. Wang C. Zhu Y. Lin Y. Wyatt C. N. Parrington J. Ma J. Evans A. M. Galione A. Zhu M. X. (2009 ) NAADP mobilizes calcium from acidic organelles through two-pore channels . Nature 459 , 596 –600 19387438 27. Pitt S. J. Funnell T. M. Sitsapesan M. Venturi E. Rietdorf K. Ruas M. Ganesan A. Gosain R. Churchill G. C. Zhu M. X. Parrington J. Galione A. Sitsapesan R. (2010 ) TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+ . J. Biol. Chem . 285 , 35039 –35046 20720007 28. Tugba Durlu-Kandilci N. Ruas M. Chuang K. T. Brading A. Parrington J. Galione A. (2010 ) TPC2 proteins mediate nicotinic acid adenine dinucleotide phosphate (NAADP)- and agonist-evoked contractions of smooth muscle . J. Biol. Chem . 285 , 24925 –24932 20547763 29. Ruas M. Rietdorf K. Arredouani A. Davis L. C. Lloyd-Evans E. Koegel H. Funnell T. M. Morgan A. J. Ward J. A. Watanabe K. Cheng X. Churchill G. C. Zhu M. X. Platt F. M. Wessel G. M. Parrington J. Galione A. (2010 ) Purified TPC isoforms form NAADP receptors with distinct roles for Ca2+ signaling and endolysosomal trafficking . Curr. Biol . 20 , 703 –709 20346675 30. Hooper R. Churamani D. Brailoiu E. Taylor C. W. Patel S. (2011 ) Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation . J. Biol. Chem . 286 , 9141 –9149 21173144 31. Zong X. Schieder M. Cuny H. Fenske S. Gruner C. Rötzer K. Griesbeck O. Harz H. Biel M. Wahl-Schott C. (2009 ) The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores . Pflugers Archiv . 458 , 891 –899 19557428 32. Rietdorf K. Funnell T. M. Ruas M. Heinemann J. Parrington J. Galione A. (2011 ) Two-pore channels form homo- and heterodimers . J. Biol. Chem . 286 , 37058 –37062 21903581 33. Davis L. C. Morgan A. J. Chen J. L. Snead C. M. Bloor-Young D. Shenderov E. Stanton-Humphreys M. N. Conway S. J. Churchill G. C. Parrington J. Cerundolo V. Galione A. (2012 ) NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing . Curr. Biol . 22 , 2331 –2337 23177477 34. Brailoiu E. Churamani D. Cai X. Schrlau M. G. Brailoiu G. C. Gao X. Hooper R. Boulware M. J. Dun N. J. Marchant J. S. Patel S. (2009 ) Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling . J. Cell Biol . 186 , 201 –209 19620632 35. Churamani D. Hooper R. Brailoiu E. Patel S. (2012 ) Domain assembly of NAADP-gated two-pore channels . Biochem. J . 441 , 317 –323 21992073 36. Zhang F. Li P. L. (2007 ) Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats . J. Biol. Chem . 282 , 25259 –25269 17613490 37. Zhang F. Jin S. Yi F. Li P. L. (2009 ) TRP-ML1 functions as a lysosomal NAADP-sensitive Ca2+ release channel in coronary arterial myocytes . J. Cell Mol. Med . 13 , 3174 –3185 18754814 38. Beck A. Kolisek M. Bagley L. A. Fleig A. Penner R. (2006 ) Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes . FASEB J . 20 , 962 –964 16585058 39. Dammermann W. Zhang B. Nebel M. Cordiglieri C. Odoardi F. Kirchberger T. Kawakami N. Dowden J. Schmid F. Dornmair K. Hohenegger M. Flügel A. Guse A. H. Potter B. V. (2009 ) NAADP-mediated Ca2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist . Proc. Natl. Acad. Sci. U.S.A . 106 , 10678 –10683 19541638 40. Guse A. H. (2012 ) Linking NAADP to ion channel activity. A unifying hypothesis . Sci. Signal . 5 , pe18 22534131 41. Lin-Moshier Y. Walseth T. F. Churamani D. Davidson S. M. Slama J. T. Hooper R. Brailoiu E. Patel S. Marchant J. S. (2012 ) Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells . J. Biol. Chem . 287 , 2296 –2307 22117075 42. Walseth T. F. Lin-Moshier Y. Jain P. Ruas M. Parrington J. Galione A. Marchant J. S. Slama J. T. (2012 ) Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg . J. Biol. Chem . 287 , 2308 –2315 22117077 43. Gómez-Suaga P. Luzón-Toro B. Churamani D. Zhang L. Bloor-Young D. Patel S. Woodman P. G. Churchill G. C. Hilfiker S. (2012 ) Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP . Hum. Mol. Genet . 21 , 511 –525 22012985 44. Pereira G. J. Hirata H. Fimia G. M. do Carmo L. G. Bincoletto C. Han S. W. Stilhano R. S. Ureshino R. P. Bloor-Young D. Churchill G. Piacentini M. Patel S. Smaili S. S. (2011 ) Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes . J. Biol. Chem . 286 , 27875 –27881 21610076 45. Neely Kayala K. M. Dickinson G. D. Minassian A. Walls K. C. Green K. N. Laferla F. M. (2012 ) Presenilin-null cells have altered two-pore calcium channel expression and lysosomal calcium. Implications for lysosomal function . Brain Res . 1489 , 8 –16 23103503 46. Yu P. L. Zhang Z. H. Hao B. X. Zhao Y. J. Zhang L. H. Lee H. C. Zhang L. Yue J. (2012 ) A novel fluorescent cell membrane-permeable caged cyclic ADP-ribose analogue . J. Biol. Chem . 287 , 24774 –24783 22661714 47. Li S. Hao B. Lu Y. Yu P. Lee H. C. Yue J. (2012 ) Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) . PloS ONE 7 , e31905 22384096 48. Yue J. Ferrell J. E. Jr. (2006 ) Mechanistic studies of the mitotic activation of Mos . Mol. Cell. Biol . 26 , 5300 –5309 16809767 49. Yue J. Wei W. Lam C. M. Zhao Y. J. Dong M. Zhang L. R. Zhang L. H. Lee H. C. (2009 ) CD38/cADPR/Ca2+ pathway promotes cell proliferation and delays nerve growth factor-induced differentiation in PC12 cells . J. Biol. Chem . 284 , 29335 –29342 19696022 50. Wei W. J. Sun H. Y. Ting K. Y. Zhang L. H. Lee H. C. Li G. R. Yue J. (2012 ) Inhibition of cardiomyocytes differentiation of mouse embryonic stem cells by CD38/cADPR/Ca2+ signaling pathway . J. Biol. Chem . 287 , 35599 –35611 22908234 51. Mi S. Hu B. Hahm K. Luo Y. Kam Hui E. S. Yuan Q. Wong W. M. Wang L. Su H. Chu T. H. Guo J. Zhang W. So K. F. Pepinsky B. Shao Z. Graff C. Garber E. Jung V. Wu E. X. Wu W. (2007 ) LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis . Nat. Med . 13 , 1228 –1233 17906634 52. Ying Q. L. Stavridis M. Griffiths D. Li M. Smith A. (2003 ) Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture . Nat. Biotechnol . 21 , 183 –186 12524553 53. Davis-Kaplan S. R. Ward D. M. Shiflett S. L. Kaplan J. (2004 ) Genome-wide analysis of iron-dependent growth reveals a novel yeast gene required for vacuolar acidification . J. Biol. Chem . 279 , 4322 –4329 14594803 54. Bankers-Fulbright J. L. Kephart G. M. Bartemes K. R. Kita H. O'Grady S. M. (2004 ) Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils . J. Cell Sci . 117 , 5749 –5757 15507482 55. Parkesh R. Lewis A. M. Aley P. K. Arredouani A. Rossi S. Tavares R. Vasudevan S. R. Rosen D. Galione A. Dowden J. Churchill G. C. (2008 ) Cell-permeant NAADP. A novel chemical tool enabling the study of Ca2+ signalling in intact cells . Cell Calcium . 43 , 531 –538 17935780 56. Naylor E. Arredouani A. Vasudevan S. R. Lewis A. M. Parkesh R. Mizote A. Rosen D. Thomas J. M. Izumi M. Ganesan A. Galione A. Churchill G. C. (2009 ) Identification of a chemical probe for NAADP by virtual screening . Nat. Chem. Biol . 5 , 220 –226 19234453 57. Bjørkøy G. Lamark T. Johansen T. (2006 ) p62/SQSTM1. A missing link between protein aggregates and the autophagy machinery . Autophagy 2 , 138 –139 16874037 58. Yamamoto A. Tagawa Y. Yoshimori T. Moriyama Y. Masaki R. Tashiro Y. (1998 ) Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells . Cell Struct. Funct . 23 , 33 –42 9639028 59. Kirkin V. McEwan D. G. Novak I. Dikic I. (2009 ) A role for ubiquitin in selective autophagy . Mol. Cell 34 , 259 –269 19450525 60. Kimura S. Noda T. Yoshimori T. (2007 ) Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3 . Autophagy 3 , 452 –460 17534139 61. Zhang Z. H. Lu Y. Y. Yue J. (2013 ) Two pore channel 2 differentially modulates neural differentiation of mouse embryonic stem cells . Plos ONE 8 , e66077 23776607 62. Sancak Y. Bar-Peled L. Zoncu R. Markhard A. L. Nada S. Sabatini D. M. (2010 ) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids . Cell 141 , 290 –303 20381137 63. Zoncu R. Bar-Peled L. Efeyan A. Wang S. Sancak Y. Sabatini D. M. (2011 ) mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase . Science 334 , 678 –683 22053050 64. Korolchuk V. I. Saiki S. Lichtenberg M. Siddiqi F. H. Roberts E. A. Imarisio S. Jahreiss L. Sarkar S. Futter M. Menzies F. M. O'Kane C. J. Deretic V. Rubinsztein D. C. (2011 ) Lysosomal positioning coordinates cellular nutrient responses . Nat. Cell Biol . 13 , 453 –460 21394080 65. Yu L. McPhee C. K. Zheng L. Mardones G. A. Rong Y. Peng J. Mi N. Zhao Y. Liu Z. Wan F. Hailey D. W. Oorschot V. Klumperman J. Baehrecke E. H. Lenardo M. J. (2010 ) Termination of autophagy and reformation of lysosomes regulated by mTOR . Nature 465 , 942 –946 20526321 66. Cang C. Zhou Y. Navarro B. Seo Y. J. Aranda K. Shi L. Battaglia-Hsu S. Nissim I. Clapham D. E. Ren D. (2013 ) mTOR regulates lysosomal ATP-sensitive two-pore Na+ channels to adapt to metabolic state . Cell 152 , 778 –790 23394946 67. Morgan A. J. Galione A. (2007 ) NAADP induces pH changes in the lumen of acidic Ca2+ stores . Biochem. J . 402 , 301 –310 17117921 68. Morgan A. J. Galione A. (2007 ) Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca2+ stores in sea urchin eggs . J. Biol. Chem . 282 , 37730 –37737 17959608 69. Ohkuma S. Poole B. (1978 ) Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents . Proc. Natl. Acad. Sci. U.S.A . 75 , 3327 –3331 28524 70. Mindell J. A. (2012 ) Lysosomal acidification mechanisms . Annu Rev. Physiol . 74 , 69 –86 22335796 71. DiCiccio J. E. Steinberg B. E. (2011 ) Lysosomal pH and analysis of the counter ion pathways that support acidification . J. Gen. Physiol . 137 , 385 –390 21402887 72. Graves A. R. Curran P. K. Smith C. L. Mindell J. A. (2008 ) The Cl−/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes . Nature 453 , 788 –792 18449189 73. Deriy L. V. Gomez E. A. Zhang G. Beacham D. W. Hopson J. A. Gallan A. J. Shevchenko P. D. Bindokas V. P. Nelson D. J. (2009 ) Disease-causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages . J. Biol. Chem . 284 , 35926 –35938 19837664 74. Christensen K. A. Myers J. T. Swanson J. A. (2002 ) pH-dependent regulation of lysosomal calcium in macrophages . J. Cell Sci . 115 , 599 –607 11861766 75. Morgan A. J. Platt F. M. Lloyd-Evans E. Galione A. (2011 ) Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease . Biochem. J . 439 , 349 –374 21992097 76. Patel S. Docampo R. (2010 ) Acidic calcium stores open for business. Expanding the potential for intracellular Ca2+ signaling . Trends Cell Biol . 20 , 277 –286 20303271 77. Guse A. H. (2009 ) Second messenger signaling. Multiple receptors for NAADP . Curr. Biol . 19 , R521 –R523 19602416 78. Wang X. Zhang X. Dong X. P. Samie M. Li X. Cheng X. Goschka A. Shen D. Zhou Y. Harlow J. Zhu M. X. Clapham D. E. Ren D. Xu H. (2012 ) TPC proteins are phosphoinositide- activated sodium-selective ion channels in endosomes and lysosomes . Cell 151 , 372 –383 23063126 79. Churamani D. Hooper R. Rahman T. Brailoiu E. Patel S. (2013 ) The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP . Biochem. J . 453 , 147 –151 23634879 80. Sulem P. Gudbjartsson D. F. Stacey S. N. Helgason A. Rafnar T. Jakobsdottir M. Steinberg S. Gudjonsson S. A. Palsson A. Thorleifsson G. Pálsson S. Sigurgeirsson B. Thorisdottir K. Ragnarsson R. Benediktsdottir K. R. Aben K. K. Vermeulen S. H. Goldstein A. M. Tucker M. A. Kiemeney L. A. Olafsson J. H. Gulcher J. Kong A. Thorsteinsdottir U. Stefansson K. (2008 ) Two newly identified genetic determinants of pigmentation in Europeans . Nat. Genet . 40 , 835 –837 18488028 81. Ho H. Ganesan A. K. (2011 ) The pleiotropic roles of autophagy regulators in melanogenesis . Pigment Cell Melanoma Res . 24 , 595 –604 21777401 82. Lloyd-Evans E. Morgan A. J. He X. Smith D. A. Elliot-Smith E. Sillence D. J. Churchill G. C. Schuchman E. H. Galione A. Platt F. M. (2008 ) Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium . Nat Med . 14 , 1247 –1255 18953351	23836916	PMC3745369	CC BY	2021-01-04 23:04:50	yes	J Biol Chem. 2013 Aug 16; 288(33):24247-24263
==== Front Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 10.1155/2013/157456Research ArticleMechanism of Hepatoprotective Effect of Boesenbergia rotunda in Thioacetamide-Induced Liver Damage in Rats Salama Suzy M. 1 Abdulla Mahmood A. 1 AlRashdi Ahmed S. 1 Hadi A. Hamid A. 2 1Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia2Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, MalaysiaMahmood A. Abdulla: mahmood955@yahoo.comAcademic Editor: Sedigheh Asgary 2013 7 8 2013 2013 15745623 3 2013 2 6 2013 17 6 2013 Copyright © 2013 Suzy M. Salama et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Researchers focused on developing traditional therapies as pharmacological medicines to treat liver cirrhosis. Objectives. Evaluating the hepatoprotective activity of Boesenbergia rotunda (BR) rhizome ethanolic extract on thioacetamide-induced liver cirrhosis in rats. Methods. Male Sprague-Dawley rats were intraperitoneally injected with 200 mg/kg TAA 3 times/week and daily oral administration of 250 mg/kg, 500 mg/kg of BR extract, and 50 mg/kg of the reference drug Silymarin for 8 weeks. At the end of the experiment, Masson's trichrome staining was used to measure the degree of liver fibrosis. Hepatic antioxidant enzymes (CAT and GPx), nitrotyrosine, cytochrome (P450 2E1), matrix metalloproteinase (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-1), and urinary 8-hydroxyguanosine were measured. Serum levels of transforming growth factor TGF-β1, nuclear transcription factor NF-κB, proinflammatory cytokine IL-6, and caspase-3 were evaluated. Serum protein expression and immunohistochemistry of proapoptotic Bax and antiapoptotic Bcl-2 proteins were measured and confirmed by immunohistochemistry of Bax, Bcl-2, and proliferating cell nuclear antigen (PCNA). Results. BR treatment improved liver histopathology, immunohistochemistry, and biochemistry, triggered apoptosis, and inhibited cytokines, extracellular matrix proteins, and hepatocytes proliferation. Conclusion. Liver cirrhosis progression can be inhibited by the antioxidant and anti-inflammatory activities of BR ethanolic extract while preserving the normal liver status. ==== Body 1. Introduction Pharmaceutical drugs that depend on compounds such as colchicine and interferons are currently available for the treatment of liver cirrhosis but with either unreliable efficacies or high situations of side effects [1]. A number of natural compounds produced from vegetation offer alternative healthcare options that are more effective and safe [2]. Ingredients from recently found or already known plant varieties are regularly being examined on experimental animals [3]. The prospective tasks and efficiency of plant extracts in liver diseases are yet to be analyzed. The ethanolic extract of Curcuma longa rhizomes extract was recently proved to exhibit hepatoprotective efficacy against TAA intoxication in rats [4]. A large number of medicinal plants with hepatoprotective activity have been reported by several researchers [5, 6]. Boesenbergia rotunda (BR) rhizomes is one that is awaiting more analysis of its part in the pathology of the liver. Boesenbergia rotunda is one of the species that belongs to family Zingiberaceae and is traditionally used in folk medicine in Southeast Asia and commonly known as temu kunci. Medical research in the past used different solvents in preparing extracts from BR such as methanol, chloroform, and hexane. The methanol extract of BR was found to contain Quercetin and Kaempferol, which are well-known antioxidant and anti-inflammatory [7]. Chloroform or hexane extract of BR was found to contain other important flavanones and chalcones which are highly antioxidant compounds [8]. Furthermore, previous studies have revealed various bioactivities of BR rhizome extract such as anticancer [9], antibacterial [10], wound healing activity [11], and antiulcerogenic [12]. Our previous study examined the effectiveness of the ethanol-based extract of BR as a preventive therapy on a rat model of liver cirrhosis induced by thioacetamide, and our results revealed that BR extract exhibited significant hepatoprotective activity [13]. As an extension of our initiatives, in this study, we studied the mechanism of the protective effect of BR ethanol extract in TAA intoxication. Silymarin was also used in our study as a well-known hepatoprotectant reference drug extracted from the seeds of Silybum marinum plant [14]. 2. Materials and Methods 2.1. Animals Thirty male Sprague-Dawley rats (180–250 g) were used in the study. The rats were held in cages with wire bottoms at 25 ± 2°C, given tap water and conventional pellet, and revealed to a 12 hours of light-dark cycle at 50–60% moisture in well-prepared animal house. Throughout the test, all animals obtained individual proper care according to the requirements defined in the “Guide for the Care and Use of Laboratory Animals” readified by the Nationwide Academia of Sciences and released by the National Institution of Health. The research was accepted by the Committee Panel for Animal Analysis, Faculty of Medicine, University of Malaya, Malaysia PM/28/08/2009/MAA. 2.2. Experiment Animal groups were divided into 5 categories of 6 rats each (Table 1). TAA was ready by complete dissolving of TAA crystals (Sigma-Aldrich, USA) in sterile distilled water [15]. Silymarin (International Clinical, USA) was used as a reference drug and prepared by complete dissolving in 10% Tween-20. At the end of the experimental period, the rats were sacrificed, and blood was collected as mentioned in our previous work [13]. Liver tissues were excised, cleaned with ice-cold normal saline, and blotted, and some tissues were prepared for histopathology and immunohistochemistry evaluation. Some other liver tissues were washed in 0.02 mol/L PBS (pH 7.0–7.2) to get rid of excess blood. One gram of each liver was sampled and homogenized (10% w/v) in 50 mM cold potassium phosphate buffer (pH 7.4) by using teflon homogenizer (Polytron, Heidolph RZR 1, Germany). The resulting tissue homogenate was allowed to centrifuge at 3500 rpm for 10 minutes at 4°C in a centrifuge (Heraeus, Germany). The collected supernatant was divided into aliquots and then kept at −80°C till being assayed. The protein content in the liver tissue homogenate collected from all animals was assayed by using bovine serum albumen (BSA) according to Lowry method [16]. Blood samples were collected from all rats into carefully labeled tubes containing activated gel and allowed to clot and centrifuge at 3000 rpm for 10 min at 4°C. The collected serum samples were divided into aliquots and then kept at −80°C. Clean urine samples from all animals were collected 24 hours before sacrifice and kept at −80°C before assaying for levels of 8-hydroxy-deoxyguanosine. 2.3. Liver Tissue Content of CYP2E1 The cytochrome enzyme (CYP2E1) plays a crucial role in the metabolism of TAA in the liver microsome [17]. Aliquots of the tissue homogenate from all rats were tested for the level of CYP2E1 enzyme using microtiter plate precoated with monoclonal antibody specific to rat CYP2E1 and following the instructions of Uscn Life Science sandwich enzyme immunoassay (E90988Ra, China). 2.4. Evaluation of Urine 8-OH-dG and Hepatic Nitrotyrosine The free radicals generated from biotransformation of TAA in the liver result in oxidative damage via their covalent binding to the macromolecules of hepatocytes including DNA, protein, and lipid molecules causing necrosis of hepatocytes [18]. In our previous study, the level of malondialdehyde has been evaluated in the liver tissue homogenate [13]. In this study, the hepatic level of nitrotyrosine was measured as a marker for protein oxidation [19] utilizing a multiclonal anti-nitrotyrosine antibody and nitrotyrosine-horseradish conjugate as per the company's manuals (MyBiosource MBS722419, USA). Urine level of 8-hydroxy-deoxyguanosine (8-OH-dG) was evaluated as a marker for DNA oxidation [20] using monoclonal antibody specific to 8-OH-dG and following the kit instructions (Genox KOG-HS10E, USA). The principle of the assay is based on a competitive enzyme-linked immunoabsorbent method for measuring the quantity of 8-hydroxy-2′-deoxyguanosine adduct resulting from DNA oxidation. 2.5. Evaluation of Antioxidant Enzymes (CAT and GPx) Endogenous antioxidants, SOD, CAT, and GPx signify the first array of protection against the harms of free radicals and are essential for avoiding or at least reducing the occurrence and development of diseases [21]. In our previous experiment, we evaluated the level of superoxide dismutase (SOD) in the liver tissue homogenate of rats. In this study, the levels of catalase (CAT) and glutathione peroxidase (GPx) enzymes were assayed in the liver tissue homogenate collected from all animals following the instructions of Cayman kits (Sigma Cat no. 707002 and no. 703102, resp.). In brief, CAT activity was evaluated by Purpald Chromagen measuring the formaldehyde produced by the reaction of CAT enzyme with methanol in the presence of H2O2. The principle of GPx assay depends on the indirect measure of GPx activity by a coupled reaction. GPx reduces hydroperoxide into the oxidized form glutathione (GSSG) which is recycled by NADPH into its reduced form glutathione reductase (GR). All assays were performed in triplicate. 2.6. Assessment of Cytokines and Chemokines Sera aliquots collected from all rats were assayed for transforming growth factor-beta (TGF-β1) as a fibrogenesis-driving cytokine [22] using an ELISA microtiter plate precoated with rat TGF-β1 specific-specific polyclonal antibodies as per the manufacturers' instructions (Abnova, KA0416, USA). The nuclear transcription factor NF-κB and the proinflammatory cytokine interleukin IL-6 as important signals in liver injury [23] were assayed by enzyme-linked immunosorbent assay using ELISA microtiter plates precoated with antibodies specific to NF-κB or IL-6 according to the instructions of the manufacturer (Uscn Life Science E91824Ra, China, for NF-κB and E90079Ra for IL-6). 2.7. Assessment of Caspase-3, Proapoptotic Bax, and Antiapoptotic Bcl-2 Based on many studies, it was found that the triad Bax, Bcl-2, and caspase-3 are involved in the apoptosis of hepatocytes during liver injury [24]. To demonstrate the effect of BR rhizome extract on the apoptosis of liver cells, rat Bax ELISA kit (Uscn Life Science E91824Ra, China), rat Bcl-2 ELISA kit (Uscn Life Science E90778Ra, China), and rat caspase-3 ELISA kit (Uscn Life Science E90626Ra, China) were applied utilizing 96-well plates precoated with monoclonal antibody specific to Bax, Bcl-2, or caspase-3 for the evaluation of the levels of Bax, Bcl-2, and caspase-3 respectively, in the sera aliquots collected from all rats. 2.8. Evaluation of Matrix Metalloproteinase Enzymes (MMP-2 and MMP-9) and TIMP-1 Matrix metalloproteinase enzymes (MMP-2 and MMP-9) and their partial regulator tissue inhibitor of metalloproteinase (TIMP-1) play a role in liver damage [25]. For this purpose, tissue homogenates were assayed for the level of these markers by monoclonal antibodies specific to the tested antigen following the kits' manuals (Uscn Life Science E90100Ra, E90553Ra, and E90552Ra, China). 2.9. Histopathological Analysis Five μm thick sections of liver samples were prepared for Masson's trichrome (Sigma, USA) staining as a marker for detecting the degree of fibrosis [26] and observing the collagen fibers developed in liver tissues [27]. Examination of the slides was performed under a light microscope, and digital images were captured using a Nikon microscope (Y-THS, Japan) (magnification ×20). 2.10. Immunohistochemistry Using poly-L-lysine-coated slides, liver sections were prepared and heated in an oven (Venticell, MMM, Einrichtungen, Germany) for 25 minutes at 60°C. After heating, liver sections were deparaffinized in xylene and rehydrated in graded alcohol. Sodium citrate buffer of concentration 10 mM was heated till boiling in a microwave for antigen retrieval. Immunohistochemistry staining was applied following the manual of the company (DakoCytomation, USA). Succinctly, 0.03% hydrogen peroxide sodium azide was used to block the endogenous peroxidase for 5 min followed by washing the tissue sections carefully using wash buffer and then incubated with Bcl-2-associated X protein (Bax) (1 : 500), proliferating cell nuclear antigen (PCNA) (1 : 200), and antiapoptotic protein Bcl-2 (1 : 50) (Santa Cruz Biotechnology Inc., California, USA) biotinylated primary antibodies for 15 minutes. After incubation, tissue sections were carefully rewashed with washing buffer and conserved in the buffer bath. After adding streptavidin-HRP, sections were kept for 15 minutes incubated and then washed. Diaminobenzidine substrate chromagen was applied to the sections and reincubated for over 8 min followed by careful washes and hematoxylin counterstaining for 5 seconds. Weak ammonia (0.037 mol/L) was used for dipping the sections 10 times and then washed and cover slipped. Light microscopy was used to examine the brown-stained positive antigens. 2.11. Statistical Analysis Statistical analysis of the results was performed using one-way ANOVA followed by Tukey post hoc test analysis using SPSS (Version 18, SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was considered statistically significant between the measurements of the two compared groups. All values were reported as mean ± SEM. 3. Results 3.1. Hepatic Level of CYP2E1 The result of the effect of BR extract on the hepatic cytochrome enzyme CYP2E1 is shown in Figure 1. Animals from cirrhosis group had significantly (P < 0.01) higher levels (2.85 ± 0.12 ng/mL) of CYP2E1 compared with normal group (0.96 ± 0.36 ng/mL) and reference group (1.14 ± 0.05 ng/mL). Low dose and high dose BR-treated rats showed significantly (P < 0.01) lower levels (1.37 ± 0.15 and 1.10 ± 0.09 ng/mL, resp.) when compared with cirrhosis group. 3.2. Level of Oxidative Stress Oxidative stress markers (hepatic nitrotyrosine and urinary 8-OH-dG) are shown in Table 2. Generally, cirrhosis rats had significantly higher levels of oxidative stress parameters (P < 0.001) than normal rats and the treated groups. Remarkably, low dose and high dose-BR treated rats showed significantly lower levels (P < 0.001) of liver nitrotyrosine and urinary 8-OH-dG compared to cirrhosis group. Moreover, no significant differences were observed in the tested oxidative stress parameters between the animals treated with the plant extract and those treated with silymarin. These results suggest that BR treatment may protect liver tissues from progressive damage during liver injury. 3.3. Hepatocellular Endogenous Enzymes Hepatocytes loss in the cirrhotic livers of rats was analyzed in another way by the activity of antioxidant enzymes CAT and GPx and the results are shown in Table 3. CAT and GPx, results were similar to that of the oxidative stress parameters but in the opposite manner, so the values of CAT and GPx in the rats of the cirrhosis group were lower than that in normal rats. These results revealed the occurrence of extensively injured hepatocytes of cirrhotic livers. Daily oral administration of low and high dose BR extract significantly (P < 0.01) increased the levels of CAT and GPx and induced the survival of hepatocytes. These results conjointly with our previous results of SOD [13] support the approach that treatment with BR extract could provide a healthy status for protecting the hepatic cells from advanced damage. 3.4. Cytokines and Chemokines Assessment The serum levels of TGF-β1, NF-κB, and IL-6 from the samples collected from all sacrificed rats are shown in Figure 2. Results showed that the serum levels of TGF-β1, NF-κB, and IL-6 were significantly elevated (P < 0.05) in the samples from cirrhosis group (100.00 ± 10.00 pg/mL, 2.86 ± 0.06 ng/mL and 288.58 ± 11.15 pg/mL, resp.) compared to the other animal groups. Administration of BR extract to animals attenuated the levels of the fibrogenesis agent TGF-β1 to 81.67 ± 1.67 pg/mL and the inflammatory mediators NF-κB and IL-6 to 1.93 ± 0.05 ng/mL and 148.12 ± 15.61 pg/mL, respectively, in the low dose BR-treated rats and 58.33 ± 4.41 pg/mL, 1.43 ± 0.07 ng/mL and 129.50 ± 3.79 pg/mL, respectively, in the high dose BR-treated rats. Levels of TGF-β1, NF-κB, and IL-6 from the high dose BR-treated group approached the values obtained from the reference group Silymarin (63.33 ± 1.67 pg/mL, 1.49 ± 0.06 ng/mL, and 125.35 ± 5.06 pg/mL, resp.) compared to the higher value in the low dose BR-treated group. Results suggest that BR extract has dose-dependent nature in its inhibitory effect to the profibrogenic and proinflammatory cytokines and NF-κB chemokine. 3.5. Serum Level of Caspase-3, Bax, and Bcl-2 The levels of the proapoptotic protein Bax and the antiapoptotic protein Bcl-2 in the rat sera are shown in Figure 3(a), and the ratio of Bax/Bcl-2 is shown in Figure 3(b). Bax results showed no significance between cirrhosis and normal group rats (1.61 ± 0.15 and 1.07 ± 0.04 ng/mL resp.). On the other hand, there was significant increase (P < 0.05) in the level of Bax from reference and high dose BR-treated groups (4.95 ± 0.11 and 4.68 ± 0.19 ng/mL, resp.) compared to cirrhosis group. On the contrary, the level of antiapoptotic protein Bcl-2 showed significant increase (P < 0.05) in the cirrhosis group compared to normal group (2.57 ± 0.23 and 0.89 ± 0.09 ng/mL, resp.), whereas no significance observed between any of the treated groups when compared with cirrhosis group indicating enhanced apoptosis in silymarin and BR-treated groups as confirmed by the ratio Bax/Bcl-2 in Figure 3(b). Furthermore, the serum level of caspase-3 in the cirrhosis group was not significantly higher (2.11 ± 0.48 ng/mL) compared to normal group (1.43 ± 0.33 ng/mL) as illustrated in Figure 3(c). Treating the cirrhotic livers with silymarin or high dose BR elevated the caspase-3 value to reach 5.54 ± 0.27 and 5.83 ± 0.19 ng/mL, respectively. The similarity observed in the results of Bax and caspase-3 confirms the enhancement of apoptosis by BR extract in the same manner as the reference drug, silymarin. Figure 3(d) shows the relation between Bax, Bcl-2, and caspase-3 in all the experimental groups. Results showed parallel increase in the serum level of Bax and caspase-3 in silymarin and BR groups, whereas Bcl-2 level showed reversed serum level suggesting increased apoptosis in the treated groups compared to cirrhosis control group. 3.6. Hepatic TIMP-1, MMP-9, and MMP-2 The results of the effect of BR extract treatment on the liver tissue homogenate level of MMP-2, MMP-9, and TIMP-1 collected from all experimental animals are illustrated in Table 4. From the results, the level of the tested enzymes were significantly high (P < 0.05) in the cirrhosis group rats compared to all other groups. On the other hand, administration of BR extract to the animals significantly (P < 0.05) attenuated the enzymatic levels of MMP-2, MMP-9, and TIMP-1 to approach the values of the reference control group with the exception of the low dose BR-treated group which did not show significance to cirrhosis group indicating the efficacy of the plant extract treatment in a dose-dependent manner. Results in Table 4 showed parallel decrease in the hepatic level of MMP-2, MMP-9, and TIMP-1 from BR-treated groups. 3.7. Masson's Trichrome Staining The degree of fibrosis determined by Masson's trichrome staining of the liver sections from all the treated groups is shown in Figure 4. Liver sections from normal rats appeared normal without signs of collagen deposition. Liver sections from the cirrhosis rats of cirrhosis group revealed increased deposition of collagen fibers around the congested central vein indicating severe fibrosis. Liver tissues from the reference group showed minimal collagen deposition indicating minimal fibrosis. Livers from rats treated with low dose BR extract showed moderate deposition of collagen fibers and moderate congestion around the central vein, while those from rats treated with high dose BR extract showed mild collagen deposition and mild congestion around the central vein. This measurement of the degree of fibrosis confirms the previous findings that treatment with BR extract might protect the animals' liver from development of fibrosis. 3.8. Immunohistochemistry Bax, Bcl-2, and PCNA staining of liver cells from all animal groups are shown in Figures 5(a), 5(b), and 6. Hepatocytes of liver tissues from cirrhosis group rats showed downregulation of Bax staining with upregulation of Bcl-2-positive hepatocytes and more PCNA staining indicating severe necrosis with high cell proliferation to repair the damaged hepatocytes. Hepatocytes from the reference group (Silymarin-treated rats) showed upregulated Bax expression, downregulated Bcl-2 expression, and few PCNA staining indicating lower levels of proliferation of necrotized hepatocytes proliferation than apoptosis. Liver tissues treated with low dose and high dose BR extract induced hepatocyte apoptosis as indicated by the upregulated Bax expression, downregulated Bcl-2, and downregulated proliferation of necrotic hepatocytes as indicated by reduced PCNA staining. These findings support the idea of BR extract-induced hepatoprotective activities against progressive liver damage by increasing apoptosis of damaged hepatocytes and ameliorating their proliferation. 4. Discussion Liver cirrhosis has become a serious health problem because of the wider use of prescribed medications with adverse reactions in modern life of today or the drug misuse. Consequently, the current research has targeted on finding new therapeutic alternatives and analyzing their mechanism to get rid of the signaling routes and reduce the loss induced on the liver [28]. Beyond the techniques with artificial pharmacology, the search also chases alternative techniques that depend on natural products. In particular, it targets those plants with known medical history or confirmed prospective of positive results against the illnesses of the liver or other body parts [29]. To aid these initiatives, in this study, we analyzed the mechanism of ethanol extract of BR rhizomes as a promising therapy for treating liver cirrhosis. Thioacetamide is biotransformed by CYP2E1 enzymes located in the microsomes of liver cells and convert it to a highly reactive toxic intermediate known as thioacetamide sulphur dioxide through oxidation [30], inducing hepatotoxicity in experimental animals and different grades of liver damage including nodular cirrhosis, production of pseudolobules, proliferation of hepatic cells, and necrosis of parenchyma cells [31]. All experimental animals in the present study were tested for CYP2E1 enzyme level in liver homogenate tissues (Figure 1). Efficacy of BR extract in comparison with silymarin control was able to inhibit metabolism of TAA, which might be one of the significant factors in hepatoprotective activity by blocking the release of toxic metabolites and ROS responsible for inducing damage of hepatocytes [32]. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) production increase during liver injury by transformation of xenobiotics such as CCl4, ethanol, and acetaminophen in the liver and play a crucial role in the death of hepatocytes [33]. TAA induces hepatocyte damage via its metabolite, TASO2, which damages the macromolecules of hepatocytes causing damage of DNA molecules, oxidation of protein molecules, and peroxidation of the cell membrane biomolecules [34, 35]. This study evaluated the oxidative stress markers and observed the degree of liver cells damage. The levels of urine 8-OH-dG and nitrotyrosine (Table 2) were high in the cirrhosis group compared to the treated groups. This supported the crude extract antioxidants mechanism in downregulating ROS, inhibiting DNA damage, and attenuating protein and lipid oxidation [36]. Furthermore, the antioxidant constituents of BR extract helped in boosting the hepatocellular endogenous enzymes (CAT and GPx) as shown in Table 3. Toxins target metabolically effective hepatic cells causing dysfunction of hepatocytes and the discharge of inflammation-related cytokines as NF-κB and IL-6 and fibrogenic mediators as TGF-β1. Many researchers have recommended that part of liver injury brought on by TAA is mediated through oxidative stress and the effect of cytokines via fat peroxidation [37, 38]. The free radicals released as the consequences of TAA metabolism in liver cells activate myofibroblasts that secrete fibrinogen and growth factors [39]. The present study revealed that BR extract treatment attenuated level of the prominent profibrogenic cytokine TGF-β1 (Figure 2) indicating the inhibitory activity of BR extract to the proliferative activity of HSCs which might be confirmed by the less collagen deposition in the liver tissues of animals treated with BR extract (Figure 4). Similarly, the high levels of proinflammatory cytokines IL-6 and NF-κB (Figure 2) observed in the cirrhosis group rats were reversed in the rats treated with BR extract indicating the anti-inflammatory activity of BR extract. A number of studies have focused on the molecular regulation of apoptosis. One of the characteristic features of liver diseases is the death of hepatocytes as a response to drug/toxicant-induced liver injury. Cytotoxic drugs and cellular stress activate the intrinsic mitochondrial apoptotic pathway [40] so that the inhibitors of Bcl-2 activate caspase-3 initiating apoptosis, whereas inducers of Bcl-2 induce necrosis of cells [41]. In vivo, necrotic death is often associated with extensive damage in the tissue resulting in necro-inflammatory response [42]. Although toxicity induced by TAA was reported to cause upregulation of Bax protein and downregulation of the antiapoptotic protein Bcl-2 and its translocation into the mitochondria, causing apoptosis [43] but other studies suggested that the ROS produced from thioacetamide biotransformation causes centrolobular necrosis [44]. Some Plant extracts such as Curcuma longa extract [4] and some antioxidants such as α-lipoic acid [45], curcumin, and curcuminoids [46] were proved to induce apoptosis. In the current study, we observed significant increase in the serum level of Bax protein and caspase-3 and decrease in Bcl-2 protein in the BR-treated and silymarin-treated animals compared to cirrhosis group animals as shown in the relation between Bax, Bcl-2, and caspase-3 in Figure 3(d). This was confirmed by the ratio Bax/Bcl-2 (Figure 3(b)) which was high in the treated groups compared to cirrhosis group and the immunohistochemistry staining of Bax and Bcl-2 (Figure 5). The ethanolic extract of BR rhizomes might contain active compounds such as the chalcone Boesenbergin A [47] and the sesquiterpene zerumbone [48] which were reported recently to induce apoptosis of liver cancer cells HepG2 through the release of cytochrome C and the formation of caspase-3 modifying the necrotic effect due to TAA intoxication to apoptosis. This modification in vivo would scale down the release of inflammatory mediators that would prevent progressive liver damage. Furthermore, feeding of rats daily with BR extract along with TAA injections 3 times/week for 8 weeks inhibited proliferation of hepatocytes as indicated by the significant reduction of PCNA staining in the liver sections from the plant extracts-treated groups similar to that in the Silymarin-treated group (Figure 6) [49]. Downregulation of cell proliferation in BR-treated rats may be attributed to the reduced damage in the BR-treated livers compared to the higher damage of hepatocytes and the upregulation of PCNA in the cirrhosis animals to regenerate the necrotic effect of TAA to hepatocytes. In liver fibrosis, there is an imbalance between excess deposition and/or a decrease in the extracellular matrix (ECM) removal with consequent scarring damage [50]. ECM is mainly controlled by matrix metalloproteinases (MMPs), which are a group of proteolytic enzymes that are able to degrade the ECM [51, 52]. MMP-9 and TIMP-1 were verified as the molecular signatures during the progress of liver cirrhosis induced by TAA [53]. Park et al. observed that TAA increased MMP-2 expression in the liver tissues of rats and reported that the balance of MMPs and TIMPs is the key factor of liver fibrogenesis [50]. In this research, administered animals with plants extract showed significant downregulation in the hepatic level of TIMP-1, MMP-9 and MMP-2 similar to silymarin reference group (Table 4). Results suggest that the efficacy of BR extract might be due to the inhibition of HSCs and kupffer cells activity and their secretion to MMP-2 and MMP-9, respectively. In addition, reduced activity of HSCs as indicated by the decreased level of collagen deposition in the BR-treated liver tissues has led to down-regulation of TIMP-1 [54]. Furthermore, the reduced activity of kupffer cells was indicated by the significant inhibitory effect of BR extract to the mediators TGF-β1, IL-6, and NF-κB levels in rat serum. Consequently, the low level of these mediators might have contributed to HSCs activation and reduced liver injury [19]. Figure 7 summarizes the effect of BR treatment on the liver damage induced by TAA intoxication. Due to the crucial role played by HSCs in liver fibrosis via their resistance to apoptosis, recent treatment strategies of liver diseases are to inhibit their proliferation or induce their apoptosis. The biochemical findings of our study were confirmed by the histopathological examinations of rat liver tissues (Figure 4) showing that livers from BR-treated rats had nearly normal liver architecture with significant reduction in collagen synthesis. This was probably due to the inhibitory effect of the plant extract on hepatic stellate cell activation. Inhibition of HSC activation and downregulation of collagen-I in the livers treated with BR extract might be due to the activity of polyphenol constituents in BR extract [55]. Conflict of Interests The authors declare that they have no financial conflict of interests. Acknowledgments This study was financially supported by the University of Malaya through University Malaya Research Grant PV042-2011A and HIR Grant (F000009-21001). The authors are thankful to the staffs of Department of Molecular Medicine and Clinical Diagnostic Laboratory of University Malaya. Figure 1 Effect of B. rotunda (BR) extract on hepatic levels of cytochrome enzyme CYP2E1 in rats at the end of the experiment. Data were expressed as mean ± SEM. *P < 0.01 compared with the normal group. P < 0.01 compared with cirrhosis group. Figure 2 Effect of BR extract on the serum level of TGF-β1, NF-κB, and IL-6 in rats at the end of the experiment. (a) TGF-β1, (b) NF-κB, and (c) IL-6. Data were expressed as mean ± SEM. Means among groups (n = 6 rats/group) show significant difference. *P < 0.001 compared to the normal group. P < 0.001 compared to cirrhosis group. # P < 0.01 compared to cirrhosis group. Figure 3 Effect of BR extract on the serum level of (a) Bax and Bcl-2, (b) Bax/Bcl-2 ratio, (c) caspase-3, and (d) relation between Bax, Bcl-2, and caspase-3 at the end of the experiment. Data were expressed as mean ± SEM. P < 0.05 compared with the cirrhosis group. P < 0.05 compared to the normal group. Figure 4 Masson's trichrome staining of representative livers sampled from rats in different experimental groups. (a) Normal liver did not show signs of collagen deposition in livers from a normal rat. (b) Severe collagen deposition (arrow) and severe fibrosis were seen in the livers from a cirrhosis rat. (c) Minor collagen deposition in the liver of a hepatoprotected rat treated with Silymarin. (d) Moderated collagen deposition and moderate congestion around the central vein in the liver of rats treated with low dose BR extract. (e) Mild collagen deposition was observed in the livers of rats treated with high dose BR extract (original magnification ×20). Figure 5 Immunohistochemistry staining of (a) Bax and (b) Bcl-2 of representative livers sampled from rats in different experimental groups. Less apoptosis indicated by (ia) few Bax-positive hepatocytes (white arrow) and (ib) more Bcl-2-positive hepatocytes (black arrow) in liver tissues from a cirrhosis rat group. (iia) High numbers of Bax-positive hepatocytes (White arrow) and (iib) very less Bcl-2-positive hepatocytes (black arrow) in the liver from a hepatoprotected rat treated with Silymarin. Moderate apoptosis as indicated by (iiia) moderate Bax staining (white arrow) and (iiib) moderate Bcl-2 staining (black arrow) indicating moderate apoptosis in the liver from the rats treated with low dose BR extract. (iva) High numbers of Bax-positive cells (white arrow) with severe apoptosis and (ivb) very less Bcl-2-positive hepatocytes (black arrow) were observed in the liver of the rats treated with high dose BR extract (original magnification ×40). Figure 6 Immunohistochemistry staining of PCNA of livers sampled from rats in different experimental groups. (a) Normal livers did not show signs of PCNA expression in hepatocytes from control rats. (b) Cirrhosis control liver showed severe fibrosis with greater PCNA expression in the necrotized hepatocytes. (c) Silymarin-treated liver showed less PCNA-stained hepatocytes (arrow) indicating less hepatocyte proliferation. (d) The low dose BR-treated liver showed moderate hepatocyte proliferation as indicated by moderate PCNA staining (arrow) in the hepatocytes. (e) High dose BR-treated livers showed minor PCNA expression (arrow) with few proliferated necrotized hepatocytes which were observed in the liver (original magnification ×40). Figure 7 Possible mechanism of hepatoprotective effect of Boesenbergia rotunda on TAA-induced liver damage in rats. Table 1 Experimental design. Group Number of animals Intraperitoneal injection (three times/week) Oral administration (5 mL/kg) Durations (weeks) Normal control 6 Sterile distilled water (1 mL/kg) 10% Tween-20 8 Cirrhosis control 6 TAA 10% Tween-20 8 Reference control 6 TAA Silymarin 50 mg/kg 8 Low dose B. rotunda 6 TAA B. rotunda 250 mg/Kg 8 High dose B. rotunda 6 TAA B. rotunda 500 mg/kg 8 Table 2 Effect of BR extract on urine OH-dG and liver tissue homogenate level of nitrotyrosine from the rats at the end of the experiment. Group 8-OH-dG ng/mL Nitrotyrosine (ng/mL) Normal group 2.17 ± 0.33 1.06 ± 0.07 Cirrhosis group 5.40 ± 0.34* 3.87 ± 0.13** Reference group 2.80 ± 0.15* 1.67 ± 0.07* Low dose BR 2.83 ± 0.33* 1.40 ± 0.20* High dose BR 2.37 ± 0.88* 1.33 ± 0.13* 8-OH-DG; 8-hydroxy-deoxyguanosine. Data are expressed as mean ± SEM. Means among groups (n = 6 rats/group) show significant difference. P < 0.001 compared with cirrhosis group. P < 0.001 compared with normal group. Table 3 Effect of BR extract on the liver tissue homogenate level of CAT and GPx from the rats at the end of the experiment. Group CAT (nmoL/min/mg protein) GPx (nmoL/min/mg protein) Normal control 54.00 ± 0.36 860.37 ± 19.32 Cirrhosis control 27.49 ± 1.67* 451.00 ± 89.68## Reference control 52.76 ± 3.29* 926.11 ± 36.42* Low dose BR 41.34 ± 2.74# 902.59 ± 69.09# High dose BR 51.42 ± 1.34* 975.05 ± 54.08* CAT: catalase, GPx: glutathione peroxidase. Data are expressed as mean ± SEM. Means among groups (n = 6 rats/group) show significant difference. P < 0.001 compared with cirrhosis group. P < 0.001 compared with normal group. # P < 0.01 compared with cirrhosis group. ## P < 0.01 compared with normal group. Table 4 Effect of BR extract on the liver tissue homogenate level of MMP-2, MMP-9, and TIMP-1 from the rats at the end of the experiment. Group MMP-2 (ng/mL) MMP-9 (ng/mL) TIMP-1 (ng/mL) Normal control 2.65 ± 1.07 9.01 ± 2.34 1.42 ± 0.55 Cirrhosis control 8.43 ± 0.86* 45.61 ± 1.88 6.09 ± 1.35 Reference control 3.34 ± 0.13* 16.72 ± 2.43* 1.77 ± 0.06* Low dose B. rotunda 5.86 ± 0.26 30.21 ± 4.43* 2.06 ± 0.19* High dose B. rotunda 3.86 ± 0.69* 14.80 ± 2.30* 1.82 ± 0.05* MMP-2: matrix metalloproteinase-2, MMP-9: matrix metalloproteinase-9, TIMP-1: metalloproteinase inhibitor-1. Data are expressed as mean ± SEM. Means among groups (n = 6 rats/group) show significant difference. P < 0.05 compared to cirrhosis group. *P < 0.05 compared with normal group. ==== Refs 1 Raison CL Demetrashvili M Capuron L Miller AH Neuropsychiatric adverse effects of interferon-α : recognition and management CNS Drugs 2005 19 2 105 123 2-s2.0-14644393659 15697325 2 Yang H Sung SH Kim YC Two new hepatoprotective stilbene glycosides from Acer mono leaves Journal of Natural Products 2005 68 1 101 103 2-s2.0-12944263651 15679328 3 Wang N Li P Wang Y Hepatoprotective effect of Hypericum japonicum extract and its fractions Journal of Ethnopharmacology 2008 116 1 1 6 2-s2.0-38649108149 18178045 4 Salama SM Abdulla MA AlRashdi AS Ismael S Alkiyumi SS Hepatoprotective effect of ethanolic extract of Curcuma longa on thioacetamide induced liver cirrhosis in rats BMC Complementary and Alternative Medicine 2013 13, articel 56 5 Alshawsh MA Abdulla MA Ismail S Amin ZA Hepatoprotective effects of Orthosiphon stamineus extract on thioacetamide-induced liver cirrhosis in rats Evidence-based Complementary and Alternative Medicine 2011 2011 2-s2.0-79959241786 103039 6 Amin ZA Bilgen M Alshawsh MA Ali HM Hadi AHA Protective role of Phyllanthus niruri extract against thioacetamide-induced liver cirrhosis in rat model Evidence-Based Complementary and Alternative Medicine 2012 2012 9 pages 241583 7 Rho HS Ghimeray AK Yoo DS Kaempferol and kaempferol rhamnosides with depigmenting and anti-inflammatory properties Molecules 2011 16 4 3338 3344 2-s2.0-79955537045 21512441 8 Ching AYL Tang SW Sukari MA Lian GEC Rahmani M Characterization of flavonoid derivatives from Boesenbergia rotunda (L.) The Malaysian Journal of Analytical Sciences 2007 11 154 159 9 Shindo K Kato M Kinoshita A Kobayashi A Koike Y Analysis of antioxidant activities contained in the Boesenbergia pandurata Schult. rhizome Bioscience, Biotechnology and Biochemistry 2006 70 9 2281 2284 2-s2.0-33749035986 10 Voravuthikunchai SP Phongpaichit S Subhadhirasakul S Evaluation of antibacterial activities of medicinal plants widely used among AIDS patients in Thailand Pharmaceutical Biology 2005 43 8 701 706 2-s2.0-30344481917 11 Mahmood A Mariod AA Abdelwahab SI Ismail S Al-Bayaty F Potential activity of ethanolic extract of Boesenbergia rotunda (L.) rhizomes extract in accelerating wound healing in rats Journal of Medicinal Plants Research 2010 4 1570 1576 12 Abdelwahab SI Mohan S Abdulla MA The methanolic extract of Boesenbergia rotunda (L.) Mansf. and its major compound pinostrobin induces anti-ulcerogenic property in vivo : possible involvement of indirect antioxidant action Journal of Ethnopharmacology 2011 137 2 963 970 2-s2.0-80052036098 21771650 13 Salama SM Bilgen M Al Rashdi AS Abdulla MA Efficacy of Boesenbergia rotunda treatment against thioacetamide-induced liver cirrhosis in a rat model Evidence-Based Complementary and Alternative Medicine 2012 2012 12 pages 137083 14 Pradhan SC Girish C Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine Indian Journal of Medical Research 2006 124 491 504 2-s2.0-33846519345 17213517 15 Aydın AF Küskü-Kiraz Z Doğru-Abbasoğlu S Güllüoğlu M Uysal M Effect of carnosine against thioacetamide-induced liver cirrhosis in rat Peptides 2010 31 67 71 19958806 16 Lowry OH Rosebrough NJ Farr AL Randall RJ Protein measurement with the Folin phenol reagent The Journal of Biological Chemistry 1951 193 1 265 275 2-s2.0-71849104860 14907713 17 Amali AA Rekha RD Lin CJ-F Thioacetamide induced liver damage in zebrafish embryo as a disease model for steatohepatitis Journal of Biomedical Science 2006 13 2 225 232 2-s2.0-33645337369 16456712 18 Sindhu RK Koo J-R Sindhu KK Ehdaie A Farmand F Roberts CK Differential regulation of hepatic cytochrome P450 monooxygenases in streptozotocin-induced diabetic rats Free Radical Research 2006 40 9 921 928 2-s2.0-33749240977 17015271 19 Bruck R Ashkenazi M Weiss S Prevention of liver cirrhosis in rats by curcumin Liver International 2007 27 3 373 383 2-s2.0-33947277886 17355460 20 Wu LL Chiou C-C Chang P-Y Wu JT Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics Clinica Chimica Acta 2004 339 1-2 1 9 2-s2.0-0345735389 21 Pham-Huy LA He H Pham-Huy C Free radicals, antioxidants in disease and health International Journal of Biomedical Science 2008 4 2 89 96 2-s2.0-57849121240 23675073 22 Gressner OA Weiskirchen R Gressner AM Evolving concepts of liver fibrogenesis provide new diagnostic and therapeutic options Comparative Hepatology 2007 6, article 7 2-s2.0-34848830380 23 Mandrekar P Szabo G Signalling pathways in alcohol-induced liver inflammation Journal of Hepatology 2009 50 6 1258 1266 2-s2.0-67349255858 19398236 24 Elsharkawy AM Oakley F Mann DA The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis Apoptosis 2005 10 5 927 939 2-s2.0-24644488751 16151628 25 Okazaki I Ninomiya Y Kyuichi T Friedman SI Extracellular Matrix and the Liver: Approach To Gene Therapy 2003 Academic Press 26 Zeisberg M Yang C Martino M Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition Journal of Biological Chemistry 2007 282 32 23337 23347 2-s2.0-34548145815 17562716 27 Edgtton KL Gow RM Kelly DJ Carmeliet P Kitching AR Plasmin is not protective in experimental renal interstitial fibrosis Kidney International 2004 66 1 68 76 2-s2.0-3042720541 15200414 28 Daly AK Donaldson PT Bhatnagar P HLA-B5701 genotype is a major determinant of drug-induced liver injury due to flucloxacillin Nature Genetics 2009 41 7 816 819 2-s2.0-67649859295 19483685 29 Khanna D Sethi G Ahn KS Natural products as a gold mine for arthritis treatment Current Opinion in Pharmacology 2007 7 3 344 351 2-s2.0-34248334669 17475558 30 Kim K-H Bae J-H Cha S-W Han S-S Park KH Jeong TC Role of metabolic activation by cytochrome P450 in thioacetamide-induced suppression of antibody response in male BALB/c mice Toxicology Letters 2000 114 1-3 225 235 2-s2.0-0034599391 10713488 31 Sadasivan S Latha PG Sasikumar JM Rajashekaran S Shyamal S Shine VJ Hepatoprotective studies on Hedyotis corymbosa (L.) Lam Journal of Ethnopharmacology 2006 106 2 245 249 2-s2.0-33646791535 16495024 32 Karamanakos PN Trafalis DTP Geromichalos GD Inhibition of rat hepatic CYP2E1 by quinacrine: molecular modeling investigation and effects on 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mutagenicity Archives of Toxicology 2009 83 6 571 580 2-s2.0-67651172890 18754103 33 Muriel P Role of free radicals in liver diseases Hepatology International 2009 3 4 526 536 2-s2.0-73349136974 19941170 34 Chilakapati J Korrapati MC Hill RA Warbritton A Latendresse JR Mehendale HM Toxicokinetics and toxicity of thioacetamide sulfoxide: a metabolite of thioacetamide Toxicology 2007 230 2-3 105 116 2-s2.0-33846280472 17187915 35 Djordjević VB Free radicals in cell biology International Review of Cytology 2004 237 57 89 2-s2.0-4544345529 15380666 36 Mori A Yokoi I Noda Y Willmore LJ Natural antioxidants may prevent posttraumatic epilepsy: a proposal based on experimental animal studies Acta Medica Okayama 2004 58 3 111 118 2-s2.0-3242772244 15471432 37 Mehendale HM Tissue repair: an important determinant of final outcome of toxicant-induced injury Toxicologic Pathology 2005 33 1 41 51 2-s2.0-13444269608 15805055 38 Okuyama H Shimahara Y Nakamura H Araya S Kawada N Thioredoxin prevents thioacetamide-induced acute hepatitis Comparative Hepatology 2004 3 Supplement 1 p. S6 39 Bassiouny AR Zaky AZ Abdulmalek SA Kandeel KM Ismail A Moftah M Modulation of AP-endonuclease1 levels associated with hepatic cirrhosis in rat model treated with human umbilical cord blood mononuclear stem cells International Journal of Clinical and Experimental Pathology 2011 4 7 692 707 2-s2.0-84855558471 22076170 40 Kang MH Reynolds CP BcI-2 Inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy Clinical Cancer Research 2009 15 4 1126 1132 2-s2.0-63149129655 19228717 41 Emi M Kim R Tanabe K Uchida Y Toge T Targeted therapy against Bcl-2-related proteins in breast cancer cells Breast Cancer Research 2005 7 R940 R952 16280040 42 Choudhury JD Kumar S Mayank V Mehta J Bardalai D A review on apoptosis and its different pathway International Journal of Biological and Pharmaceutical Research 2012 3 848 861 43 Chen L-H Hsu C-Y Weng C-F Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells World Journal of Gastroenterology 2006 12 32 5175 5181 2-s2.0-33748501055 16937528 44 Sarkar MK Sil PC Hepatocytes are protected by herb Phyllanthus niruri protein isolate against thioacetamide toxicity Pathophysiology 2007 14 2 113 120 2-s2.0-35048894799 17913477 45 Moungjaroen J Nimmannit U Callery PS Reactive oxygen species mediate caspase activation and apoptosis induced by lipoic acid in human lung epithelial cancer cells through Bcl-2 down-regulation Journal of Pharmacology and Experimental Therapeutics 2006 319 3 1062 1069 2-s2.0-33751180574 16990509 46 Wang M-E Chen Y-C Chen I-S Hsieh S-C Chen S-S Chiu C-H Curcumin protects against thioacetamide-induced hepatic fibrosis by attenuating the inflammatory response and inducing apoptosis of damaged hepatocytes Journal of Nutritional Biochemistry 2012 23 1352 1366 2-s2.0-84855164829 22221674 47 Ng -B K Bustamam A Sukari MA Abdelwahab SI Mohan S Induction of selective cytotoxicity and apoptosis in human T4-lymphoblastoid cell line (CEMss) by boesenbergin a isolated from Boesenbergia rotunda rhizomes involves mitochondrial pathway, activation of caspase 3 and G2/M phase cell cycle arrest BMC Complementary and Alternative Medicine 2013 13, article 41 48 Muhammad Nadzri N Abdul AB Sukari MA Abdelwahab SI Eid EE Inclusion Complex of Zerumbone with Hydroxypropyl-beta-Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio Evidence-Based Complementary and Alternative Medicine 2013 2013 16 pages 810632 49 Sakr SA Shalaby SY Metiram-induced histological and histochemical alterations in Liver and kidney of pregnant mice Life Science 2012 9 1 50 Park SY Shin HW Lee KB Lee M-J Jang J-J Differential expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in thioacetamide-induced chronic liver injury Journal of Korean Medical Science 2010 25 4 570 576 2-s2.0-77953986092 20358000 51 Abraham D Ponticos M Nagase H Connective tissue remodelling: cross-talk between endothelins and matrix metalloproteinases Current Vascular Pharmacology 2005 3 4 369 379 2-s2.0-24644489710 16248781 52 Wang Y Zhang J-S Huang G-C Cheng Q Zhao Z-H Effects of adrenomedullin gene overexpression on biological behavior of hepatic stellate cells World Journal of Gastroenterology 2005 11 23 3549 3553 2-s2.0-21344457713 15962372 53 An JH Seong J Oh H Kim W Han KH Paik YH Protein expression profiles in a rat cirrhotic model induced by thioacetamide The Korean Journal of Hepatology 2006 12 1 93 102 2-s2.0-33745389088 16565610 54 Arthur MJP Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis The American Journal of Physiology 2000 279 2 G245 G249 2-s2.0-0033858063 55 Kim HK Yang T-H Cho H-Y Antifibrotic effects of green tea on in vitro and in vivo models of liver fibrosis World Journal of Gastroenterology 2009 15 41 5200 5205 2-s2.0-73449123006 19891020	23997791	PMC3749608	CC BY	2021-01-05 10:52:01	yes	Evid Based Complement Alternat Med. 2013 Aug 7; 2013:157456
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23990930PONE-D-13-1677610.1371/journal.pone.0071127Research ArticleBiologyAnatomy and PhysiologyReproductive SystemReproductive PhysiologyBiochemistryProteinsChaperone ProteinsComputational BiologyMolecular GeneticsGene ExpressionGeneticsGene ExpressionMolecular Cell BiologyGene ExpressionMedicineAnatomy and PhysiologyReproductive SystemReproductive PhysiologyDiagnostic MedicinePathologyGeneral PathologyMolecular PathologyObstetrics and GynecologyPregnancyWomen's HealthHeat Shock Protein 27 Is Spatially Distributed in the Human Placenta and Decreased during Labor HSP 27 Expression in the Placenta during LaborAbdulsid Akrem Fletcher Alexander Lyall Fiona * University of Glasgow School of Medicine, Institute of Medical Genetics, Yorkhill Hospital, Glasgow, United Kingdom Zakar Tamas Editor John Hunter Hospital, Australia * E-mail: fiona.lyall@glasgow.ac.ukCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: FL AA. Performed the experiments: AA AF. Analyzed the data: FL AA. Contributed reagents/materials/analysis tools: FL. Wrote the paper: FL AA. Helped design PCR methods: AF. 2013 22 8 2013 8 8 e7112725 4 2013 1 7 2013 © 2013 Abdulsid et al2013Abdulsid et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Placental oxidative stress is a feature of human labor. Heat shock proteins (HSPs) play a key role in cellular stress. We hypothesized that placental expression of the small HSP 27 would be altered during labor and expression would vary in different regions of the placenta. Six women in labor who delivered vaginally and 6 women not in labor, who were delivered by Cesarean section, were recruited. Four equally spaced pieces were sampled from the inner, middle and outer regions of each placenta (total 12 samples per placenta). HSP 27 expression was investigated by Western blot analysis and RT-PCR. For non-labor, there was less HSP 27 protein in the inner placenta region compared with both the middle region (p<0.05) and outer region (p<0.05). For labor, there was also less HSP 27 protein in the inner region compared with both the middle (p<0.02) and outer region (p<0.01). When the 3 regions of the placenta were compared for non-labor versus labor there was less HSP 27 in the labor group at both the inner (p<0.05) and middle regions (p<0.005) compared to non-labor. Similar to HSP 27 protein, there was less HSP 27 mRNA in the labor group in both the inner region (p<0.05) and middle region (p<0.02) compared to non-labor. This study suggests that placental HSP 27 may play a role in labor and is spatially controlled. The results have important implications for how data obtained from studies in the placenta can be influenced by sampling methods. This work was supported by a PhD scholarship to E Abdulside from Libyan Government- administered By University of Glasgow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The mechanisms that are involved in maintaining a human pregnancy to term and the changes that lead to a normal pregnancy outcome or indeed an adverse outcome such as miscarriage, preeclampsia, fetal growth restriction or preterm labor are complex but the role of the placenta is crucial to them all [1]–[4]. When the production of reactive oxygen species overwhelms the intrinsic anti-oxidant defenses oxidative stress occurs. It can induce a range of cellular responses depending upon how severe the insult is and the cellular compartment in which reactive oxidative species are generated [4], [5]. The contractions that occur during labor are associated with intermittent utero-placental perfusion and could lead to an ischemia-reperfusion type injury to the placenta. Indeed Doppler ultrasound studies have demonstrated a linear inverse relationship between uterine artery resistance and the intensity of the uterine contractions during labor [6]. Labor is also associated with placental alterations in several pathways linked to oxidative stress [7]. Heat-shock proteins (HSPs) are a family of proteins expressed by all cells. They have many important physiological functions, one of the most important being to help cells to cope with stressful situations. Some HSPs are expressed constitutively while others are induced by a range of damaging insults including heat shock, ischemia, hypoxia, oxidative stress and physical injury [8]. HSPs are named according to their molecular weight. HSP 27 belongs to the family of small heat shock proteins (15–30 kDa). In response to stress, changes in expression of HSP 27 occurs and, like many proteins, HSP 27 function can also be regulated by at the post-translational level [9]. The functions of HSP 27 include protein chaperone, control of apoptosis, regulation of cell glutathione levels, inhibition of actin polymerisation as well as protection against heat shock, oxidative stress and mechanical stress [9]. HSP 27 also plays a role in atherosclerosis [10], in regulation of cytokine production from monocytes as well as expression of toll-like receptors [11]. Since HSP 27 plays a role in oxidative stress and inflammation, both features of labor, we hypothesised that HSP 27 expression would alter during labor in the placenta. Thus the aim of this study was to examine the spatial expression of HSP 27 in placentae obtained from women who delivered by cesarean section and were not in labor and secondly to compare the expression of each zone with the equivalent zone of placentas obtained from women who delivered vaginally following an uncomplicated labor. Materials and Methods Subjects Human term placentae were collected from pregnant women at the Southern General Hospital, Glasgow. All ethics protocols were followed as per Declaration of Helsinki. The study was approved by “Yorkhill ethics committee”. Signed patient consent was obtained prior to delivery. Patients were handed an information sheet telling them about the study before being handed the consent sheet. The information and consent sheets were also approved by the ethics committee. All signed consent sheets were stored incase of the need for audit. Placentae were collected from: (i) women who had uncomplicated pregnancies and delivered at term either vaginally (labor group, n = 6) or by caesarean section (non-labor group, n = 6). The labor group were all spontaneous labor and were a tight group (labor time minimum 3 hours maximum 8 hours). All placentae were free of infection, confirmed by the pathology report of every placenta. The non-labor were group were all definitely without labor. All were planned Caesarean sections performed for obstetric reasons: breach presentation (2) previous caesarean section (2) or maternal request (2). The groups studied had no underlying maternal conditions such as hypertension, preeclampsia, diabetes or gestational diabetes or any other medical disorders. There was no fetal pathology such as fetal growth restriction. The details of patients recruited are shown in Table 1. 10.1371/journal.pone.0071127.t001Table 1 Demographics of patients used for placenta collection. Category Non-labor (n = 6) Labor (n = 6) p value Maternal age (years) 28.33±5.71 26±2.28 >0.05 Placenta weight (g) 594.7±110.5 589.5±75 >0.05 Birth weight (g) 3443±537 3719±347 >0.05 Gestation age at delivery (weeks) 39.3±1.0 40.31±1.4 >0.05 No. primigravid 2 4 >0.05 No. Smokers 2 0 >0.05 Sample Collection For each patient (6 patients per group), placental samples (∼1 cm3) were obtained from three sites by taking measurements from the cord insertion point: inner third closest to cord insertion point (inner zone), middle of placenta (middle zone) and outer third of placenta (outer zone) of placenta. Within each zone four separate samples were obtained representing the four quadrants (Figure 1). Placentae had a central cord insertion. Samples were rinsed and immediately flash frozen in liquid nitrogen. For this study we had performed a power analysis using G*Power 3.1 for Macintosh and based the numbers on our previous published work [12]. 10.1371/journal.pone.0071127.g001Figure 1 Diagrammatic representation showing where samples were obtained from each individual placenta. Chemicals All chemicals were purchased from Sigma-Aldrich (U.K.) unless stated otherwise. Tissue Homogenizing For Western Blot Samples were recovered from storage at −70°C and ground in liquid nitrogen to a fine powder using a mortar and pestle. Tissues was homogenised in the presence of protease inhibitors as described previously [12]. Placenta homogenates were spun at 5000 g for 10 minutes at 4°C to remove debris then supernatants were collected and divided into aliquots and stored at −70°C. Protein concentrations were determined by Bradford analysis using bovine serum albumin as a standard. Western Blotting Western blotting was performed as described previously [12] with some modifications. A volume corresponding to 50 µg of each sample was separated by SDS-PAGE electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide resolving gels. Pre-stained low range molecular weight markers (BioRad) were loaded onto each gel. Transfer of proteins to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech) was carried out at 22V and 200 mA for 30 min. Membranes were blocked in 5% donkey serum (Serotec) in TBSTB buffer (20 mM TRIS pH 7.5, 0.5 M NaCl, 0.4% Tween and 0.25% bovine serum albumin) for 1 h at room temperature (RT). Primary antibodies were pre-absorbed in 5% human serum in TBSTB at RT during the blocking process. Membranes were incubated for 1 h at RT with primary antibody solution. The HSP 27 (mouse monoclonal antibody) was obtained from Cell Signalling Technology® (number 2402) and used at concentration of 1∶1000. Membranes were washed and then incubated for 1 h at RT with horseradish peroxidase conjugated donkey anti-mouse secondary antibody (Abcam (ab6820) diluted 1∶1000 in TBSTB. Membranes were rinsed with TBSTB (2×5 min) and once with distilled water. The same samples were exposed to a β-actin antibody (Sigma) to confirm even protein loading as shown previously [12]. Immunologically reactive proteins were visualised and quantified as described previously [12]. A standard curve was performed for different blot exposures and densitometry was performed when bands were on the linear part of the loading graph as described previously [12]. For each group of experiments the same loading control placenta sample was added to every gel and the densitometry units were normalized to that. We previously confirmed that this method of analysis gives similar findings to other quantitative methods of densitometry [12]. Statistical analysis was performed using MiniTab on a PC using analysis of variance. Comparison of groups was performed by the Mann Whitney test. Graphs show median values along with mean absolute deviation range, a robust measure of the variability of the data. Quantitative Rt-Pcr Total RNA was isolated using the RNeasy® Midi Kit (Qiagen, 75142). RNA (100ng) was reverse transcribed into cDNA. Buffers and primers were obtained from the QuantiTect® Kit (Qiagen, 205310) and GoScript™ reverse transcriptase from Promega (A501C). HSP 27 expression was analyzed by RT-PCR using validated TaqMan® Gene Expression assays with StepOnePlus (Applied Biosystems). β-actin was used as an endogenous control. A positive control human placenta cDNA (Primer design) was used. The relative target gene levels were calculated by comparative CT (ΔΔCT). Statistical analysis was performed as described above. Results Table 1 shows the demographics of the patients. Western Blotting The first set of experiments was designed to test whether there was a difference in HSP 27 expression within an individual placenta in either labor or non-labor. Figure 2 shows HSP 27 expression in the four zones of the inner, middle and outer area sampled from the cord insertion point. The upper panel shows a placenta obtained from a non-laboring caesarean section delivery. The bottom panel shows a placenta obtained from a women who was in labor and delivered vaginally. Figure 3 shows the combined analysis of all the placentae for either non-labor (upper graph) or labor (lower graph). Overall there was a significant difference between the 3 areas of the placenta for both the non-labor group and labor group (ANOVA p<0.05). For the non-labor group there was less HSP 27 in the inner compared with both the middle (p<0.05) and outer area (p<0.05). For the labor group there was also less HSP 27 in both the inner compared with the middle (p<0.02) and outer area (p<0.01). Thus HSP 27 is expressed in a spatial manner within the placenta and the distribution patterns are similar in labor and non-labor. 10.1371/journal.pone.0071127.g002Figure 2 Representative Western blot analysis of HSP 27 expression in the four quadrants of each zone of the placenta in a patient not in labor (upper panel) and a patient in labor (lower panel). (n = 6 patients in each group for entire study). Also shown is a representative β-actin loading control for one set of samples showing equal protein loading. 10.1371/journal.pone.0071127.g003Figure 3 Median optical densities for HSP 27 expression in three different placenta zones for all patients. The upper panel shows non-labor (n = 6 patients) and the lower panel shows labor (n = 6 patients). Samples were compared with the Mann Whitney U test. Values are shown as median and median absolute deviation. n..s., non-significant. The next set of experiments was designed to test whether there was a difference in HSP 27 expression between labor and non-labor groups for each of the three sites. Figure 4 shows one representative blot of non-labor versus labor for each of the three different areas of the placenta (upper panel; inner, middle panel; middle and lower panel; outer). Each of the 3 blots were performed on separate days so only labor with non-labor can be compared for this. Figure 5 shows the combined analysis for each of the three groups. Overall there was a significant difference between the 3 areas of the placenta when comparing each non-labor group and labor group in each zone (ANOVA p<0.05). When individual zones were compared there was less HSP 27 in the labor group at both the inner (p<0.05) and middle zones (p<0.005). 10.1371/journal.pone.0071127.g004Figure 4 Western blot analysis of HSP 27 expression in non-labor versus labor measured at three distances from the cord insertion point of the placenta: inner zone (top panel), middle zones (middle panel) and outer zone (bottom panel). N = 6 in each group of patients. 10.1371/journal.pone.0071127.g005Figure 5 Combined analysis of HSP 27 expression in non-labor versus labor for each of the three zones of the placenta shown in Figure 4. Samples were compared with the Mann Whitney U test. Shown is the median and median absolute deviation. n.s., non-significant. The final set of experiments was to determine whether the protein changes found in Figure 5 were reflected by changes at the mRNA level. The results are shown in Figure 6. As for HSP 27 protein there was less HSP 27 mRNA in the labor group in both the inner zone (p<0.05) and middle zone (p<0.02). 10.1371/journal.pone.0071127.g006Figure 6 Analysis of HSP 27 mRNA expression in non-labor versus labor for each of the three zones of the placenta. Samples were compared with the Mann Whitney U test. Shown is the median and median absolute deviation. n.s., non-significant. Discussion This study shows for the first time that HSP 27 is expressed in a spatial manner in the placenta with the highest expression being in the 2–4 cm (middle) area in both labour and non-labour groups. It therefore shows the importance of using a systematic method to sample the placenta. Most previous reports of placental protein expression do not take this into account. Taking a single or a few samples or averaging protein expression of several samples may well mask possible changes in expression. The study also shows that HSP 27 protein and mRNA are reduced during labor at defined zones. Apart from the reported changes and their link to placental pathology the results have important implications for how results in placental disease (and perhaps other organs) can be influenced by sampling methods. The key finding of this study was the fall in both HSP 27 mRNA and protein at the inner and middle zones of the placenta during labor and which was particularly striking in the middle zone. Many HSPs are increased to protect against stress in disease states [13] however in the present study HSP 27 was reduced. One reason for this may be that a fall in HSP 27 may be necessary to facilitate the inflammatory steps of labor which is, after all, a normal physiological process, not a disease. HSP27 protects against apoptosis, decreases oxidative stress, reduces the pro-inflammatory cytokine balance, stabilizes actin, and inhibits NFκB activation [13] but during labor the opposite effect of these events needs to occur. Previous publications of HSP 27 expression in the placenta are few. One study examined the expression of HSP 27 in placenta in labor and non-labor [7]. A different approach was taken: since different regions of the placenta were not compared, it is impossible to directly compare the present study with that one. In another study HSP 27 was reported to be unaltered in the placenta in samples from labor and non-labor. However only one biopsy was taken from each placenta and no quantification analysis was performed or presented [14]. Small HSPs can be modified by phosphorylation. HSP27 can be phosphorylated at serine 15, 78 and 82 by MAPKAPK-2 and 3 [13]. Phosphorylation favours small oligomers to form whereas de-phosphorylation favours formation of large oligomer [13]. Small and large forms may have different functions for example larger forms are important in chaperone and anti-oxidant roles whereas smaller forms are important in actin regulation [13]. The HSP 27 gene contains two functional HSE binding sites, a cAMP response element as well as HSF-1 and 2 binding sites [13]. In view of the findings of this study future studies will be directed to understand whether any changes in phosphorylation of HSP 27 or MAPKAPK-2 or 3 occurs at defined zones during labor. We have previously examined the expression of HSP 70 in the placenta [12]. In non-labor HSP 70 was reduced in the outer area of the placenta compared with the middle area. In contrast in the present study HSP 27 was reduced in the inner and middle areas compared with the outer area. With regard to labor our previous study showed that HSP 70 was increased in the inner and middle areas, the opposite of the findings for HSP 27. At this stage it is only possible to speculate why such zonal differences exist but may relate to the functions of HSP 27 and 70, some of which differ and some overlap. Placental separation is an important part of labor. Herman et al [15] showed that the process of placental separation from the uterine wall can be divided into three distinct phases i.e latent, contraction/detachment and expulsion. They showed that placental separation is accomplished by means of an orderly multiphasic process with a definite direction and sequence. They found in most cases the placenta separated from the uterine wall in a “down-up” separation i.e initiating from the lower pole. Interestingly cases with a previous Cesarean section had a higher rate of up-down separation. In contrast in the case of “fundal placentae” separation started at the placental poles (bipolar separation) and the central area of the placenta was the last to separate. It would be therefore of interest in a future study to investigate whether there was a link between the zonal distribution of HSP 27 or HSP 70 and the method of placental separation. Placentas collected at term by cesarean section are not subjected to the stress of labor however one possibility is that zonal differences in HSPs might reflect the fact that labor is not far off and that the molecular steps to allow labor to proceed have started. Thus it would be interesting to compare placentas from the second trimester where labor is not close to determine if such zonal differences still exist. In contrast at labor zonal differences in HSPs may be linked to the response to the stress of labor, extent of exposure to hypoxia or may contribute to the process that allows the placenta to separate at delivery. Wataba et al [16] showed that HSP 27 and 70 were increased in syncytial knots, avascular villi and the presence of thrombus whereas both were reduced in the presence of infarction suggesting different stresses evoke different responses in HSPs in the placenta and the response may very depending on the area of the placenta exposed to the stress. It has been shown that HSP 27 regulates apoptosis through key components of the apoptotic signalling pathway, in particular, those involved in caspase activation and apoptosis [17]. HSP 70 can also inhibit caspase 3 and 9 [18]. HSP 70, via the TLR-2 receptor, can increase IL-10 production; IL-10 can be pro-inflammatory at labor which may accelerate parturition [19]. This may also explain why HSP 70 increases at labor when HSP 27 decreases. Of interest is the observation that reduced matrix metalloproteinase 2 activity has been shown to be linked to reduced HSP 27 [20]. Whether this is linked to the zonal distribution requires further investigation. The expression of HSP 27 was also reported to be reduced in placentae from SGA neonates although zonal distribution was not investigated [21]. Small HSPs have been studied in myometrium during labor. The myometrium undergoes substantial remodeling at the time of labor including rearrangement of the cellular contractile machinery. Since HSP 27 can modulate actin polymerisation one study investigated changes in small HSPs in the myometrium at labor [22]. A 69% decrease in the small HSP αB-crystallin was found in the myometrium at labor plus multiple isoforms of HSP 27. Immunoblotting using phosphospecific HSP 27 antibodies (HSP 27-serine15, −78, and −82) detected marked changes in HSP 27 phosphorylation at labor. HSP 27-Ser15 was 3.0-fold higher in laboring myometrium. In contrast, levels of HSP 27-Ser82 were 85% less in laboring myometrium. There was no significant change in HSP 27-Ser78. It was proposed that decreased expression of αB-crystallin at the time of labor liberates HSP 27 enabling it to participate in other cellular events such as cytoskeletal remodeling. Clearly the functions and structure of the myometrium and placenta are different, however since both play a role in labor future work should investigate the expression of αB-crystallin within the placenta during labor. Also that particular study highlights how different changes in HSP 27 can occur depending on the cellular event to be targeted. In summary HSP 27 is expressed in a spatial manner in the human placenta and changes in expression occur during labor suggest that HSP 27 may be part of the signaling process of labor and thus warrants further investigation particularly with regard to a role on pre-term labor. We are grateful to Dr Kevin Hanretty for support during patient recruitment and to the Libyan Government for funding A. Abdulisid with a PhD scholarship. ==== Refs References 1 Petraglia F , Imperatore A , Challis JR (2010 ) Neuroendocrine mechanisms in pregnancy and parturition . Endocrin Rev 31 : 783 –816 . 2 Challis JRG , Mathews SG , Gibb W , Lye SJ (2000 ) Endocrine and Paracrine Regulation of Birth at Term and Preterm . Endocrin Rev 21 : 514 –550 . 3 Roberts JM , Escudero C (2012 ) The placenta in preeclampsia . Preg Hypertens 2 : 72 –83 . 4 Burton JG , Janiaux E (2011 ) Oxidative Stress . Best Practice Res Clin Obstet Gynecol 25 : 287 –299 . 5 Myatt L , Cui X (2004 ) Oxidative stress in the placenta Histochem Cell Biol . 122 : 369 –382 . 6 Brar HS , Platt LD , DeVore GR , Horenstein J , Medearis AL (1988 ) Qualitative assessment of maternal uterine and fetal umbilical artery blood flow and resistance in laboring patients by Doppler velocimetry . Am J Obstet Gynecol 158 : 952 –956 .2966588 7 Cindrova-Davies T , Yung HW , Johns J , Spasic-Boskovic O , Korolchuk S , et al (2007 ) Oxidative stress, gene expression, and protein changes induced in the human placenta during labor . Am J Pathol 171 : 1168 –1179 .17823277 8 Lanneau D , Wettstein G , Bonniaud P , Garrido C (2010 ) Heat shock proteins: cell protection through protein triage . Sci World J 3 : 1543 –1552 . 9 Ghayour-Mobarhan M , Saber H , Ferns GA (2012 ) The potential role of heat shock protein 27 in cardiovascular disease . Clin Chim Acta 413 : 15 –24 .21514288 10 Martin-Ventura JL , Duran MC , Blanco-Colio LM , Meilhac O , Leclercq A (2004 ) Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis . Circulation 110 : 2216 –2219 .15249501 11 De AK , Kodys KM , Yeh BS , Miller-Graziano C (2000 ) Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heat-shock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus . J Immunol 165 : 3951 –3958 .11034403 12 Abdulsid A , Hanretty K , Lyall F (2013 ) Plos one. Heat Shock Protein 70 Expression Is Spatially Distributed in Human Placenta and Selectively Upregulated during Labor and Preeclampsia . PLoS ONE 8(1) : e54540 .23382911 13 Garrido C , Paul C , Seigneuric R , Kampinga HH (2012 ) The small heat shock proteins family: The long forgotten chaperones . Intl J Cell Biol 44 : 1588 –1592 . 14 Li DG , Gordon CB , Stagg CA , Udelsman R (1996 ) Heat shock protein expression in human placenta and umbilical cord . Shock 5 : 320 –323 .9156786 15 Herman A , Zimerman A , Arieli S , Tovbin Y , Bezer M , et al (2002 ) Down–up sequential separation of the placenta . Ultrasound Obstet Gynecol 19 : 278 –281 .11896951 16 Wataba K , Saito T , Takeuchi M , Nakayama M , Suehara N , et al (2004 ) Changed expression of heat shock proteins in various pathological findings in placentas with intrauterine fetal growth restriction . Med Electron Microscop 37 : 170 –6 . 17 Concannon CG , Gorman AM , Samali A (2003 ) On the role of Hsp27 in regulating apoptosis . Apoptosis 8 : 61 –70 .12510153 18 Borges TJ , Wieten L , van Herwijnen MJC , Broere F , van der Zee R , et al (2012 ) The anti-inflammatory mechanisms of Hsp70 . Front Immunol 95 : 1 –12 . 19 Gibb WL, Lye SJ, Challis JRG (2006) Parturition. Knobil, Neill (Eds.), Physiology of reproduction, Elsevier Inc. 2925–2974. 20 Matalon ST , Drucker L , Fishman A , Ornoy A , Lishner M (2008 ) The Role of heat shock protein 27 in extravillous trophoblast differentiation . J Cell Biochem 103 : 719 –729 .17661346 21 Cañete P , Monllor A , Pineda A , Hernández R , Tarín JJ , et al (2012 ) Levels of heat shock protein 27 in placentae from small for gestational age newborns . Gynecol Obstet Invest 73 : 248 –251 .22414777 22 MacIntyre DA , Tyson EK , Read M , Smith R , Yeo G , et al (2008 ) Contraction in Human Myometrium Is Associated with Changes in Small Heat Shock Proteins, Endocrinol . 149 : 245 –252 .	23990930	PMC3750034	CC BY	2021-01-05 17:37:53	yes	PLoS One. 2013 Aug 22; 8(8):e71127
==== Front Eur J Med ResEur. J. Med. ResEuropean Journal of Medical Research0949-23212047-783XBioMed Central 2047-783X-18-262389558310.1186/2047-783X-18-26ResearchEffects of uric acid on endothelial dysfunction in early chronic kidney disease and its mechanisms Wang Yu 1wangyu_syd@sina.comBao Xiaorong 1jinshankidney_js@126.com1 Department of Nephrology, Jinshan Hospital affiliated to Fudan University, No.1508 Longhang Road, Jinshan District, Shanghai 201508, China2013 30 7 2013 18 1 26 26 21 2 2013 18 7 2013 Copyright © 2013 Wang and Bao; licensee BioMed Central Ltd.2013Wang and Bao; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background An increase in serum uric acid (UA) occurs during the early and middle stages of chronic kidney disease (CKD) and aggravates the deterioration of kidney function. This study aims to explore the relation between UA and endothelial dysfunction in early CKD and its mechanisms in a murine model. Methods The experimental animals were randomly divided into three groups (n = 10): sham-operation group (control group), right nephrectomy only group (CKD group) and right nephrectomy with oxonic potassium group (CKD with hyperuricemia group). Furthermore, we analyzed the relation between UA and endothelial dysfunction indices in early CKD as well as its mechanisms. Results Linear regression analysis showed that the level of serum UA had a significant positive correlation with serum endothelin-1 and the percentage of collagen I positive area, but a negative correlation with serum nitric oxide (NO) and NO/endothelin-1 ratio. In addition, the level of serum UA had significant positive correlations with serum malonaldehyde, serum C-reactive protein, serum oxidatively-modified low-density lipoprotein and serum low-density lipoprotein, but a negative correlation with serum superoxide dismutase. Conclusions Endothelial dysfunction in the CKD group was significant and had a positive correlation with the level of serum UA. Endothelial dysfunction in early CKD with hyperuricemia is perhaps related to oxidative stress, micro-inflammation and lipid oxidation. Early chronic kidney diseaseEndothelial dysfunctionLipid oxidationMicro-inflammationOxidative stressUric acid ==== Body Background Since previous studies have highlighted the role of serum uric acid (UA) in coronary heart disease [1], the effect and mechanism of UA in cardiovascular disease (CVD) has aroused widespread concern. Serum UA level is not only the most significant predictor of occurrence of primary hypertension [2], but it is also associated with cardiovascular morbidity and mortality [3-5]. Chronic kidney disease (CKD) affects 10–13% of the general population. CKD patients have an extremely high risk of developing CVD compared with the general population. Patients in the early stages of CKD are more likely to convert into CVD rather than progress towards end-stage renal disease [6]; CVD is the major cause of death in patients with CKD [7]. The increase in serum UA occurs in the early and middle stages of CKD and aggravates with the deterioration of kidney function [8]. At present, no publication has demonstrated the effect and mechanism of UA in early CKD with CVD patients. Our earlier clinical study found that serum UA was increased in patients with stage 2–3 CKD and was related to CVD (e.g. left ventricular hypertrophy), indicating that hyperuricemia was associated with CVD in early CKD. Endothelial dysfunction is an early occurrence in CVD. Vascular endothelial cells play an important role in cellular functions, such as modulating angiokinesis, the proliferation and migration of vascular smooth muscle cells, anti-platelet aggregation, and extracellular matrix generation. Pathological changes in blood vessels, including intima hyperplasia, lumen straightness and atherosclerosis, are a consequence of endothelial dysfunction. In the clinic, endothelial dysfunction is associated with CVD, and specifically with hypertension, coronary heart disease, thrombosis, and cardiac insufficiency [9-11]. Endothelial cells can synthesize and excrete many important substances, the serum levels of which will change in the condition of endothelial dysfunction, such as decreased nitric oxide (NO) level, increased endothelin-1 (ET-1) level, and decreased NO/ET-1 ratio [12,13]. Thus, the content of NO and ET-1 is important for evaluating the function of endothelial cells. Collagen I deposition in the artery is also related to the function of endothelial cells; endothelial cell dysfunction increases the excretion of transforming growth factor-β (TGF-β) and other substances of collagenous protein synthesis, leading to the deposition of abundant collagen I in the artery. Endothelial cell injury is generally related to genetic factors, lifestyle, age, obesity, smoking, blood pressure (pulse pressure), heart dysfunction, fasting hyperglycemia (impaired glucose tolerance) and insulin resistance. The unique status of CKD, namely oxidative stress, micro-inflammation, and lipid oxidation [14], can also cause endothelial cell injury. At present, studies exploring the mechanisms of endothelial cell injury in CKD patients mainly focus on end-stage renal disease patients. The mechanism of UA-induced endothelial damage in early CKD is not well known. Despite recent advances in the treatment of CKD, the disease remains an important public health challenge [15]. Traditional risk factors, such as hypertension and hypercholesterolemia, cannot explain the excess cardiovascular mortality in CKD patients. Identifying and treating risk factors of early CKD may be the best approach to prevent and delay adverse outcomes [16]. Through the establishment of early CKD animal models with elevated serum UA, we explored the relationship between UA and vascular endothelial cell damage, and further investigated the mechanisms of injury in order to elucidate intervening CVD risk factors as early as possible in CKD patients. Methods Reagents and antibodies NO, superoxide dismutase (SOD), and malondialdehyde (MDA) detection kits were purchased from Nanjing KeyGEN Biotech. Co. Ltd. (Nanjing, China). Oxidatively modified low-density lipoprotein (ox-LDL), ET-1, and C-reactive protein (CRP) detection kits were purchased from ADL (Adlitteram Diagnostic Laboratories, USA). Diaminobenzidine chromogenic kit, rabbit anti-mouse collagen I polyclonal antibody, and goat anti-rabbit polyclonal antibody were purchased from Fuzhou Maixin Biotechnology Co. Ltd. (Fujian, China). Beckman CX9 biochemical analyzer and Beckman supporting reagents were purchased from Beckman Coulter (USA). The establishment of early CKD animal model with hyperuricemia Thirty male Sprague-Dawley rats, weighing 187 g to 232 g and 6 to 7 weeks old, obtained from Xipuer-bikai experimental animal company (Shanghai, China), were employed in the present study. All experimental procedures were conducted in accordance with the Guiding Principles for the Care and Use of Animals in Research and Teaching, approved by the Institutional Animal Care and Use Committee of Jinshan Hospital affiliated to Fudan University, China. The experimental animals were randomly divided into three groups (n = 10): sham-operation group (control group, Group A), right nephrectomy-only group (CKD group, Group B), and right nephrectomy with oxonic potassium group (CKD with hyperuricemia group, Group C). The rats were housed in standard plastic cages; food and water were freely available. The experimental animals were anesthetized using an intraperitoneal injection at a dose of 5% ketamine (100 mg/kg). The surgical region was shaved and the shin was cleaned with 75% alcohol. The right kidney was exposed through a longitudinal incision under the right costal arch (proximal to the right side of the spine). For Groups B and C, the right kidney was resected. The entire procedure was performed in the sham group, but nephrectomy was not applied. After one week of normal feeding, the rats in all three groups were in good condition. The experimental group was fed with uricase inhibitor (oxonic potassium) twice a day (800 mg/kg, at 8 a.m. and 5 p.m.) by gavage. During the experiment, rats were weighed every two weeks and the administered dose was adjusted based on body weight. Unilateral nephrectomy group and the sham-operation group were fed with the same amount of saline. Sample collection and management After ten weeks of gavage administration, rats were killed and blood samples were collected from the heart into non-heparinized tubes. The blood sera were then collected via centrifugation and stored at −70°C for detection of UA, serum creatinine (Scr), NO, ET-1, CRP, MDA, SOD, ox-LDL, and LDL. For light microscopic examination, left kidney tissues from each group were fixed with 10% formalin, stored at 4°C for 14 to 16 hours, and then embedded with paraffin. After routine processing, paraffin sections of each tissue were cut into 4-μm thickness and stained with periodic acid-Schiff. The aorta tissue from descending aorta was cut, washed with 0.9% saline, and embedded in paraffin. After routine processing, paraffin sections of each tissue were cut into 4-μm thickness for hematoxylin-eosin (HE) staining and determination of collagen I. The detection of serologic indexes The detection of Scr, UA, serum NO, serum ET-1, serum SOD, serum MDA, serum CRP, serum LDL, and ox-LDL was performed according to the instructions in the kits. Statistical analysis The proportion of collagen I positive area was measured by randomly selecting three fields in each slide and dividing each field into 1,564 parts by Photoshop. The number of positive points were counted; the proportion was the number of positive points/1,564. Data were calculated by two examiners and the average values were calculated. All data were expressed by x̄ ± s. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for windows, version 13.5). Comparisons between groups were analyzed using the t-test. Variable comparisons were assessed using one-way analysis of variance (ANOVA) and multiple stepwise regression analysis. A P value < 0.05 was considered as significant. Results Early CKD animal model with hyperuricemia Group A (sham operation) and group B (right nephrectomy only) served as controls. Group C (right nephrectomy with oxonate potassium) was the experimental group. Periodic acid-Schiff staining of kidney tissues from each group showed mild glomerular mesangial proliferation in groups B and C, compared to group A (Figure 1A,C,E). No obvious pathological change in renal tubule and renal interstitium was visible in the three groups. Meanwhile, there was no renal tubular epithelial cells necrosis, no inflammatory cells in the interstitium, and no small vessel lesions in groups B and C (Figure 1D,F). In the experimental group, there was no urate crystal deposition. Compared with groups A and B, the experimental group had a significantly higher level of UA. However, there was no obvious difference in Scr among the three groups (Table 1), which was in accordance with the characteristics of early CKD. The above results indicate the establishment of an early CKD animal model with hyperuricemia. Figure 1 Pathological pictures of rat kidneys from groups A, B, and C. (A) The normal renal glomerulus of group A (×400). (B) No obvious pathological change in renal tubules of group A (×400). (C, E) Mild glomerular mesangial proliferation in renal glomerulus of groups B and C (×400). (D, F) There were no obvious pathological changes in renal tubules in groups B and C (×400). Table 1 Concentration of Src, UA, PCIPA, NO, ET-1 and NO/ET-1 ratio in three groups (x̄±s) Group Scr (μmol/L) UA (μmol/L) PCIPA (%) NO (μmol/L) ET-1 (pg/mL) NO/ET-1 A 30.20 ± 6.01 53.10 ± 8.62 12.90 ± 2.31 47.55 ± 5.39 5.89 ± 1.67 8.18 ± 2.32 B 31.70 ± 4.72 53.70 ± 11.52 12.97 ± 2.71 45.34 ± 4.76 5.92 ± 1.56 8.90 ± 3.55 C 30.80 ± 5.90 161.40 ± 28.04* 22.38 ± 3.14* 36.71 ± 3.45* 7.50 ± 1.06△ 5.07 ± 1.19* Note: : P <0.01 group C vs. groups A and B. △: P <0.05 group C vs. groups A and B. PCIPA: Percentage of collagen I positive area. Vascular endothelial cell injury in the experimental group In the light microscope, endothelial cells of group A were arranged closely under the vascular intima and inflammatory cells did not accumulate in the vascular wall (Figure 2A); smooth muscle cells were arranged in order with a spindle shape and an almost uniform morphology (Figure 2B). However, in group C, a foam-like interstitial edema of endothelial cells was visible (Figure 2E). Partial endothelial cells was shed from the vessel wall and the gap between them was broadened (Figure 2F). Further, inflammatory cells accumulated in the vascular intima (Figure 2G,H) and several inflammatory cells infiltrated within the membrane (Figure 2I,J). The thickness of the blood vessel wall increased. Medial smooth muscle cells proliferated and thickened with an irregular shape and a disordered arrangement (Figure 2K). The pathological change of the right-side nephrectomy group (group B) was similar to the experimental group, but less marked (Figure 2C). Smooth muscle cell proliferation was not obvious and it was well arranged (Figure 2D). The results confirmed significant vascular injury in the experimental group. Figure 2 Pathological images of rat arteries from groups A, B, and C. (A) Endothelial cells arranged in order under the vascular intima of group A (×400). (B) There was no obvious proliferation of medial smooth muscle cells in the vascular wall of group A (×200). (C) The morphology of endothelial cells of group B was slightly abnormal (×400). (D) There was no obvious proliferation of medial smooth muscle cells in the vascular wall of group B (×200). (E) Endothelial cells of group C had a foam-like change (×400). (F) Endothelial cells of group C shed from the vessel wall (×400). (G, H) Inflammatory cells accumulated in the vascular intima of group C (×400). (I) Neutrophil granulocytes were seen around the endothelial cells of group C (×400). (J) Mononuclear cells were seen around the endothelial cells of group C (×400). (K) Smooth muscle cells of group C proliferated, thickened, and had no order (×200). Collagen I staining of the vascular wall in the three groups is shown in Figure 3. Several collagen I depositions were visible in the vascular wall of groups A and B, while the collagen I component was significantly increased in group C. Statistical analysis showed that the percentage of collagen I positive area in the vessel wall of the experimental group was significantly higher than that of group A and group B (group C vs. group A, P < 0.01; group C vs. group B, P < 0.01) (Table 1). Figure 3 Collagen I staining of the vascular wall in groups A, B, and C. (A,B) A small amount of collagen I deposition was seen in the vascular wall of group A (×400). (C) Collagen I component was significantly increased in group C (×400). Blood NO and ET-1 were important values to reflect the function of endothelial cells. Compared to groups A and B, in the experimental group, serum NO level was low (P < 0.01), serum ET-1 level was high (P < 0.05), and the NO/ET-1 ratio was low (P < 0.01). The above results verified endothelial cell dysfunction and significant injury of vascular endothelial cells. Uric acid-induced vascular endothelial cell injury in early CKD The possibility of a direct correlation between elevated UA level and vascular disease as well as endothelial injury was also investigated. Linear regression analysis showed that the level of serum UA had a significant positive correlation with the percentage of collagen I positive area (r = 0.8403, P < 0.01) and serum ET-1 (r = 0.9374, P < 0.01), but a negative correlation with serum NO (r = −0.9462, P < 0.01) and NO/ET-1 ratio (r = −0.9230, P < 0.01) (Table 2). The percentage of collagen I positive area had a significant positive correlation with serum ET-1 (r = 0.8737, P < 0.01), but a negative correlation with serum NO (r = −0.9171, P < 0.01) and NO/ET-1 ratio (r = −0.8707, P < 0.01) (Table 3). The results indicated that UA induced vascular endothelial cell injury in early CKD and the production of collagen I in the vascular wall. Meanwhile, the production of collagen I was relative to the injury of endothelial cells. Table 2 Correlation between serum uric acid and PCIPA, NO, ET-1, and NO/ET-1 ratio in the vascular wall Serum uric acid (μmol/L) r value P value PCIPA (%) 0.8403 0.0023 NO (μmol/L) −0.9462 0.0000 ET-1 (pg/mL) 0.9374 0.0001 NO/ET-1 ratio −0.9230 0.0001 Table 3 Correlation between PCIPA in the vascular wall and NO, ET-1, and NO/ET-1 ratio PCIPA (%) r value P value NO (μmol/L) −0.9171 0.0002 ET-1 (pg/mL) 0.8737 0.0010 NO/ET-1 ratio −0.8707 0.0010 Uric acid caused vascular endothelial injury in early CKD by oxidative stress, micro-inflammation, and lipid oxidation mechanisms The above experiments showed that UA was involved in early CKD vascular endothelial cell injury; however, the mechanism is unclear. Basic research demonstrated that UA crystals deposited in the intima could directly cause endothelial cell damage. Whether there are other important mechanisms apart from this remains to be investigated. Our research showed that the experimental group had lower level of serum SOD (U/mL) compared with groups A and B (group C vs. group A, P < 0.01; group C vs. group B, P < 0.01) and higher level of serum MDA (nmol/mL) (group C vs. group A, P < 0.01; group C vs. group B, P < 0.05) (Table 4). Linear regression analysis showed that the level of serum UA had a significant positive correlation with serum MDA (r = 0.8195, P < 0.01), but a negative correlation with serum SOD (r = −0.6885, P < 0.05), which indicated that UA might lead to oxidative stress in early CKD. Further, the level of serum NO had a significant positive correlation with serum SOD (r = 0.8179, P < 0.01), but a negative correlation with serum MDA (r = −0.9171, P < 0.01). The level of serum ET-1 had a significant positive correlation with serum MDA (r = 0.8658, P < 0.01), but a negative correlation with serum SOD (r = −0.7793, P < 0.01). NO/ET-1 ratio had a significant positive correlation with serum SOD (r = 0.8143, P < 0.01), but a negative correlation with serum MDA (r = −0.9143, P < 0.01). SOD entered the multiple stepwise regression equation of NO and ET-1, indicating that oxidative stress can cause vascular endothelial dysfunction in early CKD (Table 5). Table 4 Concentration of serum SOD, MDA, CRP, ox-LDL, and LDL in the three groups (x̄±s) Group SOD MDA CRP ox-LDL LDL (U/mL) (nmol/mL) (μg/mL) (mmol/L) (mmol/L) A 249.80 ± 9.83 4.06 ± 0.28 10.43 ± 1.68 47.50 ± 11.51 0.18 ± 0.06 B 243.60 ± 8.11 4.04 ± 0.41 12.27 ± 2.76 53.31 ± 12.38 0.18 ± 0.06 C 224.40 ± 6.47# 4.40 ± 0.23△# 14.68 ± 2.01△# 65.22 ± 10.91△# 0.25 ± 0.06△# Note: *: P <0.01 group C vs. group B. △: P <0.05 group C vs. group B. #: P <0.01 group C vs. group A. Table 5 Correlation between serum UA, NO, ET-1, and NO/ET-1 ratio as well as PCIPA, SOD, MDA, CRP, ox-LDL, and LDL Serum uric acid (μmol/L) Serum NO (μmol/L) Serum ET-1 (pg/mL) NO/ET-1 ratio PCIPA (%) r value P value r value P value r value P value r value P value r value P value SOD (U/mL) −0.6885 0.0277 0.8179 0.0038 −0.7793 0.0079 0.8143 0.0041 −0.8180 0.0000 MDA (nmol/mL) 0.8195 0.0037 −0.9171 0.0002 0.8658 0.0012 −0.9143 0.0002 0.8015 0.0053 CRP (μg/mL) 0.7251 0.0177 −0.7554 0.0115 0.7447 0.0135 −0.8042 0.0050 0.6752 0.0322 ox-LDL (mmol/L) 0.8479 0.0019 −0.7459 0.0132 0.7900 0.0065 −0.7949 0.0060 0.5266 0.1179 LDL (mmol/L) 0.6356 0.0483 −0.5080 0.1339 0.5734 0.0831 −0.4947 0.1460 0.5902 0.0725 Note: NO as dependent variable and SOD, MDA, CRP, ox-LDL, LDL as independent variables, multiple stepwise regression analysis showed that serum SOD and CRP entered the equation, and the equation was y = 0.315 × 1-0.802 × 2-22.120 (y = NO, ×1 = SOD, ×2 = CRP; -22.120 was a constant). ET-1 as dependent variable and SOD, MDA, CRP, ox-LDL, and LDL as independent variables, multiple stepwise regression analysis showed that serum ox-LDL and SOD entered the equation, and the equation was y = 0.051 × 1-0.082 × 2 + 22.517 (y = ET-1, ×1 = ox-LDL, ×2 = SOD; 22.517 was a constant). NO/ET-1 ratio as dependent variable and SOD, MDA, CRP, ox-LDL, LDL as independent variables, multiple stepwise regression analysis showed that serum SOD and CRP entered the equation, and the equation was y = 0.102 × 1-0.316 × 2-13.151 (y = NO/ET-1 ratio, ×1 = SOD, ×2 = CRP; 13.151 was a constant). PCIPA as dependent variable and SOD, MDA, CRP, ox-LDL, LDL as independent variables, multiple stepwise regression analysis showed that serum SOD entered the equation, and the equation was y = 111.437-0.397 × 1 (y = PCIPA, ×1 = SOD; 111.437 was a constant). Our research also found that the experimental group had a higher level of serum CRP (μg/mL) compared with groups A and B (group C vs. group A, P < 0.01; group C vs. group B, P < 0.05) (Table 4), which indicated that the experimental group had a more obvious micro-inflammation state. Linear regression analysis showed that the level of serum UA had a significant positive correlation with serum CRP (r = 0.7251, P < 0.05), which indicated that UA was involved in the formation of micro-inflammation in early CKD. Further, the level of serum NO had a significant negative correlation with serum CRP (r = −0.7554, P < 0.05). Serum ET-1 level had a significant positive correlation with serum CRP (r = 0.7447, P < 0.05). NO/ET-1 ratio had a significant negative correlation with serum CRP (r = −0.8042, P < 0.01). CRP entered the multiple stepwise regression equation of NO and NO/ET-1 ratio, indicating that micro-inflammation was involved in the formation of vascular endothelial dysfunction in early CKD (Table 5). By studying the levels of serum LDL and ox-LDL, we found that the experimental group had a higher level of serum LDL (mmol/L) than groups A and B (group C vs. group A, P < 0.01; group C vs. group B, P < 0.05) and a higher level of serum ox-LDL (mmol/L) (group C vs. group B, P < 0.05) (Table 4). Linear regression analysis showed that the level of serum UA had a significant positive correlation with serum ox-LDL (r = 0.8479, P < 0.01) and serum LDL (r = 0.6356, P < 0.05), which indicated that UA can cause lipid metabolic disorder in early CKD. Further, the level of serum NO had a significant negative correlation with serum ox-LDL (r = −0.7459, P < 0.05), but no significant correlation with serum LDL (r = −0.5080, P > 0.05). Serum ET-1 levels had a significant positive correlation with serum ox-LDL (r = 0.7900, P < 0.01), but no significant correlation with serum LDL (r = 0.5734, P > 0.05). NO/ET-1 ratio had a significant negative correlation with serum ox-LDL (r = −0.7949, P < 0.01), but no significant correlation with serum LDL (r = −0.4947, P > 0.05). Serum ox-LDL entered the multiple stepwise regression equation of ET-1 (Table 5). The results indicated that ox-LDL had strong endothelial cell toxicity. In addition, the percentage of collagen I positive area in the vascular wall had significant positive correlations with serum MDA (r = 0.8015, P < 0.01) and serum CRP (r = 0.6752, P < 0.05), a negative correlation with serum SOD (r = −0.8180, P < 0.01), and no significant correlation with serum ox-LDL (r = 0.5266, P > 0.05) or serum LDL (r = 0.5902, P > 0.05) (Table 5). Multiple stepwise regression analysis showed that serum SOD entered the equation. According to the results, it was found that oxidative stress and micro-inflammation could lead to an increase of collagen I deposition in the vessel wall. However, whether lipid metabolism disorders had a relation with increased collagen deposition in the vessel wall was not determined. In conclusion, UA caused endothelial dysfunction in early CKD via mechanisms involved in oxidative stress, micro-inflammation, and abnormal lipid metabolism. Discussion In this study, serum UA levels in rats were elevated with potassium oxonate by gavage. In 1965, it was verified that potassium oxonate had a strong ability to inhibit uricase activity both in vivo an in vitro[17]. Many reports have established a hyperuricemia animal model with potassium oxonate [18]. In this study, we compared the morphology and biochemical changes of kidney cells between the experimental group and the control group, as well as a unilateral nephrectomy group. The experimental group and the unilateral nephrectomy group had no obvious glomerular lesions; only some of the glomerulus presented mild mesangial proliferation. No obvious abnormality of renal tubules and renal interstitium was visible. Meanwhile, hyperuricemia kidney disease caused by urate crystal deposition was not visible in the experimental group. In serology, serum creatinine levels among the three groups had no significant difference (P > 0.05). Nevertheless, the serum UA level of the experimental group was significantly higher than the other two groups (about three times). Therefore, we believe that an early-CKD animal model with hyperuricemia was successfully established. Endothelial dysfunction is prevalent in CKD patients, and in particular end-stage renal disease patients [19]. Many factors lead to endothelial dysfunction, the mechanisms of which have not been clearly elucidated as yet. Because of abnormal changes in UA excretion through the kidney, serum UA levels are higher in CKD patients compared to the normal population and serum UA continues to rise with deterioration of renal function. Many researchers reported that UA was elevated in CKD and might play a role in the pathophysiology of CKD progression through endothelial dysfunction, such as activation of local renin-angiotensin system, increased oxidative stress, and proinflammatory and proliferative actions [20-22]. This conclusion, although controversial, was supported in in vitro experimental studies showing the relationship of UA with NO production and depletion [23,24]. Given that contention, it was necessary to study the relationship of UA with early CKD vascular endothelial injury through animal experiments. Our experiment showed that the endothelial cells of the experimental rats presented obvious morphological changes. In normal circumstances, vascular endothelial cells inhibit inflammatory cell adhesion and anti-smooth muscle cell proliferation and migration. However, in experimental rats, aggregation of inflammatory cells was observed in the vessel wall, which was even infiltrated into the intima, accompanied by vascular wall was thickening. Further, smooth muscle cells were hyperplastic, thickened, irregular, and disorganized. In addition, immunohistochemical methods demonstrated that collagen I increased significantly in the vessel wall. Hence, the above vasculopathy can be regarded as a consequence of endothelial injury. Endothelial cells, an important endocrine organ, can secret many important active substances that play important roles in the cardiovascular system, such as NO, prostacyclin hormone, endothelin, angiotensin, antithrombin III, and plasminogen activator. These substances are useful for homeostasis in the normal case; when the homeostasis balance is broken, the secretion is abnormal. Therefore, to a certain extent, endothelial cell function can be assessed by detecting the concentration of these substances in the serum. In our study, the substances of contraction and relaxation of vascular NO and ET-1 [25], synthesized and secreted by endothelial cells respectively, were used to assess endothelial cell function. The study found that serum NO concentration of the experimental group was significantly low and ET-1 was significantly high compared to the unilateral nephrectomy group, indicating that NO and ET-1 secretion were out of balance. Therefore, we believe that there is a significant endothelial dysfunction in the experimental group. Further, line correlation analysis showed that serum UA level was significantly correlated with the endothelial function indicators NO and ET-1, indicating that UA has a relation to vascular endothelial cell dysfunction and participates in early CKD vascular endothelial cell injury. Increased oxidative stress and reduced antioxidant capacity are prevalent in CKD patients [26]. Even in patients with mild renal impairment, oxidative stress level has increased more significantly than the normal population. The level of oxidative stress will intensify as renal deterioration. Oxidative stress plays an important role in endothelial cell damage and functional changes [27]. At present, studies on oxidative stress and endothelial injury mainly focus on end-stage renal disease patients [28]. The present study focuses on early CKD, and found that the experimental group has a significant increased oxidative stress level related to serum UA concentration, indicating that UA is involved in the formation of the early CKD oxidative stress status. Systemic micro-inflammation is widespread in CKD patients, even accompanied by mild renal dysfunction [29]. At present, the degree of inflammatory response is closely related to the incidence and mortality of cardiovascular events in CKD patients [30,31]. CRP is an acute phase protein synthetized by liver in inflammation, which is recognized as a reliable marker reflecting the inflammatory state. Our study found that the CRP level of the experimental group was significantly higher than the unilateral nephrectomy group, indicating that the micro-inflammatory state of the experimental group was more obvious. CRP level was significantly correlated with serum UA level, showing that UA is involved in the formation of micro-inflammation in early CKD. Further analysis showed that serum CRP level was significant correlated with NO, ET-1, and NO/ET-1 ratio, all of which are indicators of endothelial function. CRP entered the NO and ET-1 multiple regression equation, indicating that micro-inflammation is involved in vascular endothelial cell damage of in early CKD. CKD is often accompanied by lipid metabolic disorders, including increased plasma lipoprotein concentrations and/or lipoprotein composition changes [32]. Our study found that in the experimental group LDL levels, as well as its oxidized form ox-LDL, were increased compared to the unilateral nephrectomy group, both of which were significantly correlated with UA, suggesting that UA leads to early CKD lipid metabolic disorders. Further analysis showed that LDL was not significantly correlated with endothelial function indicators NO and ET-1, but its oxidized form was significantly associated with these indicators. Lipid metabolism disorders might be involved in endothelial cell damage. It is believable that only ox-LDL has strong endothelial cell toxicity compared to LDL. The higher the degree of oxidation, the stronger the damage is. Our findings also confirmed this. Finally, a correlation study about the percentage of collagen I positive area of the vessel wall and serum UA levels, endothelial function indicators NO, ET-1, and NO/ET-1 ratio, as well as the activities of SOD, MDA, CRP, LDL, and ox-LDL indicated that the percentage of collagen I positive area of the vessel wall was significantly associated with serum UA level, NO, ET-1, NO/ET-1 ratio, SOD, MDA, and CRP, but had no obvious correlation with LDL and ox-LDL. Based on the above, we believe that UA causes vascular endothelial cell injury in early CKD and leads to collagen proliferation in the vessel wall. Meanwhile, collagen hyperplasia has a relation to endothelial cell injury. We found that oxidative stress and micro-inflammation can lead to increased collagen I deposition in the vessel wall, but whether lipid metabolism disorders are associated with increased collagen component deposition in the vessel wall is not yet determined. With the percentage of collagen I positive area as a dependent variable, multiple stepwise regression analysis showed that serum SOD entered the equation. Based on these results, we speculate that micro-inflammation can lead to collagen I proliferation, but oxidative stress may act in the final passage leading to increased collagen deposition in the vessel wall. Conclusions In summary, this study successfully established an early-CKD animal model with elevated serum UA by unilateral nephrectomy plus potassium oxonate intragastrically. With the animal model, we found UA had a role in early CKD vascular endothelial cell injury. UA is involved in the formation of oxidative stress, micro-inflammatory state, and abnormal lipid metabolism in early CKD, based on which UA may lead to early CKD vascular endothelial injury. Therefore, strengthened control the UA level in early CKD aiming to correct oxidative stress, micro-inflammatory state, and lipid metabolism disorders, could improve vascular endothelial function, delay the process of atherosclerosis, and improve quality of life for CKD patients. Abbreviations CKD: Chronic kidney disease; CRP: C-reactive protein; CVD: Cardiovascular disease; ET-1: Endothelin-1; LDL: Low density lipoprotein; MDA: Malonaldehyde; NO: Nitric oxide; ox-LDL: Oxidatively modified low-density lipoprotein; Scr: Serum creatinine; SOD: Super oxide dismutase; TGF-β: Transforming growth factor-β; UA: Uric acid. Competing interests The authors declare that they have no competing interests. Authors’ contributions YW: Designed the experiments, and acquired and analyzed the data. XRB: Designed the experiments and drafted the manuscript. Both authors read and approved the final manuscript. ==== Refs Gertler MM Garn SM Levine SA Serum uric acid in relation to age and physique in health and in coronary heart disease Ann Intern Med 1951 34 1421 1431 14838504 Jossa F Farinaro E Panico S Krogh V Celentano E Galasso R Mancini M Trevisan M Serum uric acid and hypertension: the Olivetti heart study J Hum Hypertens 1994 8 677 681 7807497 Freedman DS Williamson DF Gunter EW Byers T Relation of serum uric acid to mortality and ischemic heart disease: the NHANES I epidemiologic follow-up study Am J Epidemiol 1995 141 637 644 7702038 Liese AD Hense HW Lowel H Doring A Tietze M Keil U Association of serum uric acid with all-cause and cardiovascular disease mortality and incident myocardial infarction in the MONICA Augsburg cohort: World Health Organization monitoring trends and determinants in cardiovascular diseases Epidemiology 1999 10 391 397 10.1097/00001648-199907000-00009 10401873 Niskanen LK Laaksonen DE Nyyssonen K Alfthan G Lakka HM Lakka TA Salonen JT Uric acid level as a risk factor for cardiovascular and all-cause mortality in middle-aged men: a prospective cohort study Arch Intern Med 2004 164 1546 1551 10.1001/archinte.164.14.1546 15277287 Hajhosseiny R Khavandi K Goldsmith DJ Cardiovascular disease in chronic kidney disease: untying the Gordian knot Int J Clin Pract 2013 67 14 31 10.1111/j.1742-1241.2012.02954.x 22780692 Foley RN Parfrey PS Sarnak MJ Clinical epidemiology of cardiovascular disease in chronic renal disease Am J Kidney Dis 1998 32 S112 S119 10.1053/ajkd.1998.v32.pm9820470 9820470 Ohno I Relationship between hyperuricemia and chronic kidney disease Nucleosides Nucleotides Nucleic Acids 2011 30 1039 1044 10.1080/15257770.2011.611484 22132954 Loomans CJ De Koning EJ Staal FJ Rabelink TJ Zonneveld AJ Endothelial progenitor cell dysfunction in type 1 diabetes: another consequence of oxidative stress? Antioxid Redox Signal 2005 7 1468 1475 10.1089/ars.2005.7.1468 16356109 Reinhart K Bayer O Brunkhorst F Meisner M Markers of endothelial damage in organ dysfunction and sepsis Crit Care Med 2002 30 S302 S312 10.1097/00003246-200205001-00021 12004252 Goon PK Lip GY Boos CJ Stonelake PS Blann AD Circulating endothelial cells, endothelial progenitor cells, and endothelial microparticles in cancer Neoplasia 2006 8 79 88 10.1593/neo.05592 16611400 Drexler H Hornig B Endothelial dysfunction in human disease J Mol Cell Cardiol 1999 31 51 60 10.1006/jmcc.1998.0843 10072715 Thorin E Webb DJ Endothelium-derived endothelin-1 Pflugers Arch 2010 459 951 958 10.1007/s00424-009-0763-y 19967386 Abe M Maruyama N Okada K Matsumoto S Matsumoto K Soma M Effects of lipid-lowering therapy with rosuvastatin on kidney function and oxidative stress in patients with diabetic nephropathy J Atheroscler Thromb 2011 18 1018 1028 10.5551/jat.9084 21921413 Liao MT Sung CC Hung KC Wu CC Lo L Lu KC Insulin resistance in patients with chronic kidney disease J Biomed Biotechnol 2012 2012 691369 22919275 Muntner P He J Hamm L Loria C Whelton PK Renal insufficiency and subsequent death resulting from cardiovascular disease in the United States J Am Soc Nephrol 2002 13 745 753 11856780 Fridovich I The competitive inhibition of uricase by oxonate and by related derivatives of S-triazines J Biol Chem 1965 240 2491 2494 14304858 Mazzali M Kanellis J Han L Feng L Xia YY Chen Q Kang DH Gordon KL Watanabe S Nakagawa T Lan HY Johnson RJ Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-independent mechanism Am J Physiol Renal Physiol 2002 282 F991 F997 11997315 Choi JH Kim KL Huh W Kim B Byun J Suh W Sung J Jeon ES Oh HY Kim DK Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure Arterioscler Thromb Vasc Biol 2004 24 1246 1252 10.1161/01.ATV.0000133488.56221.4a 15155385 Edwards NL The role of hyperuricemia in vascular disorders Curr Opin Rheumatol 2009 21 132 137 10.1097/BOR.0b013e3283257b96 19339923 Filiopoulos V Hadjiyannakos D Vlassopoulos D New insights into uric acid effects on the progression and prognosis of chronic kidney disease Ren Fail 2012 34 510 520 10.3109/0886022X.2011.653753 22260409 Jalal DI Chonchol M Chen W Targher G Uric acid as a target of therapy in CKD Am J Kidney Dis 2013 61 1 134 146 10.1053/j.ajkd.2012.07.021 23058478 Gersch C Palii SP Kim KM Angerhofer A Johnson RJ Henderson GN Inactivation of nitric oxide by uric acid Nucleosides Nucleotides Nucleic Acids 2008 27 967 978 10.1080/15257770802257952 18696365 Zharikov S Krotova K Hu H Baylis C Johnson RJ Block ER Patel J Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells Am J Physiol Cell Physiol 2008 295 C1183 C1190 10.1152/ajpcell.00075.2008 18784379 Furchgott RF Zawadzki JV The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine Nature 1980 288 373 376 10.1038/288373a0 6253831 Annuk M Zilmer M Lind L Linde T Fellstrom B Oxidative stress and endothelial function in chronic renal failure J Am Soc Nephrol 2001 12 2747 2752 11729244 Fenster BE Tsao PS Rockson SG Endothelial dysfunction: clinical strategies for treating oxidant stress Am Heart J 2003 146 218 226 10.1016/S0002-8703(02)94796-4 12891188 Vaziri ND Ni Z Oveisi F Liang K Pandian R Enhanced nitric oxide inactivation and protein nitration by reactive oxygen species in renal insufficiency Hypertension 2002 39 135 141 10.1161/hy0102.100540 11799092 Kundhal K Lok CE Clinical epidemiology of cardiovascular disease in chronic kidney disease Nephron Clin Pract 2005 101 c47 c52 10.1159/000086221 15942250 Arici M Walls J End-stage renal disease, atherosclerosis, and cardiovascular mortality: is C-reactive protein the missing link? Kidney Int 2001 59 407 414 10.1046/j.1523-1755.2001.059002407.x 11168922 Menon V Greene T Wang X Pereira AA Marcovina SM Beck GJ Kusek JW Collins AJ Levey AS Sarnak MJ C-reactive protein and albumin as predictors of all-cause and cardiovascular mortality in chronic kidney disease Kidney Int 2005 68 766 772 16014054 Majumdar A Wheeler DC Lipid abnormalities in renal disease J R Soc Med 2000 93 178 182 10844882	23895583	PMC3750429	CC BY	2021-01-05 01:53:03	yes	Eur J Med Res. 2013 Jul 30; 18(1):26
==== Front Allergy Asthma Clin ImmunolAllergy Asthma Clin ImmunolAllergy, Asthma, and Clinical Immunology : Official Journal of the Canadian Society of Allergy and Clinical Immunology1710-14841710-1492BioMed Central 1710-1492-9-262385578010.1186/1710-1492-9-26Short ReportArticle removed: Severe contact dermatitis due to camomile: a common complementary remedy with potential sensitization risks Dogan Sibel 1sibel.dogan@hacettepe.edu.tr1 Ankara Numune Training and Research Hospital, Clinic of Dermatology and Venerology, Ankara 06100, Turkey2013 15 7 2013 9 1 26 26 21 5 2013 30 6 2013 Copyright © 2013 Dogan; licensee BioMed Central Ltd.2013Dogan; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.A retraction article was published for this article. It is available from the following link; http://www.aacijournal.com/content/9/1/28. ==== Body This article has been retracted by the authors. Although the patients originally gave consent to publication of their cases and images, they withdrew this consent shortly after publication. The article is no longer available online in order to protect patient confidentiality. The author apologizes for the inconvenience.	23855780	PMC3750701	CC BY	2021-01-05 01:12:34	yes	Allergy Asthma Clin Immunol. 2013 Jul 15; 9(1):26
==== Front Diagn PatholDiagn PatholDiagnostic Pathology1746-1596BioMed Central 1746-1596-8-1042380004210.1186/1746-1596-8-104ResearchRNAi targeting CXCR4 inhibits proliferation and invasion of esophageal carcinoma cells Wang Tao 12wang0371@126.comMi Yanfang 3myf00079@126.comPian Linping 4plp932@163.comGao Ping 1wang37127070@tom.comXu Hong 5wang37127070@126.comZheng Yuling 6zhengyuling1@sina.comXuan Xiaoyan 7xuanxiaoyanzzu@sina.com1 Department of Hemato-tumor, The First Affiliated Hospital of Henan College, University of TCM, Zhengzhou, P.R. China2 The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, P.R. China3 Department of Otolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou P.R. China4 Department of Ultrasound, The First Affiliated Hospital College, TCM of Henan, Zhengzhou P.R. China5 Henan Tumor Institute, Zhengzhou, P.R. China6 Henan University of TCM, Zhengzhou, P.R. China7 College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, P.R. China2013 24 6 2013 8 104 104 23 5 2013 3 6 2013 Copyright © 2013 Wang et al.; licensee BioMed Central Ltd.2013Wang et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.CXC chemokine receptor 4 was found to be expressed by many different types of human cancers and its expression has been correlated with tumor aggressiveness, poor prognosis and resistance to chemotherapy. However the effect of CXCR4 on the esophageal carcinoma cells remains unclear, the present study explored the effects of CXCR4 siRNA on proliferation and invasion of esophageal carcinoma KYSE-150 and TE-13 cells. Two siRNA sequence targeting CXCR4 gene were constructed and then were transfected into KYSE-150 and TE-13 cells by Lipofectamine™2000. Changes of CXCR4 mRNA and protein were analyzed by qRT-PCR and Western blot. Effect of CXCR4 siRNA on KYSE-150 and TE-13 cells proliferation was determined by MTT. Transwell invasion assay was used to evaluate the invasion and metastasis of KYSE-150 and TE-13 cells. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. qRT-PCR and Western blot demonstrate that the expression level of CXCR4 gene were obviously decreased in KYSE-150 and TE-13 cells transfected with CXCR4 targeting siRNA expression vectors. The average amount of cells transfected with CXCR4 siRNA penetrating Matrigel was significantly decreased (p<0.05). Injection of CXCR4 siRNA transfected cells inhibited tumor growth in a xenograft model compared with blank and negative control groups (p <0.05). CXCR4 silenced by siRNA could suppress the proliferation, invasion and metastasis of esophageal carcinoma cell lines KYSE-150 and TE-13 in vitro and in vivo. The results provide a theoretical and experimental basis for the gene therapy of ESCC using RNAi technology based on CXCR4 target site. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3502376691001138 Esophageal carcinomaCXCR4ProliferationInvasion ==== Body Introduction ESCC is one of the most common malignant tumors in China, and the early invasion is the main reason for its poor prognosis [1-3]. In recent years, the role of chemokine receptor in cancer cell invasion and metastasis have been concerned widely all over the world. CXCR4 (CXC chemokine receptor 4, CXCR4) is a receptor of SDF-1 (stromal cell – derived factor-1, SDF-1). CXCR4 was found to be expressed in many different types of human cancers and its expression has been correlated with tumor aggressiveness, poor prognosis and resistance to chemotherapy [4-6]. These findings suggest that CXCR4 is a potentially attractive therapeutic target. However the effect of CXCR4 on the esophageal carcinoma cells remains unclear, the present study examined the effect of CXCR4 on the proliferation and invasion of the esophageal carcinoma cell lines KYSE-150 and TE-13. Materials and methods Cells and cell culture The human esophageal carcinoma cell line KYSE-150 and TE-13 were purchased from the Chinese Academy of Sciences Cell Bank. All cells were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, USA) and grown in a 37°C, 5% CO2 incubator. Oligonucleotides and cell transfection The oligonucleotides used in this study were chemically synthesized by Sangon Co. Ltd (Shanghai, China). The sequences used were as follows: siRNA1: sense, 5'-UAAAAUCUUCCUGCCCACCdTdT-3', siRNA2: sense, 5'-GGAAGCUGUUGGCUGAAAAdTdT-3' Negative control siRNA: sense, 5′ GGUGAACUGUCUGGAUAAG 3′.For transfection, 2×105 cells were seeded into each well of six well plates and grown overnight until they were 50–80% confluent. Cells were washed, placed in serum-free medium and transfected with siRNA using Lipofectamine™2000 according to the manufacturer’s instructions (Invitrogen). After 6 h, the medium was changed to complete medium, and cells were cultured at 37°C in 5% CO2. Four groups were generated for all experiments, a blank control group (blank control); a negative control group (negative control); and a siRNA1 group (siRNA1); a siRNA2 group (siRNA2). Real time PCR analysis Total RNA was extracted using the Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcription and amplification were performed using a qPCR Quantitation Kit. The ABI 7300 HT Sequence Detection system (Applied Biosystems, Foster City, CA, USA) was used to detect the relative levels of CXCR4 in siRNA-transfected cells. The amplification reaction (40 μl) included 2× real-time Buffer (20 μl), the special primer set (0.8 μl), ddH2O (18 μl), cDNA (1 μl), and 5 U/μl Taq DNA polymerase (0.2 μl). For CXCR4 (470 bp), forward: 5'-ACCGAGGAAATGGGCTCAGGG-3'; reverse: 5'-ATAGTCAGCAGGAGGGCAGGGA -3'. The reaction conditions were as follows: stage 1, 95°C for 3 min (1 cycle); stage 2, 95°C for 14 s followed by 65°C for 45 s (40 cycles); stage 3, from 62°C up to 95°C followed by 0.2°C for 2 s (1 cycle). The results of real-time PCR were analyzed by the DDCt method. Quantitative CXCR4 expression data were calculated using 2-Ct. The experiments were performed independently four times. Western blot analysis The four experimental groups of cells were lysed for total protein extraction. The protein concentration was determined by the BCA method (KeyGEN, China), and 30 μg of protein lysates were subjected to SDS-PAGE. The electrophoreses proteins were transferred to nitrocellulose membranes (Whatman, USA), which were blocked in 5% non-fat milk and incubated overnight at 4°C with diluted antibodies against CXCR4 (1:800, Cell Signaling Technology, USA), Membranes were then incubated with HRP-conjugated secondary antibody (1:2,500, Santa Cruz, USA). After washing with PBST buffer (PBS containing 0.05% Tween-20), membranes were probed using ultra-enhanced chemiluminescence western blotting detection reagents. GAPDH was used as the internal reference. The experiments were performed independently four times. Transwell invasion assay Transwell filters (Costar, USA) were coated with matrigel (3.9 μg/μl, 60–80 μl) on the upper surface of the polycarbonic membrane (6.5 mm in diameter, 8 μm pore size). After 30 min of incubation at 37°C, the matrigel solidified and served as the extracellular matrix for tumor cell invasion analysis. Cells were harvested in 100 μl of serum free RPMI-1640 medium and added to the upper compartment of the chamber. The cells that had migrated from the matrigel into the pores of the inserted filter were fixed with 100% methanol, stained with hematoxylin, mounted, and dried at 80°C for 30 min. The number of cells invading the matrigel was counted from three randomly selected visual fields, each from the central and peripheral portion of the filter, using an inverted microscope at 200× magnification. The experiments were performed independently four times. MTT assay Cell proliferation was assessed by the MTT (Moto-nuclear cell direc cytotoxicityassay) [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]assay. The four experimental groups of cells were plated at 1 × 103 cells/well on 96-well plates. 20 μL of MTT (5 mg/mL) was added to each well after 48 h and then sub-cultured in the medium with 100 μL DMSO. The absorbance of each well was determined at 490 nm. The experiments were performed independently four times. Nude mouse tumor xenograft model Forty immunodeficient female BALB/C nude mice, 5–6 weeks old (purchased from the Experimental Animal Center of the Henan province, China) were randomly divided into eight groups (four groups per cell line, five mice per group). They were bred under aseptic conditions and maintained at constant humidity and temperature. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Zhengzhou University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Zhengzhou University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Mice in the different groups were subcutaneously injected in the dorsal scapular region with the corresponding cells from each experimental condition. Tumors were harvested after four weeks. Nude mouse tumor xenograft immunohistochemical analysis Select the four groups of tumor tissue respectively, formalin-fixed, paraffin-embedded specimens and tissue sections. HE staining observed the pathological changes of the tumor under a microscope. Observe the expression of CXCR4 protein with the method of immunohistochemical SP staining. The positive color is brown particles. Each film were counted 5 high power field (400 x), each of the visual observation of 200 cells and recording the value of the positive cells as a result of statistical analysis, calculate the average number of positive cells in the cells of each array was 200. No coloration is negative. Statistical analysis SPSS17.0 was used for statistical analysis. One-way analysis of variance (ANOVA) and the χ2 test were used to analyze the significance between groups. Multiple comparisons between the parental and control vector groups were made using the Least Significant Difference test when the probability for ANOVA was statistically significant. All data represent mean±SD. Statistical significance was set at p<0.05. Results CXCR4 siRNA inhibited significantly the mRNA expression of CXCR4 After siRNA interference KYSE-150 for 48 h, compared with negative control and blank control group, CXCR4 mRNA expression was inhibited in CXCR4 siRNA1 group and CXCR4 siRNA2 group. TE-13 cells using the targeting CXCR4 siRNA interference after 48 h, RT-PCR results also showed that CXCR4 mRNA expression in CXCR4 siRNA1 group and siRNA2 group was inhibited compared to negative control and blank control group as shown in Figure 1, the targeted CXCR4 siRNA inhibited significantly the mRNA expression of CXCR4 gene. Figure 1 CXCR4 siRNA inhibited significantly the mRNA expression of CXCR4. Cells were incubated with different synthetic oligonucleotides as described in the materials and methods section, and CXCR4 mRNA was quantified by real time PCR. The targeted CXCR4 siRNA inhibited significantly the expression of CXCR4 gene (p <0.05). CXCR4 siRNA inhibited significantly the protein expression of CXCR4 After siRNA1 and siRNA interference KYSE-150 for 48 h, Western-Blot results showed that CXCR4 protein was decreased by 50.6% and 45.6% respectively, compared with negative control group. TE-13 cells using siRNA1 and siRNA interference after 48h, Western-Blot results also showed that CXCR4 protein was decreased by 54.1% and 44.4% compared with negative control group (Figure 2). The results showed that CXCR4 siRNA1 and siRNA2 not only effectively inhibit the expression of CXCR4 mRNA, but also play an effective inhibition for CXCR4 protein expression, and targeting CXCR4 siRNA1 interference effect is more obvious. Figure 2 CXCR4 siRNA inhibited significantly the protein expression of CXCR4. Cells were incubated with different synthetic oligonucleotides as described in the materials and methods section, and CXCR4 protein was quantified by Western-Blot. The targeted CXCR4 siRNA inhibited significantly the protein expression of CXCR4 gene (p <0.05). siRNA CXCR4 reduced esophageal carcinoma cells invasion in vitro In order to detect the effects of CXCR4 on invasion of esophageal carcinoma, we used transwell invasion assay and found that KYSE-150 and TE-13 penetrating cells transfected with CXCR4 siRNA1 and CXCR4 siRNA2 reduced significantly compared to negative control and blank control group (Figure 3). It is no significant difference between negative control and blank control group. siRNA CXCR4 gene significantly inhibited esophageal squamous cell carcinoma KYSE-150 and TE-13 cells invasion ability in vitro. Figure 3 siRNA CXCR4 reduced esophageal carcinoma cells invasion in vitro. Cells were incubated with different synthetic oligonucleotides as described in the materials and methods section, and cells invasion ability was deteced by transwell invasion assay. The targeted CXCR4 siRNA reduced esophageal carcinoma cells invasion ability (p <0.05). siRNA CXCR4 inhibited esophageal carcinoma cells proliferation in vitro In order to explore the effects of CXCR4 on proliferation of esophageal carcinoma, we used MTT experiment. The results showed that absorbance value (A value) of KYSE-150 and TE-13 cells transfected with the CXCR4 siRNA1 and CXCR4 siRNA2 decreased significantly (Figure 4). It can be seen that inhibition of CXCR4 expression in vitro could not only decreased the invasion and metastasis but also inhibit the proliferation. Figure 4 siRNA CXCR4 inhibited esophageal carcinoma cells proliferation in vitro. A. TE-13 cells were incubated with different synthetic oligonucleotides as described in the materials and methods section, and cells proliferation was detected by MTT assay. The targeted CXCR4 siRNA inhibited esophageal carcinoma TE-13 cell proliferation. B. KYSE-150 cells were incubated with different synthetic oligonucleotides as described in the materials and methods section, and cells proliferation was detected by MTT assay. The targeted CXCR4 siRNA inhibited esophageal carcinoma KYSE-150 cell proliferation. The growth inhibitory effects of CXCR4 were examined in vivo using a xenograft model of esophageal cancer Nude mice were inoculated KYSE-150 and TE-13 cells after about one week, all nude mice have tumor, inoculation site appears small subcutaneous nodules. At first, tumor was oval, later becoming irregular, uneven surface. After the mice died four weeks later, strip nude mice tumor and weigh by electronic balance. The results showed that tumor weight of CXCR4 siRNA1 and CXCR4 siRNA2 group was significantly lower than that negative control and blank control group in the nude mouse, the difference have a statistically significant (p <0.05) (Figure 5). There was no significant difference (p > 0.05) of tumor weight between negative control and blank control group. The results showed that the siRNA of targeting CXCR4 inhibited tumor growth in a xenograft model. Figure 5 The growth inhibitory effects of CXCR4 were examined in vivo using a xenograft model of esophageal cancer. A. Nude mice were inoculated KYSE-150. Four weeks later, strip nude mice tumor and weigh by electronic balance. The targeted CXCR4 siRNA inhibited tumor growth in a xenograft model (p <0.05). B. Nude mice were inoculated TE-13 cells. Four weeks later, strip nude mice tumor and weigh by electronic balance. The targeted CXCR4 siRNA inhibited tumor growth in a xenograft model. CXCR4 siRNA inhibited xenografts tumor tissue CXCR4 mRNA expression effectively in vivo In nude mice esophageal carcinoma cell xenografts model, expression of CXCR4 mRNA in CXCR4 siRNA1 and CXCR4 siRNA2 group were lower significantly than in negative control and blank control group (Figure 6), The results showed that CXCR4 siRNA inhibited xenografts tumor tissue CXCR4 mRNA expression effectively in vivo. Figure 6 CXCR4 siRNA inhibited xenografts tumor tissue CXCR4 mRNA expression effectively in vivo. In nude mice esophageal carcinoma cell xenografts model, xenografts tumor tissue CXCR4 mRNA expression was quantified by real time PCR. CXCR4 siRNA inhibited xenografts tumor tissue CXCR4 mRNA expression in vivo (p <0.05). The histopathologic changes of nude mice tumor tissues The histopathologic changes of tumor tissues from two nude mice models bearing esophageal carcinoma were observed using HE stains. The tumor cells in group CXCR4 siRNA1 and CXCR4 siRNA2 had apoptosis and necrosis. Morphology was different from each other, furthermore, nuclei showed pathological division and the giant tumor cells were rare (Figure 7). While the tumor cells in negative control and blank control group were sheet arranged, size were different from each other, nuclei were large and showed multi morphology, pathological cell division and giant tumor cells were in much with marked atypia. Figure 7 The histopathologic changes of nude mice tumor tissues. The histopathologic changes of tumor tissues from two nude mice models bearing esophageal carcinoma were observed using HE stains. The tumor cells in group CXCR4 siRNA1 and CXCR4 siRNA2 had apoptosis and necrosis, morphology was different from each other, nuclei showed pathological division and the giant tumor cells were rare (400 x). CXCR4 protein expression of CXCR4 siRNA1 group was reduced in nude mice xenografts Compared with negative control and blank control group, CXCR4 protein expression of CXCR4 siRNA1 and CXCR4 siRNA2 group was significantly reduced in the two types of esophageal cell xenografts model in nude mice, the difference was significant (p <0.05)(Table 1 and Figure 8). Figure 8 CXCR4 siRNA inhibited significant CXCR4 protein expression. The expression of CXCR4 protein was observed with the immunohistochemical SP staining. The positive color is brown particles. CXCR4 siRNA inhibited significant CXCR4 protein expression. Table 1 The positive expression number of CXCR4 protein in nude mice xenografts Groups n KYSE-150 nude mice TE-13 nude mice CXCR4siRNA1 200 81±5 86±5* CXCR4siRNA2 200 97±4* 92±6* negative control 200 140±8 132±7 blank control 200 143±4 148±3 The positive color is brown particles. Each film were counted 5 high power field (400 x), each of the visual observation of 200 cells and recording the value of the positive cells as a result of statistical analysis, calculate the average number of positive cells in the cells of each array was 200. No coloration is negative. Compared with negative control and blank control group, CXCR4 protein expression of CXCR4 siRNA1 and CXCR4 siRNA2 group was significantly reduced in the two types of esophageal cell xenografts model in nude mice, the difference was significant (* p <0.05). Discussion Chemokine receptors are parts of G protein-coupled receptor superfamily whose function is changing by cell types and activities. A large amount of chemokine and receptor reactions are produced by the different combination of ligand and receptor. CXCR4 is a 7-transmembrane G-coupled receptor which belongs to the chemokine receptors family, and is expressed by variety of cells during development and adult life. Its unique ligand is SDF-1, CXCL12 in systematic name. SDF-1 and CTCR4 have a high affinity and their specific binding, SDF-1–CXCR4 axis, is the molecular basis of the biological function which plays an important role in cell communication and cell migration [4,5]. Chemokine receptor, CXCR4, was first reported high expression in human breast tumor and its’ metastasis by Mullar A etc. [7]. In breast cancer cell, polymerization of reactive protein and formation of pseudopod were mediated by CXCR4 signal transduction pathway who leads chemotaxis and invade reactions latter [8]. As the research of CXCR4 gene continues, 23 kinds of cancer cells, at least, until now, were found that have selective high expression about it, including ovarian cancer, renal cell carcinoma, colorectal cancer, melanoma, esophageal cancer and so on [9-23]. Nowadays, RNAi has become a powerful strategy for knockdown and for understanding gene function. RNAi is a general mechanism for the sequence-specific gene-silencing induced by double stranded RNA [24]. RNAi, mediated by small interfering RNA (siRNA), is a double stranded form of RNA that is about 21–23 nucleotide long and is specific for the sequence of its target [25]. For siRNA to be a useful tool in gene knockdown experiments and ultimately for therapeutic purposes, siRNA-mediated transcriptional silencing must be specific. Many studies [26-30] have shown that siRNA mediated gene silencing can be a reliable and valuable approach for large-scale screening of gene function and drug-target identification and validation. Esophageal cancer is one of the most common malignant tumors in China. Though the diagnosis and treatment of esophageal carcinoma have developed much these years, the esophageal carcinoma runs quickly in clinical and has a worse prognosis for its invasion and metastasis in the early stage [31,32]. For the further study about the effect of SDF-1–CXCR4 axis on the invasion and metastasis of ESCC and provide the new target spot to remedy ESCC, the siRNA for CXCR4 were designed to research the effects of CXCR4 on proliferation and invasion of esophageal carcinoma. Two siRNA vectors aiming to CXCR4 gene were constructed. CXCR4 siRNA was transfected into KYSE-150 and TE-13 cells by Lipofectamine™2000. Changes of CXCR4 mRNA and protein were analyzed by RT-PCR and Western blot. The results showed that the two siRNA sequence target gene of CXCR4 could both restrain the expression of ECSS CXCR4 gene and the interference effect of siRNA 1 was better. It means that different target sites make different interference effects. The transwell results indicated that the numbers of penetrating cells transfected with CXCR4 siRNA1 and CXCR4 siRNA2 reduced significantly compared to negative control and blank control group, which showed CXCR4 silenced by siRNA could suppress the proliferation, invasion and metastasis of esophageal carcinoma cell lines KYSE-150 and TE-13. For the purpose of verifying the function of CXCR4 siRNA in vivo, the animal model of xenograft tumor of carcinoma of esophagus was structured using KYSE-150 and TE-13. In the xenograft tumor, it showed that tumors of the CXCR4 siRNA group were restrained obviously. The further experiment indicated that the CXCR4 mRNA and the protein expression were obviously lower than the control groups, and the tumor cells had apoptosis and necrosis. Morphology were different from each other, furthermore, nuclei showed pathological division and the giant tumor cells were rare. CXCR4 siRNA could not only, in vivo, interfere with CXCR4 gene and affect cell proliferation of KYSE-150 and TE-13 cell, but also decrease the expression of CXCR4 and suppress tumor growth in vivo. The results provide a theoretical and experimental basis for the gene therapy of ESCC using RNAi technology based on CXCR4 target site. Conclusions CXCR4 silenced by siRNA could suppress the proliferation, invasion and metastasis of esophageal carcinoma cell lines KYSE-150 and TE-13 in vitro and in vivo. The results provide a theoretical and experimental basis for the gene therapy of ESCC using RNAi technology based on CXCR4 target site. Competing interests The authors declare that they have no competing interests. Authors’ contributions XYX, LPP and TW: conceived of the study, and participated in its design and coordination and helped to draft the manuscript. PG, HX and YLZ: carried out part of experiments and wrote the manuscript. LPP and TW performed the statistical analysis. All authors read and approved the final manuscript. Acknowledgments This study was supported by the Education Department of Henan province science and technology research projects (12A36009). ==== Refs Siegel R Naishadham D Jemal A Cancer statistics CA Cancer J Clin 2012 62 1 10 29 10.3322/caac.20138 22237781 Parkin DM Pisani P Ferlay J Estimates of the worldwide incidence of 25 major cancers in 1990 Int J Cancer 1990 80 827 841 10074914 Montesano R Hollstein M Hainant P Genetic alterations in esophageal cancer and their relevance to etiology and pathogenesis: a review Int J Cancer 1996 69 225 235 10.1002/(SICI)1097-0215(19960621)69:3<225::AID-IJC13>3.0.CO;2-6 8682592 Burger JA Kipps TJ CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment Blood 2006 107 1761 1767 10.1182/blood-2005-08-3182 16269611 Murdoch C CXCR4: chemokine receptor extraordinaire Immunol Rev 2000 177 175 184 10.1034/j.1600-065X.2000.17715.x 11138774 Duda DG Kozin SV Kirkpatrick ND Xu L Fukumura D CXCL12 (SDF1-α) - CXCR4/CXCR7 pathway inhibition: an emerging sensitizer for anticancer therapies Clin Cancer Res 2011 17 2074 2080 10.1158/1078-0432.CCR-10-2636 21349998 Muller A Homey B Soto H Ge N Catron D Involvement of chemokine receptors in breast cancer metastasis Nature 2001 410 50 56 10.1038/35065016 11242036 Liang Z Yoon Y Votaw J Goodman MM Williams L Silencing of CXCR4 blocks breast cancer metastasis Cancer Res 2005 65 967 971 15705897 Berghuis D Santos SJ Baelde HJ Taminiau AH Egeler RM Pro-inflammatory chemokine-chemokine receptor interactions within the Ewing sarcoma microenvironment determine CD8(+) T-lymphocyte infiltration and affect tumour progression J Pathol 2011 223 347 357 10.1002/path.2819 21171080 Wang L Wang Z Yang B Yang Q Wang L CXCR4 nuclear localization follows binding of its ligand SDF-1 and occurs in metastatic but not primary renal cell carcinoma Oncol Rep 2009 22 1333 1339 19885584 Wang SC Lin JK Wang HS Yang SH Li AF Nuclear expression of CXCR4 is associated with advanced colorectal cancer Int J Colorectal Dis 2010 25 1185 1191 10.1007/s00384-010-0999-1 20607251 Namlos HM Kresse SH Muler CR Henriksen J Holdhus R Global gene expression profiling of human osteosarcomas reveals metastasis-association chemokine pattern Sarcoma 2012 2012 639038 22518090 Baumhoer D Smida J Zillmer S Rosemann M Atkinson MJ Strong expression of CXCL12 is associated with a favorable outcome in osteosarcoma Mod Pathol 2012 25 522 528 10.1038/modpathol.2011.193 22173290 Grymula K Tarnowski M Wysoczynski M Drukala J Barr FG Overlapping and distinct role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 axes in regulating metastatic behavior of human rhabdomyosarcomas Int J Cancer 2010 127 2554 2568 10.1002/ijc.25245 20162608 Furusato B Mohamed A Uhlen M Rhim JS CXCR4 and cancer Pathol Int 2010 60 497 505 10.1111/j.1440-1827.2010.02548.x 20594270 Tanaka T Bai Z Srinoulprasert Y Yang BG Hayasaka H Chemokines in tumor progression and metastasis Cancer Sci 2005 96 317 322 10.1111/j.1349-7006.2005.00059.x 15958053 Redjal N Chan JA Segal RA Kung AL CXCR4 inhibition synergizes with cytotoxic chemotherapy in gliomas Clin Cancer Res 2006 12 6765 6771 10.1158/1078-0432.CCR-06-1372 17121897 Taichman RS Cooper C Keller ET Pienta KJ Taichman NS Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone Cancer Res 2002 62 1832 1837 11912162 Azab AK Runnels JM Pitsillides C Moreau AS Azab F CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy Blood 2009 113 4341 4351 10.1182/blood-2008-10-186668 19139079 Kurtova AV Tamayo AT Ford RJ Burger JA Mantle cell lymphoma cells express high levels of CXCR4, CXCR5, and VLA-4 (CD49d): importance for interactions with the stromal microenvironment and specific targeting Blood 2009 113 4604 4613 10.1182/blood-2008-10-185827 19228923 Li JK Yu L Shen Y Zhou LS Wang YC Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro World J Gastroenterology 2008 14 2308 2313 10.3748/wjg.14.2308 Lee HJ Kim SW Kim HY Li S Yun HJ Chemokine receptor CXCR4 expression, function, and clinical implications in gastric cancer Int J Oncol 2009 34 2 473 480 19148483 Peled A Wald O Burger J Development of novel CXCR4-based therapeutics Expert Opin Investig Drugs 2012 21 341 353 10.1517/13543784.2012.656197 22283809 Hannon GJ A interference Nature 2002 418 244 251 10.1038/418244a 12110901 Tuschl T Borkhardt A Small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy Mol Interventions 2002 2 158 167 10.1124/mi.2.3.158 Semizarov D Frost L Sarthy A Kroeger P Halbert DN Specificity of short interfering RNA determined through gene expression signatures Proc Natl Acad Sci 2003 11 6347 6352 12746500 Jackson AL Bartz SR Schelter J Kobayashi SV Burchard J Expression profiling revealsoff-target gene regulation by RNAi Nat Biotechnol 2003 21 635 637 10.1038/nbt831 12754523 Ghildiyal M Zamore PD Small silencing RNAs: an expanding universe Nat Rev Genet 2009 10 94 108 10.1038/nrg2504 19148191 Kim VN Han J Siomi MC Biogenesis of small RNAs in animals Nat Rev Mol Cell Biol 2009 10 126 139 10.1038/nrm2632 19165215 Czech B Hannon GJ Small RNA sorting: matchmaking for Argonautes Nat Rev Genet 2001 12 19 31 21116305 Kastenmeier A Gonzales H Gould JC Robotic applications in the treatment of diseases of the esophagus Surg Laparosc Endosc Percutan Tech 2012 22 4 304 309 10.1097/SLE.0b013e318258340a 22874678 Palanivelu C Prakash A Senthilkumar R Senthilnathan P Parthasarathi R minimally invasive esophagectomy: thoracoscopic mobilization of the esophagus and medi-astinal lymphadenectomy in prone position-experience of 130 patients J Am Coll Surg 2006 203 1 7 16 10.1016/j.jamcollsurg.2006.03.016 16798482	23800042	PMC3751032	CC BY	2021-01-05 01:30:33	yes	Diagn Pathol. 2013 Jun 24; 8:104
==== Front BMC GastroenterolBMC GastroenterolBMC Gastroenterology1471-230XBioMed Central 1471-230X-13-1262393745410.1186/1471-230X-13-126Research ArticleHigh expression level and nuclear localization of Sam68 are associated with progression and poor prognosis in colorectal cancer Liao Wen-Ting 13Liaowt2002@gmail.comLiu Jun-Ling 4liujl@sysucc.org.cnWang Zheng-Gen 5wangzghd@yahoo.com.cnCui Yan-Mei 13355341388@qq.comShi Ling 2sling100@163.comLi Ting-Ting 13vilition@163.comZhao Xiao-Hui 2Xiaohuizhao27@gmail.comChen Xiu-Ting 2Xiuting2005@163.comDing Yan-Qing 13dyq@fimmu.comSong Li-Bing 2lb.song1@gmail.com1 Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China2 State Key Laboratory of Oncology in Southern China, Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, P.R. China3 Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China4 State Key Laboratory of Oncology in South China, Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, China5 Department of Gastroenterology, the Second Affiliated Hospital, University of South China, HengYang, Hunan 421000, China2013 9 8 2013 13 126 126 25 8 2012 2 8 2013 Copyright © 2013 Liao et al.; licensee BioMed Central Ltd.2013Liao et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Src-associated in mitosis (Sam68; 68 kDa) has been implicated in the oncogenesis and progression of several human cancers. The aim of this study was to investigate the clinicopathologic significance of Sam68 expression and its subcellular localization in colorectal cancer (CRC). Methods Sam68 expression was examined in CRC cell lines, nine matched CRC tissues and adjacent noncancerous tissues using reverse transcription (RT)-PCR, quantitative RT-PCR and Western blotting. Sam68 protein expression and localization were determined in 224 paraffin-embedded archived CRC samples using immunohistochemistry. Statistical analyses were applied to evaluate the clinicopathologic significance. Results Sam68 was upregulated in CRC cell lines and CRC, as compared with normal tissues; high Sam68 expression was detected in 120/224 (53.6%) of the CRC tissues. High Sam68 expression correlated significantly with poor differentiation (P = 0.033), advanced T stage (P < 0.001), N stage (P = 0.023) and distant metastasis (P = 0.033). Sam68 nuclear localization correlated significantly with poor differentiation (P = 0.002) and T stage (P =0.021). Patients with high Sam68 expression or Sam68 nuclear localization had poorer overall survival than patients with low Sam68 expression or Sam68 cytoplasmic localization. Patients with high Sam68 expression had a higher risk of recurrence than those with low Sam68 expression. Conclusions Overexpression of Sam68 correlated highly with cancer progression and poor differentiation in CRC. High Sam68 expression and Sam68 nuclear localization were associated with poorer overall survival. Sam68BiomarkerPrognosisColorectal cancer ==== Body Background Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide. Although advances have been made in diagnostic and therapeutic techniques, the prognosis of CRC patients with distant metastases still remains poor [1]. Thus, characterization of the molecular mechanism that involves in progression and metastasis of CRC could help to identify specific biomarkers which may facilitate efficient therapeutic stratification, prediction and disease prevention. Src-associated in mitosis, 68 kDa (Sam68) belongs to the signal transduction and activation of RNA (STAR) family of K homology (KH) domain-containing RNA binding proteins [2] and is originally identified as a substrate for Src kinase phosphorylation during mitosis [3,4]. Sam68 is ubiquitously expressed and plays important roles in signaling transduction, gene transcription, and alternative splicing [2,5]. Sam68 has been suggested to act as an adaptor in signal transduction by binding to SH3- and SH2-containing proteins, through its proline-rich regions [6]. Additionally, Sam68 can interact with signaling proteins, such as Src, Grb2, Grap, SHP-1, PLCγ1/Fyn, BRK and PI3K, and has been implicated in T-cell receptor and insulin receptor signaling, as well as Ras and PI3K kinase pathways [7-11]. Moreover, Sam68 is usually a nuclear protein and plays a major role in the regulation of RNA metabolism, including mRNA transcription, alternative splicing and nuclear export [12-17]. The alternative splicing of multiple genes regulated by Sam68, including those involved in oncogenesis, such as CD44, Bcl-xl, Sgce, SMN2, SF2/ASF and Cyclin D1 [6,14-19]. The above mentioned biological functions of Sam68 closely linked this protein to oncogenic properties. First, Sam68 is involved in promotion of cell cycle progression, cell proliferation, transformation, tumorigenesis and metastasis in different cellular context [20-25]. Second, a series of published articles in the recent decade have demonstrated that Sam68 participates in transcriptional and post-transcriptional regulation of gene expressions that are relevant to human cancer [14,15,17,18,20,26]. However, deregulation of Sam68 in human cancer tissues has only been observed in limited cancer types, including prostate cancer, renal cell carcinoma, breast and cervical cancer [23,24,26-28]. Whether the deregulation of Sam68 is a prevalent event in human cancer needs further investigation. To explore the deregulation of Sam68 in human colorectal cancer, we investigated the expression patterns of Sam68 in human CRC tissues, and the correlation between Sam68 expression levels and the clinicopathologic features of CRC. Our current study indicates that the expression and localization of Sam68 may act as independent biomarkers of prognosis in CRC, suggesting that Sam68 has potential as a novel therapeutic target for the treatment of CRC. Methods Cell lines Colorectal cancer cell lines including LS174t, Colo205, SW480, HT29, HCT116 and SW620 cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 μg/μL streptomycin and 100 μg/μL penicillin in a humidified incubator containing 5% CO2 at 37°C. Patients and tissue specimens For the use of clinical materials for research purposes, prior patients' consents and approval were obtained from the Sun Yat-sen University and Cancer Center Institutional Board. All samples were collected and analyzed with prior written informed consents from the patients. A total of 224 paraffin-embedded colorectal cancer samples, which were histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center between the year 2000 to 2003, were examined. All of the patients had received chemotherapy after surgery. Prior patient consent and approval were obtained from the Institutional Research Ethics Committee. The clinicopathological features of the patients are summarized in Additional file 1: Table S1. The final study population included 97 female and 127 male patients (age range, 23–82 years). Follow-up was recorded from the date of surgery until death. Patients who died of cancer (or other causes) were classified as dead. The median follow-up time for all patients was 58.47 months. 43 corresponding metastatic lymph nodes were also obtained from the above mentioned patients. Four pairs of CRC biopsies and the matched adjacent non-cancerous colon tissues were obtained from each patient during surgery, and immediately frozen and maintained in liquid nitrogen until further use. RNA extraction, reverse transcription-polymerase chain reaction and quantitative Real-time polymerase chain reaction Total RNA was extracted from the cultured cells and surgical tissues using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (Q-PCR) analysis of Sam68 expression were performed as previously described [29], using previously published primers and probes [28]. Sam68 expression was analyzed using the 2-∆∆Ct method as described by Livak et al. [30] and normalized to the geometric mean expression level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative change in Sam68 expression was calculated by pair-wise comparison of the normalized Sam68 expression level in the tumor samples with the adjacent non-cancerous tissue samples from the same patient. Western blotting Tissue and cell lysates were prepared using SDS lysis buffer and the protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were separated by electrophoresis on a 10.5% sodium dodecyl sulfate polyacrylamide gel and electrotransferred from the gel to a nitrocellulose membrane. After blocking with 5% milk solution in Tris-buffered saline with Tween (TBST) for 1 hour, the membrane was incubated with primary antibody against rabbit antibody Sam68 (sc-333, dilution, 1:500; Santa Cruz Biotechnology, CA, USA) and rabbit anti-α-Tubulin (1:1000, Sigma, Saint Louis, MI, USA) primary antibodies. After washing with TBS-T, the membrane was incubated with secondary antibody against rabbit immunoglobulin G or mouse immunoglobulin G; then, it was examined with the enhanced chemiluminescence detection system (Amersham Biosciences Europe, Freiberg, Germany) according to the manufacturer’s instructions. Immunohistochemistry Paraffin sections were deparaffinized with xylene and rehydrated, then submerged into EDTA antigenic retrieval buffer and microwaved for antigenic retrieval. The sections were then treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation with 1% BSA to block the non-specific binding. The sections were incubated with rabbit anti-Sam68 (sc-333, dilution, 1:500; Santa Cruz Biotechnology, CA, USA) overnight at 4°C. As negative controls, rabbit anti-Sam68 antibody was replaced with normal goat serum, or the rabbit anti-Sam68 antibody was blocked by co-incubation with a recombinant Sam68 polypeptide at 4°C overnight prior to the immunohistochemical staining. The staining intensity was scored on a scale of 0 to 3 as 0 (no staining), 1 (weak staining ~ light yellow), 2 (moderate staining ~ yellowish brown) or 3 (strong staining ~ brown). Tumors with a staining intensity ≥ 2 in which at least 50% of the malignant cells were Sam68-positive were classified as high expression; tumors with a staining intensity < 2 or in which less than 50% of the malignant cell were Sam68-positive were classified as low expression. Statistical analysis All statistical analyses were carried out using SPSS 10.0 (Chicago, IL, USA). The significance of the differences between the normal and tumor tissue Q-PCR results were assessed using the Student’s two-tailed t-test. The association between Sam68 expression and clinicopathological variables were assessed using the Mann–Whitney U test. Survival curves were plotted using the Kaplan-Meier method. The Cox proportional hazards regression model was used for univariate and multivariate analysis. Two-tailed P values < 0.05 were considered significant. Results Expression of Sam68 in colorectal cancer cell lines We examined the expression of Sam68 using Western blotting in seven human colon cancer cell lines and two cases of normal intestine tissues. The results displayed that Sam68 protein expression level was much higher in CRC cell lines than that in normal intestine tissues (Figure 1A). We next measured the expression of Sam68 mRNA in the CRC cell lines using RT-PCR (Figure 1B) and (Figure 1C). In agreement with the protein expression levels, the Sam68 mRNA expression level was much higher in CRC cell lines than that in normal intestine tissues. Figure 1 Analysis of Sam68 protein and mRNA expression in colorectal cancer (CRC) cell lines and normal intestine tissues. (A) Analysis of Sam68 protein expression in CRC cell lines (LS174t, Colo205, SW480, HT29, HCT116, SW620) and two cases of normal intestine tissues (N1 and N2) by Western blotting. (B) Analysis of Sam68 mRNA expression by RT-PCR. (C) Analysis of Sam68 mRNA expression in CRC cell lines and normal intestine tissues by Q-PCR, the average ratio of Sam68 expression normalized to GAPDH is shown; values are the mean ± SD of three parallel experiments. Sam68 is upregulated in primary human CRC lesions Western blotting and RT-PCR analyses were performed to determine the expression of Sam68 in nine paired primary CRC tissues and the matched adjacent non-cancerous tissues. Sam68 was significantly upregulated at both the protein (Figure 2A) and mRNA levels (Figure 2B) in all nine of the CRC tissues tested, compared to the matched adjacent normal tissues from the same patient. Q-PCR results confirmed that Sam68 mRNA was upregulated in the tumor samples by up to 18.3-fold (Sam68 tumor/normal [T/N] ratio; Figure 2C; P < 0.01, Student’s t-test). Figure 2 Sam68 is upregulated in primary colorectal cancer (CRC) tissues compared with the adjacent normal tissues. (A, B) Analysis of Sam68 mRNA expression in primary CRC tissues (T) and the paired adjacent normal tissues (N) by RT-PCR (A) and Q-PCR (B). GAPDH was used as loading control. (C, D) Analysis of Sam68 protein expression in primary CRC tissues and the paired adjacent normal tissues by Western blotting (C) and immunohistochemistry (D). In agreement with the Western blotting results, immunohistochemical analysis confirmed that Sam68 was overexpressed in all nine of the CRC tissues tested, compared with the paired adjacent normal tissues (Figure 2D). Taken together, these results indicated that Sam68 is upregulated in CRC lesions at both transcriptional and translational levels. We further performed immunohistochemical analysis to determine the expression patterns of Sam68 in 224 paraffin-embedded CRC tissues and 43 lymph node metastatic tissues. Negative to moderate Sam68 staining was detected in the adjacent normal tissues (Figure 3A-D); however, positive Sam68 staining was detected in 206 of the 224 (92%) tumor tissues. The tumors could be divided into a low Sam68 expressing group (104 cases) and a high Sam68 expressing group (120 cases, Additional file 1: Table S1). Additionally, two main patterns of Sam68 protein expression were observed in the tumors: cytoplasmic localization (Figure 3E-F) and nuclear localization (Figure 3G-J). As shown in Additional file 1: Table S1, 61.6% (138/224) of the tumor samples displayed nuclear staining and 38.4% (86/224) displayed cytoplasmic staining. Moreover, positive expression of Sam68 was detected in 81.4% (35/43) of the lymph node metastases (Figure 4) and 65.1% (28/43) of lymph node metastases were classified as high Sam68 expressing. Figure 3 Representative images of Sam68 immunohistochemical analysis in colorectal cancer (CRC) tissues. (A – D) Negative (A, 100X; B, 400X) or moderate (C, 100X; D, 400X) Sam68 staining was detected in the adjacent non-cancerous tissues. (E – H) Cytoplasmic Sam68 staining (E, 100X; F, 400X) or nuclear Sam68 staining (G, 100X; H, 400X) was observed in primary CRC tissues. (I – J) Sam68 was upregulated in CRC compared to the paired adjacent non-cancerous tissues (I, 100X; J, 400X). Figure 4 Immunohistochemical analysis of Sam68 expression in metastatic lymph node tissues from colorectal cancer (CRC) patients. Negative, moderate or strong Sam68 staining was observed in CRC metastatic lymph node tissues. Correlations between increased expression of Sam68 and clinical aggressiveness in CRC Statistic analyses were performed to evaluate the expression patterns of Sam68 and the clinicopathological features of CRC. As shown in Table 1, the high Sam68 expression level was strongly correlated with poor tumor differentiation (P = 0.033), advanced T stage (P < 0.001), N stage (P = 0.023), and distant metastasis (P = 0.033) in this cohort of 224 cases of CRC. In addition, high expression level of Sam68 was significantly associated with the nuclear localization of Sam68 (P = 0.012). Moreover, Nuclear localization of Sam68 correlated significantly with tumor differentiation (P = 0.002) and advanced T stage (P = 0.021). These observations suggest that increased expression of Sam68 or nuclear localization of Sam68 was closely associated with aggressive phenotypes of CRC. Table 1 Correlation between clinicopathologic features and Sam68 expression levels and localization Characteristics Sam68 levels P values Sam68 localization R values P values Low High Nuclear Cytoplasm Age ≤ mean(56) 50 56 0.833 66 40 0.848 > mean (56) 54 64 72 46 Gender Male 61 66 0.583 76 51 0.535 Female 43 54 62 35 Histology Columnar adenocarcinoma 79 102 110 71 0.733 Mucinous adenocarcinoma 14 11 0.083 19 6 Others 11 7 9 9 Differentiation Well and moderate 87 86 0.033 97 76 0.002 Poor and undifferentiated 17 34 41 10 T stage 1 ~ 2 27 13 18 22 0.021 3 58 64 <0.001 77 45 4 19 43 43 19 N stage 0 68 59 0.023 72 55 0.072 1 27 48 50 25 2 9 13 16 6 Distant metastasis 0 (no) 78 74 0.033 89 63 0.173 1 (yes) 26 46 49 23 Sam68 localization Nuclear 55 83 0.012 Cytoplasm 49 37 Sam68 is associated with poor prognosis in CRC patients In univariate Cox regression analysis, tumor differentiation, T stage, N stage and distant metastasis were significant prognostic factors in this cohort of CRC patients (Additional file 2: Table S2; P < 0.001). Kaplan-Meier survival analysis demonstrated that patients with low levels of Sam68 expression had significantly longer median survival than patients with low Sam68 expression (Figure 5A, upper panel; 71 vs. 51 months; P = 0.024, log-rank test). The 5-year survival rate for patients with low Sam68 expression was 70% (95% confidence interval, 0.609-0.779), compared to 54% (95% confidence interval, 0.448-0.635) for patients with high Sam68 expression. Figure 5 Influence of Sam68 expression on overall survival in colorectal cancer (CRC). (A) Kaplan–Meier curves showing that patients with high Sam68 expression had poorer overall survival than patients with low Sam68 expression; analysis of 224 primary CRC tissues (P = 0.003). (B) Kaplan–Meier curves showing that patients with high Sam68 expression in metastatic lymph node tissues had poorer overall survival than patients with low Sam68; analysis of 43 metastatic lymph node tissues (P = 0.029). (C) Kaplan–Meier curves showing that patients with nuclear Sam68 expression had poorer overall survival than patients with cytoplasmic Sam68 expression; analysis of 224 primary CRC tissues (P = 0.005). Next, we analyzed the relationship between the expression of Sam68 in metastatic lymph nodes and survival. Kaplan-Meier analysis indicated that the expression level of Sam68 in the metastatic lymph nodes had a significant impact on survival (Figure 5B; P =0.029, log-rank test), as the median overall survival time of patients expressing low levels of Sam68 in the metastatic lymph nodes was significantly longer than patients expressing high levels of Sam68 in the metastatic lymph nodes (log-rank test, P = 0.029). We also analyzed the prognostic value of the subcellular localization of Sam68 in CRC. Spearman’s rank correlation revealed that nuclear localization of Sam68 in CRC tumors correlated significantly with poorer survival (Spearman Rho −0.232, P = 0.001). Additionally, Kaplan-Meier survival analysis confirmed that the median survival time for patients with Sam68 nuclear localization was significantly shorter than patients with Sam68 cytoplasmic localization (Figure 5C; 54 vs. 73 months, P = 0.024, log-rank test). The 5-year survival rate for patients with Sam68 cytoplasmic localization was 70% (95% confidence interval, 0.609-0.779), compared to 54% (95% confidence interval, 0.448-0.635) for patients with Sam68 nuclear localization. The Sam68 expression level, subcellular localization of Sam68, pathological stage and N stage were identified as independent prognostic factors for overall survival in CRC in multivariate survival analysis (Additional file 3: Table S3 and Additional file 4: Table S4). Furthermore, in the subgroups of CRC patients without distant metastasis (M0) or with well/moderately differentiated tumors, both the Sam68 expression level (Figure 6A and C) and subcellular localization of Sam68 (Figure 6E and G) correlated significantly with overall survival. However, no such correlations were observed in the subgroup of patients with distant metastasis or poorly differentiated tumors (Figure 6B,D,F and H). Figure 6 Overall survival curves for colorectal cancer (CRC) patients stratified by the Sam68 expression level, according to M classification and tumor differentiation. In the M0 classification (A) and moderately differentiated tumor subgroups (C), patients with low Sam68 expression had significantly better overall survival than patients with high Sam68 expression (P = 0.01). In the M1 classification (B) and poorly differentiated tumor subgroups (D), the overall survival of patients with high and low Sam68 expression was not significantly different. In the M0 classification (E) and moderately differentiated tumor subgroups (G), patients with Sam68 cytoplasmic localization had significantly better overall survival (P = 0.027 and P = 0.024, respectively) than patients with Sam68 nuclear localization. In the M1 classification (F) and poorly differentiated subgroups (H), the overall survival of patients with Sam68 cytoplasmic and nuclear localization was not significantly different. Discussion Sam68 is a substrate of the oncogenic Src kinase, which is often activated in human cancers [4]. Previous researches suggested that two opposing functions of Sam68 were reported in different cellular contexts. On one hand, a few studies indicated that Sam68 acted as a tumor suppressor. For example, Sam68 deficiency resulted in neoplastic transformation of murine NIH3T3 fibroblasts. Reduction of Sam68 was associated with anchorage-independent growth, defective contact inhibition, and the ability to form metastatic tumors in nude mice [31], while overexpression of Sam68 in NIH-3 T3 fibroblasts led to both cell cycle arrest and apoptosis [21]. On the other hand, a large proportion of recent reports demonstrated that Sam68 played an oncogenic role. Sam68 knockdown in polyoma middle T-antigen (PyMT) oncogene transformed cell lines delayed tumorigenesis and metastasis formation in nude mice [25]. Busà R and colleagues have demonstrated that Sam68 was upregulated in prostate cancer at both protein and mRNA levels. Additionally, downregulation of Sam68 in prostate cancer cells delayed cell cycle progression and reduced the proliferation rate [23]. Sam68 is also upregulated and its upregulation is correlated with shorter survival rates in breast cancer, cervical cancer, renal cell carcinoma [24,27,28]. The present study demonstrated that Sam68 was elevated in CRC tissues and the high Sam68 expression level was significantly correlated with the characteristics of aggressive CRC (including poor differentiation of tumors, advanced T stage, lymph node involvement and distant metastasis). Additionally, high Sam68 expression level was a significant predictor of poor prognosis in CRC patients. Thus, our results raised the evidence that suggested that Sam68 might promote development and progression of human CRC, supporting the pro-oncogene role of Sam68 in human cancer. Sam68 is a ubiquitously expressed protein and resides in both cytoplasm and nuclei [2]. Posttranslational modifications of Sam68, such as phosphorylation and methylation, can affect its subcellular localization, interaction with signaling proteins, as well as affinity for target RNAs [2,15,32-35]. In most cells, Sam68 predominantly resided within the nucleus and is involved in gene transcription, alternative splicing, and nuclear export [12-19]. Sam68 has been observed to exist in dynamic nuclear foci termed Sam68 nuclear bodies (SNBs), also called stress nuclear bodies [12,36]. Genes regulated by Sam68 include CD44, Bcl-xl, Sgce, SMN2, SF2/ASF, Cyclin D1, and so on, which are all involved in oncogenesis [6,14-19]. In the present study, Sam68 was found to localize to both the nuclei and cytoplasm of cancer cells. It is particularly noteworthy that the subgroup of patients with advanced clinical stage CRC often exhibited nuclear localization of Sam68, while CRC patients with well differentiated or early stage tumors often displayed cytoplasmic Sam68 staining. In addition, patients with cytoplasmic Sam68 localization had a better clinical outcome than patients with Sam68 nuclear localization. These researches suggested that nuclear Sam68 might play a dominant role in oncogenesis of CRC. However, distinguished from our results, cytoplasmic localization of Sam68 was significantly correlated with cancer progression and poor prognosis in human renal cell carcinoma and breast cancer [24,27]. It could be due to the functions of Sam68 in multiple signaling pathways, since it can be expressed in both the cytoplasm and nucleus. In the cytoplasm, Sam68 interacts with signaling molecules such as Src, Grb2, Grap [7-11] and stimulates oncogenic pathways, including the epidermal growth factor pathway, ERK and AKT pathways [37,38]. In renal cell carcinoma and breast cancer, the oncogenic role of Sam68 was closely associated with its activation of Akt/GSK-3β pathway [24,27]. Taken together, these researches suggested that cytoplasmic and nuclear localization of Sam68 might contribute to neoplastic transformation or tumor progression through different molecular mechanisms in different cancer types or cellular contexts. This study provides the first evidence to indicate that both high expression level and nuclear localization of Sam68 correlate significantly with invasiveness and aggressiveness characteristics in CRC, and poorer survival of CRC patients. Taken together, this study suggests that Sam68 may represent a novel indicator of progression and prognosis in CRC. Conclusions In conclusion, Sam68 was upregulated in primary human CRC, and high Sam68 expression levels in CRC were associated with the clinical features of aggressive disease and poorer patient prognosis. Nuclear localization of Sam68 in CRC was identified as an independent predictor of poor prognosis. However, further characterization of the mechanisms by which Sam68 is involved in the transformation and progression of human CRC is required. Competing interests The authors declare that they have no competing interests. Authors’ contributions WTL participated in the design of the study and drafted the manuscript. JLL and ZGW carried out the experiment of cell culture and molecular biology. LS supported the statistical analysis. YMC and TTL supported the evaluation of the immunohistochemical results. XHZ and XTC participated in collecting the clinical samples. LBS and YQD participated in the design of the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-230X/13/126/prepub Supplementary Material Additional file 1: Table S1 Clinicopathologic variables for patient cohort (n = 224). Click here for file Additional file 2: Table S2 Univariate Cox regression analysis of potential prognostic factors for CRC patients. Click here for file Additional file 3: Table S3 Multivariate Cox regression analysis of Sam68 levels and other potential prognostic factors for CRC patients. Click here for file Additional file 4: Table S4 Multivariate Cox regression analysis of Sam68 localization and other potential prognostic factors for CRC patients. Click here for file Acknowledgements This study was supported by: The Natural Science Foundation of China (No. 30901791, 81172055); Medical Science and Technology Research Fund Projects of Guangdong Province(N0.A2011199); Guangdong Provincial Natural Science Foundation of China (No. S2012010009643); Zhu Jiang Science&Technology New Star Foundation in Guangzhou city (2012046). ==== Refs Weitz J Koch M Debus J Hohler T Galle PR Buchler MW Colorectal cancer Lancet 2005 365 9454 153 165 10.1016/S0140-6736(05)17706-X 15639298 Lukong KE Richard S Sam68, the KH domain-containing superSTAR Biochim Biophys Acta 2003 1653 2 73 86 14643926 Taylor SJ Shalloway D An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis Nat 1994 368 6474 867 871 10.1038/368867a0 Fumagalli S Totty NF Hsuan JJ Courtneidge SA A target for Src in mitosis Nat 1994 368 6474 871 874 10.1038/368871a0 Bielli P Busa R Paronetto MP Sette C The RNA-binding protein Sam68 is a multifunctional player in human cancer Endocr Relat Cancer 2011 18 4 R91 R102 10.1530/ERC-11-0041 21565971 Richard S Torabi N Franco GV Tremblay GA Chen T Vogel G Morel M Cleroux P Forget-Richard A Komarova S Ablation of the Sam68 RNA binding protein protects mice from age-related bone loss PLoS Genet 2005 1 6 e74 10.1371/journal.pgen.0010074 16362077 Paronetto MP Venables JP Elliott DJ Geremia R Rossi P Sette C Tr-kit promotes the formation of a multimolecular complex composed by Fyn, PLCgamma1 and Sam68 Oncogene 2003 22 54 8707 8715 14647465 Huot ME Brown CM Lamarche-Vane N Richard S An adaptor role for cytoplasmic Sam68 in modulating Src activity during cell polarization Mol Cell Biol 2009 29 7 1933 1943 10.1128/MCB.01707-08 19139276 Derry JJ Richard S Valderrama Carvajal H Ye X Vasioukhin V Cochrane AW Chen T Tyner AL Sik (BRK) phosphorylates Sam68 in the nucleus and negatively regulates its RNA binding ability Mol Cell Biol 2000 20 16 6114 6126 10.1128/MCB.20.16.6114-6126.2000 10913193 Sanchez-Margalet V Najib S Sam68 is a docking protein linking GAP and PI3K in insulin receptor signaling Mol Cell Endocrinol 2001 183 1–2 113 121 11604231 Fusaki N Iwamatsu A Iwashima M Fujisawa J Interaction between Sam68 and Src family tyrosine kinases, Fyn and Lck, in T cell receptor signaling J Biol Chem 1997 272 10 6214 6219 10.1074/jbc.272.10.6214 9045636 Chen T Boisvert FM Bazett-Jones DP Richard S A role for the GSG domain in localizing Sam68 to novel nuclear structures in cancer cell lines Mol Biol Cell 1999 10 9 3015 3033 10.1091/mbc.10.9.3015 10473643 Lin Q Taylor SJ Shalloway D Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains J Biol Chem 1997 272 43 27274 27280 10.1074/jbc.272.43.27274 9341174 Matter N Herrlich P Konig H Signal-dependent regulation of splicing via phosphorylation of Sam68 Nat 2002 420 6916 691 695 10.1038/nature01153 Paronetto MP Achsel T Massiello A Chalfant CE Sette C The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x J Cell Biol 2007 176 7 929 939 10.1083/jcb.200701005 17371836 Chawla G Lin CH Han A Shiue L Ares M JrBlack DL Sam68 regulates a set of alternatively spliced exons during neurogenesis Mol Cell Biol 2009 29 1 201 213 10.1128/MCB.01349-08 18936165 Paronetto MP Cappellari M Busa R Pedrotti S Vitali R Comstock C Hyslop T Knudsen KE Sette C Alternative splicing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68 Cancer Res 2011 70 1 229 239 20028857 Valacca C Bonomi S Buratti E Pedrotti S Baralle FE Sette C Ghigna C Biamonti G Sam68 regulates EMT through alternative splicing-activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene J Cell Biol 2010 191 1 87 99 10.1083/jcb.201001073 20876280 Pedrotti S Bielli P Paronetto MP Ciccosanti F Fimia GM Stamm S Manley JL Sette C The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy Embo J 2010 29 7 1235 10.1038/emboj.2010.19 20186123 Babic I Cherry E Fujita DJ SUMO modification of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis Oncogene 2006 25 36 4955 4964 10.1038/sj.onc.1209504 16568089 Taylor SJ Resnick RJ Shalloway D Sam68 exerts separable effects on cell cycle progression and apoptosis BMC Cell Biol 2004 5 5 10.1186/1471-2121-5-5 14736338 Barlat I Maurier F Duchesne M Guitard E Tocque B Schweighoffer F A role for Sam68 in cell cycle progression antagonized by a spliced variant within the KH domain J Biol Chem 1997 272 6 3129 3132 10.1074/jbc.272.6.3129 9013542 Busa R Paronetto MP Farini D Pierantozzi E Botti F Angelini DF Attisani F Vespasiani G Sette C The RNA-binding protein Sam68 contributes to proliferation and survival of human prostate cancer cells Oncogene 2007 26 30 4372 4382 10.1038/sj.onc.1210224 17237817 Song L Wang L Li Y Xiong H Wu J Li J Li M Sam68 up-regulation correlates with, and its down-regulation inhibits, proliferation and tumourigenicity of breast cancer cells J Pathol 2010 222 3 227 237 10.1002/path.2751 20662004 Richard S Vogel G Huot ME Guo T Muller WJ Lukong KE Sam68 haploinsufficiency delays onset of mammary tumorigenesis and metastasis Oncogene 2008 27 4 548 556 10.1038/sj.onc.1210652 17621265 Rajan P Gaughan L Dalgliesh C El-Sherif A Robson CN Leung HY Elliott DJ Regulation of gene expression by the RNA-binding protein Sam68 in cancer Biochem Soc Trans 2008 36 Pt 3 505 507 18481990 Li Z Yu CP Zhong Y Liu TJ Huang QD Zhao XH Huang H Tu H Jiang S Zhang Y Sam68 expression and cytoplasmic localization is correlated with lymph node metastasis as well as prognosis in patients with early-stage cervical cancer Ann Oncol 2012 23 3 638 10.1093/annonc/mdr290 21700735 Zhang Z Li J Zheng H Yu C Chen J Liu Z Li M Zeng M Zhou F Song L Expression and cytoplasmic localization of SAM68 is a significant and independent prognostic marker for renal cell carcinoma Cancer Epidemiol Biomarkers Prev 2009 18 10 2685 2693 10.1158/1055-9965.EPI-09-0097 19755649 Liao WT Wang X Xu LH Kong QL Yu CP Li MZ Shi L Zeng MS Song LB Centromere protein H is a novel prognostic marker for human nonsmall cell lung cancer progression and overall patient survival Cancer 2009 115 7 1507 1517 10.1002/cncr.24128 19170237 Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method Methods 2001 25 4 402 408 10.1006/meth.2001.1262 11846609 Liu K Li L Nisson PE Gruber C Jessee J Cohen SN Neoplastic transformation and tumorigenesis associated with sam68 protein deficiency in cultured murine fibroblasts J Biol Chem 2000 275 51 40195 40201 10.1074/jbc.M006194200 11032831 Sette C Post-translational regulation of star proteins and effects on their biological functions Adv Exp Med Biol 2010 693 54 66 10.1007/978-1-4419-7005-3_4 21189685 Sette C Messina V Paronetto MP Sam68: a new STAR in the male fertility firmament J Androl 2010 31 66 74 10.2164/jandrol.109.008136 19875495 Lukong KE Larocque D Tyner AL Richard S Tyrosine phosphorylation of sam68 by breast tumor kinase regulates intranuclear localization and cell cycle progression J Biol Chem 2005 280 46 38639 38647 10.1074/jbc.M505802200 16179349 Cote J Boisvert FM Boulanger MC Bedford MT Richard S Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1 Mol Biol Cell 2003 14 1 274 287 10.1091/mbc.E02-08-0484 12529443 Denegri M Chiodi I Corioni M Cobianchi F Riva S Biamonti G Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors Mol Biol Cell 2001 12 11 3502 3514 10.1091/mbc.12.11.3502 11694584 Martin-Romero C Sanchez-Margalet V Human leptin activates PI3K and MAPK pathways in human peripheral blood mononuclear cells: possible role of Sam68 Cell Immunol 2001 212 2 83 91 10.1006/cimm.2001.1851 11748924 Locatelli A Lange CA Met receptors induce Sam68-dependent cell migration by activation of alternate extracellular signal-regulated kinase family members J Biol Chem 2011 286 24 21062 21072 10.1074/jbc.M110.211409 21489997	23937454	PMC3751151	CC BY	2021-01-05 01:15:57	yes	BMC Gastroenterol. 2013 Aug 9; 13:126
==== Front BMC Pregnancy ChildbirthBMC Pregnancy ChildbirthBMC Pregnancy and Childbirth1471-2393BioMed Central 1471-2393-13-1602393767810.1186/1471-2393-13-160Research ArticleFolic acid supplementation, dietary folate intake during pregnancy and risk for spontaneous preterm delivery: a prospective observational cohort study Sengpiel Verena 1verena.sengpiel@obgyn.gu.seBacelis Jonas 1jonas.bacelis@gu.seMyhre Ronny 2ronny.myhre@fhi.noMyking Solveig 2solveig.myking@fhi.noPay Aase Devold 3aaseserinedevold.pay@fhi.noHaugen Margaretha 4margaretha.haugen@fhi.noBrantsæter Anne-Lise 4anne.lise.brantsaeter@fhi.noMeltzer Helle Margrete 4helle.margrete.meltzer@fhi.noNilsen Roy M 5roy.nilsen@mfr.uib.noMagnus Per 6per.magnus@fhi.noVollset Stein Emil 7vollset@uib.noNilsson Staffan 8staffan@chalmers.seJacobsson Bo 12bo.jacobsson@obgyn.gu.se1 Department of Obstetrics and Gynaecology, Sahlgrenska Academy, Sahlgrenska University Hospital/Östra, SE-416 85 Göteborg, Sweden2 Department of Genes and Environment, Division of Epidemiology, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, NO-0403 Oslo, Norway3 Department of Obstetrics, Oslo University Hospital, P.O. Box 4950, Nydalen, NO-0424 Oslo, Norway4 Department of Exposure and Risk Assessment, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, NO-0403 Oslo, Norway5 Department of Public Health and Primary Health Care, University of Bergen, NO-5018 Bergen, Norway6 Division of Epidemiology, Norwegian Institute of Public Health, P.O. Box 4404, NO-0403 Oslo, Norway7 Norwegian Institute of Public Health and University of Bergen, Kalfarveien 31, NO-5018 Bergen, Norway8 Mathematical Sciences, Chalmers University of Technology, SE-412 96 Göteborg, Sweden2013 12 8 2013 13 160 160 8 2 2013 5 8 2013 Copyright © 2013 Sengpiel et al.; licensee BioMed Central Ltd.2013Sengpiel et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Health authorities in numerous countries recommend periconceptional folic acid to pregnant women to prevent neural tube defects. The objective of this study was to examine the association of folic acid supplementation during different periods of pregnancy and of dietary folate intake with the risk of spontaneous preterm delivery (PTD). Methods The Norwegian Mother and Child Cohort Study is a population-based prospective cohort study. A total of 65,668 women with singleton pregnancies resulting in live births in 1999–2009 were included. Folic acid supplementation was self-reported from 26 weeks before pregnancy until week 24 during pregnancy. At gestational week 22, the women completed a food frequency questionnaire, which allowed the calculation of their average total folate intake from foods and supplements for the first 4–5 months of pregnancy. Spontaneous PTD was defined as the spontaneous onset of delivery between weeks 22+0 and 36+6 (n = 1,628). Results The median total folate intake was 266 μg/d (interquartile range IQR 154–543) in the overall population and 540 μg/d (IQR 369–651) in the supplement users. Eighty-three percent reported any folic acid supplementation from <8 weeks before to 24 weeks after conception while 42% initiated folic acid supplementation before their pregnancy. Cox regression analysis showed that the amount of folate intake from the diet (hazard ratio HR 1.16; confidence interval CI 0.65-2.08) and from the folic acid supplements (HR 1.04; CI 0.95-1.13) was not significantly associated with the risk of PTD. The initiation of folic acid supplementation more than 8 weeks before conception was associated with an increased risk for PTD (HR 1.19; CI 1.05-1.34) compared to no folic acid supplementation pre-conception. There was no significant association with PTD when supplementation was initiated within 8 weeks pre-conception (HR 1.01; CI 0.88-1.16). All analyses were adjusted for maternal characteristics and socioeconomic, health and dietary variables. Conclusions Our findings do not support a protective effect of dietary folate intake or folic acid supplementation on spontaneous PTD. Pre-conceptional folic acid supplementation starting more than 8 weeks before conception was associated with an increased risk of PTD. These results require further investigation before discussing an expansion of folic acid supplementation guidelines. PregnancyPreterm deliveryPreterm birthGestational lengthFolateFolic acid supplementation ==== Body Background Folate is a B-vitamin essential for one-carbon metabolism and takes part in amino acid metabolism as well as DNA synthesis, repair and methylation [1,2]. Women are especially susceptible to folate deficiency during pregnancy, which is a period of rapid fetal growth, organ differentiation and high rates of cell division [1,3,4]. Since the 1950s, folic acid supplementation has been known to prevent megaloblastic anemia during pregnancy [5]. In the 1990s, large randomized trials demonstrated that peri-conceptional folic acid supplementation can prevent neural tube defects (NTD) in the newborn infant [6-8]. Today, national health authorities in many countries recommend periconceptional folic acid supplementation, and some countries have introduced mandatory folate fortification of foods [1,3,4,9,10]. In Norway, folic acid supplementation of 400 μg/d is recommended from the time of planning a pregnancy to gestational week 12 [2,11], as is a daily folate intake of 500 μg/d. This is in line with the Nordic Nutrition Recommendations [2]. Maternal folate status has also been associated with other adverse pregnancy outcomes such as preeclampsia, malformations such as orofacial clefts, spontaneous abortion, fetal death, fetal growth restriction and preterm delivery (PTD), although these results still remain inconclusive [1]. PTD, defined by the World Health Organization (WHO) as birth occurring before 37 weeks of gestation, is considered a major global health problem and is strongly associated with neonatal mortality as well as short- and long-term morbidity [12-14]. Spontaneous PTD is a common, complex condition with a prevalence of approximately 7% in the Norwegian population [15]. However, the effect of any single environmental factor is difficult to measure without large-scale studies [15]. Modern obstetrics are still not able to predict, prevent or treat PTD [16]. Progesterone substitution, the only promising intervention identified to date, has been shown to reduce the chance of spontaneous PTD in high-risk pregnancies, but such cases account for only a small proportion of all pregnancies [17-19]. In the past decade, some observational studies have found that folic acid supplementation reduces the risk of PTD [20-22]. In some studies, this effect has been documented with an extended folic acid supplementation scheme or dosage compared with schemes based on NTD prevention, e.g., pre-conceptional folic acid supplementation for one year or longer [21] or third-trimester folic acid supplementation [22]. The most recent Cochrane review, based on data from 21 studies including one of the largest randomized controlled trials (RCT), as well as a recent meta-analysis of all RCTs published to date, could not confirm any effect of the maternal folate status on the gestational length or the risk of spontaneous PTD [23,24]. The comparability and generalizability of these earlier studies, which focused on the association of folate status and folic acid supplementation with pregnancy outcome, is limited because folic acid supplementation was assessed without considering other folate sources, the study populations had different levels of dietary folate intake, inadequate sample sizes, limited adjustment for important confounders, and/or retrospective study designs with folate data collection only after delivery [1,4]. Although PTD is a heterogeneous pregnancy outcome with different etiologies (early vs. late or iatrogenic vs. spontaneous), previous studies have mostly treated PTD as one entity, obscuring the differences in risk among PTD subtypes [25]. The Norwegian Mother and Child Cohort Study (MoBa) can meet a number of these challenges in study design, a requirement for addressing the inconsistencies in the field. MoBa includes 106,707 pregnancies, enabling the investigation of common complex pregnancy outcomes such as PTD. A detailed prospective assessment of folic acid supplementation starting from 6 months before conception throughout the pregnancy period, data regarding dietary folate intake and comprehensive information about lifestyle habits, health and socioeconomic status provide a unique opportunity to study the association between folate intake and PTD. For example, the effect of folic acid supplementation can be compared between women with low and high dietary folate intakes. By taking into account the amount of dietary folate and folic acid supplementation during different periods of pregnancy, it might be possible to define the folic acid supplementation scheme most likely to affect PTD. The aim of this study was to examine the association of maternal folate intake from both supplemental and dietary sources with the risk of spontaneous PTD, with sub-analyses of early and late spontaneous PTD. The association of folic acid supplementation with PTD was studied in a stratified sample of women with low and high dietary folate intakes (</≥170 μg/d). Methods Study population The dataset is part of the MoBa cohort, initiated by and maintained at the Norwegian Institute of Public Health [15]. In brief, MoBa is a nation-wide pregnancy cohort that has included more than 106,000 pregnancies in the years from 1999 to 2009. The women were recruited through a postal invitation in connection with a routine ultrasound examination offered to all pregnant women in Norway approximately 17 weeks of gestation. Overall, 38.5% of the invited women participated. They were asked to fill in questionnaires focused on overall health status, lifestyle behavior and diet at gestational weeks 15–17 (Q1) and 30 (Q3). At week 22, they completed a food frequency questionnaire (FFQ). All questionnaires are available from the homepage of the Norwegian Institute of Public Health [26]. The present study used data from version 5 of the quality-assured data files made available for research in 2010. The MoBa database is linked to pregnancy and birth records from the Medical Birth Registry of Norway (MBRN) [27]. Informed written consent was obtained from each participant. The Regional Committee for Medical Research and the Norwegian Data Inspectorate approved the study. Of 106,707 pregnancies included in the MoBa version 5, 103,921 pregnancies resulted in live-born singletons. Complete data for all 3 questionnaires including information about folate intake were available for 89,032 pregnancies. Women reporting improbable energy intakes of <4.5 MJ or >20 MJ were excluded [28], leaving 87,565 pregnancies. After exclusion of women with diabetes mellitus, hypertension, autoimmune diseases, inflammatory bowel diseases, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, other immune-compromised conditions and those that underwent in-vitro fertilization, as well as those with pregnancy-related complications such as preeclampsia, hypertension, gestational diabetes, placental abruption, placenta previa, cervical cerclage and serious fetal malformations, 75,916 pregnancies were included in the study. Pregnancies with a gestational length of <22+0 or >42+6 weeks were excluded from further analysis, leaving 75,718 remaining. If a woman participated for more than one pregnancy, only her first pregnancy enrolled was included, leaving 65,668 pregnancies for analyses. Outcome The gestational age in days was determined with a second-trimester ultrasound in 98.3% of the pregnancies and was based on the last menstrual period in the remaining cases. Spontaneous PTD was defined as birth after preterm labor or pre-labor rupture of the membranes between 22+0 and 36+6 weeks. Spontaneous PTD subgroups for early (22+0-33+6) and late (34+0-36+6) PTD were analyzed. Exposure Amount of folate intake The amount of folate intake was calculated from the MoBa FFQ, a semi-quantitative questionnaire designed to yield information regarding the dietary habits and intake of dietary supplements during the first 5 months of pregnancy. The questionnaires were read optically, and the nutrient and energy intakes were calculated using FoodCalc [29] and the Norwegian Food Composition Table [30]. For the calculation of nutrients in the dietary supplements, an Access database (Microsoft Office 2003) containing the nutrient values of more than 1,000 dietary supplements was created and continuously updated. Dietary supplements commonly sold in Norway were registered based on information provided by the respective manufacturer, whereas nutritional information concerning dietary supplements bought from the Internet or abroad were obtained from the manufacturer’s or supplier’s homepage. A data program connected to the Access database read all food supplements recorded by the MoBa participants. The process of extracting dietary and supplement data is described in detail elsewhere [31,32]. Dietary folate was defined as 60% of the reported folate intake from foods, as only approximately 60% may be biologically accessible in comparison to that from the synthetic folic acid in supplements [1,3]. The total folate intake was thus calculated as supplemental folic acid + 0.6 × folate intake from foods. The daily folate intake was also categorized into 4 groups: <170 μg/d (corresponding to the earlier WHO recommendation for all women to prevent anemia [10,33]), 170–500 μg/d (corresponding to the current Nordic Nutrition Recommendations for pregnant women for the prevention of NTDs [2]), 500–1000 μg/d (corresponding to the tolerable upper limit for folic acid [2]) and >1000 μg/d. Time of folic acid supplementation The women reported their folic acid supplement use from 26 weeks before conception until gestational week 24 in 4-week intervals, including the period and frequency of supplementation. A woman was defined as a folic acid supplement user if she reported folic acid supplementation more than once a week in a registered 4-week period. Folic acid could be consumed either in the form of a folic acid supplement or as part of multivitamins. The most commonly used folic acid supplements for pregnant women in Norway contain 400 μg of folic acid, while the most commonly used multivitamin supplements contain 200 μg of folic acid. For this study, the start of folic acid supplementation was categorized as start during 26–9 weeks before conception, start during 8–0 weeks before conception, and no pre-conceptional folic acid supplementation. Covariates Information regarding the maternal age at delivery as well as the child’s sex is available from the MBRN. Parity was based on data from both the MoBa and MBRN and categorized according to the number of previous pregnancies of ≥22+0 weeks’ duration. Marital status was defined as either married/cohabitant or not. The self-reported pre-pregnancy heights and weights were used to calculate the pre-pregnancy body mass index (BMI) and were grouped according to the WHO classification as underweight (<18.5 kg/m2), normal (18.5-24.9 kg/m2), overweight (25.0-29.9 kg/m2) and obese (≥30.0 kg/m2). Maternal education was categorized as ≤12 y, 13–16 y and ≥17 y. The history of previous PTD at 22+0-36+6 weeks of gestation, as well as the history of spontaneous abortion as registered in the MBRN, were taken into account as dichotomous variables. Women reported smoking habits during pregnancy in Q1 and were categorized as non-smokers, occasional or daily smokers. The alcohol intake from different sources was self-reported in the FFQ (glasses/d, week or month) and calculated as g/d. The household income was classified as both the participant and her partner having <300,000 Norwegian Kroner (NOK)/y, as either the participant or her partner having ≥300,000 NOK/y or as the participant and her partner both having ≥300,000 NOK/y. Vitamin A supplementation was registered and categorized in the same manner as described for folic acid. These variables were used as a proxy for multivitamin supplementation, as there are no products on the Norwegian market containing vitamin A alone, and vitamin A is part of all common multivitamin preparations. In MoBa more than 99% of the participants are of Caucasian ethnicity; hence, ethnicity was not a relevant confounder. Statistical methods All statistical analyses were performed using IBM SPSS Statistics 19.0 and R 2.13.1 software. Total dietary folate intake from foods and supplements (median (IQR)) in relation to the maternal characteristics was studied with the Kruskal-Wallis test. The start of folic acid supplementation in relation to the maternal characteristics was studied with Pearson's chi-squared test. The association of total dietary folate intake and spontaneous PTD was estimated as a hazard ratio (HR) with a 95% confidence interval (CI) by using Cox regression both in an unadjusted model and adjusted for the above-mentioned covariates. In these models, the event was defined as a spontaneous PTD; all iatrogenic deliveries and deliveries after the preterm (≥37+0 weeks) or early preterm (≥34+0 weeks) period were censored. The proportional hazards assumption was investigated by testing and inspecting scaled Schoenfeld residuals using R function cox.zph [34]. Statistical significance was assumed for 2-sided p-values of <0.05. Results Folate intake and folic acid supplementation in the study population The median total dietary folate intake during the first five months of pregnancy and start of folic acid supplementation according to maternal characteristics are presented in Table 1. Dietary folate intake was higher in women who were older, did not smoke, had normal BMI (18.5-24.9 kg/m2), who were having their first child, were married/cohabitant, who had higher education levels and family incomes. Women having experienced PTD, as well as women with a history of spontaneous abortion, had significantly lower total folate intakes. While women with a history of spontaneous abortion had more often started folic acid supplementation early, no comparable pattern in women with a history of PTD was found (Table 1). Table 1 Folate variables and maternal characteristics Total folate intake (μg/d) Initiation of preconceptional folic acid supplementation, n (%) n (%) median (IQR) p1 >8 w 0-8 w no p2 Total 65668 (100) 266 (153–543) 15471 (24) 12308 (19) 37889 (58) <25 7583 (12) 228 (143–504) 842 (11) 879 (12) 5862 (77) Maternal age 25-29 22745 (35) 275 (154–547) <0.0001 4990 (22) 4605 (20) 13150 (58) <0.0001 in years 30-34 27782 (42) 270 (155–546) 7475 (27) 5662 (20) 14645 (53) >34 7558 (12) 276 (160–547) 2164 (29) 1162 (15) 4232 (56) Pre- <18.5 2004 (3) 294 (160–554) 439 (22) 344 (17) 1221 (61) pregnancy 18.5-24.9 43198 (66) 278 (157–549) <0.0001 10611 (25) 8388 (19) 24199 (56) <0.0001 BMI in 25-30 13448 (21) 248 (148–533) 2999 (22) 2483 (18) 7966 (59) kg/m2 ≥30 5341 (8) 229 (141–515) 1146 (21) 822 (15) 3373 (63) missing 1677 (3) 226 (144–491) 276 (16) 271 (16) 1130 (67) 0 33501 (51) 320 (165–566) 8753 (26) 6031 (18) 18717 (56) 1 20743 (32) 233 (146–526) <0.0001 4581 (22) 4466 (22) 11696 (56) <0.0001 Parity 2 9199 (14) 211 (144–480) 1752 (19) 1525 (17) 5922 (64) 3+ 2140 (3) 200 (140–388) 370 (17) 272 (13) 1498 (70) missing 85 (0,1) 183 (121–401) 15 (18) 14 (16) 56 (66) Marital yes 63134 (96) 268 (154–544) <0.0001 15184 (24) 12101 (19) 35849 (57) <0.0001 status no 2534 (4) 232 (147–493) 287 (11) 207 (8) 2040 (81) Maternal <13 20618 (31) 214 (138–483) 3237 (16) 2774 (13) 14607 (71) education 13 - 16 27249 (41) 291 (157–553) <0.0001 6841 (25) 5601 (21) 14807 (54) <0.0001 in >16 16415 (25) 331 (171–569) 5096 (31) 3718 (23) 7601 (46) years missing 1386 (2) 243 (144–528) 297 (21) 215 (16) 874 (63) History of no 63433 (97) 267 (154–543) <0.0001 14980 (24) 11879 (19) 36574 (58) 0.18 preterm yes 2155 (3) 237 (151–521) 472 (22) 411 (19) 1272 (59) delivery missing 80 (0,1) 252 (144–554) 19 (24) 18 (23) 43 (54) History of no 53123 (81) 269 (154–544) <0.0001 11655 (22) 10112 (19) 31356 (59) <0.0001 abortion yes 12545 (19) 251 (150–540) 3816 (30) 2196 (18) 6533 (52) never 59678 (91) 277 (156–548) 14752 (25) 11691 (20) 33235 (56) Smoking occasionally 1879 (3) 218 (141–453) <0.0001 255 (14) 231 (12) 1393 (74) <0.0001 habits daily 3733 (6) 190 (128–390) 399 (11) 319 (9) 3015 (81) missing 378 (1) 221 (140–451) 65 (17) 67 (18) 246 (65) Alcohol no alcohol 55368 (84) 289 (159–551) 13360 (24) 10672 (19) 31336 (57) consumption <0.5 6565 (10) 245 (150–528) <0.0001 1403 (21) 1162 (18) 4000 (61) <0.0001 in units/week ≥0.5 3735 (6) 149 (117–209) 708 (19) 474 (13) 2553 (68) Partners with 0 19439 (30) 225 (143–507) <0.0001 3246 (17) 2853 (15) 13340 (69) income of 1 26938 (41) 265 (154–542) 6287 (23) 5253 (20) 15398 (57) <0.0001 >300,000 2 17472 (27) 337 (169–572) 5687 (33) 3939 (23) 7846 (45) NOK/year missing 1819 (3) 227 (147–511) 251 (14) 263 (14) 1305 (72) Baby's male 33445 (51) 262 (152–541) 0.003 7875 (24) 6198 (19) 19372 (58) 0.3 sex female 32223 (49) 270 (155–545) 7596 (24) 6110 (19) 18517 (57) Tertiles of 1 21890 (33) 179 (115–503) 5251 (24) 4031 (18) 12608 (58) energy intake 2 21890 (33) 263 (155–548) <0.0001 5397 (25) 4307 (20) 12186 (56) <0.0001 in MJ 3 21888 (33) 325 (203–591) 4823 (22) 3970 (18) 13095 (60) Amount of total daily folate intake (FFQ data) and initiation of folic acid supplementation (Q1 data) according to maternal characteristics, from 65,668 participants in the Norwegian Mother and Child Cohort Study (2002 – 2009). 1 p-value, estimated with Kruskal-Wallis test. 2 p-value, estimated with Pearson's chi-squared test. Figure 1 illustrates the pattern of folic acid supplementation in the study population compared to vitamin A supplementation (as a proxy for multivitamin consumption) over the course of pregnancy. 83% of all women in the study reported folic acid supplementation at some point before and/or during pregnancy (Figure 1). While 42% began folic acid supplementation prior to conception, nearly 75% used supplements containing folic acid in the first trimester with decreasing use towards the end of pregnancy. At the same time, vitamin A supplementation was much more stable over the whole length of the pregnancy. The folic acid content of the reported supplements varied considerably, with only 2 women consuming folic acid amounts of >5000 μg/d, 575 consuming >1000 μg/d (1%), 6,160 consuming >500 μg/d (9%), 9,589 consuming >400 μg/d (15%), 24,337 consuming >200 μg/d (37%) and 30,708 consuming >100 μg/d (47%). Among supplement users, the median daily folic acid supplementation was 400 μg/d (interquartile range IQR 200–429). As presented in Table 2, folic acid from supplements was the main folate source in supplement users, while the main source was dietary folate in the whole population. There is no mandatory folate fortification of foods in Norway, and only 0.4% (n = 272) of the study population reached the Nordic Nutrition Recommendation of 500 μg/d with their dietary folate intake (2.8%, n = 1,854, if the dietary folate was not adjusted for bio-availability). Of the study participants, 31%, n = 20,369, achieved the recommended levels with their total folate intake (39.6%, n = 26,015, if the dietary folate was not adjusted for bio-availability). Figure 1 Prevalence of folic acid and vitamin A supplementation during pregnancy. Prevalence of folic acid and vitamin A supplementation during pregnancy (Q1 and Q3 data) in women with spontaneous term or preterm delivery (22+0-36+6 weeks, n = 1,628) among 65,668 participants in the Norwegian Mother and Child Cohort Study (2002 – 2009). Table 2 Folate intake from diet and supplements All Spontaneous term delivery Spontaneous PTD1 Folate (μg/d) Median IQR Median IQR Median IQR All women n = 65668 n = 50301 n = 1628 Diet 155 (123–193) 155 (123–193) 155 (123–194) Supplements 67 (0–400) 57 (0–400) 100 (0–400) Total intake 266 (154–543) 262 (153–542) 280 (154–557) Supplement users n = 35510 n = 27007 n = 891 Diet 158 (127–196) 158 (127–196) 159 (128–197) Supplements 400 (200–429) 400 (200–429) 400 (200–457) Total intake 530 (354–636) 530 (354–635) 540 (369–651) Folic acid supplementation, dietary and total folate intake during the first half of pregnancy (FFQ data) for all 65,668 participants, as well as in folic acid supplement users (n = 35,510), from the Norwegian Mother and Child Cohort Study (2002–2009). 1Preterm delivery defined as delivery between 22+0-36+6 weeks of gestation. Folate intake from different sources and risk for spontaneous PTD The median gestational age in this study population was 282 days (IQR 245–288), with 2,236 cases of PTD and 1,628 cases of spontaneous PTD. Of these, 264 babies were delivered before 34+0 weeks of gestation. Thus the PTD rate in our study population is 3.4%, approximately half of the PTD rate in the general Norwegian population as we excluded all risk-pregnancies due to maternal disease, pregnancy-complications or fetal malformation. There was no significant association between the amount of folate intake from the diet or supplements and the risk of spontaneous PTD (Table 3). Likewise, no association was found when the total folate intake was categorized according to the former WHO recommendation for women (>170 μg/d), the current Nordic Nutrition Recommendations for pregnant women (>500 μg/d) and the tolerable upper limit for folic acid supplementation (<1000 μg/d) (Additional file 1: Table S1). Cox regression for the sub-groups of early and late spontaneous PTD did not reveal any significant associations between the folate intake and pregnancy outcome (not shown). Testing the proportional hazards assumptions revealed a slight misfit for the parity variable. Therefore all analyses were also run with stratified Cox regression using parity as strata. The changes from the unstratified analyses were however marginal and did not change any pattern or conclusion. Table 3 Folate intake from different sources and risk of spontaneous preterm delivery (PTD) Unadjusted Adjusted2 Folate (μg/d) HR1 (CI) p HR1 (CI) p Diet 1.10 (0.73–1.66) 0.66 1.16 (0.65–2.08) 0.61 Supplements 1.06 (0.97–1.16) 0.17 1.04 (0.95–1.13) 0.43 Total intake 1.06 (0.98–1.15) 0.16 1.04 (0.95–1.14) 0.39 Amount of folic acid supplementation, dietary and total folate intake (FFQ data) and hazard ratios for spontaneous PTD (22+0-36+6 weeks, n = 1,628). Cox regression for 65,668 participants in the Norwegian Mother and Child Cohort Study (2002–2009). Iatrogenic deliveries have been censored in the regression model. 1 HR per 500 μg extra folate/d. 2 Cox regression, adjusted for maternal age, prepregnancy BMI, parity, history of PTD and spontaneous abortion, child’s sex, smoking habits and alcohol consumption during pregnancy, maternal education, marital status, household income, energy intake. Mutual adjustment for dietary and supplemental folate intake. Initiation of folic acid supplementation and risk for spontaneous PTD The initiation of folic acid supplementation more than 8 weeks before conception was associated with a marginally increased risk of spontaneous PTD also after adjusting for potential confounders (Table 4 and Additional file 2: Figure S1a; HR 1.19; confidence interval CI 1.05-1.34). The initiation of supplementation more than 8 weeks before conception was significantly associated with early (<34+0 weeks) but not late spontaneous PTD (Additional file 3: Figure S1b) however the difference in HR was not significant. After stratification for the total dietary folate intake from foods, the initiation of folic acid supplementation more than 8 weeks before conception was significantly associated with an increased risk of spontaneous PTD for those women with low dietary folate intakes (HR 1.22; CI 1.04-1.45). The interaction between dietary folate intake and initiation of supplementation was not significant, though. The same association was found in the subgroup of early but not late spontaneous PTD (Additional file 4: Table S2). Table 4 Initiation of pre-conceptional folic acid supplementation and risk of spontaneous preterm delivery (sPTD) sPTD Initiation of folic acid Unadjusted Adjusted1 Adjusted2 supplementation n HR (CI) p HR (CI) p HR (CI) p No 919 1 1 1 All 0-8 w preconceptional 281 0.94 (0.82–1.07) 0.36 1.01 (0.88–1.16) 0.91 1.02 (0.88–1.17) 0.82 >8 w preconceptional 428 1.14 (1.02–1.28) 0.02 1.19 (1.05–1.34) 0.01 1.19 (1.04–1.35) 0.01 No 139 1 1 1 Early 0-8 w preconceptional 46 1.02 (0.73–1.42) 0.92 1.15 (0.82–1.62) 0.43 1.10 (0.77–1.57) 0.59 >8 w preconceptional 79 1.39 (1.06–1.84) 0.02 1.53 (1.14–2.04) 0.004 1.45 (1.05–1.99) 0.02 No 780 1 1 1 Late 0-8 w preconceptional 235 0.93 (0.8–1.07) 0.30 0.98 (0.85–1.14) 0.83 1.00 (0.86–1.17) 0.98 >8 w preconceptional 349 1.10 (0.97–1.25) 0.14 1.13 (0.99–1.29) 0.07 1.14 (0.99–1.32) 0.07 Initiation of pre-conceptional folic acid supplementation (Q1 data) and hazard ratios for spontaneous PTD (n = 1,628 for 22+0-36+6 weeks, n = 264 for early (22+0-33+6 weeks), n = 1,364 for late (34+0-36+6 weeks)). Cox regression for 65,668 participants in the Norwegian Mother and Child Cohort Study (2002 – 2009). Iatrogenic deliveries have been censored in the regression model. 1 Cox regression, adjusted for maternal age, prepregnancy BMI, parity, history of PTD and spontaneous abortion, child’s sex, smoking habits and alcohol consumption during pregnancy, maternal education, marital status, household income, energy intake and dietary folate intake. 2 Adjustment as above as well as for first-trimester folic acid supplementation and pre-conceptional and first-trimester vitamin A supplementation. A history of earlier adverse pregnancy outcomes could be a motive for the early initiation of folic acid supplementation in subsequent pregnancies. However, the analysis of the subgroup of primi-gravidae (n = 23,919, 36%) showed the same overall results, with even larger HRs for the early initiation of folic acid supplementation (HR 1.32; CI 1.11-1.57). The early initiation of folic acid supplementation could characterize women that planned a pregnancy but did not become pregnant during the first months, thus including a subgroup of sub-fertile women [35]. In MoBa, the women were asked to report the number of months with regular intercourse without contraception before becoming pregnant, and these data were classified as follows: <1 month (n = 12,912, 20%), 1–2 months (n = 14,818, 23%) and >2 months (n = 21,883, 33%). Stratification for this variable still showed increased HRs for the early initiation of folic acid supplementation in the subgroup that became pregnant within the first month (HR 1.39; CI 1.02-1.89), again with larger HRs for the subgroup with early PTD (HR 2.32; CI 1.20-4.48). Pregnancy period of folic acid supplementation and risk for spontaneous PTD The time of folic acid supplementation was represented by four variables corresponding to the following periods: 26–9 weeks before conception, 0–8 weeks before conception, first trimester and second trimester. If the Cox regression included all of the confounders and the outcome of spontaneous PTD was analyzed, the prediction of the model improved after introducing all four folic acid supplementation variables (p = 0.005). Folic acid supplement use more than 8 weeks pre-conception was associated with an increased HR for spontaneous PTD, even after adjusting for the supplementation at all other time points studied (Table 5). The marginally significant association with decreased HRs for first-trimester supplementation was no longer significant if the model was adjusted for vitamin A supplementation (data not shown). Table 5 Timing of folic acid supplementation and risk of spontaneous preterm delivery (sPTD) sPTD Time of olic acid Unadjusted Adjusted1 supplementation n HR (CI) p HR (CI) p >8 w pre-conceptional 428 1.16 (1.04–1.30) 0.01 1.16 (1.02–1.31) 0.02 All 0-8 w pre-conceptional 572 1.02 (0.93–1.13) 0.64 1.04 (0.92–1.17) 0.52 1st trimester 1185 0.94 (0.84–1.05) 0.25 0.88 (0.78–0.99) 0.04 2nd trimester 795 1.11 (1.01–1.23) 0.03 1.10 (0.99–1.21) 0.08 >8 w pre-conceptional 79 1.39 (1.07–1.80) 0.01 1.34 (0.99–1.80) 0.06 Early 0-8 w pre-conceptional 103 1.21 (0.94–1.55) 0.13 1.18 (0.88–1.58) 0.26 1st trimester 199 1.07 (0.81–1.42) 0.62 0.94 (0.69–1.28) 0.67 2nd trimester 137 1.26 (0.99–1.60) 0.06 1.18 (0.91–1.51) 0.21 >8 w pre-conceptional 349 1.12 (0.99–1.27) 0.07 1.13 (0.98–1.29) 0.09 Late 0-8 w pre-conceptional 469 0.99 (0.89–1.11) 0.88 1.01 (0.89–1.15) 0.84 1st trimester 986 0.91 (0.81–1.03) 0.14 0.87 (0.76–0.99) 0.04 2nd trimester 658 1.09 (0.98–1.21) 0.13 1.08 (0.97–1.21) 0.17 Folic acid supplementation at different times (Q1 and Q3 data) and hazard ratios for spontaneous PTD (n = 1,628 for 22+0-36+6 weeks, n = 264 for early (22+0-33+6), n = 1,364 for late (34+0-36+6)). Cox regression for 65,668 participants in the Norwegian Mother and Child Cohort Study (2002 – 2009). Iatrogenic deliveries have been censored in the regression model. 1 Cox regression, adjusted for maternal age, prepregnancy BMI, parity, history of PTD and spontaneous abortion, child’s sex, smoking habits and alcohol consumption during pregnancy, maternal education, marital status, household income, energy intake and dietary folate intake. Mutual adjustment for folic acid supplementation at other time points. Discussion In this large prospective national birth cohort study, we did not find any statistically significant association between the amount of folate intake from the diet or supplements and spontaneous PTD in uncomplicated pregnancies. Folic acid supplementation starting more than 8 weeks before conception was associated with an increased HR for spontaneous PTD. When interpreting the results, the selection of the study population has to be kept in mind: all known risk-pregnancies due to maternal disease, pregnancy complications or fetal malformation have been excluded from the analysis. There might be an association between the amount of folate intake from diet or supplements and spontaneous PTD in those pregnancies excluded. Our results, demonstrating no significant protective effect of the maternal folate intake or folic acid supplementation on the spontaneous PTD risk, support a number of earlier observational studies [36-41] and RCTs [6,42,43]. A reanalysis of the most recent Cochrane review, based on data from 21 studies and one of the largest RCTs as well as a recent meta-analysis of all RCTs published to date, could not confirm the effect of the maternal folate status on the gestational length or risk of PTD [23,24]. Shaw et al. found a comparable association of increased PTD risk and pre-conceptional folic acid supplementation when analyzing data from the US National Birth Defects Study [41]. In addition, extensive supplementation with multivitamins with a major folic acid component was associated with an increased risk of PTD in a study by Alwan et al. [36]. However, some recent observational studies have found that folic acid supplementation reduces the risk of PTD [20-22]. In some cases, this association was linked to pre-conceptional folic acid supplementation for 1 year or longer [21] or third-trimester folic acid supplementation [22], raising questions about extended supplementation schemes compared to the NTD prevention scheme. A protective effect of folic acid supplementation was supported by a modest reduction in the PTD rate after the introduction of folate fortification of foods [9]. One of the most obvious explanations for these conflicting results could be the dosage of folic acid. While most of the studies finding an association with gestational length or PTD were based on comparably high doses of folic acid (≥5000 μg/d [20,22,44], ≥2500 μg/d [22,45] and ≥500 μg/d [46-48]), very few women in our study population consumed as much as 5000 μg/d of supplemental folic acid, while only 9% consumed >500 μg/d and 15% consumed >400 μg/d. However, the Hungarian RCT, one of the biggest performed so far, did not find any effect of a high dosage of 8000 μg/d of folic acid supplementation on PTD [6]. Unfortunately, the folic acid dosage was not indicated in all of the studies [21,35]. The assessment of folate intake from supplementation alone or when studying populations with different dietary folate intakes are additional factors compromising comparability and generalizability between studies. While recent US studies are performed against the background of mandatory folate fortification of food [21,41], other studies have examined supplementation effects in folate-deficient populations [44]. Few studies have assessed the effects of both dietary folate and folic acid supplementation separately [41] or combined [20,47,49], and adjustments for bioavailability are rare. In this Norwegian study population, only 0.4% of the participants reached the Nordic Nutrition Recommendation of 500 μg/d with their dietary folate intake (adjusted with a factor of 0.6 for bioavailability; 2.8% without adjustment), and 31% of the participants achieved the recommended level with their total folate intake (39.6% without adjustment). After stratification for dietary folate intake, the early initiation of folic acid supplementation was significantly associated with spontaneous PTD in the subgroup of women with low but not high dietary folate intakes. There were no significant associations between high total folate intakes and PTD risk in the MoBa study population. Confounding is always an issue when assessing the effect of a single environmental factor on a complex outcome like PTD. For example, it is well established that women with high levels of education, privileged socioeconomic status and healthier overall diets are more likely to use supplements during pregnancy [50,51] and less likely to experience PTD than women without these characteristics. Some observational studies failed to adjust for these confounders, and the effect attributed to folic acid supplementation might in fact be confounded by overall health and lifestyle behaviors. While the strength of the significance was moderate, the association with the early onset of folic acid supplementation in the current study remained significant even after extensive adjustment for maternal characteristics such as socioeconomic and life-style parameters as well as obstetric anamnesis. Associations with the early start of supplementation should be studied with particular caution. The early start of folic acid supplementation might partially identify a group of women with a history of adverse pregnancy outcomes who want to optimize conditions for their current pregnancy. As presented in Table 1, women who had previously experienced spontaneous abortions were more likely to initiate folic acid supplementation early in their subsequent pregnancies. However, the same association of the early initiation of folic acid supplementation and spontaneous PTD was found in primi-gravidae. Folic acid supplementation starting more than 8 weeks prior to conception might characterize women who planned a pregnancy but did not become pregnant during their first 2 cycles, thus constituting a subgroup of women with suboptimal fertility [35]. The same association of the early start of supplementation was found in the group of women that became pregnant within the first month. Women who choose to start early with folic acid supplementation might be distinguished by some other characteristic that could be the causal link to spontaneous preterm delivery so that we cannot exclude confounding. In addition to the amount of folic acid, the composition of supplements is another point of discussion. In some countries like Greece and Norway, commonly used supplements contain folic acid and/or iron only [20]. In other countries, folic acid is mainly consumed in the form of multivitamins, making it difficult to differentiate the effects of multivitamin use and folic acid supplementation [21,35,41,48]. Vitamins other than folic acid might explain the association between multivitamin use and PTD. Catov et al. found that in the Danish birth cohort, multivitamin use was associated with modestly decreased PTD rates, while there was no association with folic acid supplementation [37]. As seen from Figure 1, vitamin A consumption (as a proxy for multivitamin supplementation) differed considerably from folic acid supplementation. However, the MoBa FFQ allowed us to calculate folic acid separately from other supplements, and adjusting for vitamin A consumption did not change the results for pre-conceptional folic acid supplementation. Apart from the amount, timing and composition of folate exposure, differences in the definition of pregnancy outcomes hinder comparability. Most studies defined PTD as delivery at <37+0 weeks of gestation without indicating the range of gestational age. This information might be important, especially if the risk of early PTD is found to be associated with folate status, as suggested by this study and that of Bukowski et al. [21]. Although PTD is a heterogeneous pregnancy outcome with distinct etiologies for different subgroups [25], not all studies analyzed clearly defined subgroups such as spontaneous PTD [20,21,37,40,47,52]. Strengths and weaknesses With a sample size of 65,668 pregnancies, this was a well-powered study for investigating the association of folate intake and pregnancy outcomes. Due to the large study sample, there were 1,628 cases defined as spontaneous PTD and 264 and 1,364 cases in the subgroups of early and late spontaneous PTD, respectively. The estimation of the gestational length by the second-trimester ultrasounds and the definition of a clear spontaneous PTD phenotype distinguishes this study [25]. The MoBa participation rate is 38.5%, and a demographic comparison with the MBRN in 2002 showed that single women and women <25 y of age are underrepresented in MoBa. Regarding PTD (7.2% in MoBa and 7.7% in MBRN), the differences are minor, and even the sub-group composition is similar to the distribution in the total population, with spontaneous PTD accounting for 42% of all PTD [15]. Additionally, a recent study found no bias in 8 selected exposure-outcome associations [53]. The assessment of folate from both the diet and supplements is a clear strength of this study. Although all dietary assessment methods have limitations, the MoBa FFQ has been extensively validated in a sub-population of 119 MoBa participants using a 4-day weighed food diary and biological markers in the blood and urine as reference measures [54]. The dietary supplement use was evaluated specifically. The total folate intake by the FFQ showed good agreement with the folate intake detailed by the food diary and was significantly reflected by the serum folate concentrations [31]. In a subsample of an earlier MoBa version (2934 singleton pregnancies), Nilsen et al. did not find any significant associations of dietary folate intake, folic acid supplementation or plasma folate with PTD. This study also reported good agreement between the folate intake (dietary and supplements) by the MoBa FFQ and plasma folate concentration (r = 0.44, CI: 0.41-0.47) [38]. As the relevant window of susceptibility for folate effects regarding pregnancy outcomes other than NTD is not yet known, the assessment of folate intake at different time points is a further strength of this study. The prospective design ensured that the women’s answers were not influenced by their knowledge of pregnancy outcomes. Conclusions The amount of dietary folate and supplemental folic acid intake in uncomplicated 65,668 singleton pregnancies from the Norwegian Mother and Child Cohort Study was not associated with a risk of spontaneous PTD, at least not at the relatively low intake levels of dietary folate (median 155 μg/d corrected for bio-availability, uncorrected 258 μg/d) and supplemental folic acid (median 400 μg/d) in this healthy study population. The initiation of folic acid supplementation more than 8 weeks prior to conception was associated with an increased risk for overall and early spontaneous PTD in both the overall analyses and in the strata of women with low dietary folate intake. Even if MoBa allows adjustment for a variety of confounders, the presence of residual confounding cannot be ruled out. However, our results require careful investigation regarding dosage and timing of folic acid supplementation, such as in the form of an RCT, before discussing a change of the current guidelines. Abbreviations BMI: Body mass index; CI: Confidence interval; FFQ: Food frequency questionnaire; HR: Hazard ratio; IQR: Interquartile range; MoBa: The norwegian mother and child cohort study; MBRN: Medical birth registry of norway; NOK: Norwegian crowns, currency; NTD: Neural tube defect; PTD: Preterm delivery; Q1, Q3: Questionnaire 1, 3; RCT: Randomized controlled trial; WHO: World health organization. Competing interests The authors declare that they have no competing interests. Authors’ contributions All authors planned the study. VS, JB and SN analyzed the data. VS, RM, SM, BJ selected preterm deliveries. MH calculated on folate intake from Q2. All authors contributed with interpretation of results and writing of the paper. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2393/13/160/prepub Supplementary Material Additional file 1: Table S1 Folate intake according to official recommendations and risk of spontaneous PTD. Click here for file Additional file 2: Figure S1a Initiation of pre-conceptional folic acid supplementation and risk of spontaneous PTD. Initiation of pre-conceptional folic acid supplementation (Q1 data) and cumulative risk of spontaneous PTD (22+0-36+6 weeks, n = 1,628). Cox regression for 65,668 participants in the Norwegian Mother and Child Cohort Study (2002 – 2009), adjusted for maternal age, prepregnancy BMI, parity, history of PTD and spontaneous abortion, child’s sex, smoking habits and alcohol consumption during pregnancy, maternal education, marital status, household income, energy intake and dietary folate intake. Iatrogenic deliveries have been censored in the regression model. Click here for file Additional file 3: Figure 1b Initiation of pre-conceptional folic acid supplementation and risk of early spontaneous PTD. Initiation of pre-conceptional folic acid supplementation (Q1 data) and cumulative risk of early spontaneous PTD (22+0-33+6 weeks, n = 264). Cox regression for 65,668 participants in the Norwegian Mother and Child Cohort Study (2002 – 2009), adjusted for maternal age, prepregnancy BMI, parity, history of PTD and spontaneous abortion, child’s sex, smoking habits and alcohol consumption during pregnancy, maternal education, marital status, household income, energy intake and dietary folate intake. Iatrogenic deliveries have been censored in the regression model. Click here for file Additional file 4: Table S2 Initiation of pre-conceptional folic acid supplementation and risk of spontaneous preterm delivery (sPTD), depending on dietary folate intake. Click here for file Acknowledgments We are grateful to all families in Norway who are participating in this ongoing cohort study. Statement of financial support: This work was supported by grants from the Norwegian Research Council (FUGE 183220/S10, FRIMEDKLI-05 ES236011), the Swedish Medical Society (SLS 2008–21198) and Swedish government grants to researchers in public health service (ALFGBG-2863, ALFGBG-11522). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs Tamura T Picciano MF Folate and human reproduction Am J Clin Nutr 2006 83 993 1016 16685040 Nordic Council of Ministers Folate Nordic Nutrition Recommendations 2004 Integrating nutrition and physical activity 2004 Copenhagen: Norden 287 296 World Health Organization; Food and Agricultural Organization of the United Nations Folate and Folic acid Vitamin and mineral requirements in human nutrition 2004 Rome: FAO 53 62 Scholl TO Johnson WG Folic acid: influence on the outcome of pregnancy Am J Clin Nutr 2000 71 5 Suppl 1295S 1303S 10799405 Israels MC Da Cunha FA Megaloblastic anaemia of pregnancy Lancet 1952 2 214 215 14939891 Czeizel AE Dudas I Metneki J Pregnancy outcomes in a randomised controlled trial of periconceptional multivitamin supplementation. Final report Arch Gynecol Obstet 1994 255 131 139 10.1007/BF02390940 7979565 Czeizel AE Dudas I Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation The New England journal of medicine 1992 327 1832 1835 10.1056/NEJM199212243272602 1307234 MRC Vitamin Study Research Group Prevention of neural tube defects: results of the Medical Research Council Vitamin Study Lancet 1991 338 131 137 1677062 Shaw GM Carmichael SL Nelson V Selvin S Schaffer DM Occurrence of low birthweight and preterm delivery among California infants before and after compulsory food fortification with folic acid Public Health Rep 2004 119 170 173 15192904 de Bree A van Dusseldorp M Brouwer IA van het Hof KH Steegers-Theunissen RP Folate intake in Europe: recommended, actual and desired intake Eur J Clin Nutr 1997 51 643 660 10.1038/sj.ejcn.1600467 9347284 Kostholdsråd gravide (Dietary recommendation for pregnant women, in Norwegian) http://www.helsedirektoratet.no/folkehelse/ernering/kostholdsrad/gravide/Sider/default.aspx WHO Expert Committee The prevention of perinatal mortality and morbidity. Report of a WHO Expert Committee World Health Organ Tech Rep Ser 1970 457 1 60 4993526 Moster D Lie RT Markestad T Long-term medical and social consequences of preterm birth N Engl J Med 2008 359 262 273 10.1056/NEJMoa0706475 18635431 Bryce J Boschi-Pinto C Shibuya K Black RE WHO estimates of the causes of death in children Lancet 2005 365 1147 1152 10.1016/S0140-6736(05)71877-8 15794969 Magnus P Irgens LM Haug K Nystad W Skjaerven R Stoltenberg C Cohort profile: the Norwegian Mother and Child Cohort Study (MoBa) Int J Epidemiol 2006 35 1146 1150 10.1093/ije/dyl170 16926217 Lockwood CJ Predicting premature delivery–no easy task N Engl J Med 2002 346 282 284 10.1056/NEJM200201243460412 11807155 Hassan SS Romero R Vidyadhari D Fusey S Baxter JK Khandelwal M Vijayaraghavan J Trivedi Y Soma-Pillay P Sambarey P Dayal A Potapov V O'Brien J Astakhov V Yuzko O Kinzler W Dattel B Sehdev H Mazheika L Manchulenko D Gervasi MT Sullivan L Conde-Agudelo A Phillips JA Creasy GW Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial Ultrasound in obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics and Gynecology 2011 38 18 31 23946514 Dodd JM Flenady VJ Cincotta R Crowther CA Progesterone for the prevention of preterm birth: a systematic review Obstet Gynecol 2008 112 127 134 10.1097/AOG.0b013e31817d0262 18591318 Romero R Nicolaides K Conde-Agudelo A Tabor A O'Brien JM Cetingoz E Da Fonseca E Creasy GW Klein K Rode L Soma-Pillay P Fusey S Cam C Alfirevic Z Hassan SS Vaginal progesterone in women with an asymptomatic sonographic short cervix in the midtrimester decreases preterm delivery and neonatal morbidity: a systematic review and metaanalysis of individual patient data American journal of obstetrics and gynecology 2012 206 124 e121-119 22284156 Papadopoulou E Stratakis N Roumeliotaki T Sarri K Merlo DF Kogevinas M Chatzi L The effect of high doses of folic acid and iron supplementation in early-to-mid pregnancy on prematurity and fetal growth retardation: the mother-child cohort study in Crete, Greece (Rhea study) European journal of nutrition 2013 52 327 336 10.1007/s00394-012-0339-z 22430980 Bukowski R Malone FD Porter FT Nyberg DA Comstock CH Hankins GD Eddleman K Gross SJ Dugoff L Craigo SD Timor-Tritsch IE Carr SR Wolfe HM D'Alton ME Preconceptional folate supplementation and the risk of spontaneous preterm birth: a cohort study PLoS Med 2009 6 e1000061 10.1371/journal.pmed.1000061 19434228 Czeizel AE Puho EH Langmar Z Acs N Banhidy F Possible association of folic acid supplementation during pregnancy with reduction of preterm birth: a population-based study European journal of obstetrics, gynecology, and reproductive biology 2010 148 135 140 10.1016/j.ejogrb.2009.10.016 19926391 Charles DH Ness AR Campbell D Smith GD Whitley E Hall MH Folic acid supplements in pregnancy and birth outcome: re-analysis of a large randomised controlled trial and update of Cochrane review Paediatr Perinat Epidemiol 2005 19 112 124 10.1111/j.1365-3016.2005.00633.x 15787886 Fekete K Berti C Trovato M Lohner S Dullemeijer C Souverein OW Cetin I Decsi T Effect of folate intake on health outcomes in pregnancy: a systematic review and meta-analysis on birth weight, placental weight and length of gestation Nutrition journal 2012 11 75 10.1186/1475-2891-11-75 22992251 Pennell CE Jacobsson B Williams SM Buus RM Muglia LJ Dolan SM Morken NH Ozcelik H Lye SJ Relton C Genetic epidemiologic studies of preterm birth: guidelines for research American journal of obstetrics and gynecology 2007 196 107 118 10.1016/j.ajog.2006.03.109 17306646 Norwegian Mother and Child Study http://www.fhi.no/eway/default.aspx?pid=238&trg=MainArea_5811&MainArea_5811=5895:0:15,3046:1:0:0:::0:0 Irgens LM The Medical Birth Registry of Norway. Epidemiological research and surveillance throughout 30 years Acta Obstet Gynecol Scand 2000 79 435 439 10.1080/j.1600-0412.2000.079006435.x 10857866 Meltzer HM Brantsaeter AL Ydersbond TA Alexander J Haugen M Methodological challenges when monitoring the diet of pregnant women in a large study: experiences from the Norwegian Mother and Child Cohort Study (MoBa) Matern Child Nutr 2008 4 14 27 18171404 Lauritzen J FoodCalc http://www.ibt.ku.dk/jesper/FoodCalc/Default.htm Norwegian Food Safety Authority, Norwegian Directorate of Health, Department of Nutrition - University of Oslo Matvaretabellen (The Norwegian Food Table, in Norwegian) http://www.matvaretabellen.no/ Brantsaeter AL Haugen M Hagve TA Aksnes L Rasmussen SE Julshamn K Alexander J Meltzer HM Self-reported dietary supplement use is confirmed by biological markers in the Norwegian Mother and Child Cohort Study (MoBa) Ann Nutr Metab 2007 51 146 154 10.1159/000103275 17536192 Haugen M Brantsaeter AL Alexander J Meltzer HM Dietary supplements contribute substantially to the total nutrient intake in pregnant Norwegian women Ann Nutr Metab 2008 52 272 280 10.1159/000146274 18645244 FAO/WHO Expert Consultation Requirements of Vitamin A, Iron, Folate and Vitamin B12 Report of a Joint FAO/WHO Expert Consultation (FAO Fisheries Report). World Health Organization, 1988 Rome Grambsch PM Therneau TM Proportional hazards tests and diagnostics based on weighted residuals Biometrika 1994 81 515 526 10.1093/biomet/81.3.515 Callaway L Colditz PB Fisk NM Folic acid supplementation and spontaneous preterm birth: adding grist to the mill? PLoS Med 2009 6 e1000077 10.1371/journal.pmed.1000077 19434229 Alwan NA Greenwood DC Simpson NA McArdle HJ Cade JE The relationship between dietary supplement use in late pregnancy and birth outcomes: a cohort study in British women BJOG : an international journal of obstetrics and gynaecology 2010 117 821 829 10.1111/j.1471-0528.2010.02549.x 20353456 Catov JM Bodnar LM Olsen J Olsen S Nohr EA Periconceptional multivitamin use and risk of preterm or small-for-gestational-age births in the Danish National Birth Cohort The American journal of clinical nutrition 2011 94 906 912 10.3945/ajcn.111.012393 21795441 Nilsen RM Vollset SE Monsen AL Ulvik A Haugen M Meltzer HM Magnus P Ueland PM Infant birth size is not associated with maternal intake and status of folate during the second trimester in Norwegian pregnant women The Journal of nutrition 2010 140 572 579 10.3945/jn.109.118158 20089778 Timmermans S Jaddoe VW Hofman A Steegers-Theunissen RP Steegers EA Periconception folic acid supplementation, fetal growth and the risks of low birth weight and preterm birth: the Generation R Study The British journal of nutrition 2009 102 777 785 10.1017/S0007114509288994 19327193 Dunlop AL Taylor RN Tangpricha V Fortunato S Menon R Maternal Micronutrient Status and Preterm Versus Term Birth for Black and White US Women Reproductive sciences 2012 19 939 948 10.1177/1933719112438442 22527984 Shaw GM Carmichael SL Yang W Siega-Riz AM Periconceptional intake of folic acid and food folate and risks of preterm delivery American journal of perinatology 2011 28 747 752 10.1055/s-0031-1280855 21681695 Fleming AF Martin JD Stenhouse NS Pregnancy anaemia, iron and folate deficiency in Western Australia Med J Aust 1974 2 479 484 4431351 Fletcher J Gurr A Fellingham FR Prankerd TA Brant HA Menzies DN The value of folic acid supplements in pregnancy J Obstet Gynaecol Br Commonw 1971 78 781 785 10.1111/j.1471-0528.1971.tb00338.x 5097161 Baumslag N Edelstein T Metz J Reduction of incidence of prematurity by folic acid supplementation in pregnancy Br Med J 1970 1 16 17 10.1136/bmj.1.5687.16 5460838 Rolschau J Kristoffersen K Ulrich M Grinsted P Schaumburg E Foged N The influence of folic acid supplement on the outcome of pregnancies in the county of Funen in Denmark. Part I Eur J Obstet Gynecol Reprod Biol 1999 87 105 110 discussion 103–104 10.1016/S0301-2115(99)00084-6 10597955 Scholl TO Hediger ML Bendich A Schall JI Smith WK Krueger PM Use of multivitamin/mineral prenatal supplements: influence on the outcome of pregnancy Am J Epidemiol 1997 146 134 141 10.1093/oxfordjournals.aje.a009244 9230775 Siega-Riz AM Savitz DA Zeisel SH Thorp JM Herring A Second trimester folate status and preterm birth Am J Obstet Gynecol 2004 191 1851 1857 10.1016/j.ajog.2004.07.076 15592264 Furness DL Yasin N Dekker GA Thompson SD Roberts CT Maternal red blood cell folate concentration at 10–12 weeks gestation and pregnancy outcome The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians 2012 25 1423 1427 10.3109/14767058.2011.636463 23921867 Scholl TO Hediger ML Schall JI Khoo CS Fischer RL Dietary and serum folate: their influence on the outcome of pregnancy Am J Clin Nutr 1996 63 520 525 8599315 Bailey RL Dodd KW Gahche JJ Dwyer JT McDowell MA Yetley EA Sempos CA Burt VL Radimer KL Picciano MF Total folate and folic acid intake from foods and dietary supplements in the United States: 2003–2006 The American journal of clinical nutrition 2010 91 231 237 10.3945/ajcn.2009.28427 19923379 Nilsen RM Vollset SE Gjessing HK Magnus P Meltzer HM Haugen M Ueland PM Patterns and predictors of folic acid supplement use among pregnant women: the Norwegian Mother and Child Cohort Study Am J Clin Nutr 2006 84 1134 1141 17093167 Bergen NE Jaddoe VW Timmermans S Hofman A Lindemans J Russcher H Raat H Steegers-Theunissen RP Steegers EA Homocysteine and folate concentrations in early pregnancy and the risk of adverse pregnancy outcomes: the Generation R Study BJOG : an international journal of obstetrics and gynaecology 2012 119 739 751 10.1111/j.1471-0528.2012.03321.x 22489763 Nilsen RM Vollset SE Gjessing HK Skjaerven R Melve KK Schreuder P Alsaker ER Haug K Daltveit AK Magnus P Self-selection and bias in a large prospective pregnancy cohort in Norway Paediatr Perinat Epidemiol 2009 23 597 608 10.1111/j.1365-3016.2009.01062.x 19840297 Brantsaeter AL Haugen M Alexander J Meltzer HM Validity of a new food frequency questionnaire for pregnant women in the Norwegian Mother and Child Cohort Study (MoBa) Matern Child Nutr 2008 4 28 43 18171405	23937678	PMC3751653	CC BY	2021-01-05 01:22:11	yes	BMC Pregnancy Childbirth. 2013 Aug 12; 13:160
==== Front Diagn PatholDiagn PatholDiagnostic Pathology1746-1596BioMed Central 1746-1596-8-1232390264610.1186/1746-1596-8-123ResearchNeuropathological and neuroprotective features of vitamin B12 on the dorsal spinal ganglion of rats after the experimental crush of sciatic nerve: an experimental study Hobbenaghi Rahim 1r.hobbenaghi@gmail.comJavanbakht Javad 2javadjavanbakht@ut.ac.irHosseini Ehan 3hosseiniehsan460@gmail.comMohammadi Shahin 4korzan2008@gmail.comRajabian Mojtaba 5rajabian_m@yahoo.comMoayeri Pedram 6Dr.moayeri@ut.ac.irAghamohammad hassan Mehdi 7aghamh@ut.ac.ir1 Department of Pathology, Faculty of Veterinary Medicine, University of Urmia, Urmia, Iran2 Department of Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran3 Faculty of Para Veterinary Medicine, Ilam University, Ilam, Iran4 Graduate Faculty of Veterinary Medicine, University of Urmia, Urmia, Iran5 Food Hygiene Department, University of Shahekord, Shahekord, Iran6 Resident of Large Animal Internal Medicine Department, University of Shahekord, Shahekord, Iran7 Department of Clinical Science, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran2013 31 7 2013 8 123 123 3 6 2013 18 6 2013 Copyright © 2013 Hobbenaghi et al.; licensee BioMed Central Ltd.2013Hobbenaghi et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Spinal motoneuron neuroprotection by vitaminB12 was previously reported; the present study was carried out to evaluate neuroprotectivity in the dorsal root ganglion sensory neuron. Methods In present study thirty-six Wister-Albino rats (aged 8–9 weeks and weighing 200–250 g) were tested. The animals were randomly divided into 6 groups which every group contained 6 rats. Group A: received normal saline (for 42 days); Group B: vitamin B12 was administered (0.5 mg/kg/day for 21 days); Group C: received vitamin B12 (1 mg/kg/day for 21days); Group D: received vitamin B12 (0.5 mg/kg/day for 42 days); Group E; received vitamin B12 (1 mg/kg/day for 42 days); Group F; received no treatment. The L5 Dorsal Root Ganglion (DRG) neurons count compared to the number of left and right neurons .Furthermore, DRG sensory neurons for regeneration were evaluated 21 or 42 days after injury (each group was analyzed by One-Way ANOVA test). Results (1): The comparison of left crushed neurons (LCN) number with right non-crushed neurons in all experimental groups (B, C, D and C), indicating a significant decline in their neurons enumeration (p<0/05). (2): The comparison of test group’s LCN with the control group’s LCN revealed a significant rise in the number of experimental group neurons (p<0/05). (3): Moreover, comparing the number of right neurons in experimental groups with the number of neurons in crushed neurons indicated that the average number of right neurons showed a significant increase in experimental groups (p<0/05). Conclusion Consequently, the probability of nerve regeneration will be increased by the increment of the administered drug dosage and duration. On the other hand, the regeneration and healing in Dorsal Spinal Ganglion will be improved by increase of administration time and vitamin B12 dose, indicating that such vitamin was able to progress recovery process of peripheral nerves damage in experimental rats. Finally, our results have important implications for elucidating the mechanisms of nerve regeneration. Moreover, the results showed that vitaminB12 had a proliferative effect on the dorsal root ganglion sensory neuron. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7395141841009256 Dorsal root ganglionRatSurgerySciatic nerveVitamin B12 ==== Body Background Unlike the central nervous system (CNS), neurons the peripheral nervous system (PNS) can regenerate by its own after physical injury because of activation of the intrinsic growth capacity of neurons [1]. The sciatic nerve, innervating the hind paw of the rat, is frequently used to study peripheral nerve regeneration. After crush injury, the fibers distal to the lesion undergo Wallerian degeneration: the axon and myelin degenerate and are ingested by Schwann cells and invading macrophages. Schwann cells surrounding the distal fibers proliferate so that the endoneurial tubes surrounding the original nerve fibers remain intact, providing the environment through which the regenerating axons can grow [2,3]. Peripheral nerve injury results in a loss of sensory and motor function and nerve repair often results in poor functional recovery. This is, in part, due to dorsal root ganglion (DRG) cell death, regeneration errors or failure to regenerate [4-6]. The sciatic nerve, comprising a mixed population of motor and sensory axons, is a commonly used model for studying nerve regeneration and it regeneration is accompanied by a variety of changes in the DRG neurons cell bodies and regeneration is associated with the expression of new genes and proteins [2,7]. The DRG consists of heterogeneous population of neurons. During development neurons must receive appropriate neurotrophic support to survive which is achieved by establishing appropriate peripheral target connections via their neuritis [8]. Injury to DRG neuron cellular body or central axon results in somatosensory defects that do not recover spontaneously. Lost DRG neurons are not replaced. Central axon response to injury is weak [9-11], leading to slow axonal regrowth [12], Moreover, the proximal and distal axonal processes of sensory neurons exhibit different regeneration rates: Dorsal root axons regenerate at a lower rate as compared to distal axon regrowth into the lesioned spinal or sciatic nerve [12-15]. The reasons for this discrepancy are not clear. The sensory neurons extending into the sciatic nerve are located in the L4–L6 dorsal root ganglion (DRGs). After sciatic nerve injury (SNI), the damaged neurons suffer important changes to switch the neuro-transmitter state to a pro-regenerative state [16]. B-vitamins were reported to attenuate degenerating processes in the nervous system and therefore have been clinically administered in a combination of B1 (thiamine), B6 (pyridoxine), and B12 (cobalamine;Cbl)[17]. Ganglion count in rat after SNI Vitamin B12(vit B12) is a micronutrient that plays significant roles in numerous biological processes. It acts as a coenzyme, which is required for metabolism of folate and biosynthesis of nucleotide [18]. It helps maintaining normal functions of the brain. vit B12deficient neuropathy is well established in humans, and has also been described in animal models [19]. The CNS and especially the spinal cord are severely damaged by vit B12deficiency [20]. Vit B12 is transported in the blood bound to transcobalamin (TC) and internalized into cells by its receptor, (TC-R, also named CD320). In the cells, vit B12 is transferred to the cytoplasm by the lysosomal membrane vit B12 transporter (LMBRD1) and serves as cofactor for methyl malonylCoA mutase (Mut) and methionine synthase (MS) [21]. Futhermore, vit B12 deficiency leads to methionine deficiency, which is required for the synthesis of both phospholipids and myelin. In addition, vitamin B12 has been shown to exert antioxidant properties [22], which result from direct and indirect effects [23]. In parallel of this research several studies regarding vit B12 effects on nerves healing have been conducted. Yagihashi et al. (1982) suggested that continuous treatment with CH3-B12 had an ameliorative effect on the peripheral nerve lesions in experimental diabetic neuropathy [24]. In addition, Okada et al. (2010) showed that vit B12may provide a basis for more beneficial treatments of nervous disorders through effective systemic or local delivery of high doses of methylcobalamin to target organs [25]. Watanabe in 1994 tested a high dosage of vit B12 (500 mg/kg) on repairing the damaged nerves of rats, somehow the potential return of muscles action was significantly faster than that recorded in control group, which treated with high dose of vitamin B12, rats [26]. Swett et al. have studied on neurons dorsal root ganglion count in rat after SNI [27]. In the present study, we investigated the neuroprotective effects of vit B12on dorsal spinal ganglion of rats after the experimental sciatic nerve crush. Methods Animals All experimental protocols were approved by the local animal care committee in accordance with Faculty of Urmia Veterinary Medicine office regulations. In current study, 36 Wister-albino rats of both sexes, weighing 200–250 g, with averagely 6 weeks old were selected. The animals were kept two by two in individual propylene cages under standard laboratory conditions by the dimensions of 30×50×25 cm3. Rats were maintained on a 12 hour light/dark cycle at 22±1°C and 50±10% humidity. The animals were kept in standard room conditions and fed with standard rat diet and water ad libitum. Experimental protocol The rats were divided into six groups and randomly allotted into one of six experimental groups each group contained six animals. Control animals in group A received normal saline (for 42 days) (n=6), and test animals in group B received vit B12following surgery (0.5 mg/kg/day for 21 days) (n=6); however, test animals of group C received vit B12following surgery (1 mg/kg/day for 21 days) (n=6). Test animals in group D received vit B12following surgery (0.5 mg/kg/day for 42 days) (n=6), whereas test animals in group E received vit B12following surgery (1 mg/kg/day for 42 days) (n=6), and in F: Sham group had no surgery and vit B12injection, and were euthanized after 42 days (n=6) in order to necropsy examination. In addition, the vit B12in experimental group was injected intraperitoneally. Surgical technique DRG of the spinal cord segment L5 which mainly sciatic nerve is originated from is located in L1 vertebral column of the rat. Therefore, to remove it, according to the method of the mentioned article, spinal segment, dorsal root and related ganglion were removed from L1 of the vertebral column [28]. All of the operations were performed under microscope by same surgeon. The left lateral thigh was operated after shaving and preparing the skin with 10% povidone iodine. The sciatic nerve was exposed by opening the fascial plane between the gluteal and femoral musculature via a longitudinal incision (2–3 cm). All surgical procedures were carried out under ketamine hydrochloride (75 mg /kg) and Xylazine hydrochloride (5 mg/kg) anesthetic solutions, the sciatic nerve of such 30 rats was exposed at mid-thigh level and either crushed for 5 minutes with a pair of hemostatic forceps (9–10 mm size). Subsequently, the muscles and skin were separately sutured by 4/0 catgut thread and allowed to breathe room air. The surgery was followed by the animals’ special care until recovery. Sample collection and microscopic examination In both group animals (the control and experimental), the left sciatic nerve had been crushed before the treatment was started and then sutured. The animals were anaesthetized with IP injection of sodium thiopenthal after 21 and 42 days. The left and right dorsal root ganglia-L5 and sections from distal parts of the right (non-operated) and left (operated) sciatic nerves were collected, fixed, and prepared for light microscopic examination. A 10 mm-long sample of the right sciatic nerve segment was removed without any injury, fixed, and prepared for histopathological examination. Tissue fragments were fixed in 10% neutral buffered formalin solution (for 72 hours), upon stability embedded in paraffin, sectioned at 5 μm thickness and stained with hematoxylin and eosin (H&E). Method of calculating the number of neurons To determine the total number of neurons in the spinal dorsal root node, the researchers used series of cross-sectional counting method. In this method, through examination of each neuronal population the sensory neurons, one of every 5 or 10 sections, were counted and the total cells were multiplied by 5 or 10, respectively to give an estimate of total cell numbers and neurons, containing clear nuclear or nucleus were counted. Finally, the total number of neurons in the dorsal root node was compared to that of different groups. The neuronal counting method was explained by Clarke [29]. Briefly, the left and right L5 DRG were removed and post-fixed in 4% paraformaldehyde then 30% sucrose, both at 4°C for 24 h. the ganglia were blocked in tissue freezing medium and stored at −80°C. Each entire ganglion was cut into serial 15-μm cryosections and 1 from 4 sections mounted onto gelatin-coated glass slides and dried overnight. Neuron counts were performed using light microscopy. By a camera (DP11 camera) mounted on top of the microscope, images from the sections were prepared at magnifications of ×100, ×400, and ×1000, normal and clear neuronal nuclei or nucleus were counted. Neuron loss was calculated by subtracting the number of neurons in ipsilateral ganglion from that in their contralateral controls. Loss was then expressed as a percentage of the neuron count in the control ganglia. Statistical analysis The data were expressed as mean ± standard deviation (SD), and analyzed by repeated measures of variance. In order to comparing number of counted neurons between different groups a one-way ANOVA test and to evaluate time interventions and drug dosage, Duncan test were used. Experimental results were considered to be significantly different from control values with p-value set at.05 (SPSS). Results The procedures for reconstructing the regenerated DRG neuron populations were identical to those used in an earlier study, describing the normal sciatic DRG neuron populations in the rat [30]. The results of the present study indicated that there was a significant decrease in the number of DRG neurons in all groups with SNI compared to early stages of injury of the sham and control groups. Furthermore, the results of this study demonstrated that compared to the number of neurons in left ganglions of crushing sciatic nerve (LGCSN) with neurons in right ganglions of non-crushing sciatic nerve (RGNCSN) in all experimental groups (EGs) (0/5 and 1 mg/kg, 21 and 42 days) a significant decrease in the number of EGs neurons was recorded (p<0/05). This decrease was 7015± 132 to 6336± 142 respectively (p<0/05) (Table 1). Table 1 Indicates of the average number of neurons of left crushing sciatic nerve of all groups (A) compared to the right non-crushing sciatic nerve of the same groups (B) Groups Mean ±SD A decrease in the number of neurons of A group in relation to B and their comparison with sham group 21-day (0.5 mg/kg) 7015± 132 21-day (1 mg/kg) 7002± 109 42-day (0.5 mg/kg) 6749± 124 42-day (1 mg/kg) 6336± 142 Sham 8815± 114 Values are means ± SD (n=5). p<0.05 comparison to sham group. Moreover, comparison the number of neurons in LGCSN (of each EG) with neurons in same side of the control group (CG) (p<0/05) revealed a significant increase in the frequency of EG neurons (p<0/05). The increase was 7079± 102 to7779± 123 respectively (p<0/05) (Table 2). Table 2 Indicates of the average increase in the number of neurons of left crushing sciatic nerve of experimental group (A) compared to the right non-crushing sciatic nerve of the control group (B) Groups Mean ±SD An increase in the number of neurons of A group in relation to B and their comparison with control group 21-day (0.5 mg/kg) 7079± 102 21-day (1 mg/kg) 7069± 117 42-day (0.5 mg/kg) 7435± 200 42-day (1 mg/kg) 7779± 123 Control 7025± 173 Values are means ± SD (n=5). p=<0.05 comparison to the right non-crushing sciatic nerve of the control group. On the other hand, comparison the number of right neurons in EGs with the number of neurons in CG indicated a significant increase in the average number of right EGs neurons (p<0/05). This increase was 7023± 164 to 7095± 171 respectively (p<0/05) (Table 3). Table 3 Indicates of the average increase in the number of neurons of right non-crushing sciatic nerve of the experimental group (A) compared to the non-crushing sciatic nerve of the control group (B) Groups Mean ±SD An increase in the number of neurons of A group in relation to B 21-day (0.5 mg/kg) 7023± 164 21-day (1 mg/kg) 7028± 163 42-day (0.5 mg/kg) 7447± 119 42-day (1 mg/kg) 7095± 171 Values are means ± SD (n=4). p<0.05 comparison to the non-crushing sciatic nerve of the control group. In the treatment groups (0.5 mg/kg, 21 days) the number of DRG neurons after SNI revealed a significant rise in the number of neurons compared to that of the CG (p<0.05). Also the same significant difference was available in the number of neurons in other treatment groups (1 mg/kg, 21 days) (p<0.05). The comparison between two experimental 21-day groups of 0.5 mg/kg and 1 mg/kg showed no significant difference. Additionally, in the treatment group (1 mg/kg, 42 days), the numbers of neurons solely demonstrated significant rise in comparison with CG (p<0.05). The number of neurons between 42-day group (0.5 mg/kg) and CG did not show remarkable increase. In addition, there was not any significant difference in 0.5 and 1 mg/kg 42-day groups (Figure 1). Figure 1 Histopathological evaluation of dorsal Root Ganglion neurons in the treatment and control groups. A: Death of dorsal Root Ganglion (DRG) neurons in the control group that treated with normal saline, there were not nucleolus and nuclei (arrow), (H&G, x400). B: Note that of dorsal Root Ganglion (DRG) neurons in the experimental groups that treated vitamins B12, there were nucleolus and nuclei (arrow), (H&Gx400). In order to investigate whether the neuroprotective effect of vit B12 is dose-dependent, we administered one higher dose of the vitamin (i.e., 1 mg/kg vit B12) 42 d after SNI surgery. As shown in Figure 2B, higher doses of the vitamin alleviated the neuroprotective behavior more effectively than lower doses (compare to Figure 2A), and stronger effects were again observed after the second drug injection which was given 24 h after the first injection. Thus, SNI-induced neuroprotective behavior was dose-dependently inhibited by systemic administration of vitamins B12. Figure 2 Investigation neuron increase and decrease percentage in experiment groups compared to the right ganglions of non-crushing sciatic nerve and control group. In order to examining the impact and simultaneous interaction of both factors (time and treatment dosage) in the number of neurons, a two-way ANOVA and Duncan test were used. The comparison between the control groups at 21 days after surgery represented a significant difference with treatment groups both in 21 and 42 days treatment groups. On the other hand, the number of neurons in control groups at 42 days after surgery was similar the number of neurons at 21-day groups for both dosages. The results indicated that neuron injury begins to repair 42 days after surgery while vit B12 accelerated such process conspicuously. Discussion Peripheral nerve regeneration is a complex biological process involving interactions among multiple cells, neurotrophic factors and extracellular matrice proteins [31]. This report led us to believe that a systemic examination of DRG neuron responses to vit B12 is advantageous to better understand the role of vitamin B12 in peripheral nerve regeneration. The present study evaluated the protective effects of vit B12on the number of DRG cells recovery after inducing SNI in the rats. In a serious trauma like SNI, a short period of localized ischemia is followed by evident increase in the pressure of endoneural fluid and defect of the normal capillary blood flow in the endoneurium [32]. Subsequently, Wallerian degeneration may arise distal to the lesion [14,17]. It has been shown that sciatic nerve crush leads to histological changes in the DRG cells number [33]. Loss of the neurons occurs and the nerve cells become less after peripheral nerve injury in the DRG [15,18]. This finding is in accordance with our results in which we showed the number of the cells were decreased. Histological changes, such as decrease in the number of DRG cells after SNI, have been reported by other researchers [34-36]. Our results are in accordance with these findings, in which we demonstrated that the total number of L5-DRG cells after SNI were decreased. Vit B12is also a good scavenger of the reactive oxygen species and is suggested to be a good neuroprotectant. It can pass through the blood brain barrier, which is an evidence of amplification of its neuroprotectant potential in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases [37]. In addition, it has been reported that vit B12 has protective effects after spinal cord injuries. The previous studies have stressed the anti-apoptotic and anti-necrotic effects of the vit B12 on the neurons [38]. These reports may explain the beneficial effects of vit B12 in the present study after SNI. These properties including anti-inflammatory, antioxidant, anti-apoptotic and anti-necrotic might be effective in the present study. In previous reports on the effects of vit B12 on neurons in vivo, high levels of vit B12 improved nerve conduction and regeneration in streptozotocin-diabetic rats [17], and experimental acrylamide neuropathy [26]. We used a rat SNI model in the present study to evaluate the effects of vit B12in vivo. However, we used continuous administration of doses of vit B12 (0.5 and 1 mg/kg/day) higher than that used in previous reports (500 μg/kg/day). Our morphological and histological evaluation offers a possible explanation of this phenomenon. Vit B12increased the regeneration of axons. We consider that repair discrepancy is due to the differences in the doses of vit B12and time of administration. In our study, vit B12had not remarkable affect on neurons at low-dose concentrations, especially with low-dose administration or short time, because the concentration of vit B12declines immediately [39] and the absorption of vit B12from the ileum and entire intestine is regulated by limitation of binding to gastric intrinsic factor and passive diffusion [40,41]. From the results of our study, we consider that it is necessary to devise new clinical methods, such as high-dose systemic or long time, to deliver high doses of vit B12 to target organs to treat nervous disorders more effectively. Thus, the high-dose vit B12 treatment together with long time might have the potential to treat not only peripheral nerve injury but also central nervous injury. The results of our study indicated that the experimental groups, under treatment with vit B12, were prevented from reducing the number of neurons in DRG (Table. 2). Moreover, the DRG neurons decrease of left side of the operated experimental group compared to non-operated right side of the same group was slightly, especially it reached to the lowest degree (6336± 142) in 1mg/kg dosage in 42-day, and the result was significant compared to the other groups (p<0/05). On the other hand, the left neurons of 0/5mg/kg group in 21-day shows an increase of 7779± 123 compared to the same ganglion of the control group. Also, according to Table 3, the neuronal mean increase shows a significant rise in association with the increase of vit B12 dosage and time that such variation in 0/5 and1 mg/kg group reached to 7023± 164 to 7095± 171respectively (Table 3). In addition, the decrease in the neurons death percentage of the right side of the non-crushing sciatic nerves compared to the same side of the control group was 20/32, 20/27, 20/05 and 19/51 percent (Table 3 and Figure 1 ). On the other hand, the regeneration and healing in Dorsal Spinal Ganglion will be improved by increase of administration time and vit B12 dose, indicating that such vitamin was able to progress recovery process of peripheral nerves damage in experimental rats. In parallel with our research several studies have been conducted. Yamatsu evaluated the effect of vitaminB12 on the nerve repair after a crush injury. Vit B12 demonstrated significant increase in the regeneration and improvement on SNI tests suggesting an inhibitory effect on the nerve degeneration and facilitating the regeneration after an injury [42]. Okada et al. evaluated the effectiveness of vit B12 on nerve regeneration and found that it demonstrated the greatest improvement on nerve regeneration [25]. Yamazaki also demonstrated that vit B12 improves the neuromuscular junction by decreasing nerve degeneration and improving nerve regeneration in the motor nerve terminals in a gracile axonal dystrophy mice model [43]. In addition to, the results of this study confirm the results of one-way ANOVA and represent that the neurons construction and repairing started by time passage. Because the average number of neurons in under-prescription groups by vit B12 in 0.5mg/Kg and 1mg/Kg doses increased in 42-day experimental groups from 7435± 200 to7779± 123, and this difference was obvious and significant between the control group and 42-day experimental group in 1mg/kg dosage (p<0/05). In agreement with our study, Yagihushi et al., 1976 suggested that a constant 16-week cures by vit B12 (500 microgram/kg) had a completely repairing effect on the injured peripheral nerves in experimental diabetic neuropathy of rats. In another study, Taniguchi et al., 1987 indicated that vit B12 cured neuropathy in uromicina patients. They advised that vitamin B12 could be used as a cure in such forms of neuropathy [44]. In addition, another study by, Hasegawa et al. (1978) studies showed that the vitamin B complex facilitated functional recovery from nerve injury faster than its components, and showed that B1 and B12 had significant facilitating effects on the functional recovery as well [45]. In this study, we showed that the vitamin B12 was the most effective in promoting neuronal survival in DRG neurons. These results suggest that the metabolic pathway of vit B12, is associated with neuronal survival, but further understanding of this role and development of an effective delivery system for vitamin B12 may enable us to treat several nervous disorders and to obtain new insights into nerve regeneration. Conclusion Our study suggests that vitamin B12 has neuroprotective and restorative effects on secondary pathochemical events after sciatic nerve injury in rats. These restorative effects have been mainly observed on neuronal numbers. We believe that further preclinical research into the utility of Cbl may indicate its usefulness as a potential treatment on neurodegeneration after trauma in rats but more and detailed experimental studies are needed to determine the effects of Cbl on the detrimental results of secondary sciatic nerve injury in human and animal. Furthermore, this study revealed that the regeneration and healing in Dorsal Spinal Ganglion will be improved by increase of administration time and vitamin B12 dose, indicating that such vitamin was able to progress recovery process of peripheral nerves damage in experimental rats. Competing interests The authors declare that they have no conflict of interest. Authors’ contributions RH and SHM participated in the neurohistopathological evaluation, performed the literature review, acquired photomicrographs and drafted the manuscript and gave the final histopathological diagnosis. JJ performed sequencing alignment and manuscript writing .EH, MR,PM and MAMH edited the manuscript and made required changes. All authors have read and approved the final manuscript. Acknowledgements The authors thank staff of the Veterinary Teaching Hospital, Urmia University for their valuable technical assistance. ==== Refs Rishal I Fainzilber M Retrograde signaling in axonal regeneration Exp Neurol 2010 223 5 10 10.1016/j.expneurol.2009.08.010 19699198 Fawcett JW Keynes RJ Peripheral nerve regeneration Ann Rev Neurosci 1990 13 43 60 10.1146/annurev.ne.13.030190.000355 2183684 Bridge PM Ball DJ Mackinnon SE Nakao Y Brandt K Nerve crush injuries a model for axonotmesis Exp Neurol 1994 127 284 90 10.1006/exnr.1994.1104 8033968 Gordon T Kelly ME Sanders EJ Shapiro J Smith PA The effects of axotomy on bullfrog sympathetic neurons J Physiol 1987 392 213 229 2833598 Lundborg G A 25-year perspective of peripheral nerve surgery: evolving neuroscientific concepts and clinical significance J Hand Surg 2000 25 391 414 10.1053/jhsu.2000.4165 Navarro X Valero A Gudiño G Forés J Rodríguez FJ Verdú E Pascual R Cuadras J Ensheathing glia transplants promote dorsal root regeneration and spinal reflex restitution after multiple lumbar rhizotomy Ann Neurol 1999 45 207 215 10.1002/1531-8249(199902)45:2<207::AID-ANA11>3.0.CO;2-K 9989623 Fu SY Gordon T The cellular and molecular basis of peripheral nerve regeneration Mol Neurobiol 1997 14 67 116 10.1007/BF02740621 9170101 Andres C Hasenauer J Allgower F Threshold-Free Population Analysis Identifies Larger DRG Neurons to Respond Stronger to NGF Stimulation PLoS One 2012 7 3 e34257 10.1371/journal.pone.0034257 22479579 Andersen LB Schreyer DJ Constitutive expression of GAP-43 correlates with rapid, but not slow regrowth of injured dorsal root axons in the adult rat Exp Neurol 1999 155 157 164 10.1006/exnr.1998.6903 10072292 Chong MS Woolf CJ Turmaine M Intrinsic versus extrinsic factors in determining the regeneration of the central processes of rat dorsal root ganglion neurons: the influence of a peripheral nerve graft J Comp Neurol 1996 370 97 104 10.1002/(SICI)1096-9861(19960617)370:1<97::AID-CNE9>3.0.CO;2-G 8797160 Jenkins R McMahon SB Bond AB Expression of c-Jun as a response to dorsal root and peripheral nerve section in damaged and adjacent intact primary sensory neurons in the rat Eur J Neurosci 1993 5 751 759 10.1111/j.1460-9568.1993.tb00539.x 8261145 Wujek JR Lasek RJ Correlation of axonal regeneration and slow component B in two branches of a single axon J Neurosci 1983 3 243 251 6185656 Chong MS Woolf CJ Haque NS Axonal regeneration from injured dorsal roots into the spinal cord of adult rats J Comp Neurol 1999 410 42 54 10.1002/(SICI)1096-9861(19990719)410:1<42::AID-CNE5>3.0.CO;2-F 10397394 Oblinger MM Lasek RJ A conditioning lesion of the peripheral axons of dorsal root ganglion cells accelerates regeneration of only their peripheral axons J Neurosci 1984 4 1736 1744 6204020 Komiya Y Axonal regeneration in bifurcating axons of rat dorsal root ganglion cells Exp Neurol 1981 73 824 826 10.1016/0014-4886(81)90215-6 6167462 Guan Y Wacnik PW Yang F Spinal cord stimulation-induced analgesia: electrical stimulation of dorsal column and dorsal roots attenuates dorsal horn neuronal excitability in neuropathic rats Anesthesiology 2010 113 1392 1405 10.1097/ALN.0b013e3181fcd95c 21068658 Corinne G Mizisin LM Austin N B vitamins alleviate indices of neuropathic pain in diabeticrats Eur J Pharmacol 2009 612 1–3 41 47 19393643 Carr DF Whiteley G Alfirevic A Investigation of inter-individual variability of the one-carbon folate pathway: a bioinformatic and genetic review Pharmacogenomics J 2009 9 291 305 10.1038/tpj.2009.29 19581920 Scalabrino G Corsi MM Veber D Cobalamin (vitamin B(12)) positively regulates interleukin-6 levels in rat cerebrospinal fluid J Neuroimmunol 2002 127 1–2 37 43 12044973 Scalabrino G The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: Lessons learned from its deficiency Prog Neurobiol 2009 88 3 203 220 10.1016/j.pneurobio.2009.04.004 19394404 Hemendinger RA Edward J Armstrong III, Benjamin Rix Brooks’s.Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death Toxicol Appl Pharmacol 2011 251 3 217 225 10.1016/j.taap.2011.01.003 21237187 Edward R Vitamin B12, folic acid, and the nervous system The Lancet Neurol 2006 5 11 949 960 10.1016/S1474-4422(06)70598-1 Jean-Louis G Maatem CF Shyuefang B Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase Biochimie 2013 95 5 1033 1040 10.1016/j.biochi.2013.01.020 23415654 Yagihashi S Invivo effect of methylcobalamin on the peripheral nerve structure in streptozotocin Diabetic Rats Hom metab Res 1982 14 9 10 13 Okada K Tanaka H Methylcobalamin increases Erk1/2 and Akt activities through the methylation cycle and promotes nerve regeneration in a rat sciatic nerve injury model Exp Neurol 2010 222 2 191 203 10.1016/j.expneurol.2009.12.017 20045411 Watanabe TE Ultrahigh dose Methylcobalamin prometes nerve Regeneration in experimental acrylamid neuropathy J Neurol Sci 1994 12 2 140 143 8021696 Swett JE Hong CZ Most dorsal root ganglion neurons of the adult rat survive nerve crush injury Somatosens Mot Res 1995 12 177 189 10.3109/08990229509093656 8834296 Swett JE Torigoe Y Sensory neurons of the rat sciatic nerve Exp Neurol 1991 114 82 103 10.1016/0014-4886(91)90087-S 1915738 Clarke PG Neuron death in vertebrate development: in vitro methods Methods Cell Biol 1995 46 277 321 7609654 Kuwabara S Nakazawa R Intravenous Methyl cobalamin for uremic and diabetic neuropathy in chronic hemodialisis patients Intern Med 1999 38 6 472 475 10.2169/internalmedicine.38.472 10411351 David JB Litchfield CR Jeffrey MV Spatiotemporal expression profiling of proteins in rat sciatic nerve regeneration using reverse phase protein arrays Proteome Sci 2012 10 9 10.1186/1477-5956-10-9 22325251 Kurtoglu Z Ozturk AH Bagdatoglu C Turac A Camdeviren H Uzmansel D Aktekin M Effects of trapedil on the sciatic nerve with crush injury: a light microscopic study Neuroanatomy 2004 3 54 58 McKay Hart A Brannstrom T Wiberg M Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat: timecourse of cell death and elimination Exp Brain Res 2002 142 308 318 10.1007/s00221-001-0929-0 11819038 Gigo-Benato D Russo TL Tanaka EH Assis L Effects of 660 and 780 nm low-level laser therapy on neuromuscular recovery after crush injury in rat sciatic nerve Lasers Surg Med 2010 42 673 682 20976807 Gjurasin M Miklic P Zupancic B Peptide therapy with pentadecapeptide BPC 157 in traumatic nerve injury Regul Pept 2010 160 33 41 10.1016/j.regpep.2009.11.005 19903499 Yin ZS Zhang H Bo W Erythropoietin promotes functional recovery and enhances nerve regeneration after peripheral nerve injury in rats Am J Neuroradiol 2010 31 509 515 10.3174/ajnr.A1820 20037135 Sakly G Hellara O Trabelsi A Neuropathie périphérique réversible liée au déficit en vitamineB12 Clin Neurophysiol 2005 35 5–6 149 153 Karem A Omar K Noor H Evaluation of vitamin B12 effects on DNA damage induced by pioglitazone Mutation Research/Genetic Toxicol Environ Mutagen 2012 748 2 48 51 Nava-Ocampo AA Pastrak A Cruz T Pharmacokinetics of high doses of cyanocobalamin administered by intravenous injection for 26 weeks in rats Clin Exp Pharmacol Physiol 2005 32 13 18 10.1111/j.1440-1681.2005.04145.x 15730428 Kuzminski AM Del Giacco EJ Allen RH Effective treatment of cobalamin deficiency with oral cobalamin Blood 1998 92 1191 1198 9694707 Wolters M Strohle A Hahn A Cobalamin: a critical vitamin in the elderly Prev Med 2004 39 1256 1266 10.1016/j.ypmed.2004.04.047 15539065 Yamatsu K Kaneko T Pharmacological studies on degeneration and regeneration peripheralnerves. E ffects of methylcobalamin and cobalamin on EMG patterns and loss of muscle weight in rats with crush sciatic nerve Nippon Yakurigaku Zasshi 1976 72 2 256 268 Yamazak K oda K Methylcobalamin (Methyl 1 – B 42) promotes regeneration of motor nerve terminals degenerating in anterior gracile muscle of gracile axonal dystrophy (GAD) mutant mohse Neuroscil Lett 1994 170 195 197 10.1016/0304-3940(94)90272-0 Taniguchi H Ejiri K Improverment of aut onomic neuropathy after methylcobalamin treatment in uremic patients on hemodialysis Clin Ther 1987 9 6 604 614 Hasegawa K Mikuni N Effects of a vitamin B complex on functional recovery after nerve injury (author's transl) Nihon Yakurigaku Zasshi 1978 74 6 721 34 10.1254/fpj.74.721 711031	23902646	PMC3751865	CC BY	2021-01-05 01:30:35	yes	Diagn Pathol. 2013 Jul 31; 8:123
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24009705PONE-D-13-1954310.1371/journal.pone.0072791Research ArticleInterferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation Synergy Mechanism of IFN-α and RBVPanigrahi Rajesh 1 Hazari Sidhartha 1 Chandra Sruti 1 Chandra Partha K. 1 Datta Sibnarayan 1 Kurt Ramazan 1 Cameron Craig E. 4 Huang Zhuhui 5 Zhang Haitao 1 Garry Robert F. 2 Balart Luis A. 3 Dash Srikanta 1 * 1 Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America 2 Micribiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America 3 Gastroenterology, Hepatology and Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America 4 Department of Biochemistry and Molecular Biology, Penn State University, University Park, United States of America 5 Hepatitis Research Program, Southern Research Institute, Frederick, Maryland, United States of America Ray Ranjit Editor Saint Louis University, United States of America * E-mail: sdash@tulane.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SH PKC RFG SD. Performed the experiments: RP SH SC PKC SND RK. Analyzed the data: RP SH PKC SND ZH HZ SD. Contributed reagents/materials/analysis tools: CEC HZ. Wrote the manuscript: RFG LAB SD. 2013 23 8 2013 8 8 e7279113 5 2013 12 7 2013 © 2013 Panigrahi et al2013Panigrahi et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Purpose Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture. Methods The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software. Results RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation. Conclusions We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH. This work was supported from National Institutes of Health grant CA127481, CA089121, AI 103106 and bridge funding received from Tulane University Health Sciences Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction HCV infection leads to a fast progression to chronic liver disease, liver cirrhosis and hepatocellular carcinoma [1]. There are 160 million people infected with HCV representing a major public health problem worldwide [2]. HCV is an enveloped positive-stranded RNA virus that belongs to the Flaviviridae family. This family includes yellow fever and dengue viruses, which also affect humans [3]. The genome of HCV is organized into a highly conserved 5’-untranslated region (5’ UTR), a large open reading frame (ORF) and a 3’-untranslated region (3’ UTR). The 5’ UTR of HCV genome binds to the host ribosome using the internal ribosome entry site (IRES) mechanism that facilitates translation of HCV protein [3,4]. The HCV genome contains a large open reading frame (ORF) that encodes for a polyprotein 3011 amino acid long. The polyprotein is proteolytically processed in the endoplasmic reticulum (ER) membrane into 10 different mature viral proteins by the cellular and viral protease [3]. The core protein and the two glycoproteins E1 and E2 are structural proteins; they are required for the formation of the viral particle, as well as assembly, export and infection. The non-structural (NS) proteins include the p7 ion channel, the NS2 protease, the NS3 serine protease and RNA helicase, the NS4A polypeptide (a cofactor for NS3 protease), the NS4B, the NS5A protein, and the NS5B RNA-dependent RNA polymerase, which are required for replication of the viral genome. The NS proteins (protease and polymerase) have been the targets of intense research efforts for the development of antiviral drugs against HCV. The highly conserved 3’ UTR present at the very end of the HCV genome is important for the initiation of viral RNA replication [5]. HCV infection is initiated by the attachment and entry of virus particles into the host cells by receptor mediated endocytosis [6]. IFN-α and RBV, along with one of the protease inhibitors, is the standard-of-care for chronic HCV 1a infection [7]. Recently the FDA approved two protease inhibitors (Telaprevir and Boceprevir) that are specific to HCV genotype 1 virus NS3 sequences. IFN-α in combination with RBV is still used as the standard treatment for other HCV genotypes. Ribavirin is a guanosine analogue used for the treatment of a number of RNA viruses including the respiratory syncytial virus (RSV), Lass fever virus and HCV [8]. IFN-α and RBV combination therapy is more effective in the treatment of chronic HCV infection than treatment with a single agent [9]. Ribavirin is a synthtic guanosine nucleoside analogue (1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) which has been shown to be metabolized intracellularly into ribavirin mono (RMP), di- (RDP) and triphosphate (RTP) [10]. Although RBV is extensively used to treat patients with HCV-infection the direct antiviral mechanism by which the compound inhibits viral replication remains largely elusive [8]. Furthermore, the mechanism by which the combination of RBV and IFN-α combination improves the treatment response is unclear [11]. Understanding the synergistic antiviral mechanisms of IFN-α and RBV action using the improved HCV cell culture system is important and may open new therapeutic interventions to improve the clinical response. In our present study, we addressed the mechanism of IFN-α and RBV combination synergy by using full-length infectious cell culture, replicon model and a sub-genomic HCV IRES expression model. We observed that IFN-α and RBV each directly inhibited translation of HCV IRES by blocking polyribosome formation. Our results suggest that IFN-α and RBV each activate PKR and eIF-2α phosphorylation which blocks HCV IRES mediated translation and synergistically inhibits HCV replication. Furthermore, RBV mediated inhibition of IMPDH activity also contributes to the blockadge of polyribosome loading. Materials and Methods Cell culture and reagents Human hepatoma cell lines, Huh-7 and Huh-7.5 were maintained in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), non-essential amino acids and sodium pyruvate. The stable S3-GFP replicon cell line (HCV JFH1 2a) was maintained in DMEM and 10% FBS supplemented with G-418 (1 µg/mL) as described in our previous study [12]. Full-length Renilla luciferase (Rluc) reporter based JFH-∆V3-Rluc clone used in our infectivity assay was a kind gift from Curt H. Hagedorn Laboratory [13]. A replication defective adenovirus that expresses T7 RNA polymerase (AdexCAT7) was a kind gift from Tatsuo Miyamura, National Institute of Infectious Disease, Tokyo, Japan [14]. Cell culture derived infectious HCV stocks were prepared from the supernatants of Huh-7.5 cells as described previously [15]. Recombinant IFN-α 2b (Intron-A) was purchased from Schering-Plough (New Jersey, USA). Ribavirin, Cycloheximide and Guanosine were purchased from Sigma Chemical Company (St. Louis, MO, USA). Interferon lambda 1 (IL-29) was obtained from Peprotech, Rocky Hills, NJ, USA. IFN-α and RBV combination treatment in sub-genomic replicon cell line and in infected HCV cell culture S3-GFP replicon cells were cultured in presence of different concentrations of IFN-α and RBV alone and in combination for 72 hours. The antiviral effect of IFN-α and RBV combination treatment was evaluated by GFP expression under a fluorescence microscope and quantified by flow cytometric analysis. Huh-7.5 cells were infected with JFH-∆V3-Rluc virus (MOI 0.1) using a standard protocol [15]. After 48 hours, infected cultures were treated with increasing concentrations of IFN-α or RBV alone or in combination. After 72 hours, the antiviral effect of IFN-α and RBV treatment was measured by NS5A-Rluc activity. Total protein concentration was measured by the Bradford method and luciferase activity was expressed in per micro-gram of total protein. HCV core protein expression was also measured by immunocytochemistry using the following protocol. Infected Huh-7.5 cells with or without IFN-α treatment were mounted onto a glass slide via the cytospin method. The cells were washed in PBS, fixed in chilled acetone for 15 minutes and then permeabilized by Reveal Decloaker RTU (Biocare Medical, RV 100) reagent for 25 minutes. Slides were cooled for 25 minutes and blocking was performed with Background Sniper (Biocare Medical, BS966) for 10 minutes at room temperature. The cells were incubated with monoclonal anti-core antibody (Thermo Scientific, Pierce HCV-core antigen specific mouse monoclonal antibody, Ma1-080) at 1:200 diluted with Da Vinci Green Diluent (Biocare Medical, PD900) for 1 h at room temperature. Cells were washed three times in Tris-buffered saline (TBS) pH 8.0, and incubated with MACH 4 mouse probe (Biocare Medical, UP534) for 10 minutes and then incubated with MACH4 HRP Polymer (Biocare Medical, MRH534) for 30 minutes. Next, the cells were treated with diaminobenzidine (DAB) chromogen (Dako Cytomation, Carpinteria, CA) for 5 minutes. The slides were counterstained with hematoxylin for 30s and Tacha’s bluing Solution (Biocare Medical, HTBLU) for 30 s, dehydrated, mounted and observed by light microscopy. IFN-α and RBV effect on HCV-IRES mediated translation A chimeric sub-genomic clone of HCV IRES and GFP (pHCV IRES-GFP) was used to determine the antiviral mechanisms of IFN-α and RBV as described previously [16]. Plasmids pEGFP-N1 and pDsRed-N1 expressing GFP and RFP (red fluorescence protein) respectively from a human cytomegalovirus (CMV) promoter by a non-IRES dependent mechanism were used as a control (BD Biosciences, Clonetech, Palo Alto, CA). Huh-7 cells (1X104 cells/well) were infected with AdexCAT7 (10 pfu/cell) for 2 hours at 370C, and then transfected with pHCVIRES-GFP clone using the X-tremeGENE 9 transfection reagent (Roche Diagnostics, Indianapolis, IN). The pEGFP-N1 and pDsRed-N1 plasmid was transfected without addition of AdexCAT7 to the cells. Huh-7 cells were first transfected with 1µg of HCV IRES-GFP or pEGFPN1 or pDsRedN1 plasmid and treated with IFN-α (10-1000 IU/mL) and RBV (10-40 µg/mL). After 24 hrs, GFP expression was monitored under a fluorescence microscope (Olympus IX 70, Germany) and quantified by flow cytometric analysis. Transfected cells were examined with a fluorescent microscope at 484 nm for the expression of GFP and at 340 nm for Hoechst 33342 stain. Nuclear stain was superimposed over cytoplasmic GFP using Adobe Photoshop computer software generated the images. Evaluation of synergy interaction at the level of HCV-IRES mediated translation A Sub-genomic construct (pHCV-IRES-RLuc) with T7 promoter, HCV IRES-Rluc fusion, 3’ UTR of HCV, a cDNA copy of the autolytic ribozyme from antigenomic strand of the hepatitis delta virus and T7 transcriptional terminator sequences was used to study HCV-IRES mediated translation. Huh-7 cells were transfected with pHCV-IRES-RLuc plasmid using the same procedure described above. The cells were treated with IFN-α (10-1000 IU/mL) and RBV (10-80 µg/mL) alone and combination immediately after transfection and incubated at 37oC for 24 hours. After 24 hours, the cells were washed with PBS, lysed and Renilla luciferase activity was measured (Luman LB9507, EG & G, Berthold, Berlin, Germany). The consistency of the results was maintained by quantifying emissions from triplicate wells for each treatment. IFN-λ also shows good antiviral effect against HCV (unpublished data). To determine the combinatory effect of IFN-α, IFN-λ and RBV, Huh-7 cells transfected with pHCV-IRES-RLuc plasmid were treated with 0, 10, 100, 1000 IU/mL of IFN-α 0 10 20 50 100 μg/mL IFN-λ and 0, 10, 20, 40 µg/mL of RBV. Renilla luciferase values were analyzed by using the CalcuSyn software (Biosoft). This program uses the median-effect principle to delineate the interaction between these two drugs. For each combination, the program generates a combination index (CI) based on the equation below described by Chou et al [17,18]. A combination index (CI) of <1 means synergism, CI=1 means additive and CI>1 means antagonism. Drug–drug combination analysis of IFN-α and RBV was performed with the MacSynergy II program [19,20]. Polyribosome fractionation and Northern blot analysis To examine whether IFN-α and RBV treatment inhibits translation by preventing the loading of polyribosomes on the IRES-GFP mRNA, polysome analysis was performed using sucrose density gradient centrifugation. Huh-7 cells were transfected with pHCV IRES-GFP clone using a two-step transfection procedure. After transfection, cells were treated with IFN-α (1000 IU/mL) or RBV (40 µg/mL) and after 24 hours, the expression of GFP was examined. The polysome analysis was performed using a protocol described earlier [21]. Briefly, transfected Huh-7 cells were washed twice with ice-cold PBS pH-7.2 containing 100µg/mL cycloheximide. The cells were lysed by 200 µl of polysomes lysis buffer containing 100 mM KCl, 5mM MgCl2, 10 mM HEPES, pH7.4, 100 µg/mL cycloheximide, 0.5% Nonidet P-40, and 1000 units/mL RNase inhibitor (Ambion Inc, Austin, TX). The cell lysate was passed four times through a 27-gauge needle to ensure complete cell lysis. Nuclei were pelleted by centrifugation at 12,000 rpm for 5 minutes. The supernatant was collected and centrifuged an additional time to ensure the removal of any nuclei. The resulting supernatant was layered on a linear 15-60% (w/v) sucrose gradient in polysome gradient buffer (100 mM KCl, 5 mM Mg Cl2 and 10 mM HEPES pH 7.4) and centrifuged at 36,000 rpm for 2 hours at 4°C in a Beckman SW41 rotor. The distribution of ribosomal RNA along the sucrose density gradient fractions was determined using a polysome fractionator (Teledyne ISCO, Brandel, Inc, Gaithersburg, Maryland). Total RNA was isolated from the sucrose density fractions by treating with proteinase K solution (0.2 M Tris-HCl, pH 7.5, 25 mM EDTA, 0.3 M NaCl, 2% SDS, and 250 µg/mL proteinase K, RNase free DNase-I 10 U/mL). Half of the RNA samples were subjected to Northern blot analysis to examine the distribution of HCV IRES-GFP mRNA in each fraction. Identical experiments were performed to determine the effect of RBV on the distribution of HCV IRES-GFP mRNA in the polysome fractions. Control experiments were performed using the pEGFP-N1 plasmid to determine the effect of IFN-α on the distribution of mRNA in the polysome fraction whose translation occurs via cap-dependent mechanism. To detect the HCV-IRES mRNA in the polysome fractions an anti-sense 32P labeled riboprobe (106 cpm/mL) targeted to the highly conserved 5’ UTR of HCV genome was used. To detect EGFP mRNA in the polysome fractions, an anti-sense 32P labeled RNA probe (106 cpm/mL) targeted to the GFP was used. Northern hybridization was performed using the ULTRAhyb reagent (Ambion Inc, Austin, TX) at 680C for 16 hours. Blots were then washed twice for 15 min each at 370C using a washing solution (0.1X SSC-0.1% SDS), followed by two 15 min washes at 370C using a washing solution (0.1X SSC-0.1% SDS). The membrane was exposed for autoradiography using Bio Max X-ray film (Kodak imaging system). Proteins bound to polyribosomes were isolated using a standard protocol [22]. Briefly, sucrose gradient fractions were precipitated by an addition of cold trichloroacetic acid to a final volume of 10% and were incubated on ice for 30 minutes. This step was followed by centrifugation at 20,000g for 15 min at 4°C. Pellets were washed once with 5% TCA and once with cold acetone. Finally, protein pellets were resuspended in a sample buffer (50 mM Tris at pH 6.8, 2% SDS, 2% glycerol) and 1% β-mercaptoethanol or 1 mM DTT, heated at 65°C, and processed for SDS-PAGE. Protein in the or transferred to nitrocellulose for Western blot. Western blot analysis Protein lysates from cells were prepared after treatment with IFN-α and RBV for 24 hours. Equal amounts of protein were resolved on SDS-PAGE gels. The antibodies to PKR, eIF2α, peIF2α (Ser51), β-actin, PKR, anti-mouse IgG, and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling, Beverly, MA. Antibody to p-PKR (pT446) was obtained from Epitomics, Burlingame, CA. Antibody to IMPDH was obtained from Santa Cruz, Dallas, USA. Twenty microgram of proteins were resuspended in sample buffer (50 mM Tris at pH 6.8, 2% SDS, 2% glycerol) and 1% β-mercaptoethanol or 1 mM DTT, heated at 65°C, and processed for SDS-PAGE. Proteins were transferred to nitrocellulose membrane and Western blotting was performed using a standard protocol. Knockdown of PKR and IMPDH mRNA siRNA duplexes targeting the coding regions of human IMPDH1 (Qiagen, catalog no. SI02781044), PKR (Qiagen, SI02223018) and unrelated control siRNA were obtained from Qiagen. Huh7 cells were transfected with the indicated siRNA duplexes using Oligiofectamine (Invitrogen, CA). After 6 hours of siRNA treatment the cells were transfected with the IRES-GFP sub-genomic construct and then treated with either IFN-α or RBV to examine translational inhibition. Results IFN-α and RBV synergistically inhibit HCV replication in replicon and full-length infectious cell culture models The genomic and sub-genomic clones for HCV genotype 2a used to develop the HCV replication model are shown (Figure S1A ). We first measured the cytotoxic effects of IFN-α and RBV treatment alone and in various combinations using Huh-7 cells and S3-GFP replicon cell line by a MTT assay. Ribavirin up to 200 µg/mL did not show any cytotoxicity (Figure S2A ). The viability of S3-GFP cells were more than 90% at 48 hours when treated with RBV (10-60 µg/mL) and IFN-α (10-1000 IU/mL) alone (Figure S2B ) or in combination (Figure S2C ). Based on the MTT assay results, concentration of RBV (10-40µg/ml) permitting high viability was used for subsequent antiviral assays. The antiviral effect of IFN-α and RBV combination treatment in S3-GFP replicon cells after 72 hours was confirmed by the measurement of GFP expression under a fluorescence microscope (Figure 1A ) and the expressed GFP was quantified by flow cytometric analysis (Figure 1B ). The sub-genomic replicon system does not produce infectious virus due to lack of the structural proteins. Antiviral effect of IFN-α and RBV combination treatment was measured using an infectious cell culture model using the JFH1-Rluc chimera virus. The IFN-α and RBV treatment gradually reduced the RLuc activity in a dose dependent manner (Figure 1C ). The inhibition of HCV replication was significant at RBV 20µg/mL with IFN-α (100 IU/mL) and RBV 40µg/mL with IFN-α (250 IU/mL). We verified the antiviral effect of combination treatment by measuring HCV core protein expression by immunostaining (Figure 1D ). The number of HCV core positive cells in five different high power fields (hpf) were counted and compared with untreated control (Figure 1E ). 10.1371/journal.pone.0072791.g001Figure 1 Antiviral effect of IFN-α and RBV combination treatment using a sub-genomic replicon cell line (S3-GFP) and HCV infected Huh-7.5 cells. (A) S3-GFP cells were treated with IFN-α and RBV for 72 hours. GFP expression was examined under a fluorescence microscope. (B) GFP positive cells were quantified by flow cytometric analysis. (C) Infected Huh-7.5 cells were treated with IFN-α alone, RBV alone and combination for 72 hours. Renilla Luciferase activity of infected cells was measured and normalized with 1µg of cellular protein. (D) Expression of HCV core protein was measured by immunostaining and (E) core positive cells in five different high power fields (hpf) at 40X magnification were counted under a light microscope. Quantitative assessment of the number of HCV positive cells with mean and standard deviation of the combination treatment are compared. IFN-α, IFN-λ1 and RBV combination treatment synergistically inhibit HCV IRES mediated translation Previously we reported that type I and Type II IFN inhibit HCV replication by targeting the 5’ UTR of HCV RNA genome used for IRES mediated translation [23]. Here we examined whether IFN-α and RBV combination treatment could also inhibit the HCV IRES mediated translation. The mechanisms of IFN-α and RBV action on HCV translation were examined using HCV IRES-GFP or HCV IRES-RLuc based subgenomic clones (Figure S1B ). Plasmid clones pEGFP-N1 and pDsRed-N1 were used as controls to examine the effect of IFN-α and RBV treatment on the expression of GFP or RFP by non-IRES mechanisms (Figure S1B ). High-level expression of GFP from HCV IRES in Huh-7 cells was achieved by using two-step transfection procedures that first involve infection with replication defective adenovirus that expresses T7 RNA polymerase (AdexCaT7), followed by transfection with a transcription plasmid (Figure S1C ). The HCV IRES mediated translation of GFP was inhbited by both IFN-α and RBV at increasing concentration of both the drugs as evidenced by fluorescence imaging (Figure 2A ) and Western blot analysis (Figure 2B ). The cap dependent translation of GFP or RFP was not inhibited by addition of these two drugs (Figures S3A and S3B ). IFN-α and RBV show maximum HCV IRES inhibition at 1000 IU/mL and 40 µg/mL respectively. Results of Northern blot analysis indicate that the intracellular IRES GFP mRNA is relatively stable in the IFN-α and RBV treatment. There is no significant difference in the stability of HCV IRES-GFP mRNA in Huh-7 cells treated with increasing concentrations of IFN-α (10 to 1000 IU/mL) compared to GAPDH mRNA level used as a control (data not shown). These results indicate that IFN-α and RBV treatment inhibit translation of HCV IRES-GFP without altering the stability of intracellular HCV IRES sub-genomic mRNA. Interferon lambda (IFN-λ1) is a type III IFN, which has been found to have a sustained antiviral activity against HCV (unpublished results). We quantified the relative antiviral activity of IFN-α, IFN-λ1 and RBV at the level of HCV IRES translation using a HCV IRES Rluc plasmid (Figure S1B ). Huh-7 cells were transfected with HCVIRES-Rluc plasmid and then treated with different concentrations of IFN-α, IFN-λ1 and RBV alone and in combination. The antiviral activity of combination treatment was measured by Renilla luciferase activity per microgram of cellular protein. The results presented in Figure 3A show that IFN-α, IFN-λ1 and RBV each inhibits HCV replication in a dose-dependent manner. The combination of IFN-α and IFN-λ1 at the level of HCV-IRESRLuc expression was examined (Figure 3B ). Combination treatment of IFN-α with RBV (Figure 3C ) and IFN-λ1 with RBV (Figure 3D ) showed a stronger inhibitory effect on HCV IRES-Rluc expression. Determination of a synergistic, additive or antagonstic effect of IFN-α and RBV combination was performed according to the median effect principle using the CalcuSyn computer program. The combination treatment of IFN-α and RBV was highly synergistic with CI values of <1. Results using CalcuSyn software revealed synergistic interactions across the entire range of RBV with either IFN-α or IFN-λ1 combinations tested (Figure 4A and 4B ). IFN-α and IFN-λ1 combination treatment did not show synergistic inhibition of the HCV IRES-translation (Figure 4C ). Analysis of IFN-α, IFN-λ1 and RBV treatment was subsequently performed with the MacSynergy II program. The MacSynergy II program calculated the theoretical additive interactions of the drugs based on the Bliss Independence mathematical definition of expected effects for drug–drug interactions. The additive interactions were calculated from the dose–response. If the interactions are additive, the resulting surface appeared as horizontal plane at 0% above the calculated additive surface in the resulting difference plot. Peaks above this plane is an indicative of synergy, while depression below the horizontal plane is an indication of antagonism. This analysis revealed that RBV treatment in combination with either IFN-α or IFN-λ1 had resulted in strong synergistic interactions (Figure 4G and 4H ). In contrast, IFN-α and IFN-λ1 combination treatment show slightly antagonistic interactions (Figure 4C and 4I ). Average cell inhibition was shown in Figure 4D, 4E and 4F. In conclusion, synergistic interactions between RBV and IFN treatments were observed at physiologically relevant concentrations. 10.1371/journal.pone.0072791.g002Figure 2 IFN-α and RBV each inhibited the internal ribosome entry site (IRES) mediated translation of green fluorescence protein (GFP). Huh-7 cells were infected with T7-expressing adenovirus. After 2 hrs, HCV IRES-GFP plasmid was transfected and then treated with indicated concentration of IFN-α and RBV. (A) HCV IRES mediated GFP expression was monitored under fluorescent microscopy. (B) Inhibition of GFP expression was further confirmed by Western blot analysis in both IRES and non-IRES mechanisms. β-actin is used as loading controls. 10.1371/journal.pone.0072791.g003Figure 3 Different combinations of IFN-α, RBV, and IFN-λ inhibits HCV IRES Rluc mediated translation. Huh-7 cells were infected with T7-expressing adenovirus. After 2 hrs, HCV IRES-RLuc plasmid was transfected and then treated with indicated concentration of IFN-α, IFN-λ and RBV. The concentration dependent inhibition of Renilla luciferase activity by (A) IFN-α, RBV, and IFN-λ single treatment;(B) Combination of IFN-α + IFN-λ; (C) Combination of IFN-α + RBV and (D) Combination of IFN-λ + RBV. 10.1371/journal.pone.0072791.g004Figure 4 Analysis for synergistic effect of IFN-α + IFN-λ, IFN-α + RBV, and IFN-λ + RBV using Calcusyn and MacSynergyII software. (A) CalcuSyn software analysis show that IFN-α + RBV combination treatment has a very strong synergy antiviral activity against HCV IRES mediated inhibition with combination index, CI<1. (B) IFN-λ + RBV combination treatment also has a very strong synergy antiviral activity with CI<1. (C) IFN-α + IFN-λ treatment are either additive or slightly antagonistic. Three dimensional inhibition plots of (D) IFN-α + RBV, (E) IFN-λ + RBV and (F) IFN-α + IFN-λ treatment against HCV IRES mediated inhibition of Rluc at 95% confidence interval synergy plot. Three dimensional synergy plot of (G) IFN-α + RBV, (H) IFN-λ + RBV, and (I) IFN-λ + IFN-α. IFN-α and RBV treatment prevents loading of polyribosome to HCV IRES containing mRNA The translation of HCV genomic RNA is initiated by the binding of the host cell ribosome to a highly conserved RNA sequence called the internal ribosome entry site (IRES), located in the 5’ UTR. We examined whether inhibition of GFP expression in the HCV IRES subgenomic clone could have occurred due to a differential loading of polyribosome. The upper panel (Figure 5A ) shows the separation of 40S, 60S and 80S and polyribosme in the sucrose density gradient using a polysome fractionator (Teledyne ISCO, BRANDEL). Total RNA from each gradient fraction was isolated and analyzed by agarose gel electrophoresis. The location of monosomes and polysomes was determined by ethidium bromide staining (Figure 5B ). Polysome fractionation of IRES-GFP transfected Huh-7 cells after treatment with IFN-α or RBV was performed to examine distribution of HCV IRES-GFP mRNA in the monosome and polysome fractions. The amount of HCV IRES containing GFP mRNA associated with each ribosome fraction was determined by Northern blot analysis using an antisense RNA probe targeted to the 5’ UTR. Northern analysis of transfected cells revealed that under a normal translation condition without treatment, the distribution of HCV IRES-GFP mRNA gradually increased from monosome to polysome, suggesting an increased efficiency of ribosome loading and continued translation. In contrast, IFN-α treatment (IFN+) resulted in an arrest of the majority of HCV IRES-GFP mRNA in the monosome peaks and reduction in the polysome fractions (Figure 5C , lanes 12-14). Similar results were consistently achieved in three separate experiments. A Similar mechanism is also operative in the case of RBV treatment. Polysome analysis was performed using HCV IRES-GFP transfected cells treated with RBV. The distribution of IRES-GFP mRNA in the RBV treated (RBV+) cells was found in monosome peaks and reduction in polysome peaks (Figure 5D , lanes 10-14). To address the specificity of IFN-α action on the IRES-GFP mRNA translation, we examined mRNA distribution using EGFP mRNA after IFN-α or RBV treatment. The distribution of GFP mRNA in the polysome fractions was measured by Northern blot analysis using a GFP specific antisense riboprobe. We found no significant difference in the distribution of EGFP mRNA between the monosome and polysome fractions between cells with or without IFN-α (Figure 5E ). To correlate the results of HCV IRES-GFP mRNA distribution profiles in the polysome fractions in the transfected cells with and without IFN treatment, we performed comparative analysis by measuring the density of bands seen in the Northern blot analysis. The band intensity of Northern blots was measured using TatalLab (TL120) software and the values were expressed as a percentage of total RNA recovered in the gradient (Figure 5F ). This analysis clearly shows that both IFN-α and RBV the inhibited loading of polyribosomes to the HCV-IRES containing mRNA. This type of alternation in the mRNA distribution was not observed using control mRNA that is translated via non-IRES mechanism (Figure 5G ). 10.1371/journal.pone.0072791.g005Figure 5 The distribution of HCV IRES-GFP mRNA in the monosome and polysomes fractions in the Huh-7 cells with (+) and without (-) IFN-α and RBV treatment. (A) Illustrates the separation of monosome and polysomes along the sucrose gradient fractions (1 to 14). The values indicate the spectrophotometry of optical density of the polysome fractions at 260 nm wavelengths. The point arrow shows the 60S, 80S and separation between monosomes and polysomes in the gradient fractions. (B) Formaldehyde agarose gel electrophoresis and ethidium bromide staining of RNA samples isolated from the corresponding gradient fractions of untreated Huh-7 cells. The 18S and 28S band appears on the gel throughout the fractions and it become more intense on the 80S fractions of the gradient as expected. (C) Shows the distribution of HCV IRES-GFP mRNA in the monosome and polysome fractions by Northern blot analysis using a riboprobe targeted to the 5’ UTR. In the untreated IFN (-) cells the IRES-GFP mRNA efficiently translated and formed polyribosome complexes (Lane 11-14). But the IFN treatment (+) prevented polysome formation on IRES-GFP mRNA (Lane 11-14). (D) In the RBV untreated Huh-7 cells, the IRES-GFP mRNA efficiently translated and formed polyribosome complexes (Lane 11-14). RBV treatment (+) prevented polysome formation on IRES-GFP mRNA (Lane 10-14). (E) Similar experiment was performed where the effect of IFN-α or RBV treatment on the distribution of EGFP mRNA was examined by Northern blotting using RNA probe specific to GFP. IFN-α treatment did not alter the distribution of EGFP mRNA that translates by non-IRES dependent mechanism. (F) Comparison of the relative amount of HCV IRES and non-IRES mRNAs in monosome and polysome fractions in the sucrose density gradient analysis generated from the transfected cells. Density of the Northern blot was measured using an image analysis computer software (Total Lab, TL120). Values are expressed as percentage of total mRNA recovered from the gradient versus the mRNA present in each fraction. (G) The formation of polyribosome of EGFP mRNA was not altered by IFN-α treatment. RBV treatment altered the association of IMPDH and protein kinase R (PKR) with polysome fractions To determine whether IMPDH levels could be associated differently in the polysome fractions after RBV treatment which is why inhibited the HCV IRES mediated GFP translation, protein extracts were prepared from monosome and polysome fractions and Western blot analysis was performed. Untreated cells IMPDH, PKR, and pPKR were detectable throughout the gradient (Figure 6A ). IFN-α treatment induced PKR activation and eIF2α phosphorylation. The phosphorylated eIF2α protein was accumulated in the monosome and disome fractions but absent in the polysome fractions (Figure 6B , lane 6-10). Ribavirin treatment accumulated the IMPDH levels in the monosome and disome fractions but not in the polysome fractions (Figure 6C ). Activated pPKR and peIF2α were also detected in the monosome and disome fractions but not in the polysome fractions (Figure 6C , lanes 7-10). These results indicated that RBV treatment inhibited distribution of cellular IMPDH, which accumulated in the lower density ribosome fractions. 10.1371/journal.pone.0072791.g006Figure 6 Western blot analysis of polyribosome fractions of HCV IRES-GFP transfected cells. (A) Untreated HCV IRES-GFP transfected cells showing IMPDH, PKR, pPKR is bound to the ribosome throughout the gradient. (B) PKR induced peIF2α protein is found in the monosome-containing fractions (lanes 1-5) and absent in the higher density polyribosome fractions (lanes 6-10) due to IFN-α treatment. (C) IMPDH, pPKR and peIF2α proteins were found in the monosome fractions (lanes 1-6) but excluded from the high-density polyribosome fractions (lanes 6-10). The level of actin was detected throughout the gradient. PKR and IMPDH are required for IFN-α and RBV mediated inhibition of HCV IRES-GFP translation We found the phosphorylation of PKR and eIF2α was increased due to IFN-α or RBV treatment (Figure 7A ). Ribavirin is a synthetic nucleoside analog and known inhibitor of IMPDH enzyme. Ribavirin or IFN-α treatment did not increase or decrease the expression of IMPDH level (Figure 7A ). Inhibition of IMPDH and PKR levels by siRNA restored the inhibitory action of RBV on HCV IRES-GFP translation (Figure 7B and C ). Inhibition of IMPDH activity by RBV is known to decrease the intracellular level of guanosine nucleotide pools resulting in the antiviral activity. Pretreatment with increased concentration of guanoside indeed neutralized the RBV mediated IRES-GFP translation (Figure 7D ). Depletion of the GTP pool caused by the inhibition of IMPDH enzyme activity due to RBV contributes to the inhibition of HCV IRES-GFP translation. We also verified that the inhibition of PKR by siRNA prevented IFN-α mediated inhibition of HCV IRES-GFP translation (Figure 7E and F ). These results suggest that PKR and IMPDH are involved in the IFN-α and RBV mediated synergistic inhibition of HCV IRES mediated translation. 10.1371/journal.pone.0072791.g007Figure 7 IFN-α and RBV synergy antiviral mechanism involves the activation of PKR, eIF2α and inhibition of cellular IMPDH. (A) IFN-α and RBV each induced phosphorylation of PKR and eIF2α. (B) Flow cytometric analysis showing RBV show a dose dependent inhibition of HCV IRES-GFP translation. (C) Inhibition of IMPDH and PKR levels by siRNA prevented RBV antiviral action against HCV IRES-GFP translation determined by flow cytometric analysis. (D) Dose dependent prevention of RBV action due to increasing concentration of guanosine was determined by flow cytometric analysis. (E) IFN-α inhibits HCV IRES-GFP translation. (F) Inhibition of PKR by siRNA prevented IFN-α mediated inhibition of HCV IRES-GFP translation. Discussion Molecular studies for determining IFN-α antiviral mechansims against HCV are possible due to the availability of highly efficient HCV cell culture systems. Many investigators, including our laboratory, have shown that IFN-α effectively inhibits HCV replication in cell culture model [23,24]. IFN-α binds to the cell surface receptors leading to the activation of Janus kinase signal transducer and activator of transcription (Jak-Stat) pathway. Activation of cellular Jak-Stat pathway results in the phosphorylation and nuclear translocation of the Stat-IRF9 complex to initiate antiviral gene transcription [25]. A number of key antiviral proteins are induced through the activation of the Jak-Stat pathway including the double stranded RNA-activated protein kinase (PKR), 2’5’-oligoadenylate synthethase (2’5’ OAS) and MxA. Studies have shown that IFN-α induced antiviral activity is mediated by interferon inducible ISGs [26]. Mechanisms of IFN-α antiviral activity through the inhibition of HCV IRES mediated translation are supported by a number of studies [27–31]. The newly discovered type III IFN called IFN-λ also inhibits IRES mediated translation of HCV and hepatitis A [32]. There is an agreement that Type I, Type II and Type III IFN inhibit HCV replication by blocking at the level of HCV IRES mediated translation that involves the PKR induced phosphorylation of eIF2α [27,28]. The eIF2α is an eukaryotic initiation factor required for protein translation [33]. This eIF2 protein exists as heterotrimer consisting of eIF-α, eIF-beta and eIF-gamma. The eIF2 protein complexes with GTP and the initiator t-RNA to form the 43S pre-initiation complex. The 43S pre-initiation complex binds to AUG codon on the target mRNA to initiate protein translation. The dissociation of the complex occurs when the eIF2 to hydrolyzes its GTP by eIF5 (a GTPase-activating protein). This conversion causes the eIF-2-GDP to be released from the 48S complex and translation to begin after recruitment of 60S ribosome used and formation of 80S initiation complex. With the help of guanine nucleotide exchange factor eIF2-beta, the eIF2-GDP is exchanged to eIF2-GTP, which initiates another round of translation. The phosphorylation of eIF2α inhibits recycling of this initiation factor and blocks protein synthesis [33]. The antiviral activity of RBV against HCV is mediated through a number of mechanisms which include: (i) inhibition of cellular IMPDH required for de novo synthesis of guanosine triphosphate, (ii) RBV triphosphate directly inhibits HCV RNA polymerase activity, (iii) RBV can be incorporated into viral genome by HCV RNA polymerase causing mutation in the viral genome, (iv) RBV enhances IFN-α signaling by inducing the expression of interferon-stimulated genes (ISG), (v) RBV also inhibits cellular eIF4E activity required for translation of viral genome, and (vi) RBV helps to clear the virus by stimulating the T helper 1 response of host. Among these candidate mechanisms inhibition of cellular IMPDH by RBV has been verified by a number of laboratories using HCV and other virus infection models [34–37]. Molecular studies of RBV action against HCV are possible due to the availability of in vitro cell culture systems. A number of new studies support the RBV antiviral mechanism against HCV replication through inhibition of cellular IMPDH and reduction of GTP pool [37–40]. Mori et al [37] reported that the predominant antiviral mechanism of RBV against HCV is through the inhibition of inosine monophosphate dehydrogenase (IMPDH) not though the error catastrophe, the IFN signaling or oxidative stress. This study is supported by results of other investigators who showed that decrease in GTP also leads to suppression of HCV RNA synthesis by NS5B RNA polymerase [38]. The mechanism of IMPDH inhibition by RBV is supported by the report of Zhou et al [39] indicating that exogenous guanosine suppressed the RBV effect where as potent IMPDH inhibitors MPA and VX-497 enhanced RBV antiviral effect. IMPDH modulates intracellular guanosine nucleotide levels. Therefore it affects a number of cellular processes involved in translation, cell proliferation and RNA/DNA synthesis. IMPDH catalyzes the important step in guanine nucleotide biosynthesis. IMPDH has been shown to be associated with polyribosome, suggesting that this house-keeping gene plays an important role in translational regulation [41]. In our study we found that the distribution of IMPDH is halted in monosome and disome fraction and absent in polysome fractions supporting the role IMPDH in HCV IRES mediated translation. Ribavirin in combination with IFN-α showed a marked improvement in the sustained antiviral response in chronic HCV infection. The synergistic antiviral mechanism of IFNα and RBV combination therapy is not known. Only a few studies have been published which explain why RBV and IFN-α combination treatment is highly effective against HCV replication [42–46]. Thomas et al [42] showed that RBV enhanced the IFN-α antiviral activity by inducing the expression of interferon inducible genes (ISGs) and interferon regulatory factor (IRF-7) and (IRF-9). Stevenson et al [43] showed that RBV enhanced IFN-α induced phosphorylation of Stat1, Stat3 and MxA expression and enhanced IFN-α induced cellular Jak-Stat pathway. Liu et al [44] showed that RBV enhances IFN-α signaling through activation of separate antiviral signaling by inducing the expression of cellular p53. This finding is supported by a report indicating that p53 plays an important role in host antiviral defense mechanisms and directly inhibits HCV replication [45]. A previous report by Liu et al [46] indicates that RBV enhances the IFN-α antiviral activity through the up-regulation of PKR activity. None of these studies have shown the synergistic antiviral effect of IFN-α and RBV combination treatment using HCV cell culture. Our results indicate IFN-α and RBV combination treatment synergistically inhibit HCV replication in replicon and infected cell culture models. We show here for the first time that the synergy antiviral action of IFN-α and RBV combination therapy is at the level of inhibition of HCV IRES mediated translation. IFN-α directly inhibits HCV IRES translation by preventing polyribosome loading through PKR mediated eIF2α phosphorylation. Ribavirin inhibits HCV IRES translation by preventing the polyribosome loading of HCV IRES mRNA. Ribavirin mediated blockage of polyribosome loading involves two important mechanisms that involve PKR and IMPDH. Ribavirin mediated PKR and eIF2α phosphorylation inhibits the recycling of eIF2α and inhibits HCV IRES translation. Ribavirin mediated inhibition of IMPDH activity decreases the cellular GTP pool, which inhibits the HCV-IRES translation by preventing polyribosome loading. This is supported by the results showing that pretreatment of guanosine prevented RBV mediated HCV IRES-GFP translation. Based on these observations, we propose a model explaining how RBV mediated depletion of GTP pool and activation of PKR by IFN-α and RBV combination treatment could be playing an important role in the synergy antiviral mechanism (Figure 8 ). The detailed mechanism how IFN-α and RBV combination treatment leads to efficient translation arrest of HCV IRES mRNA will be the topic of future investigation. 10.1371/journal.pone.0072791.g008Figure 8 Diagram summarized the proposed IFN-α and RBV synergy antiviral mechanisms against HCV IRES-GFP translation. IFN-α binds to the cell surface receptor, which activates the cellular Jak-Stat pathway leading to the activation of PKR. The activated PKR phosphorylates the eIF-2α. Phosphorylation of eIF-2α inhibits the recycling of initiation factors and translation initiation. On the other hand, RBV activates the PKR and eIF2 α phosphorylation and inhibits the translation initiation. Ribavirin inhibits HCV IRES translation by inhibiting IMPDH and GTP pool. Supporting Information Figure S1 HCV genomic and sub-genomic constructs and cell culture models used to study IFN-α and RBV antiviral synergy mechanisms. (A) HCV full-length JFH1-RLuc chimera clone (13) used in the infectivity assay and sub-genomic HCV RNA used to generate stable S3-GFP replicon cell line (12). (B) Structure of pHCV IRES-GFP and pHCV-IRES-Rluc plasmid clone used for this study. The HCV-IRES-sequences were transcribed from T7 promoter and the 3’ UTR sequences were added at the 3’ end of GFP or RLuc. pEGFP-N1 and pDsRed-N1 plasmids were used as controls. (C) Shows the steps used to express HCV IRES-GFP or HCV IRES-RLuc using a recombinant adenovirus expressing T7 RNA polymerase. (TIF) Click here for additional data file. Figure S2 MTT assay showing the effect of IFN-α and RBV combination treatment on viability of Huh-7 and S3-GFP cells. (A) Huh-7 cells were treated with increasing concentration of RBV (10-200 µg/mL) for 48 hours and the viability was measured. (B) S3-GFP cells were treated with indicated concentrations of RBV or IFN-α and cell viability was determined at 48 hours. (C) Cell viability of combination treatment of IFN-α and RBV at various combinations. S3-GFP cells were treated with different concentration of RBV with one concentration of IFN-α for 48 hours and cell viability was determined by MTT assay. (TIF) Click here for additional data file. Figure S3 Effect of IFN-α and RBV treatment on (A) GFP and (B) Red fluorescence protein (RFP) expression by non-IRES mechanism. (TIF) Click here for additional data file. We thank Daniel Hoskins and Michelle McCarthy for critically reviewing this manuscript. The authors thank Charles M Rice for providing Huh-7.5 cells, and Shuanghu Liu and Curt H Hagedorn, University of Utah School of Medicine, for providing the JFH-∆V3-Rluc plasmid. The authors acknowledge Krzysztof Moroz for taking the picture of immunostaining slides. ==== Refs References 1 Lavanchy D (2009 ) The global burden of hepatitis C . Liver Int 29 (Suppl 1 ): 74 –81 . doi:10.1111/j.1478-3231.2008.01702.x. PubMed: 19207969. 2 Shepard CW , Finelli L , Alter MJ (2005 ) Global epidemiology of hepatitis C virus infection . Lancet Infect Dis 5 : 558 -567 . doi:10.1016/S1473-3099(05)70216-4. PubMed: 16122679.16122679 3 Moradpour D , Penin F , Rice CM (2007 ) Replication of hepatitis C virus . Nat Rev Microbiol 5 (6): 453 –463 . doi:10.1038/nrmicro1645. PubMed: 17487147.17487147 4 Qi ZT , Kalkeri G , Hanible J , Prabhu R , Bastian F et al. (2003 ) Stem-loop structures (II-IV) of the 5’ untranslated sequences are required for the expression of the full-length hepatitis C virus genome . Arch Virol 148 [3]: 449 -467 . doi:10.1007/s00705-002-0933-0. PubMed: 12607098.12607098 5 Friebe P , Bartenschlager R (2002 ) Genetic analysis of the sequences in the 3’ UTR of hepatitis C virus that are important for RNA replication . J Virol 76 : 5326 -5338 . doi:10.1128/JVI.76.11.5326-5338.2002. PubMed: 11991961.11991961 6 Blanchard E , Belouzard S , Goueslain L , Wakita T , Dubuisson J et al. (2006 ) Hepatitis C virus entry depends on clathrin-mediated endocytosis . J Virol 80 : 6964 -6972 . doi:10.1128/JVI.00024-06. PubMed: 16809302.16809302 7 Hofmann WP , Zeuzem S (2011 ) A new standard of care for the treatment of chronic HCV infection . Nat Rev Gastroenterol Hepatol 8 : 257 –264 . PubMed: 21468124.21468124 8 Paeshuyse J , Dallmeier K , Neyts J (2011 ) RBV for the treatment of chronic hepatitis C virus infection: a review of the proposed mechanisms of action . Curr Opin Virol 1 (6): 590 -598 . doi:10.1016/j.coviro.2011.10.030. PubMed: 22440916.22440916 9 Davis GL , Esteban-Mur R , Rustgi V , Hoefs J , Gordon SC et al. (1998 ) Interferon alfa-2b alone or in combination with RBV for the treatment of relapse of chronic hepatitis C . N Engl J Med 339 : 1493 –1499 . doi:10.1056/NEJM199811193392102. PubMed: 9819447.9819447 10 Sidwell RW , Witkowsk JT , Allen LB , Robins RK , Khare GP et al. (1972 ) Broad-spectrum antiviral activity of virazole-1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide . Science 177 : 705 -706 . doi:10.1126/science.177.4050.705. PubMed: 4340949.4340949 11 Feld JJ , Hoofnagle JH (2005 ) Mechanism of action of interferon and RBV in treatment of hepatitis C . Nature 436 (7053): 967 -972 . doi:10.1038/nature04082. PubMed: 16107837.16107837 12 Hazari S , Chandra PK , Poat B , Datta S , Garry RF et al. (2010 ) Impaired antiviral activity of IFN-α in Huh-7 cells with defective Jak-Stat pathway . Virol J 7 : 36 . doi:10.1186/1743-422X-7-36. PubMed: 20149251.20149251 13 Liu S , Nelson CA , Xiao L , Lu L , Seth PP et al. (2011 ) Measuring antiviral activity of benzimidazole molecules that alter IRES RNA structure with an infectious hepatitis C virus chimera expressing Renilla luciferase . Antiviral Res 89 : 54 -63 . doi:10.1016/j.antiviral.2010.11.004. PubMed: 21075143.21075143 14 Aoki Y , Aizaki H , Shimoike T , Tani H , Ishii K et al. (1998 ) A human liver cell line exhbits efficient translation of HCV RNA produced by a recombinant adenovirus expressing T7 RNA polymerase . Virology 250 : 140 -150 . doi:10.1006/viro.1998.9361. PubMed: 9770428.9770428 15 Datta S , Hazari S , Chandra PK , Samara M et al. (2011 ) Mechanisms of HCV’s resistance to IFN-alpha in cell culture involve expression of functional IFN-alpha receptor 1 . Virol J 8 : 351 . doi:10.1186/1743-422X-8-351. PubMed: 21756311.21756311 16 Hazari S , Patil A , Joshi V , Sullivan DE , Fermin CD et al. (2005 ) Alpha interferon inhibits translation mediated by the internal ribosome entry site of six different hepatitis C virus genotypes . J Gen Virol 86 : 3047 -3053 . doi:10.1099/vir.0.81132-0. PubMed: 16227227.16227227 17 Chou TC , Talalay PA (1977 ) Simple generalized equation for the analysis of multiple inhibitors of Michaelis-Menten kinetic system . J Biol Chem 252 : 6438 -6442 . PubMed: 893418.893418 18 Chou TC , Talalay P (1984 ) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors . Adv Enzyme Regul 22 : 27 -55 . doi:10.1016/0065-2571(84)90007-4. PubMed: 6382953.6382953 19 Prichard MN , Aseltine KR , Shipman C Jr MacSynergy II. Version 1.0 User manual. Ann Arbor : University of Michigan . 20 Prichard MN , Shipman Jr C (1996 ) Analysis of combinations of antiviral drugs and design of effective multidrug therapies . Antiviral Therapies 1 :9 -20 . 21 Taha C , Liu Z , Jin J , AL-Hasani H , Sonenberg N et al. (1999 ) Opposite translational control of Glut 1 and Glut 4 glucose transporter mRNA in response to insulin . J Biol Chem 274 : 33085 -33091 . doi:10.1074/jbc.274.46.33085. PubMed: 10551878.10551878 22 Lerner RS , Nicchitta CV (2006 ) mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation . RNA 12 : 775 -789 . doi:10.1261/rna.2318906. PubMed: 16540694.16540694 23 Dash S , Shah K , Prabhu R , Deluca C , Bastian F et al. (2005 ) IFNα, beta, gamma each inhibits hepatitis C virus replication at the levels of internal ribosome entry site mediated translation . Liver Int 25 [3]: 580 -594 . doi:10.1111/j.1478-3231.2005.01082.x. PubMed: 15910496.15910496 24 Guo JT , Bichko VV , Seeger C (2001 ) Effect of alpha interferon on the hepatitis C virus replicon . J Virol 75 : 8516 -8523 . doi:10.1128/JVI.75.18.8516-8523.2001. PubMed: 11507197.11507197 25 Goodbourn S , Didcock L , Randall RE (2000 ) Interferons: cell signaling, immune modulation, antiviral responses and virus countermeasures . J Gen Virol 81 : 2341 -2364 . PubMed: 10993923.10993923 26 Jiang D , Guo H , Xu C , Chang J , Gu B et al. (2008 ) Identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis C virus . J Virol 82 [4]: 1665 -1678 . doi:10.1128/JVI.02113-07. PubMed: 18077728.18077728 27 Wang C , Pflugheber J , Sumpter R , Sodora DL , Hui D et al. (2003 ) Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication . J Virol 77 : 3898 -3912 . doi:10.1128/JVI.77.7.3898-3912.2003. PubMed: 12634350.12634350 28 Rivas-Estilla AM , Svitkin Y , Lastra ML , Hatzoglou M , Sherker A et al. (2002 ) PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone . J Virol 76 : 10637 -10653 . doi:10.1128/JVI.76.21.10637-10653.2002. PubMed: 12368306.12368306 29 Shimazaki T , Honda M , Kaneko S , Kobayashi K (2002 ) Inhibition of internal ribosomal entry site-directed translation of HCV by recombinant IFN-alpha correlates with a reduced La protein . Hepatology 35 : 199 -208 . doi:10.1053/jhep.2002.30202. PubMed: 11786977.11786977 30 Koev G , Duncan RF , Lai MM (2002 ) Hepatitis C virus IRES-dependent translation is insensitive to an eIF2alpha-independent mechanism of inhibition by interferon in hepatocyte cell lines . Virology 297 : 195 -202 . doi:10.1006/viro.2002.1455. PubMed: 12083818.12083818 31 Kato J , Kato N , Moriyama M , Goto T , Taniguchi H et al. (2002 ) Interferons specifically suppress the translation from the internal ribosome entry site of hepatitis C virus through a double-stranded RNA- activated protein kinase-independent pathway . J Infect Dis 186 : 155 -163 . doi:10.1086/341467. PubMed: 12134250.12134250 32 Kanda T , Wu S , Kiyohara T , Nakamoto S , Jiang X et al. (2012 ) Interleukin-29 suppresses hepatitis A and hepatitis C viral internal ribosome entry site-mediated translation . Viral Immunol 25 (5): 379 -386 . doi:10.1089/vim.2012.0021. PubMed: 23035851.23035851 33 Dabo S , Meurs EF (2012 ) DsRNA-dependent protein kinase PKR and its role in stress, signaling and HCV infection . Viruses 4 (11): 2598 -2635 . PubMed: 23202496.23202496 34 Leyssen P , Balzarini J , De Clercq E , Neyts J (2005 ) The predominant mechanism by which RBV exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase . J Virol 79 : 1943 –1947 . doi:10.1128/JVI.79.3.1943-1947.2005. PubMed: 15650220.15650220 35 Shu QN , Nair V (2008 ) Inosine monophosphate dehydrogenase (IMPDH) as a target in drug discovery . Med Res Rev 28 : 219 –232 . doi:10.1002/med.20104. PubMed: 17480004.17480004 36 Leyssen P , De Clercq E E , J Neyts J (2006) The anti-yellow fever virus activity of RBV is independent of error-prone replication. Mol Pharmacol 69: 1461–1467 doi:10.1124/mol.105.020057. PubMed: 16421290. 37 Mori K , Ikeda M , Ariumi Y , Dansako H , Wakita T et al. (2011 ) Mechanism of action of RBV in a novel hepatitis C virus replication cell system . Virus Res 157 : 61 –70 . doi:10.1016/j.virusres.2011.02.005. PubMed: 21320556.21320556 38 Simister P , Schmitt M , Geitmann M , Wicht O , Danielson UH et al. (2009 ) Structural and functional analysis of hepatitis C virus strain JFH1 polymerase . J Virol 83 : 11926 -11939 . doi:10.1128/JVI.01008-09. PubMed: 19740982.19740982 39 Zhou S , Liu R , Baroudy BM , Malcolm BA , Reyes GR (2003 ) The effect of RBV and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA . Virology 3 10: 333 -342 . 40 Lau JY , Tam RC , Liang TJ , Hong Z (2002 ) Mechanism of action of RBV in the combination treatment of chronic HCV infection . Hepatology 35 (5): 1002 -1009 . doi:10.1053/jhep.2002.32672. PubMed: 11981750.11981750 41 Mortimer SE , Xu D , McGrew D , Hamaguchi N , Lim HC et al. (2008 ) IMP dehydrogenise type I associates with polyribosomes translating rhodopsin mRNA . J Biol Chem 283 : 36354 -36360 . doi:10.1074/jbc.M806143200. PubMed: 18974094.18974094 42 Thomas E , Feld JJ , Li Q , Hu Z , Fried MW et al. (2011 ) RBV potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis C virus cell culture models . Hepatology 53 [1]: 32 -41 . doi:10.1002/hep.23985. PubMed: 21254160.21254160 43 Stevenson NJ , Murphy AG , Bourke NM , Keogh CA , Hegarty JE et al. (2011 ) RBV enhances IFN-alpha signaling and MxA expression: a novel immune modulation mechanism during treatment of HCV . PLOS ONE 6 : e27866 . doi:10.1371/journal.pone.0027866. PubMed: 22114715.22114715 44 Liu WL , Su WC , Cheng CW , Hwang LH , Wang CC et al. (2007 ) RBV upregulates the activity of double stranded RNA-activated protein kinase and enhances the action of IFNα against HCV virus . J Infect Dis 196 : 425 -434 . doi:10.1086/518894. PubMed: 17597457.17597457 45 Dharel N , Kato N , Muroyama R , Taniquichi H , Otsuka M et al. (2008 ) Potential contribution of tumor suppressor p53 in the host defence against hepatitis C virus . Hepatology 47 : 1136 -1149 . PubMed: 18220274.18220274 46 Liu WL , Yang HC , Su WC , Wang CC , Chen HL et al. (2012 ) RBV enhances the action of IFNα against hepatitis C virus by promoting the p53 activity through ERK1/2 pathway . PLOS ONE 9 : e43824 .	24009705	PMC3751885	CC BY	2021-01-05 17:37:55	yes	PLoS One. 2013 Aug 23; 8(8):e72791
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23991193PONE-D-13-0560710.1371/journal.pone.0073405Research ArticleBiologyBiochemistryMetabolismGeneticsHuman GeneticsGenetic Association StudiesPopulation GeneticsGenetic PolymorphismMedicineClinical GeneticsPersonalized MedicineClinical ImmunologyImmune SystemCytokinesImmunologic SubspecialtiesTransplantationDrugs and DevicesPharmacokineticsDrug MetabolismPharmacogeneticsNovel Single Nucleotide Polymorphisms in Interleukin 6 Affect Tacrolimus Metabolism in Liver Transplant Patients Association Study of IL6 in Tacrolimus MetabolismChen Dawei 1 Fan Junwei 1 Guo Feng 1 Qin Shengying 2 Wang Zhaowen 1 * Peng Zhihai 1 * 1 Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China 2 Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Bio-X Institutes, Ministry of Education, Shanghai Jiao Tong University; Shanghai Genomepilot Institutes for Genomics and Human Health, Shanghai, China Man Kwan Editor The University of Hong Kong, Hong Kong * E-mail: zhaowenw@163.com (ZW); pengzhsh@hotmail.com (ZP)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: DC JF ZW ZP. Performed the experiments: FG SQ. Analyzed the data: DC JF. Contributed reagents/materials/analysis tools: SQ. Wrote the paper: DC. 2013 26 8 2013 8 8 e734055 2 2013 22 7 2013 © 2013 Chen et al2013Chen et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background Tacrolimus is the first-line immunosuppressant after organ transplantation. It is mainly metabolized by cytochrome P450, family 3, subfamily A (CYP3A) enzymes, but there are large individual differences in metabolism. Interleukin 6 (IL6) has been shown to cause a pan-suppression of mRNA levels of ten major CYP enzymes in human hepatocyte cultures. IL6 has been shown to provide hepatoprotection in various models of liver injury. Rs1800796 is a locus in the IL6 gene promoter region which regulates cytokine production. We speculated that IL6 rs1800796 polymorphisms may lead to individual differences in tacrolimus metabolism by affecting CYP3A enzymes levels and liver function after liver transplantation. Methodology/Principal Findings Ninety-six liver transplant patients receiving tacrolimus were enrolled in the study. Two single nucleotide polymorphisms (SNP), CYP3A5 rs776746 and IL6 rs1800796, were genotyped in both donors and recipients. The effects of SNPs on tacrolimus concentration/dose (C/D ratio) at four weeks after transplantation were studied, as well as the effects of donor IL6 rs1800796 polymorphisms on liver function. Both donor and recipient CYP3A5 rs776746 allele A showed association with lower C/D ratios, while donor IL6 rs1800796 allele G showed an association with higher C/D ratios. Donor CYP3A5 rs776746 allele A, IL6 rs1800796 allele C, and recipient CYP3A5 rs776746 allele A were associated with fast tacrolimus metabolism. With increasing numbers of these alleles, patients were found to have increasingly lower tacrolimus C/D ratios at time points after transplantation. Donor IL6 rs1800796 allele G carriers showed an association with higher glutamic-pyruvic transaminase (GPT) levels. Conclusions Combined analysis of donor CYP3A5 rs776746, IL6 rs1800796, and recipient CYP3A5 rs776746 polymorphisms may distinguish tacrolimus metabolism better than CYP3A5 rs776746 alone. IL6 may lead to individual differences in tacrolimus metabolism mainly by affecting liver function. This work was supported by the National Nature Science Foundation of China (81170446) and the foundation for combination of medicine and engineering research of Shanghai Jiao Tong University (YG2012MS05). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Tacrolimus is the first-line immunosuppressant after organ transplantation, reducing rejection and improving graft and recipient survival. However, it is also characterized by a narrow therapeutic window, and large individual differences in metabolism [1], [2]. Indeed, after administration of “standard” doses, some patients show no therapeutic effects or serious side effects. Individualization of medical treatment could result in great improvement in therapeutic efficacy, and reduction of side effects, as well as reduction of the cost of treatment [3], [4]. At present, it is difficult to institute individualized medicine in the early postoperative period. Genetic factors such as polymorphisms can be closely related to drug metabolism. There is growing interest in the field of pharmacogenomics which focuses on the relationship between host genetics and drug metabolism [5]. Large clinical studies have shown that genetic factors can guide individualized medication of warfarin and clopidogrel [6], [7]. Pharmacogenomics research on tacrolimus could contribute to individualized medication in the early postoperative period of liver transplantation. CYP3A enzymes, which are mainly expressed in liver and intestine, are the major metabolic enzymes of tacrolimus [8], [9]. The rs776746 polymorphisms in intron 3 of CYP3A5 have been correlated with altered gene expression due to a splicing defect. These CYP3A5 rs776746 GG genotype carriers are associated with slow tacrolimus metabolism [10], [11]. However, the effect by CYP3A5 rs776746 still does not completely explain individual differences in tacrolimus metabolism [12], [13]. IL6 has been demonstrated to cause a pan-suppression of mRNA of ten major CYP enzymes in human hepatocyte cultures [14]. IL6 could promote hepatic survival by stimulating liver regeneration and providing hepatoprotection in various models of liver injury [15], so it may be relevant to liver function after liver transplantation. The rs1800796 locus, which is in the IL6 gene promoter region, could regulate cytokine production and has been proved to be a functional SNP [16], [17]. The aim of the study was to investigate the relationship between tacrolimus metabolism and IL6 rs1800796 in a large liver transplant cohort to evaluate the possibility of individualizing tacrolimus treatment in the early postoperative period of liver transplantation. Materials and Methods Patients A total of 96 patients (16 female and 80 male) who underwent liver transplantation at Shanghai Jiao Tong University Affiliated First People's Hospital between July 2007 and February 2011 were enrolled in this study. One patient was excluded from further analysis because the genotyping failed. The patients were all Han Chinese. The average age of the patients was 47.8±8.3 years, and the average weight was 63.0±10.4 kg. All of the patients received tacrolimus-based immunosuppressive regimens. Tacrolimus (Prograf, Astellas Pharma, Japan) was administered orally twice daily at an initial dose of 0.06 mg/kg/d. The dose of tacrolimus was adjusted according to the target blood concentration of 7 to10 ng/ml during the first month after transplantation. Ethics Statement This research was approved by the Ethics Committee of Shanghai Jiao Tong University, and informed written consent was obtained according to the Declaration of Helsinki and its amendments. Data Collection Blood samples were collected half an hour before tacrolimus was administered, and trough concentrations (ng/ml) were then detected by PRO-Trac™ II Tacrolimus ELISA kit (Diasorin, USA) with microparticle enzyme immunoassay (ELx800NB analyzer, BioTek, USA). C/D ratio was calculated with trough concentration and weight standardized 24-hour tacrolimus dose (mg/kg/d). We calculated C/D ratios at four weeks after transplantation, and we used the median of C/D ratios at weeks 1 to 4 to measure weekly changes in tacrolimus metabolism. We also used the median of glutamic-pyruvic transaminase levels (U/L) at weeks 1 to 4 after transplantation to measure weekly liver function. Extracting genomic DNA and genotyping Using an AllPrep DNA/RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions, genomic DNA was isolated from both donor and recipient liver tissue, where were previously stored at −80°C. Genotyping of SNPs was conducted by the Sequenom MassARRAY SNP genotyping platform (Sequenom, USA) [18]. The protocols included DNA and primer preparation, PCR amplification, SAP treatment, primer extension, resin cleanup, spotting primer extension products on SpectroCHIP, and detection primer extension products by mass spectrometer. Statistical Analysis Hardy-Weinberg equilibrium and allele frequency were analyzed using PLINK v1.07 (http://pngu.mgh.harvard.edu/purcell/plink/). Quantitative data between two groups were compared using Mann-Whitney U tests, and among several groups by Kruskal-Wallis. Non-parametric tests were performed in SPSS v17.0 (SPSS, USA). Two-sided tests were used in all analysis, and P<0.05 was considered statistically significant. Results Gene polymorphisms For CYP3A5, rs776746 allele A (28.4%) was found to be the minor allele, while allele G (71.6%) was the major allele. For IL6, rs1800796 allele G (30.3%) was found to be the minor allele, while allele C (69.7%) was the major allele. Both SNP frequencies were in accordance with Hardy-Weinberg equilibrium (P>0.05). There were no differences in allele frequencies of the two SNPs between donors and recipients. Genotype frequencies of the two SNPs are shown in Table 1. 10.1371/journal.pone.0073405.t001Table 1 Genotype frequencies of CYP3A5 rs776746 and IL6 rs1800796. Genotypes Frequency of donors Frequency of recipients SNPs Refa Mutb Ref Mut Ref Mut rs776746 GG GA+AA 46(0.484) 49(0.516) 50(0.526) 45(0.474) rs1800796 CC CG+GG 41(0.432) 54(0.568) 49(0.516) 46(0.484) a “Ref” is for referenced genotype, which is constituted of major allele and major allele. b“Mut” is for mutated genotypes, which are constituted of major allele and minor allele or minor allele and minor allele. Associations between CYP3A5 rs776746, IL6 rs1800796 polymorphisms and tacrolimus C/D ratios The effects of donor CYP3A5 rs776746 and IL6 rs1800796 polymorphisms on tacrolimus C/D ratios at four weeks after transplantation are shown in Table 2. Tacrolimus C/D ratios of donor CYP3A5 rs776746 allele A carriers at weeks 1, 3, and 4 were 199.0, 88.7, and 85.6, respectively, while C/D ratios of non-carriers were 295.1, 121.2, and 148.8, respectively. These differences between donor CYP3A5 rs776746 allele A carriers and non-carriers were significant (P = 0.006, 0.028, 0.001, respectively). Tacrolimus C/D ratios of donor IL6 rs1800796 allele G carriers at weeks 2 and 3 were 132.3 and 127.4, respectively, while C/D ratios of non-carriers were 105.2 and 97.4, respectively, and the differences were significant (P = 0.032, 0.021, respectively). Thus, donor CYP3A5 rs776746 allele A and IL6 rs1800796 allele C are associated with fast tacrolimus metabolism. 10.1371/journal.pone.0073405.t002Table 2 The effects of donor CYP3A5 rs776746 and IL6 rs1800796 polymorphisms on tacrolimus C/D ratios. Week 1 Week 2 Week 3 Week 4 SNPs Genotypes N C/D ratio P C/D ratio P C/D ratio P C/D ratio P rs776746 GG 46 295.1±413.2 0.006 130.8±130.9 0.085 121.2±81.1 0.028 148.8±126.9 0.001 GA+AA 49 199.0±209.8 102.8±101.1 88.7±102.5 85.6±75.5 rs1800796 CC 41 221.2±275.0 0.314 105.2±91.1 0.032 97.4±76.1 0.021 95.1±83.7 0.113 CG+GG 54 240.9±318.7 132.3±119.0 127.4±125.3 133.2±119.3 The effects of recipient CYP3A5 rs776746 and IL6 rs1800796 polymorphisms on tacrolimus C/D ratios at four weeks after transplantation are shown in Table 3. Tacrolimus C/D ratios of recipient CYP3A5 rs776746 allele A carriers at weeks 1, 2, 3, and 4 were 179.0, 100.6, 97.0, and 90.2, respectively, while C/D ratios of non-carriers were 290.5, 133.2, 123.7, and 136.5, respectively, and the differences were significant (P = 0.003, 0.018, 0.030, 0.017, respectively). There was no significant difference in tacrolimus C/D ratio between recipient IL6 rs1800796 allele G carriers and non-carriers. Thus, recipient CYP3A5 rs776746 allele A is also associated with fast tacrolimus metabolism. 10.1371/journal.pone.0073405.t003Table 3 The effects of recipient CYP3A5 rs776746 and IL6 rs1800796 polymorphisms on tacrolimus C/D ratios. Week 1 Week 2 Week 3 Week 4 SNPs Genotypes N C/D ratio P C/D ratio P C/D ratio P C/D ratio P rs776746 GG 50 290.5±283.4 0.003 133.2±107.8 0.018 123.7±113.0 0.030 136.5±114.0 0.017 GA+AA 45 179.0±237.5 100.6±105.7 97.0±82.9 90.2±83.0 rs1800796 CC 49 282.6±338.3 0.217 133.3±142.5 0.344 110.6±110.8 0.604 133.1±144.4 0.135 CG+GG 46 208.7±260.8 115.0±64.7 109.6±90.4 95.7±94.0 Associations between combined polymorphisms and tacrolimus C/D ratios Donor CYP3A5 rs776746 allele A, IL6 rs1800796 allele C, and recipient CYP3A5 rs776746 allele A were shown to be associated with fast tacrolimus metabolism as stated above. Therefore, these three alleles were further investigated in a combination analysis. The associations between the number of alleles associated with fast metabolism and tacrolimus C/D ratios are shown in Table 4. With increasing numbers of alleles associated with fast metabolism, patients were found to have increasingly lower tacrolimus C/D ratios at all time points through the four weeks (P = 0.001, 0.001, <0.001, <0.001, respectively). 10.1371/journal.pone.0073405.t004Table 4 Combined analysis of donor CYP3A5 rs776746 allele A, IL6 rs1800796 allele C, and recipient CYP3A5 rs776746 allele A on tacrolimus C/D ratios. Week 1 Week 2 Week 3 Week 4 Numa N C/D ratio P C/D ratio P C/D ratio P C/D ratio P ≤1 19 460.0±420.9 0.001 186.5±121.7 0.001 177.7±140.8 <0.001 172.5±177.6 <0.001 2−3 61 242.4±261.0 117.2±96.4 109.6±88.7 107.2±81.1 ≥4 15 91.8±167.9 59.0±93.6 67.7±49.0 73.4±47.9 a “Num” indicates the number of alleles associated with fast metabolism patients carrying. Associations between donor IL6 rs1800796 polymorphisms and GPT The effects of donor IL6 rs1800796 polymorphisms on GPT at four weeks after transplantation were studied. GPT of donor IL6 rs1800796 allele G carriers at weeks 1, 2, 3, and 4 were 167.0, 45.5, 30.0, and 34.5, respectively, while GPT of non-carriers were 142.5, 29.0, 20.0, and 20.0, respectively. The differences between donor IL6 rs1800796 allele G carriers and non-carriers at weeks 2, 3, and 4 were significant (P = 0.004, 0.002, 0.006, respectively), but not at week 1 (P = 0.141). Discussion The CYP3A enzymes including three functional enzymes as CYP3A4, CYP3A5, and CYP3A7 are responsible for the oxidative metabolism of over 50% of the drugs in widespread use [19]. CYP3A4 is responsible for most CYP3A-mediated drug metabolism [20]. There have been previous studies on the associations between CYP3A4 gene polymorphisms and tacrolimus metabolism [21]–[23], but there are no definitive conclusions comparing with CYP3A5 rs776746. CYP3A7 is predominantly expressed in fetal liver and may have little role in adults [19]. CYP3A5 is the major metabolic enzyme for tacrolimus. CYP3A5 rs776746 allele A non-carriers produce truncated, nonfunctional CYP3A5 enzyme because of a splicing defect, so these patients metabolize tacrolimus slower than carriers [10], [11]. This dose-modifying effect in Eastern Asian populations is higher than in Caucasian populations [10]. In a study by Birdwell et al, eight SNPs in the CYP3A4 gene and one in the CYP3A7 gene were found to be associated with tacrolimus metabolism, but these SNPs were in linkage disequilibrium with CYP3A5 rs776746 [12]. CYP3A5 enzyme is expressed both in liver and intestine [24], so tacrolimus metabolism is associated with both donor and recipient CYP3A5 gene polymorphisms in liver transplant patients [10], [11]. Our study has verified that both donor and recipient CY3A5 rs776746 were associated with tacrolimus metabolism in a transplant population. It has been shown previously that CYP3A5 rs776746 could predict tacrolimus metabolism to a certain extent, but could not completely explain the individual differences [12], [13]. Presently, about two hundred cytokines produced by many cell types have been found, and they usually act by autocrine and paracrine mechanisms [25]. In recent years, more attention has been paid to relationships between cytokines and tacrolimus metabolism. There have been reports that donor and recipient IL10 gene polymorphisms were associated with tacrolimus metabolism and IL18 was thought to reduce tacrolimus C/D ratios through up regulation of P-glycoprotein [8], [26], [27]. Our study is the first to demonstrate that donor IL6 rs1800796 polymorphisms are associated with tacrolimus metabolism. Previous studies have shown that cytokines affect pharmacokinetic and pharmacodynamic behaviors of drugs [25], [28]. IL6 has been shown to suppress mRNA of CYP3A5 in human hepatocyte cultures [14]. Cytokines also play a central role in immunologic events that occur after transplantation [29]. The liver often undergoes ischemia/reperfusion and immune injury during the transplantation and postoperative time. In this regard, IL6 activates the STAT3 signaling pathway through the gp130-IL6R complex on hepatocytes, and this process promotes liver regeneration, the acute-phase response, and hepatoprotection against Fas and toxic damage in liver [15]. Cytokine production may differ among different people due to gene polymorphisms [30], [31]. The rs1800796 locus in IL6 promoter, which can regulate its production, was shown to be a functional SNP [16], [17]. The C to G variation at IL6 rs1800796 has been shown to decrease transcriptional activity of the IL6 promoter, and carriers of CC genotype are high-expressors of IL6 [16], [17], [32]. Thus, we speculate that carriers of CC genotype have higher levels of IL6, leading to increased suppression of CYP3A5 expression and hepatoprotection. The current study showed that carriers of donor IL6 rs1800796 CC genotype were associated with faster recovery of liver function after transplantation, which is consistent with the fact that IL6 is a key molecule for liver regeneration and repair [33]. Suppression of CYP3A5 expression and hepatoprotection have opposite effects on tacrolimus metabolism. In the current study, carriers of donor IL6 rs1800796 CC genotype had a lower tacrolimus C/D ratios compared with non-carriers, indicating that the hepatoprotection effect of IL6 was greater than the suppression effect on CYP3A5 in liver transplant patients in the early postoperative period. The ability of donor IL6 rs1800796 to predict tacrolimus metabolism was less than CYP3A5 rs776746. The reason for this observation may be the opposing effects on tacrolimus metabolism of IL6 which could suppress CYP3A5 expression or provide hepatoprotection. The current study demonstrated that donor IL6 gene polymorphisms were associated with tacrolimus metabolism, not the recipient. Donor IL10 gene polymorphisms have been reported to be associated with tacrolimus metabolism [8]. Expression of IFNG and IL10 has been found to be up regulated in hepatocytes from allograft tissue after orthotopic liver transplantation [34]. There is evidence that hepatocytes can express IL6 in the HBV-infected liver microenvironment [35]. The liver has the unique capacity to regulate its growth and mass after liver injury [36]. The current data are consistent with the concept that hepatocyte secretion of IL6 may facilitate liver repair and protection after transplantation. Li et al [27] have found that serum IL18 levels were associated with tacrolimus C/D ratios and that recipient IL18 gene polymorphisms were not associated with tacrolimus C/D ratios and serum IL18 levels. We suspect that donor IL18 gene polymorphisms may be associated with tacrolimus metabolism as has been shown for IL6 in the current study. At present, genotyping of CYP3A5 rs776746 polymorphisms has been used in clinical applications, however, it could not predict tacrolimus metabolism accurately. Our study showed that genotyping of donor IL6 rs1800796 polymorphisms could assist CYP3A5 rs776746 to predict tacrolimus metabolism more effectively. Various populations in other regions of the world may differ in certain features of genetic architecture. Whether IL6 rs1800796 is applicable to other populations with regards to tacrolimus metabolism requires further investigation. The gene-drug observations in the current study suggest that genetic factors may also affect liver transplant patient outcomes including graft and recipient survival rates. Proof of such associations requires further study. Conclusions Donor gene polymorphisms of IL6 play a more important role than those of recipient. Combined polymorphisms of donor CYPA5 rs776746, IL6 rs1800796, and recipient CYP3A5 rs776746 have a greater effect on tacrolimus metabolism than CYP3A5 rs776746 alone. IL6 levels may lead to individual differences in tacrolimus metabolism mainly by affecting liver function. ==== Refs References 1 Bowman LJ , Brennan DC (2008 ) The role of tacrolimus in renal transplantation . Expert Opin Pharmacother 9 : 635 –643 .18312164 2 Staatz CE , Tett SE (2004 ) Clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ transplantation . Clin Pharmacokinet 43 : 623 –653 .15244495 3 Yu X , Xie H , Wei B , Zhang M , Wang W , et al (2011 ) Association of MDR1 gene SNPs and haplotypes with the tacrolimus dose requirements in Han Chinese liver transplant recipients . PLoS One 6 : e25933 .22110582 4 Wei-lin W , Jing J , Shu-sen Z , Li-hua W , Ting-bo L , et al (2006 ) Tacrolimus dose requirement in relation to donor and recipient ABCB1 and CYP3A5 gene polymorphisms in Chinese liver transplant patients . Liver Transpl 12 : 775 –780 .16628701 5 Wang L , McLeod HL , Weinshilboum RM (2011 ) Genomics and drug response . N Engl J Med 364 : 1144 –1153 .21428770 6 Mega JL , Close SL , Wiviott SD , Shen L , Hockett RD , et al (2009 ) Cytochrome p-450 polymorphisms and response to clopidogrel . N Engl J Med 360 : 354 –362 .19106084 7 Klein TE , Altman RB , Eriksson N , Gage BF , Kimmel SE , et al (2009 ) Estimation of the warfarin dose with clinical and pharmacogenetic data . N Engl J Med 360 : 753 –764 .19228618 8 Zhang X , Wang Z , Fan J , Liu G , Peng Z (2011 ) Impact of interleukin-10 gene polymorphisms on tacrolimus dosing requirements in Chinese liver transplant patients during the early posttransplantation period . Eur J Clin Pharmacol 67 : 803 –813 .21359536 9 Jacobson PA , Oetting WS , Brearley AM , Leduc R , Guan W , et al (2011 ) Novel polymorphisms associated with tacrolimus trough concentrations: results from a multicenter kidney transplant consortium . Transplantation 91 : 300 –308 .21206424 10 Tang HL , Xie HG , Yao Y , Hu YF (2011 ) Lower tacrolimus daily dose requirements and acute rejection rates in the CYP3A5 nonexpressers than expressers . Pharmacogenet Genomics 21 : 713 –720 .21886016 11 Uesugi M , Masuda S , Katsura T , Oike F , Takada Y , et al (2006 ) Effect of intestinal CYP3A5 on postoperative tacrolimus trough levels in living-donor liver transplant recipients . Pharmacogenet Genomics 16 : 119 –127 .16424824 12 Birdwell KA , Grady B , Choi L , Xu H , Bian A , et al (2012 ) The use of a DNA biobank linked to electronic medical records to characterize pharmacogenomic predictors of tacrolimus dose requirement in kidney transplant recipients . Pharmacogenet Genomics 22 : 32 –42 .22108237 13 de Jonge H , Metalidis C , Naesens M , Lambrechts D , Kuypers DR (2011 ) The P450 oxidoreductase *28 SNP is associated with low initial tacrolimus exposure and increased dose requirements in CYP3A5-expressing renal recipients . Pharmacogenomics 12 : 1281 –1291 .21770725 14 Dickmann LJ , Patel SK , Rock DA , Wienkers LC , Slatter JG (2011 ) Effects of interleukin-6 (IL-6) and an anti-IL-6 monoclonal antibody on drug-metabolizing enzymes in human hepatocyte culture . Drug Metab Dispos 39 : 1415 –1422 .21555507 15 Taub R (2004 ) Liver regeneration: from myth to mechanism . Nat Rev Mol Cell Biol 5 : 836 –847 .15459664 16 Zhang G , Zhou B , Wang W , Zhang M , Zhao Y , et al (2012 ) A functional single-nucleotide polymorphism in the promoter of the gene encoding interleukin 6 is associated with susceptibility to tuberculosis . J Infect Dis 205 : 1697 –1704 .22457277 17 Okada R , Wakai K , Naito M , Morita E , Kawai S , et al (2012 ) Pro-/anti-inflammatory cytokine gene polymorphisms and chronic kidney disease: a cross-sectional study . BMC Nephrol 13 : 2 .22230215 18 Gabriel S, Ziaugra L, Tabbaa D (2009) SNP genotyping using the Sequenom MassARRAY iPLEX platform. Curr Protoc Hum Genet Chapter 2: Unit 2 12. 19 MacPhee IA (2012 ) Pharmacogenetic biomarkers: cytochrome P450 3A5 . Clin Chim Acta 413 : 1312 –1317 .22037511 20 Daly AK (2006 ) Significance of the minor cytochrome P450 3A isoforms . Clin Pharmacokinet 45 : 13 –31 .16430309 21 Gervasini G , Garcia M , Macias RM , Cubero JJ , Caravaca F , et al (2012 ) Impact of genetic polymorphisms on tacrolimus pharmacokinetics and the clinical outcome of renal transplantation . Transpl Int 25 : 471 –480 .22369694 22 Cho JH , Yoon YD , Park JY , Song EJ , Choi JY , et al (2012 ) Impact of cytochrome P450 3A and ATP-binding cassette subfamily B member 1 polymorphisms on tacrolimus dose-adjusted trough concentrations among Korean renal transplant recipients . Transplant Proc 44 : 109 –114 .22310591 23 Elens L , Bouamar R , Hesselink DA , Haufroid V , van der Heiden IP , et al (2011 ) A new functional CYP3A4 intron 6 polymorphism significantly affects tacrolimus pharmacokinetics in kidney transplant recipients . Clin Chem 57 : 1574 –1583 .21903774 24 Lown KS , Kolars JC , Thummel KE , Barnett JL , Kunze KL , et al (1994 ) Interpatient heterogeneity in expression of CYP3A4 and CYP3A5 in small bowel. Lack of prediction by the erythromycin breath test . Drug Metab Dispos 22 : 947 –955 .7895614 25 Zidek Z , Anzenbacher P , Kmonickova E (2009 ) Current status and challenges of cytokine pharmacology . Br J Pharmacol 157 : 342 –361 .19371342 26 Li D , Zhu JY , Gao J , Wang X , Lou YQ , et al (2007 ) Polymorphisms of tumor necrosis factor-alpha, interleukin-10, cytochrome P450 3A5 and ABCB1 in Chinese liver transplant patients treated with immunosuppressant tacrolimus . Clin Chim Acta 383 : 133 –139 .17568575 27 Li Y , Zou Y , Cai B , Yang B , Ying B , et al (2012 ) The associations of IL-18 serum levels and promoter polymorphism with tacrolimus pharmacokinetics and hepatic allograft dysfunction in Chinese liver transplantation recipients . Gene 491 : 251 –255 .22008665 28 Prandota J (2005 ) Important role of proinflammatory cytokines/other endogenous substances in drug-induced hepatotoxicity: depression of drug metabolism during infections/inflammation states, and genetic polymorphisms of drug-metabolizing enzymes/cytokines may markedly contribute to this pathology . Am J Ther 12 : 254 –261 .15891270 29 Liu F , Li B , Wang WT , Wei YG , Yan LN , et al (2012 ) Interleukin-10-1082G/A polymorphism and acute liver graft rejection: a meta-analysis . World J Gastroenterol 18 : 847 –854 .22371646 30 Awad MR , El-Gamel A , Hasleton P , Turner DM , Sinnott PJ , et al (1998 ) Genotypic variation in the transforming growth factor-beta1 gene: association with transforming growth factor-beta1 production, fibrotic lung disease, and graft fibrosis after lung transplantation . Transplantation 66 : 1014 –1020 .9808485 31 Turner DM , Williams DM , Sankaran D , Lazarus M , Sinnott PJ , et al (1997 ) An investigation of polymorphism in the interleukin-10 gene promoter . Eur J Immunogenet 24 : 1 –8 . 32 Komatsu Y , Tai H , Galicia JC , Shimada Y , Endo M , et al (2005 ) Interleukin-6 (IL-6)—373 A9T11 allele is associated with reduced susceptibility to chronic periodontitis in Japanese subjects and decreased serum IL-6 level . Tissue Antigens 65 : 110 –114 .15663749 33 Karp SJ (2009 ) Clinical implications of advances in the basic science of liver repair and regeneration . Am J Transplant 9 : 1973 –1980 .19563334 34 Alfrey EJ, Most D, Wang X, Lee LK, Holm B, et al.. (1995) Interferon-gamma and interleukin-10 messenger RNA are up-regulated after orthotopic liver transplantation in tolerant rats: evidence for cytokine-mediated immune dysregulation. Surgery 118: 399–404; discussion 404–395. 35 Xiang WQ , Feng WF , Ke W , Sun Z , Chen Z , et al (2011 ) Hepatitis B virus X protein stimulates IL-6 expression in hepatocytes via a MyD88-dependent pathway . J Hepatol 54 : 26 –33 .20937539 36 Fausto N , Campbell JS (2003 ) The role of hepatocytes and oval cells in liver regeneration and repopulation . Mech Dev 120 : 117 –130 .12490302	23991193	PMC3753270	CC BY	2021-01-05 17:37:57	yes	PLoS One. 2013 Aug 26; 8(8):e73405
==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2013/372646Research ArticleDesigning a Bioengine for Detection and Analysis of Base String on an Affected Sequence in High-Concentration Regions http://orcid.org/0000-0003-0140-9644Bhattacharyya Debnath 1 Mandal Bijoy Kumar 1 Kim Tai-hoon 2 1Department of Computer Science & Engineering, Faculty of Engineering and Technology, NSHM Knowledge Campus, Durgapur 713212, India2Department of Convergence Security, Sungshin Women's University, 249-1, Dongseon-dong 3-ga, Seoul 136-742, Republic of KoreaTai-hoon Kim: taihoonn@daum.netAcademic Editor: Sabah Mohammed 2013 13 8 2013 2013 3726464 7 2013 13 7 2013 Copyright © 2013 Debnath Bhattacharyya et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.We design an Algorithm for bioengine. As a program are enable optimal alignments searching between two sequences, the host sequence (normal plant) as well as query sequence (virus). Searching for homologues has become a routine operation of biological sequences in 4 × 4 combination with different subsequence (word size). This program takes the advantage of the high degree of homology between such sequences to construct an alignment of the matching regions. There is a main aim which is to detect the overlapping reading frames. This program also enables to find out the highly infected colones selection highest matching region with minimum gap or mismatch zones and unique virus colones matches. This is a small, portable, interactive, front-end program intended to be used to find out the regions of matching between host sequence and query subsequences. All the operations are carried out in fraction of seconds, depending on the required task and on the sequence length. ==== Body 1. Introduction It is known that viroids are the smallest replicating pathogenic agents (see [1] for relevant references), which is entirely composed of RNA with genome sizes in the range of 330–380 nucleotides [2], that is 10 times smaller than the smallest bacteriophage of Escherichia coli [3]. It is also known that they infect a wide variety of plants and produce severe disease symptoms in many plants [4–12], but there is no evidence for the existence of a protective protein coat for viroids. The molecular mechanisms by which viroids replicate and interact with their hosts are not yet understood. In its most severe form, the disease [5, 6] caused by potato spindle tuber viroid (PSTV) causes general stunting of potato plant growth, deformity of the upper foliage, and production of disfigured potatoes [5]. Mild strains of PSTV which produce barely detectable symptoms have also been isolated [7]. Furthermore, plants infected with mild strains are somehow protected from developing symptoms following subsequent inoculation with severe strains [8, 9]. The sequence of the 247 nucleotide residues of the single strand circular RNA of avocado sunblotch viroid (ASBV) was determined using partial enzymes cleavage methods on overlapping viroid fragments obtained by partial ribonucleic digestion followed by 32p-labelling in vitro at their 5′-ends. ASBV is much smaller than potato spindle tuber viroid (PSTV; 359 residues) and chrysanthemum stunt viroid (CSV; 356 residues). The sequences of the viroid progeny and the cloned DNA were identical. In vitro mutagenesis of infectious PSTV cDNAs will allow systematic investigation of the role of specific sequences in viroid replication and pathogenesis [10]. A complex of considerable stability is possible between the 5′-end of U1 RNA and a specific nucleotide sequence of the potato spindle tuber viroid complement. Small nuclear RNAs (snRNAs) that are associated with ribonucleoprotein particles are believed by some to be involved in the processing of the primary transcription products of split genes. The 5′-end of one such RNA, U1, has been shown to exhibit complementarity with the ends of introns, and it is believed that this affords a mechanism ensuring correct excision of the intron sequences and accurate joining of the coding sequences [11]. The invention provides a novel retroviral packaging system, in which retroviral packaging constructs and packageable vector transcripts are produced from high-expression plasmids by replicating in a human's cell via the enzyme reverse transcriptase to produce DNA from its RNA genome. Retroviruses are enveloped viruses that belong to the viral family retroviridae. High titers of recombinant retrovirus are produced in infected cells. The methods of the invention include the use of the novel retroviral constructs to transduce primary human cells, including T cells and human hematopoietic stem cells, with foreign genes by cocultivation at high efficiencies. The invention is useful for the rapid production of high viral supernatants, and to transduce with high-efficiency cells that are refractory to transduction by conventional means [12]. 2. Basis of the Algorithm There are four issues which are focused mainly to provide for detection of a fixed base string on an affected sequence. 2.1. Similarity To define similarity, perhaps it is useful to first introduce the notion of “distance” between two strings. The distance between two strings is zero if they are exactly the same. The distance between two strings increases if they get more dissimilar. One way of defining distance between two strings is to look at the amount of change they needed to do to one to obtain the other. They could go on to introduce other changes, insert, and delete. Insert “happens” when they inserted some letter into the sequence (at some position), and delete happens when they deleted some letter at some position. 2.2. Edit Distance This is defined as the minimum number of changes to be performed on one sequence to make it exactly the same as another. 2.3. Alignment of Sequence For every two sequences, there are huge permutations of possible alignments (cubic in the length of sequences). Alignment procedure itself can be visualized as a series of insert, delete operations. 2.4. Scoring Function A scoring function determines this notion of goodness of alignment. They could compute the distance between alignments in such a way that the cost of a match is 0 (when the sequence on top and below has the same ith character). Cost of a mismatch is that they could choose different scoring schemes. Another sample scoring scheme could give lesser weights for replacement of A by T, and G by C (and vice versa) as against replacement of A by G or the others. Domain knowledge is used while determining scoring schemes. 3. Designing of the Algorithm There are basic steps that constitute the whole process of analysis for high-concentration regions (HCR) detection of a fixed base string on an affected sequence and those steps are as follows. 3.1. Match Occurs in the following Way Q[i] = H[j] to H[m − L + 1]. As for example, Q[1] = H[1] first match found. Next Q[2] match with H[1] to H[m − L + 1]. This process will continue at the end of query sequence. This process is repeated at the end of query sequence, until all possible matches are found. Match found then Q[i] = H[j]. 3.2. Analysis of Matching Method The analysis of matching method is done in four different parts. 3.2.1. Consider a DNA Sequence and Their Related Changes 1 2 3 4 5 6 7 8 9 10 11 12…………n DNA CG G A A C T A A A C T C …………n n RNA CG G A A C U A A A C U C …………n n cDNA G C C T T G A T T T G A G …………n n cRNA GC C U U G A U U U G A G …………n n, where, n is the number of bases in the nucleotide sequence. n n is the nth (i.e., last) base (A/T/G/C) in host and query genome sequences, which consist of bases A, T, G, and C (note that T is replaced with U in the case of the RNA). This example is applicable both in host and query sequences, and n is the length of the sequence in both cases, but they are the same or do not depend on user. 3.2.2. Generating the Query Subsequence from Input Sequence They broke the host and query sequence into user requirement subsequences length for easy implementation of Figure 1. From Figure 1 pictorial representation, it is clear that for ith subsequence W i (called colons): i is the starting position of the subsequence and j = (i − 1) + L is end position of the subsequence, where L is the subsequence length (word size). For example, if word size is 4, then: For W1 starting position (i) = 1 and (end position) j = (1 − 1) + 4 = 4, W2 starting position (i) = 2 and (end position) j = (2 − 1) + 4 = 5 and W3 starting position (i) = 3 and (end position) j = (3 − 1) + 4 = 6 and so on. The clones with word size less than 3 (three) has no importance in matching context and hence we considered the clones with word size in the range: 3 ≤ L ≤ n. Therefore, ranges for i and j are as 3 ≤ i ≤ n − L + 1 and L + 1 ≤ j ≤ n, respectively. The subsequence generation time, both in host and query sequences cases, at the end (subsequence length − 1) number of nucleotide base pair (a, t, g, and c) remains as it is. This is the reason why probability of infection decreases. To solve this problem, we have to find the result in reverse order. The host sequence is defined by H and query sequence is defined by Q; each of the sequences must have the same or different lengths. So, we could write H = ATGCTAGCAGTAGACGATAGC………n, n > 0 and T = TGCAGTAGCAGATGAC…………m, m > 0, where n and m are the length of host and query sequences. After subsequence division, they could get the result as follows. So, they could rewrite H[i] = H[1]H[2]………H[n − L + 1], 1 ≤ i ≤ n − L + 1 and Q[j] = Q[1] Q[2]………Q[m − L + 1], 1 ≤ j ≤ m − L + 1. If the subsequence length or word size is L (3 < L ≤ n − L + 1). If the number of subsequence is S, the total number of subsequences is generated in case that host sequence is 1 ≤ S ≤ n − L + 1 and case that query sequences is 1 ≤ S ≤ m − L + 1. This subsequence method is required to reduce the complexity of the program execution. 3.2.3. Matching between Host and Query Sequence Let us look for matches in between Host sequence and Query sequence in Table 1. Here, host sequence is the virus sequence and Query sequence is the Tomato chloroplast,… and so forth, complete genome sequence of the Tomato plant and Root sequence. 16 possible matches may occur, and matches found are shown in the following: DNA versus DNA DNA versus RNA DNA versus cDNA DNA versus cRNA RNA versus DNA RNA versus RNA RNA versus cDNA RNA versus cRNA cDNA versus DNA cDNA versus RNA cDNA versus cDNA cDNA versus cRNA cRNA versus DNA cRNA versus RNA cRNA versus cDNA cRNA versus cRNA. In these cases, the value of i is incremented by i = no. of unmatched character + no. of substring match × 3; similarly j is incremented by this same procedure. Otherwise Q[i] ≠ H[j]; that is, unmatched occurs, the value of i and j is incremented by one. At the end, we could get the result as Table 2. Host and Query sequence infections are calculated by \|NBM\|/\|\|TL\| where NBM is the total no of base pair match, which is equivalent to total number word match multiplied by word size, is divided by length of host sequence in case of virus infection, length of query sequence in case of plant infection. 3.2.4. Threshold Value Proving this hypothesis, we have considered a threshold value, on this threshold value we can take the decision as described as follows.Infectivity “HIGH” means that the virus is highly infectious on target sequence; that is, chloroplast of the tomato plant is infected by PSTVd virus from head to tail. In this situation, the infection between the source (PSTVd) and the target sequence (tomato chloroplast) is very high. Infectivity “NEGLIGIBLE” means that the virus is infected on target sequence; that is, chloroplast of the tomato plant is infected by PSTVd virus from head to tail are not infected. In this situation, the infection between the source (PSTVd) and the target sequence (tomato chloroplast) is infected, but it is not harmful. Infectivity “LOW” means the virus infection is found, but not so called infectious on target sequence; that is, chloroplast of the tomato plant is infected by PSTVd virus from head to tail are not infected. In this situation, the infection between the source (PSTVd) and the target sequence (tomato chloroplast) is noninfectious. 4. Experimental Data 4.1. Matches between Host Sequence and Query Sequence This aspect is given in Figure 2. 4.2. Alignment Demo The matter of alignment is shown in Figure 3. 4.3. Pictorial Representation Shows That Match Region The pictorial representation of matched region is shown in Table 3 (word size 3). 4.4. Highest Matching Word The highest matched word is given in Table 4. 5. Project Spectrum We have the following:A base program to detect the HCRs in a target sequence for a given viral sequence. A method to locate the start and end positions of infection and isolate the infected regions. A method to identify the longest infected region or the largest HCR. An extension to allow all 4 possible transforms of the viral sequence (i.e., DNA, RNA, cDNA, and cRNA). An extension to allow scanning of all possible transforms of the normal plant (target) sequence, that is, DNA, RNA, cDNA, and cRNA. A total of 4×4 scan orientations. An extension to identify successive regions of Edit Distance = 1. An extension to detect and report all such extrapolated infection regions and locate the largest of them. 6. Architecture of Process The required architecture for the whole process is shown in Figure 4. 6.1. Inputs The Inputs Taken are normal plant sequence: a steam of DNA bases in FASTA format, that is, a text file containing an DNA sequence. limitations: none. viral sequence: a steam of RNA bases in fasta format, that is, a text file containing an RNA sequence. limitations: size of file should be less than 400 Kbytes. 6.2. Codon Generator Codon Generator is shown in Figure 5. 6.3. Codon Tree The structure of codon tree is given in Figure 6. 6.4. Transforms The process of transformation is shown in Figure 7. 6.5. Sequence Analyzer The process of sequence analyzer is given in Figure 8. 7. Complexity The algorithm uses an M-array tree to structure the input sequence and then allows the target to “pour through” the root and fit in place. Thus, the target sequence looks at a match, rather than the other way round. Here, M = 5 so the time complexity of the program is O(n 1 log⁡M O(n 1 log⁡5 n 2)n 2) O (n 1 log⁡5 n 2) n 1: size of viral sequence n 2: size of plant sequence. 8. Analysis A comparison of a variant of the same program, using the strcmp() library function yielded the following timings. This is tabulated in Table 5. 9. Performance The program was tested with real inputs and the time spent is tabulated in Table 6. 10. Conclusion This algorithm shows that virus and normal plant interaction was found only in between virus RNA with normal plant cDNA and RNA stand only. The virus and plant interaction was found only in normal in nature, no such other orientation is applicable. The colon size varies from 3 to 9. The lower the subsequence size, the higher the interaction rate. This algorithm also can apply on any type of virus and any type of normal plant genome sequences. In future, an attempt will be made to apply this software in real-life example such as Potato Spindle Tuber Viroid infected only chloroplast of the Tomato plant not in their root. Figure 1 Figure 2 Matches between Host Sequence and Query Sequence. Figure 3 Alignment demo. Figure 4 Architecture of process. Figure 5 Codon generator. Figure 6 Codon tree. Figure 7 Process of transformation. Figure 8 Sequence analyzer. Table 1 Source sequence Target sequence DNA → DNA RNA → RNA cDNA → cDNA cRNA → cRNA Table 2 H[1] H[5]H[6] ………………H[n − L + 1] Source sequence S[i] : CGG C U AAAC………………….n Target sequence T[i] : CG G A A C U A A A C U C ………m T[1] T[4]T[5]………….T[m − L + 1] Total word match = 3 Table 3 Pictorial representation for showing the match region. Position Match position Total base pair match Gap Highest match position without gap Highest match position with gap 1st position 1–6 (1–3 and 4–6) 6 0 2nd position 8–10 3 1 3rd position 12–14 3 1 4th position 17–22 6 2 5th position 25–36 12 2 25–33 6th position 38–39 3 1 25–39 Table 4 Highest matching word. Words/colones Repeat numbers ATG 3 TTT 5 TAT 1 TGC 1 Table 5 Analysis of present algorithm. Target input with fixed base sequence, 349 bytes Time with strcmp() Time with this Algorithm 200 KB 200 seconds 25 milliseconds 1 MB 7 minutes 456 milliseconds 1.5 MB 15 minutes 1-2 second (s) >2 MB The computer hanged ~15 seconds Table 6 Performance of viruses of different size. Virus (in KB) Plant (in KB) Time taken <400 bytes <5 ~0.5 milliseconds 500–1024 bytes <5 ~0.5 milliseconds 1–5 <100 ~90 milliseconds 1–5 200–1024 ~400 milliseconds 10–100 1024–5 MB ~1–4 seconds 10–100 5–7 MB ~5–10 seconds 100–300 ~10 ~15–20 seconds ==== Refs 1 Diener TO Viroids and Viroid Diseases 1979 New York, NY, USA Wiley 2 Gross HJ Domdey H Lossow C Nucleotide sequence and secondary structure of potato spindle tuber viroid Nature 1978 273 5659 203 208 2-s2.0-0017821271 643081 3 Gross HJ Krupp G Domdey H Nucleotide sequence and secondary structure of citrus exocortis and chrysanthemum stunt viroid European Journal of Biochemistry 1982 121 2 249 257 2-s2.0-0020006795 7060550 4 Gross HJ Liebl U Alberty H A severe and a mild potato spindle tuber viroid isolate differ in three nucleotide exchanges only Bioscience Reports 1981 1 3 235 241 2-s2.0-0019540763 6271277 5 Haseloff J Symons RH Chrysantemum stunt viroid: primary sequence and secondary structure Nucleic Acids Research 1981 9 12 2741 2752 2-s2.0-0019729258 7279660 6 Visvader JE Gould AR Bruening GE Symons RH Citrus exocortis viroid: nucleotide sequence and secondary structure of an Australian isolate FEBS Letters 1982 137 2 288 292 2-s2.0-0020073725 15768484 7 Symons RH Avocado sunblotch viroid: primary sequence and proposed secondary structure Nucleic Acids Research 1981 9 23 6527 6537 2-s2.0-0019878340 7322921 8 Haseloff J Mohamed NA Symons RH Viroid RNAs of cadang-cadang disease of coconuts Nature 1982 299 5881 316 321 2-s2.0-0019922899 9 Van Wezenbeek P Vos P van Boom J van Kammen A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA) Nucleic Acids Research 1982 10 794 797 10 Gross HJ Riesner D Viroids: a class of subviral pathogens Angewandte Chemie 1980 19 4 231 243 2-s2.0-0018823791 6155812 11 Diener TO Are viroids escaped introns? Proceedings of the National Academy of Sciences of the United States of America 1981 78 8 I 5014 5015 2-s2.0-0019792938 16593072 12 Dickson E A model for the involvement of viroids in RNA splicing Virology 1981 115 1 216 221 2-s2.0-0019637521 7292989	24000321	PMC3755424	CC BY	2021-01-05 10:05:32	yes	Biomed Res Int. 2013 Aug 13; 2013:372646
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24015313PONE-D-13-2536510.1371/journal.pone.0074022Research ArticleNewcastle Disease Virus Fusion Protein Is the Major Contributor to Protective Immunity of Genotype-Matched Vaccine Genotype-Matched NDV VaccineKim Shin-Hee 1 Wanasen Nanchaya 1 Paldurai Anandan 1 Xiao Sa 1 Collins Peter L. 2 Samal Siba K. 1 * 1 Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America 2 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, United States of America Meng Xiang-Jin Editor Virginia Polytechnic Institute and State University, United States of America * E-mail: ssamal@umd.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SHK PLC SKS. Performed the experiments: SHK NW AP SX. Analyzed the data: SHK AP SX. Contributed reagents/materials/analysis tools: PLC SKS. Wrote the manuscript: SHK PLC SKS. 2013 28 8 2013 8 8 e7402218 6 2013 25 7 2013 2013This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein. This research was supported by NIAID contract N01A060009 (85% support) and the NIAID, NIH, Intramural Research Program (15% support). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Newcastle disease (ND) is a highly contagious avian disease with worldwide distribution [1]. The causative agent, Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxoviridae. The genome of NDV consists of six transcriptional units (3′-N–P-M-F–HN-L-5′) [2]. The F and HN proteins form spike-like projections on the outer surface of the viral envelop and are the neutralizing and protective antigens of NDV. The F protein is synthesized as an inactive precursor (F0) that is cleaved by host cell protease into two biologically active F1 and F2 subunits [3]. The cleavage of the F protein is a prerequisite for virus entry and cell-to-cell fusion. The sequence of the F protein cleavage site is a well-characterized, major determinant of NDV pathogenicity in chickens. Homotypic interaction between the F and HN proteins is necessary for initiation of the fusion process [4,5]. All NDV strains belong to a single serotype [6,7]. However, genetic and antigenic diversity are recognized for NDV isolates, and the detection of progressive changes in strains isolated over successive years indicates that NDV is a continually evolving virus [8,9]. Based on genome length and sequence of the F gene, NDV strains have been classified into classes I (nine genotypes, 1-9) and II (eleven genotypes, I-XI). Class I strains are usually avirulent. A recent study also proposes that class I viruses comprise a single genotype, while class II contains 15 genetic groups including 10 previously established (I–IX, and XI) and five new genotypes (X, XII, XIII, XIV and XV) [10]. Class II contains both virulent and avirulent strains and the avirulent vaccine strains LaSota and B1 presently in worldwide use. Currently, the circulating strains associated with disease outbreaks worldwide predominantly are from genotypes V, VI, and VII of class II [11–14]. In contrast, the LaSota and B1 vaccine strains were isolated some 60 years ago and belong to genotype II in class II. Although the current vaccines offer substantial protection against disease, they do not completely prevent infection or virus shedding, and disease can occur in vaccinated birds [8,12]. Recent studies indicated that inactivated vaccines or live attenuated vaccines developed from currently circulating genotype strains had increased effectiveness against current virulent strains, particularly in preventing shedding of challenge virus [15,16]. Recently, we developed a reverse genetics system for a highly virulent NDV strain Banjarmasin/010/10 (Ban/010) that was isolated from diseased chickens during an outbreak in Indonesia in 2010 [17]. The Ban/010 virus is classified in genotype VII of class II and has only 89 and 87% amino acid identity for the F and HN proteins, respectively, with the LaSota and B1 vaccine strains [18]. A mutant virus, named recombinant Ban/AF (rBan/AF), was generated in which the virulent F protein cleavage site motif “RRQKR↓F” was modified to be identical to that of strain LaSota “GRQGR↓L” by three amino acid substitution (underlined) [17]. The rBan/AF virus was completely avirulent, and was genetically stable during 10 consecutive passages in chickens. Serological analysis showed that rBan/AF induced higher neutralization and hemagglutination inhibition antibody titers against the prevalent viruses than did the commercial vaccines B1 or LaSota. Both rBan/AF and the commercial vaccines provided protection against clinical disease and mortality after challenge with virulent NDV strain Ban/010 (genotype VII) or GB Texas (genotype II). However, rBan/AF significantly reduced challenge virus shedding from the vaccinated birds compared to the B1 vaccine. These results confirm that a genotype-matched vaccine generated by reverse genetics can provide better protection than commercial vaccines [15–17]. However, development of a reverse genetics system for a circulating NDV strains can be costly and time consuming. An alternative strategy for producing genotype-matched vaccine is to replace the F and HN genes of a recombinant commercial vaccine strain with those of circulating strains in which the virulent F protein cleavage site motif was modified to an avirulent motif. Although this strategy is cost-effective and less time consuming, there are concerns that a chimeric virus may not be genetically stable and growth retarded due to gene incompatibility. Furthermore, a chimeric genotype-matched vaccine has never been directly compared with a same genotype-matched vaccine made by full-genome reverse genetics. Therefore, the purpose of this study was to compare the protective immunity of genotype VII vaccines developed by chimeric and by full-genome reverse genetics approaches. In order for this study, we generated three recombinant chimeric LaSota viruses in which the F and/or HN ORF was replaced with that of rBAN/AF, the attenuated version of Ban/010. These chimeric viruses were compared to the parental recombinant LaSota (rLaSota) and rBan/AF viruses for replication in vivo and in vitro, and for immunogenicity and protective efficacy against challenge with virulent GB Texas or Ban/010 viruses. This study also evaluated the relative contributions of F versus HN in the superior protection provided by vaccine virus that is homologous to the challenge virus. These results will be useful for development of genotype-matched NDV vaccines. Materials and Methods Viruses and cells The chicken embryo fibroblast cell line (DF1) and human epidermoid carcinoma cell line (HEp-2; ATCC, Manassas, VA, USA) were grown in Dulbecco’s minimal essential medium (DMEM) with 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS. The modified vaccinia virus strain Ankara (MVA) expressing T7 RNA polymerase was kindly provided by Dr. Bernard Moss (NIAID, NIH) and propagated in primary chicken embryo fibroblast cells in DMEM with 2% FBS. In experiments that required supplementations of exogenous protease for the cleavage of the F protein, normal SPF chicken egg allantoic fluid was added to a concentration of 10%. NDV strains rLaSota [19] and rBan/AF [17] and the three recombinant chimeric viruses generated in this study were grown in the allantoic cavities of 9-day-old specific pathogen free (SPF) embryonated chicken eggs. Virus stocks were quantified by hemagglutination (HA) assay with chicken erythrocytes. All experiments involving virulent NDV were performed in our USDA approved enhanced Biosafety Level-3 (BSL-3+) facility following the guidelines and approval of the Animal Care and Use Committee (IACUC), University of Maryland. All experiments were approved by the IACUC (protocol number R-09-81) and conducted following the guidelines. All animal care and handling, including euthanasia were conducted according to the procedures of Animal Care and Use committee and guideline of the American Veterinary Medical Association. All efforts were made to minimize discomfort and pain. The personnel conducting this experiment examined infected birds three times a day for clinical symptoms following the well-established scoring system. Birds that show a total score of 0 were considered "normal" while birds showing scores of 1 to 8 were considered "sick." If a bird presents a score of 2 or 3 in any of the categories above, we increased the monitoring frequency to 3 times daily. Supportive care was provided for animals that show scores of 2 or 3. If the condition worsens or does not improve after it has reached a score of 3, it was euthanized as it is considered that the bird has reached a moribund state. If necessary, facility veterinarian in the department was contacted to determine whether the bird needs to be euthanized or requires supportive care. Supportive care was provided if it does not interfere with the objective of this study. Birds were anesthetized and killed by an overdose of isoflurane, the inhalant anesthetic. Briefly, sterile cotton gauze was placed in the bottom of a sterile bell jar and was covered with a wire mesh. Approximately, 1 to 2 ml of isoflurane was added into the cotton gauze. The bird was placed in the jar and the lid was closed quickly. The bird was removed from the jar after cessation of breathing. Generation of recombinant chimeric NDV strain LaSota in which the F and/or HN gene ORFs were replaced by those of the avirulent Indonesian strain rBan/AF The F and HN ORFs of the avirulent strain rBan/AF were placed individually or together into a full-length antigenomic cDNA of NDV strain LaSota in place of the corresponding LaSota F and HN ORF(s) (Figure 1). These manipulations were facilitated by the presence of unique restriction enzyme sites (PacI, MluI, and AgeI) created in the untranslated regions (UTRs) flanking the F and HN ORFs in the NDV cDNA. The F and HN ORFs of rBan/AF were modified to be flanked by 5´ and 3´ UTRs of the respective rLaSota F or HN gene along with the compatible restriction enzyme sites by overlapping PCR. The engineered rBan/AF F and/or HN genes were digested with the appropriate restriction enzymes and used to replace the corresponding genes in the full-length LaSota cDNA, resulting in three full-length chimeric NDV cDNAs (Figure 1). Thus, the LaSota gene-start and gene-end signals flanking the F and HN genes remained undisturbed. 10.1371/journal.pone.0074022.g001Figure 1 Construction and recovery of three chimeric versions of the LaSota vaccine strain in which the complete F and/or HN gene ORF was replaced with that of rBan/AF, an attenuated version of the circulating virulent Indonesian Ban/010 strain. (A) Genome maps of the parental rLaSota and rBan/AF strains and the three chimeric derivatives. ORFs are shown as rectangles that are filled for those derived from rLaSota and are open for those from rBan/AF. The F protein cleavage sites are shown. (B) SDS-PAGE analysis of purified virions of strains rLaSota (lane 1), Ban/AF (lane 2), rLaSota-Ban/AF F (lane 3), rLaSota-Ban/AF HN (lane 4), and rLaSota-Ban/AF F HN (lane 5). The viruses were harvested from allantoic fluids, purified through a 30% sucrose cushion, and analyzed on an 8% SDS-PAGE gel under reducing conditions, and stained with Coomassie brilliant blue. Infectious viruses were generated using a reverse genetics system established in our laboratory [20]. Briefly, HEp-2 cells were transfected with three plasmids individually encoding the N, P, and L proteins (2.0 µg, 1.0 µg, and 0.5 µg per single well of a six-well dish, respectively) and a fourth plasmid encoding the full-length antigenome (5.0 µg) using Lipofectamine (Invitrogen) and simultaneously infected with vaccinia MVA expressing T7 RNA polymerase at a multiplicity of infection (MOI) of 1 PFU/cell. Two days after transfection, alloquot of transfection mixture was inoculated into the allantoic cavity of 9-day-old embryonated chicken eggs. Following incubation for 2 days, allantoic fluid was harvested and recovery of the virus was confirmed by hemagglutination assay using 1% chicken red blood cells. The recovered viruses were plaque purified. The absence of adventitious mutations in the F and/or HN genes of Indonesian strain in the recovered viruses were confirmed by nucleotide sequencing analysis using primers targeting a downstream of the M gene and an upstream of the L gene. Cleavage of the F protein of chimeric viruses To evaluate cleavage of the F proteins of the chimeric viruses, DF1 cells were infected at an MOI of 0.1, and cell lysates were collected at 24 h post-infection (hpi), denatured under reducing conditions, subjected to electrophoresis on an 8% polyacrylamide gel, and subjected to Western blot analysis separately with anti-NDV F rabbit polyclonal antiserum [21] and an anti-NDV HN monoclonal antibody. Growth characteristics of parental and chimeric NDVs in DF1 cells The ability of the chimeric viruses to produce plaques was tested in DF1 cells under 0.8% methylcellulose overlay. The plaques were immunostained using polyclonal antibody raised against the N protein of NDV [22]. The multicycle growth kinetics of the F and HN chimeric viruses, along with their respective parental viruses, was evaluated in DF1 cells in the presence of 10% chicken egg allantoic fluid. Virus titers in the collected supernatants were quantified in DF1 cells by limiting dilution in the presence of added allantoic fluid and expressed as 50% tissue culture infectious dose (TCID50/ml) by the end-point method of Reed and Muench [23]. Mean death time in embryonated chicken eggs The pathogenicity of parental and chimeric viruses was determined by the mean death time (MDT) test in 9-day-old SPF embryonated chicken eggs [1]. Briefly, a series of 10-fold dilutions of infected allantoic fluid (0.1 ml) was inoculated into the allantoic cavities of five 9-day-old eggs per dilution and incubated at 37 °C. The eggs were examined once every 8 h for 7 days, and the time of embryo death was recorded. The MDT was determined as mean time (h) for the minimum lethal dose of virus to kill all the inoculated embryos. The criteria for classifying the virulence of NDV isolates are: <60 h, virulent strains; 60 to 90 h, intermediate virulent strains; and >90 h, avirulent strains. Immunization of chickens with parental and chimeric NDVs Two-week-old SPF chickens in groups of eleven (11 birds each) were immunized with each virus (200 µl of each, 106 EID50) by the intranasal route. One group of chickens remained as unvaccinated controls. Three birds from each group were sacrificed at 4 days post-infection (dpi) and tissues samples (lung and trachea) were collected for vaccine virus titration. Virus titers were determined by limiting dilution and immunostaining in DF1 cells as described before. The presence of virus in the tissue samples also were determined by inoculation into 9-day-old SPF embryonated chicken eggs, which provides a more sensitive means of detection but does not provide a titer. In general, we obtained HA titers ranging from 24 to 27 from positive samples. At 3 dpi, the allantoic fluids were tested for virus growth by HA assay. For analysis of antibody responses, serum samples (3 ml from each chicken) were collected on weeks 2, 3, 4, and 5. Serum antibody titers were determined by hemagglutination inhibition (HI) assay using rLaSota or rBan/AF as antigen [17]. Challenge of immunized chickens with virulent NDV strains Five weeks post immunization, the remaining chickens (eight per immunizing virus) were transferred to a USDA-certified BSL3containment facility for NDV challenge. The birds in each group were challenged with 100 chicken 50% lethal dose (CLD50) of virulent NDV strains GB-Texas (four birds) or Ban/010 (four birds) through the oculo-nasal route [17]. Oral and cloacal swabs were collected from three birds at 4 and 7 dpi, and shedding of the challenge virus was determined by inoculating clarified swab samples into 9-day-old SPF embryonated chicken eggs and conducting HA assay as described above. Three chickens from each group were sacrificed at 4 days post-challenge (dpc) to evaluate challenge virus replication in different organs. Tissue samples (brain, trachea, lungs, and spleen) were collected, and the challenge virus titers in homogenized tissue samples were determined by a limiting end point dilution assay as described above. Statistical Analysis Statistically significant differences in serological analysis between different immunized chicken groups were evaluated by one-way analysis of variance (ANOVA) (SPSS 13.0 for Windows, SPSS Inc, Chicago, IL). Results Generation of recombinant chimeric LaSota viruses with the F and/or HN gene ORF replaced by that of a circulating Indonesian NDV strain We used reverse genetics to make three chimeric derivatives of the LaSota vaccine strain in which the complete ORF of the F and/or HN genes was replaced with that of the rBan/AF strain (Figure 1A), which is a version of the highly virulent Ban/010 strain that was rendered avirulent solely by three amino acid substitutions in the cleavage site of the F protein. Specifically, the F protein cleavage site of rBan/AF, and of the three chimeric viruses, was identical to that of strain LaSota. Infectious viruses were recovered from all of the three chimeric cDNAs (rLaSota-Ban F, rLaSota-Ban HN, and rLaSota-Ban F HN). To evaluate genetic stability, the viruses were passaged five times in 9-day-old embryonated chicken eggs. All the viruses were able to replicate well in the eggs (> 28 HAU/ml) and the sequences of the F and HN genes were confirmed, showing that the introduced ORFs were maintained without any adventitious mutations. We further evaluated incorporation of heterologous glycoproteins into chimeric virions, since the presence of the compatible cytoplasmic tails and transmembrane regions is important for NDV replication [24]. The viral proteins in partially purified parental and chimeric viruses [25] were analyzed by SDS-PAGE and visualized by Coomassie blue staining (Figure 1B). This confirmed a similar pattern of the major structural proteins into each virus, with the expected sizes and amounts, suggesting that the substitutions in rLaSota backbone did not affect virion assembly (Figure 1B). Syncytium formation and cleavage efficiency of the F protein of chimeric viruses in vitro Strain LaSota lacks a polybasic sequence or furin motif at the F protein cleavage site, and depends on extracellular protease for cleavage [19]. In our previous study, we modified the highly virulent Indonesian Ban/010 strain by changing its cleavage site sequence (R RQK R↓F) to that of LaSota (GRQGR↓L), resulting in the avirulent rBan/AF derivative. Whereas the Ban/010 parent induced extensive syncytia and plaque formation in cell culture in the absence of added protease, the rBan/AF derivative caused only single-cell infections without syncytia or plaques in the presence or absence of extracellular protease [17]. Therefore, in the present study, the ability of the chimeric viruses to form syncytia and plaques in cell culture was determined. DF1 cells were infected with the parental (rLaSota and rBan/AF) and the three chimeric viruses at an MOI of 0.01 in the presence or absence of 10% allantoic fluid as protease supplementation. The cells were visualized 48 hpi by photomicroscopy directly (Figure 2A) and following immunostaining with antiserum against the NDV N protein (Figure 2B). In parallel, the ability of the viruses to produce plaques was evaluated on DF1 cells under 0.8% methylcellulose overlay (not shown). In the presence of added protease, the rLaSota virus produced syncytia (Figure 2A and B) and plaques (not shown), whereas the rBan/AF virus produced neither. Among the three chimeric viruses, only the one containing F from the LaSota and HN from the rBan/AF strain (rLaSota-Ban/AF HN) produced syncytia and plaques, similar to those of rLaSota. In contrast, the other two chimeric viruses (rLaSota-Ban/AF F and rLaSota-Ban/AF F HN) produced neither syncytia (Figure 2A and B) nor plaques (not shown). Thus, the formation of syncytia and plaques in the presence of protease was associated with the presence of the LaSota F protein. Evaluation of the cleavage efficiency of the F proteins by Western blot analysis showed that the LaSota-derived F protein present in both rLaSota and rLaSota-Ban/AF HN was cleaved more efficiently than the rBan/AF-derived F protein present in the other viruses (Figure 3). These results suggest that the greater efficiency of cleavage of the LaSota-derived F protein was required for syncytium and plaque formation. 10.1371/journal.pone.0074022.g002Figure 2 Production of syncytia by parental and chimeric viruses. DF1 cells in six-well plates were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.01 PFU/cell, incubated for 72 h, and visualized by photomicroscopy directly (A) or following immunoperoxidase staining using antiserum against the N protein of NDV, with viral antigen stained red (B). 10.1371/journal.pone.0074022.g003Figure 3 Cleavage of the F0 proteins of parental and chimeric viruses. (A) Proteolytic cleavage of the F0 proteins of parental and chimeric viruses in infected DF1 cells analyzed by Western blot. (B) The positions of the precursor protein F0, the cleavage product F1, and the HN protein are indicated. Lanes: 1. rLaSota, 2. rBan/AF, 3. rLaSota-Ban/AF F, 4. rLaSota-Ban/AF HN, and 5. rLaSota-Ban/AF F HN (B) The relative levels of the F0 and F1 proteins in the Western blot experiment in part C were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F1 by the amount of F1 plus F0. Each bar represents mean and standard error of the mean of triplicate samples. Growth characterization of chimeric NDVs in vitro The growth kinetics of parental and chimeric NDVs was evaluated in DF1 cells after infection at an MO1 of 0.01 in the presence of exogenous protease (Figure 4). Parental rLaSota replicated more efficiently than rBan/AF in the first 24 h of infection, but the titers were similar thereafter. Among the chimeric viruses, rLaSota-Ban/AF HN grew to higher titer than the two parental viruses and the other two chimeric viruses, reaching a maximum titer of 1.3 × 107 TCID50/ml at 40 hpi. The other two chimeric viruses (rLaSota-Ban/AF F and rLaSota-Ban/AF F HN) replicated less efficiently than the other tested viruses and reached their maximum titers (106 TCID50/ml) at 56 hpi. The pattern of in vitro replication of chimeric viruses also showed the correlation with their ability to form syncytia in DF1 cells (Figure 2). 10.1371/journal.pone.0074022.g004Figure 4 In vitro multicycle growth of parental and chimeric viruses in chicken embryo fibroblast DF1 cells following infection with an MOI of 0.01 PFU/cell. Exogenous protease was provided in the infected cells. The viral titers were determined by limiting dilution on DF1 cells. Pathogenicity of the chimeric viruses The pathogenicity of chimeric viruses was evaluated by a standard, internationally-accepted pathogenicity test for NDV, namely the MDT assay in embryonated chicken eggs (Table 1). Of the two parental viruses, rBan/AF (122 h) was somewhat more attenuated than the rLaSota virus (112 h). For the three chimeric viruses, the order of increasing attenuation was rLaSota-Ban/AF HN (129 h), rLaSota-Ban/AF F (148 h), and rBan/AF F HN (>168 h). Thus, introduction of either of the two rBan/AF glycoproteins conferred attenuation, with F having a greater effect than HN, and the effect was greatest when both rBan/AF glycoproteins were introduced. At least in the case of the rBan/AF HN glycoprotein, this attenuation was not due to a direct defect in the replication of the chimeric virus, since the chimeric rLaSota-Ban/AF HN virus replicated the most efficiently in cell culture (Figure 4). Table 1 Pathogenicity of parental and chimeric viruses in embryonated eggs. Virus MDT (h)a rLaSota 112 rBan/AF 122 rLaSota-Ban/AF F 148 RLaSota-Ban/AF HN 129 rLaSota-Ban/AF F HN >168 a Mean embryo death time (MDT): the mean time (h) for the minimum lethal dose of virus to kill all of the inoculated embryos. Pathotype definition: virulent strains, <60 h; intermediate virulent strains, 60 to 90 h; avirulent strains, >90 h. Replication, immunogenicity, and protective efficacy of chimeric NDVs To evaluate the protective efficacy of the chimeric viruses, 2-week-old chickens were immunized with each virus by intranasal route. Three birds from each group were sacrificed at 4 dpi and tissue samples (brain, trachea, lungs, and spleen) were collected for virus titration. Virus titration of the homogenates by limiting dilution assay showed that detection of parental and chimeric viruses was restricted to the trachea, and their titers were low, ranging from 1.6 to 2.4 log10 TCID50/g, with slight differences between the parental and chimeric viruses (data not shown). To enhance the sensitivity of virus detection, the tissue homogenates also were inoculated into eggs (Table 2). Replication of rLaSota and rBan/AF was confirmed in the trachea and lungs, whereas replication of the three chimeric viruses was mostly found in the trachea of the immunized birds. None of the viruses were detected in the brain and spleen. Table 2 Replication of parental and chimeric viruses in 2-week-old immunized chickens. Virus replication in embryonated eggsa Virus Brain Trachea Lung Spleen rLaSota 0/3 3/3 2/3 0/3 rBan/AF 0/3 3/3 2/3 0/3 rLaSota-Ban/AF F 0/3 3/3 0/3 0/3 rLaSota-Ban/AF HN 0/3 3/3 1/3 0/3 rLaSota-Ban/AF F HN 0/3 3/3 1/3 0/3 a Groups of 2-week-old chickens were inoculated with each virus by the intranasal route. Three birds from each group were sacrificed on day 4, and tissues samples (brain, trachea, lung, and spleen) were collected and homogenized. To confirm the virus replication, each sample (100 µl, containing approximately 10 µg of tissue) of homogenized tissue was inoculated into each of three eggs, and allantoic fluids were collected on 3 dpi. Virus replication was determined by hemagglutination assay. The immunogenicity of the chimeric viruses was determined by collecting sera at 2, 3, 4, and 5 weeks post-immunization and evaluating antibody responses using HI assay (log2) against the two different parental viruses, namely the rLaSota (Figure 5A) and rBan/AF (Figure 5B) strains. The highest titers were obtained from sera collected at 2 (rBan/AF-specific assay) or 4 (rLaSota-specific assay) weeks post-immunization, after which the titers decreased. In the rLaSota-specific assay, antisera raised against either parental virus tended to have higher HI titers than those raised against the chimeric viruses, especially at 2 weeks post-immunization. In the rBan/AF-specific assay, antisera raised against the parental rBan/AF virus generally had the highest titers at the various time points, whereas antisera against the parental rLaSota virus generally had the lowest titers. Among the sera raised against the three chimeric viruses, there was no clear pattern of reactivity associated with the presence of the homologous versus the heterologous HN protein. 10.1371/journal.pone.0074022.g005Figure 5 Induction of serum antibodies in 2-week-old chickens in response to infection with parental and chimeric viruses. Chickens were inoculated with each virus (64 HA units) by the intranasal route in the same experiment as Table 2. Sera were collected at 2, 3, 4, and 5 weeks post-infection. Virus-specific antibodies were measured by a hemagglutination inhibition assay using rLaSota (A) or rBan/AF (B) virus and chicken erythrocytes. To evaluate the protective efficacy of chimeric NDVs, at 5 weeks post-immunization, the remaining immunized chickens in each immunization group were divided into two challenge groups. Each challenge group (8 birds each) was challenged with 100 CLD50 per chicken of the virulent GB Texas (genotype II, homologous to the LaSota vaccine) or the virulent wild-type Indonesian strain (Ban/010, genotype VII, the parent of the rBan/AF strain) via the oculo-nasal route. In addition, three chickens each from the unimmunized group were challenged with either of the two challenge NDVs. For the unimmunized chickens, challenge with the GB Texas virus resulted in clinical symptoms (at 4 day post-challenge (dpc), and 100% of mortality at 5 dpc, whereas challenge with the Ban/010 virus resulted in 100% of mortality at 3 dpc (data not shown). The virus titers in the trachea of dead chickens were >7.0 log10 TCID50/g. In contrast, all of the previously-immunized chickens (5 chickens for each group) were completely protected from clinical disease and mortality against the GB-Texas and Ban/010 strains. Shedding and replication of the GB-Texas and Ban/010 challenge viruses in the immunized chickens were evaluated. Oral and cloacal swabs (3 birds each) were collected at 4 and 7 dpc and inoculated into eggs for sensitive detection of infectious virus. In addition, 3 birds from each challenge group were sacrificed at 4 dpi and tissue samples (brain, trachea, lungs, and spleen) were collected and tissue homogenates prepared. These were evaluated for the presence of virus by inoculation into eggs, and virus titers were determined by limiting dilution assay, as above. In the GB-Texas challenged groups, virus shedding was not detected in most swab samples (Table 3), and no virus was detected in any of the collected tissue samples (data not shown). This indicated that all the immunized groups of the chickens were efficiently protected against GB-Texas. However, in the Ban/010-challenged group, virus shedding was detected in both the oral and cloacal swabs in all of the chickens immunized with rLaSota (Table 3), and high virus titers were detected in the trachea (2.9 × 106 TCID50/g) and lungs (1.8 × 105 TCID50/g) (Figure 6). Similarly, virus shedding was detected in chickens immunized with rLaSota containing the Ban/AF HN (rLaSota-Ban/AF HN). In contrast, virus shedding was present in only 1 of 3 in chickens immunized with a genotype-matched vaccine, rBan/AF, and only in the oral swab. At 7 dpi, no oral or coacal shedding of challenge viruses was detected in all of the collected swab samples (data not shown). Table 3 Oral and cloacal shedding of NDV challenge viruses. NDV strain GBT challenge NDV strain Indonesia challenge Chicken groupa Oral Cloacal Oral Cloacal rLaSota 0/3 0/3 3/3 3/3 rBan/AF 1/3 0/3 1/3 0/3 rLaSota-Ban/AF F 1/3 0/3 0/3 0/3 rLaSota-Ban/AF HN 1/3 0/3 3/3 2/3 rLaSota-Ban/AF F HN 0/3 0/3 1/3 0/3 a Groups of 2-week-old chickens were inoculated with each virus by the intranasal routes and challenged with NDV strain GBT or Indonesia. Oral (A) and cloacal (B) swabs were collected from the 3 birds in each group on day 4 and 7 post challenge. To confirm the shedding of challenge virus, aliquots (100 µl each, out of a total of 1 ml of swab fluid) of the collected samples were inoculated into three eggs, and allantoic fluids were collected on 3 dpi. Virus replication was determined by hemagglutination assay. On day 7 post challenge, challenge virus shedding was not detected in all of the collected samples. 10.1371/journal.pone.0074022.g006Figure 6 Shedding of challenge Ban/010 virus in chickens previously immunized with parental and chimeric viruses. From the challenge experiment in Table 3, tissue samples were harvested from the 3 birds in each group on day 4 post-challenge, and virus titers were determined by a limiting dilution assay on DF-1 cells. Data from challenge with the GB Texas strain are not shown because no virus was detected. The two chimeric LaSota viruses containing rBan/AF F and F and HN together showed comparable levels of protective efficacy to that of rBan/AF. Our findings showed that rBan/AF vaccine and the two chimeric viruses containing the genotype-matched F gene (rLaSota-Ban/AF F and rLaSota-Ban/AF F HN) effectively prevented shedding of Indonesian challenge virus, whereas viruses lacking the Indonesian F gene, namely rLaSota and chimeric rLaSota-Ban/AF HN, were less effective in preventing shedding of Indonesian challenge virus. These results show that the genotype-matched vaccines provide better protection than genotype mismatched vaccines, and this superior efficacy is due to the F protein. Discussion All NDV isolates belong to a single serotype. Consistent with this, currently used vaccines, such as strains B1 and LaSota, are known to protect against morbidity and mortality caused by NDV isolates in different parts of the world [26]. However, recent studies have suggested that NDV strains currently in circulation represent genotypes that differ from that of the vaccine strains [8], and the current vaccines allow considerable breakthrough infection, shedding, and disease by the presently circulating genotype viruses. Infection and shedding permits recirculation of the virus in the environment and provides a setting in which the viral population may acquire mutations and adaptive changes in response to immune pressure [27]. Therefore, the use of genotype-matched vaccines has been suggested for better control of NDV [15,16,28]. Our previous study also demonstrated that an attenuated vaccine virus (rBan/AF) generated by reverse genetics from a circulating virulent virus in Indonesia provided better protection against genotype-matched isolates than that provided by the genotype-mismatched vaccine stains LaSota and B1 [17]. However, generation of a genotype-matched vaccine by reverse genetics can be time consuming. Therefore, in this study, we generated recombinant chimeric viruses, using an existing LaSota-based reverse genetics system, by replacing the ORF of F and HN genes of LaSota with those from rBan/AF, which is a recombinant attenuated derivative of the highly virulent Indonesian Ban/010 strain of genotype VII. The attenuated phenotype of the rBan/AF strain was derived solely by the introduction of three amino acid substitutions in the F protein cleavage site of the virulent Ban/010 parent, which changed the site to be identical to that of the LaSota strain. In our study, all the chimeric viruses were readily recovered, and the incorporation of the surface glycoproteins into virions and the yield of virions were similar between parental and chimeric viruses. This indicated that the amino acid sequence differences between the F and HN proteins of rLaSota versus rBan/AF did not detectably affect virus assembly and recovery. However, in vitro characterization of the parental and chimeric viruses showed that the F protein of the LaSota strain was cleaved more efficiently, in the presence of added allantoic fluid as a source of protease, than the F protein of rBan/AF, and the presence of this protein was associated with the formation of syncytia and plaques as well as better growth in vitro. This was true for both the parental and chimeric viruses. Thus, the rBan/AF HN virus had efficient cleavage of the F protein, induced syncytium and plaque formation, and had relatively efficient replication in DF1 cells, similar to rLaSota. Nonetheless, the virus was somewhat more attenuated (MDT 129 h) than rLaSota (MDT 112 h). In contrast, rLaSota-Ban/AF F and rLaSota-Ban/AF F HN showed inefficient cleavage of the F protein, single cell infection without syncytium or plaque formation, and reduced replication in infected DF1 cells. The finding that cleavage of the rBan/AF F protein was less efficient than that of the rLaSota F protein, even though their cleavage activation sequences were identical, indicated that structural features of the rBan/AF F protein additional to the cleavage site affect proteolytic processing of the F protein. Chimeric LaSota containing rBan/AF F and HN together resulted in a greater attenuation of the virus in ovo (MDT > 168 h) than either glycoprotein alone, showing that the rBan/AF HN protein also contributed to attenuation in ovo. One possible explanation could be that the glycoproteins from rBan/AF may not be perfectly compatible with the LaSota background. However, this did not appear to be true for the rBan/AF HN protein, at least, since the rLaSota-Ban/AF HN virus replicated more efficiently in vitro than either parent, and thus did not appear to be impaired. Immunization of 2-week-old chickens confirmed that all the chimeric viruses were highly attenuated. Replication of the three chimeric viruses was mostly limited to the trachea (Table 2), and thus they were somewhat more attenuated in birds than the LaSota vaccine strain, consistent with the results of the MDT assay. Thus, they should be safe vaccines. Despite this attenuation, the chimeric viruses induced relatively good serum antibody responses. In general, sera from chickens immunized with parental and chimeric viruses had higher HI titers against rLaSota than rBan/AF (p<0.05). This was associated with greater restriction of the GB-Texas challenge strain (homologous to LaSota) compared to the Ban/010 challenge strain. In the Ban/AF-specific HI assay, the highest HI titers were observed with parental rBan/AF, followed by rLaSota containing rBan/AF F protein (rLaSota-Ban/AF F), whereas the lowest titers were observed with rLaSota (p<0.05). Our challenge study showed that chickens vaccinated by rLaSota-Ban/AF F and rLaSota-Ban/AF F HN completely prevented shedding of the Ban/010 challenge virus, whereas rLaSota-Ban/AF HN did not prevent shedding of Ban/010 (Figure 5B). Thus, the presence of the rBan/AF F protein in the immunizing virus was associated with complete restriction of the Ban/010 challenge virus. It is noteworthy that this greater efficacy was not associated with more efficient replication in vitro (in fact, the rLaSota-Ban/AF F and rLaSota-Ban/AF F HN viruses replicated less efficiently in vitro than the others; Figure 4) or in vivo (Table 2). These results indicate that, although the F and HN proteins of NDV are known to be the virus neutralizing antigens and the major protective antigens [29–31], the presence of the homologous F protein was more important in restricting the homologous challenge virus. Previously, cross protection studies have suggested that chickens vaccinated with a live LaSota vaccine displayed disease symptoms after being challenged with antigenic variants of genotype VII [32]. Inactivated vaccines and live attenuated vaccines developed from currently circulating genotype strains were shown to have increased effectiveness in preventing virus shedding [15,16]. In contrast, several studies showed that current vaccines have good protective efficacy for morbidity and mortality against viruses circulating in Asia [33,34]. However, shedding of challenge virus was not evaluated in these studies. It has been thought that the extent of the vaccine failure in the commercial farm group may be due to poor vaccination practices, field environmental and/or immunosuppressive factors affecting the efficacy of the vaccine [35]. However, our previous study with the Ban/010 strain circulating in Indonesia suggested that complete prevention of shedding of genotype VII can be achieved by a recombinant genotype matched vaccine generated by reverse genetics [17]. In the present study, evaluation of recombinant chimeric viruses confirmed our previous findings that a genotype-matched vaccine generated either by chimeric or full-genome approach is needed for prevention of virus shedding. Further our study identified the F protein as the major protective antigen for efficient protection of chickens against genotype VII NDV strain. This study also needs to be verified by evaluating protective efficacy in broiler chickens, which are older (slaughtered at 5-16 weeks). We thank Daniel Rockemann, Girmay Gebreluul, Yonas Araya, Andrea Ferrero-Perez, and our laboratory members for excellent technical assistance; and Dr. Bernard Moss (NIAID, NIH) for providing the vaccinia T7 recombinant virus and the pTM1 plasmid [36]. ==== Refs References 1 Alexander DJ (1989 ) Newcastle disease , In: Purchase HG Arp LH Domermuth CH Pearson JE A laboratory manual for the isolation and identification of avian pathogens , 3rd ed. The American Association of Avian Pathologists, Kendall/Hunt Publishing Company pp. 114 -120 . 2 Lamb R , Parks G (2007 ) Paramyxoviridae: the viruses and their replication , In: Knipe DM Howley PM Griffin DE Lamb RA Martin MA Philadelphia : Lippincott Williams & Wilkins pp. 1449 –1496 . 3 Lamb RA , Paterson RG , Jardetzky TS (2006 ) Paramyxovirus membrane fusion: lessons from the F and HN atomic structures . Virology 344 : 30 -37 . doi:10.1016/j.virol.2005.09.007. PubMed: 16364733.16364733 4 Deng RZ , Wang A , Mirza M , Iorio RM (1995 ) Localization of a domain on the paramyxovirus attachment protein required for the promotion of cellular fusion by its homologous fusion protein spike . Virology 209 : 457 -469 . doi:10.1006/viro.1995.1278. PubMed: 7778280.7778280 5 Melanson VR , Iorio RM (2004 ) Amino acid substitutions in the F-specific domain in the stalk of the Newcastle disease virus HN protein modulate fusion and interfere with its interaction with the F protein . J Virol 78 : 13053 -13061 . doi:10.1128/JVI.78.23.13053-13061.2004. PubMed: 15542657.15542657 6 Pedersen JC , Senne DA , Woolcock PR , Kinde H , King DJ et al. (2004 ) Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003 outbreak in California and other recent outbreaks in North America . J Clin Microbiol 42 : 2329 -2334 . doi:10.1128/JCM.42.5.2329-2334.2004. PubMed: 15131226.15131226 7 Samal SK (2011 ) Newcastle disease and related avian paramyxoviruses , In: Samal SK The biology of paramyxoviruses . Caister Academic Press pp. 69 -114 . 8 Miller PJ , Decanini EL , Afonso CL (2010 ) Newcastle disease: evolution of genotypes and the related diagnostic challenges . Infect Genet Evol 10 : 26 -35 . doi:10.1016/j.meegid.2009.09.012. PubMed: 19800028.19800028 9 Kim SH , Nayak S , Paldurai A , Nayak B , Samuel A et al. (2012 ) Complete genome sequence of a novel Newcastle disease virus strain isolated from a chicken in West Africa . J Virol 86 : 11394 -11395 . doi:10.1128/JVI.01922-12. PubMed: 22997417.22997417 10 Diel DG , da Silva LHA , Liu H , Wang Z , Miller PJ et al. (2012 ) Genetic diversity of avian paramyxovirus type 1: Proposal for a unified nomenclature and classification system of Newcastle disease virus genotypes . Infect Genet Evol 12 : 1770 -1779 . doi:10.1016/j.meegid.2012.07.012. PubMed: 22892200.22892200 11 Aldous EW , Fuller CM , Mynn JK , Alexander DJ (2004 ) A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1 virus (PPMV-1) responsible for the 1978 to present panzootic in pigeons . Avian Pathol 33 : 258 -269 . doi:10.1080/0307945042000195768. PubMed: 15276997.15276997 12 Cattoli G et al. (2010 ) Emergence of a new genetic lineage of Newcastle disease virus in West and Cwntral Africa-implications for diagnosis and control . Vet Micobiol 142 : 168 -176 . 13 Khan ST , Rehmani , Rue C , Miller P , Afonso CL (2010 ) Phylogenetic and pathological characterization of Newcastle disease virus isolates from Pakistan . J Clin Microbiol 48 : 1892 –1894 . doi:10.1128/JCM.00148-10. PubMed: 20237105.20237105 14 Liu XF , Wan HQ , Ni XX , Wu YT , Liu WB (2003 ) Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985–2001 . Arch Virol 148 : 1387 –1403 . PubMed: 12827467.12827467 15 Miller PJ , King DJ , Afonso CL , Suarez DL (2007 ) Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge . Vaccine 25 : 7238 –7246 . doi:10.1016/j.vaccine.2007.07.017. PubMed: 17719150.17719150 16 Miller PJ , Estevez C , Yu Q , Suarez DL , King DK (2009 ) Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses . Avian Dis 53 : 39 –49 . doi:10.1637/8407-071208-Reg.1. PubMed: 19432002.19432002 17 Xiao S , Nayak B , Samuel A , Paldurai A , Kanabagattebasavarajappa M, et al. (2012 ) Generation by reverse genetics of an effective, stable, live-attenuated Newcastle disease virus vaccine based on a currently circulating, highly virulent Indonesian strain. PLOS ONE 7: e52751 18 Xiao S , Paldurai A , Nayak B , Samuel A , Bharoto EE et al. (2012 ) Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia . J Virol 86 : 5969 -5970 . doi:10.1128/JVI.00546-12. PubMed: 22532534.22532534 19 Huang Z , Krishnamurthy S , Panda A , Samal SK (2001 ) High-level expression of a foreign gene from the 3´ proximal first locus of a recombinant Newcastle disease virus . J Gen Virol 82 : 1729 -1736 . PubMed: 11413385.11413385 20 Krishnamurthy S , Huang Z , Samal SK (2000 ) Recovery of a virulent strain of Newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation . Virology 278 : 168 -182 . doi:10.1006/viro.2000.0618. PubMed: 11112492.11112492 21 Samal S , Kumar S , Khattar SK , Samal SK (2011 ) A single amino acid change Q114R in cleavage site sequence of Newcastle disease virus fusion protein attenuates viral replication and pathogennicity . J Gen Virol 92 : 2333 -2338 . doi:10.1099/vir.0.033399-0. PubMed: 21677091.21677091 22 Kim SH , Xiao S , Shive H , Collins PL , Samal SK (2012 ) Replication, neurotropism, and pathogenicity of avian paramyxovirus serotypes 1-9 in chickens and ducks . PLOS ONE 7 : e34927 . doi:10.1371/journal.pone.0034927. PubMed: 22558104.22558104 23 Reed LJ , Muench HA (1938 ) A simple method of estimating fifty percent endpoints . Am J Hyg 27 : 493 -497 . 24 Kim SH , Subbiah M , Samuel AS , Collins PL , Samal SK (2011 ) Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses . J Virol 85 : 8582 -8596 . doi:10.1128/JVI.00652-11. PubMed: 21680512.21680512 25 Peter HD , Robinson WS (1965 ) Isolation of the Newcastle disease virus . Proc Natl Acad Sci U S A 54 : 794 -800 . doi:10.1073/pnas.54.3.794. PubMed: 5217460.5217460 26 Cho SH , Kwon HJ , Kim TE , Kim JH , Yoo HS et al. (2008 ) Characterization of a recombinant Newcastle disease vaccine strain . Clin Vaccine Immunol 15 : 1572 –1579 . doi:10.1128/CVI.00156-08. PubMed: 18768673.18768673 27 Perozo F , Marcano R , Afonso CL (2012 ) Biological and Phylogenetic Characterization of a Genotype VII Newcastle Disease Virus from Venezuela: Efficacy of Field Vaccination . J Clin Microbiol 50 : 1204 -1208 . doi:10.1128/JCM.06506-11. PubMed: 22238433.22238433 28 Hu S , Ma H , Wu Y , Liu W , Wang X et al. (2009 ) vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics . Vaccine 27 : 904 -910 . doi:10.1016/j.vaccine.2008.11.091. PubMed: 19095026.19095026 29 Boursnell ME et al. (1990 ) A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) protects chickens against challenge by NDV . Virology 78 : 297 –300 . PubMed: 2167557. 30 Cosset FL et al. (1991 ) Newcastle disease virus (NDV) vaccine based on immunization with avian cells expressing the NDV hemagglutinin-neuraminidase glycoprotein . Virology 185 : 862 –866 . doi:10.1016/0042-6822(91)90560-X. PubMed: 1660204.1660204 31 Karaca K et al. (1998 ) Recombinant fowlpox viruses coexpressing chicken type I IFN and Newcastle disease virus HN and F genes: influence of IFN on protective efficacy and humoral responses of chickens following in ovo or post-hatch administration of recombinant viruses . Vaccine 16 : 1496 –1503 . doi:10.1016/S0264-410X(97)00295-8. PubMed: 9711795.9711795 32 Qin ZM , Tan LT , Xu HY , Ma BC , Wang YL et al. (2008 ) Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996 to 2005 J . Clin Microbiol 46 : 601 -611 . doi:10.1128/JCM.01356-07. 33 Jeon WJ , Lee EK , Lee YJ , Jeong OM , Kim YJ et al. (2008 ) Protective efficacy of commercial inactivated Newcastle disease virus vaccines in chickens against a recent Korean epizootic strain . J Vet Sci 9 : 295 -300 . doi:10.4142/jvs.2008.9.3.295. PubMed: 18716450.18716450 34 Liu XF , Wan HQ , Ni XX , Wu YT , Liu WB (2003 ) Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985–2001 . Arch Virol 148 : 1387 –1403 . PubMed: 12827467.12827467 35 Dortmans JC , Peeters BP , Koch G (2012 ) Newcastle disease virus outbreaks: vaccine mismatch or inadequate a p p l i c a t i o n ? Vet Microbiol 160 : 17 -22 . doi:10.1016/j.vetmic.2012.05.003. PubMed: 22655976.22655976 36 Moss B , Elroy-Stein O , Mizukami T , Alexander WA , Fuerst TR (1990 ) Product review. New mammalian expression vectors . Nature 348 : 91 –92 . doi:10.1038/348091a0. PubMed: 2234068.2234068	24015313	PMC3755997	CC0	2021-01-05 17:38:17	yes	PLoS One. 2013 Aug 28; 8(8):e74022
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24023916PONE-D-13-1905110.1371/journal.pone.0073952Research ArticleDepletion of CD4+ CD25+ Regulatory T Cells Promotes CCL21-Mediated Antitumor Immunity Antitumor Effects of CCL21 Combined with Anti-CD25Zhou Shuang * Tao Huihong Zhen Zhiwei Chen Haixia Chen Guolin Yang Yaoqin * Department of Histology and Embryology, Tongji University School of Medicine, Shanghai, China Mosley R. Lee Editor University of Nebraska Medical center, United States of America * E-mail: shuangzhou@tongji.edu.cn (SZ); yaoqiny@163.com (YY)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SZ YY. Performed the experiments: SZ HT ZZ. Analyzed the data: SZ HC GC. Contributed reagents/materials/analysis tools: HT ZZ HC. Wrote the manuscript: SZ YY. 2013 2 9 2013 8 9 e739528 5 2013 24 7 2013 © 2013 Zhou et al2013Zhou et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.CCL21 is known to attract dendritic cells (DCs) and T cells that may reverse tumor-mediated immune suppression. The massive infiltration of tumors by regulatory T cells (Tregs) prevents the development of a successful helper immune response. In this study, we investigated whether elimination of CD4+ CD25+ Tregs in the tumor microenvironment using anti-CD25 monoclonal antibodies (mAbs) was capable of enhancing CCL21-mediated antitumor immunity in a mouse hepatocellular carcinoma (HCC) model. We found that CCL21 in combination with anti-CD25 mAbs (PC61) resulted in improved antitumor efficacy and prolonged survival, not only inhibited tumor angiogenesis and cell proliferation, but also led to significant increases in the frequency of CD4+, CD8+ T cells and CD11c+ DCs within the tumor, coincident with marked induction of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) at the local tumor site. The intratumoral immune responses were accompanied by the enhanced elaboration of IL-12 and IFN-γ, but reduced release of the immunosuppressive mediators IL-10 and TGF-β1. The results indicated that depletion of Tregs in the tumor microenvironment could enhance CCL21-mediated antitumor immunity, and CCL21 combined with anti-CD25 mAbs may be a more effective immunotherapy to promote tumor rejection. This work was supported by National Natural Science Foundation of China (No. 31000527), Shanghai Health Bureau Research Fund for young investigator (to Dr. Shuang Zhou) and Hong Kong Scholar program (No. XJ2011025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The induction of an effective antitumor immune response requires both antigen-presenting cells (APCs) and activated T cells. One might speculate that a stronger immune response could be achieved by attracting larger numbers of effector T cells and mature dendritic cells (DCs) to the tumor site. Increasing evidence shows that chemokines play an integral role in the initiation of a specific immune response [1,2]. CCL21, formerly known as secondary lymphoid tissue chemokine (SLC), is a CC chemokine that is capable of recruiting DCs, naive T cells and B cells via its specific receptor CCR7 (CC chemokine receptor type 7) found on these cell types [3–5]. Based on its expression pattern and that of its receptor, CCL21 could serve as a potent agent in cancer immunotherapy. Previous studies have demonstrated that CCL21 administered intratumorally elicits tumor rejection in murine models of established tumors [6,7]. We and others have also shown that vaccination with CCL21 modification is an effective strategy to stimulate antitumor immune responses in a mouse hepatocellular carcinoma (HCC) model [8–11]. The CCL21-mediated antitumor response is dependent on both CD4+ and CD8+ lymphocyte subsets, also accompanied by DCs infiltration [6]. However, it should be noted that CCL21 elicits a substantial infiltration of DCs and naive T cells into the tumor, as well as the naturally occurring regulatory T cells (Tregs) by means of CCR7 [12–14]. Tregs are thought to control key aspects of immunological tolerance to self-antigens. They are broadly identified as a small proportion of CD4+ T cells that highly express CD25 (IL-2Rα-chain) on their surface [15,16]. It has also been shown that Tregs specifically express Foxp3 (forkhead box P3) [17]. CD4+ CD25+ Tregs act in a regulatory capacity by suppressing the activation and function of other immunocytes, they can control immune responses induced by DCs in vivo [18], also prevent CD8+ T cell maturation by inhibiting CD4+ Th cells at tumor sites [19]. Tregs are present in high frequencies among tumor-infiltrating lymphocytes supposedly facilitating tumor development [20]. Thus, Tregs accumulate in the tumor microenvironment and inhibit antitumor immunity, presenting a major obstacle for developing effective and therapeutic cancer vaccines. This notion could explain anti-CD25 monoclonal antibodies (mAbs) treatment inducing tumor rejection in animal models [21,22]. A potential problem associated with the use of CD25-specific antibodies is the simultaneous depletion of conventional CD25+ effector T cells, whose loss may compromise the beneficial effect of depleting the Tregs [23]. Previous studies have indicated that treatment of mice with anti-CD25 mAbs is only beneficial within a limited time window, in the later time points anti-CD25 mAbs will not only deplete Tregs, but also affect the effector cells that are involved in tumor rejection [24–26]. It is undeniable that the beneficial effect of Tregs depletion in tumor regression is abrogated when CD4+ helper cells are also depleted. Therefore, a combination of anti-CD25 and vaccination may be necessary and provide an improved immunotherapeutic approach for tumors. In this study, we performed a combination treatment of CCL21 and anti-CD25 mAbs (PC61) in a mouse HCC model. This approach attempts to attract mature host DCs and activated T cells at the tumor site, meanwhile, the suppressive effects of Tregs can be reduced. Our results suggested that CCL21-mediated antitumor immunity was strengthened when combined with anti-CD25 mAbs administration, characterized by increasing the frequency of tumor-specific CD8+ T cells and CD11c+ DCs, and enhancing the production of IL-12 and IFN-γ within the tumor, leading to improved antitumor efficacy. Materials and Methods Animals C57BL/6J (H-2b) female mice, 6 to 8 weeks of age, were purchased from the Chinese Academy of Science and housed at the Animal Maintenance Facility of Tongji University. The protocol was approved by the Animal Ethics Committee of Tongji University. All animal experiments were performed under specific pathogen-free conditions in accordance with institutional guidelines. Tumor cell line The murine Hepal-6 hepatocellular carcinoma cell line (CRL-1830) was from American Type Culture Collection (ATCC). Hepa1-6 cell line was transfected with pRRL-CMV lentiviral-luciferase vector to creat a stable luciferase expression clone selected by limited dilution. Cells (Luc-Hepa1-6) were propagated in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (GIBCO-BRL), 0.1 mM nonessential amino acids, 1 µM sodium pyruvate, 2 mM fresh L-glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin, 50 µg/ml gentamicin, and 0.5 µg/ml fungizone, and maintained at 37°C in humidified atmosphere containing 5% CO2 in air. Treatment of established tumors A total of 3×106 Luc-Hepal-6 cells diluted in 200 µl of serum-free RPMI 1640 medium were injected subcutaneously into the right flank of C57BL/6J mice to inoculate tumors. On day 8 after inoculation, tumor-bearing mice were randomly divided into different treatment groups as follows (n = 8 for each group): (1) control group, intraperitoneal (ip) injection with 0.5 mg normal rat IgG1 in 200 µl PBS; (2) CCL21 treatment group, subcutaneous (sc) injection with 0.5 µg recombinant murine CCL21 (PeproTech, Rocky Hill, NJ) in 50 µl PBS in the right flank based on the previous study [6]; (3) anti-CD25 treatment group, ip injection with 0.5 mg purified anti-CD25 mAbs (PC61) in 200 µl PBS as previously described [22,27]; (4) combination treatment group, sc injection with 0.5 µg CCL21 in 50 µl PBS and ip injection with 0.5 mg anti-CD25 mAbs in 200 µl PBS. Anti-CD25 mAbs for in vivo administration and rat IgG1 control antibodies were obtained from Accurate Chemical and Scientific Corporation (Westbury, NY). CCL21 injections were administered one time every other day for five times. The administration of anti-CD25 mAbs or control antibodies was performed by a single injection on day 8 after inoculation. Tumor sizes were monitored every other day for 9 days with Vernier calipers after the start of treatment. Tumor volume was calculated by the formula: V (in mm3) = 0.5(ab 2), where a is the long diameter and b is the short diameter. The mice were subjected to imaging on day 7 after the start of treatment. On day 9 after treatment initiation, mice in all groups were sacrificed. Tumors were removed and weights were determined. Survival curve analysis and the following experiments were respectively done in the independent treatment groups. Bioluminescence imaging in vivo The bioluminescence imaging was performed using an animal imaging system (NightOWL LB 983 Molecular Imaging System, Berthold, Germany). For in vivo imaging, the mice received an ip injection of 150 mg/kg D-luciferin potassium salt (Caliper Life Sciences, USA) in 200 µl DPBS. After 5 minutes of luciferin injection, the mice were anesthetized via ip injection of pentobarbital (50 mg/kg). Each mouse was placed in a left lateral decubitus position and a digital grayscale animal image was acquired, followed by the acquisition and overlay of a pseudocolor image representing the spatial distribution of detected photons emerging from active luciferase within the animal. Photons emitted from specific regions were quantified using a IndiGo software (Berthold). Regions of interest (ROI) were drawn around the tumor sites and quantified as photon counts per second. Flow cytometry The tumors were mechanically dissociated in cold filter-sterilized PBS-3% BSA and gently mashed through a 70-µm-pore-size nylon mesh to produce a single cell suspension. Samples were stained with Alexa 488-conjugated anti-mouse CD25 (eBioscience, USA). Subsequently, the cells were fixed and permeabilized followed by PE-conjugated anti-mouse Foxp3 (eBioscience). Isotype controls for each antibody were also included. For analysis of effector cells infiltration, samples from day 7 after treatment initiation were stained with FITC-conjugated antibodies to mouse CD4, CD8 and PE-conjugated anti-mouse CD11c (eBioscience). All the samples were acquired on a FACSCalibur (Becton Dickinson, USA) counting of 20,000 lymphocyte-sized events and analyzed with WinMDI 2.9 software. Total cell populations for CD4+, CD8+, CD25+ Foxp3+ T cells and CD11c+ DCs were calculated by multiplying the percentage of occurrence in a dot plot of a cell population by the total number of cells counted. Immunohistochemistry Tumor tissues were fixed in periodate-lysine-paraformaldehyde (PLP), embedded in O.C.T (Sakura Finetek, USA), and cut into 10 µm sections. Then, the sections were stained with specific antibodies for analysis. Tregs within the progressive tumor were double-stained with FITC-CD4 and Cy3-Foxp3 antibody (eBioscience). Nuclei were counterstained with DAPI (Sigma, USA). Isotype-matched antibodies were used as a control. Microvessel density and CD8+ T cells in tumor tissues were detected with rat anti-mouse CD31 antibody (BD Pharmingen, USA) and rat anti-mouse CD8 (eBioscience), followed by HRP-conjugated rabbit anti-rat IgG (Invitrogen, USA) and DAB liquid substrate system (Sigma), respectively. According to the method of Weidner et al [28], the quantification of microvessel density (MVD) was assessed. Cell proliferation was performed on the frozen tumor sections with rat anti-mouse Ki-67 antibody (Biolegend, USA) followed by FITC-conjugated rabbit anti-rat IgG (Invitrogen). Results were expressed as the percentage of Ki-67 positive cells ± SEM per ×400 magnification. A total of ten ×400 fields were examined and counted from three tumors in each of the treatment groups. The Ki-67 proliferation index was calculated according to the following formula: the number of Ki-67 positive cells/total cell count ×100%. Western blot analysis Tumor tissues were homogenized with lysis buffer (RIPA) (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% sodium Deoxycholate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin) on ice. After centrifugation at 14,000 rpm at 4°C for 30 min, supernatants were collected and total protein concentrations were determined by BCA assay (Pierce, USA). Equal amounts of denatured proteins were loaded onto 10% SDS-PAGE gel and transferred on PVDF membrane (Millipore, USA). Membranes were blocked with 5% nonfat milk in TBST (1×TBS containing 0.1% Tween 20), and then incubated with primary antibodies to CCR7 and Foxp3 (eBioscience) overnight, After washing with TBST three times, HRP-conjugated secondary antibodies (KPL, USA) were bound and performed with chemiluminescence using SuperSignal West Pico substrate (Pierce). Band intensities were quantified using Band Leader software. Cytokine ELISA Levels of intratumoral cytokines (IL-12, IFN-γ, IL-10, and TGF-β1) were determined using commercially available ELISA kits (eBioscience). Frozen tumor tissue samples from day 7 after treatment initiation were accurately weighed and placed in cold RIPA buffer at a ratio of 100 mg tissue per milliliter. Samples were homogenized and subjected to one round of freeze-thaw, sonicated for 10 min, and incubated at 4 °C for one hour. The final homogenates were centrifuged at 14,000 rpm at 4°C for 30 min. Tissue supernatants were used for cytokine determination following the manufacturer’s instruction and the data were expressed at pg/mg tumor tissue. Statistical analysis Statistical significance was determined using one-way ANOVA or Student’s t test, as appropriate. P < 0.05 was considered statistically significant, and * P < 0.01 would be highly statistically significant. Results Accumulation of Tregs and different expression patterns of CCR7 and Foxp3 within the progressive tumor To explain the importance of Tregs depletion, Tregs in the tumor site were double-stained with CD4 and Foxp3 antibodies. The expression patterns of CCR7 and Foxp3 within the progressive tumor were identified by western blot. The results showed that CD4+ Foxp3+ Tregs gradually accumulated in the tumor tissue of early-stage HCC on day 8 and 14 after Hepa1-6 sc inoculation (Figure 1A), CCR7 downregulation and Foxp3 upregulation in the development of HCC from day 8 to 29 were also verified (Figure 1B). 10.1371/journal.pone.0073952.g001Figure 1 Accumulation of Tregs and different expression patterns of CCR7 and Foxp3 within the progressive tumors. (A) CD4+ Foxp3+ Tregs were increased in the tumor tissues of early-stage HCC on day 8 and 14 after Hepa1-6 subcutaneous inoculation. Serial 5-µm-thick cryostat sections were double-stained with CD4 (green) and Foxp3 (red) antibodies. Scale bar, 50 µm. (B) CCR7 downregulation and Foxp3 upregulation in the development of HCC from day 8 to 29 were verified by western blot. β-actin was used as an internal control. Protein levels were determined by densitometry analysis and were expressed as ratios to β-actin (below each blot). The ratio obtained from the first lane was set as 1. All data were representative of at least two independent experiments. Maximal inhibition of tumor growth by combination therapy of CCL21 and anti-CD25 mAbs The effects of combination treatment on the growth of Hepa1-6 tumors were evaluated in tumor-bearing mice models. Schedule of experimental procedures was showed in Figure 2A. Although CCL21 and anti-CD25 alone also significantly inhibited tumor growth after treatment, the combination therapy of CCL21 and anti-CD25 mAbs resulted in a more robust inhibition of tumor development, and had the most significant delay in tumor growth as determined by tumor volume after treatment initiation (Figure 2D) and tumor weight on day 9 when treatment was stopped (Figure 2E). Bioluminescence imaging analysis of tumor on day 7 after the start of treatment confirmed the antitumor effects (Figure 2B and C). Similarly, the increased tumor growth inhibition in the combination therapy was translated into the prolonged survival according to the Kaplan-Meier analysis and the mice received the combination therapy remained 100% survival at the end of observation period (Figure 2F). 10.1371/journal.pone.0073952.g002Figure 2 Schedule of experimental procedures and antitumor effects of combination therapy with CCL21 and anti-CD25 mAbs in HCC model. (A) Schematic representation of experiment protocol described in materials and methods. Animals were divided into four groups (n = 8 for each group). (B) Representative bioluminescence images of Luc-Hepa1-6 tumors on day 7 after the start of treatment. (C) Imaging analysis (photons per second) depicting the tumor volumes of mice using the IndiGo imaging analysis software (n = 5 for each group). (D) CCL21 combined with anti-CD25 mAbs treatment resulted in maximally inhibition of tumor growth. Tumor sizes were monitored on day 1 to 9 after treatment initiation (n = 8 for each group). (E) The combination treatment significantly reduced tumor weight. Tumor weights were measured on day 9 after treatment initiation when harvested (n = 8 for each group). (F) Survival curves were constructed in the independent treatment groups according to the Kaplan-Meier method (n = 8 for each group). P < 0.05, P < 0.01, compared with the control group. Depletion of Tregs by anti-CD25 mAbs in CCL21-treated mice To determine whether ip injection of anti-CD25 mAbs (PC61) led to a selective loss of Tregs in CCL21-treated mice, the population of CD25+ Foxp3+ Tregs within the tumors was assessed by flow cytometry respectively on day 1 to 9 after treatment initiation. Representative percentages of CD25+ Foxp3+ Tregs were shown in density plots (Figure 3A). The results from all test and control mice were summarized and provided in a curve diagram (Figure 3B). On day 1 to 7 after treatment initiation, intratumoral CD25+ Foxp3+ Tregs percentages remained essentially constant at a significantly lower level (P < 0.01) in CCL21 combined with anti-CD25 treated mice, as well as anti-CD25 treatment group, whereas even 6-fold lower mumbers of cells compared with control group. But there was a linear increase (range 1% to 4.8%) in CCL21 treatment group. Notably, we used two different Alexa 488-anti-mouse CD25 mAbs (PC61 or 7D4) to get analogous detection results for Tregs depletion confirmation in mouse spleen after a single ip injection of 0.5 mg anti-CD25 mAbs (data not shown). 10.1371/journal.pone.0073952.g003Figure 3 Depletion of Tregs by anti-CD25 mAbs in CCL21-treated mice. Respectively on day 1, 3, 5, 7, 9 after treatment initiation in the independent treatment groups, mice were sacrificed and tumors were harvested for quantification of CD25+ Foxp3+ Tregs. (A) Flow cytometry analysis for the population of CD25+ Foxp3+ Tregs within the tumors on day 1 to 9 after treatment initiation. Representative percentages of CD25+ Foxp3+ Tregs were shown in density plots. (B) A curve diagram was summarized for the results from all test and control mice (n = 3 for each group). P < 0.05, *P < 0.01, compared with the control group. Similar results were obtained in three independent experiments. Dynamic changes of CCR7 and Foxp3 expression after combination treatment The chemotaxis effect of CCL21 can be measured by the level of intratumoral CCR7 expression, and depletion of Tregs can be defined by Foxp3 expression. The results demonstrated that coadministration of CCL21 with anti-CD25 mAbs resulted in dynamic changes of CCR7 and Foxp3 expression, upregulation of CCR7 and inhibition of Foxp3 expression on day 1 to 7 after treatment initiation (Figure 4). 10.1371/journal.pone.0073952.g004Figure 4 Dynamic changes of CCR7 and Foxp3 expression after combination treatment. CCR7 and Foxp3 expressions in tumor tissue lysates on day 1 to 9 after treatment initiation were determined by western blot. β-actin was used as a an internal control. Protein levels were determined by densitometry analysis and were expressed as ratios to β-actin (below each blot). The ratio obtained from the first lane was set as 1. All data were representative of at least two independent experiments. Inhibition of tumor angiogenesis and cell proliferation To estimate angiogenesis within the tumor tissue, microvessel counts were determined by immunohistochemical staining for CD31. The combination therapy resulted in a more obvious inhibition of the angiogenesis in tumors compared with the control and monotherapy groups. The average number of microvessels per high-power field from the section on day 7 after treatment initiation was highly statistically significant in the combination group (P < 0.01, Figure 5A and B). Immunohistochemical analysis of cell proliferation was performed on the frozen tumor sections on day 7 after treatment initiation with Ki-67 antibody. The Ki-67 proliferation index was calculated and showed a significant suppression in CCL21 treatment groups, which was 6-fold decrease in the combination group compared with the control group (P < 0.01, Figure 5A and C). 10.1371/journal.pone.0073952.g005Figure 5 Inhibition of angiogenesis and proliferation within the tumors after combination treatment. (A) Representative images of CD31 positive microvessels and Ki-67 positive cells in tumor tissues on day 7 after treatment initiation estimated by immunohistochemical staining. Scale bar, 100 and 50 µm. (B) and (C) Microvessel density and percentage of Ki-67 positive cells were determined by counting the number of the positive staining per high-power field in the section, as described in “Materials and Methods”. P < 0.05, *P < 0.01, compared with the control group. Enhancement of the frequency of CD4+, CD8+ T cells and CD11c+ DCs at the tumor site after combination treatment To quantify the tumor-infiltrating CD4+, CD8+ T cells and CD11c+ DCs, the tumor samples on day 7 after treatment initiation, a representative time-point for Tregs depletion and tumor growth inhibition, were analyzed by flow cytometry. The results indicated that sc injection of CCL21 could increase the infiltration of CD4+, CD8+ and CD11c+ effector cells at the tumor site. Compared with the control group, there were significant increases in the frequency of CD4+ T cells in CCL21 treatment group or combination treatment group. Despite a small proportion loss of CD4+ T cells by anti-CD25 mAbs in the combination treatment group, there were significantly more infiltrating CD8+ T cells and CD11c+ DCs into the tumor (P < 0.01, Figure 6B), especially marked induction of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) at the local tumor site (Figure 6A). 10.1371/journal.pone.0073952.g006Figure 6 The increased frequency of CD4+, CD8+ T cells and CD11c+ DCs at the tumor site after combination treatment. The tumor samples on day 7 after treatment initiation were assayed (n = 3 for each group). (A) Immunohistochemical analysis showed that marked infiltration of tumor-specific CD8+ CTLs into tumor tissues from combination-treated mice. a: control group; b: CCL21 treated group; c: anti-CD25 treated group; d: CCL21 combined with anti-CD25 treated group. Scale bar, 100 µm. (B) Flow cytometry analysis revealed that the combination treatment enhanced the frequency of effector cells at the tumor site, significantly more tumor-infiltrating CD4+, CD8+ T cells and CD11c+ DCs. P < 0.05, *P < 0.01, compared with the control group. Similar results were obtained in three independent experiments. Evaluation of intratumoral cytokines production To further evaluate the antitumor immune responses, intratumoral cytokines secretion after different treatments was detected by ELISA. Compared with the control and monotherapy groups, the combination treatment promoted significantly enhanced elaboration of IL-12 and IFN-γ, whereas revealed significantly reduced release of immunosuppressive mediators IL-10 and TGF-β1 (P < 0.01, Figure 7A–D). 10.1371/journal.pone.0073952.g007Figure 7 Evaluation of intratumoral cytokines production after combination treatment. ELISA analysis for the intratumoral cytokine secretion profiles on day 7 after treatment initiation (n = 3 for each group). The combination treatment significantly enhanced the levels of IL-12 (A) and IFN-γ (B), but significantly reduced the levels of IL-10 (C) and TGF-β1 (D). P < 0.05, **P < 0.01, compared with the control group. Similar results were obtained in three independent experiments. Discussion The generation of an antitumor immune response is a complex process dependent on coordinate interaction of different subsets of effector cells, including both DCs and lymphocyte effectors. However, most tumors are not efficiently rejected despite their recognition by CD4+ and CD8+ T cells [29]. Tumor cells interfere with host DCs maturation, function and infiltration into the tumor [30,31]. Therefore, an implicit goal of immunotherapeutic approach designed to elicit an immune response against malignant cells is the migration of effector cells to the tumor site. It is believed that the most effective immunotherapies against solid tumors will be those that result in a large, sustained tumor infiltration by tumor-specific effector cells. CCL21 can attract DCs and naive T cells to evoke effective antitumor immunity by binding to CCR7, which makes it a good therapeutic candidate against cancer. We have evaluated the antitumor responses in mice cancer models after administration of DCs genetically modified to express CCL21 [8,9]. More recent studies revealed a potential major role of Tregs in controlling the immune responses against tumors. It has been suggested that the presence of Tregs can explain the poor clinical efficacy of immunotherapeutic protocols in human tumors [32,33]. Although the precise mechanisms of suppression by Tregs remain to be determined, these cells can inhibit immune cell functions either directly through cell-cell contact or indirectly through the secretion of immunosuppressive mediators, such as IL-10 and TGF-β1 [18]. Hence, it is possible that by removing tumor-specific Tregs, antitumor immunity could be enhanced. Many studies in mice have shown that removal or inhibition of this subset of cells can enhance antitumor immune responses [34–37]. Accordingly, new immunotherapeutic strategies for tumors have increasely aimed at inhibition or depletion of Tregs. Depletion of Tregs by anti-CD25 mAbs could represent an important adjunct to cancer immunotherapy. However, solely depleting Tregs might not always result in tumor regression. Anti-CD25 mAbs will not only deplete Tregs, but also affect the effector cells expressing early activation marker CD25. Therefore, approaches combining Tregs depletion with other immunologic interventions might be more beneficial. The recruitment or expansion of Tregs is closely correlated with early tumor growth [38]. Our findings also indicated that CD4+ CD25+ Foxp3+ Tregs gradually accumulated in the tumor tissue of mouse early-stage HCC. The presence of Tregs at the tumor sites emphasized their potential role to down-regulate the functions of effector T cell subsets. In this study, we investigated whether elimination of CD4+ CD25+ Tregs using anti-CD25 mAbs (PC61) was capable of enhancing CCL21-mediated antitumor immunity in a mouse HCC model. We showed that ip injection of anti-CD25 mAbs was capable of significantly eliminating CD25+ Foxp3+ Tregs within the tumor. Although anti-CD25 mAbs in the combination treatment group resulted in a proportion loss of activated CD25+ T cells, CCL21-mediated recruitment of T cells repaired and enhanced the effects of activated T cells. One resolution may be that the levels of CD25 appear higher and more stable on Tregs than activated effector cells. In addition, it is well documented that Tregs express higher levels of CD25 than activated T cells [39]. Nevertheless, the combination treatment with CCL21 and anti-CD25 mAbs maximally reduced CD25+ Foxp3+ Tregs from established tumors. The results demonstrated that coadministration of CCL21 with anti-CD25 mAbs resulted in dynamic changes of CCR7 and Foxp3 expression, upregulation of CCR7 and inhibition of Foxp3 expression on day 1 to 7 after treatment initiation. The expression levels of two proteins were usually determined by the increased numbers of mature DCs and naive T cells and reduced number of Tregs within the tumor. In contrast to Tregs with a low CCR7 expression, mature DCs and naive T cells could be easily attracted to the tumor site by CCL21 due to their high CCR7 expression [14]. Tumor-associated DCs generally show an immature phenotype with low CCR7, which tends to induce immune tolerance and Tregs [20]. As a control, Foxp3 was markedly upregulated within the growing tumors, but CCR7 expression was inhibited at a low level. The results indicated that CCR7 expression inhibition might be associated with tumor poor immune status compared with Foxp3 expression increase in tumor progression. It should be noted here that CCR7 was not expressed on Hepal-6 cells (data not shown). Therefore, CCR7 could probably be acted as a prognosis for immunotherapies against tumors in our model. A variety of reports have indicated that, apart from chemotaxis, CCR7 controls the cytoarchitecture, the rate of endocytosis and the migratory speed of the DCs [40], also protects of circulating CD8+ T cells and DCs from apoptosis [41,42]. We have found that CCL21 contributes to the maturation of bone marrow-derived DCs (BMDCs), and strengthens their APC functions and induces secretion of IL-12 and IFN-γ [8]. In the present study, these findings supported and extended our previous studies demonstrating that the maturation of BMDCs stimulated by CCL21. Our current work is focused on the mechanism of CCL21 stimulating BMDCs maturation, the preliminary results suggest NF-κB signal may be involved in the process [43]. The functional axis encompassing CCL21/CCR7 makes up a key component in the initiation of the adaptive immune response. The interaction of DCs with CCL21 is essential for the function of these cells in tumor-bearing mice and may provide tools for novel therapeutic strategies. We found that CCL21 in combination with anti-CD25 mAbs resulted in improved antitumor efficacy and prolonged survival, as well as blockade of tumor angiogenesis and cell proliferation. Apart from CCR7, mouse CCL21 also interacts with another receptor CXCR3, through which it can block angiogenesis in vivo [44]. Such bioactivity of CCL21 is conducive to induce antitumor immune responses. We believe that the inhibition of angiogenesis plays an important role in enhancing the antitumor effects of the combination treatment group. Further studies revealed that the combination treatment increased the frequency of tumor-specific CD4+, CD8+ T cells and CD11c+ DCs at the tumor site. Despite a small proportion loss of CD4+ T cells by anti-CD25 mAbs in the combination treatment group, there were significantly more tumor-infiltrating CD8+ CTLs and CD11c+ DCs. These data suggested that functional capacity of tumor-infiltrating effector cells was markedly enhanced after Tregs depletion in CCL21-treated mice, infiltration of activated CD4+ T cells and DCs in the Tregs-deprived tumor microenvironment might promote CD8+ T cells function, leading to effective T-cell priming and the generation of powerful antitumor immune responses. After combination treatment, the tumor site cellular infiltrates were accompanied by the enhanced elaboration of IL-12 and IFN-γ, but reduced release of the immunosuppressive mediators IL-10 and TGF-β1. IL-12 and IFN-γ mediate a range of biological effects that facilitate antitumor immunity [45], whereas IL-10 and TGF-β have been implicated in the induction or conversion of Tregs [46]. These results showed that the combination treatment was more effective in generating systemic antitumor responses. It can be seen that CCL21-mediated antitumor response could be further boosted by depletion of Tregs. The precise mechanism involved in this process remains to be fully elucidated. We believe that CCL21 administration directionally attracts more mature DCs and naive T cells to the tumor site, ip injection of anti-CD25 mAbs both targets the suppressive activity of Tregs and reduces any risks of developing a systemic neutralizing antibody response [47]. In conclusion, our results demonstrated that in the Tregs-deprived tumor microenvironment, CCL21-mediated antitumor immunity was strongly improved, leading to a maximal therapeutic efficacy. The approach used here could also potentially be employed for an effective immunotherapeutic strategy against established tumors. ==== Refs References 1 Sallusto F , Mackay CR , Lanzavecchia A (2000 ) The role of chemokine receptors in primary, effector, and memory immune responses . Annu Rev Immunol 18 : 593 –620 . doi:10.1146/annurev.immunol.18.1.593. PubMed: 10837070.10837070 2 Franciszkiewicz K , Boissonnas A , Boutet M , Combadière C , Mami-Chouaib F (2012 ) Role of chemokines and chemokine receptors in shaping the effector phase of the antitumor immune response . Cancer Res 72 : 6325 –6332 . doi:10.1158/0008-5472.CAN-12-2027. PubMed: 23222302.23222302 3 Saeki H , Moore AM , Brown MJ , Hwang ST (1999 ) Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes . J Immunol 162 : 2472 –2475 . PubMed: 10072485.10072485 4 Yoshida R , Nagira M , Kitaura M , Imagawa N , Imai T et al. (1998 ) Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemokine receptor CCR7 . J Biol Chem 273 : 7118 –7122 . doi:10.1074/jbc.273.12.7118. PubMed: 9507024.9507024 5 Gunn MD , Tangemann K , Tam C , Cyster JG , Rosen SD et al. (1998 ) A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes . Proc Natl Acad Sci U S A 95 : 258 –263 . doi:10.1073/pnas.95.1.258. PubMed: 9419363.9419363 6 Sharma S , Stolina M , Luo J , Strieter RM , Burdick M et al. (2000 ) Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo . J Immunol 164 : 4558 –4563 . PubMed: 10779757.10779757 7 Sharma S , Stolina M , Zhu L , Lin Y , Batra R et al. (2001 ) Secondary lymphoid organ chemokine reduces pulmonary tumor burden in spontaneous murine bronchoalveolar cell carcinoma . Cancer Res 61 : 6406 –6412 . PubMed: 11522634.11522634 8 Liang CM , Ye SL , Zhong CP , Zheng N , Bian W et al. (2007 ) More than chemotaxis: A new anti-tumor DC vaccine modified by rAAV2-SLC . Mol Immunol 44 : 3797 –3804 . doi:10.1016/j.molimm.2007.03.026. PubMed: 17521735.17521735 9 Liang CM , Zhong CP , Sun RX , Liu BB , Huang C et al. (2007 ) Local expression of secondary lymphoid tissue chemokine delivered by adeno-associated virus within the tumor bed stimulates strong anti-liver tumor Immunity . J Virol 81 : 9502 –9511 . doi:10.1128/JVI.00208-07. PubMed: 17567706.17567706 10 Nguyen-Hoai T , Baldenhofer G , Ahmed Sayed, Pham-Duc M, Vu MD, Lipp M et al. (2012 ) CCL21 (SLC) improves tumor protection by a DNA vaccine in a Her2/neu mouse tumor model . Cancer Gene Ther 19 : 69 –76 . doi:10.1038/cgt.2011.69. PubMed: 21997231.21997231 11 Kar UK , Srivastava MK , Andersson A , Baratelli F , Huang M , Kickhoefer VA et al. (2011 ) Novel CCL21-vault nanocapsule intratumoral delivery inhibits lung cancer growth . PLOS ONE 6 : e18758 . doi:10.1371/journal.pone.0018758. PubMed: 21559281.21559281 12 Schneider MA , Meingassner JG , Lipp M , Moore HD , Rot A (2007 ) CCR7 is required for the in vivo function of CD4+ CD25+ regulatory T cells . J Exp Med 204 : 735 –745 . doi:10.1084/jem.20061405. PubMed: 17371928.17371928 13 Förster R , Davalos-Misslitz AC , Rot A (2008 ) CCR7 and its ligands: balancing immunity and tolerance . Nat Rev Immunol 8 : 362 –371 . doi:10.1038/nri2297. PubMed: 18379575.18379575 14 Menning A , Höpken UE , Siegmund K , Lipp M , Hamann A et al. (2007 ) Distinctive role of CCR7 in migration and functional activity of naive- and effector/memory-like Treg subsets . Eur J Immunol 37 : 1575 –1583 . doi:10.1002/eji.200737201. PubMed: 17474155.17474155 15 Sakaguchi S , Sakaguchi N , Asano M , Itoh M , Toda M (1995 ) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases . Immunol 155 : 1151 –1164 . 16 O’Garra A , Vieira P (2004 ) Regulatory T cells and mechanisms of immune system control . Nat Med 10 : 801 –805 . doi:10.1038/nm0804-801. PubMed: 15286781.15286781 17 Hori S , Nomura T , Sakaguchi S (2003 ) Control of regulatory T cell development by the transcription factor Foxp3 . Science 299 : 1057 –1061 . doi:10.1126/science.1079490. PubMed: 12522256.12522256 18 Nizar S , Meyer B , Galustian C , Kumar D , Dalgleish A (2010 ) T regulatory cells, the evolution of targeted immunotherapy . Biochim Biophys Acta 1806 : 7 –17 . PubMed: 20188145.20188145 19 Chaput N , Guillaume DJ , Bergot AS , Cordier C , Stacie NA et al. (2007 ) Regulatory T cells prevent CD8 T cell maturation by inhibiting CD4 Th cells at tumor sites . J Immunol 179 : 4969 –4978 . PubMed: 17911581.17911581 20 Wang HY , Wang RF (2007 ) Regulatory T cells and cancer . Curr Opin Immunol 19 : 217 –223 . doi:10.1016/j.coi.2007.02.004. PubMed: 17306521.17306521 21 Shimizu J , Yamazaki S , Sakaguchi S (1999 ) Induction of tumor immunity by removing CD4+ CD25+ T cells: a common basis between tumor immunity and autoimmunity . J Immunol 163 : 5211 –5218 . PubMed: 10553041.10553041 22 Onizuka S , Tawara I , Shimizu J , Sakaguchi S , Fujita T et al. (1999 ) Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody . Cancer Res 59 : 3128 –3133 . PubMed: 10397255.10397255 23 Betts GJ , Clarke SL , Richards HE , Godkin AJ , Gallimore AM (2006 ) Regulating the immune response to tumours . Adv Drug Deliv Rev 58 : 948 –961 . doi:10.1016/j.addr.2006.05.006. PubMed: 17070961.17070961 24 Teng MW , Swann JB , von Scheidt B , Sharkey J , Zerafa N et al. (2010 ) Multiple antitumor mechanisms downstream of prophylactic regulatory T-cell depletion . Cancer Res 70 : 2665 –2674 . doi:10.1158/0008-5472.CAN-09-1574. PubMed: 20332236.20332236 25 Zelenay S , Lopes-Carvalho T , Caramalho I , Moraes-Fontes MF , Rebelo M et al. (2005 ) Foxp3+ CD25- CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion . Proc Natl Acad Sci U S A 102 : 4091 –4096 . doi:10.1073/pnas.0408679102. PubMed: 15753306.15753306 26 Oliver MG , Stefan N , Erik B , Toonen LWJ , Louis B et al. (2007 ) CD4+ FoxP3+ regulatory T cells gradually accumulate in gliomas during tumor growth and efficiently suppress antiglioma immune responses in vivo . Int J Cancer 121 : 95 –105 . doi:10.1002/ijc.22607. PubMed: 17315190.17315190 27 Wei WZ , Jacob JB , Zielinski JF , Flynn JC , Shim KD et al. (2005 ) Concurrent induction of antitumor immunity and autoimmune thyroiditis in CD4+ CD25+ regulatory T cell-depleted mice . Cancer Res 65 : 8471 –8478 . doi:10.1158/0008-5472.CAN-05-0934. PubMed: 16166327.16166327 28 Weidner N , Semple JP , Welch WR , Folkman J (1991 ) Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma . N Engl J Med 324 : 1 –8 . doi:10.1056/NEJM199101033240101. 29 Flavell RA , Sanjabi S , Wrzesinski SH , Licona-Limón P (2010 ) The polarization of immune cells in the tumour environment by TGFbeta . Nat Rev Immunol 10 : 554 –567 . doi:10.1038/nri2808. PubMed: 20616810.20616810 30 Kerkar SP , Restifo NP (2012 ) Cellular constituents of immune escape within the tumor microenvironment . Cancer Res 72 : 3125 –3130 . doi:10.1158/0008-5472.CAN-11-4094. PubMed: 22721837.22721837 31 Palucka K , Banchereau J (2012 ) Cancer immunotherapy via dendritic cells . Nat Rev Cancer 12 : 265 –277 . doi:10.1038/nrc3258. PubMed: 22437871.22437871 32 deLeeuw RJ , Kost SE , Kakal JA , Nelson BH (2012 ) The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature . Clin Cancer Res 18 : 3022 –3029 . doi:10.1158/1078-0432.CCR-11-3216. PubMed: 22510350.22510350 33 Predina J , Eruslanov E , Judy B , Kapoor V , Cheng G et al. (2013 ) Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery . Proc Natl Acad Sci U S A 110 : E415 –E424 . doi:10.1073/pnas.1211850110. PubMed: 23271806.23271806 34 Byrne WL , Mills KH , Lederer JA , O’Sullivan GC (2011 ) Targeting regulatory T cells in cancer . Cancer Res 71 : 6915 –6920 . doi:10.1158/0008-5472.CAN-11-1156. PubMed: 22068034.22068034 35 Baba J , Watanabe S , Saida Y , Tanaka T , Miyabayashi T , Koshio J et al. (2012 ) Depletion of radio-resistant regulatory T cells enhances antitumor immunity during recovery from lymphopenia . Blood 120 : 2417 –2427 . doi:10.1182/blood-2012-02-411124. PubMed: 22806892.22806892 36 Pedroza-Gonzalez A , Verhoef C , Ijzermans JN , Peppelenbosch MP , Kwekkeboom J et al. (2013 ) Activated tumor-infiltrating CD4+ regulatory T cells restrain antitumor immunity in patients with primary or metastatic liver cancer . Hepatology 57 : 183 –194 . doi:10.1002/hep.26013. PubMed: 22911397.22911397 37 Girardin A , McCall J , Black MA , Edwards F , Phillips V , Taylor ES et al. (2013 ) Inflammatory and regulatory T cells contribute to a unique immune microenvironment in tumor tissue of colorectal cancer patients . Int J Cancer 132 : 1842 –1850 . doi:10.1002/ijc.27855. PubMed: 23002055.23002055 38 Needham DJ , Lee JX , Beilharz MW (2006 ) Intra-tumoural regulatory T cells: A potential new target in cancer immunotherapy . Biochem Biophys Res Commun 343 : 684 –691 . doi:10.1016/j.bbrc.2006.03.018. PubMed: 16563349.16563349 39 Baecher-Allan C , Brown JA , Freeman GJ , Hafler DA (2001 ) CD4+ CD25high regulatory cells in human peripheral blood . J Immunol 167 : 1245 –1253 . PubMed: 11466340.11466340 40 Sánchez-Sánchez N , Riol-Blanco L , Rodríguez-Fernández JL (2006 ) The multiple personalities of the Chemokine Receptor CCR7 in dendritic cells . J Immunol 176 : 5153 –5159 . PubMed: 16621978.16621978 41 Sánchez-Sánchez N , Riol-Blanco L , de la Rosa G , Puig-Kröger A , García-Bordas J et al. (2004 ) Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cells . Blood 104 : 619 –625 . doi:10.1182/blood-2003-11-3943. PubMed: 15059845.15059845 42 Kim JW , Ferris RL , Whiteside TL (2005 ) Chemokine Receptor CCR7 expression and protection of circulating CD8+ T lymphocytes from apoptosis . Clin Cancer Res 11 : 7901 –7910 . doi:10.1158/1078-0432.CCR-05-1346. PubMed: 16278415.16278415 43 Zhou S , Li R , Qin J , Zhong C , Liang C (2010 ) SLC/CCR7 stimulates the proliferation of BMDCs by the pNF-kappaB p65 pathway . Anat Rec (Hoboken) 293 : 48 –54 . doi:10.1002/ar.21015. PubMed: 19899121.19899121 44 Soto H , Wang W , Strieter RM , Copeland NG , Gilbert DJ et al. (1998 ) The CC chemokine 6Ckine binds the CXC chemokine receptor CXCR3 . Proc Natl Acad Sci U S A 95 : 8205 –8210 . doi:10.1073/pnas.95.14.8205. PubMed: 9653165.9653165 45 Del Vecchio M , Bajetta E , Canova S , Lotze MT , Wesa A et al. (2007 ) Interleukin-12: biological properties and clinical application . Clin Cancer Res 13 : 4677 –4685 . doi:10.1158/1078-0432.CCR-07-0776. PubMed: 17699845.17699845 46 Banchereau J , Pascual V , O’Garra A (2012 ) From IL-2 to IL-37: the expanding spectrum of anti-inflammatory cytokines . Nat Immunol 13 : 925 –931 . doi:10.1038/ni.2406. PubMed: 22990890.22990890 47 Ko K , Yamazaki S , Nakamura K , Nishioka T , Hirota K et al. (2005 ) Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+ CD25+ CD4+ regulatory T cells . J Exp Med 202 : 885 –891 . doi:10.1084/jem.20050940. PubMed: 16186187.16186187	24023916	PMC3759453	CC BY	2021-01-05 17:38:31	yes	PLoS One. 2013 Sep 2; 8(9):e73952
==== Front Front PhysiolFront PhysiolFront. Physiol.Frontiers in Physiology1664-042XFrontiers Media S.A. 10.3389/fphys.2013.00240PhysiologyReview ArticlePhysiology and pharmacology of the cardiovascular adrenergic system Lymperopoulos Anastasios Laboratory for the Study of Neurohormonal Control of the Circulation, Department of Pharmaceutical Sciences, Nova Southeastern University College of PharmacyFort Lauderdale, FL, USAEdited by: Giuseppe Rengo, Salvatore Maugeri Foundation, Italy Reviewed by: Maurizio Taglialatela, University of Molise, Italy; Michele Ciccarelli, Università degli Studi di Salerno, Italy Correspondence: Anastasios Lymperopoulos, Laboratory for the Study of Neurohormonal Control of the Circulation, Department of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, 3200 S. University Dr., HPD (Terry) Bldg/Room 1338, Fort Lauderdale, FL, 33328-2018, USA e-mail: al806@nova.eduThis article was submitted to Clinical and Translational Physiology, a section of the journal Frontiers in Physiology. 13 8 2013 04 9 2013 2013 4 24030 7 2013 14 8 2013 Copyright © 2013 Lymperopoulos.2013This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Heart failure (HF), the leading cause of death in the western world, ensues in response to cardiac injury or insult and represents the inability of the heart to adequately pump blood and maintain tissue perfusion. It is characterized by complex interactions of several neurohormonal mechanisms that get activated in the syndrome in order to try and sustain cardiac output in the face of decompensating function. The most prominent among these neurohormonal mechanisms is the adrenergic (or sympathetic) nervous system (ANS), whose activity and outflow are greatly elevated in HF. Acutely, provided that the heart still works properly, this activation of the ANS will promptly restore cardiac function according to the fundamental Frank-Starling law of cardiac function. However, if the cardiac insult persists over time, this law no longer applies and ANS will not be able to sustain cardiac function. This is called decompensated HF, and the hyperactive ANS will continue to “push” the heart to work at a level much higher than the cardiac muscle can handle. From that point on, ANS hyperactivity becomes a major problem in HF, conferring significant toxicity to the failing heart and markedly increasing its morbidity and mortality. The present review discusses the role of the ANS in cardiac physiology and in HF pathophysiology, the mechanisms of regulation of ANS activity and how they go awry in chronic HF, and, finally, the molecular alterations in heart physiology that occur in HF along with their pharmacological and therapeutic implications for the failing heart. adrenergic nervous systemheart failurecardiac myocyteadrenal glandcatecholamineadrenergic receptor ==== Body Introduction Heart failure (HF) is a clinical syndrome that develops in response to a cardiac injury or insult that causes decline in the pumping capacity (contractile function) of the heart. It is marked by a perpetual interplay between the underlying myocardial dysfunction and the compensatory neurohumoral mechanisms that are activated in an effort to maintain cardiac output in the face of declining heart function. Among these neurohormonal mechanisms, elevated activities of the adrenergic (or sympathetic) nervous system (ANS), of the renin-angiotensin-aldosterone system (RAAS), and of several cytokines, play central roles (Mann and Bristow, 2005; Mudd and Kass, 2008). These systems get activated in an effort to compensate for the depressed myocardial function and preserve cardiovascular homeostasis. Upon long-term presence of the initial insult to the heart muscle, however, cardiac function ultimately succumbs to their deleterious effects on cardiac structure and performance, leading to cardiac decompensation, and this progressively worsening function renders the heart unable to sustain daily life activities. The present review will discuss the role of the ANS in cardiac physiology and pathophysiology. ANS and cardiac function The ANS exerts a wide variety of cardiovascular effects, including heart rate acceleration (positive chronotropy), increase in cardiac contractility (positive inotropy), accelerated cardiac relaxation (positive lusitropy), accelerated atrioventricular conduction (positive dromotropy), decrease in venous capacitance, and constriction of resistance and cutaneous vessels (Figure 1). All of these effects aim to increase cardiac performance to prepare and enable the body for the so-called “fight or flight response.” Conversely, the mirror branch of the autonomic nervous system, the parasympathetic (cholinergic) nervous system, slows the heart rate (bradycardia) through vagal nerve impulses, with minimal or no effect on cardiac contractility. This is because the cardiac ventricles, responsible for contraction, receive almost exclusively adrenergic fiber innervations, whereas the cholinergic system fibers run with the vagus nerve subendocardially, after it crosses the atrioventricular groove, and reach mainly the atrial myocardium with minimal connections to the ventricular myocardium (Zipes, 2008; Triposkiadis et al., 2009). Therefore, whereas heart rate can be controlled (in opposing fashion) by both autonomic branches, cardiac contraction/relaxation is controlled practically solely by the ANS (Figure 1). Figure 1 Overview of the cardiovascular ANS. In contrast to the ANS which innervates both atria and ventricles of the heart, the cholinergic (parasympathetic) nervous system mainly innervates cardiac atria only. See text for more details. CSAR, cardiac sympathetic afferent reflex; H, hypothalamus; M, medulla oblongata. The ventricular ANS innervation is characterized by a gradient from base to apex (Pierpont et al., 1985). The cardiac neuronal system is composed of cell stations comprising afferent, efferent, and interconnecting neurons behaving as a control system (Armour, 2004). The ANS outflow to the heart and to the peripheral circulation is regulated by cardiovascular reflexes. Afferent fibers project to the central nervous system by the autonomic nerves, whereas efferent impulses travel from the central nervous system to peripheral organs. The main reflex responses originate from the aortic arch and the carotid baroreceptors (ANS inhibition), cardiopulmonary baroreceptors (diverse reflexes including the Bezold-Jarisch reflex, ANS inhibition), cardiovascular low-threshold polymodal receptors (ANS activation), and peripheral chemoreceptors (ANS activation) (Malliani et al., 1983; Triposkiadis et al., 2009). ANS activation in the cardiovascular system translates into release of the two catecholamines that mediate its effects, i.e., norepinephrine (NE or noradrenaline) and epinephrine (Epi or adrenaline), and this can occur via the following mechanisms (Figure 2): (a) NE released by cardiac sympathetic nerve terminals, resulting in an increase in heart rate and shortening of atrioventricular conduction, and in an increase in contractile strength, (b) Epi (and to a much lesser extent NE) released into the circulation by the adrenal medulla, affecting both the myocardium and peripheral vessels, and, finally, (c) local release of NE and Epi by various peripheral adrenergic nervous systems that can synthesize and release these catecholamines in an autocrine/paracrine manner and are located in blood vessels and in cardiac myocytes themselves (Lymperopoulos et al., 2007, 2012). Figure 2 ANS-dependent regulation of cardiac function. See text for details. Gi/o, inhibitory or other G protein; Gs, stimulatory G protein; Ach, Acetylcholine; NET, NE transporter; Aldo, Aldosterone; MR, Mineralocorticoid Receptor. Adrenergic receptors (ARs) in the cardiovascular system The ANS neurotransmitters NE and Epi mediate their effects in cells and tissues by binding to specific cell surface ARs, which belong to the superfamily of G protein-coupled receptors (GPCRs) or seven transmembrane-spanning receptors or heptahelical receptors (7TMRs). Approximately 80% of NE released by ANS nerve terminals is recycled by the NE transporter (NET) type 1, whereas the remainder spills over into the circulation (Leineweber et al., 2002). The receptors for both ANS catecholamines are divided into three types and 9 total different subtypes, as follows: three α1AR subtypes (α1A, α1B, α1D), three α2AR subtypes (α2A, α2B, α2C), and three βAR subtypes (β1, β2, β3) (Bylund et al., 1994). All ARs primarily signal through heterotrimeric G proteins. The human heart contains all three βAR subtypes (Lymperopoulos et al., 2012). β1AR is the predominant subtype in the (normal, healthy) myocardium, representing 75–80% of total βAR density, followed by β2AR, which comprises about 15–18% of total cardiomyocyte βARs and the remaining 2–3% is β3ARs (under normal conditions) (Brodde, 1993). The principal role of βARs in the heart is the regulation of cardiac rate and contractility in response to NE and Epi. Stimulation of β1ARs (mainly) and of β2ARs (to a lesser extent) increases cardiac contractility (positive inotropic effect), frequency (positive chronotropic effect), and rate of relaxation (lusitropic effect) as well as accelerates impulse conduction through the atrioventricular node (positive dromotropic effect) and pacemaker activity from the sinoatrial node (Colucci et al., 1986). β3ARs are predominantly inactive during normal physiologic conditions (Skeberdis et al., 2008); however, their stimulation seems to produce a negative inotropic effect opposite to that induced by β1ARs and β2ARs, involving the nitric oxide synthase (NOS) pathway (Gauthier et al., 1998), thus acting as a “fuse” against cardiac adrenergic overstimulation (Rozec et al., 2009). Agonist-induced activation of βARs catalyzes the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the G α subunit of heterotrimeric G proteins, resulting in the dissociation of the heterotrimer into active Gα and free Gβγ subunits (always associated together, i.e., a heterodimer that functions as a monomer) which can transduce intracellular signals independently of each other (Lohse et al., 2003). The most powerful physiologic mechanism to increase cardiac performance is activation of cardiomyocyte β1ARs and β2ARs, which, in turn, activates Gs proteins (stimulatory G proteins). Gs protein signaling stimulates the effector adenylate cyclase (AC), which converts adenosine triphosphate (ATP) to the second messenger adenosine 3′,5′-monophosphate or cyclic AMP (cAMP), which in turn binds to and activates the cAMP-dependent protein kinase (protein kinase A, PKA). PKA is the major effector of cAMP (there is also Epac, exchange protein directly activated by cAMP, which can be activated by cAMP independently of PKA and whose precise roles in the heart are currently unknown), and, by phosphorylating a variety of substrates, including plasmalemmal L-type calcium channels and the sarco/endoplasmic reticulum calcium ATPase regulator phospholamban, it ultimately results in significant raise in free intracellular Ca2+ concentration, which is the master regulator of cardiac muscle contraction Of note, β2AR also mediates the effects of catecholamines in the heart, but in a qualitatively different manner from β1AR, as it can also couple to the AC inhibitory G protein (Gi). In fact, this switching of β2AR signaling from Gs to Gi proteins is postulated to be induced by the phosphorylation of the β2AR by PKA (Daaka et al., 1997). Nonetheless, it is now generally accepted that in the heart, β2AR signals and functions in a substantially different way compared to β1AR (Communal et al., 1999; Chesley et al., 2000; Zhu et al., 2001). Importantly, whereas β1AR activation enhances cardiomyocyte apoptosis, β2AR exerts antiapoptotic effects in the heart (Communal et al., 1999; Dorn et al., 1999; Chesley et al., 2000; Zhu et al., 2001). This essential difference between the two receptor subtypes is ascribed to the signal of β2AR through Gi proteins (Chesley et al., 2000). Studies using transgenic mice, β2AR-selective stimulation and adenoviral-mediated β2AR overexpression, have demonstrated the protective effects of β2AR signaling in the myocardium, including improved cardiac function and decreased apoptosis. Conversely, hyperstimulation or overexpression of β1AR has detrimental effects in the heart (Dorn et al., 1999; Liggett et al., 2000). Both α2- and βARs, like the majority of GPCRs, are subject to agonist-promoted (homologous) desensitization and downregulation, a regulatory process that diminishes receptor response to continuous or repeated agonist stimulation (Ferguson, 2001; Reiter and Lefkowitz, 2006). At the molecular level, this process is initiated by receptor phosphorylation by a family of kinases, termed GPCR kinases (GRKs), followed by binding of βarrestins (βarrs) to the GRK-phosphorylated receptor (see below). The βarrs then uncouple the receptor from its cognate G proteins, sterically hinder its further binding to them (functional desensitization) and subsequently target the receptor for internalization (Ferguson, 2001; Reiter and Lefkowitz, 2006). Across all mammalian species, GRK2 and GRK5 are the most physiologically important members of the GRK family because they are expressed ubiquitously and regulate the vast majority of GPCRs. They are particularly abundant in neuronal tissues and in the heart (Arriza et al., 1992; Rockman et al., 2002). Of note, the differences between the two predominant cardiac βARs, i.e., β1AR & β2AR, in terms of their signaling properties, might take a quite different shape and have a much bigger bearing on pathophysiologic implications in the setting of human HF: for instance, and as discussed in more detail in subsequent sections, β1AR is selectively downregulated (i.e., functional receptor number reduced) in human HF, thus shifting the above mentioned stoichiometry of β1AR:β2AR toward 50:50 in the failing heart from ~75:~20% in the normal, healthy heart (Bristow et al., 1982, 1986). However, β2AR is also non-functional and does not signal properly in the failing heart (Bristow et al., 1982, 1986; Rockman et al., 2002). In addition, emerging evidence suggests that β2AR signaling in the failing heart is quite different from that in the normal heart, i.e., is more diffuse and non-compartmentalized and resembles more the pro-apoptotic “diffuse” cAMP signaling pattern of the β1AR (Nikolaev et al., 2010). Therefore, this stoichiometric shift in favor of the supposedly “good” β2AR in HF appears unable to help the heart improve its structure and function. The human heart also expresses α1A- and α1BARs, albeit at much lower levels than βARs (~20% of total βARs) (Woodcock et al., 2008). The importance of cardiac α1ARs in cardiac physiology is still a matter of debate. In contrast, their role in regulation of blood flow by inducing constriction in the smooth muscle wall of major arteries (e.g., aorta, pulmonary arteries, mesenteric vessels, coronary arteries, etc.) is well known and indisputable (Shannon and Chaudhry, 2006). The α1ARs couple to the Gq/11 family of heterotrimeric G proteins, thereby activating phospholipase C (PLC)-β. PLCβ generates the second messengers inositol [1,4,5]-trisphosphate (IP3) and 2-diacylglycerol (DAG) from the cell membrane component phospholipid phosphatidylinositol (Pierpont et al., 1985; Triposkiadis et al., 2009)-bisphosphate (PIP2). IP3 binds specific receptors in the SR membrane which cause release of Ca2+ from intracellular stores, whereas DAG activates protein kinase C (PKC) and transient receptor potential (TRPV) channels. The end result is again raised intracellular [Ca2+], which leads to contraction (vasoconstriction). Finally, regarding α2AR subtypes, α2BARs are known to be present in vascular smooth muscle causing constriction of certain vascular beds, while centrally located α2AARs can inhibit sympathetic outflow (presynaptic inhibitory autoreceptors) and thus lower systemic blood pressure (Philipp et al., 2002; Philipp and Hein, 2004). The release of NE from cardiac sympathetic nerve terminals is controlled by both presynaptic α2A- and α2CARs (Hein et al., 1999), and genetic deletion of both of these α2AR subtypes leads to cardiac hypertrophy and HF due to chronically enhanced cardiac NE release, as well as enhanced NE and Epi secretion from the adrenal medulla (Brede et al., 2002, 2003; Lymperopoulos et al., 2007). Regulation of ANS outflow & activity in health and in chronic HF There are several mechanisms by which the ANS controls cardiac function. The first one to be documented historically is through the aortic arch and carotid sinus (high pressure) and cardiopulmonary (low pressure) baroreceptor reflexes (Kaye and Esler, 2005). Aside from these baroreceptor inputs, additional factors that act within the central nervous system play a role in regulation of cardiac ANS activity. In particular, suprabulbar subcortical monoaminergic neurons and brainstem angiotensin II have attracted interest courtesy of their ability to regulate ANS outflow in HF (Figure 2). NE turnover in subcortical regions in HF is significantly higher than that in the cortex and than in healthy subjects (Aggarwal et al., 2002). Moreover, the rate of subcortical NE release correlates well with global ANS activity, as measured by total body NE plasma spillover. Angiotensin II-dependent ANS activation plays an important role in adverse hemodynamic and left ventricular remodeling responses to myocardial infarction, possibly through superoxide formation (Lindley et al., 2004; Wang et al., 2004). Thus, part of the benefit of RAAS modulators in HF might derive from centrally-mediated suppression of ANS activity. As the heart becomes progressively unresponsive to the stimulatory effects of catecholamines, chronic stimulation of cardiac ANS nerve terminals leads to chronically elevated NE release in the heart (increased NE spillover). Presynaptic α2ARs present on cardiac ANS nerve terminals and acting as NE release-inhibiting autoreceptors play a crucial role in regulation of cardiac NE release from sympathetic nerves (Philipp et al., 2002; Philipp and Hein, 2004). Indeed, knockout (KO) mice lacking either the α2A- or α2CAR subtype show significantly enhanced cardiac ANS activity and circulating catecholamine levels, as well as significantly worse heart function and clinical indices, during the course of surgical pressure overload (by means of transverse aortic constriction, TAC)-induced HF compared with age-matched wild-type HF mice (Hein et al., 1999; Brede et al., 2002). Moreover, double α2A/α2CAR KO mice exhibit even worse cardiac phenotypes than single α2AAR KO mice and, by 4 months of age, they spontaneously develop cardiomyopathy (without stress or any specific insult) (Brum et al., 2002). In HF patients, the expected inhibitory effects of α2AR stimulation on NE spillover are markedly blunted, thereby contributing to the increase in cardiac NE spillover observed in chronic HF (Aggarwal et al., 2001). Thus, presynaptic inhibitory α2-adrenergic autoreceptors crucially regulate ANS cardiac nerve activity and NE release into the heart and any dysfunction of these receptors either due to genetic polymorphisms or enhanced desensitization/downregulation (see below) translate into increased morbidity and mortality in chronic HF (Figure 2). Perhaps the crucial role of presynaptic α2ARs in regulating NE release from cardiac ANS nerves stems from the fact that they are the only presynaptic ARs that can inhibit NE release; presynaptic βARs (of the β2AR subtype, mainly) are facilitatory autoreceptors enhancing NE release at sympathetic nerve terminals (Docherty, 2002), a phenomenon whose inhibition may contribute to the therapeutic benefit of β-blockers in HF (see below) (Figure 2). Circulating Epi and NE derive from two major sources in the body: the cardiac sympathetic nerve endings, which release NE directly onto the cardiac muscle, and the adrenal medulla, whose chromaffin cells synthesize, store and release Epi (mainly) and NE upon acetylcholine stimulation of the nicotinic cholinergic receptors (nAChRs) present on their cell membranes (Figure 2; Lymperopoulos et al., 2007). Epi represents approximately 80% of the total adrenal catecholamine secretion under normal conditions, with NE the rest ~20% (Eaton and Duplan, 2004). However, these percentages vary widely depending on the physiological condition of the adrenal gland and of the whole body. Thus, all of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla, and the total amount of catecholamines presented to cardiac ARs at any given time is composed of these circulating NE & Epi plus NE released locally from sympathetic nerve terminals within the heart (Lymperopoulos et al., 2007). The secretion of catecholamines from the adrenal glands is regulated in a complex manner by a variety of cell membrane receptors present in chromaffin cells. Many of these receptors are GPCRs, including, similarly to cardiac ANS nerve endings, α2ARs that inhibit secretion (inhibitory presynaptic autoreceptors), and βARs that enhance it (facilitatory presynaptic autoreceptors) (Figure 2; Hein et al., 1999; Brede et al., 2002; Philipp and Hein, 2004; Lymperopoulos et al., 2007). Of note, although various presynaptic auto- and heteroreceptors, facilitate (increase) adrenal catecholamine secretion, e.g., βARs, muscarinic cholinergic receptors (mAChRs), angiotensin II-ergic, histaminergic, and adrenomedullin receptors, the α2ARs are the only receptor type reported to date to inhibit adrenal catecholamine secretion (Brede et al., 2003; Moura et al., 2006; Lymperopoulos et al., 2007). An increase in GRK2 expression and activity (see above) has been documented in several cardiovascular diseases, including increased cardiac expression in HF (Rengo et al., 2011, 2012a; Lymperopoulos and Bathgate, 2012) and increased expression in some vascular beds in hypertension (Penn et al., 2000). Recently, we reported that GRK2 expression and activity are increased also in the adrenal medulla during HF (Lymperopoulos et al., 2007). Specifically, our studies over the past few years have established that adrenal GRK2 upregulation is responsible for severe adrenal α2AR dysfunction in chronic HF, which causes a loss of the sympathoinhibitory function of these receptors in the adrenal gland, and catecholamine secretion is thus chronically elevated (Figure 2; Lymperopoulos et al., 2007, 2008, 2010; Rengo et al., 2010, 2012b). This emerging crucial role for adrenal GRK2 in HF is underlined by the fact that its specific inhibition, via adenoviral-mediated βARKct adrenal gene delivery, leads to a significant reduction in circulating catecholamine levels, restoring not only adrenal, but also cardiac function in HF (Lymperopoulos et al., 2007). Additional evidence for the crucial role of adrenal GRK2-regulated α2ARs in regulating adrenal ANS tone in HF comes from the phenylethanolamine-N-methyl transferase (PNMT)-driven GRK2 KO mice (Lymperopoulos et al., 2010). These mice, which do not express GRK2 in their adrenal medullae from birth, display decreased ANS outflow and circulating catecholamines in response to myocardial infarction, which translates into preserved cardiac function and morphology over the course of the ensuing HF (Lymperopoulos et al., 2010). Of note, elevated GRK2-dependent α2AR dysfunction during HF might also occur in other peripheral sympathetic nerve terminals of the heart (Figure 2) and of other organs, thus contributing to the increased NE release and spillover, as well as to the presynaptic α2AR dysfunction in ANS neurons observed in chronic HF (see above) (Lang et al., 1997; Aggarwal et al., 2001). Effects of ANS overactivity in chronic HF Myocardial systolic dysfunction is associated with neurohormonal hyperactivity as a compensatory mechanism to maintain cardiac output in the face of declining cardiac function. The neuronal part of this response is represented by ANS cardiac nerve terminals, whereas the hormonal (or humoral) part is represented by increased secretion, and elevated circulating levels of certain hormones, the most prominent being Epi & NE, along with the RAAS hormones (i.e., angiotensin II & aldosterone) (Dzau et al., 1981). ANS hyperactivity is evidenced by increased plasma NE & Epi levels, elevated (central) sympathetic outflow, and heightened NE spillover from activated cardiac sympathetic nerve terminals into the circulation (Pepper and Lee, 1999). Cardiac NE spillover in untreated HF patients can reach up to 50-fold higher levels than those of healthy individuals under maximal exercise conditions (Morris et al., 1997). The information on chronic ANS activation in HF with preserved left ventricular ejection fraction (i.e., diastolic HF) is very limited. In patients with hypertension, ANS hyperactivity may contribute to the development of left ventricular diastolic dysfunction and thus increase HF risk (Hogg and McMurray, 2005). In systolic HF, patients may actually have decreased ANS neuronal density & function, resulting in decreased NE concentration within the cardiomyocytes, in addition to decreased postsynaptic βAR density, due to depletion of cardiac ANS neuronal NE stores and decreased NE presynaptic reuptake secondary to NE transporter downregulation (Regitz et al., 1991; Backs et al., 2001). With regards to the other major AR type expressed in the heart, α1ARs in HF may function in a compensatory fashion to maintain cardiac inotropy, but their involvement in cardiac pathophysiology appears limited to situations of cardiac hypertrophy that ultimately lead to HF (Knowlton et al., 1993). For instance, in the presence of pressure overload, cardiac α1AARs get activated and promote cardiomyocyte survival (i.e., block apoptosis), protecting against adverse remodeling and decompensation to HF (Du et al., 2006; Huang et al., 2007). Conclusions/future perspectives A vast number of studies over the past few decades have established the crucial role of activated ANS in the compensatory response of the circulation to retain its hemodynamic stability in the face of a cardiac insult, and when this fails, its excessive activation that accelerates HF progression and poses severe toxicity on the chronically failing heart. Additionally, the benefits of β-blockers and other therapeutic modalities that mitigate or protect the heart against this ANS hyperactivity are also well established. Among the several basic research developments aiming at reducing the activity and/or the detrimental effects of the ANS on the failing heart are sympatholytic agents (α2AR agonists), polymorphic variants of cardiac ARs that confer better prognosis in HF or better responses to current HF treatments, new sympathomimetics that seek to augment the function of the seemingly “cardioprotective” β1AR while simultaneously blocking the “cardiotoxic” β1AR (e.g., clenbuterol), activation of the cardiac parasympathetic nervous system, and, last but not least, augmentation of cardiac βAR-dependent function without the accompanying elevation of ANS activity/outflow. The latter is pursued with the very promising GRK2 inhibition therapeutic approach, which improves both cardiac adrenergic and inotropic reserves, while keeping the ANS outflow in check by restoring or augmenting central, cardiac and adrenal sympatho-inhibitory α2AR function. Future studies will most certainly help ascertain the magnitude of the therapeutic potential these ANS activity-targeting approaches hold for the fight against HF and other cardiovascular diseases. Conflict of interest statement The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ==== Refs References Aggarwal A. Esler M. D. Lambert G. W. Hastings J. Johnston L. Kaye D. M. (2002 ). Norepinephrine turnover is increased in suprabulbar subcortical brain regions and is related to whole-body sympathetic activity in human heart failure . Circulation 105 , 1031 –1033 10.1161/hc0902.105724 11877349 Aggarwal A. Esler M. D. Socratous F. Kaye D. M. (2001 ). Evidence for functional presynaptic alpha-2 adrenoceptors and their down-regulation in human heart failure . J. Am. Coll. Cardiol . 37 , 1246 –1251 10.1016/S0735-1097(01)01121-4 11300430 Armour J. A. (2004 ). Cardiac neuronal hierarchy in health and disease . Am. J. Physiol. Regul. Integr. Comp. Physiol . 287 , R262 –R271 15271675 Arriza J. L. Dawson T. M. Simerly R. B. Martin L. J. Caron M. G. Snyder S. H. (1992 ). The G-protein-coupled receptor kinases betaARK1 and betaARK2 are widely distributed at synapses in rat brain . J. Neurosci . 12 , 4045 –4055 1403099 Backs J. Haunstetter A. Gerber S. H. Metz J. Borst M. M. Strasser R. H. (2001 ). The neuronal norepinephrine transporter in experimental heart failure: evidence for a post-transcriptional downregulation . J. Mol. Cell. Cardiol . 33 , 461 –472 10.1006/jmcc.2000.1319 11181015 Brede M. Nagy G. Philipp M. Sorensen J. B. Lohse M. J. Hein L. (2003 ). Differential control of adrenal and sympathetic catecholamine release by alpha2-adrenoceptor subtypes . Mol. Endocrinol . 17 , 1640 –1646 10.1210/me.2003-0035 12764077 Brede M. Wiesmann F. Jahns R. Hadamek K. Arnolt C. Neubauer S. (2002 ). Feedback inhibition of catecholamine release by two different alpha2-adrenoceptor subtypes prevents progression of heart failure . Circulation 106 , 2491 –2496 10.1161/01.CIR.0000036600.39600.66 12417548 Bristow M. R. Ginsburg R. Minobe W. Cubicciotti R. Sageman W. S. Lurie K. (1982 ). Decreased catecholamine sensitivity and β-adrenegic receptor density in failing human hearts . N. Engl. J. Med . 307 , 205 –211 10.1056/NEJM198207223070401 6283349 Bristow M. R. Ginsburg R. Umans V. Fowler M. Minobe W. Rasmussen R. (1986 ). β1-and β2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective β1-receptor down-regulation in heart failure . Circ. Res . 59 , 297 –309 10.1161/01.RES.59.3.297 2876788 Brodde O. E. (1993 ). Beta-adrenoceptors in cardiac disease . Pharmacol. Ther . 60 , 405 –430 10.1016/0163-7258(93)90030-H 7915424 Brum P. C. Kosek J. Patterson A. Bernstein D. Kobilka B. (2002 ). Abnormal cardiac function associated with sympathetic nervous system hyperactivity in mice . Am. J. Physiol. Heart Circ. Physiol . 283 , H1838 –H1845 12384461 Bylund D. B. Eikenberg D. C. Hieble J. P. Langer S. Z. Lefkowitz R. J. Minneman K. P. (1994 ). International union of pharmacology nomenclature of adrenoceptors . Pharmacol. Rev . 46 , 121 –136 7938162 Chesley A. Lundberg M. S. Asai T. Xiao R. P. Ohtani S. Lakatta E. G. (2000 ). The β2-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through Gi-dependent coupling to phosphatidylinositol 3-kinase . Circ. Res . 87 , 1172 –1179 10.1161/01.RES.87.12.1172 11110775 Colucci W. S. Wright R. F. Braunwald E. (1986 ). New positive inotropic agents in the treatment of congestive heart failure. Mechanisms of action and recent clinical developments . N. Engl. J. Med . 314 , 290 –299 10.1056/NEJM198601303140506 2867470 Communal C. Singh K. Sawyer D. B. Colucci W. S. (1999 ). Opposing effects of β1- and β2-aadrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein . Circulation 100 , 2210 –2212 10.1161/01.CIR.100.22.2210 10577992 Daaka Y. Luttrell L. M. Lefkowitz R. J. (1997 ). Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A . Nature 390 , 88 –91 10.1038/36362 9363896 Docherty J. R. (2002 ). Age-related changes in adrenergic neuroeffector transmission . Auton. Neurosci . 96 , 8 –12 10.1016/S1566-0702(01)00375-7 11911505 Dorn G. W. II. Tepe N. M. Lorenz J. N. Koch W. J. Liggett S. B. (1999 ). Low- and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in Gα q-overexpressing mice . Proc. Natl. Acad. Sci. U.S.A . 96 , 6400 –6405 10.1073/pnas.96.11.6400 10339599 Du X. J. Gao X. M. Kiriazis H. Moore X. L. Ming Z. Su Y. (2006 ). Transgenic alpha1A-adrenergic activation limits post-infarct ventricular remodeling and dysfunction and improves survival . Cardiovasc. Res . 71 , 735 –743 10.1016/j.cardiores.2006.06.015 16859660 Dzau V. J. Colucci W. S. Hollenberg N. K. Williams G. H. (1981 ). Relation of the renin-angiotensin-aldosterone system to clinical state in congestive heart failure . Circulation 63 , 645 –651 10.1161/01.CIR.63.3.645 7006851 Eaton M. J. Duplan H. (2004 ). Useful cell lines derived from the adrenal medulla . Mol. Cell. Endocrinol . 228 , 39 –52 10.1016/j.mce.2003.02.001 15541571 Ferguson S. S. (2001 ). Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling . Pharmacol. Rev . 53 , 1 –24 11171937 Gauthier C. Leblais V. Kobzik L. Trochu J. N. Khandoudi N. Bril A. (1998 ). The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle . J. Clin. Invest . 102 , 1377 –1384 10.1172/JCI2191 9769330 Hein L. Altman J. D. Kobilka B. K. (1999 ). Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission . Nature 402 , 181 –184 10.1038/46040 10647009 Hogg K. McMurray J. (2005 ). Neurohumoral pathways in heart failure with preserved systolic function . Prog. Cardiovasc. Dis . 47 , 357 –366 10.1016/j.pcad.2005.02.001 16115515 Huang Y. Wright C. D. Merkwan C. L. Baye N. L. Liang Q. Simpson P. C. (2007 ). An alpha1A-adrenergic extracellular signal-regulated kinase survival signaling pathway in cardiac myocytes . Circulation 115 , 763 –772 10.1161/CIRCULATIONAHA.106.664862 17283256 Kaye D. Esler M. (2005 ). Sympathetic neuronal regulation of the heart in aging and heart failure . Cardiovasc. Res . 66 , 256 –264 10.1016/j.cardiores.2005.02.012 15820194 Knowlton K. U. Michel M. C. Itani M. Shubeita H. E. Ishihara K. Brown J. H. (1993 ). The alpha 1A-adrenergic receptor subtype mediates biochemical, molecular, and morphologic features of cultured myocardial cell hypertrophy . J. Biol. Chem . 268 , 15374 –15380 8393439 Lang C. C. Stein C. M. Nelson R. A. He H. B. Belas F. J. Blair I. A. (1997 ). Sympathoinhibitory response to clonidine is blunted in patients with heart failure . Hypertension 30 , 392 –397 10.1161/01.HYP.30.3.392 9314422 Leineweber K. Wangemann T. Giessler C. Bruck H. Dhein S. Kostelka M. (2002 ). Age-dependent changes of cardiac neuronal noradrenaline reuptake transporter (uptake1) in the human heart . J. Am. Coll. Cardiol . 40 , 1459 10.1016/S0735-1097(02)02168-X 12392837 Liggett S. B. Tepe N. M. Lorenz J. N. Canning A. M. Jantz T. D. Mitarai S. (2000 ). Early and delayed consequences of beta(2)-adrenergic receptor overexpression in mouse hearts: critical role for expression level . Circulation 101 , 1707 –1714 10.1161/01.CIR.101.14.1707 10758054 Lindley T. E. Doobay M. Sharma R. Davisson R. (2004 ). Superoxide is involved in the central nervous system activation and sympathoexcitation of myocardial infarction induced heart failure . Circ. Res . 94 , 402 –409 10.1161/01.RES.0000112964.40701.93 14684626 Lohse M. J. Engelhardt S. Eschenhagen T. (2003 ). What is the role of beta-adrenergic signaling in heart failure? Circ. Res . 93 , 896 –906 14615493 Lymperopoulos A. (2012 ). Ischemic emergency? endothelial cells have their own “adrenaline shot” at hand . Hypertension 60 , 12 –14 10.1161/HYPERTENSIONAHA.112.197020 22665125 Lymperopoulos A. Bathgate A. (2012 ). Pharmacogenomics of the heptahelical receptor regulators G-protein-coupled receptor kinases and arrestins: the known and the unknown . Pharmacogenomics 13 , 323 –341 10.2217/pgs.11.178 22304582 Lymperopoulos A. Rengo G. Koch W. J. (2007 ). Adrenal adrenoceptors in heart failure: fine-tuning cardiac stimulation . Trends Mol. Med . 13 , 503 –511 17981507 Lymperopoulos A. Rengo G. Koch W. J. (2012 ). GRK2 inhibition in heart failure: something old, something new . Curr. Pharm. Des . 18 , 186 –191 10.2174/138161212799040510 22229578 Lymperopoulos A. Rengo G. Funakoshi H. Eckhart A. D. Koch W. J. (2007 ). Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure . Nat. Med . 13 , 315 –323 10.1038/nm1553 17322894 Lymperopoulos A. Rengo G. Gao E. Ebert S. N. Dorn I. I. G. W. Koch W. J. (2010 ). Reduction of sympathetic activity via adrenal-targeted GRK2 gene deletion attenuates heart failure progression and improves cardiac function after myocardial infarction . J. Biol. Chem . 285 , 16378 –16386 10.1074/jbc.M109.077859 20351116 Lymperopoulos A. Rengo G. Zincarelli C. Soltys S. Koch W. J. (2008 ). Modulation of adrenal catecholamine secretion by in vivo gene transfer and manipulation of G protein-coupled receptor kinase-2 activity . Mol. Ther . 16 , 302 –307 10.1038/sj.mt.6300371 18223549 Malliani A. Pagani M. Pizzinelli P. Furlan R. Guzzetti S. (1983 ). Cardiovascular reflexes mediated by sympathetic afferent fibers . J. Auton. Nerv. Syst . 7 , 295 –301 10.1016/0165-1838(83)90082-6 6875194 Mann D. L. Bristow M. R. (2005 ). Mechanisms and models in heart failure: the biomechanical model and beyond . Circulation 111 , 2837 –2849 10.1161/CIRCULATIONAHA.104.500546 15927992 Morris M. J. Cox H. S. Lambert G. W. Kaye D. M. Jennings G. L. Meredith I. T. (1997 ). Region-specific neuropeptide Y overflows at rest and during sympathetic activation in humans . Hypertension 29 , 137 –143 10.1161/01.HYP.29.1.137 9039093 Moura E. Afonso J. Hein L. Vieira-Coelho M. A. (2006 ). Alpha2-adrenoceptor subtypes involved in the regulation of catecholamine release from the adrenal medulla of mice . Br. J. Pharmacol . 149 , 1049 –1058 10.1038/sj.bjp.0706950 17075569 Mudd J. O. Kass D. A. (2008 ). Tackling heart failure in the twenty-first century . Nature 451 , 919 –928 10.1038/nature06798 18288181 Nikolaev V. O. Moshkov A. Lyon A. R. Miragoli M. Novak P. Paur H. (2010 ). Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation . Science 327 , 1653 –1657 10.1126/science.1185988 20185685 Penn R. B. Pronin A. N. Benovic J. L. (2000 ). Regulation of G protein-coupled receptor kinases . Trends Cardiovasc. Med . 10 , 81 –89 10.1016/S1050-1738(00)00053-0 11150735 Pepper G. S. Lee R. W. (1999 ). Sympathetic activation in heart failure and its treatment with beta-blockade . Arch. Intern. Med . 159 , 225 –234 10.1001/archinte.159.3.225 9989534 Philipp M. Hein L. (2004 ). Adrenergic receptor knockout mice: distinct functions of 9 receptor subtypes . Pharmacol. Ther . 101 , 65 –74 10.1016/j.pharmthera.2003.10.004 14729393 Philipp M. Bred M. Hein L. (2002 ). Physiological significance of alpha(2)-adrenergic receptor subtype diversity: one receptor is not enough . Am. J. Physiol. Regul. Integr. Comp. Physiol . 283 , R287 –R295 12121839 Pierpont G. L. DeMaster E. G. Reynolds S. Pederson J. Cohn J. N. (1985 ). Ventricular myocardial catecholamines in primates . J. Lab. Clin. Med . 106 , 205 –210 4020248 Regitz V. Leuchs B. Bossaller C. Sehested J. Rappolder M. Fleck E. (1991 ). Myocardial catecholamine concentrations in dilated cardiomyopathy and heart failure of different origins . Eur. Heart J . 12 Suppl. D , 171 –174 1915450 Reiter E. Lefkowitz R. J. (2006 ). GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling . Trends Endocrinol. Metab . 17 , 159 –165 10.1016/j.tem.2006.03.008 16595179 Rengo G. Leosco D. Zincarelli C. Marchese M. Corbi G. Liccardo D. (2010 ). Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure . Am. J. Physiol. Heart Circ. Physiol . 298 , H2032 –H2038 10.1152/ajpheart.00702.2009 20304818 Rengo G. Lymperopoulos A. Leosco D. Koch W. J. (2011 ). GRK2 as a novel gene therapy target in heart failure . J. Mol. Cell. Cardiol . 50 , 785 –792 10.1016/j.yjmcc.2010.08.014 20800067 Rengo G. Lymperopoulos A. Zincarelli C. Femminella G. Liccardo D. Pagano G. (2012a ). Blockade of β-adrenoceptors restores the GRK2-mediated adrenal α (2) -adrenoceptor-catecholamine production axis in heart failure . Br. J. Pharmacol . 166 , 2430 –2440 10.1111/j.1476-5381.2012.01972.x 22519418 Rengo G. Perrone-Filardi P. Femminella G. D. Liccardo D. Zincarelli C. de Lucia C. (2012b ). Targeting the β-adrenergic receptor system through g-protein-coupled receptor kinase 2: a new paradigm for therapy and prognostic evaluation in heart failure: from bench to bedside . Circ. Heart Fail . 5 , 385 –391 10.1161/CIRCHEARTFAILURE.112.966895 22589366 Rockman H. A. Koch W. J. Lefkowitz R. J. (2002 ). Seven-transmembrane-spanning receptors and heart function . Nature 415 , 206 –212 10.1038/415206a 11805844 Rozec B. Erfanian M. Laurent K. Trochu J. N. Gauthier C. (2009 ). Nebivolol, a vasodilating selective beta(1)-blocker, is a beta(3)-adrenoceptor agonist in the nonfailing transplanted human heart . J. Am. Coll. Cardiol . 53 , 1532 –1538 10.1016/j.jacc.2008.11.057 19389564 Shannon R. Chaudhry M. (2006 ). Effect of alpha1-adrenergic receptors in cardiac pathophysiology . Am. Heart J . 152 , 842 –850 10.1016/j.ahj.2006.05.017 17070143 Skeberdis V. A. Gendviliene V. Zablockaite D. Treinys R. Macianskiene R. Bogdelis A. (2008 ). Beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current . J. Clin. Invest . 118 , 3219 –3227 18704193 Triposkiadis F. Karayannis G. Giamouzis G. Skoularigis J. Louridas G. Butler J. (2009 ). The sympathetic nervous system in heart failure physiology, pathophysiology, and clinical implications . J. Am. Coll. Cardiol . 54 , 1747 –1762 10.1016/j.jacc.2009.05.015 19874988 Wang H. Huang B. S. Ganten D. Leenen F. H. H. (2004 ). Prevention of sympathetic and cardiac dysfunction after myocardial infarction in transgenic rats deficient in brain angiotensinogen . Circ. Res . 94 , 843 –849 10.1161/01.RES.0000120864.21172.5A 15061159 Woodcock E. A. Du X. J. Reichelt M. E. Graham R. M. (2008 ). Cardiac alpha 1-adrenergic drive in pathological remodelling . Cardiovasc. Res . 77 , 452 –462 10.1093/cvr/cvm078 18032391 Zhu W. Z. Zheng M. Koch W. J. Lefkowitz R. J. Kobilka B. K. Xiao R. P. (2001 ). Dual modulation of cell survival and cell death by β2-adrenergic signalling in adult mouse cardiomyocytes . Proc. Natl. Acad. Sci. U.S.A . 98 , 1607 –1612 10.1073/pnas.98.4.1607 11171998 Zipes D. P. (2008 ). Heart-brain interactions in cardiac arrhythmias: role of the autonomic nervous system . Cleve. Clin. J. Med . 75 , S94 –S96 10.3949/ccjm.75.Suppl_2.S94 18540155	24027534	PMC3761154	CC BY	2021-01-04 23:08:39	yes	Front Physiol. 2013 Sep 4; 4:240
==== Front PLoS One PLoS One plos PLoS ONE 1932-6203 Public Library of Science San Francisco, USA 24039815 PONE-D-13-14400 10.1371/journal.pone.0072895 Research Article Drug-Eluting Stents for Acute Coronary Syndrome: A Meta-Analysis of Randomized Controlled Trials A Meta-Analysis of DES for ACS Wang Lishan 1 2 1 Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, P.R. China 2 FengHe (ShangHai) Information Technology Co., Ltd, Shanghai, P.R. China Lipinski Michael Editor University of Virginia Health System, United States of America Competing Interests: The authors have declared that no competing interests exist. Performed the experiments: LW. Analyzed the data: LW. Wrote the paper: LW. 2013 5 9 2013 8 9 e728958 4 2013 15 7 2013 © 2013 Lishan Wang 2013 Lishan Wang https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Drug-eluting stents (DES) are increasingly used for treatment of acute coronary syndrome (ACS). However, clinical efficacy and safety of various types of DES is not well established in these subjects. We therefore evaluated clinical utility of second-generation and first-generation DES in patients with ACS by conducting a meta-analysis. Methods A search of Medline, Embase, the Cochrane databases, and Web of Science was made. Randomized controlled trials (RCTs) which compared second-generation DES (everolimus-eluting stents [EES] or zotarolimus-eluting stents [ZES]) versus first-generation DES (sirolimus-eluting stents [SES] or paclitaxe-eluting stents [PES]) in patients with ACS and provided data on clinical efficacy or safety endpoints were included. Pooled estimates were calculated using random-effects model. Result A total of 2,757 participants with ACS in 6 RCTs were included. Compared with first-generation one, second-generation DES trended to be associated with the decreased incidence of definite or probable stent thrombosis in ACS patients (risk ratio [RR] = 0.60, 95% confidence intervals [CI] 0.33 to 1.07, p = 0.09). However, the rate of target lesion revascularization (TLR) significantly increased in second-generation DES (RR = 2.08, 95%CI 1.25 to 3.47, p = 0.005). There were no significant differences in the incidence of major adverse cardiac events (MACEs), all-cause death, cardiac death, and recurrent myocardial infarction between the two arms (all p>0.10). The second-generation EES showed a tendency towards lower risk of MACEs (p = 0.06) and a beneficial effect on reducing stent thrombosis episodes (p = 0.009), while the second-generation ZES presented an increased occurrence of MACEs (p = 0.02) and TLR (p = 0.003). Conclusion Second-generation DES, especially EES, appeared to present a lower risk of stent thrombosis, whereas second-generation ZES might increase the need for repeat revascularization in ACS patients. During coronary interventional therapy, DES class should be adequately considered in order to maximize clinical benefit of DES implantation in these specific subjects. See accompanying retraction notice. ==== Body pmcIntroduction Drug-eluting stents (DES) are increasingly used for treatment of acute coronary syndrome (ACS). Previous randomized controlled trials (RCTs) and meta-analysis have demonstrated that DES were superior to bare-metal stents in minimizing the occurrence of stent restenosis and reducing the need for revascularization in patients with ACS [1], [2], [3], [4], [5], which was the major drawback of percutaneous coronary interventions (PCI) in bare-metal stents era. In patients in stable condition the newer second-generation DES, eluting with everolimus (EES) or zotarolimus (ZES), has shown promise in improving further the clinical outcomes compared with the first-generation sirolimu- or paclitaxe-eluting stent (SES or PES) [6], [7], [8]. However, the issue that whether clinical utility of various types of DES in treating ACS settings with the higher possible thrombotic coronary lesions is identical remains uncertain. To date there is a limited number of registry studies and RCTs comparing the second-generation versus first-generation DES in ACS patients and delivering conflicting results. Korea Acute Myocardial Infarction Registry (KAMIR) study showed that the first-generation SES had the lower 1 year incidences of major adverse cardiac events (MACEs) and target lesion revascularization (TLR) than the second-generation ZES in patients with ST-segment elevation myocardial infarction undergoing primary PCI [9]. However, the benefit of the first-generation DES was not shown in an early small-scale study [10] and a randomized trial [11]. In contrast, the second-generation EES appeared to be associated with lower incidences of MACEs [12] and definite and/or probable stent thrombosis in patients with ST-segment elevation myocardial infarction [13]. These inconsistent findings confused interventional cardiologists' stent selection decisions beyond consideration of characteristics of device performance. As thus, here we conducted a meta-analysis of RCTs to evaluate the clinical outcomes of ACS patients treated with the second- versus the first-generation DES. Materials and Methods Eligible criteria The clinical studies were eligible for inclusion if 1) study design involved patient randomization; 2) they compared second-generation DES (EES or ZES) versus first-generation DES (SES or PES) in patients with ACS (unstable angina, non-ST segment elevation acute myocardial infarction, and ST segment elevation acute myocardial infarction); 3) the information on clinical efficacy or safety endpoints (e.g. MACEs, all-cause death, cardiac death, recurrent myocardial infarction, TLR, or definite and/or probable stent thrombosis) was available; 4) follow-up duration was no less than 6 months. We restricted our analyses to the DES approved by the US Food and Drug Administration (FDA). Trials would be excluded if the data on patient and procedural characteristics was not available, and post-hoc analyses of RCTs were also excluded. Study identification We performed an electronic search of Medline, Embase, the Cochrane Central Register of Controlled Trials, the Cochrane Database of Systematic Reviews, and ISI Web of Science until December 2012 for the eligible trials. Complex search strategies were formulated using the following terms: everolimus-eluting stent, zotarolimus-eluting stent, second-generation eluting stent, sirolimus-eluting stent, paclitaxel-eluting stent, first-generation eluting stent, unstable angina, acute myocardial infarction, and acute coronary syndrome. We also checked the references and citations of the eligible studies from the potential eligible articles to ensure that no clinical trials were missed. The search was restricted to English-language literature. Study enrollment, data collection, and quality assessment Two investigators (W.L., Z.W.) assessed trial eligibility using predefined eligibility criteria in duplicate and independently. The data, such as participant characteristics, lesion and procedural characteristics, and follow-up duration from each study, were extracted. The occurrence of clinical outcomes was also recorded. Any disagreements were resolved through consensus. Also all the eligible trials were assessed by the following quality criteria recommended by the Cochrane Collaboration: sequence generation of the allocation; concealment of allocation; blinding of participants, personnel, and outcome assessors; use of intention to treat analysis; description of withdrawals and dropouts. A numerical score between 0 and 5 was assigned as a measure of study design and reporting quality with 0 being the weakest and 5 designated the strongest, based on the validated scale put forward by Jadad and colleagues [14]. Statistical analyses Treatment effects were reported as risk ratio (RR) with 95% confidence intervals (CI). Pooled estimates were calculated with random-effects model. For studies with no event of interest in a treatment group, 1.0 was added to all cells for continuity correction [15]. Statistical homogeneity was quantified with the I2 statistic with a scale of 0% to 100% (>75% represented very large between-study inconsistency) [16]. Subgroup analysis was performed to test the potential influence of clinical factors including ACS classification, mean age, time from pain to angioplasty, percentage of TIMI grade 0/1, type of DES, stent length, stent size, and follow-up duration. For verification of the robustness of the results, sensitivity analyses were conducted by alternatively using fixed-effect model, and by omitting each trial at a time from analysis and then computing overall estimates for the remaining studies. The potential publication bias was qualitatively assessed using funnel plot method. The significance level was set at p<0.05. The pooling analyses were performed using Review Manager 5.1 software (Cochrane Collaboration, Copenhagen, Denmark). The present work was performed as the guidelines proposed by Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (Checklist S1). Results Results from our literature search were detailed in Figure 1. Briefly, our initial search yielded 880 potential literature citations from the electronic databases. Of them, 734 were excluded by removing duplicate literatures and through review of citations. Abstracts from 146 articles were reviewed and an additional 79 trials were excluded, leaving 67 studies for full publication review. Thereafter 61 were excluded (43 were non-randomized studies, 12 used DES which were not approved by FDA, 3 had no data on clinical characteristics, 1 was post-hoc analysis of RCTs, 2 were pooled analysis of RCTs) and no additional relevant study was identified from the references and citations of eligible articles. Finally, 6 studies were found to conform to the predefined inclusion criteria [11], [12], [13], [17], [18], [19]. 10.1371/journal.pone.0072895.g001 Figure 1 Flow chart of studies selection. DES = drug-eluting stents; RCTs = randomized controlled trials; FDA = the US Food and Drug Administration. The demographic, clinical, and procedural characteristics of the 6 trials were shown in Table 1 and Table 2. A total of 2,757 participants with ACS were enrolled in the meta-analysis. Among them 1,302 were randomly allocated to receive second-generation DES implantation and 1,455 to receive first-generation DES treatment. Of the enrolled 6 trials, two [11], [17] were three-arm trials (ZES vs. PES vs. SES), but the rest were two-arm trials (two [12], [13] for EES vs. SES; two [18], [19] for ZES vs. SES). Five [11], [12], [13], [17], [18] focused on patients with acute myocardial infarction, and one [19] on unrestricted ACS in which only 16.2% ST-segment elevation acute myocardial infarction was involved. Four trials reported 30 day follow-up clinical outcomes [11], [12], [13], [17]; 5 reported 6–12 month data [11], [12], [13], [17], [18]; and 2 reported 18 month data [17], [19]. The majority of participants was male and the mean age ranged from 59.7 years to 65.3 years. Total stent length per patient ranged from 22.5 mm to 31.6 mm and mean size of stents from 3.14 mm to 3.27 mm. All of the enrolled patients received dual antiplatelet therapy no less than 12 months or to the end of the follow-up. Additionally, the level of evidence for each article was graded with a score of 3 to 4 according to the Jadad quality score (Table S1). 10.1371/journal.pone.0072895.t001 Table 1 Baseline patient characteristics of randomised controlled trials included in the meta-analysis. Study, year No. enrolled, randomization ratio Comparisons Study design Mean age Male % Diabetes, % Current smoker, % Pain to angioplasty, h NS TEACS, % STEAMI, % Target vessel (LAD/LCX/ RCA/LM), % Primary end points Follow-up methods Follow-up duration KOMER, 2011 611, 1:1:1 ZES vs. PES vs. SES single-blind, multicentre 59.7 79 20.8 54.8 5.3 0 100 53.8/6/37.2/0 MACEs Angiography 30d, 12m,18m Sawada T, 2012 35, 2:1 EES vs. SES single-blind, single center 65.3 78.8 42.4 42.4 NA 0 100 60.6/0.03/37.7/0 NIT and stent thrombosis OCT, angioscopy, angiography 30d, 7m SEZE, 2012 121, 1:1 ZES vs. SES single-blind, multicentre 60.9 81 22.3 53.8 5.3 0 100 58/9/33/0 Late lumen loss Angiography, IVUS 12m SORT OUT III ACS, 2012 1052, 1:1 ZES vs. SES open-label, multicentre 63.1 73.1 13.6 38.5 NA 83.8 16.2 40.95/27.25/2 9.95/1.65 MACEs Angiography 18m XAMI 2012 625, 2:1 EES vs. SES single-blind, multicentre 61.5 73.7 9.7 54.7 2.85 4 96 40.1/19/40.4/0.2 MACEs Angiography 30d, 12m ZEST AMI, 2009 328, 1:1:1 ZES vs. PES vs. SES single-blind, multicentre 59.7 82.3 25.9 56.7 4.75 0 100 46.3/11.6/42.1/0 MACEs Angiography 30d, 12m MACEs was defined as cardiac death, recurrent myocardial infarction, and target vessel or lesion revascularization. EES = everolimus-eluting stents. IVUS = intravascular ultrasound; LAD = left anterior descending artery; LCX = left circumflex artery; LM = left main artery; MACEs = major adverse cardiac events; NA = not available; NIT = neointimal thickness; NSTEACS = non-ST-segment elevation acute coronary syndrome; OCT = optical coherence tomography; PES = paclitaxel-eluting stents. RCA = right coronary artery; SES = sirolimus-eluting stents; STEAMI = ST-segment elevation acute myocardial infarction; ZES = zotarolimus-eluting stent. 10.1371/journal.pone.0072895.t002 Table 2 Baseline lesion and procedural characteristics. Study, year Reference vessel diameter, mm Lesion length, mm No. of lesions per patient No. of stents per patient Total stent length per patient, mm Stent size (mm) Multivessel disease Initial TIMI grade flow, 0/1/2/3, % Final TIMI grade flow after procedure, 0/1/2/3, % Max inflation pressure, atm DAPT durati on, m Use of glycoprotein IIb/IIIa inhibitors, % KOMER, 2011 2.97 19.5 1.0 1.2 24.1 3.27 42.9 54.3/11.3/16.5/17.9 0/0.1/6.2/93.7 NA ≥12 25 Sawada T, 2012 2.94 NA NA NA 22.5 3.14 0 90.9/0/9.1/0 NA 17.8 7 0 SEZE 2012 2.79 23.4 NA 1.15 28.6 3.16 67 63/7.5/11.5/18 0/0/6.5/93.5 15.5 12 12.4 SORT OUT III ACS, 2012 NA NA 1.42 NA 28.1 3.2 NA NA NA NA 12 22.15 XAMI 2012 NA NA NA 1.35 26 NA 47.3 55.4/6.1/17.1/21.4 NA NA 12 75.6 ZEST AMI 2009 2.96 27.11 1.22 1.51 31.6 3.25 45.1 58.5/10.1/15.5/15.9 0.3/0.6/10.3/88.8 15.2 ≥12 19.8 DAPT = dual antiplatelet therapy; NA = not available; TIMI = Thrombolysis In Myocardial Infarction. Meta-analytic pooling for the incidence of MACEs, all-cause death, and cardiac death showed that the second-generation DES did not provide a greater advantage compared with the first-generation DES in ACS patients (MACEs: RR = 1.13, 95% CI 0.73 to 1.76, p = 0.53, I2 = 57%, Figure 2A; all-cause death: RR = 0.88, 95% CI 0.56 to 1.38, p = 0.59, I2 = 57%, Figure 2B; cardiac death: RR = 0.82, 95% CI 0.35 to 1.92, p = 0.65, I2 = 12%, Figure 2C). Moreover, second-generation DES did not show the superiority in lowering the risk of recurrent myocardial infarction (RR = 0.83, 95% CI 0.27 to 2.61, p = 0.75, I2 = 53%, Figure 3A). Notably, the risk for TLR in ACS patients receiving second-generation DES treatment was over 2 times higher than the first-generation DES (RR = 2.08, 95%CI 1.25 to 3.47, p = 0.005, I2 = 0%, Figure 3B). Conversely, the second-generation DES trended to be associated, albeit nonsignificantly, with decreased incidence of definite or probable stent thrombosis (RR = 0.60, 95%CI 0.33 to 1.07, p = 0.09, I2 = 15%, Figure 3C). 10.1371/journal.pone.0072895.g002 Figure 2 Pooled risk ratios of second-generation versus first-generation drug-eluting stents for acute coronary syndrome for major adverse cardiac events (A), all-cause mortality (B), and cardiac death (C). CI = confidence intervals; DES = drug-eluting stents; M–H = Mantel-Haenszel. 10.1371/journal.pone.0072895.g003 Figure 3 Pooled risk ratios of second-generation versus first-generation drug-eluting stents for acute coronary syndrome for recurrent myocardial infarction (A), target lesion revascularization (B), and definite or probable stent thrombosis (C). Abbreviations as Figure 2. In addition, in acute myocardial infarction (AMI) subgroup, there were no significant differences in the occurrence of MACEs and TLR between the two arms (Table 3). Nevertheless, compared with the first-generation DES, the second-generation DES might dramatically lower the risk of stent thrombosis by 54% (RR = 0.46, p = 0.01). However, when the analysis was restricted to unselected ACS patients, in which only one study (SORT OUT III ACS trial [19]) was enrolled, pooled results showed that the second-generation DES was inferior to the first-generation one in reducing the incidence of MACEs (p = 0.02) and TLR (p = 0.01). Nevertheless, the second-generation DES did not increase the risk of stent thrombosis compared with the first-generation one (p = 0.48). In ZES subgroup the second-generation DES showed an increased occurrence of MACEs (RR = 1.45, p = 0.02) and TLR (RR = 2.31, p = 0.003), while in EES subgroup a tendency to lower the risk of MACEs (RR = 0.55, p = 0.06) and a benefit in reducing stent thrombosis episodes were found (RR = 0.39, p = 0.009). With the prolongation of follow-up duration, the unfavorable effects of the second-generation DES on MACEs and TLR became statistically significant at 18 months post stent implantation (MACEs: RR = 1.62, p = 0.01; TLR: RR = 2.66, p = 0.002). Nevertheless, the second-generation DES showed a tendency toward lowering the risk of stent thrombosis at 30 days (RR = 0.35, p = 0.06), and the benefit became significant statistically at 6 to 12 months after stent implantation (RR = 0.48, p = 0.01). Moreover, in ACS patients with lower TIMI grade (TIMI 0/1) the second-generation DES might show the more beneficial effect on lowering the risk of stent thrombosis in comparison to the first-generation one (RR = 0.36, p = 0.02). In addition, time from symptom to angioplasty had little impact on the above clinical endpoints (Table 3). 10.1371/journal.pone.0072895.t003 Table 3 Subgroup analyses based on the data on MACEs, TLR, and stent thrombosis. MACEs TLR Stent thrombosis Subgroups No. of studies RR (95% CI) P value No. of studies RR (95% CI) P value No. of studies RR (95% CI) P value Study on AMI 4 0.97 [0.61, 1.54] 0.90 4 1.92 [0.88, 4.15] 0.10 5 0.46 [0.26, 0.83] 0.01 Study on unclassified ACS 1 1.76 [1.11, 2.80] 0.02 1 2.45 [1.22, 4.93] 0.01 1 1.51 [0.48, 4.73] 0.48 Age ≥60 3 0.98 [0.45, 2.13] 0.95 4 1.88 [1.07, 3.28] 0.03 4 0.58 [0.27, 1.22] 0.15 Age <60 2 1.36 [0.85, 2.20] 0.20 1 3.42[1.01,11.56] 0.05 2 0.54 [0.08, 3.53] 0.52 Pain to angioplasty>5h 2 1.17 [0.69, 1.99] 0.56 2 2.10 [0.78, 5.63] 0.14 2 0.86 [0.28, 2.61] 0.79 Pain to angioplasty<5h 2 0.85 [0.35, 2.07] 0.73 1 1.37 [0.27, 7.01] 0.70 2 0.38 [0.13, 1.14] 0.08 TIMI 0/1≥70% 1 0.89 [0.37, 2.15] 0.79 2 1.07 [0.34, 3.35] 0.91 2 0.36 [0.15, 0.84] 0.02 TIMI 0/1<70% 3 1.00 [0.54, 1.83] 0.99 2 2.47 [0.93, 6.55] 0.07 3 0.59 [0.26, 1.31] 0.19 EES 1 0.55 [0.29, 1.03] 0.06 2 1.06 [0.26, 4.26] 0.94 2 0.39 [0.19, 0.79] 0.009 ZES 4 1.45 [1.06, 1.98] 0.02 3 2.31 [1.34, 3.99] 0.003 4 0.97 [0.45, 2.09] 0.93 30 day follow-up 3 0.73 [0.40, 1.35] 0.32 3 0.82 [0.17, 3.96] 0.80 4 0.35 [0.12, 1.04] 0.06 6–12 month follow-up 4 0.95 [0.61, 1.49] 0.83 4 1.58 [0.72, 3.46] 0.25 5 0.48 [0.26, 0.86] 0.01 18 month follow-up 2 1.62 [1.11, 2.37] 0.01 2 2.66 [1.45, 4.88] 0.002 2 1.23 [0.54, 2.79] 0.63 ACS = acute coronary syndrome; AMI = acute myocardial infarction; CI = confidence intervals; EES = everolimus-eluting stents; MACEs = major adverse cardiac events; RR = risk ratios; TIMI = Thrombolysis In Myocardial Infarction; TLR = target lesion revascularization; ZES = zotarolimus-eluting stent. In sensitivity analysis, when the XAMI study [12] was omitted from the analysis on MACEs, and the SORT OUT III ACS study [19] from TLR and stent thrombosis, the corresponding original results were reversed (MACEs: RR = 1.45, 95%CI 1.06 to 1.98, p = 0.02, I2 = 0%; TLR: RR = 1.73, 95%CI 0.83 to 3.64, p = 0.15, I2 = 0%; stent thrombosis: RR = 0.46, 95%CI 0.26 to 0.83, p = 0.01, I2 = 0%). Except for the process, omission of each trial one at a time from the analysis or alternatively using fixed-effect model did not have any relevant influence on other overall results in the meta-analysis. Funnel plots were performed for all outcomes and did not show symmetry (Figure S1), suggesting that there exist the substantial publication bias in the meta-analysis. Discussion The present study, to our knowledge, was the first meta-analysis based on the currently available data from RCTs to comparing the clinical values of second-generation versus first-generation DES in patients with ACS. It revealed that ACS subjects treated with the second-generation DES had the similar incidences of MACEs, all-cause death, cardiac death, and recurrent myocardial infarction as those treated with the first-generation DES. However, the second-generation DES was associated with increased risk of repeat revascularization in comparison to the first-generation DES, with the relative risk of TLR of 2.08. Nevertheless, the second-generation DES had a trend toward lower the risk of definite or probable stent thrombosis in overall ACS patients. And the second-generation DES reduced the incidence of stent thrombosis by 54% in patients with acute myocardial infarction. Second-generation ZES might be associated with increased occurrence of MACEs and TLR. Conversely, in patients with acute myocardial infarction, receiving EES implantation, or the lower TIMI grade, the second-generation DES might be the more beneficial in reducing the risk of stent thrombosis than the first-generation one. Newer second-generation DES was primarily conceived to further improve clinical utility of DES on the basis of first-generation one. Unfortunately, the current study did not show the differences in reducing the incidences of MACEs, all-cause mortality, cardiac death, and recurrent myocardial infarction between the two generations DES in ACS patients. Nevertheless, the overall results in the current study showed that the incidences of the above clinical outcomes were low in both arms. These findings may also reflect the progress over the last few years in ACS patient treatment. Unexpectedly, the TLR rate in ACS patients undergoing the second-generation DES implantation was higher than that receiving first-generation DES. Of note, of 5 trials enrolled in the analysis of the clinical endpoint, two showed the superiority of first-generation DES [17], [19], and the other 3 did not present intergroup differences. Both KOMER trial [17] and SORT OUT III ACS trials [19] compared clinical efficacy and safety of second-generation ZES versus first-generation SES or/and PES in ACS patients. The two studies consistently demonstrated ZES did not have the superiority and even presented the inferiority in reducing the risk of repeat revascularization to the first-generation DES. Furthermore, the ENDEAVOR III study [20], a prospective, randomized, single-blinded multicenter trial, comparing ZES and SES in patients with stable coronary disease undergoing elective PCI, also indicated that ZES was associated with significantly higher late lumen loss and binary restenosis at 8 month angiographic follow-up. Based on these findings, we presumed that the use of ZES might be the major cause responsible for the unfavorable overall result on TLR. Indeed, when the analysis was restricted to subjects receiving ZES implantation, it showed that the use of the second-generation DES was associated with the higher incidence of TLR. Nevertheless, it was notable that the second-generation ZES included in the meta-analysis has a phosphorylcholine coating polymer that is a synthetic copy of the predominant phospholipid in the outer membrane of red blood cells. The unfavorable finding was not extrapolated automatically to the newer generation of ZES, such as Endeavor Resolute DES, which uses a proprietary new biocompatible polymer called BioLinx. Recently a clinical study [21], comparing the long-term clinical outcomes of the two ZES, indicated that Endeavor Resolute ZES significantly reduced the angiographic in-stent late lumen loss and had a lower 2 year incidence of TLR in patients with coronary heart disease. However, compared with the first-generation DES, the use of the second-generation EES in ACS settings did not provide a significant impact on this clinical endpoint. That is to say, among the second-generation DES, EES should be recommended with priority when the rate of repeat revascularization was regarded as prime target of coronary interventional therapy in ACS patients undergoing PCI. The propensity for stent thrombosis following first-generation DES implantation after discontinuation of dual antiplatelet therapy has raised safety concerns [22], [23]. Recently a pooled patient-level meta-analysis demonstrated that among patients with ST-segment elevation ACS undergoing primary PCI, the first-generation DES (SES and PES) are associated with the increased risk of very late stent thrombosis and recurrent myocardial infarction compared with bare-metal stents [24]. The development of newer second-generation DES aims mainly to address the issue. A comprehensive network meta-analysis by Palmerini T et al. [25], in which 50,844 patients with unclassified coronary heart diseases were enrolled, showed that the second-generation DES, EES but not ZES, had the lower rate of stent thrombosis within 2 years of implantation than bare-metal stents and first-generation DES. The beneficial effect of EES was also confirmed by another small-scale meta-analysis performed by Alazzoni A et al [26]. However, another meta-analysis of 19 trials including 16,924 unrestricted coronary artery disease subjects did not find the differences in stent thrombosis between the overall second-generation DES and the overall first-generation those during the first year after stent implantation [27]. Unfortunately, these previous consistently focused on the patients with unrestricted coronary heart diseases and did not further perform a pooled analysis on the specific subsets. As thus, safety value of second-generation DES in patients with coronary artery diseases, especially with ACS, was yet not well established. The current meta-analysis investigated the issue and showed a beneficial tendency of the second-generation DES toward lowering the incidence of stent thrombosis compared with the first-generation DES. Moreover, after omitting the SORT OUT III ACS study [19] from the pooling analysis, the intergroup difference became significant. As thus, the original nonsignificant difference might be mainly caused by the enrollment of SORT OUT III ACS study in the meta-analysis. Causally, clinical design of SORT OUT III ACS study differed from that of the others included in the meta-analysis. The high percentage of patients with non-ST segment elevation ACS (83.8%) was recruited in the trial [19]. Non-ST segment elevation ACS was characterized by lower possible thrombotic coronary lesions than ST-segment elevation ACS. That is to say, in terms of lowering the risk of stent thrombosis the second-generation DES might have the more superiority in patients with higher possible thrombotic lesions. Indeed, in acute myocardial infarction subgroup we did find the benefit associated with second-generation DES implantation. Notably, the significant reduction in the occurrence of stent thrombosis was achieved under dual antiplatelet therapy with recommended duration by corresponding clinical guidelines. It was highly commendable for second-generation DES to provide an additional benefit. Methodologically, the use of random-effect model and relatively low statistical heterogeneities among the included trials might ensure the robustness of conclusions from the current study. Moreover, major results in the present study were further confirmed with sensitivity analyses. However, due to the limited sample size, the findings in the subgroup analyses, especially in the EES subgroup, were not solid enough and should be interpreted with caution. Larger-scale studies will be needed to further verify the findings and conclusions in the subgroup analyses of the current study. In addition, the other limitation of our study was that there existed a substantial publication bias which might influence the overall results. As thus, the publication of negative data should be encouraged to elaborate the true effects of the second-generation DES on the ACS subjects. In summary, this meta-analysis based on the currently available data from RCTs did not show significant differences in the incidence of MACEs, all-cause death, cardiac death, and recurrent myocardial infarction between the second-generation and the first-generation DES. Nevertheless, the second-generation DES, especially EES, appeared to present a lower risk of stent thrombosis in ACS patients, especially in acute myocardial infarction. However, the second-generation DES, mainly referring to ZES, seemed to increase the need for repeat revascularization compared with the first-generation one. Therefore, in process of interventional therapy for these specific subjects, DES class and ACS classification should be adequately considered in order to maximize clinical benefit of DES implantation. Supporting Information Figure S1 Publication bias analysis using funnel plot method. (TIF) Click here for additional data file. Table S1 Quality assessment of the enrolloed trials. (DOC) Click here for additional data file. Checklist S1 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. (DOC) Click here for additional data file. ==== Refs References 1 Stone GW , Lansky AJ , Pocock SJ , Gersh BJ , Dangas G , et al . (2009) Paclitaxel-eluting stents versus bare-metal stents in acute myocardial infarction. N Engl J Med 360 : 1946–1959.19420364 2 Di Lorenzo E , De Luca G , Sauro R , Varricchio A , Capasso M , et al . (2009) The PASEO (PaclitAxel or Sirolimus-Eluting Stent Versus Bare Metal Stent in Primary Angioplasty) Randomized Trial. JACC Cardiovasc Interv 2 : 515–523.19539255 3 Valgimigli M , Percoco G , Malagutti P , Campo G , Ferrari F , et al . (2005) Tirofiban and sirolimus-eluting stent vs abciximab and bare-metal stent for acute myocardial infarction: a randomized trial. JAMA 293 : 2109–2117.15870414 4 Sabate M , Cequier A , Iniguez A , Serra A , Hernandez-Antolin R , et al . (2012) Everolimus-eluting stent versus bare-metal stent in ST-segment elevation myocardial infarction (EXAMINATION): 1 year results of a randomised controlled trial. Lancet 380 : 1482–1490.22951305 5 Greenhalgh J, Hockenhull J, Rao N, Dundar Y, Dickson RC, et al .. (2010) Drug-eluting stents versus bare metal stents for angina or acute coronary syndromes. Cochrane Database Syst Rev: CD004587. 6 Kedhi E , Joesoef KS , McFadden E , Wassing J , van Mieghem C , et al . (2010) Second-generation everolimus-eluting and paclitaxel-eluting stents in real-life practice (COMPARE): a randomised trial. Lancet 375 : 201–209.20060578 7 Stone GW , Rizvi A , Newman W , Mastali K , Wang JC , et al . (2010) Everolimus-eluting versus paclitaxel-eluting stents in coronary artery disease. N Engl J Med 362 : 1663–1674.20445180 8 Park DW , Kim YH , Yun SC , Kang SJ , Lee SW , et al . (2010) Comparison of zotarolimus-eluting stents with sirolimus- and paclitaxel-eluting stents for coronary revascularization: the ZEST (comparison of the efficacy and safety of zotarolimus-eluting stent with sirolimus-eluting and paclitaxel-eluting stent for coronary lesions) randomized trial. J Am Coll Cardiol 56 : 1187–1195.20883925 9 Kim HK , Jeong MH , Ahn YK , Kim JH , Chae SC , et al . (2010) Comparison of outcomes between Zotarolimus- and sirolimus-eluting stents in patients with ST-segment elevation acute myocardial infarction. Am J Cardiol 105 : 813–818.20211324 10 Choi CU , Rha SW , Chen KY , Li YJ , Poddar KL , et al . (2009) Lack of clinical benefit of improved angiographic results with sirolimus-eluting stents compared with paclitaxel and zotarolimus-eluting stents in patients with acute myocardial infarction undergoing percutaneous coronary intervention. Circ J 73 : 2229–2235.19789418 11 Lee CW , Park DW , Lee SH , Kim YH , Hong MK , et al . (2009) Comparison of the efficacy and safety of zotarolimus-, sirolimus-, and paclitaxel-eluting stents in patients with ST-elevation myocardial infarction. Am J Cardiol 104 : 1370–1376.19892052 12 Hofma SH , Brouwer J , Velders MA , van't Hof AW , Smits PC , et al . (2012) Second-generation everolimus-eluting stents versus first-generation sirolimus-eluting stents in acute myocardial infarction. 1-year results of the randomized XAMI (XienceV Stent vs. Cypher Stent in Primary PCI for Acute Myocardial Infarction) trial. J Am Coll Cardiol 60 : 381–387.22835668 13 Sawada T, Shinke T, Otake H, Mizoguchi T, Iwasaki M, et al .. (2012) Comparisons of detailed arterial healing response at seven months following implantation of an everolimus- or sirolimus-eluting stent in patients with ST-segment elevation myocardial infarction. Int J Cardiol doi: 10.1016/j.ijcard.2012.10.043. 14 Jadad AR , Moore RA , Carroll D , Jenkinson C , Reynolds DJ , et al . (1996) Assessing the quality of reports of randomized clinical trials: is blinding necessary? Control Clin Trials 17 : 1–12.8721797 15 Zhang S , Ge J , Yao K , Qian J (2011) Meta-analysis of early versus deferred revascularization for non-ST-segment elevation acute coronary syndrome. Am J Cardiol 108 : 1207–1213.21872193 16 Ioannidis JP , Patsopoulos NA , Evangelou E (2007) Uncertainty in heterogeneity estimates in meta-analyses. BMJ 335 : 914–916.17974687 17 Kang WC , Ahn T , Lee K , Han SH , Shin EK , et al . (2011) Comparison of zotarolimus-eluting stents versus sirolimus-eluting stents versus paclitaxel-eluting stents for primary percutaneous coronary intervention in patients with ST-elevation myocardial infarction: results from the Korean Multicentre Endeavor (KOMER) acute myocardial infarction (AMI) trial. EuroIntervention 7 : 936–943.21959255 18 Chung WY , Kang J , Cho YS , Park HJ , Yang HM , et al . (2012) A randomized, prospective, two-center comparison of sirolimus-eluting stent and zotarolimus-eluting stent in acute ST-elevation myocardial infarction: the SEZE trial. Chin Med J (Engl) 125 : 3373–3381.23044291 19 Thim T , Maeng M , Kaltoft A , Jensen LO , Tilsted HH , et al . (2012) Zotarolimus-eluting vs. sirolimus-eluting coronary stents in patients with and without acute coronary syndromes: a SORT OUT III substudy. Eur J Clin Invest 42 : 1047–1054.22624990 20 Kandzari DE , Leon MB , Popma JJ , Fitzgerald PJ , O'Shaughnessy C , et al . (2006) Comparison of zotarolimus-eluting and sirolimus-eluting stents in patients with native coronary artery disease: a randomized controlled trial. J Am Coll Cardiol 48 : 2440–2447.17174180 21 Tada T , Byrne RA , Cassese S , King L , Schulz S , et al . (2013) Comparative efficacy of 2 zotarolimus-eluting stent generations: resolute versus endeavor stents in patients with coronary artery disease. Am Heart J 165 : 80–86.23237137 22 Stone GW , Moses JW , Ellis SG , Schofer J , Dawkins KD , et al . (2007) Safety and efficacy of sirolimus- and paclitaxel-eluting coronary stents. N Engl J Med 356 : 998–1008.17296824 23 Pfisterer M , Brunner-La Rocca HP , Buser PT , Rickenbacher P , Hunziker P , et al . (2006) Late clinical events after clopidogrel discontinuation may limit the benefit of drug-eluting stents: an observational study of drug-eluting versus bare-metal stents. J Am Coll Cardiol 48 : 2584–2591.17174201 24 De Luca G , Dirksen MT , Spaulding C , Kelbaek H , Schalij M , et al . (2012) Drug-eluting vs bare-metal stents in primary angioplasty: a pooled patient-level meta-analysis of randomized trials. Arch Intern Med 172 : 611–621.22529227 25 Palmerini T , Biondi-Zoccai G , Della Riva D , Stettler C , Sangiorgi D , et al . (2012) Stent thrombosis with drug-eluting and bare-metal stents: evidence from a comprehensive network meta-analysis. Lancet 379 : 1393–1402.22445239 26 Alazzoni A , Al-Saleh A , Jolly SS (2012) Everolimus-Eluting versus Paclitaxel-Eluting Stents in Percutaneous Coronary Intervention: Meta-Analysis of Randomized Trials. Thrombosis 2012 : 126369.22655192 27 Martin-Reyes R , Moreno R , Sanchez-Recalde A , Navarro F , Franco J , et al . (2012) Comparison of the safety between first- and second-generation drug eluting stents: meta-analysis from 19 randomized trials and 16,924 patients. Int J Cardiol 160 : 181–186.21546100	24039815	PMC3764147	CC BY	2023-06-01 23:50:15	yes	PLoS One. 2013 Sep 5; 8(9):e72895
==== Front Cancer Cell IntCancer Cell IntCancer Cell International1475-2867BioMed Central 1475-2867-13-792393769310.1186/1475-2867-13-79Primary ResearchDiagnosis, classification and grading of canine mammary tumours as a model to study human breast cancer: an Clinico-Cytohistopathological study with environmental factors influencing public health and medicine Shafiee Radmehr 1rshafiee1366@gmail.comJavanbakht Javad 2javadjavanbakht@ut.ac.irAtyabi Nahid 2natyabi@yahoo.comKheradmand Pegah 3pegahkheradmand@yahoo.comKheradmand Danial 4danialkheradmand@yahoo.comBahrami Alimohammad 5am.bahrami@ilam.ac.irDaraei Hasti 6Hdaraei@gmail.comKhadivar Farshid 1farshid.khadivar@gmail.com1 Faculty of Veterinary Medicine, Tehran University, Tehran, Iran2 Department of Pathology, Faculty of Veterinary Medicine, Tehran University, Tehran, Iran3 Semnan University of Medical Science, Faculty of Medicine, Semnan, Iran4 MD, Graduate Islamic Azad University of Mashhad, Faculty of Medicine, Mashhad, Iran5 MD, Graduate Paraveterinary Faculty of Ilam, University of Ilam, Ilam, Iran6 Department of Environmental Health Engineering, Alborz University of Medical Sciences, Karaj, Iran2013 9 8 2013 13 79 79 15 7 2013 5 8 2013 Copyright © 2013 shafiee et al.; licensee BioMed Central Ltd.2013shafiee et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background The human “Elston and Ellis grading method” was utilized in dogs with mammary tumor to examine its relation to prognosis in this species, based on a 2-year follow-up period. Although cytopathology is widely used for early diagnosis of human neoplasms, it is not commonly performed in veterinary medicine. Our objectives in this study were to identify cytopathology criteria of malignancy for canine mammary tumors and the frequency of different types of mammary lesions and their relationship with histologic grade was investigated. Another aim of this study was to differentiate the simple and adenocarcinoma tumors from the complex or mixed tumor described by Elston and Ellis grading method. Methods The study was performed in 15 pure or mixed-breed female dogs submitted to surgical resections of mammary tumours. The mammary tumours were excised by simple mastectomy or regional mastectomy, with or without the superficial inguinal lymph nodes. Female dogs were mainly terriers (9 dogs) or mixed (3 dogs), the 3 other animals were a German shepherd, Dachshund and Pekingese. Before surgical excision of the tumour, FNAC was performed using a 0.6 mm diameter needle attached to a 10 ml syringe held in a standard metal syringe holder. The cytological sample was smeared onto a glass slide and either air-dried for May-Grünwald-stain, or ethanol-fixed for Papanicolaou stain and masses were surgically removed, the tumours were grossly examined and tissue samples were fixed in 10%-buffered-formalin and embedded in paraffin. Sections 4 μm thick were obtained from each sample and H&E stained. Results We obtained a correct cytohistological correlation in 14/15 cases (93.3%) when all cytopathological examinations were considered. Of the 15 cases examined, 2(13.3%) had well-differentiated (grade I), 6(40%) had moderately differentiated (grade II) and 7(46.7%) had poorly differentiated (grade III) tumours. Classification of all canine mammary gland lesions revealed 13(86.7%) malignant and 2(13.3%) benign tumors. The histological examination showed that the most common tumor types of mammary glands in bitches were: complex carcinoma, adenocarcinoma, malignant mixed tumour, benign mixed tumour, simple carcinoma– (5/15; 33.3%), (3/15; 20%), (3/15; 20%) and (2/15;13.3%), respectively. Simple carcinoma and cystic hyperplasia were less common - (1/15; 6.7%), and (1/15; 6.7%), respectively. Moreover, the most often tumors occur in inguinal mammary (60%) and abdominal (27%) glands. Conclusions Our results demonstrate that, because of the similarity of the cytohistopathological findings in the human and canine mammary gland tumours, it is possible to use the same cytopathological criteria applied in human pathology for the diagnosis of canine mammary gland tumours. Furthemoer, routine use of this human grading method would help the clinician to make a more accurate prognosis in the interests of post-surgical management in dogs with mammary carcinomas. Furthermore, this research will allow a more discriminating classification of mammary tumors and probably has a bearing on cytohistopathology, epidemiology, pathogenesis and prognosis. The most often tumors occur in inguinal mammary (60%) and abdominal (27%) glands. This interesting regional difference may be due to a) the duration of the growth before the diagnosis; b) the age of the dogs; and c) high prevelance of unspayed animals. Moreover, the most common type of tumor was complex carcinoma – 33.3% (5 cases). Grading of tumoursCytohistopathologyDogMammary glandsTumour ==== Body Background Mammary gland tumours are one of the most common neoplasms in female dogs [1]. These spontaneous tumors are very similar to those in humans, but the incidence rate among bitches is 3 times larger than in women [2], and there are some molecular and biological similarities between canine and human mammary tumours [1-4]. Malignant cases occur in approximately half of canine mammary tumours (CMTs) [5,6], with a 3-fold higher incidence when compared with breast tumours in human females.2 Similar to their human counterparts, these tumours occur almost exclusively in females, and only rarely in male dogs [2]. Generally, in canine malignant mammary tumours (CMMT), metastatic spread occurs via lymphatic vessels to the regional lymph nodes or haematogenously and the lungs are the most frequent site of distant metastases [7]. A number of new methods are therefore tested within tumor pathology to improve the diagnostic and prognostic accuracy. To make a diagnosis, a number of clinical and paraclinical tests are used because it is essential to find the most aggressive malignant tumors that need radical surgery and supplementary treatment as soon as possible. After excision, a hematoxylin-eosin stain is often used to provide a histopathological diagnosis, but even though this is performed by experienced pathologists, the disorderly structure of some mammary tumors may cause confusion and may lead to an erroneous diagnosis of malignancy [8-11]. Anamnesis and physical examination coupled with epidemiological findings have an important role in the diagnosis and prognosis. Radiographic screening, surgical biopsy or aspiration biopsy are beneficial tools for early diagnosis of tumours. Although the first attempt is to make a differential diagnosis with inflammation or hyperplasia these tools should be used to understand the biological behavior of the mass that also contribute to the prognosis [12]. Cytological examination has important benefits in clarifying some aspects in early diagnosis of mammary lesions. This procedure is commonly used in palpable lesions such as mammary glands, thyroid, lymph nodes and salivary glands. It also prevents the need for a surgical attempt and complications that might occur during surgery [13]. Fine needle aspiration cytology (FNAC) is a method largely used to obtain samples for cytological diagnosis in several organs. When applied to mammary gland lesions, the method shows good diagnostic accuracy. Moreover, it is a simple, rapid and low cost method, with minimal risk for the patient [11-13]. Paget (1854) [14] was the first to use samples of aspirated mammary tumours for microscopic examination. In veterinary medicine, the use of cytology as a diagnostic method is very recent and has been growing in the last 20 years [7]. Our objectives in this study were to identify cytopathology criteria of malignancy for canine mammary tumors and the frequency of different types of mammary lesions and their relationship with histologic grade was investigated. Another aim was to differentiate the simple tumor from the complex or mixed tumor described by Elston and Ellis grading method. Results Macroscopic details and findings The macroscopic specifications of the multifarious mammary masses were summarized in the Figure 1 and Table 1. The preferential localisation of mammary neoplasms were the inguinal lobes (60% of cases), abdominal lobes (%27) and thoracic lobes (%13). Furthermore, 45% of the inguinal masses affected the cranio-inguinal lobe, %33 caudo-abdominal lobe and %22 cranio and caudo abdominal lobes, whereas thoracic masses were found in two cases and abdominal masses in four of cases. Eventually, % 67 of tumour masses were found in the left mammary lobes and %33 in the right mammary lobes. Eighty % of mammary tumours exhibited a small size, with weight inferior to 50 g but a relatively high proportion of these masses (20%) weighted more than 100 g, even reaching 110 g and 180 g (cases n° 13 and 17 respectively). The majority of the tumours showed a hard or an elastic consistency but some of them appeared fluctuant (cases n°3, 6 and 8). In the great majority of cases (93%), the aspect of the tumour on the cut surface was grayish-white and lobed. Some cystic structures or blood spot districts areas were also often found (in 40% and 20% of cases, respectively) (Figure 1). Figure 1 The relative risk of neoplasms of the mammary gland in female dogs in individual age and tumor size categories. Table 1 Cytological and histopathological analysis of pre-operative and sampled during surgery from the mammary neoplasms of the 15 females dogs together with signalment of bitches included in the this study The number of cases of mammary tumours in 15 female dogs Tumour localisation Cytological type Cytological classification Histopathological type Histopathological classification 1 Right cranio and caudo inguinal lobes / left caudo-inguinal lobe carcinoma Malignant Complex carcinoma Malignant 2 Left thoracic lobe carcinoma Malignant Malignant mixed tumour Malignant 3 Left cranio abdominal lobe Benign secretory Benign Benign mixed tumour Benign 4 Left cranio and caudo inguinal lobes carcinoma Malignant Complex carcinoma Malignant 5 Right cranio inguinal lobe carcinoma Malignant Complex carcinoma Malignant 6 Left caudo inguinal lobe carcinoma Malignant Simple carcinoma Malignant 7 Left cranio inguinal lobe adenocarcinoma Malignant Malignant mixed tumour Malignant 8 Left cranio and caudo inguinal lobe and right caudo-inguinal lobe carcinoma Malignant Complex carcinoma Malignant 9 Right caudo inguinal lobe Benign secretory Benign Benign mixed tumour Benign 10 Left cranio inguinal lobe carcinoma Malignant Complex carcinoma Malignant 11 Left cranio and caudo abdominal lobes carcinoma Malignant Solid adenocarcinoma Malignant 12 Right cranio inguinal lobe hyperplasia Benign Cystic hyperplasia Malignant 13 Left cranio inguinal lobe carcinoma Malignant Malignant mixed tumour Malignant 14 Right cranio-abdominal lobe adenocarcinoma Malignant Papillary adenocarcinoma Malignant 15 Left thoracic lobe adenocarcinoma Malignant Solid adenocarcinoma Malignant Cytology findings All the tumor masses were divided into four cytologic groups: hyperplasia (one case), adenocarcinoma (2 cases), carcinoma (9cases), benign secretory (3 cases) (Figure 2). Figure 2 Cytological analysis of pre-operative fine needle aspirates and histopathological analysis of mammary tumour masses sampled during surgery in the 15 females dogs. In all cases with malign characteristics (malign (dogs n°2,4,5,6,8,10,11,12,13 and 15), and malign suspected masses(dogs n°1,7 and 14)), clusters of cells with anisocytosis, anisokaryosis and hyperchromasia were observed (Figure 3A). In the other malignant tumours, some nuclear anomalies were identified such as double nucleus in 8 samples (53%) of malignant tumours (Figure 3B, 3C, 3E and 3F), giant nucleus in 8 samples (53%), mitotic figures in 7 samples (46%) and abnormal chromatin structures in 4 samples (26%). In 5 cases spindle shaped cells were associated with tumour cells (33%) (Figure 3E and 3G). In the 2 benign tumours (dog n°3 and 9), the mammary gland structure remained uniform (Figure 3H). Figure 3 Evaluation of accuracy of fine needle aspiration cytology for diagnosis of canine mammary tumours. A: Fine-needle aspirate from a mammary carcinoma in adog. Variation in cell (anisocytosis) and nuclear. (anisokaryosis) size are present, May-Grunwald-Giemsa staining method, 1000X. B: Adenocarcinoma: Malignant mammary epithelial cells: Fine needle aspirate with hypercellular pleomorphic, large hyperchromatic naked cells with coarse and abundant chromatin granules and vacuolar changes, May-Grunwald-Giemsa staining method, 1000X. C: Malignant multinucleated mammary epithelial cell; nuclei exhibit nuclear criteria of malignancy; nuclei superimposed and in different focal planes. May-Grunwald-Giemsa staining method, 1000X. D: Cytological appearance of spindle shape cells, May-Grunwald-Giemsa staining method, 400X. E: Myoepithelial cells (spindle shape) with abundant chromatin granules (red arrows) in adenocarcinoma, May-Grunwald-Giemsa staining method, 1000X. F and G: This cluster of cells shows multinucleated cells containing several irregularly sized nucleiare found in some cases, May-Grunwald-Giemsa staining method, 1000X. H: Fine-needle aspiration biopsy. Benign mammary tumor epithelial cell cluster. Cytological appearance of uniform epithelial cells, May-Grunwald-Giemsa staining method, 400X. Histopathology findings All the tumor samples were divided into six histopathologic groups: hyperplasia (one cases), adenocarcinoma (3 cases), complex carcinoma (5), simple carcinoma (1), benign mixed tumor (2 cases), and malignant mixed tumor (3cases) (Figure 4A), adenocarcinomas was further divided into papillary (one adenocarcinomas), and solid (two adenocarcinomas) types. According to their maximum diameter, the tumours were classified as T1 in 8/15 (53.3%), T2 in 3/15 (20%) and T3 in 4/15 (26.7%) dogs. The most frequently represented tumour type was complex carcinoma (5/15; 33.3%), followed by adenocarcinoma (3/15; 20%), malignant mixed tumor (3/15; 20%), benign mixed tumor (2/15;13.3% cases), simple carcinoma (1/15; 6.7%)and cystic hyperplasia (1/15; 6.7%), as presented in Table 1 (Figure 2). The histological grades of the 15 cases were as follows: grade I, 2(13.3%); grade II, 6(40%); grade III, 7(46.7%) with high mitotic index. The relationship between tumour grading and histological type is presented in Table 2. Of the 15 dogs in which mammary examination was performed, 2 had well-differentiated grade I tumours (Cystic hyperplasia and benign mixed tumour), 6 had moderately differentiated grade II (1 papillary adenocarcinoma, 3 complex carcinoma, 1 benign mixed tumour and 1 malignant mixed tumor) and 7 had poorly differentiated grade III (1 solid adenocarcinoma, 1 papillary adenocarcinoma, 1 simple carcinoma, 2 complex carcinoma and 2 malignant mixed tumor) (Table 2). Figure 4 Histopathological evaluation of mammary gland tumours in dogs. A: Solid adenocarcinoma; The presence of neoplastic, mitotic and inflammatory cells (H&E, X 200.), B: Cartilage tissue cells due to metaplasia in malignant mixed tumour H&E, X 400. C: Myoepithelial reaction included with spindle shape cells in carcinoma, H&E, X 400. D and E: A cystic tumour of adenocarcinoma showing local invasion of interlobular connective tissue and note the high mitotic index and also different mitotic figures (H&E, (H&E, × 400 and 200). F: Papillary adenocarcinoma proliferating neoplastic epithelial cells in the form of papillary projections and presence of mitotic figures. H&E, 200×, G and H: Beinign mixed mammay tumour. Tumour tissue showing epithelial and myoepithelial components, 400×, I: The presence of neoplastics emboli within the dermal lympathic vessels, which was occasionally observed with some of the most aggressive adenocarcinoma, leads to blockage of the superficial dermal lymphatic drainage (H&E, X 200), J: Cholesterol cleft in malignant mixed tumour, (H&E, X 400). K: Cells exhibit variable numbers of mitoses are found; or, the second population of cells may have oval to fusiform vesicular nuclei with an extensive amount of eosinophilic cytoplasm and distinct cells margins (H&E, X 400) L: Haemorrhage foci and the central necrotic areas are interpreted as an indication that the neoplastic cells are growing faster and that there is therefore a higher risk of progression to invasive carcinoma (H&E, X 400). Table 2 Relationship between histological grading and tumour type together with number and percentage of cases in 15 dogs with mammary tumour Histological type Grade I Grade II Grade III Total Cystic hyperplasia 1(6.7%) _ _ 1(6.7%) Solid adenocarcinoma _ _ 1(6.7%) 1(6.7%) Papillary adenocarcinoma _ 1(6.7%) 1(6.7%) 2(13.3%) Simple carcinoma _ _ 1(6.7%) 1(6.7%) Complex carcinoma _ 3(20%) 2(13.3%) 5(33.3%) Benign mixed tumour 1(6.7%) 1(6.7%) _ 2(13.3%) Malignant mixed tumor _ 1(6.7%) 2(13.3%) 3(20%) Total 2(13.3%) 6(40%) 7(46.7%) 15(100%) Of the 15 canine mammary cancers (CMTs) included, 13 of the 15 cases exhibited a range of morphologies, a highly pleomorphic cell population and polygonal were a prominent feature of all neoplasms, accounting for greater than 86.7%of the tumour cell population in most cases. Also, in the group of CMTs, 46.7% (7/15) of cases were composed of highly cellular areas with a homogeneous population of spindle cells (Figure 4B, 4C and 4D). On the other hand, a high mitotic rate (More than three mitotic figures per high-power field (400×) was identified in 73.37% (11/15cases), with atypical mitoses conspicuous in all tumours (Figure 3E and 4E). In addition, 11 of the 15 (73.37%) CMTs cases showed necrotic foci and10 of the 15 (66. 7%) CMTs cases showed infiltrates of various numbers inflammatory foci primarily consisting of lymphocytes, plasma cells, and neutrophils cells (Figure 4G). Furthermore, in the group of CMTs, 66.7% (10/15) of cases exhibited haemorrhage localized in the different regions of the tumor tissue (Figure 4L). Most CMTs this study increased mitotic activity, cellularity, nuclear pleomorphism and the presence of lesional necrosis are ominous features and suggest an increased risk of local recurrence (Figure 4I). According to local invasiveness, 33.3% of the tumours (5 out of 15) were found (Figure 3K and 4J). In addition, the neoplastic cells within the blood vessels were observed as well (Figure 4I). Moreover, 7 of the 15(46.7%) CMTs cases revealed that these tissue sections were comprised of cancerous epithelial cells characterized by hyperchromasia, enlarged nuclei, prominent nucleoli of mammary gland. Also, 4 of the 15(26.7%) CMTs cases exhibited cholesterol clefts in the lumina of the ducts (Figure 4J). Discussion Clinical and cytopathological similarities between canine mammary tumours and human breast cancer have been described in recent decades [7]. Considering the breed distribution, cross breeds, terrier, mixed, German shepherd, Dachshund and Pekingese were predominant, which is similar as in other studies [15-17]. The age at diagnosis ranged from 6 to 14 years, with a median of 10 years. This interval of risk age is in agreement with other studies [17-20]. Mammary tumors are the most common neoplasms in female dogs1. Malignant tumors may carry a poor prognosis and necessitate surgery. Few data are available on the value of cytologic examination as a diagnostic or prognostic tool for mammary tumors in dogs. FNAC is considered a fast, accurate and cost-effective method for the diagnosis of human mammary tumours [8-11]. However, the evaluation of its accuracy is poorly reported in veterinary medicine. There is a difference concerning the frequency of lesions diagnosed in human versus the canine mammary gland. We performed a validity study to further characterize sensitivity and specificity values, as well as the accuracy of FNAC in the diagnosis of CMTs. In our study, we found 93.3% cytological and histological diagnostic agreement. In previously reported studies of the canine mammary lesions, the agreement between the cytological and histological diagnosis ranged from 25% to 47% [5,21-23]. These results are low when compared with results of studies of human breast lesions published by Choi et al. [24] and Ciatto et al. [25]. They described high levels of agreement between cytological and histological diagnoses, ranging from 64.8% to 74.1%. In addition, when the authors excluded the inconclusive cases, their level of agreement increased to 93.1 and 96.7%, respectively. The results were at variance with findings of Simeonov and Stoikov [26], who reported 84.6% of correlation between cytological and histological diagnoses of mammary tumours. In some studies, the fine needle aspiration cytology specimens contained many individual bizarre, multi-nucleated, and/or giant cells having hyperchromatic pleomorphic nuclei, prominent nucleoli, and relatively abundant cytoplasm, admixed with numerous mitotic figures in a hemorrhagic or inflammatory background in human. A small amount of sheet-like or three-dimensional clusters of malignant cells coexisted [20,22]. Histopathologic examination is considered the gold standard for the diagnosis of CMTs. The histological analysis of CMTs usually includes a spindle cell component. However, according to Allen et al. 1986, the presence of spindle cells in cytological samples of breast neoplasms is not restricted to mixed tumours, as these cells may be observed in other breast lesions, including myofibroblastomas, fibromatoses and even spindle cell carcinomas [27]. Despite the similar cytological and histological features between canine and human mixed tumours of mammary gland, in canines, these tumours are very common, while in humans they are very rare. Most of the canine mammary tumours are benign or malign mixed tumours that are composed with epithelial and myoepithelial proliferations with generally cartilage, bone and squamous metaplasia [28]. Allen et al. 1986 reported that the existence of spindle shaped cells within cytological aspirates should not be limited to mixed tumours as these cells might also exist in other mammary lesions such as myofibroblastoma . Haziroglu et al., 2010, present spindle shaped cells reported in one case of malignant mixed tumour and in one case of complex carcinoma [29]. In the present study, spindle shaped cells were encountered in two cases of malignant mixed tumour, in two cases of complex carcinoma and in one cases of solid adenocarcinoma, agreeing in this way with the previous reports. Histopathological examination of the biopsy specimens was established as the most reliable diagnostic approach and revealed the characteristics of the tumour in many terms, which included pleomorphism, mitotic index, differentiation level, presence of necrosis, and the stromal invasion (the infiltration with neoplastic cells of the blood and lymph vessels and the cutaneous and soft tissue and the sur-gical margins). This data have been accepted as a golden standard in diagnosis due to its great importance in terms of the biological behaviour and the prognostic outcome of the neoplasia [30]. According to some authors, [31-34] tumors might have the potential to feed themselves via alternative pathways by vascular channels covered by deregulated neoplastic cells. The presence of neoplastic emboli within the dermal lymphatic vessels, which was occasionally observed with some of the most aggressive CMTs, leads to blockage of the superficial dermal lymphatic drainage. The outcome is a clinical presentation that resembles an inflammatory process (inflammatory mammary cancer), which has a poor prognosis and a rapid, fatal clinical course, since all the available treatments are usually palliative [35-37]. The central necrotic areas are interpreted as an indication that the neoplastic cells are growing faster and that there is therefore a higher risk of progression to invasive carcinoma [38,39]. Histopathological diagnosis of CMTs is crucial in prediction of tumour behaviour after surgical excision. Moreover, histopathologic typing of the tumour is also important in establishing a post-operative chemotherapy plan to increase the survival time following the surgery since several protocols have been used with success in dogs [40]. Various classification systems [41-43] have been developed to estimate the prognosis of the disease. Several studies revealed that half (42.0-55.0%) of the surgically removed mammary tumors in bitches were malignant 7. Meuten reported that about 20.0-40.0% of bitches with mammary tumors developed malignant kinds. 49 Although Simeonov and Stoikov reported that only 19.0% benign and 81.0% mammary tumors were malignant [26]. Moreover, Tavasoly et al., 2013 reported that all samples (n = 37) were malignant. In the present study only 13.3% benign and 86.7% mammary tumors were malignant [44]. Tavasoly et al., 2013 reported, 86.5% (n = 32), and 13.5% (n = 5) of mammary tumors were carcinomas and sarcomas, respectively. The most frequently represented tumor type was simple carcinoma 56.8% (n = 21), followed by complex carcinoma 13.5% (n = 5), sarcoma 13.5% (n = 5), carcinoma arising from benign tumor 10.8% (n = 4) and special type of carcinoma 5.4% (n = 2). Rezaie et al. found that 70.6% of bitches had tubulopapillary carcinoma, 23.5%- solid carcinoma, and 5.9% - cribriform carcinoma [45] . Ežerskytė et al. indicated that the most common tumor types of mammary glands in bitches were simple carcinoma, complex carcinoma and carcinosarcoma 46.0%, 27.0% and 13.0%, respectively [46]. In the present study, the most frequently represented neoplasm type was complex carcinoma (5/15; 33.3%), followed by adenocarcinoma (3/15; 20%), malignant mixed tumor (3/15; 20%), simple carcinoma (1/15; 6.7%)and cystic hyperplasia (1/15; 6.7%). However, in spite of this high percentage of malignant mammary tumors, according to WHO classification, the vast majority of malignant tumors were well differentiated adenocarcinomas, mostly complex and tubulopapillary, whereas special types of carcinomas and sarcomas were rare, which is similar as in other studies [16,17,47-49]. The measurement of only one of the parameters (variations of nucleus dimensions, giant nucleus formation, nucleus / cytoplasm distortion and rate, nuclear pleomorphism, changes in chromatin structures (altered dimensions, irregular chromatin shapes in nucleus, clearing of the parachromatin areas), variation in nucleolus number, abnormal nucleolus shape and presence of macronucleolus, mitotic count) associated with histological grade is unlikely to provide powerful prognostic information [50-52]. Complete histological grading is therefore preferable to nuclear grading for accurate prognosis. In the present study the main criteria retained to determine malignancy were tubule formation, the nucleus pleomorphism and dimension and a significant variation in the mitotic rates. Most grading systems of mammary carcinomas in dogs are a modification of the numeric method of Elston and Ellis. In the present study, a correlation between histological type and grade was evident. Carcinomas with a comparatively favourable prognosis, such as Complex carcinomas [30,53], were usually of grade II or III. On the other hand, simple carcinoma (the most malignant type) was usually grade III. Similar observations were reported in human patients by Elston and Ellis [51]. Because of the diversity of histological typing criteria, grading methods and endpoints used in different studies on the prognostic value of histological grading in canine mammary cancer, the results of such studies are difficult to compare [5,42,53]. In the only study 58 similar to ours(Due to the high percentage of tumor growth in the grade III), 50% of dogs with grade I mammary tumours, 64% with grade II, and 79% with grade III died within 2 years of surgical treatment. These results differed from our findings, possibly due to the use of a less refined grading method and the inclusion of sarcomas, which have the least favorable prognosis of all mammary tumours [50,54]. Conclusions Our results demonstrate that, because of the similarity of the cytological findings in human and canine mammary gland tumours, it is possible to use the same cytological criteria applied in human pathology for the diagnosis of canine mammary gland tumours. This study is hoped to open the way up for further cytopathology studies. This study demonstrated that the Elston and Ellis method of histological grading in canine mammary tumor is a reliable prognostic factor. That is correlated with histopathological classification. Histological grading of canine mammary carcinomas by the Elston and Ellis method was significantly related to prognosis, especially in cases of simple carcinoma. Its routine use should be helpful in indicating appropriate post-surgical treatment. The estimation of the proliferative activity of tumours by well standardized mitotic counting techniques should have a central position in histopathology research and practice. Tumors of the mammary glands were most common in 6 – 14 year old bitches. The most often tumors occur in inguinal mammary (60%) and abdominal (27%) glands. This interesting regional difference may be due to a) the duration of the growth before the diagnosis; b) the age of the dogs; and c) high prevelance of unspayed animals. The most common type of tumor was complex carcinoma – 33.3% (5 cases). Materials and methods Animals characteristics The study was performed in 15 pure or mixed-breed female dogs submitted to surgical resections of ‘mammary tumours’ in the Veterinary School Hospital of Tehran University Faculty with the complaints of mass existence in different mammary lobes. The animals, aged 6–14 years (mean ± SD = 10.5 ± 1.8), showed with or without clinical or radiological evidence of distant metastasis. Female dogs were mainly terriers (9 dogs) or mixed (3 dogs), the 3 other animals were a German shepherd, Dachshund and Pekingese. They were selected from cases treated surgically between July 2011 and February 2013. The mammary tumours were excised by simple mastectomy or regional mastectomy [50], with or without the superficial inguinal lymph nodes. Cytological evaluation Before surgical excision of the tumour, Fine needle aspiration cytology (FNAC) was performed using a 0.6 mm diameter needle attached to a 10 ml syringe held in a standard metal syringe holder. The cytological sample was smeared onto a glass slide and either air-dried for May-Grünwald-stain, or ethanol-fixed for Papanicolaou stain. Subsequently, dogs were induced with propofol (4 mg/kg, IV, Propofol, Abbott) and anaesthetized with isoflurane (2-3%, Isoflurane, Adeka) and masses were surgically removed, the tumours were grossly examined and tissue samples were fixed in 10%-buffered-formalin and embedded in paraffin. Sections 4 μm thick were obtained from each sample and H&E stained. The cytopathological criteria adopted were those proposed by Bibbo [55] and for histopathological analysis of tumors it was used the Veterinary [30] and Human [56] classification. We considered the histopathological diagnosis as the ‘gold standard. Clinical and histopathological evaluation Tumour size: Mammary neoplasms were classified by size according to the World Health Organization Clinical Staging System TNM 19, as T1 (<3 cm maximum diameter), T2 (3–5 cm maximum diameter) and T3 (>5 cm maximum diameter). In cases of multiple tumours, the largest one was used as the basis for classification. Tumour type: Representative sections of each tumour (from the central core to periphery) and the excised lymph nodes were fixed in 10% buffered formalin, processed by routine methods, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin (HE). Histopathological findings were recorded and used to classify the tumours according to the criteria of a recently validated system [30]. In cases with multiple tumours, the most malignant one as defined by Misdorp [50] was recorded. Tumour grade: Histological grading was performed on HE-stained sections. According to the Elston and Ellis method [51], the grade for each case was derived from an assessment of (1) tubule formation, (2) nuclear pleomorphism, and (3) mitotic counts, each feature being scored 1 to 3 points. The scores were then added to obtain the tumour grade, as follows: 3–5 points, well-differentiated (grade I); 6–7 points, moderately differentiated (grade II); 8–9 points, poorly differentiated (grade III). Grading was carried out by one veterinary pathologist and, without prior knowledge of the results, confirmed by a second pathologist. Classification of tumours The final diagnosis was classified in the protocol according to the following five categories: (1) benign, (2) suspicious-probably benign, (3) suspicious-probably malignant, (4) malignant and (5) insufficient/inadequate material for the diagnosis. However, to establish a comprehensive histological correlation, the two categories of suspicious cases were classified in a generic group entitled ‘suspicious-not otherwise specified. Abbreviations CMTs: Canine mammary tumours; CMMT: Canine malignant mammary tumours; FNAC: Fine needle aspiration cytology; HE: Haematoxylin and eosin. Competing interests The authors declare that they have no competing interests. Authors’ contributions NA and RSH participated in the histopathological evaluation, performed the literature review, acquired photomicrographs and drafted the manuscript and gave the final histopathological diagnosis and designed and carried out all the experiments. JJ is the principal investigator of the laboratory in which the research was performed and contributed to the interpretation of the data and writing of the manuscript. PKH, DKH, AMB, HD and FKH edited the manuscript and made required changes and wrote the manuscript. All authors have read and approved the final manuscript. Acknowledgements The authors thank staff of the Department of pathology, Faculty of Veterinary Medicine, Tehran University for their valuable technical assistance. ==== Refs Moe L Population-based incidence of mammary tumours in some dog breeds J Reprod Fertil Suppl 2001 57 439 443 11787188 Egenvall A Bonnett BN Ohagen P Olson P Hedhammar A von Euler H Incidence of and survival after mammary tumors in a population of over 80,000 insured female dogs in Sweden from 1995 to 2002 Prev Vet Med 2005 69 109 127 10.1016/j.prevetmed.2005.01.014 15899300 Rivera P Biochemical Markers and Genetic Risk Factors in Canine Tumors 2010 Uppsala: Doctoral Thesis Swedish University of Agricultural Sciences http://www.diss-epsilon.slu.se:8080/archive/00002280/01/rivera_p_100503.pdf. Accessed 2010;29.8.11 Desantis C Siegel R Bandi P Jemal A Breast cancer statistics CA Cancer J Clin 2011 61 409 418 21969133 Brody RS Goldschmidt NH Roszel JR Canine mammary gland neoplasms J Am Anim Hosp Assoc 1983 35 5 1961 1990 Brunelle M Cyclooxygenase-2 expression in normal and neoplastic canine mammary cell lines Vet Pathol 2006 43 5 656 666 10.1354/vp.43-5-656 16966442 Sorenmo K Canine mammary gland tumors Vet Clin Small Anim 2003 33 573 596 10.1016/S0195-5616(03)00020-2 Cyrta J Andreiuolo F Azoulay S Balleyguier C Bourgier C Mazouni C Mathieu MC Delaloge S Vielh P Pure and mixed mucinous carcinoma of the breast: fine needle aspiration cytology findings and review of the literature Cytopathology 2012 32 6 e64 8 de Graaf H Willemse P Laddé BE Van den Bergen HA Krebber M Tjabbes T Sluiter WJ Evaluation of a cytological scoring system for predicting histological grade and disease-free survival in primary breast cancer Cytopathology 1994 5 5 294 300 10.1111/j.1365-2303.1994.tb00433.x 7819514 Bofin AM Lydersen S Isaksen C Hagmar BM Interpretation of fine needle aspiration cytology of the breast: a comparison of cytological, frozen section, and final histological diagnoses Cytopathology 2004 15 6 297 304 10.1111/j.1365-2303.2004.00159.x 15606361 Gupta K Sood NK Uppal SK Mohindroo J Mahajan S Raghunath M Singh K Epidemiological Studies on Canine Mammary Tumour and its Relevance for Breast Cancer Studies IOSR Journal of Pharmacy 2012 2 2 322 333 10.9790/3013-0220322333 Baker R Lumsden JH Baker R, Lumsden JH The mammary gland Color Atlas of Cytology of the Dog and Cat 2000 St Louis: Mosby 253 262 Paget J: Apud McCARTHY E Giant-cell tumor of mammary: a historical perspective Histopathology 1854 153 1980 14 25 Eisenberg AJ Hajdus I Wilhelmus J Melamed MR Kinne D Preoperative aspiration cytology of breast tumors Acta Cytol 1986 30 135 146 3008470 Zatloukal J Lorenzova J Tichy F Nečas A Kecova H Kohout P Breed and age as a risk factors for canine mammary tumours Acta Vet Brno 2005 74 103 109 10.2754/avb200574010103 Hsu WL Huang HM Liao JW Wong ML Chang SC Increased survival in dogs with malignant mammary tumours overexpressing HER-2 protein and detection of a silent single nucleotide polymorphism in the canine HER-2 gene Vet J 2009 180 116 123 10.1016/j.tvjl.2007.10.013 18061495 Morris JS Nixon C King OJA Morgan IM Philbey AW Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERα and p53 Vet J 2009 179 422 429 10.1016/j.tvjl.2007.10.025 18314357 Nieto A Pena L Perez-alenza MD Sanchez MA Flores JM Castano M Immunohistologic detection of estrogen receptor alpha in canine mammary tumors: clinical and pathologic associations and prognostic signi fi cance Vet Pathol 2000 37 239 247 10.1354/vp.37-3-239 10810988 Sarli G Preziosi C Benazzi G Castellani P Marcato S Prognostic value of histologic stage and proliferative activity in canine malignant mammary tumors J Vet Diagn Invest 2002 14 25 34 10.1177/104063870201400106 12680640 Yang WY Liu CH Chang CJ Lee CC Chang KJ Lin CT Proliferative activity, apoptosis and expression of oestrogen receptor and Bcl-2 oncoprotein in canine mammary tumours J Comp Pathol 2006 134 70 79 10.1016/j.jcpa.2005.07.002 16423573 Karayannopoulou M Kaldrimidou E Dessiris A Some epidemiological aspects of canine mammary tumors treatment and prognosis. Bull. Helen Vet Med Soc 1989 40 111 121 Fidler IJ Brodey SR A necropsy study of canine malign mammary neoplasm J Am Vet Med Assoc 1967 151 710 715 5233810 Pamukcu M Erturk E Distribution of tumor types in dogs in Ankara Vet J Ankara Univ 1962 9 1 9 Choi YD Choi YH Lee JH Nam JH Juhng SW Analysis of Fine needle aspiration cytology of the breast Acta Cytol 2004 48 801 806 10.1159/000326449 15581165 Ciatto S Morrone D Catarzi S Del Turco MR Bianchi S Ambrogetti D Cariddi A Radial scars of the breast: review of 38 consecutive mammographic diagnoses Radiology 1993 187 3 757 60 8388568 Simeonov R Stoikov D Study on the correlation between the cytological and histological tests in the diagnostics of canine spontaneous mammary neop-lams Bulgarian Journal of Veterinary Medicine 2006 9 211 219 Allen SW Prasse KW Mahaffey EA Cytologic differentiation of benign from malignant canine mammary tumors Vet Pathol 1986 23 649 655 3811129 Cassali GD Gobbi H Malm C Schmitt FC Evaluation of accuracy of fine needle aspiration cytology for diagnosis of canine mammary tumours: Comparative features with human tumours Cytopathology 2007 18 191 196 17573766 Haziroglu R Yardimci B Aslan S Yildirim MZ Yumusak N Beceriklisoy H Agaoglu R Kucukaslan I Cytological Evaluation of canine mammary tumours with fine needle aspiration biopsy technique Revue Méd Vét 2010 161 5 212 218 22480111 Misdorp W Else R Hellmen E Lipscomb T Histologic classification of mammary tumors of the dog and cat 1999 Washington DC: Armed forces institute of pathology 3 6 Sood AK Seftor EA Fletcher MS Gardner LM Heidger PM Buller RE Seftor RE Hendrix MJ Molecular determinants of ovarian cancer plasticity Am J Pathol 2001 158 1279 1288 10.1016/S0002-9440(10)64079-5 11290546 Shirakawa K Tsuda H Heike Y Kato K Asada R Inomata M Sasaki H Kasumi F Absence of endothelial cells, central necrosis, and fibrosis are associated with aggressive inflammatory breast cancer Cancer Res 2001 61 445 451 11212228 Maniotis AJ Folberg R Hess A Seftor EA Gardner LM Pe’er J Trent JM Meltzer PS Hendrix MJ Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry Am J Pathol 1999 155 739 752 10.1016/S0002-9440(10)65173-5 10487832 Folberg R Hendrix MJ Maniotis AJ Vasculogenic mimicry and tumor angiogenesis Am J Pathol 2000 156 361 381 10.1016/S0002-9440(10)64739-6 10666364 Marconato L Romanelli G Stefanello D Prognostic factors for dogs with mammary inflammatory carcinoma: 43 cases (2003–2008) J Am Vet Assoc 2009 235 967 972 10.2460/javma.235.8.967 Clemente M Perez-Alenza MD Illera JC Peña L Histologic, immunologic and ultrastructural description of vasculogenic mimicry in canine mammary cancer Vet Pathol 2010 47 265 274 10.1177/0300985809353167 20106772 De M Souza CH Toledo-Piza E Amorin R Barboza A Tobias KM Inflammatory mammary carcinoma in 12 dogs: clinical features, cyclooxygenase-2 expression, and response to piroxicam treatment Can Vet J 2009 50 5 506 510 19436636 Virnig BA Tuttle TM Shamliyan T Kane RL Ductal carcinoma in situ of the breast: a systematic review of incidence, treatment, and outcomes J Natl Cancer Inst 2010 102 170 178 10.1093/jnci/djp482 20071685 Vogl G Dietze O Hauser-Kronberger C Angiogenic potential of ductal carcinoma in situ (DCIS) of human breast Histopathology 2005 47 617 624 10.1111/j.1365-2559.2005.02299.x 16324200 Karayannopoulou M Kaldrymidou E Constantinidis TC Dessiris A Adjuvant post-operative chemotherapy in bitches with mammary cancer J Vet Med A 2001 48 85 96 10.1046/j.1439-0442.2001.00336.x Hampe JF Misdorp W Tumours and dysplasias of the mammary gland. B. World. Health Organ 1974 50 111 133 Gilbertson SR Kurzman ID Zachrau RE Canine mammary epithelial neoplasms: biological implications of morphologic characteristics assessed in 232 dogs Vet Pathol 1983 20 127 142 6836870 Misdorp В Else R Hellmen E Lipscomb T Histological classification of mammary tumors of the dog and cat. WHO International Histological Classification of Tumors in Domestic Animals. 2nd series, vol. VII 2001 Washington DC: Armed Forces Institute of Pathology, American Registry of Pathology Tavasoly A Golshahi H Rezaie A Farhadi M Classification and grading of canine malignant mammary tumors Veterinary Research Forum 2013 4 1 25 30 Rezaie A Tavasoli A Bahonar A Mehrazma M Grading in canine mammary gland carcinoma J Biol Sci 2009 9 333 338 10.3923/jbs.2009.333.338 Ežerskytė A Zamokas G Grigonis A The retrospective analysis of mammary tumors in dogs Vet Med Zoot 2011 53 75 3 8 Rungsipipat A Tateyama S Yamaguchi R Immunohistochemical analysis of c-yes and c-erbB-2 oncogene products and p53 tumor suppressor protein in canine mammary tumors J Vet Med Sci 1999 61 27 32 10.1292/jvms.61.27 10027159 Millanta F Calandrella M Bari G Comparison of steroid receptor expression in normal, dysplastic and neoplastic canine and feline mammary tissues Res Vet Sci 2005 79 225 232 10.1016/j.rvsc.2005.02.002 16054892 Thuroczy J Reisvaag GJK Perge E Tibold A Szilagyi J Balogh L Immunohistochemical detection of progesterone and cellular proliferation in canine mammary tumours J Comp Pathol 2007 137 122 129 10.1016/j.jcpa.2007.05.005 17645888 Misdorp W Meuten D Tumors of the mammary gland Tumors in Domestic Animals 2002 4 Ames, Iowa, USA: Iowa State Press 575 607 Elston CW Ellis IO Pathological prognostic factors in breast cancer I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 1991 19 403 410 Karayannopoulou M Kaldrymidou E Constantinidis TC Histological grading and prognosis in dogs with mammary carcinomas: application of a human grading method J Comp Pathol 2005 133 4 246 252 10.1016/j.jcpa.2005.05.003 16202421 Benjamin SA Lee AC Saunders WJ Classification and behavior of canine mammary epithelial neoplasms based on life-span observations in beagles Vet Pathol 1999 36 423 436 10.1354/vp.36-5-423 10490210 MacEwen E Withrow S Withrow S, MacEwen E, Saunders WB Tumors of the mammary gland Small Animal Clinical Oncology 1996 2 Philadelphia pp. 356 372 Bibbo M Comprehensive cytopathology 1997 2 Philadelphia: WB Saunders Company 413 444 Rosen PP Oberman HA Tumors of mammary gland Armed Forces Institute of Pathology 1993 Washington p390	23937693	PMC3765114	CC BY	2021-01-05 01:31:08	yes	Cancer Cell Int. 2013 Aug 9; 13:79
==== Front BMC Complement Altern MedBMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central 1472-6882-13-1832386683010.1186/1472-6882-13-183Research ArticleGastroprotective effect of desmosdumotin C isolated from Mitrella kentii against ethanol-induced gastric mucosal hemorrhage in rats: possible involvement of glutathione, heat-shock protein-70, sulfhydryl compounds, nitric oxide, and anti-Helicobacter pylori activity Sidahmed Heyam Mohamed Ali 1diamondhm@hotmail.comAzizan Ainnul Hamidah Syahadah 3ainnul_azizan@yahoo.comMohan Syam 1syammohan@yahoo.comAbdulla Mahmood Ameen 2mahmood955@yahoo.comAbdelwahab Siddig Ibrahim 4Siddigroa@yahoo.comTaha Manal Mohamed Elhassan 4manalroa@yahoo.comHadi A Hamid A 3ahamid@um.edu.my.comKetuly Kamal Aziz 3KKetuly@yahoo.comHashim Najihah Mohd 1najihahmh@um.edu.myLoke Mun Fai 5lokemunfai@um.edu.myVadivelu Jamuna 5jamuna@um.edu.my1 Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia2 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia3 Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia4 Medical Research Centre, Jazan University, P.O. Box 114, Jazan, Saudi Arabia5 Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia2013 19 7 2013 13 183 183 19 11 2012 20 6 2013 Copyright © 2013 Sidahmed et al.; licensee BioMed Central Ltd.2013Sidahmed et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Mitrella kentii (M. kentii) (Bl.) Miq, is a tree-climbing liana that belongs to the family Annonaceae. The plant is rich with isoquinoline alkaloids, terpenylated dihydrochalcones and benzoic acids and has been reported to possess anti-inflammatory activity. The purpose of this study is to assess the gastroprotective effects of desmosdumotin C (DES), a new isolated bioactive compound from M. kentii, on gastric ulcer models in rats. Methods DES was isolated from the bark of M. kentii. Experimental rats were orally pretreated with 5, 10 and 20 mg/kg of the isolated compound and were subsequently subjected to absolute ethanol-induced acute gastric ulcer. Gross evaluation, mucus content, gastric acidity and histological gastric lesions were assessed in vivo. The effects of DES on the anti-oxidant system, non-protein sulfhydryl (NP-SH) content, nitric oxide (NO)level, cyclooxygenase-2 (COX-2) enzyme activity, bcl-2-associated X (Bax) protein expression and Helicabacter pylori (H pylori) were also investigated. Results DES pre-treatment at the administered doses significantly attenuated ethanol-induced gastric ulcer; this was observed by decreased gastric ulcer area, reduced or absence of edema and leucocytes infiltration compared to the ulcer control group. It was found that DES maintained glutathione (GSH) level, decreased malondialdehyde (MDA) level, increased NP-SH content and NO level and inhibited COX-2 activity. The compound up regulated heat shock protein-70 (HSP-70) and down regulated Bax protein expression in the ulcerated tissue. DES showed interesting anti-H pylori effects. The efficacy of DES was accomplished safely without any signs of toxicity. Conclusions The current study reveals that DES demonstrated gastroprotective effects which could be attributed to its antioxidant effect, activation of HSP-70 protein, intervention with COX-2 inflammatory pathway and potent anti H pylori effect. ==== Body Background Gastric ulcer is a common disease affecting many people worldwide [1]. Some factors that are identified in the etiology of this disorder include stress, cigarette smoking, alcohol consumption, nutritional deficiencies and infections [2]. However, the over-ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) and H pylori infection remains the predominant cause of peptic ulcer disease [3]. The gastric ulcer disease was observed to correlate with changes in several physiological parameters, such as Reactive oxygen species (ROS), NO, lipid peroxidation and gastric acid over secretion [4]. Treatment of gastric ulcer is considered a clinical problem due to the increasingly widespread use of NSAIDs and low-dose aspirin [5]. Despite the effectiveness of reciprocal antiulcer drugs such as the antacids, anticholinergics, proton pump inhibitors and histamine H-2 receptor antagonists, the majority of them possess adverse effects that limit their usage [6]. Nowadays, the pursuit to discover alternative therapies to treat gastric ulcer is of high concern [7]. A large number of natural antiulcer compounds have been isolated from medicinal plants and the common chemical classes of bioactive compounds that have been identified as possessing antiulcer activity are the alkaloids, saponins, xanthones, triterpenes and tannins, among others [8]. M. kentii is a tree-climbing liana which belongs to the family Annonaceae. The plant is native to Peninsular Malaysia, several parts of Indonesia including the islands of Sumatra and Borneo as well as New Guinea. In Malaysia, M. kentii is used traditionally as a drink in the form of a root decoction to treat fever [9]. Experimentally, the plant showed anti-inflammatory activity [10]. Previous chemical studies on M. kentii resulted in the isolation of isoquinoline alkaloids [11], terpenylated dihydrochalcones [12] and four other benzoic acids [10]. As a continuation of our research for biologically active compounds for the treatment of gastric ulcer from the Malaysian flora, a hexane extract of the bark of this plant was selected for phytochemical investigations. For the first time, our study led to the isolation of DES (Figure 1) from M. kenti. It is a known compound which was previously isolated from the roots of Desmos dumosus[13] and Uvaria schefferi[14]. Figure 1 Chemical structure of desmosdumotin C. It is known that ethanol induces gastric mucosa lesions and petechial bleeding in humans [15], where ethanol is found to penetrate easily and rapidly into the gastric mucosa and causes membrane damage, exfoliation of cells, erosion and ulcer formation. It is claimed that ROS are involved in the ulcer formation caused by ethanol [16]. Ethanol-induced gastric ulcer models are commonly used to study both the pathogenesis of and therapy for human ulcerative diseases [2]. DES has a unique chalcone skeleton, and it is known that naturally occurring chalcones have shown interesting bioactivities such as antimalarial, antitumor, anti-HIV and anti-oxidant effects [17]. However, the compound has not been well-studied so far to evaluate its bioactivites, except for its significant and selective in vitro cytotoxicity toward cancer cell lines [13]. Based on these prospective activities of its chemical structure, the current study is conducted to evaluate for the first time the gastroprotective effect of DES from M. kentii and possible mechanism(s) involved against ethanol-induced ulcer model in rats. Methods Plant materials The bark of M. kentii was collected in Mersing, Johor. A voucher specimen (KL 4139) is deposited at the Herbarium of Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia. Extraction and isolation of DES The dried and powdered bark (1.0 kg) of M. kentii was extracted exhaustively with hexane using Soxhlet extractor. The hexane extract was concentrated under reduced pressure to give a residue. Hexane crude extract was subjected to column chromatography (CC). The isolation and purification of DES were carried out by chromatography on a small column silica gel (0.040-0.063 mm) using n-hexane: ethyl acetate, 9:1 as a solvent system. DES, C19H20O4, was isolated as a yellow needle crystal from a n-hexane – CH2Cl2 mixture, m.p.: 93–94 °C; UV ( methanol : 380, 242, 225 nm; IRmax (cm-1, NaCl disc) : 3401, 1657, 1624, 1577, 1513, 1426, 1371, 1243, 1153, 1122, 977, 944; EIC-MS m/z [M + H]+ (%) : 312.140729 (calc. 312.3646 for C19H20O4); 1H NMR (CDCl3, TMS) ( (ppm): 8.32 (1H, d, J = 16Hz), 7.92 (1H, d, J = 16Hz), 7.66 (2H, m, Ar-2”,6”-H), 7.37 (3H, s, Ar-3”, 4”, 5”-H), 3.93 (3H, s, OCH3), 2.02 (3H, s, Ar-CH3) 1.36 (6H, s, CH3 × 2). 13C NMR (CDCl3, TMS) ( (ppm) : 198.14 (C-1), 192.48 (C-3), 187.26 (C-1’), 176.70 (C-5), 144.94 (C-2’, 3’), 135.29 (C-1”), 130.66 (C-3”, C-5”), 128.98 (C-4”), 123.30 (C-2”, 6”), 113.68 (C-2), 106.67 (C-4), 62.23 (OCH3), 50.49 (C-6), 24.44 (CH3 × 2), 9.88 (Ar-CH3). The compound was identified by comparison of their spectroscopic data with literature values. Chemicals and drugs TPTZ, DTNB, Griess reagent were purchased from Sigma-Aldrich Chemical Co. Kuala Lumpur, Malaysia. Indomethacin and omeprazole were obtained from University of Malaya Medical Center. All other used chemicals and reagents were of analytical grade. Animals Healthy ICR mice (6–8 weeks old weighing 20–30 g) and Sprague Dawley rats (200–220 g) were obtained from the Experimental Animal House, Faculty of Medicine, University of Malaya. All procedures relating to animal care and the animal research protocols conformed to the animal care guidelines of the Institutional Animal Care and Use Committee, University of Malaya. This study specifically was presented to the institutional ethical review board (UM ICUCA) for approval, and the approval was granted [Ethic No FAR/29/06/2012/HMAS (R)]. The animals were fed standard pellets and free access to water ad libitum. All animals received human care according to the criteria outlined in the “Guide for the Care and use of Laboratory Animals “prepared by the National Academy of Sciences and published by the National Institute of Health. Acute toxicity study Thirty six mice (18 male, 18 female) were assigned equally into three groups. Overnight fasted animals received DES at doses of 30 and 300 mg/kg body weight according to Organization for Economic Co-operation and Development (OECD) Guideline 420 protocol year 1992. Animals treated with 5% Tween 80 were served as a control group. The food was withheld for further 3–4 h after dosing. During14 days of treatment, the animals were observed for any mortality or physiological changes. On day 15, body weight variation was determined and all the animals anesthetized using ketamine and xylazil to collect Serum for biochemical analysis then sacrificed. the liver and kidney were excised for histology study. Induction of acute gastric lesion To avoid coprophagy, each rat was kept in a cage with a raised floor of wide mesh and all animals divided randomly into six groups (n = 6). The animals were fasted overnight prior for oral pre-treatment (5 ml/kg b.w) as mention in Table 1. Table 1 animal group with different pretreatment Animal group Pretreatment Group (A) normal control Rats pretreated with vehicle (5% Tween 80 v/v) + vehicle) (5% Tween 80 v/v). Group (B) ulcer control Rats pretreated with vehicle (5% Tween 80 v/v) + absolute ethanol). Group (C) reference control Rats pretreated with 20 mg/kg of omeprazole in vehicle + absolute ethanol). Group (D) Rats pretreated with 5 mg/kg of DES in vehicle + absolute ethanol). Group (E) Rats pretreated with 10 mg/kg of DES in vehicle + absolute ethanol). Group (F) Rats pretreated with 20 mg/kg of DES in vehicle + absolute ethanol). The pre-treatments were administered for 1 hour, subsequently; all groups except the normal group (A) received absolute ethanol (5 ml/kg). 1 h later, the animals anesthetized using ketamine & xylazil and their blood was collected from their jugular veins for serum biochemical analysis. The animals were then sacrificed and their stomachs were removed immediately [18]. Gastroprotective assessments Each stomach of the experimental animals was opened along the greater curvature and the stomachs were washed with ice normal saline. Gastric ulcer on the gastric mucosa appears as elongated bands of hemorrhagic lesions. The length (mm) and width (mm) of each band was measured using planimeter [(10 mm × 10 mm = ulcer area) under dissecting microscope (1.8×)]. The area of each ulcer lesion was measured by counting the number of small squares, 2 mm × 2 mm, covering the length and width of each hemorrhagic band. The sum of the areas of all lesions for each stomach was applied in the calculation of the ulcer area (UA) wherein the sum of small squares × 4 × 1.8 = UA mm2. The inhibition percentage (I%) was calculated by the following formula described in [19] with slight modifications: The inhibition percentageI%=UAcontrol–UAtreated/UAcontrol×100% Gastric tolerability test Each experimental stomach was observed under an illuminated magnifier (3×) to evaluate the gastric lesions according to the modified scoring system of [20] (0:no lesions; 0.5: slight hyperaemia or ≤ 5 petechiae; 1: ≤ 5 erosions ≤ 5 mm in length; 1.5: ≤ 5 erosions ≤ 5 mm in length and many petechiae; 2: 6–10 erosions ≤ 5 mm in length; 2.5: 1–5 erosions > 5 mm in length; 3: 5–10 erosions >5 mm in length; 3.5: >10 erosions >5 mm in length; 4: 1–3 erosions ≤ 5 mm in length and 0.5-1 mm in width; 4.5: 4–5 erosions ≤ 5 mm in length and 0.5-1 mm in width; 5: 1–3 erosions > 5 mm in length and 0.5-1 mm in width; 6: 4 or 5 grade 5 lesions; 7: ≥6 grade 5 lesions; 8: complete lesion of the mucosa with hemorrhage). Determination of gastric secretion The effect of DES on gastric acid output was determined following the recommended method [21]. Briefly, Sprague Dawley rats assigned equally into five groups (n = 6). After 24 h fasting, immediately after pylorus ligature, 5% Tween 80, omeprazole (30 mg/kg), and DES (5, 10 and 20 mg/kg) were administered intraduodenally. 4 hours later, all animals sacrificed by cervical dislocation, their stomachs were removed immediately and the gastric content was collected to determined gastric secretion volume (ml), pH value using digital pH meter and total acidity by titrating with 0.01 N sodium hydroxide using phenolphthalein as indicator and was expressed as mEq/l. Measurement of mucus content The gastric mucosa of each animal was gently rubbed off using a glass slide and the weight of the collected mucus was measured using precise electronic balance [22]. Serum biochemical assays Serum samples were analyzed at University of Malaya Medical Centre using Hitachi Auto-analyzer to evaluate changes in serum biochemical parameters. Histological evaluation A small fragment of the gastric wall from each animal was fixed in 10% buffered formalin solution followed by tissue dehydrated with alcohol and xylene. Then, each sample was embedded in paraffin wax, sectioned at 5 μm in slides prior for staining. Hematoxylin and eosin (H & E) stain was used for light microscopy [23]. Moreover, to evaluate mucus production, some slides were also stained by periodic acid Schiff Base (PAS) following the manufacture instruction (Sigma Periodic Acid-Schiff (PAS) Kit). For further analysis, other slides underwent for immunohistochemistry (IHC) staining using Dako ARK™ to observed immunhistochemical localization of HSP-70 (1:100) and Bax (1:50) proteins. Both proteins were purchased from Santa Cruz Biotechnology, Inc., California, USA. Preparation of gastric tissue homogenate A specimen of gastric wall from each animal was homogenized (10%) in ice cold 0.1 mol/l phosphate buffered saline (PBS). The homogenates were centrifuged at 10,000 g for 15 min at 4°C. The pure supernatant was used to quantify the gastric tissue contents of GSH, MDA, NP-SH and NO. GSH levels Total GSH content (nmol GSH/g tissue) was estimated by interaction with DTNB (5,5 -dithiobis-2-nitrobenzoic acid) and the absorbance was read in a spectrophotometer (412 nm) [24] . Thiobarbituric acid reactive substance assay Thiobarbituric acid reactive substance (TBARS) assay was used to estimate MDA content. According to [25], the gastric homogenate was added to a 0.126 ml solution containing 26 mM thiobarbituric acid, 0.26 M HCL, 15% trichloroacetic acid and 0.02% butaylated hydroxyltoluene. The mixture was incubated in a water bath at 95°C for 1 h. After cooling, the mixture was centrifuged at 3000 g for 10 min. The absorbance was read in a spectrophotometer at 532 nm and the results were expressed in μmol/g tissue MDA. Tetramthoxy propane was used as standard. Estimation of NP-SH content Gastric mucosal NP-SH (μmol/g of tissue) were measured according to the method of [26]. Briefly, aliquots of 5 ml of the gastric homogenates were mixed with a solution containing 4 ml of distilled water and 1 ml of 50% trichloroacetic acid. The mixture was vortex for 15 min and centrifuged at 3000 × g. 2 ml of supernatant was mixed with 4 ml of 0.4 M Tris Buffer at pH 8.9; 0.1 ml of DTNB [5,5 dithiobis-(2-nitrobenzoic acid)] was added and the sample was shaken. The Absorbance was recorded within 5 min of the addition of DTNB at 412 nm against a reagent blank with no homogenate. NO level NO content was quantified by measuring nitrite/nitrate concentration using Griess assay [27]. In brief, gastric homogenates were deproteinated with absolute ethanol for 48 h at 4°C, then centrifuged at 12000 g for 15 min at 4°C. To an aliquot of the supernatant, vanadium trichloride 0.8% (w/v) in 1 M HCl was added for the reduction of nitrate to nitrite, followed by the rapid addition of Griess reagent (sigma) and the absorbance at 540 nm was measured. The results were expressed as (μmol/g tissue). Sodium nitrite was used as standard. In vitro evaluation of COX-2 inhibitory activity The COX-2 inhibitory activity of DES was estimated using a COX-inhibitor screening Kit (Cayman Chemical, USA). According to the manufacturer’s instructions, DES was dissolved in DMSO at final concentration was 0–100 μg/ml. The inhibition was calculated by the comparison of compound treated to control incubations. Indomethacin was used as reference standard. Ferric-reducing antioxidant power (FRAP) assay The FRAP value of DES was estimated according to the method of [28] with slight modification. Briefly, the FRAP reagent was prepared freshly from acetate buffer (pH 3.6), 10 mM TPTZ [ 2,4,6-Tri(2-pyridyl)-s-triazine] solution in 40 mM HCl and 20 mM iron (III) chloride solution in proportions of 10:1:1 (v/v), respectively. 50 μl of the compound were added to 1.5 ml of the FRAP reagent in the dark, 4 min later the absorbance was then recorded at 593 nm. The standard curve was constructed linear (R2 = 0.9723) using iron (II) sulfate solution (100–1000 μM), and the results were expressed as μM Fe (II)/g dry weight of the compound. DPPH assay method The scavenging activity of the DES was evaluated according to the recommended method of [29]. Briefly, the compound was mixed with 0.3 mM DPPH [2,2-diphenyl-1-picrylhydrazyl] /ethanol solution to give final concentrations of the compound (50, 25, 12.5, 6.25 μg/ml in ethanol. 30 min later, the absorbance was observed at 518 nm then converted into a percentage of antioxidant activity expressed as the inhibition concentration at 50% (IC50). In vitro anti-H pylori activity H pylori strain, J99 (ATCC 700824) was cultured with brain heart infusion broth (BHI; Oxoid) supplemented with 10% horse serum (Invitrogen) incubated at 37°C in a humidified CO2 incubator (Forma Steri-Cycle) for 3 days. Minimum inhibitory concentration (MIC) was determined by a modified microtiter broth dilution method on sterile 96-well polypropylene microtitre plates with round-bottom wells (Eppendorf). Briefly, DES was dissolved and diluted in 5% DMSO to give a 10× working stock solution. H. pylori was diluted to a final concentration of 2 × 106 CFU/ml in culture medium. Aliquots of 10 μl of DES were added to 90 μl of H. pylori in a well of the microtitre plate. Concentration of DES ranged from 31.25 to 250 μg/ml. The microtiter plate was incubated for 3 days in a CO2 incubator. The plate was examined visually and measured using a microplate reader (Varioskan Flash) at 600 nm to determine the lowest concentration showing complete growth inhibition, which was recorded as the MIC. Wells containing H. pylori with 10 μl of 5% DMSO and BHI medium containing 250 μg/ml DES, were used as control and blanks respectively. The result was recorded in accordance with the Clinical and Laboratory Standards Institute [30]. Statistical analysis All tests were performed at least in triplicates and the values were represented as mean ± S.E.M (standard error mean). The statistical differences between groups were determined according to SPSS version 16.0 and Graph Pad prism 6 using ordinary one-way ANOVA followed by Dunnetts multiple comparison tests. A value of P < 0.05 was considered significant. Results Toxicity study The toxicity study showed no toxic symptoms or mortality and there were no abnormal physiological or behavioral changes, body weight alteration at any time of observation up to 300 mg/kg during the experimental period. Histological examination to the liver and kidney and the serum biochemical analysis didn’t show any differences incomparable to the control group (data not shown but available upon request). Gross evaluation Pre-treatment with DES at doses of 5, 10, 20 mg/kg b.w and omeprazole at 20 mg/kg significantly (p < 0.05) reduced the ulcer area formation by 69.77%, 90.18%, 86.56% and 79.07%, respectively, compared to the ulcer control. Table 2 shows the statistical significant differences between treatment groups subjected to ethanol induced gastric ulcer. Macroscopic observation showed that DES pre-treated groups (Figure 2D, 2E and 2F) or omeprazole group (Figure 2C) considerably reduced gastric lesion compared to the ulcer control group; where ethanol induced intense gastric mucosal damage in the form of elongated band of hemorrhages (Figure 2B). Table 2 Gastroprotective effect of desmosdumotin C against ethanol-induced ulceration and observed liver function test Animal group Pre-treatment 5 ml/kg Mucus weight Ulcer area Inhibition (%) ALT AST (IU/L) (IU/L) A Normal control 2.9 ± 0.2 * 0.00 0.00 36.57 ± 1.67* 230 ± 9.81 * B ulcer control 0.98 ± 0.3 557.28 ± 6.2 NA 56.5 ± 2.71 293 ± 2.15 C Omeprazole (20 mg/kg) 1.55 ± 0.2* 108 ± 7.7 * 79.07 48.2 ± 2.5 * 275.7 ± 6.01 * D DES (5 mg/kg) 1.37 ± 0.5 * 168.48 ± 9 * $ 69.77 51 ± 1.47 * 283.6 ± 4.39 * E DES (10 mg/kg) 2.09 ± 0.1 #$ 54.72 ± 3.8 $ 90.18 32 ± 2.8 #$ 240.04 ± 3.79 $# F DES (20 mg/kg) 1.5 ± 0.4 * 74.88 ± 10.3 $ 86.56 34.2 ± 1.6 $ 257.4 ± 9.22$ NA, not applicable; AST, Aspartate transaminase; ALT, Alanine Aminotranferase. All values are represented as mean (n = 3–5 animals) ± standard error mean, indicates (p < 0.05) compared to ulcer control. $ indicates (p < 0.05) statistical differences compared to omeprazole group. Figure 2 Gross evaluation. Macroscopic appearance of the gastric mucosa of the rats pre-treated with DES at doses 5, 10, 20 mg/kg (D,E, F) or omeprazole 20 mg/kg (C) showed reduced lesion formation when compared to the ulcer control rats (B) 2C. Ethanol-induced sever injuries to the gastric mucosa appear as elongated bands of haemorrhage (white arrow). (A) Showed normal macroscopic appearance of the intact stomach from normal group. (magnification: 1.8×). Gastric tolerability DES animal groups didn’t exhibit any significant gastric lesions. The changes observed in the range of 0–1 according to Adami scoring scale. Only few petechiae scored in rat stomach regardless of a given dose. Gastric acidity In animal model using ligated pylorus method, the treatment with DES (5, 10 and 20 mg/kg) and omeprazole (30 mg/kg), respectively, reduced the volume of gastric juice, total acidity and raised gastric pH significantly (p < 0.05) compared to the control group (Table 3). Table 3 Effects of DES and omeprazole, administered intraduodenally, on the biochemical parameters of gastric juice obtained from pylorus-ligature in rats Animal group treatment 5 ml/kg Volume (ml) pH Acid output [H+] mEq/L A Control group (5% Tween 80) 3.5 ± 0.015 3.83 ± 0.088 95 ± 0.88 B Omeprazole (30 mg/kg) 2.71 ± 0.015* 6.17 ± 0.015* 83 ± 1.15* C DES (5 mg/kg) 3.1 ± 0.12* $ 4.92 ± 0.012$ 92 ± 0.58$ D DES (10 mg/kg) 2.87 ± 0.12* 5.98 ± 0.01* 89 ± 0.88$ E DES (20 mg/kg) 2.94 ± 0.008 5.96 ± 0.01$ 90 ± 0.33$ Results are expressed as mean ± S.E.M. (n = 6 rats).* indicate p < 0.05 compared to control group. $ indicate p < 0.05 compared to omeprazole. Gastric mucus content The ulcer control group produced the lowest content of gastric mucus, while the pretreated DES groups or omeprazole group significantly (p < 0.05) increased the mucus production compared to the ulcer control group (Table 2). Serum biochemical analysis Serum analysis showed that the rats in ulcer control had increased levels of the liver enzymes; Aspartate transaminase (AST) and Alanine Aminotranferase (ALT). However, in DES pretreated animals, the serum concentration of this biomarker significantly (p < 0.05) lowered than ulcer control (Table 2). Histological evaluation Histological observation using H&E staining further confirm the ability of DES to prevent ethanol-induce gastric damage in the superficial layer of the gastric mucosa compared to the normal control group (Figure 3A). The ulcer control group showed highly extensive gastric lesion, submucosal edema and leucocytes infiltration (Figure 3B). Pre-treatment with DES (Figure 3D, 3E and 3F) and omeprazole (Figure 3C), have relatively better protection as observed by decreasing ulcer area, reduced or complete absence of edema and leucocytes infiltration and flattening of mucosal fold was also observed. Figure 3 Histological evaluation. The gastric mucosa of the rats pretreated with DES at doses 5, 10, 20 mg/kg (D, E, F) or omeprazole (C) showed improved histological appearance compared to ulcer control rats (B) which have extensive visible hemorrhagic necrosis of the gastric mucosa with edema and leucocytes infiltration of submucosa. The black arrow indicates edema in submucosa and the white arrow indicates disruption to the deep mucosa layer. (A) showed normal histological apperance of the intact stomach from normal group. (H & E stain: 20×). Mucus staining PAS staining was used to observe the glycogen level in control and pretreated animals. DES pre-treatment (Figure 4D, 4E and 4F) or omeprazole (Figure 4C) resulted into the expansion of a substantially continuous PAS-positive mucous gel layer that lining the entire gastric mucosal surface observed as a magenta color. However, gastric specimen from ulcer control group didn’t exhibit this magenta staining color of PAS (Figure 4B). Figure 4 Tissue glycoprotein. Effect of DES on gastric tissue glycoprotein-PAS staining in ethanol-induced gastric ulcer in rats where (A) normal group, (B) ulcer group, (C) omeprazole group, (D, E, F) treated DES groups at doses 5, 10 and 20 mg/kg, respectively, where the black arrows indicates the glycoprotein appear as magenta stain (PAS stain 20×). HSP-70 and Bax immunohistochemistry Using immunhistochemistry staining, the immunostained localization of HSP-70 was up regulated in DES pretreated animals more than that observed in ulcer control group (Figure 5). This result indicates the possible participation of this protein in protective effect of DES. On the other hand, the immunostained localization of the pro-apoptotic Bax protein in all experimental animals was down regulated compared to the ulcer control group (Figure 6). Hence, the suppressive effect on Bax protein in treatment group might be contributed in the gastroprotective activity of DES. The antigen site in immunohistochemistry appears as a brown-colored. Figure 5 Immunohistochemical analysis of Hsp-70 protein. HSP-70 expression in the gastric tissue of rats submitted to ethanol-induced gastric mucosal lesions at different groups where (A) normal control group, (B) ulcer control group (B), (C) omeprazole group, (D, E, F) the pre-treated groups with DES at doses 5, 10 and 20 mg/kg, respectively. The antigen site appears as a brown color (IHC: 20×). Figure 6 Immunohistochemical analysis of Bax protein. Bax expression in the gastric tissue of rats submitted to ethanol-induced gastric mucosal lesions at different groups where (A) normal control group, (B) ulcer control group, (C) omeprazole group, (D, E, F) pre-treated group with DES at doses 5, 10 and 20 mg/kg, respectively. The antigen site appears as a brown color (IHC: 20×). Effect of DES on GSH and MDA level GSH as endogenous antioxidant, its level was significantly (p < 0.05) lowered in ulcer control group than the other groups. DES in the pre-treated animals was significantly (p < 0.05) restored the GSH levels that depleted due to ethanol administration (Figure 7A). MDA was used as indicator for lipid peroxidation. Thus, TBARS assay showed that the ulcer control group significantly (p < 0.05) has higher MDA level into the gastric homogenate than the other pretreated groups. Gastric MDA level significantly (p < 0.05) decreased in DES pretreated group’s (Figure 7B). Figure 7 Effect of DES on gastric tissue homogenate content of (A) Glutathione (GSH), (B) Malondialdehyde (MDA), (C) Non protein sulfhydryl (NP-SH) and (D) Nitric oxide (NO). DES pre-treatment significantly increased GSH, decreased MDA and replenished NP-SH and NO content. Statistical analysis was assessed with ordinary one way ANOVA followed by Dunnett ’ s Multiple comparison tests. All values are represented as mean of 3 – 5 animals. ± SEM. * indicates (p < 0.05) compared to ulcer control. $ indicates (p < 0.05) statistical differences compared to omeprazole group. Effect of DES on NP-SH compounds content The ulcer control group showed the lowered NP-SH level into the gastric homogenate, while DES significantly (p < 0.05) elevated NP-SH level in pretreated animal compared to ulcer control group (Figure 7C). Effect of DES on NO level and COX-2 enzyme Ulcer control showed the lowest level of NO. DES pre-treatment significantly (p < 0.05) has increased NO level into the gastric homogenate compared to ulcer control group. However, none of the treatment was able to increase NO level near to the normal control (Figure 7D). Moreover, DES inhibited COX-2 enzyme activity by 29.5% and 34.8% at 250 and 500 ng/ml, respectively compared with standard COX-2 inhibitor, indomethacin (71.37%) (Figure 8). Figure 8 Inhibition of COX-2 enzyme. DES was observed to inhibit COX-2 catalyzed prostaglandin biosynthesis by 29.5% and 34.8% at 250 and 500 ng/ml, respectively, compared with indomethacin as COX-2 inhibitor shows inhibition of 71.37%. The results represent as mean ± SEM. Antioxidant evaluation of DES FRAP and DPPH assays were used to evaluate DES radical scavenging activity. FRAP assay showed that DES has antioxidant capacity with 120.7 ± 2.40 which is significantly (p < 0.05) lowered than the positive control used in this study those exhibiting 2562.7 ± 56.64 and 879.3 ± 10.00, for Gallic acid and Ascorbic acid, respectively (Figure 9). Meanwhile the DPPH assay showed insignificant inhibition in the dose of DES used in this study (data not shown). Therefore, it could be said that the antioxidant effect of DES is probably through indirect antioxidant mechanism. Figure 9 Ferric reducing/antioxidant power assay. The FRAP value (μM Fe (II)/g dry mass) of DES in compare with that of ascorbic acid and gallic acid. In vitro anti-Hpylori activity DES represents interesting MIC with 125 μg/ml against H. pylori J99. Discussion In this study, the gastroprotective activity of DES was evaluated on ethanol-induced ulcer model in rats. The effects of DES on the antioxidant system and COX-2 enzyme activity, as well as its anti H. pylori effect were also assessed. The ethanol model is widely used to evaluate gastroprotective activity, since ethanol is found to penetrate easily and rapidly into the gastric mucosa, causing membrane damage, exfoliation of cells and erosion. This subsequently increases mucosal permeability together with the release of vasoactive products, which result in gastric lesions and gastric ulcer formation [31]. Ethanol-induced gastric ulcer predominantly affects the glandular portion of the stomach. However, in the present study, DES pre-treatment was found to significantly attenuate ethanol induced-gastric ulcer. The purpose of the following discussion is to evaluate the possible mechanisms that underlie the observed gastroprotective effect of DES. In order to define the side effects of DES on the overall physiological function, serum biochemical parameters were evaluated. In our study, when compared to the normal group, animals in the ulcer group showed an increased serum level of the liver enzymes (AST and ALT) as an indicator of hepatic injury, since a high level of hepatic enzymes is a sign of alcoholic tissue damage due to ethanol administration [32]. However, DES pre-treatment showed a significant decrease in the elevated serum level of the liver enzymes, close to the normal control level. This finding indicates the high efficacy of the compound against ethanol-induced tissue injuries. Reactive oxygen species (ROS) are the final products generated from a normal cellular metabolic process [33]. Oxidative stress results from the accumulation of ROS and the inability of the antioxidant system to overcome them. Thus, in this situation, excessive production of ROS affects cell integrity [34] such as in gastric tissue where oxidative stress was reported earlier to contribute in the gastrointestinal mucosal lesion formation [35]. Antioxidants have been observed to protect gastric mucosa from ulceration [33], where antioxidants are compounds that have the ability to protect against tissue damage through radical scavenging mechanism [36]. A previous study proved that ethanol induced gastric tissue injury by increasing reactive species formation [37]. Subsequently, ROS accumulation depleted GSH level and increased lipid peroxidation [34]. GSH is an intracellular antioxidant that inhibits oxidative stress [38] and plays an important protective role against ethanol-induced gastric cell injury [39]. It was observed that the aggressive effect of ethanol on gastric mucosa is associated with reduced GSH level [40]. Apart from GSH, ethanol exerts its allergenic effect on gastric tissue by increasing lipid peroxidation [41] where MDA is the main product of lipid peroxidation. Therefore, MDA is considered a marker of ROS-mediated gastric lesions [42]. The present study shows that pre-treatment with DES significantly protected the gastric mucosa from ethanol-induced ulceration by restoring the depleted GSH level and reducing the elevated MDA level compared to the ulcer control group. These results showed the ability of DES to reduce oxidative stress. Hence, to further evaluate this antioxidant property, FRAP assay was used and the results indicated that the compound possesses weak radical scavenging activity. Meanwhile, there is insignificant inhibition in the DPPH assay. Therefore, it could be suggested that DES inhibited oxidative stress via the cellular antioxidant mechanism. Heat shock proteins (HSPs) are stress proteins that maintain the cellular homeostasis against stress factors [43]. HSP70 over expression occur in response to various stimuli such as heat, drug exposure or oxidative stress [44]. Acute and chronic gastric ulcers in rats were observed to be associated with HSP70 induction [43]. HSP70 expression enhances cellular protection-tolerances against high concentration of alcohol [45]. Experimentally, it was found that there is a correlation between HSP induction and mucosal protection [46]. Many compounds have been reported to protect the tissue from oxidative damage remarkably through their activities as HSPs inducers [47]. Our study observed that DES pre-treatment followed by ethanol administration resulted in HSP70 over expression in experimental gastric tissue, suggesting that induction of HSP70 might contribute to the protective effect of DES against ethanol-induced gastric injuries. Again, this result supports the hypothesis regarding the antioxidant activity of DES against oxidative stress. It was reported earlier that apoptosis or programmed cell death was believed to be one of the main factors that contributes in gastric ulcer formation. Blocking of apoptotic cell death is among the mechanisms that are implicated to control gastric lesions [48]. Apart from the antisecretory effect of omeprazole, it was recently proved to exert its antiulcer action via anti-apoptotic effect [49]. Ethanol was reported to induce gastric mucosal lesion by increasing apoptotic cell death [37]. In many experimental ulcer models, apoptosis results from the alteration of Bcl-2 anti-apoptotic and Bax pro-apoptotic proteins expression [48]. Bcl-2 Proteins inhibit most types of apoptotic cell death [50], while Bax proteins boost this process [48]. In the results presented herein, IHC assay showed that DES was able to suppress Bax protein expression when compared to the ulcer control group. Hence, as DES exerted Bax protein suppression effect, it might be suggested that anti-apoptotic effect is involved in the gastroprotective activity of DES against ethanol-induced gastric tissue injury. Gastric defensive mechanisms are based mainly on the delicate balance between aggressive and protective factors [51]. Several studies suggest that mucus gel layer is the first defensive mechanism of the mucosa against internal and external aggressive factors [52]. Ethanol tends to disrupt the gastric mucosal layer and lowers the level of tissue proteins [53]. Hence, the compound that has the ability to increase mucus production might be expected to possess gastroprotective activity [54]. To evaluate this effect, DES was subjected to PAS staining and the result revealed the capability of DES to maintain gastric mucus integrity against depletion by ethanol administration. NP-SH plays an important role in protecting gastric mucosa from aggressive agents [55]. Various ulcerogenic agents have been reported to induce tissue damage by decreasing the endogenous NP-SH level [56]. It is known that ethanol exerts its aggressive effect on the gastric mucosa by diminishing endogenous NP-SH content [57]. NP-SH participates in controlling the production and nature of the mucus in order to protect the gastric mucosa from the noxious effect of ROS formation due to ethanol administration [58]. Our study shows that the DES pre-treatment significantly inhibited ethanol-induced NP-SH depletion when compared to the ulcer control group. Therefore, it could be proposed that replenishment of the endogenous NP-SH might contribute in the gastroprotective activity of DES. Prostaglandins (PGs) play an important role in the maintenance of mucosal integrity which is formed by the COX isoenzymes, namely COX-1 and COX-2 isoforms. Recent studies have found that PGs biosynthesis in the gastrointestinal tract is exclusively catalyzed by COX-1, whereas COX-2 mainly yields PGs in pathophysiological reactions such as inflammation [59]. Independent of PGs, other protective factors involved in the maintenance of mucosal integrity include NO and heat shock proteins [60]. Under normal conditions, NO is formed by nitric oxide synthase (NOS). Neuronal NOS (nNOS) constitutively produces NO, whereas inducible NOS (iNOS) forms NO under inflammatory gastrointestinal damage [61]. On the other hand, activation of HSP-70 suppresses gastric iNO synthesis [45]. The relation between COX-2 and NO at the inflammatory condition was well documented [62] and experimentally in animal models, mucosal injury was found to be accompanied with COX-2 expression [63] as well as inducible nitric oxide (iNO) [64]. Accordingly, the control of stomach ulceration was observed to be achieved by the suppression of inflammatory mediators [65]. It is important to mention that selective COX-2 inhibitors do not damage normal gastric mucosa. However, severe gastric damage occurs when COX-2 inhibition is accompanied by suppression of NO formation or defunctionalization of the afferent nerves [59]. Thus, to evaluate the cytoprotective activity of DES, its effect on COX-2/NO system was evaluated. DES interfered with COX-2 inflammatory pathway and NO level. It is interesting to discover a compound with combined anti-inflammatory and anti-ulcer activities, taking into account the serious limitations of many anti-inflammatory agents that show deleterious effects on the stomach, resulting in gastric mucosal damage [66]. The microaerophilic bacterium H pylori is a gram negative bacilliform considered to be one of the main etiologic factors in the development of the peptic ulcer disease [67]. The bacterium infection results from its induction effect on inflammatory cells to the gastric mucosa [68], without invading the gastric epithelium [69]. Currently, common anti H pylori regimen therapies pose side effects. Therefore, the need to discover new agents with potential anti H pylori activity is of high concern [70]. A potent antibacterial compound is one that shows an MIC value of less than or equal to 250 [71]. To evaluate the antimicrobial activity of DES, the compound was examined against H pylori strains and the result of the present study showed an interesting DES MIC value of 125 μg/ml against H. pylori J99. Conclusions The current study introduces, for the first time, the isolation of DES compound from M. kentii plant and the evaluation of its gastroprotective activity against ethanol-induced gastric ulcer. The possible gastroprotective mechanism(s) of DES might be attributed to the intracellular antioxidant effect revealed by lowered MDA levels and restored GSH levels, besides HSP-70 up regulation. Moreover, DES exhibited anti-apoptotic activity marked by the down regulation of Bax protein. Furthermore, DES was found to maintain endogenous NP-SH content. The compound inhibited COX-2 activity and replenished the NO level. It also showed an interesting MIC against H Pylori bacterium. These results warrant further study on DES compound as an effective gastroprotective and therapeutic agent for gastric ulcer. Abbreviations M.kentii: Mitrella kentii; DES: Desmosdumotin C; TXB 2: Thromboxane B 2; PAF: Platelet activating factor; NP-SH: Non protein sulfhydryl; COX-2: Cycloxygenase-2 enzyme; NO: Nitric oxide; MDA: Malondialdehyde; GSH: Glutathione; HSP: Heat shock protein; Bcl2: B-cell lymphoma 2; Bax: Bcl-2–associated X protein; HE: Hematoxylene and eosin; PAS: Periodic acid schiff base; IHC: Immunhistochemistry; H pylori: Helicobacter pylori; AST: Aspartate transaminase; ALT: Alanine aminotranferase; DTNB: 5,5 -dithiobis-2-nitrobenzoic acid; TBARS: Thiobarbituric acid reactive substance; FRAP: Ferric-reducing antioxidant power; S.E.M: Standard error mean; ANOVA: Analysis of variance; MIC: Minimum inhibitory concentration; PBS: Phosphate buffered saline. Competing interests The authors declare that they have no competing interests. Authors’ contributions HMS drafted the manuscript and performed the toxicity study, gastroprotective study, COX-2 activity study and gastric homogenate contents estimation. AA, AAH and KAK carried out the extraction and isolation of the compound. SM performed the statistical analysis and revised the manuscript critically for important intellectual content. MAA, SIA, NMH participated in the design of the study. MMT performed the Immunhistochemistry staining. MFL and JV carried out the H pylori study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-6882/13/183/prepub Acknowledgements The authors would like to express their thanks and appreciation to University of Malaya (HIR grant F00009- 21001) for providing funding to perform this study. ==== Refs Anand S Nagaraju B Ahmed N Swain SR Ahmed F Padmavathi G Narendra Sharath Chandra J Shampalatha S Antiulcer activity of Trigonella foenum-graecum leaves in cold restraint stress-induced ulcer model Mol Clin Pharmacol 2012 3 1 90 99 Oyagi A Ogawa K Kakino M Hara H Protective effects of a gastrointestinal agent containing Korean red ginseng on gastric ulcer models in mice BMC complem alter med 2010 10 1 45 10.1186/1472-6882-10-45 Ramakrishnan K Salinas RC Peptic ulcer disease Am Fam Physician 2007 76 7 1005 1012 17956071 Taha MME Salga MS Ali HM Abdulla MA Abdelwahab SI Hadi AHA Gastroprotective activities of < i > Turnera diffusa</i > Willd. ex Schult. revisited: Role of arbutin. J Ethnopharmacol 2012. Taha MME, Salga MS, Ali HM, Abdulla MA, Abdelwahab SI, Hadi AHA: Gastroprotective activities of < i > Turnera diffusa</i > Willd. ex Schult. revisited: Role of arbutin J Ethnopharmacol 2012 141 1 273 281 10.1016/j.jep.2012.02.030 22374081 Malfertheiner P Chan FKL McColl KEL Peptic ulcer disease Lancet 2009 374 9699 1449 1461 10.1016/S0140-6736(09)60938-7 19683340 Ji C-X Fan D-S Li W Guo L Liang Z-L Xu R-M Zhang J-J Evaluation of the anti-ulcerogenic activity of the antidepressants duloxetine, amitriptyline, fluoxetine and mirtazapine in different models of experimental gastric ulcer in rats Eur J Pharmacol 2012 691 1–3 46 51 22789173 Santin JR Lemos M Júnior LCK Niero R De Andrade SF Antiulcer effects of < i > Achyrocline satureoides</i > (Lam.) DC (Asteraceae)(Marcela), a folk medicine plant, in different experimental models J Ethnopharmacol 2010 130 2 334 339 10.1016/j.jep.2010.05.014 20546870 de Souza Almeida ES Niero R Clasen BK Balogun SO de Oliveira Martins DT Pharmacological mechanisms underlying the anti-ulcer activity of methanol extract and canthin-6-one of < i > Simaba ferruginea</i > A St-Hil. in animal models. J Ethnopharmacol 2011 134 3 630 636 Eswani N Kudus KA Nazre M Noor AGA Ali M Medicinal plant diversity and vegetation analysis of logged over hill forest of tekai tembeling forest reserve, jerantut Pahang. J Agric Sci 2010 2 3 P189 Saadawi S Jalil J Jasamai M Jantan I Inhibitory effects of acetylmelodorinol, chrysin and polycarpol from mitrella kentii on prostaglandin E2 and thromboxane B2 production and platelet activating factor receptor binding Molecules 2012 17 5 4824 4835 22538486 Ellis J Gellert E Summons R The alkaloids of mitrella kentii (annonaceae) Aust J Chem 1972 25 12 2735 2736 10.1071/CH9722735 Benosman A Oger JM Richomme P Bruneton J Roussakis C BöschT S Ito K Ichino K Hadi AHA New terpenylated dihydrochalcone derivatives isolated from Mitrella kentii J Nat Prod 1997 60 9 921 924 10.1021/np9700331 Wu JH McPhail AT Bastow KF Shiraki H Ito J Lee KH Desmosdumotin C, a novel cytotoxic principle from < i > desmos dumosus</i> Tetrahedron Lett 2002 43 8 1391 1393 10.1016/S0040-4039(02)00026-6 Ichimaru M Nakatani N Moriyasu M Nishiyama Y Kato A Mathenge SG Juma FD ChaloMutiso PB Hydroxyespintanol and schefflerichalcone: two new compounds from Uvaria scheffleri J natural med 2010 64 1 75 79 10.1007/s11418-009-0358-0 Kim JH Choi SK Choi SY Kim HK Chang HI Suppressive effect of astaxanthin isolated from the Xanthophyllomyces dendrorhous mutant on ethanol-induced gastric mucosal injury in rats Biosci Biotechnol Biochem 2005 69 7 1300 1305 10.1271/bbb.69.1300 16041134 Mahmood A Sidik K Salmah I Suzainur K Philip K Antiulcerogenic activity of ageratum conyzoides leaf extract against ethanol-induced gastric ulcer in rats as animal model Int j mol med Adv sci 2005 1 4 402 405 Nakagawa-Goto K Wu P-C Bastow KF Yang S-C Yu S-L Chen H-Y Lin J-C Goto M Morris-Natschke SL Yang P-C Antitumor agents 283. further elaboration of desmosdumotin C analogs as potent antitumor agents: activation of spindle assembly checkpoint as possible mode of action . Bioorg Med Chem 2011 19 5 1816 1822 10.1016/j.bmc.2011.01.001 21296579 Potrich FB Allemand A Da Silva LM Dos Santos AC Baggio CH Freitas CS Mendes DAGB Andre E De Paula Werner MF Marques MCA Antiulcerogenic activity of hydroalcoholic extract of < i > Achillea millefolium</i > L.: involvement of the antioxidant system J Ethnopharmacol 2010 130 1 85 92 10.1016/j.jep.2010.04.014 20420892 Njar VCO Adesanwo JK Raji Y Methyl angolenate: the antiulcer agent from the stem bark of entandrophragma angolense Planta Med-J Med Plant Res 1995 61 1 91 92 10.1055/s-2006-958015 Adami E Marazzi-Uberti E Turba C Pharmacological research on gefarnate, a new synthetic isoprenoid with an anti-ulcer action Arch Int Pharmacodyn Ther 1964 147 113 14116921 Shay H A simple method for the uniform production of gastric ulceration in the rat Gastroenterology 1945 4 43 61 Tan PV Nyasse B Dimo T Mezui C Gastric cytoprotective anti-ulcer effects of the leaf methanol extract of < i > Ocimum suave</i > (Lamiaceae) in rats J Ethnopharmacol 2002 82 2 69 74 12241979 Mendonça FS Camargo LM Freitas SH Dória RGS Baratella-Evêncio L Evêncio Neto J Aspectos clínicos e patológicos de um surto de fotossensibilização hepatógena em ovinos pela ingestão de Brachiaria decumbens (Gramineae) no município de Cuiabá, Mato Grosso Ciência Animal Brasileira 2008 9 4 1034 1041 23977643 Rahman I Kode A Biswas SK Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method Nat Protoc 2007 1 6 3159 3165 10.1038/nprot.2006.378 17406579 Hodges DM DeLong JM Forney CF Prange RK Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds Planta 1999 207 4 604 611 10.1007/s004250050524 Sedlak J Lindsay RH Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent Anal Biochem 1968 25 1 192 4973948 Miranda KM Espey MG Wink DA A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite Nitric oxide 2001 5 1 62 71 10.1006/niox.2000.0319 11178938 Benzie IFF Strain J The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay Anal Biochem 1996 239 1 70 76 10.1006/abio.1996.0292 8660627 Michielin EMZ de Lemos Wiese LP Ferreira EA Pedrosa RC Ferreira SRS Radical-scavenging activity of extracts from < i > Cordia verbenacea</i > DC obtained by different methods J Supercrit Fluids 2011 56 1 89 96 10.1016/j.supflu.2010.11.006 Jorgensen JH Hindler JF Reller LB Weinstein MP New consensus guidelines from the clinical and laboratory standards institute for antimicrobial susceptibility testing of infrequently isolated or fastidious bacteria Clin Infect Dis 2007 44 2 280 10.1086/510431 17173232 Szabo S Brown A Prevention of ethanol-induced vascular injury and gastric mucosal lesions by sucralfate and its components: possible role of endogenous sulfhydryls Proceedings of the Society for Experimental Biology and Medicine Society for Experimental Biology and Medicine 1987 New York, NY: Royal Society of Medicine 493 497 Salga MS Ali HM Abdullah MA Abdelwahab SI Hussain PD Hadi AHA Mechanistic studies of the anti-ulcerogenic activity and acute toxicity evaluation of dichlorido-copper (II)-4-(2-5-bromo-benzylideneamino) ethyl) piperazin-1-ium phenolate complex against ethanol-induced gastric injury in rats Molecules 2011 16 10 8654 8669 Tandon R Khanna R Dorababu M Goel R Oxidative stress and antioxidants status in peptic ulcer and gastric carcinoma Indian J Physiol Pharmacol 2004 48 1 115 118 15270379 Kurose I Higuchi H Miura S Saito H Watanabe N Hokari R Hirokawa M Takaishi M Zeki S Nakamura T Oxidative stress‒mediated apoptosis of hepatocytes exposed to acute ethanol intoxication Hepatology 1997 25 2 368 378 10.1002/hep.510250219 9021949 Nanjundaiah SM Annaiah HNM Dharmesh SM Gastroprotective effect of ginger rhizome (Zingiber officinale) extract: role of gallic acid and cinnamic acid in H Evid-Based Complement Altern Med 2011 2011 10.1093/ecam/nep1060 Tachakittirungrod S Okonogi S Chowwanapoonpohn S Study on antioxidant activity of certain plants in Thailand: mechanism of antioxidant action of guava leaf extract Food Chem 2007 103 2 381 388 10.1016/j.foodchem.2006.07.034 Chen S-H Liang Y-C Chao JC Tsai L-H Chang C-C Wang C-C Pan S Protective effects of Ginkgo biloba extract on the ethanol-induced gastric ulcer in rats World J Gastroenterol 2005 11 24 3746 3750 15968732 Yoshikawa T Naito Y Kishi A Tomii T Kaneko T Iinuma S Ichikawa H Yasuda M Takahashi S Kondo M Role of active oxygen, lipid peroxidation, and antioxidants in the pathogenesis of gastric mucosal injury induced by indomethacin in rats Gut 1993 34 6 732 737 10.1136/gut.34.6.732 8314503 Mutoh H Hiraishi H Ota S Yoshida H Ivey K Terano A Sugimoto T Protective role of intracellular glutathione against ethanol-induced damage in cultured rat gastric mucosal cells Gastroenterology;(USA) 1990 98 6 1452 1459 23977452 Victor BE Schmidt KL Smith GS Miller TA Protection against ethanol injury in the canine stomach: role of mucosal glutathione Am J Physiol Gastrointest Liver Physiol 1991 261 6 G966 G973 Rozza AL Moraes TM Kushima H Tanimoto A Marques MOM Bauab TM Hiruma-Lima CA Pellizzon CH Gastroprotective mechanisms of < i > Citrus lemon</i > (Rutaceae) essential oil and its majority compounds limonene and β-pinene: involvement of heat-shock protein-70, vasoactive intestinal peptide, glutathione, sulfhydryl compounds, nitric oxide and prostaglandin E < sub > 2</sub> Chem Biol Int 2011 189 1 82 89 Kwiecien S Brzozowski T Konturek S Effects of reactive oxygen species action on gastric mucosa in various models of mucosal injury J Physiol Pharmacol 2002 53 1 39 50 11939718 Chang XR Peng L Yi SX Peng Y Yan J Association of high expression in rat gastric mucosal heat shock protein 70 induced by moxibustion pre-treatment with protection against stress injury World J Gastroenterol 2007 13 32 4355 17708611 Hartl FU Hayer-Hartl M Molecular chaperones in the cytosol: from nascent chain to folded protein Science 2002 295 5561 1852 1858 10.1126/science.1068408 11884745 Tsukimi Y Okabe S Recent advances in gastrointestinal pathophysiology: role of heat shock proteins in mucosal defense and ulcer healing Biol Pharm Bull 2001 24 1 1 9 10.1248/bpb.24.1 11201234 Yeo M Kim DK Cho SW Hong HD Ginseng, the root of Panax ginseng CA Meyer, protects ethanol-induced gastric damages in rat through the induction of cytoprotective heat-shock protein 27 Dig Dis Sci 2008 53 3 606 613 10.1007/s10620-007-9946-6 17763949 Odashima M Otaka M Jin M Konishi N Sato T Kato S Matsuhashi T Nakamura C Watanabe S Induction of a 72-kDa heat-shock protein in cultured rat gastric mucosal cells and rat gastric mucosa by zinc L-carnosine Dig Dis Sci 2002 47 12 2799 2804 10.1023/A:1021029927386 12498304 Konturek PC Brzozowski T Konturek S Pajdo R Konturek J Kwiecień S Taut A Hahn E Apoptosis in gastric mucosa with stress-induced gastric ulcers J physiol pharmacol off j Polish Physiol Soc 1999 50 2 211 Biswas K Bandyopadhyay U Chattopadhyay I Varadaraj A Ali E Banerjee RK A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical J Biol Chem 2003 278 13 10993 11001 10.1074/jbc.M210328200 12529378 Liu M-J Fei S-J Qiao W-L Du D-S Zhang Y-M Li Y Zhang J-F The protective effect of 17β-estradiol postconditioning against hypoxia/reoxygenation injury in human gastric epithelial cells Eur J Pharmacol 2010 645 1–3 151 157 20654613 El-Missiry M El-Sayed I Othman A Protection by metal complexes with SOD-mimetic activity against oxidative gastric injury induced by indometacin and ethanol in rats Ann Clin Biochem 2001 38 6 694 700 10.1258/0004563011900911 11732653 Wallace JL Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn’t the stomach digest itself? Physiol Rev 2008 88 4 1547 1565 10.1152/physrev.00004.2008 18923189 Szabo S Trier J Brown A Schnoor J Early vascular injury and increased vascular permeability in gastric mucosal injury caused by ethanol in the rat Gastroenterology 1985 88 1 Pt 2 228 3871087 Allen A Garner A Mucus and bicarbonate secretion in the stomach and their possible role in mucosal protection Gut 1980 21 3 249 262 10.1136/gut.21.3.249 6995243 Szabo S Trier JS Frankel PW Sulfhydryl compounds may mediate gastric cytoprotection Sci (New York, NY) 1981 214 4517 200 10.1126/science.7280691 Rafatullah S Galal A Al-Yahya M Al-Said M Gastric and duodenal antiulcer and cytoprotective effects of Aframomum melegueta in rats Pharm Biol 1995 33 4 311 316 10.3109/13880209509065384 Loguercio C Taranto D Beneduce F del Vecchio BC De Vincentiis A Nardi G Romano M Glutathione prevents ethanol induced gastric mucosal damage and depletion of sulfhydryl compounds in humans Gut 1993 34 2 161 165 10.1136/gut.34.2.161 8432465 Salim A Sulphydryl-containing agents: a new approach to the problem of refractory peptic ulceration Pharmacology 1992 45 6 301 306 10.1159/000139015 1488453 Peskar BM Role of cyclooxygenase isoforms in gastric mucosal defence J Physiol Paris 2001 95 1 3 9 11595412 Halter F Tarnawski A Schmassmann A Peskar B Cyclooxygenase 2—implications on maintenance of gastric mucosal integrity and ulcer healing: controversial issues and perspectives Gut 2001 49 3 443 453 10.1136/gut.49.3.443 11511570 Yamamoto H Tanaka A Kunikata T Hirata T Kato S Takeuchi K Inducible types of cyclooxygenase and nitric oxide synthase in adaptive cytoprotection in rat stomachs J Physiol Paris 1999 93 5 405 412 10.1016/S0928-4257(99)00128-X 10674917 Tian J Kim SF Hester L Snyder SH S-nitrosylation/activation of COX-2 mediates NMDA neurotoxicity Proc Natl Acad Sci 2008 105 30 10537 10540 10.1073/pnas.0804852105 18650379 Jackson LM Wu KC Mahida YR Jenkins D Hawkey CJ Cyclooxygenase (COX) 1 and 2 in normal, inflamed, and ulcerated human gastric mucosa Gut 2000 47 6 762 770 10.1136/gut.47.6.762 11076873 Ko JKS Cho CH Co-regulation of mucosal nitric oxide and prostaglandin in gastric adaptive cytoprotection Inflammation Res 1999 48 9 471 478 10.1007/s000110050489 Abdelwahab SI Mohan S Abdulla MA Sukari MA Abdul AB Taha MME Syam S Ahmad S Lee KH The methanolic extract of Boesenbergia rotunda (L) Mansf. and its major compound pinostrobin induces anti-ulcerogenic property in vivo: Possible involvement of indirect antioxidant action J Ethnopharmacol 2011 137 2 963 970 10.1016/j.jep.2011.07.010 21771650 Oliveira FA Vieira-Júnior GM Chaves MH Almeida FRC Florêncio MG Lima RCP JrSilva RM Santos FA Rao VSN Gastroprotective and anti-inflammatory effects of resin from protium heptaphyllum in mice and rats Pharmacol Res 2004 49 2 105 111 10.1016/j.phrs.2003.09.001 14643690 Fahey JW Haristoy X Dolan PM Kensler TW Scholtus I Stephenson KK Talalay P Lozniewski A Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of helicobacter pylori and prevents benzo [a] pyrene-induced stomach tumors Proc Natl Acad Sci 2002 99 11 7610 7615 10.1073/pnas.112203099 12032331 Rudi J Kuck D Strand S von Herbay A Mariani SM Krammer PH Galle PR Stremmel W Involvement of the CD95 (APO-1/Fas) receptor and ligand system in helicobacter pylori-induced gastric epithelial apoptosis J Clin Invest 1998 102 8 1506 10.1172/JCI2808 9788963 Aihara M Tsuchimoto D Takizawa H Azuma A Wakebe H Ohmoto Y Imagawa K Kikuchi M Mukaida N Matsushima K Mechanisms involved in helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45 Infect Immun 1997 65 8 3218 3224 9234778 Baker DD Chu M Oza U The value of natural products to future pharmaceutical discovery Nat product reports 2007 24 6 1225 1244 10.1039/b602241n Moraes TM Rodrigues CM Kushima H Bauab TM Villegas W Pellizzon CH Brito ARMS Hiruma-Lima CA Hancornia speciosa: indications of gastroprotective, healing and anti-Helicobacter pylori actions J Ethnopharmacol 2008 120 2 161 168 10.1016/j.jep.2008.08.001 18761076	23866830	PMC3765280	CC BY	2021-01-05 01:16:09	yes	BMC Complement Altern Med. 2013 Jul 19; 13:183
==== Front Diabetol Metab SyndrDiabetol Metab SyndrDiabetology & Metabolic Syndrome1758-5996BioMed Central 1758-5996-5-492412832510.1186/1758-5996-5-49ReviewLow birth weight: causes and consequences Negrato Carlos Antonio 1carlosnegrato@uol.com.brGomes Marilia Brito 2mariliabgomes@gmail.com1 Department of Internal Medicine, Bauru’s Diabetics Association, 17012-433 Bauru São Paulo,Brazil2 Department of Internal Medicine, Diabetes Unit, State University Hospital of Rio de Janeiro, Rio de Janeiro, Brazil2013 2 9 2013 5 49 49 6 7 2013 29 8 2013 Copyright © 2013 Negrato and Gomes; licensee BioMed Central Ltd.2013Negrato and Gomes; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.During our phylogenetic evolution we have selected genes, the so called thrifty genes, that can help to maximize the amount of energy stored from every consumed calorie. An imbalance in the amount of stored calories can lead to many diseases. In the early 80’s the distinguished English epidemiologist David Barker, formulated a hypothesis suggesting that many events that occur during the intrauterine life and early in infancy can influence the occurrence of many diseases that will develop in adulthood. This theory proposes that under-nutrition and other insult or adverse stimulus in utero and during infancy can permanently change the body’s structure, physiology and metabolism. The lasting or lifelong effects of under-nutrition will depend on the period in the development at which it occurs. The clues that led Barker to his conclusions started to be discovered when he was studying the temporal trends in the incidence of ischemic heart disease in England and Wales. Examining data found in The Hertfordshire records, collected in the beginning of the last century, he found that the rates of mortality by ischemic heart disease was much higher in children born in less affluent counties and mostly in those with low birth weight. After his initial findings a myriad of diseases have been found to be linked to low birth weight and under-nutrition in utero and in the neonatal period. These diseases were then nominated adult diseases with fetal origin. Epidemiological studies that led to these findings suggest that in utero and early postnatal life have critical importance for long-term programming of health and disease, opening unique chances for primary prevention of chronic diseases. ==== Body Introduction In order to understand what we weight from our intrauterine life until adulthood, we must understand our genes. It is supposed that at least 24.000 genes constitute the human blueprint and that over 250 of these genes may help determine our weight [1]. About 40.000 years ago, somewhere in the world, a Paleolithic couple emerged to become the great, great, great… grandparents of us all. Their genes still inhabit our cells, and we continue to pass them from one generation to the next, and these genes still shape our metabolic pathways. We must look to our Paleolithic ancestors to understand the modern-day problems of obesity, diabetes and many other diseases. We must look to these genes to know why the environment we have created is so aggressive to our health. We must understand how and why the genes that were advantageous in the past are so deleterious now [1]. Our ancestors were nomads. They used to travel long distances in search of food. The food sources were unreliable and no one ever knew exactly where or when they would get the next meal. When food was present, especially like fat and protein of animal origin, to store calories within the body was a very wise strategy [1]. Paleolithic men were possibly lean, muscular and strong. Their bodies evolved to withstand the danger that was everywhere. They used to have vigorous physical activity and insufficient food was a constant risk [1]. Women like the men used to be physically active and in order to supplement what men brought back home, they gathered nuts, berries, fruits, vegetables and roots. Obesity supposedly did not exist, because the food supply was so uncertain [1]. We cannot be certain, but possibly the Paleolithic diet was approximately 30 percent protein that came from fish and meat. We can certainly assume that the mammals our ancestors ate were also lean; they probably did not live in feedlots or graze on carefully managed pastures. They had to exert themselves to obtain food and to avoid becoming the meal of some other animal. So like the hunters who hunted them, their body composition was largely composed by muscles [1]. Our ancestors’ diet possibly consisted of up to 100 grams of fiber per day, what is 5–10 times higher than is typical today. Fat corresponded to approximately 20 percent and was mostly unsaturated since it used to come from nuts and seeds. Since the meat was also lean, Paleolithic diet was minimally composed by saturated fat [1]. Paleolithic men and women did not live long, possibly less than 20 years. If they survived birth and infancy, most died in what we consider today young adulthood. They were vulnerable to famine, predators, accidents and infections; the women faced all this plus the risk of childbirth. We can certainly assume that our ancestors did not have health problems obesity-related that plague us today. Because of their low fat intake, their blood vessels were probably free of fat deposits and stroke; heart attacks and high blood pressure were possibly rare. Diabetes and obesity were not known those days. Also, by not living so long, they would not have time to develop chronic diseases [1]. Thrifty genes The genes of our Paleolithic ancestors evolved for people who spent their days in constant physical activity and whose diet was low in calories and in saturated fat. Although, evolution selected genes that could support times of a poor food supply of a poor food supply, long winters and long times of recurrent droughts. These genes were called thrifty genes because they helped to maximize the amount of energy that could be obtained and stored from every consumed calorie [1,2]. One possible mechanism for accomplishing this was insulin resistance (IR) that was an adaptive condition for babies, before and after birth, in order to use few calories more efficiently. In times when food was more abundant, these genes would enable these babies to eat more and stock body fat for the future hard times that would certainly come. When food was scarce these genes would preserve those fat stores, slowing down metabolism and keeping the body’s energy reserves [1,2]. Thrifty genes were advantageous for life in the Paleolithic era. About 10.000 years ago, the world began to change. Many humans developed agriculture, domesticated animals, left behind their nomadic way of life, settled down and developed cities and civilizations. Famines became less frequent and the nutritional environment became altered. Humans started to eat more grains and consequently less fish, fruits and vegetables. Animals started to be raised in enclosed spaces and also became fatter. The saturated fat they contained was less favorable to the human metabolism and cardiovascular system [1,2]. It seems that these thrifty genes became less important in societies with better climate and abundant supplies. This helps to explain why obesity and diabetes for example, are more common in some parts of the world than in others [1,2]. The scars of early life In the 1980’s, Barker developed a hypothesis according which many nutritional events that occur during the intrauterine life and early in infancy will influence the development of adult diseases. This became the so called Barker hypothesis [3]. Lucas, proposed the term “programming” to describe the process by which an adverse event happening at a critical period of development, has long lasting or lifelong significance [4]. Fetal programming opened the field for extensive research into the fetal origin of adult diseases. The association between low birth weight (LBW), which reflects intrauterine nutritional status, and the development of adult diseases has been confirmed in many studies for type 2 diabetes (T2D), hypertension (HT) and ischemic heart diseases (IHD) [1-4]. These findings will be presented in this review. Barker’s hypothesis suggests that under-nutrition and other insult or adverse stimulus in utero and during infancy can permanently change the body’s structure, physiology and metabolism. The lasting or lifelong effects of under-nutrition will depend on the period in the development at which it occurs. In early gestation it will reduce body’s size permanently, whereas in late gestation it will have deep effects on body form without necessarily reducing body size. Fetuses and neonates that are rapidly growing are more vulnerable to under-nutrition. These effects include altered gene expression, reduced cell numbers, imbalance between cell types, altered organ structure, pattern of hormonal release and hormonal responses [5]. Thus, the tendencies of our body to become obese, to develop T2D and many other diseases in the adulthood are affected by our genes, our early development and our lifestyle [1]. The clues that led Barker, a distinguished epidemiologist to formulate his hypothesis started to be discovered when he was studying the temporal trends in the incidence of IHD in England and Wales [5,6]. The incidence of IHD increased rapidly in the western world in the beginning of the 20th century, and in the present days it has been growing in the so called developing countries such as China, India, Russia and in many eastern European countries. Such abrupt changes, in a so short time frame, could not be explained by genetic changes, and then the attention of the scientists was directed to the existing lifestyle in the industrialized countries. The relationship of IHD with obesity, smoking habit, high cholesterol levels and stress among many other factors have been well established [5,6]. In 1977, Forsdahl has found considerable variations in the rates of mortality from IHD in twenty Norwegian counties. He has suggested that these variations could not be explained by the current differences in standard of living existing then. Such differences did exist in the past as was shown by large variations in infant mortality. A significant positive correlation has been found between the county age-adjusted mortality from IHD in people aged between 40 and 69 years and county’s infant mortality relating to the early years in the same cohorts. His findings suggested that great poverty in childhood and adolescence followed by prosperity, was a risk factor for IHD [7]. Studying the geographic distribution of mortality by IHD, Barker has found that although the rise in IHD in England and Wales has been associated with increasing prosperity, paradoxically, mortality rates by IHD were higher in the least affluent areas. A strong geographical relation between IHD mortality rates in 1968–78 and infant mortality in 1921–25 was observed. IHD was found to be strongly correlated with both neonatal and postneonatal mortality rates. These findings led him also to suggest that poor nutrition in early life increases susceptibility to the effects of an affluent diet [3]. Barker concluded then that under-nutrition in women in childbearing age could be the origin of high rates of IHD in the next generation, since it would impair a woman’s ability to nourish her baby in utero and in early infancy [5,6]. The Hertfordshire records In order to test the hypothesis that IHD is programmed in utero, it was necessary to study people now, in middle and late life whose early growth had been recorded somewhere. Barker then undertook a search for medical records of babies born in the early 1900s, with the staff from the Medical Research Council of Britain. They searched archives and hospital record departments throughout the country. Many were found; some were in large collections preserved for many years, some had no more then a few hundred records that were kept by a clinic or a midwife. Some were found in lofts, sheds, garages, boiler rooms or flooded basements. Finally, in Hertfordshire, a county localized in the East region of England, just north of London, the appointments made by Ethel Margaret Burnside, the first county’s chief health visitor and lady inspector of midwives were found. She set up an army of trained nurses to attend women in childbearing age and to advise mothers about their babies health [5]. From 1911 onwards, when a woman in Hertfordshire had a baby, she was attended by a midwife that visited her home at regular intervals to get information about the baby’s health and development. The weight was recorded at birth and at one year of age, when visits ceased. From 1923 onwards the health visitors continued their visits until the child was five years old. The ledgers containing these information were maintained until 1945. They were found by the Medical Research Council in 1986 [5]. With the data found in Hertfordshire ledgers, Barker used records from England’s National Health Service to trace about 16.000 men and women who had been born in this county between 1911 and 1930. He then matched information about their current health status to the infant data that were collected by Burnside’s nurses. In 1989 when his study was published, 3.865 people had already died with ages ranging from 20 to 74 years. He observed that the mortality by IHD is almost two times more prevalent in those that had a birthweight ≤ 2.500 g when compared to those who had a birthweight ≥ 4.000 g. Barker discovered that men who had been small at birth, and who were still small at age one, were at the highest risk of presenting IHD. The mortality rates by any other disease had no relation to the birthweight [6]. Subsequent research throughout the world, has found similarly strong associations between LBW and IHD [8-10]. Thrifty phenotype hypothesis or the small-baby-syndrome The ‘thrifty phenotype’ hypothesis was proposed based on studies which showed that individuals who had a LBW (small babies) have an increased risk of developing symptoms of the metabolic syndrome (MS), T2D and cardiovascular diseases (CVD) later on [11]. This hypothesis, had the following two premises: 1) LBW is an indicator of maternal and, consequently, fetal under-nutrition, and 2) phenotypic characteristics that lead to a ‘saving’ of energy are beneficial for the individual in conditions of poor postnatal nutrition. Essentially, this hypothesis proposes that prenatal under-nutrition leads to decreased insulin secretion and, simultaneously, IR in the fetus which, in turn slows down prenatal weight gain. This phenotype results from active fetal adaptations and is preserved for the life span of affected individuals [11]. Accordingly, in later life such a phenotype must be ‘thrifty’ and help affected individuals to cope better with conditions of food shortage. However, under affluent conditions in modern western societies this advantage turns into a disadvantage and leads to the MS, T2D and CVD [11]. A small for gestational age (SGA) is a full term baby who is under 2.500 g at birth or born with a birth weight and/or length under two standard deviations (2 SDs) for the gestational age and sex of the population [12]. The determination of gestational age is generally difficult, being the most precise those performed with ultra-sound, while those assessed from the time of last normal menstrual period are deceivable [13,14]. The SGA babies can have their birth weight (SGA-w) affected, their birth length (SGA-l) or both (SGA-w/l). These subgroups achieve final height in different ways. SGA-w born children are mostly likely to achieve catch-up growth after the second year of life, while SGA-w/l children more frequently remain short in adulthood [15]. A growth retardation is a failure that can occur during the intrauterine development and is called intrauterine growth retardation (IUGR). If this condition is detected, both mother and fetus should undergo adequate monitoring by fetal biometry and doppler ultrasonography of uterine and fetal blood vessels [12]. The general incidence of SGA newborns is 3-10% [16-18]. In a study conducted in the USA in 2004 with 95.000 healthy newborns, 2.3% were SGA. The rate of LBW for the entire studied population (4.112.052) was 8.1% [19]. Etiology of the small-baby-syndrome or small for gestational age Most patients born SGA do not have a clear etiology for this condition to happen. Nevertheless, several maternal and fetal conditions have been identified as causative factors for the birth of a SGA baby. Maternal factors can be related to insufficient substrate supply to the fetus during development due to many different causes such as reduced maternal food intake, maternal systemic diseases such as HT and diabetes, periodontal disease, abnormal placental function that can lead to an impaired utero-placental blood supply or disruption of the placental transfer, abruption, infarction or mal-development of the placenta. The majority of these factors can influence growth during the last trimester of pregnancy and result predominantly in IUGR that refers to poor growth of a baby while in the mother’s womb during pregnancy [20-23]. Other maternal contributing factors to SGA are: parity, ethnicity, delivery at age less than 16 and more than 35 years and previous history of SGA born children. Parents size seems to be less important on the baby’s birth weight [24]. The exposure of the fetus to a toxic intrauterine milieu caused by tobacco, alcohol consumption or illicit drugs abuse increase the risk of SGA or IUGR births. Smoking during pregnancy has the most significant influence with a relative risk of 3.24 [21,25]. Several fetal factors are related to the birth of a SGA baby, like some chromosomal anomalies such as gonadal disgenesy, Edward Syndrome, Turner Syndrome, Down Syndrome and Prader-Willi Syndrome [24]. The thrifty phenotype seems to be strongly associated with the birth of a SGA baby. Several mechanisms have been proposed to explain growth retardation of the fetus and the infant. The growth is assumed to be altered both quantitatively and qualitatively by a poor nutritional environment. Metabolic disturbances depend on the period of gestation in which a famine affected the mother and the children, as a Dutch SGA study showed in examining the population who suffered from famine during the Second World War [26,27]. If fetal exposure occurs during early pregnancy it will affect lipid metabolism, but if it occurs in late pregnancy, it will affect the glucose metabolism [26]. It is supposed that an inadequate development of pancreatic beta cell mass and their function are the link between poor fetal nutrition, IR and T2D later in life. A thrifty phenotype is adapted to survive in poor nutritional circumstances. Later in adulthood, abundant food intake and decreased energy expenditure lead towards obesity, glucose intolerance and HT among many other diseases, caused by epigenetic alterations that occurred during the intrauterine life [11,28,29]. How can it matter fifty or more years late that a person was born small? Nowadays, there is compelling evidence linking epigenetic factors to many human diseases. Epigenetic factors, by different types of reactions, could mediate the interplay between genes and environment resulting in activation or repression of genetic transcription, or even silencing the genetic transcription. The most important epigenetic reactions affecting genetic transcription are acetylation and methylation. These reactions occur mainly in the tail of histones that are proteins where DNA is wrapped. Brownlee et al. have demonstrated in human aortic endothelial cells, that excessive concentration of reactive oxygen species (ROS) can induce monomethylation of lysine from histone 3 increasing the expression of the subunit p65 of nuclear factor kappa beta. This reaction is responsible for the increased transcription of vascular cell adhesion molecule 1 and monocyte chemoattractant molecule 1 that are both related to diabetes, hypertension and other components of the MS [30,31]. Plagemann et al., have recently demonstrated in animal models that the neonatally acquired adipogenic and diabetogenic phenotype can probably be caused, at least in part, by over-nutrition in pre-and/or neonatal period, that can lead to alterations of DNA methylation patterns within the promoter regions of genes whose products are involved in the hypothalamic regulation of appetite, body weight and metabolism. In the promoter region of proopiomelanocortin (POMC), the most important anorexigenic neurohormone, neonatally overfed rats develop hypermethylation of activating transcription factor binding sites, in parallel with hypomethylation at an inhibitory transcription factor binding site. The promoter region of the hypothalamic insulin receptor gene promoter was found to be hypermethylated. These studies suggest that perinatal programming of long-term increased obesity and diabetes risk due to neonatal over-nutrition may occur via altered methylation patterns of the promoter regions of central nervous body weight-regulating neuropeptides and receptors [32-34]. Consequences of the small-baby-syndrome or small for gestational age for newborns, infants and adolescents During the newborn period SGA babies present increased risk of hypoglycemia, hypothermia, hypercoagulability, hyperbilirubinemia, hypotension, necrotizing enterocolitis, respiratory distress syndrome, lower Apgar scores, umbilical artery acidosis, more intubations and complications during delivery and approximately 20 times increased risk of neonatal death than babies born with an appropriate for gestational age (AGA) weight [24,35-37]. In the first two years of life, about 90–95% of children born SGA present catch-up growth [38]. More than 80% of SGA infants achieve catch-up growth during the first six months of life [39]. Ponderal index at birth is not related to postnatal catch-up growth in infants born SGA, but birth length and target (parental) height are important. The genetic influence on catch-up growth appears to start from the onset of childhood. For SGA children, being born short or becoming short during the first two years of life is similar in terms of risk for adult short stature [40]. In the childhood years, about 10% of children born SGA do not achieve catch-up growth after the second year of life and remain short (≤ 2 SDs) during childhood, adolescence and adulthood [38,41]. The risk of short final adult height was found to be five times higher for children with LBW and seven times higher for those with low birth length compared with children with normal birth size. At age 20, men and women were 4.50 cm and 3.94 cm, respectively, shorter than those born with AGA, and also have raised insulin and proinsulin levels which could be markers of early changes in insulin sensitivity [41]. Children born SGA seem to have modest independent effects on learning, cognition, and attention in adolescence [42]. They also have a greater risk of being psychosocially disadvantaged, less socially competent and present more behavioral difficulties due to impairments of neurocognitive and educational development and also specific adaptation difficulties towards short stature [43]. These children also present low scores of alertness, mood instability, significant differences in academic and professional achievements [44]. Children born SGA present early and rapid start of puberty; the amplitude of pubertal spurt is small, and they reach their final height earlier than children born with AGA. Girls have an advanced menarche by 5–10 months and boys have more genital alterations [38]. Consequences of the small-baby-syndrome or small for gestational age for adults After Barker’s findings of the strong associations between LBW and IHD it was suggested that many diseases of adulthood could have a fetal origin. Many subsequent studies have shown the association between LBW and a higher risk of developing several adult diseases. In 1976, Ravelli et al., have conducted a historical cohort study of 300.000 19-year-old men exposed to the Dutch famine of 1944–45 and examined at military induction. They have found that outcomes were opposite depending on the time of exposure. During the last trimester of pregnancy and the first months of life, exposure to famine produced significantly lower obesity rates, suggesting that nutritional deprivation affected a critical period of development for adipose-tissue cellularity. During the first half of pregnancy, however, exposure resulted in significantly higher obesity rates consistent with the inference that nutritional deprivation affected the differentiation of hypothalamic centers regulating food intake and growth [45]. Many other conditions have been identified such as an increased risk of T2D [11,46,47] and IR with a decreased insulin-stimulated glucose uptake [48]; high death rates from IHD [49,50]; higher risk of developing MS [51-53]; CVD and HT independently of genetic factors, shared familial environment, and risk factors for HT in adulthood, including body mass index [54-57]; dyslipidemia with an atherogenic lipid profile [58-60] and obesity [61,62]. All these findings have been confirmed in many distinct populations such as in USA [63], Sweden [64], France [65], Norway [66] and Finland [67]. The association between LBW and T2D was found to be strong even after correction for many risk factors and it is independent of the degree of obesity and frequency and intensity of physical activity [68]. Epidemiological evidence of this casual relationship has been extended to many other diseases such as higher risk of breast cancer [69,70], end-stage renal disease mainly during the first 14 years of life [71,72], osteoporosis [73,74], spontaneous hypothyroidism [75], adult asthma [76], cardiac hypertrophy [77], depression [78], male reproductive health problems, including hypospadias, cryptorchidism and testicular cancer [38,79], liver cirrhosis [80], adult schizophrenia [81], adult hearing loss [82], polycystic ovary syndrome and precocious pubarche [83]. The phenotype that seems to be more strongly associated to higher risks of adult diseases is LBW followed by a fast catch-up growth [84-86]. Conclusions A clear phenomenological association has been demonstrated by many epidemiological studies between LBW and increased risk later in life, for many diseases such as IR, mortality by IHD, MS, T2D, CVD, HT, dyslipidemia, obesity, breast and testicular cancer, end-stage renal disease, osteoporosis, spontaneous hypothyroidism, adult asthma and hearing loss, cardiac hypertrophy, depression, liver cirrhosis, schizophrenia, polycystic ovary syndrome, precocious pubarche, hypospadias, cryptorchidism, low scores of alertness, mood instability, significant differences in academic and professional achievement. Probably, perinatally acquired alterations of DNA methylation patterns of gene promoters of central nervous regulators of body weight and metabolism play a key role in mediating these relationships. In conclusion, under-nutrition during neonatal life plays a critical role, beyond prenatal development, in the long-term programming of health and disease. This opens a variety of opportunities and challenges to primarily prevent chronic diseases such as stature deficits, endocrine, metabolic and neurodevelopmental disturbances during childhood and several diseases as those above mentioned, during adulthood. This should be appropriately considered in future health care policies as well as research programs. Pre and neonatal under-nutrition should be avoided to prevent long-term deleterious consequences. Further studies are suggested to evaluate epigenomic mechanisms such as alterations of DNA methylation, that potentially underlie the increased risks for all these diseases. Abbreviations IR: Insulin resistance; LBW: Low birth weight; T2D: Type 2 diabetes; HT: Hypertension; IHD: Ischemic heart diseases; MS: Metabolic syndrome; CVD: Cardiovascular disease; SGA: Small for gestational age; SGA-w: Small for gestational age with low birth weight; SGA-l: Small for gestational age with short length; SGA-w/l: Small for gestational age with low birth weight and length; IUGR: Intrauterine growth retardation; ROS: Reactive oxygen species; POMC: Proopiomelanocortin; AGA: Appropriate for gestational age. Competing interests Both authors declare that they have no competing interests. Authors’ contributions CAN and MBG drafted, reviewed and edited the manuscript. Both authors read and approved the final manuscript. ==== Refs Kaufman FR The evolution of our destruction Diabesity: the obesity-diabetes epidemic that threatens America-and what we must do to stop it 2005 1 New York: Bantam Dell Neel JV Diabetes mellitus: a “thrifty” genotype rendered detrimental by “progress”? Am J Hum Genet 1962 14 353 362 13937884 Barker DJP Osmond C Infant mortality, childhood nutrition and ischaemic heart disease in England and Wales Lancet 1986 1 1077 1081 2871345 Lucas A Bock GR, Whelan J Programming by early nutrition in man The childhood environment and adult disease 1991 Chinchester: John Wiley and Sons 38 55 Barker DJP Mothers, Babies and Health in Later Life 1998 2 Edinburgh: Churchill Livingstone Barker DJP Eriksson JG Winter PD Margetts B Simmonds SJ Weight in infancy and death from ischaemic heart disease Lancet 1989 2 577 580 2570282 Forsdahl A Are poor living conditions in childhood and adolescence an important risk factor for arteriosclerotic heart disease? Br J Prev Soc Med 1977 31 2 91 95 884401 Curhan GC Chertow GM Willett WC Spiegelman D Colditz GA Manson JE Birth weight and adult hypertension and obesity in women Circulation 1996 94 6 1310 1315 10.1161/01.CIR.94.6.1310 8822985 Leon DA Koupilova I Lithell HO Berglund L Mohsen R Vagero D Failure to realise growth potential in utero and adult obesity in relation to blood pressure in 50 year old Swedish men BMJ 1996 312 7028 401 406 10.1136/bmj.312.7028.401 8601110 Martyn CN Barker DJ Osmond C Mothers’ pelvic size, fetal growth, and death from stroke and coronary heart disease in men in the UK Lancet 1996 348 9037 1264 1268 10.1016/S0140-6736(96)04257-2 8909378 Hales CN Barker DJP Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis Diabetologia 1992 35 595 601 10.1007/BF00400248 1644236 Lee PA Chernausek SD Hokken-Koleaga AC Czernichow P International Small for Gestational Age Advisory Board consensus development conference statement: management of short children born small for gestational age April 24 – October 1, 2001 Pediatrics 2003 111 1253 1261 10.1542/peds.111.6.1253 12777538 Taipale P Hiilesmaa V Predicting delivery date by ultrasound and last menstrual period in early gestation Obstet Gynecol 2001 97 189 194 10.1016/S0029-7844(00)01131-5 11165580 Morin I Morin L Zhang X Platt RW Blondel B Bréart G Determinants and consequences of discrepancies in menstrual and ultrasonographic gestational age estimates BJOG 2005 112 145 152 10.1111/j.1471-0528.2004.00311.x 15663577 Karlberg J Albertsson-Wikland K Growth in a full term small for gestational age: from birth to final height Pediatr Res 1995 28 219 251 Alkalay AL Graham JM JrPomerance JJ Evaluation of neonates born with intrauterine growth retardation: review and practice guidelines J Perinatol 1998 18 142 151 9605307 Hediger ML Overpeck MD Maurer KR Kuczmarski RJ McGlynn A Davis WW Growth of infants and young children born small or large for gestational age: findings from the Third National Health and Nutrition Examination Survey Arch Pediatr Adolesc Med 1998 152 1225 1231 10.1001/archpedi.152.12.1225 9856434 Rapaport R Tuvemo T Growth and growth hormone in children born small for gestational age Acta Pediatr 2005 94 1348 1355 10.1080/08035250510043860 Hamilton BE Martin JA Ventura SJ Sutton PD Menacker F Births: preliminary data for 2004 Natl Vital Stat Rep 2005 54 1 17 16450552 Bryan SM Hindmarsh PC Normal and abnormal fetal growth Horm Res 2006 65 19 27 10.1159/000091502 16612110 Saenger P Czernichow P Hughes I Reiter EO Small for gestational age: short stature and beyond J Clin Endocrinol Metab 2007 28 2 219 251 Xiong X Buekens P Fraser WD Beck J Offenbacher S Periodontal disease and adverse pregnancy outcomes: a systematic review BJOG 2006 113 135 143 10.1111/j.1471-0528.2005.00827.x 16411989 Negrato CA Tarzia O Jovanovič L Chinellato LE Periodontal disease and diabetes mellitus J Appl Oral Sci 2013 21 1 1 12 10.1590/1678-7757201302106 23559105 Jancevska A Tasic V Damcevski N Danilovski D Jovanovska V Gucev Z Children born small for gestational age (SGA) Prilozi 2012 33 2 47 58 23425869 Ahluwalia IB Merritt R Beck LF Rogers M Multiple lifestyle and psychosocial risks and delivery of small for gestational age infants Obstet Gynecol 2001 97 649 656 10.1016/S0029-7844(01)01324-2 11339910 Ravelli ACJ van der Meulen JHP Michels RPJ Osmond C Barker DJ Hales CN Glucose tolerance in adults after prenatal exposure to famine Lancet 1998 351 173 177 10.1016/S0140-6736(97)07244-9 9449872 de Rooij SR Painter RC Roseboom TJ Phillips DI Osmond C Barker DJ Glucose tolerance at age 58 and the decline of glucose tolerance in comparison with age 50 in people prenatally exposed to the Dutch famine Diabetologia 2006 49 4 637 643 10.1007/s00125-005-0136-9 16470406 Ozanne SE Hales CN The long-term consequences of intrauterine protein malnutrition for glucose metabolism Proc Nutr Soc 1999 58 3 615 619 10.1017/S0029665199000804 10604194 Hales CN Barker DJP The thrifty phenotype hypothesis Br Med Bull 2001 60 5 20 10.1093/bmb/60.1.5 11809615 El-Osta A Brasacchio D Yao D Pocai A Jones PL Roeder RG Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia J Exp Med 2008 205 10 2409 2417 10.1084/jem.20081188 18809715 Negrato CA Mattar R Gomes MB Adverse pregnancy outcomes in women with diabetes Diabetol Metab Syndr 2012 4 1 41 10.1186/1758-5996-4-41 22964143 Plagemann A Harder T Brunn M Harder A Roepke K Wittrock-Staar M Hypothalamic proopiomelanocortin promoter methylation becomes altered by early overfeeding: an epigenetic model of obesity and the metabolic syndrome J Physiol 2009 587 4963 4976 10.1113/jphysiol.2009.176156 19723777 Plagemann A Roepke K Harder T Brunn M Harder A Wittrock-Staar M Epigenetic malprogramming of the insulin receptor promoter due to developmental overfeeding J Perinat Med 2010 38 393 400 20443665 Plagemann A Harder T Schellong K Schulz S Stupin JH Early postnatal life as a critical time window for determination of long-term metabolic health Best Pract Res Clin Endocrinol Metab 2012 26 5 641 653 10.1016/j.beem.2012.03.008 22980046 Bernstein IM Horbar JD Badger GJ Ohlsson A Golan A Morbidity and mortality among very-low-birth-weight neonates with intrauterine growth restriction. The Vermont Oxford Network Am J Obstet Gynecol 2000 182 198 206 10.1016/S0002-9378(00)70513-8 10649179 Clausson B Cnattingius S Axelsson O Preterm and term births of small for gestational age infants: a population-base study of risk factors among nulliparous women Br J Obstet Gynecol 1998 105 1011 1017 10.1111/j.1471-0528.1998.tb10266.x McIntire DD Bloom SL Casey BM Leveno KJ Birth weight in a relation to morbidity and mortality among newborn infants N Engl J Med 1999 340 1234 1238 10.1056/NEJM199904223401603 10210706 Clayton PE Cianfarani S Czernichow P Johannsson G Rapaport R Rogol A Consensus statement: Management of the child born small for gestational age through to adulthood: A consensus statement of the International Societies of Pediatric Endocrinology and the Growth Hormone Research Society J Clin Endocrinol Metab 2007 92 804 810 17200164 Karlberg JP Albertsson-Wikland K Kwan EY Lam BC Low LC The timing of early postnatal catch-up growth in normal, full term infants born short for gestation age Horm Res 1997 48 17 24 10.1159/000191279 9161867 Luo ZC Albertsson-Wikland K Karlberg J Length and body mass index at birth and target height influences on patterns of postnatal growth in children born small for gestational age Pediatrics 1998 102 6 E72 10.1542/peds.102.6.e72 9832600 Leger J Levy-Marchal C Bloch J Pinet A Chevenne D Porquet D Reduced final height and indications for insulin resistance in 20 year olds born small for gestational age: regional cohort study BMJ 1997 315 341 347 10.1136/bmj.315.7104.341 9270455 O’Keeffe MJ O’Callaghan M Williams GM Najman JM Bor W Learning, cognitive, and attentional problems in adolescents born small for gestational age Pediatrics 2003 112 2 301 317 10.1542/peds.112.2.301 12897278 Noeker M Neurocognitive and psychosocial development in SGA and the indication for growth hormone therapy Clin Padiatr 2006 218 5 249 259 10.1055/s-2005-836635 Strauss RS Adult functional outcome of those born small for gestational age: twenty-six-year follow-up of the 1970 British Birth Cohort JAMA 2000 283 5 625 632 10.1001/jama.283.5.625 10665702 Ravelli GP Stein ZA Susser MW Obesity in young men after famine exposure in utero and early infancy N Engl J Med 1976 295 349 353 10.1056/NEJM197608122950701 934222 Forsén T Eriksson JG Tuomilehto J Reunanen A Osmond C Barker D The fetal and chilhood growth of persons who develop type 2 diabetes Ann Intern Med 2000 133 176 182 10906831 Harder T Rodekamp E Schellong K Dudenhausen JW Plagemann A Birth weight and subsequent risk of type 2 diabetes: a meta-analysis Am J Epidemiol 2007 165 8 849 857 10.1093/aje/kwk071 17215379 Jaquet D Gaboriau A Czernichow P Levy-Marchal C Insulin resistance early in adulthood in subjects born with intrauterine retardation J Clin Endocrinol Metab 2000 85 1041 1046 Forsén T Eriksson J Tuomilehto J Teramo K Osmond C Barker DJ Mother’s weight in pregnancy and coronary heart disease in a cohort of Finnish men: follow-up study BMJ 1997 315 7112 837 840 10.1136/bmj.315.7112.837 9353502 Eriksson JG Forsén T Tuomilehto J Osmond C Barker DJ Early growth and coronary disease in later life: longitudinal study BMJ 2001 322 949 953 10.1136/bmj.322.7292.949 11312225 Barker DJ Hales CN Fall CH Osmond C Phipps K Clark PM Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidemia (syndrome x): relation to reduced fetal growth Diabetologia 1993 36 1 62 67 10.1007/BF00399095 8436255 Jaquet D Deghmoun S Chevenne D Collin D Czernichow P Lévy-Marchal C Dynamic change in adiposity from fetal to postnatal life is involved in the metabolic syndrome associated with reduced fetal growth Diabetologia 2005 48 5 849 855 10.1007/s00125-005-1724-4 15834547 Ramadhani MK Grobbee DE Bots ML Castro Cabezas M Vos LE Oren A Lower birth weight predicts metabolic syndrome in young adults: the Atherosclerosis Risk in Young Adults (ARYA) - Study Atherosclerosis 2006 184 1 21 27 10.1016/j.atherosclerosis.2005.03.022 16326169 Eriksson J Forsén T Tuomilehto J Osmond C Barker D Fetal and childhood growth and hypertension in adult life Hypertension 2000 36 790 794 10.1161/01.HYP.36.5.790 11082144 Barker DJ Birth weight and hypertension Hypertension 2006 48 3 357 358 10.1161/01.HYP.0000236552.04251.42 16880347 Barker DJP The developmental origins of adult disease J Am Coll Nutr 2004 23 588S 595S 10.1080/07315724.2004.10719428 15640511 Bergvall N Iliadou A Johansson S de Faire U Kramer MS Pawitan Y Genetic and shared environmental factors do not confound the association between birth weight and hipertension Hypertension 2007 115 2931 2938 Barker DJ Developmental origins of raised serum cholesterol Int J Epidemiol 2003 32 5 876 877 10.1093/ije/dyg293 14559766 Kajantie E Barker DJ Osmond C Forsén T Eriksson JG Growth before 2 years of age and serum lipids 60 years later: the Helsinki Birth Cohort Study Int J Epidemiol 2008 37 2 280 289 10.1093/ije/dyn012 18267964 Nair MK Nair L Chacko DS Zulfikar AM George B Sarma PS Markers of fetal onset adult diseases: a comparasion among low birthweight and normal birthweight adolescents Indian Pediatr 2009 46 S43 S47 19279368 Kensara OA Wootton SA Phillips DI Patel M Jackson AA Elia M Hertfordshire Study Group Fetal programming of body composition: relation between birth weight and body composition measured with dual energy x-ray absortiometry and anthropometric methods in older Englishmen Am J Clin Nutr 2005 82 5 980 987 16280428 Rasmussen EL Malis C Jensen CB Jensen JE Storgaard H Poulsen P Altered fat tissue distribution in young adult men who had low birth weight Diabetes Care 2005 28 1 151 153 10.2337/diacare.28.1.151 15616250 Bhargava SK Sachdev HS Fall CH Osmond C Lakshmy R Barker DJ Relation of serial changes in childhood body-mass index to impaired glucose tolerance in young adulthood N Engl J Med 2004 350 865 875 10.1056/NEJMoa035698 14985484 Whincup PH Cook DG Ashead T Taylor SJ Walker M Papacosta O Childhood size is more strongly related than size at birth to glucose and insulin levels in 10–11 years old children Diabetologia 1997 40 319 326 10.1007/s001250050681 9084971 Law CM Schiell AW Newsome CA Syddall HE Shinebourne EA Fayers PM Fetal, infant, and a childhood growth and adult pressure: a longitudinal study from birth to 22 years of age Circulation 2002 105 1088 1092 10.1161/hc0902.104677 11877360 Li C Jonhnson MS Goran MI Effects of low birth weight on insulin resistance syndrome in Caucasian and African-American children Diabetes Care 2001 24 2035 2042 10.2337/diacare.24.12.2035 11723079 Boney CM Verma A Tucker R Vohr BR Metabolic syndrome in childhood: association with birth weight, maternal obesity, and gestational diabetes mellitus Pediatrics 2005 115 290 296 10.1542/peds.2004-1808 Clausen JO Borch-Jonhsen K Pedersen O Relation between birth weight and the insulin sensitivity index in population sample of 331 young, healthy Caucasions Am J Epidemiol 1997 146 23 31 10.1093/oxfordjournals.aje.a009188 9215220 Innes K Byers T Schymura M Birth characteristics and subsequente risk for breast cancer in very young women Am J Epidemiol 2000 152 12 11121 11128 Mellemkjaer L Olsen ML Sørensen HT Thulstrup AM Olsen J Olsen JH Birth weight and risk of early-onset breast cancer (Denmark) Cancer Causes Control 2003 14 1 61 64 10.1023/A:1022570305704 12708726 Hoy WE Hugson MD Bertram JF Douglas-Denton R Amann K Nephron number, hypertension, renal disease, and renal failure J Am Soc Nephrol 2005 16 2557 2564 10.1681/ASN.2005020172 16049069 Bjorn EV Lorentz MI Torbjorn L Stein H Bjarne MI Low birth weight increases risk for end-stage renal disease J Am Soc Nephrol 2008 19 151 157 10.1681/ASN.2007020252 18057216 Antoniades L MacGregor AJ Andrew T Spector TD Association of birthweight with osteoporosis and osteoarthritis in adult twins Rheumatology 2003 42 791 796 10.1093/rheumatology/keg227 12730541 Dennison EM Sydall HE Sayer AA Gilbody HJ Cooper C Birth weight and weight at 1 year are independent determinants of bone mass in the seventh decade: the Hertfordshire cohort study Pediatr Res 2005 75 4 582 586 15695596 Kajantie E Phillips DI Osmond C Barker DJ Forsén T Eriksson JG Spontaneous hypothyroidism in adult women is predicted by small body size at birth and during childhood J Clin Endocrinol Metab 2006 91 12 4953 4956 10.1210/jc.2006-1093 16984989 Shaheen SO Sterne JA Montgomery SM Azima H Birth weight, body mass index and asthma in young adults Thorax 1999 54 5 396 402 10.1136/thx.54.5.396 10212102 Porrello ER Widdop RE Delbridge LMD Early origins of cardiac hypertrophy: does cardiomyocyte attrition programme for pathological catch-up growth of the heart? Clin Exp Pharmacol Physiol 2008 35 1358 1364 10.1111/j.1440-1681.2008.05036.x 18759854 Gale CR Martyn CN Birth weight and later risk of depression in a national birthcohort Br J Psychiatry 2004 184 28 33 10.1192/bjp.184.1.28 14702224 Main KM Jensen RB Asklund C Høi-Hansen CE Skakkebaek NE Low birth weight and male reproductive function Horm Res 2006 65 Suppl 3 116 122 16612124 Andersen AM Osler M Birth dimensions, parental mortality, and mortality in early adult age: a cohort study of Danish men born in 1953 Int J Epidemiol 2004 33 1 92 99 10.1093/ije/dyg195 15075152 Neugebauer R Accumulating evidence for prenatal nutricional origins of mental disorders JAMA 2005 294 5 621 623 10.1001/jama.294.5.621 16077059 Barrenãs ML Bratthall A Dallgren J The thrifty phenotype hypothesis and hearing problems BMJ 2003 327 7425 1199 1200 10.1136/bmj.327.7425.1199 14630755 Ibáñez L Valls C Potau N Marcos MV de Zegher F Polycystic ovary syndrome after precocious pubarche: ontogeny of the lowbirth weight effect Clin Endocrinol 2001 55 5 667 672 10.1046/j.1365-2265.2001.01399.x Adair LS Cole TJ Rapid child growth raises blood pressure in adolescent boys who were thin at birth Hypertension 2002 41 451 456 12623942 Barker DJP Eriksson JG Forsén TJ Osmond C Fetal origins of adult disease: strenght of effects and biological basis Int J Epidemiol 2002 31 1235 1239 10.1093/ije/31.6.1235 12540728 Cianfarani S Germani D Branca F Low birthweight and adult insulin resistance: the “catch-up growth” hypothesis Arch Dis Child Fetal Neonatal 1999 81 F71 73 10.1136/fn.81.1.F71	24128325	PMC3765917	CC BY	2021-01-05 01:30:58	yes	Diabetol Metab Syndr. 2013 Sep 2; 5:49
==== Front Eur J Med ResEur. J. Med. ResEuropean Journal of Medical Research0949-23212047-783XBioMed Central 2047-783X-18-292400485610.1186/2047-783X-18-29ResearchIntegrated gene network analysis and text mining revealing PIK3R1 regulated by miR-127 in human bladder cancer Xu Yahong 1xuyahong@medmail.com.cnLuo Shunwen 1luosw74@163.comLiu Yang 1liuyangjfj@sohu.comLi Jian 1apollo99101@126.comLu Yi 1luy6116@yahoo.cnJia Zhigang 1megatron118@163.comZhao Qihua 1Oliver452@163.comMa Xiaoping 1Mxp5612@sina.comYang Minghui 1langongjixian@163.comZhao Yue 2Zhaoyue1633@163.comChen Ping 3chenping452@yeah.netGuo Yu 1guoyu_215@126.com.cn1 Department of Urology, the 452nd Hospital of People’s Liberation Army, Chengdu 610021, China2 Cadre aircrew division, the 452nd Hospital of People’s Liberation Army, Chengdu 610021, China3 Nursing Department, the 452nd Hospital of People’s Liberation Army, Chengdu 610021, China2013 1 9 2013 18 1 29 29 22 1 2013 9 7 2013 Copyright © 2013 Xu et al.; licensee BioMed Central Ltd.2013Xu et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background Cancer is the result of a complex multistep process that involves the accumulation of sequential alterations of several genes, including those encoding microRNAs (miRNAs) that have critical roles in the regulation of gene expression. In this study, we aimed to predict potential mechanisms of bladder cancer related miRNAs and target genes by bioinformatics analyses. Methods Here we used the method of text mining to identify nine miRNAs in bladder cancer and adopted protein-protein interaction analysis to identify interaction sites between these miRNAs and related-target genes. Results There are two relationship types between bladder cancer and its related miRNAs: causal and unspecified. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment test showed that there were three pathways related to four miRNA targeted genes. The remaining five miRNAs annotated to disease are not enriched in the KEGG pathways. Of these, PIK3R1 is the overlapping gene among 38 genes in the cancer and bladder cancer pathways. Conclusions These findings provide new insights into the role of miRNAs in the pathway of cancer and give us a hypothesis that miR-127 might play a similar role in regulation and control of PIK3R1. Bladder cancermiRNAphosphoinositide 3-kinase ==== Body Background Bladder cancer is the 10th most common cancer worldwide, with the highest rates reported in Europe, North America and Australia compared to Eastern countries [1,2]; there are an estimated 261,000 new cases diagnosed and 115,000 deaths reported each year. The incidence of bladder cancer is highly correlated with increasing age. It rarely occurs before the age of 40 to 50 years of age, arising most commonly in the seventh decade of life [3,4]. The median ages at diagnosis are 69 years for men and 71 for women [5]. MicroRNAs (miRNAs) are a class of 17 to 27 nucleotide single-stranded RNA molecules that regulate gene expression post-transcriptionally. A large body of evidence implicates aberrant miRNA expression patterns in most, if not all, human malignancies. Indeed, cancer is the result of a complex multistep process that involves the accumulation of sequential alterations of several genes and deregulation of those encoding miRNAs [6]. An increasing number of studies have demonstrated that microRNAs can function as potential oncogenes or oncosuppressor genes, depending on the cellular context and the target genes they regulate [7]. The first evidence of alterations of microRNA genes in human cancer came from studies of chronic lymphocytic leukemia (CLL). In a large study of indolent versus aggressive CLL, Calin et al. discovered a signature of 13 microRNAs capable of distinguishing between indolent and aggressive CLL [8]. For bladder cancer, Takahiro et al. demonstrated that KRT7 mRNA was significantly down-regulated by transfection of miR-30-3p, miR-133a and miR-199a in the bladder cancer cell line (KK47), suggesting that these three miRNAs may have a tumor suppressive role via the mechanism underlying transcriptional repression of KRT7 [9]. miRNAs and their target genes are usually validated by quantitative real time polymer transcriptase chain reaction (q-RT-PCR) and Western blot in a wet lab. However, wet lab works consumes a large amount of time and may not be able to obtain the desired results. Hence, we used text mining to identify nine miRNAs in bladder cancer and adopted protein-protein interaction analysis to identify interaction sites between these miRNAs and target genes. We obtained a long list of statistically significant genes without any unifying biological theme. Functional annotation of differentially expressed genes is a necessary and critical step in the analysis of microarray data [10]. A more judicious approach offers query-based access to an integrated database that disseminates biologically rich information across large datasets and displays graphic summaries of functional information. Therefore, we hope to find the important genes that are highly associated with the biological progression of bladder cancer through the use of bioinformatics tools. Methods miRNAs in bladder cancer Bladder cancer related miRNAs were drawn from miR2Disease (http://www.mir2disease.org), which is a manually curated database providing a comprehensive resource of microRNA deregulation in various human diseases [11]. The current version of miR2Disease documents 1,939 curated relationships between 299 human microRNAs and 94 human diseases by reviewing more than 600 published papers. Known targets of miRNA TarBase database houses a manually curated collection of experimentally tested miRNA targets in human/mouse, fruit fly, worm, and zebra fish, distinguishing between those that tested positive and those that tested negative [12]. A search for experimentally proven targets of nine bladder cancer-related miRNAs was performed using the TarBase database (http://diana.cslab.ece.ntua.gr/tarbase) (approved by the 452nd Hospital of People’s Liberation Army). KEGG pathway analysis The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was determined by the Fisher exact test with a P-value less than 0.05. The KEGG pathway reveals that most target genes of miRNAs are located in the bladder cancer pathway including the Wnt pathway and cancer pathway. Based on this data, microRNA-regulated gene networks associated with bladder cancer were visualized by Cytoscape [13]. Cytoscape is an open source bioinformatics software platform for visualizing molecular interaction networks and integrating them with gene expression profiles and other state data. Only four miRNAs, including oncomiR miR-21, miR-101-3p, miR-221-3p and miR-133 were found in the miRNA-target genes network in this study. Among the remaining five miRNAs, miR-127 is usually differentially expressed as part of a miRNA cluster between normal cells and cancer cells [14]. Hence, we tried to study the functional mechanism of miR-127 in bladder cancer. Target genes prediction TargetScan is a well-known software for predicting microRNA targets from conserved UTR sequences [15], including software (miRanda) and database (microRNA.org). Additionally, starBase is a public platform for exploring miRNA-target interactions from CLIP-Seq (HITS-CLIP, PAR-CLIP) and degradome sequencing (PARE) data [16]. It also provides intersections of multiple target predictions, such as TargetScan, PicTar, miRanda, PITA, RNA22 and miRSVR. In this study, miRNA target genes were obtained from TargetScan (http://www.targetscan.org), miRanda (http://www.microrna.org), starBase (http://www.starbase.sysu.edu.cn) and miRDB (http://www.mirdb.org), respectively (Table 1). The position of the target site was set in the 3'UTR, with the context score −0.3. Table 1 Methods and resources for microRNA (miRNA) target prediction Method Type of method References Resource TargetScan seed complementarity [15] http://www.targetscan.org miRanda complementarity [17] http://www.microrna.org StarBase CLIP-Seq [16] http://www.starbase.sysu.edu.cn miRDB thermodynamicsa [18] http://www.mirdb.org Secondary structures of miRNA binding sites We analyzed miRNA targets based on the common criteria, including seed-pairing, free energy of miRNA: target duplex, and proper dynamic programing score. To avoid the incongruency brought about by different standards or outcomes of RNA secondary structure and free energy predicted by distinct algorithms, we recalculated the secondary structures of miRNA. Of note, microRNA-response elements (MREs) involved in duplexes and their free energy for all predicted miRNAs were obtained from the online database by RNAcofold, which is a program for the prediction of hybrid structures of two RNA sequences (http://www.tbi.univie.ac.at/ivo/RNA) [19,20]. Thus, we provided a relatively equal platform or prerequisite to compare the structure of all these microRNA targets. Results Bladder cancer related miRNAs There are two relationship types between bladder cancer and its related miRNAs: causal and unspecified. In this study, data were filtered by selecting causal relationships between bladder cancer and microRNA. Of note, nine miRNAs such as hsa-miR-199a*, hsa-miR-143, hsa-miR-127, hsa-miR-30-3p, hsa-miR-221, hsa-miR-21, hsa-miR-101, hsa-miR-129 and hsa-miR-133a were listed (Table 2). Table 2 Bladder cancer related microRNAs (miRNAs) in miR2Disease miRNA Reference Year hsa-miR-199a [9] 2009 hsa-miR-143 [21] 2009 hsa-miR-127 [14] 2006 hsa-miR-30-3p [9] 2009 hsa-miR-221 [22] 2009 hsa-miR-21 [23] 2009 hsa-miR-101 [24] 2009 hsa-miR-129 [25] 2009 hsa-miR-133a [9] 2009 Target genes of nine miRNAs and their roles in the KEGG pathway Target genes of nine selected miRNAs were obtained from TargetScan and starBase databases. Then, KEGG pathway analysis was applied to demonstrate the potential biological function of these target genes. Figure 1 shows that target genes of four miRNAs such as hsa-miR-221, hsa-miR-30-3p, hsa-miR-133a and hsa-miR-21 were enriched in three pathways. The remaining five miRNAs annotated to the disease were not enriched in the KEGG pathways. Figure 1 MicroRNA (miRNA) to mRNA network visualized by Cytoscape. Pink dot represents target genes and red dot represents hub genes; yellow rectangle is miRNA; blue edge indicates target gene located in the pathway of human cancer; green edge means that target gene is located in the Wnt signaling pathway and orange edge that target gene is located in the bladder cancer pathway. Target prediction of miR-127 Given that miR-127 is highly embedded in a CpG island and numerous recent studies have revealed the role of miR-127 in human cancers [14,26], we tried to focus on the study of target prediction of miR-127. Thirty eight target genes regulated by miR-127 were identified via five predicted software instruments including TargetScan, PicTar, PITA, miRanda and RNA22. We also identify the binding site of miR-127 and PIK3R1 in chromosome 5:67595672-67595693[+] (GRCh37/hg19) via CLIP-Seq datasets in the deepView genome browser (Figure 2). Figure 2 Illustrative screen shots from the deepView browser. The deepView browser provides an integrated view of CLIP-Seq data, known and predicted microRNA (miRNA) target sites, protein-coding genes, non-coding (ncRNA) genes, miRNAs, strand-specific peak clusters, genome-wide target-peaks and target-plots. In Figure 3, we compared the 38 predicted genes with cancer/bladder cancer related genes and then found that PIK3R1 is the only one gene which overlapped among 38 genes in the bladder cancer pathway (Figure 4) and pathway of cancer (Figure 5). Therefore, we tried to describe miR-127 and its target gene PIK3R1 and their mechanism of action in bladder cancer. Figure 3 Venn diagram of 38 target genes of miR-127. There are 42 genes in the bladder cancer pathway and 325 genes in the cancer pathway. Figure 4 Pathway in bladder cancer, without PIK3R1. Figure 5 Pathway in cancer, includes PIK3R1. The analysis of miRNA targets was based on the common criteria, including seed-pairing, free energy of miRNA, target duplex, and proper dynamic programing score. To avoid the incongruency brought about by different standards or outcomes of RNA secondary structure and free energy predicted by distinct algorithms, we recalculated the secondary structures of miRNA. MicroRNA-response element (MRE) duplexes and their free energy for all the predicted miRNAs were obtained from the online database by RNAcofold. The four most probably binding sites included SNP-56, −2236, −2611 and −3496. Additional file 1: Table S3. Discussion We selected known targets of the nine miRNAs and demonstrated their roles in biological process via KEGG pathway analysis. The KEGG enrichment test showed three pathways to be related to the target genes of the four miRNAs. Only four miRNAs, including miR-21, miR-101-3p, miR-221-3p and miR-133 were in the miRNA-target genes network. MiR-127, one of the remaining five miRNAs, is usually differentially expressed as part of a miRNA cluster between normal cells and cancer cells [14]. Moreover, a predicted target of miR-127, proto-oncogene BCL6, was down-regulated after treatment with chromatin-modifying drugs [14]. In the current study, we focused on the study of the gene PIK3R1 which was the only overlapping gene among 38 genes in the pathway of cancer. PIK3R1, also known as GRB1, p85α, p85-ALPHA, is one of the core members involving the phosphoinositide 3-kinase (PI3K) pathway [27]. PI3K plays a pivotal role in cell growth, proliferation and survival and inter-signaling systems via this pathway are up-regulated in many types of cancer [28,29]. It is strongly hypothesized that alterations of several pathway components can affect the normal function of the PI3K pathway. Knowles et al. has identified that alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer are significantly related to tumor phenotype and clinical behavior [30]. PIK3R1 constitutively binds and inhibits the release of catalytic subunit p110 of PI3K. Mutation of PIK3R1 has been observed in ovarian and colon cancer [31], and higher kinase activity was detected in breast cancer [32]. Our findings confirm that the role of PIK3R1 can also be extrapolated in the biological process of bladder cancer. However, no studies have investigated the role of genetic variations in this pathway in bladder cancer. In this project, we used a large case control study to evaluate the associations of a comprehensive catalog of single nucleotide polymorphisms (SNPs) in the PI3K pathway. Four binding sites of hsa-miR-127-3p including SNP-56, −2236, −2611 and −3496 were identified in the 3′ untranslated region of PIK3R1 mRNA, suggesting that single SNPs located at miRNA-binding sites are likely to affect the expression of their targets and might contribute to the pathogenesis of bladder cancer. Conclusions Our data demonstrate a significant association between miR-127 and its target gene of PIK3R1 via analysis of the CLIP-Seq data, RNA secondary structure and free energy. The results indicate that miR-127 plays an important role in regulating PIK3R1 that is involved in both the cancer and bladder cancer pathways. Abbreviations (miRNAs): microRNAs; (KEGG): Kyoto encyclopedia of genes and genomes; (CLL): Chronic lymphocytic leukemia; (MREs): microRNA-response elements; (MRE): MicroRNA-response elements; (SNPs): Single nucleotide polymorphisms. Competing interests The authors declare that they have no competing interests. Authors’ contributions SL, YL and JL collected the nine miRNAs related to bladder cancer in TarBase and participated in KEGG pathway analysis. YL, ZhJ and PC carried out target genes prediction. YG, QZ, XM participated in analysis of miRNA targets based on the common criteria. YX and MY participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1: Table S3 Binding sits of hsa-miR-127-3p. Click here for file ==== Refs Larsson SC Andersson SO Johansson JE Wolk A Diabetes mellitus, body size and bladder cancer risk in a prospective study of Swedish men Eur J Cancer 2008 44 2655 2660 10.1016/j.ejca.2008.07.012 18707871 Meliker J Nriagu J Arsenic in drinking water and bladder cancer: review of epidemiological evidence Trace Metals other Contaminants Environment 2007 9 551 584 Zeegers MPA Kellen E Buntinx F Brandt PA The association between smoking, beverage consumption, diet and bladder cancer: a systematic literature review World J Urol 2004 21 392 401 10.1007/s00345-003-0382-8 14685762 Shariat SF Milowsky M Droller MJ Bladder cancer in the elderly 2009 Elsevier 653 667 Volanis D Kadiyska T Galanis A Delakas D Logotheti S Zoumpourlis V Environmental factors and genetic susceptibility promote urinary bladder cancer Toxicology letters 2010 193 131 137 10.1016/j.toxlet.2009.12.018 20051252 Visone R Croce CM MiRNAs and cancer Am J Pathol 2009 174 1131 10.2353/ajpath.2009.080794 19264914 Iorio MV Croce CM MicroRNAs in cancer: small molecules with a huge impact J Clin Oncol 2009 27 5848 5856 10.1200/JCO.2009.24.0317 19884536 Calin GA Ferracin M Cimmino A Di Leva G Shimizu M Wojcik SE Iorio MV Visone R Sever NI Fabbri M A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia New Engl J Med 2005 353 1793 1801 10.1056/NEJMoa050995 16251535 Ichimi T Enokida H Okuno Y Kunimoto R Chiyomaru T Kawamoto K Kawahara K Toki K Kawakami K Nishiyama K Identification of novel microRNA targets based on microRNA signatures in bladder cancer Int J Cancer 2009 125 345 352 10.1002/ijc.24390 19378336 Dennis G JrSherman BT Hosack DA Yang J Gao W Lane HC Lempicki RA DAVID: database for annotation, visualization, and integrated discovery Genome Biol 2003 4 P3 10.1186/gb-2003-4-5-p3 12734009 Jiang Q Wang Y Hao Y Juan L Teng M Zhang X Li M Wang G Liu Y miR2Disease: a manually curated database for microRNA deregulation in human disease Nucleic acids res 2009 37 D98 D104 10.1093/nar/gkn714 18927107 Sethupathy P Corda B Hatzigeorgiou AG TarBase: a comprehensive database of experimentally supported animal microRNA targets Rna 2006 12 192 197 16373484 Shannon P Markiel A Ozier O Baliga NS Wang JT Ramage D Amin N Schwikowski B Ideker T Cytoscape: a software environment for integrated models of biomolecular interaction networks Genome res 2003 13 2498 2504 10.1101/gr.1239303 14597658 Saito Y Liang G Egger G Friedman JM Chuang JC Coetzee GA Jones PA Specific activation of microRNA-127 with downregulation of the proto-oncogene BCL6by chromatin-modifying drugs in human cancer cells Cancer Cell 2006 9 435 443 10.1016/j.ccr.2006.04.020 16766263 Lewis BP Burge CB Bartel DP Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets Cell 2005 120 15 20 10.1016/j.cell.2004.12.035 15652477 Yang JH Li JH Shao P Zhou H Chen YQ Qu LH starBase: a database for exploring microRNA-mRNA interaction maps from Argonaute CLIP-Seq and Degradome-Seq data Nucleic acids res 2011 39 D202 D209 10.1093/nar/gkq1056 21037263 Betel D Koppal A Agius P Sander C Leslie C Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites Genome biology 2010 11 R90 10.1186/gb-2010-11-8-r90 20799968 Wang X El Naqa IM Prediction of both conserved and nonconserved microRNA targets in animals Bioinformatics 2008 24 325 332 10.1093/bioinformatics/btm595 18048393 Hofacker IL Vienna RNA secondary structure server Nucleic acids res 2003 31 3429 3431 10.1093/nar/gkg599 12824340 Bernhart SH Tafer H Mückstein U Flamm C Stadler PF Hofacker IL Partition function and base pairing probabilities of RNA heterodimers Algorithms for Molecular Biology 2006 1 3 10.1186/1748-7188-1-3 16722605 Lin T Dong W Huang J Pan Q Fan X Zhang C Huang L MicroRNA-143 as a tumor suppressor for bladder cancer The J urology 2009 181 1372 1380 10.1016/j.juro.2008.10.149 Lu Q Lu C Zhou GP Zhang W Xiao H Wang XR MicroRNA-221 silencing predisposed human bladder cancer cells to undergo apoptosis induced by TRAIL 2010 Elsevier 635 641 Catto JWF Miah S Owen HC Bryant H Myers K Dudziec E Larré S Milo M Rehman I Rosario DJ Distinct microRNA alterations characterize high-and low-grade bladder cancer Cancer res 2009 69 8472 10.1158/0008-5472.CAN-09-0744 19843843 Friedman JM Liang G Liu CC Wolff EM Tsai YC Ye W Zhou X Jones PA The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the polycomb group protein EZH2 Cancer res 2009 69 2623 10.1158/0008-5472.CAN-08-3114 19258506 Dyrskjøt L Ostenfeld MS Bramsen JB Silahtaroglu AN Lamy P Ramanathan R Fristrup N Jensen JL Andersen CL Zieger K Genomic profiling of microRNAs in bladder cancer: miR-129 is associated with poor outcome and promotes cell death in vitro Cancer res 2009 69 4851 10.1158/0008-5472.CAN-08-4043 19487295 Lu J Getz G Miska EA Alvarez-Saavedra E Lamb J Peck D Sweet-Cordero A Ebert BL Mak RH Ferrando AA MicroRNA expression profiles classify human cancers Nature 2005 435 834 838 10.1038/nature03702 15944708 Fruman DA Cantley LC Carpenter CL Structural organization and alternative splicing of the murine phosphoinositide 3-kinase p85 (alpha) gene Genomics 1996 37 113 121 10.1006/geno.1996.0527 8921377 Cantley LC The phosphoinositide 3-kinase pathway Science Signaling 2002 296 1655 Luo J Manning BD Cantley LC Targeting the PI3K-Akt pathway in human cancer: rationale and promise Cancer Cell 2003 4 257 10.1016/S1535-6108(03)00248-4 14585353 Knowles MA Platt FM Ross RL Hurst CD Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer Cancer and Metastasis Reviews 2009 28 305 316 10.1007/s10555-009-9198-3 20013032 Terauchi Y Tsuji Y Satoh S Minoura H Murakami K Okuno A Inukai K Asano T Kaburagi Y Ueki K Increased insulin sensitivity and hypoglycaemia in mice lacking the p85α subunit of phosphoinositide 3-kinase Nature Genetics 1999 21 230 235 10.1038/6023 9988280 Sun M Paciga JE Feldman RI Yuan Z-q Coppola D Lu YY Shelley SA Nicosia SV Cheng JQ Phosphatidylinositol-3-OH kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor α (ERα) via interaction between ERα and PI3K Cancer res 2001 61 5985 5991 11507039	24004856	PMC3766679	CC BY	2021-01-05 01:53:04	yes	Eur J Med Res. 2013 Sep 1; 18(1):29
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24039784PONE-D-13-2573710.1371/journal.pone.0072593Research ArticleProtein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells PKCbeta and CD40 Ligand-Induced Monocytes AdhesionWu Zeyu 1 2 * Zhao Gang 1 2 Peng Lin 1 2 Du Jialin 1 2 Wang Sanming 1 2 Huang Yijie 1 2 Ou Jinrui 1 2 Jian Zhixiang 1 2 * 1 Department of General Surgery, Guangdong General Hospital, Guangzhou, Guangdong Province, China 2 Guangdong Academy of Medical Sciences, Guangzhou, Guangdong Province, China Mohanraj Rajesh Editor UAE University, Faculty of Medicine & Health Sciences, United Arab Emirates * E-mail: zywu1977@163.com (ZYW); JZX-118@163.com (ZXJ)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: ZYW ZXJ. Performed the experiments: ZYW GZ LP. Analyzed the data: JLD SMW YJH JRO. Contributed reagents/materials/analysis tools: ZYW ZXJ. Wrote the paper: ZYW. 2013 9 9 2013 8 9 e7259320 6 2013 15 7 2013 © 2013 Wu et al2013Wu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Accumulating evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. CD40 and its ligand CD40L are highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related molecular mechanisms are not fully understood. The present study was designed to evaluate the direct effect of CD40L on monocytic cell adhesion and gain mechanistic insight into the signaling coupling CD40L function to the proinflammatory response. Exposure of cultured human aortic endothelial cells (HAECs) to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased human monocytic THP-1 cells to adhere to them under static condition. CD40L treatment induced the expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression in HAECs. Furthermore, exposure of HAECs to CD40L robustly increased the activation of protein kinase C beta (PKCβ) in ECs. A selective inhibitor of PKCβ prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, stimulation of ECs to CD40L induced nuclear factor-κB (NF-κB) activation. PKCβ inhibition abolished CD40L-induced NF-κB activation, and NF-κB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. Our findings provide the evidence that CD40L increases VCAM-1 expression in ECs by activating PKCβ and NF-κB, suggesting a novel mechanism for EC activation. Finally, administration of CD40L resulted in PKCβ activation, increased VCAM-1 expression and activated monocytes adhesiveness to HAECs, processes attenuated by PKCβ inhibitor. Therefore, CD40L may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them. Supported by the Science and Technology Planning Project of Guangdong Province, China (No.2011B080701078, No. 20120318083). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Atherosclerosis is a complex pathological process that possesses many features of chronic inflammation and is considered an immunoinflammatory disease [1], [2]. The adhesion of circulating monocytes to endothelial cells (ECs) monolayer, which is regulated by multiple cell adhesion molecules, such as selectins, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 (VCAM-1) (which are expressed on the surface of ECs in response to inflammatory stimuli), contributes importantly to the inflammatory aspects of the progression of atherogenesis [3], [4]. Therefore, modulation of monocyte adhesion to the vascular endothelium is regarded as an important therapeutic target for the prevention and treatment of atherosclerosis. CD40 and CD40 ligand (CD40L or CD154) are members of the tumor necrosis factor (TNF) and TNF-receptor (TNFR) family and tnteraction of the multipotent immunomodulator CD40L with its receptor CD40 has emerged as an important contributor to the inflammatory process in the vessel wall [5]–[7]. CD40 and CD40L are expressed on endothelial cells, vascular smooth muscle cells, mononuclear cells, and platelets, and CD40-CD40L interaction has been shown to exhibit proinflammatory and proatherogenic effects in vitro and in vivo [8], [9]. In addition to the cell-associated form, CD40L also exists in a soluble, biologically active form (sCD40L), which has similar proinflammatory effects on vascular cells. Interestingly, sCD40L is associated with acute coronary syndromes [10], [11], as well as hypercholesterolemia [12], and elevated sCD40L levels predict an increased cardiovascular risk in healthy subjects [13]. Therefore, CD40L has been suggested as a potential therapeutic target to modulate vascular inflammation and possibly influence cardiovascular risks. However, the underlying molecular mechanism by which CD40L enhances vascular inflammation and atherogenesis is not fully understood. The present study tested the hypothesis that CD40L induce monocyte activation and subsequent adhesion to ECs. It also examined the direct effects of CD40L on signal transduction involved in these processes. Materials and Methods Materials Human recombinant CD40L was obtained from Alex Inc and was purified before use with the EndoTrap 5/1 (Profos AG) to remove contaminated bacterial endotoxins (lipopolysaccharide). Antibodies used in the present study include the following: mouse anti-PKCβ antibody (BD Biosciences), mouse anti–VCAM-1 (Chemicon International), rabbit anti–NF-κB p65 antibody, rabbit anti-IκBα antibody, rabbit anti–β-actin antibody (Cell Signaling Technology), and goat anti-CD40 antibody (Academy Biomedical). Selective PKCβ inhibitor [3-(1-(3-imidazol-1-ylpropyl)- 1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione] were purchased from Calbiochem (San Diego, CA, USA). Animals Male C57BL6 mice, 8 weeks of age, were obtained from the Jackson Laboratory (Bar Harbor, Maine). Mice were housed in temperature-controlled cages with a 12-h light-dark cycle and given free access to water and normal chows. These mice were randomly divided into sham-treated (control group) and CD40L-treated groups. CD40L (1.5 mg/g/d) was administered by tail-vein injection for 3 consecutive days, and control mice received 0.9% physiological saline injection. To identify the critical role of PKCβ, some mice also received PKCβ inhibitor (2 mg/kg) which was given by intraperitoneal injection. The mice were euthanized with inhaled isoflurane. Mice aortas were removed and immediately frozen in liquid nitrogen. The animal protocol was reviewed and approved by the institutional Animal Care and Use Committee of Guangdong Academy of Medical Sciences. Cell culture Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL). The cells were grown at 37°C in humidified 5% CO2 and used for experiments between passages 3 and 5 [14]. Human peripheral monocytes were collected under a protocol approved by the Human Research Committee of the Guangdong General Hospital and were cultured as described previously [15]. The participants provide their written informed consent to participate in this study. Static adhesion assay THP-1 monocytes were prestained with 5 µM calcein-AM (Invitrogen) at 37°C for 30 minutes. After washing in PBS, fluorescently labeled THP-1 monocytes were added onto the HAEC monolayers at the density of 106 cells/mL. To block VCAM-1 function, HAEC monolayers were incubated with blocking antibodies against VCAM-1 (25 µg/mL) for 1 h before the addition of THP-1 monocytes. Nonadherent monocytes were removed by gently washing with complete medium after 30 minutes. Fluorescence intensity (FI) was measured using the Infinite F200 Fluorescent ELISA Reader (TECAN) set at excitation and emission wavelengths of 485 and 530 nm [16]. Some experiments used freshly isolated human peripheral monocytes. Flow conditions adhesion assay Confluent HAEC monolayers, grown on 25-mm glass coverslips, were inserted into the flow chamber. THP-1 cells (0.5×106/mL) suspended in flow buffer (PBS containing 0.1% human serum albumin) were drawn through the chamber at flow rates corresponding to an estimated shear stress of 1.0 dyne/cm2. The accumulation of THP-1 cells on ECs after 2 minutes of cell perfusion by counting the number of cells in 4 different fields. Quantitative real-time polymerase chain reaction Total mRNA was isolated with TRIZOL Reagent (Invitrogen) according to manufacturer's instructions. Real-time PCR was performed with the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, Calif) on the ABI 7500 DNA Sequence Detection System with standard fluorescent chemistries by using 5′-GATACAACCGTCTTGGTCAGCCC-3′ (sense) and 5′-CGCATCCTTCAACTGGCCTT-3′ (antisense) for the VCAM-1. The correlation between the amounts of RNA used and of PCR products obtained with target gene and with the internal standard (β-actin) was examined. Small interfering RNA (siRNA) transfection Control siRNA and siRNA against CD40 were obtained from Santa Cruz Biotechnology, and HAECs were transfected according to the manufacturer's instruction. Briefly, 2.0×106 cells were seeded on 100-mm plates the day before transfection. The medium was switched to Opti-MEM and either control siRNA or CD40 siRNA in Oligofectamine was added to the culture medium for 4 h (final concentration 100 nM), after which the medium was replaced with normal culture medium. Luciferase assay For transient transfection, 0.5 µg NF-κB reporter were simultaneous transfected into cells with 0.3 µg of the β-galactosidase (β-gal) reporter as internal reference. To transfect the construct of VCAM-1 luciferase plasmids, we cloned regions spanning –1716 to –119 bp of the human VCAM-1 promoter into vector pGL3-basic (Promega). HAECs were transfected with 1 µg of the plasmids and 1 µg of the control pCMV-β-gal plasmid using LipofectAMINE Plus reagents (Invitrogen). Cell extracts were prepared 24 h after transfection, and luciferase assays were performed using the Dual-Luciferase® Reporter (DLR™) Assay System (Promega), then normalized for β-gal using the formula (luciferase activity/β-gal activity×100) and reported as relative light units of luciferase activity (RLU) [17]. NF-κB p65 DNA-binding activity Five microgram of nuclear extracts was used to determine p65 DNA-binding activity by using an ELISA-based assay, according to the manufacturer's instructions (Active Motif TransAM). Briefly, κB oligonucleotide-coated plates (in a 96-well format) were incubated for 1 hour with the nuclear extracts. Specificity was achieved through incubation with anti-p65 primary antibodies for 1 h. HRP-conjugated secondary antibodies were used for the detection of p65 bound to the κB sequences. Immunoblotting To detect PKC activation, cytosol and membrane fractions of THP-1 cell lysates were prepared as described previously [18]. To detect NF-κB nuclear translocation and IκBα cytosol degradation, cytosol and nuclear fractions of THP-1 cells (1×106/mL) were prepared with the use of Nuclear and Cytoplasmic Extraction Reagents (Pierce). An equal amount of protein (30 μg) from each fraction was subjected to 12% SDS-PAGE. Immunoreactive protein was detected with SignalFire™ ECL Reagent (Cell Signaling Technology). The β-actin was used as the loading control. Platelet preparation and activation The platelet-rich plasma obtained from healthy human subjects was collected into an equal volume of acid-citrate-dextrose buffer (38 mM citric acid, 75 mM trisodium citrate, 124 mM glucose; pH 4.5) and centrifuged at 700 g for 10 minutes. Platelets were resuspended in Tyrode's/HEPES buffer (1.8 mM CaCl2, 2.7 mM KCl, 0.5 mM MgCl2, 137 mM NaCl, 10 mM HEPES, 0.36 mM NaH2PO4, 5 mM glucose; pH 7.4), and centrifuged at 700 g for 10 minutes. After resuspension, 1.5×108 platelets were incubated with HAECs, and activated with 0.2 U/mL human thrombin (Sigma Aldrich). Cell culture dishes were centrifuged at 700 g for 2 minutes, and thrombin was neutralized after 5 minutes with 2 U/mL hirudin (Sigma Aldrich) [19]. PKCβ activity assay PKCβ was first immunoprecipitated by PKCβ-specific antibody and PKCβ activity was assayed by PKCβ-specific peptides using TruLight™ Protein Kinase Cβ Assay Kit (Calbiochem) according to the provided protocol. Preparation of sub-cellular fractions: Cellular cytosolic, membrane and nuclear fractions were prepared as described previously [18]. Statistical analyses Results are expressed as mean ± SEM. Comparison between groups was analyzed via one-way analysis of variance followed by Student-Newman-Keuls test. P<0.05 was considered significant. Nonquantitative results were representative of at least three independent experiments. Results CD40L induce the adhesion of THP-1 cells and human peripheral monocytes to vascular ECs We first used a calcein-AM fluorescence-based adhesion assay to evaluate the effect of CD40L on cell-cell adhesion between monocytes and ECs. Exposure of HAECs to CD40L for 24 hours increased THP-1 cell adhesion in a dose-dependent manner ( Figure 1A and 1B ). Adhesion of THP-1 cells to ECs significantly increased as early as 4 hours and reached a maximum at 24 hours after incubation ( Figure 1C ). CD40L also increased the adhesion of human peripheral blood monocytes to ECs ( Figure 1D ). 10.1371/journal.pone.0072593.g001Figure 1 CD40L induces the adhesion of THP-1 cells or human peripheral monocytes to ECs. (A) HAECs were incubated with the indicated concentrations of CD40L for 24 h, and static adhesion assays were performed as detailed in Methods. Attached THP-1 cells were visualized and counted on an inverted fluorescent microscopy. Magnification, ×20. (B) Quantification of fluorescence density expressed as means ± SEM. * P<0.05 vs 0 ng/mL. (C) HAECs were incubated in the presence of (40 ng/mL) for the indicated hours, and then static adhesion assays were performed. P<0.05 vs 0 h. (D) HAECs were incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed with the use of human peripheral monocytes. P<0.05 vs control. (D) Platelets were activated as described and incubated with HAECs, then THP-1 cells adhesion was analyzed by static adhesion assays. * P<0.05 vs resting platelets. Under the pathophysiological milieu, activated platelets may serve as the source of sCD40L and stimulus for activation of CD40 receptor, thus promoting CD40-induced signaling. Therefore, we performed analysis to determine the effect of activated platelets on adhesion of THP-1 cells to ECs. As shown in Figure 1E , activated platelets potently stimulated adhesion of THP-1 cells to HAECs, and this induction was largely reversed in the presence of anti-CD40L antibody, indicating the CD40L dependent. CD40L increases the expression of VCAM-1 in vascular ECs VCAM-1 is a well-known mediator of monocyte adhesion to the endothelium, leading to the infiltration of monocytes into the subendothelial area and the development of atherosclerosis [20]. To investigate the underlying mechanism of CD40L-mediated inhibition of monocyte adhesion, we then explore the role of CD40L on VCAM-1 expression in HAECs. CD40L treatment broadly and markedly stimulated the expression of VCAM-1 mRNA ( Figure 2A ) and protein ( Figure 2B ) in HAECs. Because VCAM-1 is mainly regulated at the transcriptional level, the effects of CD40L on VCAM-1 promoter activity were explored. CD40L significantly increased VCAM-1 promoter activity in HAECs ( Figure 2C ). Anti–VCAM-1 blocking antibody essentially attenuated CD40L-induced THP-1 cell adhesion to ECs, validating that VCAM-1 plays a dominant role in this process ( Figure 2D ). 10.1371/journal.pone.0072593.g002Figure 2 CD40L induces expression of VCAM-1 in ECs. (A and B) HAECs were incubated in the presence of indicated concentrations of for 24 hours. A, Total RNA was isolated and subjected to quantitative RT-PCR to analyze VCAM-1 mRNA levels. * P<0.05 or ** P<0.01 vs 0 ng/mL. (B) VCAM-1 protein expression was determined by Western blot. Total cell lysates were subjected to SDS-PAGE and immunoblotting. Blots represent 4 independent experiments with similar results. (D) HAECs were pretreated with antibodies (50 μg/mL) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed. * P<0.05 vs CD40L. Data are representative of 4 independent experiments with similar results. CD40L induces the adhesion of THP-1 cells to ECs under flow conditions Next, we examined the effects of CD40L on THP-1 cell adhesion to ECs under flow conditions. Few if any THP-1 cells accumulated on control (PBS)-treated ECs under laminar shear stress (1.0 dyne/cm2). After incubation of ECs with CD40L, THP-1 cell adhesion increased significantly. Most of accumulated THP-1 cells adhered to Ecs in response to CD40L stimulation compared with unstimulated control cells ( Figure 3A ). Accumulation of THP-1 cells induced by CD40L was attenuated significantly in ECs pretreated with anti–VCAM-1 blocking antibody ( Figure 3B ). 10.1371/journal.pone.0072593.g003Figure 3 CD40L induces the accumulation of THP-1 cells on ECs under flow conditions. (A and B) HAECs were incubated in the presence of CD40L (80 ng/mL) or PBS (control) for 24 h, and flow adhesion assays were performed at 37°C. In some experiments, HAECs were pretreated with anti–VCAM-1 antibody (50 μg/mL) for 30 minutes before assay. * P<0.05 vs CD40L. Photographs captured from microscope represent 3 independent experiments with similar results. ×20. (B) Quantification of attached THP-1 cells. * P<0.05 vs CD40L. Data are representative of 4 independent experiments with similar results. Inhibition of CD40 with siRNA blocks CD40L-enhanced VCAM-1 expression and ECs adhesion To investigate whether CD40L increased VCAM-1 expression and ECs adhesion by binding to its receptor, CD40, CD40L (40 ng/mL) was added to the HAECs, which had been pre-transfected with the specific siRNA against CD40. Transfection of the CD40 siRNA resulted in 90±5% reduction of CD40 protein expression in HAECs, as detected with immunoblotting using the antibody specific for CD40 ( Figure 4A ). Notably, transfection of the CD40-specific siRNA markedly reduced CD40L- upregulated VCAM-1 expression in HAECs ( Figure 4B ), leading to the abrogation of monocyte adhesion to ECs ( Figure 4C ). These results indicated that CD40 was required for CD40L-enhanced inflammatory response in HAECs exposed to CD40L. 10.1371/journal.pone.0072593.g004Figure 4 Interaction of CD40 with CD40L mediates CD40L-enhanced VCAM-1 expression and THP-1 adhesion. HAECs were transfected with vectors encoding the CD40 siRNA or control siRNA and then incubated with CD40L (80 ng/mL) for 24 h. (A) Representative blot showing CD40 protein expression in HAECs transfected with control or CD40 siRNA. (B) VCAM-1 protein expression and (C) THP-1 adhesion were determined as indicated. Data are shown as representative blots or are expressed as the means ± SEM by three independent assays from 4 independent experiments. * P<0.05 vs CD40L plus Control siRNA. CD40L induces NF-κB activation at the upstream of Iκ-Bα phosphorylation in vascular ECs Transcription factor NF-κB activation is mediated by phosphorylation of IκBα, an inhibitor of NF-κB, and nuclear translocation of NF-κB p65. We next examined the effect of CD40L on NF-κB activation in ECs. As shown in Figure 5A , exposure of ECs with CD40L resulted in significant induction of NF-κB luciferase activity in a concentration-dependent manner. To avoid the limitations of transient transfection systems, we further determined the function of CD40L on NF-κB transcriptional activity. CD40L treatment also increased the NF-κB DNA binding activity in a dose-dependent fashion ( Figure 5B ). 10.1371/journal.pone.0072593.g005Figure 5 CD40L induces NF-κB activation. (A) HAECs were transfected with NF-κB reporter for 24 h and then incubated with the indicated concentrations of CD40L for another 8 h. The luciferase activity was determined using β-gal as the control. Results of three independent experiments are expressed as fold of control. * P<0.05, ** P<0.01 vs 0 ng/mL. (B and C) HAECs were stimulated with the indicated concentrations of CD40L for 2 h. Cells were lysed and the protein extracts were assayed for p65 DNA-binding activity. (C) IκB-α protein expression was measured by immunoblotting. (D) HAECs were infected with Ad-IκB, or Ad-GFP for 24 h and then incubated with CD40L (80 ng/ml) for another 24 h. VCAM-1 protein expression was determined by Western blot. The results were reproducible in 4 independent experiments. * P<0.05. NF-κB activation requires the phosphorylation, ubiquitination, and degradation of its inhibitor, IκBα [21]. Cytoplasmic extracts were recovered before and 15 minutes and 2 hours after stimulation and Western blot analysis of IκBα was conducted. Exposure of HAECs with CD40L induced IκBα degradation, demonstrating that CD40-mediated NF-κB activation upstream of IκBα degradation ( Figure 5C ). To confirm the functional role of NF-κB activation, HAECs were infected with Ad-IκB and treated with sCD40L; and VCAM-1 mRNA and protein levels were studied. As shown in Figure 5D , Ad-IκB infection effectively blocked CD40L- induced upregulation of VCAM-1 protein expression. These effects were specific because Ad-GFP infection had no effect on VCAM-1 levels. Thus, NF-κB activity induced by CD40L is responsible for the increased VCAM-1 levels. PKCβ mediates CD40L-induced NF-κB activation PKCβ functions as the upstream kinase in IKK activation [22]. To establish PKCβ as a mediator for CD40L-induced expression of VCAM-1, we first determined whether PKCβ inhibitor, an anilino-monoindolylmaleimide compound that potently inhibits PKCβ without affecting other PKC isoforms, altered the effects of CD40L on NF-κB activation. PKCβ inhibitor significantly ablated CD40L-enhanced IκBα degradation and NF-κB p65 nuclear translocation ( Figure 6A ). Additional evidence for PKCβ–dependent NF-κB activation was obtained from genetic inhibition of PKCβ. As demonstrated in Figure 6B , adenoviral overexpression of PKCβ-DN, but not empty vector, abolished the effects of CD40L on NF-κB activation, whereas overexpression of PKCβ-WT significantly enhanced CD40L-induced NF-κB activation. 10.1371/journal.pone.0072593.g006Figure 6 Effect of CD40L on NF-κB activation and IκBα degradation in ECs. (A) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 2 h. Cytosol and nuclear fractions were prepared to measure NF-κB p65 and IκB-α protein expression by immunoblotting. (B) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h. Total cell lysates were prepared and subjected to immunoblotting. Blots represent 4 independent experiments with similar results. We further determine the PKCβ is responsible for cell adhesion induced by CD40L, by treating vascular ECs with PKCβ inhibitor. The PKCβ inhibitor also markedly diminished CD40L-induced VCAM-1 expression ( Figure 6C ) and monocyte adhesion ( Figure 6D ). Taken together, these results suggest that CD40L triggers PKCβ activation in ECs, which leads to activation of NF-κB and induction of VCAM-1 expression. CD40L activate PKCβ in vascular ECs We next determined whether CD40L activated PKCβ in HAECs. The phosphorylation of PKCβ at Thr642 and translocation of PKCβ from the cytosol into cytoplasmic membrane are considered critical steps in the activation of PKCβ. Thus, PKCβ phosphorylation was monitored in total cell lysates in Western blots. As shown in Figure 7A , CD40L treatment induced PKCβ Thr642 phosphorylation without altering the total PKCβ expression. Inhibition of PKCβ with PKCβ inhibitor abolished CD40L-induced PKCβ phosphorylation, indicating a specific inhibition by PKCβ inhibitor. We next assayed PKCβ activity by using incorporation in PKCβ–specific peptides. Exposure of HAECs to CD40L significantly increased PKCβ activity. Overexpression of PKCβ-DN abolished CD40L-enhanced PKCβ activation, whereas PKCβ-WT increased PKCβ activity ( Figure 7B ). These results implied that CD40L activated PKCβ. 10.1371/journal.pone.0072593.g007Figure 7 CD40L increases PKCβ phosphorylation and the translocation of PKCβ from cytosol to the membrane. Confluent HAEC were exposed to CD40L (80 ng/mL, 1 h), and the translocation of PKCβ and PKCβ phosphorylation was assayed as described in Methods. (A) CD40L increased the phosphorylation of PKCβ in HAEC. The blot is a representative of 3 blots obtained from 3 independent experiments. (B) PKCβ activity was determined as described in Methods (n = 4). * P<0.05 vs CD40L plus GFP. (C) CD40L increases the translocation of PKCβ from cytosol into the nucleus. The blot is a representative of 3 blots from 3 individual experiments. (D) Analysis of the purity of subcellular fractions. The subcellular fractions were prepared as described in Methods. Marker enzymes were detected by Western blot with the use of specific antibodies. The translocation of PKCβ is considered a critical step in PKCβ activation. Exposure of HAECs to CD40L significantly increased the presence of PKCβ in membrane fractions but lowered the amount of PKCβ in the cytosol ( Figure 7C ). The purity of these subcellular fractions was confirmed by using antibodies against specific protein marker enzymes [23], [24] of the cytosol (lactate dehydrogenase), plasma membrane (alkaline phosphatase), respectively. Lactate dehydrogenase was detected only in the cytosolic fraction, whereas alkaline phosphatase was found only in the membrane fraction ( Figure 7D ). Thus, CD40L caused cellular redistribution of PKCβ from the cytosol to membranes. CD40L-dependent monocyte adhesion Is operative in vivo In an effort to determine whether CD40L causes monocyte adhesion in vivo, recombinant CD40L (1.5 mg/kg) was administered into C56BL6J mice by tail-vein injection. Three days after being given CD40L, mice were euthanized; VCAM-1 expression and monocyte adhesion were monitored in both CD40L-infused and vehicle-treated mice. CD40L treatment significantly induced PKCβ activation ( Figure 8A ), increased VCAM-1 expression ( Figure 8B ) and enhanced the adhesiveness of monocytes to HAECs ( Figure 8C ). These effects of CD40L were almost completely abolished in the presence of PKCβ inhibitor. 10.1371/journal.pone.0072593.g008Figure 8 PKCβ mediates CD40L induced mice monocyte activation. C57BL/6J mice were injected IV with CD40L (1.5 mg/kg/d) from femoral veins. After 3 days, monocytes were isolated from plasma. In some experiments, monocytes were isolated from plasma. (A) Representative blots showing PKCβ activation in the aortas. (B) VCAM-1 expression. (C) Monocytes were isolated from plasma and adhesion was assayed as indicated. The results are representative of 6 mice in each group. Discussion In this study, we demonstrated that CD40L increases expression of adhesion molecules, especially VCAM-1, in nonactivated ECs, thus enhancing adhesion of THP-1 cells under static and laminar flow condition. Anti–VCAM-1 antibody inhibited THP-1 cell accumulation, thus validating a contribution of VCAM-1 to this process. Furthermore, we implicated that PKCβ mediates the CD40L-induced monocyte activation. Pharmacological or genetic inactivation of PKCβ reduced the response of human or mouse monocytes exposed to CD40L. Inhibition of PKCβ in HAECs not only decreased CD40L-induced NF-κB activation but also reduced CD40L-mediated VCAM-1 expression and monocyte adhesion to HAECs. Thus, cells that express PKCβ or conditions that increase expression of PKCβ may exhibit enhanced response to CD40L. Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol. PKC participates importantly via several mechanisms that promote atherogenesis [25]. In the present study, CD40L activated PKCβ in vascular ECs. PKCβ, which plays a role in inflammation in various types of cells, increases monocyte-endothelial interaction by mediating increase in VCAM-1 in ECs [26]. We found that selective inhibition of PKCβ abolished induction of VCAM-1 by CD40L, indicating its central role in CD40L-induced EC activation. NF-κB is a key transcriptional factor involved in regulating the expression of proinflammatory mediators, including adhesion molecules, thereby playing a critical role in mediating inflammatory responses [27]. To achieve its biological functions, NF-κB must undergo a variety of post-translational modifications, including acetylation [28], [29]. This study identifies NF-κB as the molecular link between CD40L-induced PKCβ activation and increased expression of VCAM-1. Distinct PKC isoforms stimulate NF-κB in different ways. Previous study reported that PKCβ activation by high glucose induces activation of NF-κB and increased expression of VCAM-1 in ECs [25]. We show here that CD40L induces IκBα degradation in the cytosol and translocation of NF-κB p65 to the nucleus in ECs. The underlying possible mechanism applied by CD40L to activate PKCβ in ECs remains unclear. Ca2+, phospholipids, and diacylglycerol activate conventional PKC enzymes including PKCβ [25]; however, we currently have little information about the direct effects of CD40L on these molecules. The exact mechanism(s) for CD40L- induced PKCβ activation in ECs will require further investigation. Our results indicate that CD40L induce VCAM-1 in ECs via a PKCβ and NF-κB activation pathway and increase THP-1 cell adhesion to ECs, suggesting a novel mechanism for EC activation by CD40 signaling. In conclusion, this study demonstrated that the PKCβ signaling pathway participates in the proinflammatory action of CD40L through inducing NF-κB activation and VCAM-1 expression in ECs and monocytes adhesion. This pathway may contribute to the diverse inflammatory responses to CD40L and the link between CD40L levels and adverse clinical outcomes and may further support the involvement of PKCβ in atherogenesis induced by proinflammatory conditions. Our observations shed new light on the molecular pathways that link inflammation, atherosclerosis, and cardiovascular events. ==== Refs References 1 Ross R (1999 ) Atherosclerosis: an inflammatory disease . N Engl J Med 340 : 115 –126 .9887164 2 Hansson GK , Libby P (2006 ) The immune response in atherosclerosis: a double-edged sword . Nat Rev Immunol 6 : 508 –519 .16778830 3 Galkina E , Ley K (2007 ) Vascular adhesion molecules in atherosclerosis . Arterioscler Thromb Vasc Biol 27 : 2292 –2301 .17673705 4 Kawakami A , Tanaka A , Nakajima K , Shimokado K , Yoshida M (2002 ) Atorvastatin attenuates remnant lipoprotein-induced monocyte adhesion to vascular endothelium under flow conditions . Circ Res 91 : 263 –271 .12169653 5 Schonbeck U , Lippy P (2001 ) CD40 signaling and plaque instability . Circ Res 89 : 1092 –1103 .11739273 6 Mach F , Schonbeck U , Libby P (1998 ) CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis 137 Suppl: S89–95 7 Phipps RP (2000 ) Atherosclerosis: the emerging role of inflammation and the CD40-CD40 ligand system . Proc Natl Acad Sci U S A 97 : 6930 –6932 .10860949 8 Mach F , Schonbeck U , Sukhova GK , Bourcier T , Bonnefoy JY , et al (1997 ) Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis . Proc Natl Acad Sci U S A 94 : 1931 –1936 .9050882 9 Karmann K , Hughes CC , Schechner J , Fanslow WC , Pober JS (1995 ) CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression . Proc Natl Acad Sci U S A 92 : 4342 –4346 .7538666 10 Forfang K , Froland SS , Gullestad L (1999 ) Enhanced levels of soluble and membrane bound CD40 ligand in patients with unstable angina: possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes . Circulation 100 : 614 –620 .10441098 11 Heeschen C , Dimmeler S , Hamm CW , van den Brand MJ , Boersma E , et al (2003 ) Soluble CD40 ligand in acute coronary syndromes . N Engl J Med 348 : 1104 –1111 .12646667 12 Garlichs CD , Johm S , Schmeißer Eskafi S , Stumpf C , Karl M , et al (2001 ) Upregulation of CD40 and CD40 ligand (CD154) in patients with moderate hypercholesterolemia . Circulation 104 : 2395 –2400 .11705814 13 Schonbeck U , Varo N , Libby P , Buring J , Ridker PM (2001 ) Soluble CD40L and cardiovascular risk in women . Circulation 104 : 2266 –2268 .11696462 14 Zhang Y , Qiu J , Wang X , Zhang Y , Xia M (2011 ) AMP-activated protein kinase suppresses endothelial cell inflammation through phosphorylation of transcriptional coactivator p300 . Arterioscler Thromb Vasc Biol 31 : 2897 –2908 .21940946 15 Kawakami A , Tanaka A , Nakajima K , Shimokado K , Yoshida M (2002 ) Atorvastatin attenuates remnant lipoprotein-induced monocyte adhesion to vascular endothelium under flow conditions . Circ Res 91 : 263 –271 .12169653 16 Kawakami A , Aikawa M , Libby P , Alcaide P , Luscinskas FW , et al (2006 ) Apolipoprotein CIII in apolipoprotein B lipoproteins enhances the adhesion of human monocytic cells to endothelial cells . Circulation 113 : 691 –700 .16461842 17 Xia M , Ling W , Zhu H , Wang Q , Ma J , et al (2007 ) Anthocyanin prevents CD40-activated proinflammatory signaling in endothelial cells by regulating cholesterol distribution . Arterioscler Thromb Vasc Biol 27 : 519 –524 .17158355 18 Xie Z , Dong Y , Scholz R , Neumann D , Zou MH (2008 ) Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells. Circulation . 117 : 952 –962 . 19 Henn V , Slupsky JR , Grafe M , Anagnostopoulos I , Forster R , et al (1998 ) CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells . Nature 391 : 591 –594 .9468137 20 Li H , Cybulsky MI , Gimbrone MA Jr, Libby P (1993 ) An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium . Arterioscler Thromb 13 : 197 –204 .7678986 21 Cooper JT , Stroka DM , Brostjan C , Palmetshofer A , Bach FH , et al (1996 ) A20 blocks endothelial cell activation through a NF-kappaB-dependent mechanism . J Biol Chem 271 : 18068 –18073 .8663499 22 Shinohara H , Yasuda T , Aiba Y , Sanjo H , Hamadate M , et al (2005 ) PKC beta regulates BCR-mediated IKK activation by facilitating the interaction between TAK1 and CARMA1 . J Exp Med 202 : 1423 –1431 .16301747 23 Hoffmann K , Blaudszun J , Brunken C , Hopker WW , Tauber R , et al (2005 ) New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues . Anal Bioanal Chem 381 : 1138 –1144 .15761741 24 Srinivas KS , Chandrasekar G , Srivastava R , Puvanakrishnan R (2004 ) A novel protocol for the subcellular fractionation of C3A hepatoma cells using sucrose density gradient centrifugation . J Biochem Biophys Methods 60 : 23 –27 .15236907 25 Koya D , King GL (1998 ) Protein kinase C activation and the development of diabetic complications . Diabetes 47 : 859 –866 .9604860 26 Kouroedov A , Eto M , Joch H , Volpe M , Luscher TF , et al (2004 ) Selective inhibition of protein kinase C{beta}2 prevents acute effects of high glucose on vascular cell adhesion molecule-1 expression in human endothelial cells . Circulation 110 : 91 –96 .15210597 27 Rao RM , Yang L , Garcia-Cardena G , Luscinskas FW (2007 ) Endothelial- dependent mechanisms of leukocyte recruitment to the vascular wall . Circ Res 101 : 234 –247 .17673684 28 Ishinaga H , Jono H , Lim JH , Kweon SM , Xu H , et al (2007 ) TGF-beta induces p65 acetylation to enhance bacteria- induced NF-kappaB activation . EMBO J 26 : 1150 –1162 .17268554 29 Chen LF , Mu Y , Greene WC (2002 ) Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-κ . B. EMBO J 21 : 6539 –6548 .12456660	24039784	PMC3767684	CC BY	2021-01-05 17:39:08	yes	PLoS One. 2013 Sep 9; 8(9):e72593
==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2013/782450Research ArticleSuperoxide-Dismutase Deficient Mutants in Common Beans (Phaseolus vulgaris L.): Genetic Control, Differential Expressions of Isozymes, and Sensitivity to Arsenic Talukdar Dibyendu 1 Talukdar Tulika 2 1Department of Botany, RPM College, University of Calcutta, Uttarpara, West Bengal, Hooghly 712 258, India2Department of Botany, Krishnagar Govt. College, University of Kalyani, West Bengal, Krishnanagar 741101, IndiaDibyendu Talukdar: dibyendutalukdar9@gmail.comAcademic Editor: Brynn Levy 2013 28 8 2013 2013 78245020 4 2013 28 7 2013 Copyright © 2013 D. Talukdar and T. Talukdar.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Two common bean (Phaseolus vulgaris L.) mutants, sodPv 1 and sodPv 2, exhibiting foliar superoxide dismutase (SOD) activity of only 25% and 40% of their mother control (MC) cv. VL 63 were isolated in EMS-mutagenized (0.15%, 8 h) M2 progeny. Native-PAGE analysis revealed occurrence of Mn SOD, Fe SOD, Cu/Zn SOD I and Cu/Zn SOD II isozymes in MC, while Fe SOD, and Mn SOD were not formed in sodPv 1 and sodPv 2 leaves, respectively. In-gel activity of individual isozymes differed significantly among the parents. SOD deficiency is inherited as recessive mutations, controlled by two different nonallelic loci. Gene expressions using qRT PCR confirmed higher expressions of Cu/Zn SOD transcripts in both mutants and the absence of Fe SOD in sodPv 1 and Mn SOD in sodPv 2. In 50 μM arsenic, Cu/Zn SODs genes were further upregulated but other isoforms downregulated in the two mutants, maintaining SOD activity in its control level. In an F2 double mutants of sodPv 1 × sodPv 2, no Fe SOD, and Mn SOD expressions were detectable, while both Cu/Zn SODs are down-regulated and arsenic-induced leaf necrosis appeared. In contrast to both mutants, ROS-imaging study revealed overaccumulation of both superoxides and H2O2 in leaves of double mutant. ==== Body 1. Introduction Superoxide radicals are ubiquitously generated in many biological oxidations within all compartments of the cell. The toxicity of superoxide radicals has been attributed to their interaction with other cellular constituents, in particular with hydrogen peroxide [1]. Superoxide dismutases (SODs; EC 1.15.1.1) are a family of metalloenzymes that catalyze the disproportionation of superoxide radicals into H2O2 and O2 and constitute the first line of defense against the toxicity of superoxide radicals [1, 2]. Based on the metal cofactor used by the enzyme, SODs are classified into three groups as iron SOD (Fe SOD), manganese SOD (Mn SOD), and copper-zinc SODs (Cu/Zn SOD) [3]. SOD isozymes are located in different cellular compartments [3]. Fe SODs are located in the chloroplast, Mn SODs in the mitochondrion and the peroxisome, and Cu/Zn SODs in the chloroplast, the cytosol, and possibly the extracellular space. Cu/Zn SOD is normally quite stable, due in large part to copper and zinc binding and oxidation of an intramolecular disulfide [3]. Copper serves as the catalyst for superoxide disproportionation, whereas zinc and the disulfide participate in proper protein folding [3]. Arsenic (As) is a ubiquitous toxic metalloid without known biological functions in higher plants [4, 5]. In recent times, the impact of irrigation with high As contaminated groundwater on soil and crops has drawn huge attention due to transfer of As to the food chain via the groundwater-soil-plant system [6–8]. As can induce oxidative stress through generation of reactive oxygen species (ROS) [4, 5], and moderate accumulation of ROS significantly affects nuclear gene expression [9]. ROS sensors could be activated to induce signaling cascades that ultimately impinge on gene expression. Alternatively, components of signaling pathways could be directly oxidized by ROS. Finally, ROS might change gene expression by targeting and modifying the activity of transcription factors [9]. Mutational strategy provides a powerful tool to study the genetic, physiological, and molecular mechanisms of plants' response to abiotic stresses [10–12]. Among the edible legumes, this tool has been successfully used in Pisum sativum L. Lens culinaris Medik. and Lathyrus sativus L. to decipher metal tolerance and accumulation [10, 11], role of ascorbate and glutathione in redox balance and stress tolerance [12, 13], to ascertain genetic basis of flavonoid deficiency [14], to assess gene-dosage effect on antioxidant defense [15], and tolerance to salinity stress [16]. Among the enzymatic defense components in edible legumes, two mutants differing in catalase deficiency have recently been isolated and their genetic basis has been characterized in lentil [11]. The importance of SOD in ROS metabolism, maintenance of DNA integrity and life span has been nicely demonstrated by analysis of mutants in microbes and animals [17, 18]. In higher plants, importance of SOD has been studied in transgenic plants overexpressing SODs [19], which often produced inconclusive results [20]. The effects of Zn availability on SOD activity were studied in novel raz (requires additional zinc) mutant of Medicago truncatula, manifesting the importance of SOD mutant to study genetics of metal tolerance in plants [21]. The importance of Mn SOD in plant growth and Fe SOD isozymes in early chloroplast development has recently been elucidated in different Arabidopsis mutants [19, 20]. Using mutagenic primers in RT PCR, mutations were induced in Cu/Zn SOD genes and a thermostable SOD was engineered in higher plant Potentilla atrosanguinea [22]. Phaseolus vulgaris L. or common bean is a widely grown food legume and is rich in antioxidant flavonoids and proteins [23]. Like many other edible legumes, beans are highly sensitive to arsenate form of arsenic [23, 24] and showed severe perturbations in different morpho-physiological, micromorphological, and biochemical parameters under As exposure [25]. An earlier study revealed significant alterations in SOD activity in leaves of common beans subjected to As treatment [25], although genetic basis of SOD activity, roles of different isozymes, and their expression pattern were not known. Information about structural and functional aspects of SOD isozymes could benefit agricultural crop production through a better understanding of the genetic programs by which plants optimize photosynthetic activity in their green tissues during diverse types of stress conditions [3, 22, 25]. As part of a broad strategy to develop novel and desirable mutants for stress response in grain legumes, induced mutagenic technique has been adopted and progeny with variant phenotype was screened for antioxidant capacity. In the process, four plants exhibiting severe SOD deficiency in leaves were isolated at EMS-induced M2 generation and advanced to next generation to perform a detail study. The objective of the present study was framed to (1) measure the foliar SOD activity, (2) identify and analyze the in-gel activity of different SOD isozymes, (3) trace the inheritance of SOD deficiency in intercrossed population, (4) investigate the gene expression pattern of mRNA transcripts of different SOD isozymes, and (5) detect the ROS accumulation in leaves of mother variety, two mutant lines, and F2-segregating progeny under untreated and As (50 μM)-treated conditions. 2. Materials and Methods 2.1. Plant Materials Fresh and healthy seeds of common bean legume (Phaseolus vulgaris L. cv. VL 63) presoaked with water (6 h) were treated with freshly prepared 0.15% aqueous solution of EMS (Sigma-Aldrich) for 8 h with intermediate shaking at 25 ± 2°C. M1 seeds were sown treatment wise in completely randomized block design as reported earlier [14, 26]. During screening of antioxidant activity of M2 plants in 2010, three variant plants showing abnormally low foliar activity of superoxide dismutase were detected. Seeds of these three variant plants (mean 120 seeds plant−1) were harvested, separately, and were sown in next season (2011) to raise M3 progeny. Leaf SOD activity of respective progeny plants (a total of 210 plants) was again confirmed at M3 generation, and based on this primary observation, the mutants were tentatively designated as Phaseolus vulgaris SOD-deficient mutant. As no phenotypic abnormalities were observed, the progeny plants derived from these three variant M2 plants were tested for SOD activity in completely separate sets. Further biochemical and molecular characterizations of SOD enzymes were performed on these progeny plants. 2.2. Culture Conditions and As-Treatment Protocols Fresh and healthy seeds of common bean legume (Phaseolus vulgaris L. cv. VL 63), mutant plants, and their intercrossed F2-progeny plants were surface sterilized with NaOCl (0.1%, w/v) and continuously washed under running tap water followed by distilled water. Seeds were allowed to germinate in the dark in two separate sets on moistened filter paper at 25°C. Germinated seedlings were randomly placed in polythene pots (10 plants pots−1) containing 300 mL of Hoagland's No 2 nutrient media and were allowed to grow for 7 d. The plants were, then, subjected to 50 μM sodium arsenate (As, MW 312.01 g/mol; technical grade, purity 98.5%, Sigma-Aldrich) keeping untreated plant as control. Untreated mother variety and mutant plants were designated as mother control (MC) and mutant control (MuC), respectively. Control and treated plants were allowed to grow for another 7 d. Nutrient solution was refreshed every alternate day to prevent depletion of nutrients as well as As in the course of the plant's exposure to the metalloid. The experiment was carried out in a completely randomized block design with three replicates in an environmentally controlled growing chamber under a 14 h photoperiod, 28/18 (±2°C), relative humidity of 70 ± 2%, and a photon flux density of 100 μmol m−2s−1. 2.3. Determination of SOD Activity and Electrophoretic Analysis of SOD Isozymes Fresh leaf tissue (250 mg) was homogenized in 1 mL of 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA, 1 mM dithiothreitol, and 2% (w/v) polyvinyl pyrrolidone (PVP) using a chilled mortar and pestle kept in an ice bath. The homogenate was centrifuged at 15,000 g at 4°C for 30 min. Clear supernatant was used for enzyme assays. All operations were performed at 0–4°C. Soluble protein content was determined using BSA as a standard [27]. SOD (EC 1.15.1.1) activity was determined by nitro blue tetrazolium (NBT) photochemical assay, according to Beyer Jr. and Fridovich [28]. In this method, 1 ml of solution containing 50 mM K-phosphate buffer (pH 7.8), 9.9 mM L-methionine, 57 μM NBT, and 0.025% triton-X-100 was added into small glass tubes, followed by 20 μL of enzyme extract. Reaction was started by adding 10 μL of riboflavin solution (0.044 mg mL−1), and the absorbance of solution was measured at 560 nm. SOD activity was expressed as U (unit) mg−1 protein. One unit of SOD was equal to that amount which causes a 50% decrease of SOD-inhibited NBT reduction. SOD isozymes were individualized by native PAGE on 10% acrylamide gels and were localized by a photochemical method [28]. Activity-staining gels were incubated for 30 min in 50 mM K-phosphate buffer at pH 7.5 containing 2 mM KCN or 5 mM H2O2 or 5 mM NaN3. Cu/Zn-SODs are inhibited by KCN and H2O2; Fe SODs are inactivated by H2O2 and NaN3 but resistant to KCN and Mn SODs are resistant to all three inhibitors. Quantification of the bands was performed using a Gel Doc system (Bio-Rad Laboratories, Chennai, TN, India) coupled with a high sensitive CCD camera. Band intensity was expressed as relative transmittance units. Based on the observed variations, isozyme bands were assigned to putative loci following the earlier adopted principles [29]. Only clearly visible bands were scored in the present study. 2.4. Genetic Control and Allelism Test of SOD-Deficient Mutations Inheritance of mutations controlling SOD deficiency was traced in segregating populations of F2 generation derived from MC × mutants. For allelism test, intercrosses were made between mutants. Chi-square test was employed to test the goodness of fit between observed and expected values for all crosses. Zymograms of SOD isoforms of leaf F1 and F2 plants were analysed and compared with parents. 2.5. RNA Isolation and Relative Gene Expression through Quantitative RT PCR Gene expression levels of different SOD isozymes of MC, MuC, F2-segregating progenies, and As-treated plants of Phaseolus vulgaris (PvSOD) were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) technique. Total RNA was isolated from the young leaves of 14d-old Phaseolus leaves (control and treated in separate sets of experiment) using the RNA isolation kit (Chromous Biotech, Bangalore, India) and treated with DNaseI (Chromous Biotech, Bangalore, India) at 37°C for 30 min. The quality of total RNA samples was determined spectrophotometrically (Systonic, Kolkata, India) and by 1% agarose gel electrophoresis with 500 bp DNA ladder. First-strand cDNA was synthesized from DNase-treated RNA with oligo-dT primer and with MmuLV reverse transcriptase enzyme kit (Chromous Biotech, Bangalore, India) following manufacturer's instructions. Quantitative RT PCR of first stand cDNA was run on ABI Step-One Real Time PCR machine. Amplification was done in a total reaction volume of 50 μL, containing template (first strand cDNA) 2.0 μL, forward primer 2.0 μL, reverse primer 2.0 μL, 2 X PCR SYBR green ready mixture (Fast Q-PCR Master Mix, Chromous Biotech, India, Cat no. QCR 05/QCR 06), 25.0 μL, and DEPC water 19.0 uL. The SOD primers for four isozymes (Cu/Zn-SODs I and II, Fe SOD, and Mn SOD) of PvSOD (Table 1) were constructed by Primer Express software with the search of available GenBank/legume databases [30]. The qRT-PCR cycling stages consisted of initial denaturation at 94°C (3 min), followed by 35 cycles of 94°C (5 s), 62°C (10 s), 72°C (10 s), and a final extension stage at 72°C (2 min). A melting curve analysis was performed after every PCR reaction to confirm the accuracy of each amplified product. Samples for qRT PCR were run in five biological replicates and two technical replicates. DEPC water for the replacement of template was used as negative control. RT-PCR reaction mixtures were loaded onto 2% agarose gels in TAE buffer. A 100 bp DNA ladder was run on every gel. The mRNA levels were normalized against a common bean ubiquitin as the housekeeping gene and the relative (to control) expression of target genes was calculated as 2−ΔΔCt; Ct is cycle threshold, following Livak and Schmittgen [31]. 2.6. Detection and Imaging of Superoxide and H2O2 Radicals by Confocal Laser Scanning Microscopy Detection and imaging of superoxide radicals in leaf sections was carried out using the fluorescence probe dihydroethidium (DHE), following the earlier method [32]. Bean leaf segments of approximately 30 mm2 were incubated for 1 h at 25°C, in darkness, with 10 μM DHE prepared in 5 mM Tris-HCl buffer at pH7.4, and samples were washed twice with the same buffer for 12 min each. After washing, leaf sections were embedded in a mixture of 15% acrylamide-bisacrylamide stock solution, and 100 mm thick sections, as indicated by the vibratome scale, were cut under 10 mM phosphate-buffered saline (PBS). Sections were then soaked in glycerol: PBS (containing azide) (1 : 1 v/v) and mounted in the same medium for examination with a (CLSM) system (Carl Zeiss, LSM 780, Bangalore, India) using standard filters and collection modalities for DHE green fluorescence (λ excitation 488 nm; λ emission 520 nm) and Chlorophyll autofluorescence (Chl a and b, λ excitation 429 and 450 nm, resp.; λ emission 650 and 670 nm, resp.) as blue. H2O2 was detected by incubation with 25 μM 2′ 7′-dichloro fluorescein diacetate (DCF-DA) (excitation 485 nm, emission 530 nm) [32]. A preinfused leaf sections with 1 mM tetramethylpiperidinooxy (TMP), a scavenger of superoxide radicals, and 1 mM ascorbate, a scavenger of H2O2 served as negative controls. 2.7. Statistical Analysis The results presented were the mean values ± standard errors obtained from at least four replicates. Statistical significance of mean values between control and treated seedlings was determined by Students t-test (two tailed) using Microsoft Excel tool pack “data analysis” 2007. Multiple comparisons among treatments were performed by ANOVA using software SPSS v. 10.0 (SPS Inc., USA), and means were separated by Duncan's multiple range test. A probability of P < 0.05 was considered significant. 3. Results and Discussions 3.1. Foliar SOD Activity and In-Gel Activity of Isozymes in Control and As-Treated Leaves In crude extracts of leaves under unstressed condition, measurable total SOD specific activity in MC plants was 40.8 (±3.9) U mg−1 protein. SOD activity in progeny mutant plants was only between 25% (10.2 ± 2.2 U mg−1 protein) and 45% (19.1 ± 2.9 U mg−1 protein) of MC plants, exhibiting severe deficiency of foliar SOD level in the mutant plants. Based on SOD activity level, mutant population could be clearly separated into two groups of progeny plants; one group of plants coming from two M2 parent (total three plants detected in M2 generation) showed mean SOD activity of only 25% of MC plants while the other group possessed SOD activity of only 45% of MC SOD level. All the mutant plants were phenotypically similar to MC plants (figure not shown). The analysis of SOD activity by native PAGE showed the presence of four isozymes in leaves of MC plants (Figure 1). Isozymes were identified with specific inhibitors (KCN and H2O2) as an Mn SOD, Fe SOD, and two Cu/Zn SODs (I and II), representing 45, 30, and 25% of the total SOD activity, respectively (Figure 2). The band showing resistance to inhibition by KCN, H2O2, and NaN3 was identified as Mn SOD (Figure 3), whereas band manifesting resistance to KCN but sensitive to H2O2 and NaN3 was detected as Fe SOD (Figure 3). The other two isozymes were sensitive to both KCN and H2O2 but resistant to NaN3. Therefore, they were assigned as Cu/Zn SODs (Figure 3) with the more anodal band was detected as Cu/Zn SOD II (Figure 1). The Cu/Zn SODs I and II were identified as faint bands in comparison with other isoforms of SOD in leaves, representing only 15% and 10% of total SOD activity, respectively (Figures 1–3). Different scenario was encountered in two groups of mutant plants. A total of three isozyme bands were clearly visualized in leaves of mutant plants (Figure 1). Band position and inhibitor studies in comparison with MC plants revealed complete absence of Fe SOD in the group showing only 25% SOD activity of MC plants, while there was no detectable Mn SOD band in other group, exhibiting 45% SOD activity of MC plants (Figures 1–3). Based on this observation, the former group of mutant plants was designated as sodPv 1 (superoxide dismutase deficient Phaseolus vulgaris mutant 1) while the latter was described as sodPv 2 (superoxide dismutase deficient Phaseolus vulgaris mutant 2). In sodPv 1 mutant, bands representing Mn SOD, Cu/Zn SOD I, and CU/ZnSOD II performed 60%, 25%, and 15% of the total SOD activity, respectively (Figure 2). On the other hand, in sodPv 2 mutant plants, major share (65%) of SOD activity was carried out by Cu/Zn SODs (45% by Cu/Zn SOD I and 20% by Cu/Zn SOD II) while rest (35%) was contributed by Fe SOD (Figure 2). Under 50 μM As exposure, foliar SOD activity in treated mother plants was increased over MC by about 3-fold (121.9 ± 5.7 U mg−1 protein), indicating generation of excess superoxide radicals due to As treatment, and it is in agreement with earlier reports on As-treated P. vulgaris, P. aureus, Lathyrus sativus, and Trigonella foenum-graecum [3, 7, 25, 33]. Besides As, heavy metal-dependent induction of SOD activity was also found in crops like Pisum sativum [32], Lens culinaris [34], Helianthus annuus [35], and Coffea arabica [36]. In the present case, the increased SOD activity in treated mother plants was accompanied with enhancement of band intensity of leaf Fe-SOD and Cu/Zn SODs (I and II) over that of MC (Figure 1), representing 38% and 52% of the total SOD activity (Figure 2). By contrast, band corresponding the expression of Mn SOD was faint (Figure 1), representing 10% of total SOD expression under As exposure (Figure 2). In the similar treatment protocol, measurable SOD level compared to MuC varied nonsignificantly (P > 0.05) in the As-treated mutants. In native PAGE gel, Cu/Zn SODs bands became intensified and thick in both mutants (representing nearly 83% of total SOD activity in sodPv 1 and 90% of that in sodPv 2 mutant), but bands representing Mn SOD (17% of total SOD) and Fe SOD (10% of total SOD) were visualized as very faint and thin in sodPv 1 and sodPv 2 mutants, respectively (Figures 1 and 2). 3.2. Genetics of SOD Deficiency and Isozyme Pattern in Phaseolus Mutants The two mutants exhibited phenotypes similar to MC. The MC plants as well as the two mutants were purely homozygous for SOD expression, as revealed by preliminary screening of their self-pollinated progeny (data not shown). Reciprocal crosses between sodPv 1 as well as sodPv 2 and MC yielded F1 plants with normal level of SOD activity (ranged 37.7–43.2 U mg−1 protein, mean 40.3 ± 5.9 U mg−1 protein). Segregation of normal and SOD-deficient plant type showed good fit [χ 2 = 0.43 at 1 df (degrees of freedom), P < 0.05] to 3 : 1 in F2, indicating recessive nature of mutant traits. In order to ascertain the allelic relationships of genes controlling SOD deficiency in Phaseolus, sodPv 1 and sodPv 2 were reciprocally crossed. All the F1 plants exhibited normal SOD activity (37.9–42.8 U mg−1 protein, mean 41.1 ± 3.7 U mg−1 protein) and zymogram phenotype identical to MC (Figure 4). However, in F2, out of 480 plants screened, 270 plants possessed SOD activity (38.3–43.7 U mg−1 protein, mean 40.9 ± 4.9 U mg−1 protein) and isozyme patterns similar to MC, while rest of the plants (210) exhibited deficiency in SOD activity and altered zymogram pattern. A careful examination of these 210 plants revealed further segregation, of which 88 plants were identified exhibiting SOD activity (mean 10.8 ± 3.3 U mg−1 protein, 25.2% of MC) and isozyme pattern (Fe SOD absent) like sodPv1, 101 plants were detected manifesting SOD activity (18.3 ± 4.9 U mg−1 protein, 44.8% of MC) and isozyme pattern (Mn SOD absent) similar to sodPv 2 mutant, and rest 21 plants were isolated with SOD activity of 12.5% (mean 5.1 ± 2.8 U mg−1 protein) of MC and occurrence of only Cu/Zn SOD I and II isozymes as faint bands (Figure 4). Inhibition study confirmed the presence of only Cu/Zn SODs isozymes, and no Fe SOD and Mn SOD bands could be visualized in these 21 plants (Figure 5(A)). The plants were self-fertile but carried distinct necrotic spots on pod wall (Figure 5(B)). Therefore, considering the whole F2 population of total 480 plants, the segregation was consistent [χ 2 = 4.09 at 3 df (degrees of freedom), P < 0.05] with 9 : 3 : 3 : 1 in F2. The results strongly suggested recessive nature of both mutations which was manifested in inheritance of zymogram pattern of respective parents and was controlled by interactions between two different nonallelic loci. The recessive nature of null mutations in SOD isozyme loci was also reported in sunflower, maize, soybean, and other plants [37–39]. Obviously, in the presence of dominant alleles of both loci (SOD Pv1-SODPV2-), SOD activity and zymograms of isozymes were normal in the present MC plants. Absence of dominant alleles in the loci (either in the form of sodPv 1-SODPv 2- or SODPv 1-sodPv 2-) led to origin of two SOD-deficient mutants, differing distinctly in total SOD activity level and isozyme banding pattern. In the last class of progeny plants, double mutant recessive to both loci (sodPv 1 and sodPv 2) resulted in extremely deficient SOD level accompanied with phenotypic anomalies. 3.3. Gene Expression of SOD Isozymes under Un-Stressed and As-Treated Conditions The expression patterns of the Cu/ZnSODs I and II, Mn SOD, and Fe SOD genes in leaves of Phaseolus seedlings and two mutant lines under unstressed, and As-treated conditions were investigated by qRT-PCR (Figure 2). Under unstressed condition, the mRNA transcripts of Mn SOD, Fe SOD, Cu/Zn SODs I, and Cu/znSOD II isoforms were amplified by PCR in MC (Table 1, Figure 6). The mRNA transcripts of Cu/Zn SODs were also detectable in both sodPv 1 and sodPv 2 mutants, but there were no detectable transcripts of Fe SOD in sodPv 1 mutant and no Mn SOD in case of sodPv 2 mutants (Figure 6). Besides Cu/Zn SODs I and II, genes encoding Mn SOD transcripts were expressed in sodPv 1 while those of Fe SOD were detected in sodPv 2 mutant leaves (Figure 6). Relative expression pattern (compared to respective gene of MC) indicated that genes encoding Mn SOD as well as Cu/Zn SODs I and II transcripts in sodPv 1 exhibited significantly (P < 0.05) higher level than those of MC values (Figure 7). Higher transcript levels of Cu/Zn SODs and Fe SOD compared to MC were also deduced in sodPv 2 mutant leaves (Figure 7). Between the two mutants, gene expression of Cu/Zn SODs transcripts was markedly higher in sodPv 2 mutant (Figures 6 and 7). Dramatic changes in gene expression pattern of SOD isozymes were observed in leaves of mother variety exposed to 50 μM As. In comparison with unstressed condition (MC), relative mRNA levels of Cu/Zn SODs, Mn SOD, and Fe SOD were increased by 2-fold, 1.5-fold, and 3-fold, respectively, in treated mother (Figures 6 and 7). The Results suggested upregulation of Cu/Zn SOD, Mn SOD, and Fe SOD genes in response to As treatment of mother variety. Compared to MC, total SOD-specific activity was markedly increased in As-treated mother variety. Certainly, enhanced activity of Cu/Zn SODs and Fe SOD isoforms mainly contributed to this rise which can be attributed to As-induced transcriptional upregulation of both Cu/Zn SOD and Fe SOD genes. However, the declining activity of Mn SOD isoform, as manifested in native gel, was not in harmony with enhanced level of its mRNA transcript, indicating possible regulation of this isozyme at posttranscriptional level. In As-treated sodPv 1 and sodPv 2 mutants, mRNA transcripts of Cu/Zn SODs as amplified in qRT-PCR (Figure 6) were increased by about 2-2.2-fold over their corresponding levels in untreated control (MuC) and >3.0-fold in relation to MC (Figure 7). By contrast, reduced level of mRNA transcripts was observed for Mn SOD in sodPv 1 and for Fe SOD in sodPv 2 mutant exposed to As treatment (Figures 6 and 7). The results indicated As-induced upregulation of Cu/Zn SODs genes in both mutants but concomitant down-regulation of Mn SOD in sodPv 1 and Fe SOD in sodPv 2 mutant. Overexpression of mRNA genes controlling Cu/Zn SODs led to higher activity of this isozyme in zymogram, which was manifested as increasing band intensity and thickness in native PAGE. Likewise, declining activity of Mn SOD in sodPv 1 and Fe SOD in sodPv 2 mutant exposed to 50 μM As treatment was presumably due to down-regulation of their respective genes governing mRNA transcripts. The results indicated differential regulation of SOD isozymes in both mutants, subjected to As treatment, presumably, balancing SOD activity in MuC levels (25% in sodPv 1 and 45% in sodPv 2 of MC). Differential regulations of SOD isoforms were also reported in cadmium-treated pea, transgenic alfalfa, salinity-induced lentils, and in many other plants experiencing stresses [32, 40–42]. Since Mn SOD and Fe SOD are proved to be mitochondrial and plastidic isoforms, respectively, their down-regulation in the present mutants of common beans could have adverse effect on plant growth, as observed in Nicotiana seedlings [43] and in transgenic Arabidopsis [19]. Interestingly, no As-induced oxidative damage and consequent retardation of plant growth was visible in any part of the present mutants, despite down-regulation of MnSOD in sodPv 1 and Fe SOD in sodPv 2 mutant under As exposure. Obviously, upregulations of Cu/Zn SOD I and II mRNA gene counterbalanced down-regulation of other SOD isoforms in the mutant plants, and presumably, are holding the key in response of both mutants to high As exposure. Compared to chloroplast, the amount of ROS produced by mitochondria is rather minor [44] and chloroplasts bear a particular risk of oxygen toxicity because molecular O2 can be photoreduced from photosystem I (PSI) [44]. The Cu/Zn SODs are the most prolific SOD isozymes in chloroplast [3, 43, 44], and therefore their overexpression has immense significance in ROS metabolism and oxidative balance in leaves of present As-treated mother plants and mutant lines. However, to what extent this SOD expression is modulating antioxidant defense components of Phaseolus seedlings and mutants in response to As treatment; further study is needed to decipher it. 3.4. Gene Expression of F2-Segregating Progeny Plants under Unstressed and As-Treated Conditions Compared to their respective parent, there were no significant alterations in mRNA levels and morphology of F2-segregating progeny plants either under untreated or As-treated conditions (data not shown) except in the double mutant. Compared to sodPv 1 and sodPv 2 single gene mutant phenotypes, relative expression of mRNA genes encoding Cu/Zn SODs I and II was significantly low in the double mutant plants under untreated condition (Figures 7 and 8(a)). Furthermore, no mRNA transcripts of Mn SOD and Fe SOD were detected. The results indicated origin of double knockout mutant for SOD deficiency in segregating mutant progeny of Phaseolus. Upon exposure of the mutant to 50 μM As for 7 days in hydroponics, Cu/Zn SODs transcript level is reduced further (Figures 7 and 8), suggesting As-induced down-regulation of the gene. Declining level of mRNA transcripts and complete absence of Mn SOD and Fe SOD resulted in extreme reduction of foliar SOD activity in the mutant, which was manifested as thin faint bands in zymogram of Cu/Zn SODs (Figures 4 and 5(a)). The plants became more stunted, and severe necrotic spots were visible on trifoliate leaves and petioles under As exposure (Figure 8(b)). It is noteworthy that necrotic spots were visible in the pod walls of the mutant even under untreated condition, and thus appearance of new necrotic spots on photosynthetic part after As treatment indicated onset of As-induced oxidative damage, triggered by severe SOD-deficiency. The result strongly confirmed incorporation of both recessive mutations in the mutant, impeding normal SOD activity through knocking out of its Mn SOD and Fe SOD isozymes and down-regulating of Cu/Zn SODs. It now seems likely that two different recessive mutations at SOD loci of Phaseolus affected two different isoforms of the same enzyme, leading to origin of SOD deficiency with differential gene expression and isozyme banding patterns in two mutant lines. Remarkably enough, the Cu/Zn SODs were normal to over-expressed in the presence of either Mn SOD (sodPv 1) or Fe SOD (sodPv 2) but were downregulated when both isoforms were absent in the double mutant. The highly contrasting phenotypes as observed between two single mutants and double mutant derived from their inter-crossed progeny are unique. The outcome is not in accordance with earlier reports on Arabidopsis mutants, manifesting severe albino phenotypes in double knockout of two Fe SOD isoforms and severe sensitivity to oxidative stress in its two single mutant parents [20]. Among the legumes, similar phenomenon was observed in catalase-deficient mutants in lentil [11], dwarf mutations, and flavonoid-deficient mutants of grass pea [14, 45]. Certainly, the dominant alleles of both loci are complimenting with each other in the present case to give normal gene expression of all four isozymes while incorporation of both mutations in homozygous recessive forms resulted in absence of both Fe SOD and Mn SOD expressions in double mutant. The results also revealed inheritance of SOD deficiency as stable mutations, null for Fe SOD in sodPv 1 and Mn SOD isozymes for sodPv 2, along with As tolerance in advanced generations. 3.5. ROS Imaging by CLSM The remarkable tolerance of sodPv 1 and sodPv 2 mutants but sensitivity of their double mutants and mother plants to As treatment was strongly evidenced by ROS imaging study. Occurrence of superoxide radicals was analyzed by CLSM using the fluorescence probe DHE. In MC, no red fluorescence due to superoxide radicals was detected (Figure 9(A)). However, in leaves of As-treated mother plants and two mutants, the red fluorescence was detected in trifoliate leaves with more severe effects on mother plants. It was not so abundant and localized only in vascular tissue, especially in xylem, and in epidermis of mutant lines (Figure 9(B)). In leaves of As-treated mother plants, the red fluorescence was detected in high amount in and around vascular regions, sclerenchyma, mesophyll, and epidermis (Figures 9(C) and 9(E)). When the leaf sections were preincubated with 1 mM TMP (a superoxide scavenger), a significant reduction of the fluorescence was observed (Figure 9(D)), thus exhibiting the specificity of DHE for detection and imaging of superoxide radicals in bean leaves. For H2O2 imaging, crosssection of MC leaves incubated with DCF-DA exhibited a bright green florescence which was highly restricted to only xylem regions of vascular tissue and sclerenchyma (Figure 9(F)). In As-treated leaves of mother plant, the green florescence was enhanced markedly in vascular bundles, sclerenchyma, mesophyll regions, and in epidermis (Figure 9(G)). Under untreated condition, DCF-DA fluorescence was localized only in xylem of two mutant lines (Figure 9(H)). No significant change in green fluorescence was observed in As-treated mutants (figure not shown). By contrast, the double mutant exhibited both red and green fluorescences, abundantly distributed in vascular tissues, epidermis, and mesophyll regions (Figures 9(I) and 9(J)). Quite interestingly, DCF-DA fluorescence was also distinctly visualized in leaf epidermal hair of As-treated double mutant (Figure 9(J)), which was not noticed in any other cases. A preincubation with 1 mM ascorbate considerably tamed the florescence (Figure 9(K)). The analysis of superoxides and H2O2 in leaf sections by CLSM showed an enhanced generation of superoxide radicals and H2O2/other peroxide and their abundant localizations in mesophylls of treated mother plant and double mutant, exposed to As. Overproduction of superoxides and H2O2 due to toxic metals/metalloids was distinctly screened by CLSM study in pea, Medicago sativa, and Lupinus luteus roots [32, 46, 47]. The absence of both red and green fluorescences in mesophyll tissues of present MC and two mutants and their abundant occurrence even in overlapping conditions with tissue autofluorescence (blue) in some cases in As-treated leaves of mother variety and double mutant plants suggested that ROS production could be associated with chloroplasts in these genotypes. It was also indicative that, despite SOD-deficiency, both mutant lines contained superoxides and H2O2 levels in their photosynthetic part/s quite effectively under As treatment. By contrast, higher magnitude of ROS generation and their accumulation in leaves of treated mother variety and double mutant plants was evidenced by enhanced florescence. This suggested foot printing of As-induced ROS generation and consequent oxidative damage to photosynthetic parts of these plant types. 4. Conclusions For the first time, two SOD-deficient mutants were isolated in P. vulgaris by EMS-induced mutagenic technique. The two mutants manifested normal growth. Native PAGE analysis and mRNA gene expression analysis revealed complete absence of Fe SOD in sodPv 1 and Mn SOD in sodPv 2 mutant leaves, while Cu/Zn SODs I and II were differentially expressed. The mother plants possessed all four isozymes. Genetic analysis revealed inheritance of SOD deficiency as recessive mutations, controlled by two different nonallelic loci; the occurrence of both alleles in recessive forms led to origin of a double mutant in F2. Under 50 μM arsenic (As) treatment for 7 days, Cu/Zn SOD isozymes were markedly up-regulated in the two mutants and mother variety. Measurable SOD activity in the two mutants, however, changed nonsignificantly as Mn SOD in sodPv 1 and Fe SOD in sodPv 2 were downregulated in response to As. This was consistent with isozyme banding pattern in native PAGE. The As-treated mutants showed normal growth, and no significant accumulation of ROS was observed in leaf as revealed by ROS-imaging study. In the double mutant, total absence of both Mn SOD and Fe SOD transcripts was accompanied with significant down-regulation of Cu/Zn SODs, resulting in ROS accumulation at high magnitude during As exposure and appearance of necrotic spots on photosynthetic organs. The isolation and genetic characterization of two SOD-deficient mutants, differing in SOD constituents and gene expressions, can be used as novel molecular tools to decipher deeper roles of SOD in abiotic stress tolerance of common beans. Furthermore, the origin of a double mutant is a unique phenomenon as the mutant in self-fertilisation and, thus, can be incorporated in different genetic backgrounds to reveal crosstalk between SOD and different antioxidant defense machinery of edible legumes. A genetic manipulation can, therefore, be facilitated in antioxidant defense components in favor of better crop growth and yield. Figure 1 Zymogram of four isozymes of superoxide dismutase (SOD) in leaves of Phaseolus vulgaris L. Cv. VL 63 (mother variety) and two SOD-deficient mutant lines, sodPV 1, and sodPv 2 under untreated (control) and 50 μM arsenic- (As-) treated conditions; lane 1: mother control, lane 2: sodPv 1 (untreated), lane 3: sodPv 2 (untreated), lane 4: treated mother, lane 5: treated sodPv 1 mutant, and lane 6: treated sodPv 2 mutant. Figure 2 Activity of individual isozymes of superoxide dismutase (SOD) in mother control (MC), two mutants, sodPv 1 and sodPv 2, and 50 μM arsenic- (As-) treated mother variety VL-63 and two mutant lines in Phaseolus vulgaris L. Band intensities were expressed as relative transmittance (T) units. The results are means ± SE of at least three replicates, and same letters (four different types to represent four isozymes) above error bars denote nonsignificant (P > 0.05) differences among means by Duncan's multiple range test. Figure 3 Inhibition (KCN, H2O2, and NaN3) study and visualization of SOD isoforms in native PAGE of leaf extracts of Phaseolus vulgaris cv. VL 63 (mother variety) and two SOD-deficient mutant lines, sodPv 1, and sodPv 2; lane 1: mother control (no inhibitor), lane 2: mother variety (KCN), lane 3: sodPv 1 (KCN), lane 4: sodPv 2 (KCN), lane 5: mother variety (H2O2), lane 6: sodPv 1 (H2O2), lane 7: sodPv 2 (H2O2), lane 8: mother variety (NaN3), lane 9: sodPv 1, and lane 10: sodPv 2. Note that complete absence of band at lane 7; Mn SOD is absent in sodPv 2 mutant, and Fe SOD and Cu/Zn SODs are inhibited by H2O2 treatment. Figure 4 In-gel activity of SOD isozymes in F2-segregating progeny derived from sodPv 1 × sodPv 2 mutants in Phaseolus vulgaris L.; lane 1: F1 progeny, and lanes 2–10: F2 progeny of which lane 2 is normal plant (all four isozymes visualized), lanes 3 and 4: progeny with sodPv 1 phenotype (Fe SOD absent), lanes 5 and 6: progeny with sodPv 2 phenotype (Mn SOD absent), lanes 7–10: double mutant (both Mn SOD and Fe SOD absent). Figure 5 (A) Inhibition study of SOD isozymes produced by double mutant obtained from crosses between sodPv 1 and sodPv 2 mutants of Phaseolus vulgaris L.; lane 1: mother control (no inhibitor), lanes 2 & 3: double mutants (no inhibitor), lane 4: double mutant (KCN), lane 5: double mutant (H2O2), and lanes 6 & 7: double mutants (NaN3). Note that both Cu/Zn SODs I and II were inhibited by KCN and H2O2 in lanes 4 and 5, while visualized as, and normal faint bands by NaN3 at lanes 6 & 7, (B) appearance of necrotic spots (→) on pod wall of double mutant. Figure 6 Representative agarose gel (2%) electrophoresis shows the amplified product of mRNA expression of SOD isozymes in mother control (MC), two SOD-deficient mutants, sodPv 1 and sodPv 2 under untreated conditions, and arsenic (50 μM sodium arsenate, As)-treated mother variety and two mutants; lane 1: Mn SOD, lane 2: Fe SOD, lane 3: Cu/Zn SOD I, lane 4: Cu/zn SOD II, lane 5: housekeeping gene, and lane 6–100 bp DNA ladder (bold white arrow 200 bp). Figure 7 Relative expressions of genes governing mRNA transcripts of four SOD isoforms in leaves of mother control (MC) variety VL 63 of Phaseolus vulgaris L., two SOD-deficient mutants, sodPv 1 and sodPv 2, and a double mutant (DM) type derived from F2 progeny of sodPv 1 × sodPv 2 mutants under untreated, and 50 μM arsenic- (As-) treated conditions were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The mRNA levels were normalized with respect to housekeeping gene ubiquitin and are expressed relative to those of control (MC) value, which were arbitrarily given a value of 1. Data are means ± SE of five biological replicates and two technical replicates. Single asterisks denote upregulation (at least 2.0-fold), and double asterisks represent down-regulation (at least 2.0-fold) of genes with respect to control values. Figure 8 (a) Representative agarose gel (2%) electrophoresis shows the amplified product of foliar mRNA expression of Cu/Zn SODs isozymes in mother control (MC, lanes 1, and 2) and a double mutant derived from sodPv 1 × sodPv 2 mutants under untreated (lanes 3 and 4) and arsenic (50 μM sodium arsenate) treated (lanes 5 and 6) conditions, and (b) normal trifoliate leaflets (MC) and leaflets showing necrosis (→) in double mutant. Figure 9 Representative imaging of superoxide radicals and H2O2 productions in Phaseolus vulgaris L. leaves by CLSM. Images are developed from several optical sections collected by confocal microscopy showing the autofluorescence (blue; excitation at 633 nm, emission at 680 nm) and fluorescence due to DHE and DCF-DA. A–E, (I) superoxide-dependent DHE fluorescence (red) in leaf cross sections from mother control (A) and arsenic (As)-treated (B–K) leaves; (B) As-treated sodPv 1 mutant, (C) As-treated mother plants, (D) for the negative control, leaves were incubated with 1 mm TMP, a superoxide scavenger, (E) magnified view of leaf mesophyll with red fluorescence and autofluorescence, (I) red fluorescence in leaf section of double mutant, F–H, J, and (K) H2O2-dependent DCF-DA fluorescence (green) in leaf cross sections; (F) MC leaves, (G) As-treated mother plants, (H) As-treated sodPv 2 mutant, (J) green fluorescence in leaf tissues along with epidermal hair of double mutant, (K) as a negative control, and leaves were incubated with 1 mm ascorbate (ASC), which acts as a H2O2 scavenger. The results are representative of at least three independent experiments. Ep: Epidermis; Hr: hair; Ms: mesophyll cells; Scl: sclerenchyma; Xyl: xylem vessels. Bars = 50 μm. Table 1 Oligonucleotides used for qRT-PCR analysis of target gene expressions of superoxide dismutase (SOD) isozymes in Phaseolus vulgaris L. mother cv. VL 63 and two of its SOD-deficient mutant lines. F1—forward primers and R1—reverse primers. Target genes Oligonucleotide sequence (5′→3′) Amplicon size (bp) Mn SOD F1-AGTCAAGTTGCAGAGTGCAATCAAGTTC- 143 R1-CAAAGTGATTGTCAATAGCCCAACCTAAAG- Fe SOD F1-AACAAGCAAATAGCCGGAACAGAACTAAC- 127 R1-AGAAATCGTGATTCCAGACCTGAGCAG- Cu/Zn SOD I F1-GGCTGTATGTCAACTGGACCTCATTTCA- 139 R1-TGTCAACAATGTTGATAGCAGCGGTG- Cu/Zn SOD II F1-GGATATATGGCATCTGTAACTCATATGC- 115 R1-GCATAAGAATGCTGATAGACAGGGTC- Ubiquitin (housekeeping gene) F1-GCTCTCCATTTGCTCCCTGTT- 144 R1-TGAGCAATTTCAGGCACCAA- ==== Refs 1 Fridovich I Superoxide radical and superoxide dismutases Annual Review of Biochemistry 1995 64 97 112 2-s2.0-0029053451 2 Halliwell B Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life Plant Physiology 2006 141 2 312 322 2-s2.0-33745662190 16760481 3 Alscher RG Erturk N Heath LS Role of superoxide dismutases (SODs) in controlling oxidative stress in plants Journal of Experimental Botany 2002 53 372 1331 1341 2-s2.0-0036001083 11997379 4 Mascher R Lippmann B Holzinger S Bergmann H Arsenate toxicity: effects on oxidative stress response molecules and enzymes in red clover plants Plant Science 2002 163 5 961 969 2-s2.0-0036852070 5 Talukdar D Bioaccumulation, growth and antioxidant defense responses of Leucaena species differing in arsenic tolerance International Journal of Botany and Research 2013 3 1 1 18 6 Azizur Rahman M Hasegawa H Mahfuzur Rahman M Mazid Miah MA Tasmin A Arsenic accumulation in rice (Oryza sativa L.): human exposure through food chain Ecotoxicology and Environmental Safety 2008 69 2 317 324 2-s2.0-36549063768 17346792 7 Talukdar D Effect of arsenic-induced toxicity on morphological traits of Trigonella foenum-graecum L. and Lathyrus sativus L. during germination and early seedling growth Current Research Journal of Biological Sciences 2011 3 2 116 123 8 Talukdar D Bioaccumulation and transport of arsenic in different genotypes of lentil (Lens culinaris Medik.) International Journal of Pharma and Bio Sciences 2013 4 1B 694 701 9 Apel K Hirt H Reactive oxygen species: metabolism, oxidative stress, and signal transduction Annual Review of Plant Biology 2004 55 373 399 2-s2.0-3242715114 10 Tsyganov VE Belimov AA Borisov AY A chemically induced new pea (Pisum sativum ) mutant SGECdt with increased tolerance to, and accumulation of, cadmium Annals of Botany 2007 99 2 227 237 2-s2.0-33947722144 17259229 11 Talukdar D Catalase-deficient mutants in lentil (Lens culinaris Medik.): perturbations in morpho-physiology, antioxidant redox and cytogenetic parameters International Journal of Agricultural Science and Research 2013 3 2 217 232 12 Talukdar D Ascorbate deficient semi-dwarf asfL1 mutant of Lathyrus sativus exhibits alterations in antioxidant defense Biologia Plantarum 2012 56 4 675 682 2-s2.0-84859718652 13 Talukdar D An induced glutathione-deficient mutant in grass pea (Lathyrus sativus L.): modifications in plant morphology, alteration in antioxidant activities and increased sensitivity to cadmium Bioremediation, Biodiversity and Bioavailability 2012 6 75 86 14 Talukdar D Flavonoid-deficient mutants in grass pea (Lathyrus sativus L.): genetic control, linkage relationships, and mapping with aconitase and S nitrosoglutathione reductase isozyme loci The Scientific World Journal 2012 2012 11 pages 345983 15 Talukdar D The aneuploid switch: extra-chromosomal effect on antioxidant defense through trisomic shift in Lathyrus sativus L. Indian Journal of Fundamental and Applied Life Sciences 2011 1 4 263 273 16 Talukdar D Isolation and characterization of NaCl-tolerant mutations in two important legumes, Clitoria ternatea L. and Lathyrus sativus L.: induced mutagenesis and selection by salt stress Journal of Medicinal Plant Research 2011 5 16 3619 3628 2-s2.0-80051923681 17 Chary P Dillon D Schroeder AL Natvig DO Superoxide dismutase (sod-1 ) null mutants of Neurospora crassa : oxidative stress sensitivity, spontaneous mutation rate and response to mutagens Genetics 1994 137 3 723 730 2-s2.0-0028360692 8088518 18 Gralla EB Valentine JS Null mutants of Saccharomyces cerevisiae Cu,Zn superoxide dismutase: characterization and spontaneous mutation rates Journal of Bacteriology 1991 173 18 5918 5920 2-s2.0-0025938799 1885557 19 Morgan MJ Lehmann M Schwarzländer M Decrease in manganese superoxide dismutase leads to reduced root growth and affects tricarboxylic acid cycle flux and mitochondrial redox homeostasis Plant Physiology 2008 147 1 101 114 2-s2.0-50649115800 18337490 20 Myouga F Hosoda C Umezawa T A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis Plant Cell 2008 20 11 3148 3162 2-s2.0-58549118439 18996978 21 López-Millán AF Ellis DR Grusak MA Effect of zinc and manganese supply on the activities of superoxide dismutase and carbonic anhydrase in edicago truncatula M wild type and raz mutant plants Plant Science 2005 168 4 1015 1022 2-s2.0-13944250252 22 Kumar A Dutt S Bagler G Ahuja PS Kumar S Engineering a thermo-stable superoxide dismutase functional at sub-zero to >50°C, which also tolerates autoclaving Scientific Reports 2012 2, article 387 2-s2.0-84860435050 23 Singh HP Batish DR Kohli RK Arora K Arsenic-induced root growth inhibition in mung bean (Phaseolus aureus Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation Plant Growth Regulation 2007 53 1 65 73 2-s2.0-34547760410 24 Stoeva N Berova M Zlatev Z Effect of arsenic on some physiological parameters in bean plants Biologia Plantarum 2005 49 2 293 296 2-s2.0-18144371232 25 Talukdar D Arsenic-induced oxidative stress in the common bean legume, Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide Physiology and Molecular Biology of Plants 2013 19 1 69 79 24381439 26 Talukdar D Genetics of pod indehiscence in grass pea (Lathyrus sativus L.) Journal of Crop Improvement 2011 25 2 161 175 2-s2.0-79953715149 27 Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Analytical Biochemistry 1976 72 1-2 248 254 2-s2.0-0017184389 942051 28 Beyer WF Jr. Fridovich I Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions Analytical Biochemistry 1987 161 2 559 566 2-s2.0-0023130150 3034103 29 Weeden NF A suggestion for the nomenclature of isozyme loci Pisum Newsletter 1988 20 44 45 30 Phaseolus gene bank 2013, http://www.ncbi.nlm.nih.gov/UniGene/lbrowse2.cgi?TAXID=3885 31 Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆C t method Methods 2001 25 4 402 408 2-s2.0-0035710746 11846609 32 Rodríguez-Serrano M Romero-Puertas MC Zabalza A Cadmium effect on oxidative metabolism of pea (Pisum sativum L.) roots. Imaging of reactive oxygen species and nitric oxide accumulation in vivo Plant, Cell and Environment 2006 29 8 1532 1544 2-s2.0-33747830121 33 Talukdar D Arsenic-induced changes in growth and antioxidantmetabolism of fenugreek Russian Journal of Plant Physiology 2013 60 5 652 660 34 Talukdar D Exogenous calcium alleviates the impact of cadmium-induced oxidative stress in Lens culinaris Medic. seedlings through modulation of antioxidant enzyme activities Journal of Crop Science and Biotechnology 2012 15 4 325 334 35 Laspina NV Groppa MD Tomaro ML Benavides MP Nitric oxide protects sunflower leaves against Cd-induced oxidative stress Plant Science 2005 169 2 323 330 2-s2.0-21444451990 36 Gomes-Junior RA Moldes CA Delite FS Antioxidant metabolism of coffee cell suspension cultures in response to cadmium Chemosphere 2006 65 8 1330 1337 2-s2.0-33748905228 16762393 37 Griffin JD Palmer RG Genetic studies with two superoxide dismutase loci in soybean Crop Science 1989 29 968 971 38 Perl-Treves R Galun E The tomato Cu,Zn superoxide dismutase genes are developmentally regulated and respond to light and stress Plant Molecular Biology 1991 17 4 745 760 2-s2.0-0026245748 1912497 39 Fambrini M Sebastiani L Rossi VD Cavallini A Pugliesi C Genetic analysis of an electrophoretic variant for the chloroplast-associated form of Cu/Zn superoxide dismutase in sunflower (Helianthus annuus L.) Journal of Experimental Botany 1997 48 310 1143 1146 2-s2.0-0030873524 40 McKersie BD Murnaghan J Jones KS Bowley SR Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance Plant Physiology 2000 122 4 1427 1437 2-s2.0-0034001533 10759538 41 Kayihan C Eyidogan F Afsar N Oktem HA Yucel M Cu/Zn superoxide dismutase activity and respective gene expression during cold acclimation and freezing stress in barley cultivars Biologia Plantarum 2012 56 4 693 698 2-s2.0-84858843352 42 Bandeoğlu E Eyidogan F Yücel M Öktem HA Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress Plant Growth Regulation 2004 42 69 77 43 Bowler C Van Camp W Van Montagu M Inze D Superoxide dismutase in plants Critical Reviews in Plant Sciences 1994 13 3 199 218 2-s2.0-0028028885 44 Foyer CH Noctor G Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria Physiologia Plantarum 2003 119 3 355 364 2-s2.0-0242309094 45 Talukdar D Dwarf mutations in grass pea (Lathyrus sativus L.): origin, morphology, inheritance and linkage studies Journal of Genetics 2009 88 2 165 175 2-s2.0-70350771205 19700854 46 Kopyra M Gwóźdź EA Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus Plant Physiology and Biochemistry 2003 41 11-12 1011 1017 2-s2.0-0344927787 47 Ortega-Villasante C Rellán-Álvarez R Del Campo FF Carpena-Ruiz RO Hernández LE Cellular damage induced by cadmium and mercury in Medicago sativa Journal of Experimental Botany 2005 56 418 2239 2251 2-s2.0-24944536376 15996984	24078924	PMC3771264	CC BY	2021-01-05 10:06:39	yes	Biomed Res Int. 2013 Aug 28; 2013:782450
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24069157PONE-D-13-1477410.1371/journal.pone.0072758Research ArticleCortisol as a Prognostic Marker of Short-Term Outcome in Chinese Patients with Acute Ischemic Stroke Cortisol and Short-Term Outcome in StrokeZi Wen-Jie Shuai Jie * Department of Neurology, Xin Qiao Hospital, Third Military Medical University, Chongqing, P. R. China Minnerup Jens Editor University of Münster, Germany * E-mail: zhangbytianjin@163.comCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: JS WJZ. Performed the experiments: JS WJZ. Analyzed the data: WJZ. Contributed reagents/materials/analysis tools: JS WJZ. Wrote the paper: JS. 2013 12 9 2013 8 9 e727589 4 2013 14 7 2013 © 2013 Zi, Shuai2013Zi, ShuaiThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background Early prediction of outcome is important for allocation of therapeutic strategies. Endocrine alterations of the hypothalamus–pituitary–axis are one of the first stress-induced alterations after cerebral ischemia. We therefore evaluated the prognostic value of serum cortisol in Chinese patients with an acute ischemic stroke. Methods In a prospective observational study, serum cortisol was measured using a solid-phase, competitive chemiluminescent enzyme immunoassay on admission in serum of 226 consecutive Chinese patients with an acute ischemic stroke. The prognostic value of serum cortisol to predict the functional outcome, mortality within 90 days, was compared with clinical variables (e.g., advanced age and the National Institutes of Health Stroke Scale [NHISS] score) and with other known predictors. Results Patients with a poor outcome and nonsurvivors had significantly increased serum cortisol levels on admission (P<0.0001, P<0.0001). There was a positive correlation between levels of cortisol and the NIHSS (r = 0.298, P<0.0001), glucose levels (r = 0.324, P<0.0001) and infarct volume (r = 0.328, P<0.0001). Cortisol was an independent prognostic marker of functional outcome and death [odds ratio 3.44 (2.58–6.23) and 4.21 (1.89–9.24), respectively, P<0.0001 for both, adjusted for age, the NIHSS and other predictors] in patients with ischemic stroke. In receiver operating characteristic curve analysis, cortisol could improve the NIHSS score in predicting short-term functional outcome (Area under the curve [AUC] of the combined model, 0.87; 95% CI, 0.82–0.92; P = 0.01) and mortality (AUC of the combined model, 0.90; 95% CI, 0.84–0.95; P = 0.01). Conclusion Cortisol can be seen as an independent short-term prognostic marker of functional outcome and death in Chinese patients with acute ischemic stroke even after correcting confounding factors. Combined model can add significant additional predictive information to the clinical score of the NIHSS. The authors have no support or funding to report. ==== Body Introduction In China, approximately 1.6 million stroke patients will die every year, which has exceeded heart disease to become the second leading cause of death and adult disability [1]. In addition, China has 2.5 million new stroke cases each year and 7.5 million stroke survivors [2]. Reliable prognostic markers available during the initial phase after acute stroke may aid clinical decision-making and allocation of healthcare resources. Numerous clinical variables (e.g., advanced age and symptom severity) have been identified as potential predictors of outcome. However, the need to identify better biomarkers as predictors of outcome in acute stroke still exists. The period following ischemic stroke can be considered as a reaction to a stressful event. The major characteristic of the stress response is the activation of the sympathetic nervous system (SNS) and the hypothalamo–pituitary–adrenal (HPA) axis [3]. In cerebral ischemia, endocrine changes of the HPA axis are one of the first measurable alterations [4]. Cortisol is a HPA axis-related hormone with a robust circadian rhythm where levels typically peak in the morning hours and decline across the day [5]. Cortisol has an important effect on the glucose, protein and fat metabolism and cardiovascular reactivity [6]. Some studies showed that high cortisol level was associated with decreased physical function [7], level of consciousness [8]. Fiorentino et al [5] reported that saliva levels of cortisol can be seen as a useful biological marker for identification of patients who are “at risk” of lower benefits from inpatient rehabilitation services. In addition, increased concentrations of cortisol have been observed in subarachnoid haemorrhage [9], and acute ischemic stroke [4]. Some studies have found that elevated plasma or urinary cortisol concentrations in acute ischemic stroke are related to greater stroke severity, larger infarct volume and/or unfavorable outcome, including death [4], [10]–[13]. For patients after acute ischemic stroke, high serum cortisol level was significantly correlated to the presence of acute confusional state [8]. We propose a hypothesis that higher levels of serum cortisol at admission could predict short-term outcomes in Chinese patients with acute ischemic stroke. The aim of this prospective cohort study was to verify this hypothesis. Subjects and Methods Patients and Study Design We conducted a prospective cohort study at the neurology department of the General Hospital, Tianjin Medical University, China. From February 2009 to September 2012, all patients with first-ever acute ischemic stroke were included. All patients were Chinese. All patients were admitted within 48 hours of experiencing a new focal or global neurological event. Brain imaging (either CT or MRI) was performed routinely within 24 to 48 hours after admission. An acute ischemic stroke was defined according to the World Health Organization criteria [14]. We excluded patients with other causes of activation of the HPA axis (eg, those with surgical procedures within the last 3 weeks or those with concomitant preexisting or nosocomial infections), intracranial hemorrhage, malignancy, febrile disorders, acute or chronic inflammatory disease at study enrollment. Patients receiving immunosuppressive agents, all types of steroids, and psychotropic drugs were also excluded. One hundred healthy people matched for age and gender were assigned to the normal control group. Records of potential controls were reviewed by a neurologist (not an author) to exclude the presence of stroke, other types of diseases. Controls receiving immunosuppressive agents, all types of steroids, and psychotropic drugs also should be excluded. The Institutional Review Committee on Human Research of Tianjin Medical University approved the study protocol. The patients or their relatives gave written informed consent prior to entering the study. Clinical Variables At baseline, demographic data (age and sex) and history of conventional vascular risk factors (hypertension, diabetes mellitus, atrial fibrillation, hyperlipidemia, smoking habit, alcohol abuse) were obtained. All these information were obtained through interviews. Routine blood and biochemical tests, ECG, and a baseline brain CT/MRI scan were performed in all patients at admission. Stroke severity was assessed on admission using the National Institutes of Health Stroke Scale (NIHSS) by a neurologist [15]. The NIHSS score ranges from 0 to 34 and higher values reflect more severe neurological damage. Whenever the NIHSS values were missing, values were estimated from chart review retrospectively, previously shown to give reliable estimates for NIHSS [16]. Stroke subtype was classified according to TOAST (Trial of Org 10172 in Acute Stroke Treatment) criteria [17]. The clinical stroke syndrome was determined by applying the criteria of the Oxfordshire Community Stroke Project: total anterior circulation syndrome (TACS); partial anterior circulation syndrome (PACS); lacunar syndrome (LACS); and posterior circulation syndrome (POCS) [18]. End Points and follow-up We also recorded time from symptom recognition to admission. We considered the following endpoints: (i) the primary end-point was functional outcome on day 90. Functional outcome was assessed by the modified Rankin Scale (mRS) [19]. A favourable functional outcome was defined as a mRS of 0–2 points, whereas an unfavourable outcome was defined as a mRS of 3–6 points; (ii) secondary end-points were all-cause mortality within 90 days. Outcome assessment was performed by one trained medical staff blinded to cortisol levels with a structured interview, if discharged, with telephone interview. Neuroimaging MRI with diffusion-weighted imaging (DWI) was available in 152 stroke patients (67.2%). In those patients, DWI lesion volumes were determined by one experienced neurologist unaware of the clinical and laboratory results. The infarct volume was calculated by using the formula 0.5×a×b×c (where a is the maximal longitudinal diameter, b is the maximal transverse diameter perpendicular to a and c is the number of 10-mm slices containing infarct) [20]. Blood Collection and Quantification Blood samples of patients and controls were obtained at 6:00 AM in the next morning of the day of admission. After centrifugation, aliquots of the samples were immediately stored at −80°C before assay. Serum cortisol concentration was measured with a solid-phase, competitive chemiluminescent enzyme immunoassay in a calibrated IMMULITE 2000 analyzer (Diagnostic Products Corporation, Los Angeles, CA, USA).The normal range of morning serum cortisol concentration in our hospital laboratory is 145 to 650 nmol/L, which is broadly the same as that of other laboratories (201–681 nmol/L [21], 138–690 nmol/L [22], 119–618 nmol/L [12]).The detection limit was 5 nmol/L. The intra-assay coefficient of variation [CV] and inter-assay CV were 1.5–3.2%, 2.1%–4.3%, respectively. For all measurements, levels that were not detectable were considered to have a value equal to the lower limit of detection of the assay Statistical Analysis Results are expressed as percentages for categorical variables and as medians (interquartile ranges, IQRs) for the continuous variables. Proportions were compared using the χ2 test, and the Mann–Whitney test to compare continuous variables between groups. Correlations were determined using Spearman critical value rankings. Multivariate analysis was performed by binary logistic regression analysis, which allows adjustment for confounding factors (age, stroke syndrome, stroke etiology, the NIHSS score, infarct volume, vascular risk factors, glucose and C-reactive protein). Results were expressed as adjusted OR (odds ratios) with the corresponding 95% CIs (confidence intervals). Receiver operating characteristic (ROC) curves were utilized to evaluate the accuracy of cortisol to predict outcomes and death. Area under the curve (AUC) was calculated as measurements of the accuracy of the tests. The AUC summary equals the probability that the underlying classifier will score a randomly drawn positive sample higher than a randomly drawn negative sample. The time to death was analyzed by Kaplan–Meier survival curves. Two-sided P values of less than 0.05 were regarded as significant. All statistical analysis was performed with SPSS for Windows, version 20.0 (SPSS Inc., Chicago, IL, USA). Results Baseline characteristics of study samples During the inclusion period, 313 patients were registered. Acute ischemic stroke was diagnosed in 237 patients (45 with transient ischemic attack, 4 with haemorrhagic stroke, 10 with onset of symptoms >48 hours, 7 without informed consent, 4 with systemic infections, 3 with malignant tumor and 3 with surgical procedures within the last 3 weeks were not analyzed) and 226 completed follow-up (8 lost to follow-up and 3 withdraw). In the study population, 149 (65.9%) were male and median age was 65 years (interquartile ranges,IQR 55–74). The median time from symptom recognition to admission to hospital was 6.5 hours (IQR 2.5–11.5), and 198 patients (87.6%) were admitted within 24 hours of symptom recognition. The median NIHSS score on admission was 6 points (IQR, 3 to 10). Hypercortisolemia was found in 64 (28.3%) patients. An unfavorable functional outcome was found in 77 patients (34.1%) with a median mRS score of 4 (IQR, 3–6). 34 patients died, thus the mortality rate was 15.0%. The baseline characteristics of the 226 patients presenting with acute ischemic stroke are described in Table 1. 10.1371/journal.pone.0072758.t001Table 1 Baseline characteristics of acute ischemic stroke patients. Characteristics All Good outcomes Poor outcomes pa (n = 226) (n = 149) (n = 77) Male sex (%) 149(65.9) 99(66.4) 50(64.9) NS Age (years), median(IQR) 65(55–74) 61(50–68) 74(62–79) <0.001 Stroke severity, median NIHSS score (IQR) 6(3–10) 3(2–7) 10(6–15) <0.001 Infarct volume(mL,IQR; n = 152) 9(5.8–17) 8(4–12)b 12.5(8.3–65) <0.001 Vascular risk factors no. (%) Hypertension 169(74.8) 105(70.5) 58(83.1) <0.01 Diabetes mellitus 63(27.9) 40(26.8) 23(29.9) NS Hypercholesterolemia 59(26.1) 43(28.9) 16(20.8) <0.01 Coronary heart disease 66(29.2) 44(29.5) 22(28.6) NS Atrial fibrillation 49(21.9) 34(22.8) 15(19.5) NS Family history for stroke 55(24.3) 39(26.2) 16(20.8) NS Smoking habit 52(23.1) 36(24.2) 16(20.8) NS Alcohol abuse 48(21.3) 32(21.5) 16(20.8) NS Clinical findings median(IQR) Systolic blood pressure(mmHg) 156(145–177) 155(143–174) 158(147–180) NS Diastolic blood pressure(mmHg) 90(82–99) 88(81–96) 91(84–100) NS Temperature (°C) 36.9(36.3–37.5) 37.0(36.4–37.5) 36.9(36.5–37.7) NS BMI (kg m−2) 25.6(23.4–27.2) 25.5(23.3–27.0) 25.6(23.5–27.7) NS Heart rate (beats min−1) 81(69–90) 80(69–89) 81(70–90) NS Laboratory findings (median, IQR) Cortisol (nmol L−1) 454(389–630) 441(367–511) 643(456–786) <0.001 Total cholesterol (mmol L−1) 4.11(3.41–4.94) 4.07(3.35–4.92) 4.13(3.44–4.99) NS HDL (mmol L−1) 1.31(1.05–1.63) 1.30(1.05–1.62) 1.31(1.07–1.66) NS LDL (mmol L−1) 2.10(1.37–2.71) 2.11(1.38–2.71) 2.10(1.36–2.71) NS Triglycerides(mmol L−1) 1.42(1.05–1.88) 1.37(1.06–1.72) 1.47(1.05–2.16) NS Glucose(mmol L−1) 5.62(4.99–6.93) 5.39(4.91–6.45) 6.06(5.12–7.59) <0.01 C-reactive protein (mg L−1) 4.1(3.2–8.4) 3.8(3.0–7.8) 4.4(3.5–8.9) <0.01 Leucocyte count (×103 m L−1) 8.4(6.2–9.7) 8.3(6.1–9.4) 8.4(6.4–9.8) NS Stroke syndrome no. (%) TACS 27(11.9) 8(5.4) 27(24.7) <0.001 PACS 87(38.5) 59(39.6) 87(36.4) NS LACS 45(19.9) 28(18.8) 17(22.1) NS POCS 67(29.7) 50(33.6) 17(22.1) <0.01 Stroke etiology no. (%) Small-vessel occlusive 42(18.6) 29(19.4) 13(16.9) NS Large-vessel occlusive 44(19.5) 30(20.1) 14(18.2) NS Cardioembolic 85(37.6) 60(40.3) 25(32.3) NS Other 14(6.2) 7(4.7) 7(9.1) NS Unknown 41(18.1) 27(18.1) 14(18.2) NS IQR, interquartile range; TACS, total anterior circulation syndrome; LACS, lacunar syndrome; PACS, partial anterior circulation syndrome; POCS, posterior circulation syndrome; NIHSS, National Institutes of Health Stroke Scale; BMI, Body mass index; HDL, High-density lipoproteins; LDL, Low-density lipoproteins. a p value was assessed using Mann-Whitney U test or χ2 test. b 104 patients were with good outcomes. Serum cortisol levels and stroke characteristics The results indicated that the serum cortisol levels were significantly (p<0.0001) higher in acutely ischemic stroke patients as compared to normal controls (456; IQR, 397–621 nmol/L and 424; IQR, 368–515 pmol/L, respectively; Figure 1.). Cortisol levels increased with increasing severity of stroke as defined by the NIHSS score. There was a positive correlation between levels of cortisol and the NIHSS (r = 0.298, P<0.0001; Figure 2a.) and glucose levels (r = 0.324, P<0.0001; Figure 2b.) There was a modest correlation between levels of serum cortisol levels and age(r = 0.157, P = 0.021). There was no correlation between levels of serum cortisol levels and sex (P = 0.230). C-reactive protein and white blood cells count were not correlated with the serum cortisol levels (p = 0.103 and p = 0.124, respectively). In patients for whom MRI data were available (n = 152), there was a positive correlation between levels of cortisol and the infarct volume (r = 0.328, P<0.0001; Figure 2c.) In addition, cortisol values were significantly higher in patients with TACS 625 nmol/L (IQR 478–812) compared with patients with PACS 487 nmol/L (IQR 356–614, P<0.001), LACS 438 nmol/L (IQR 344–613, P<0.001) or POCS 476 nmol L (IQR 335–661, P<0.001). Cortisol levels were not correlated with the C-reactive protein and Leucocyte count. 10.1371/journal.pone.0072758.g001Figure 1 Serum cortisol levels in acute ischemic stroke patients and control group. Mann–Whitney U-test. All data are medians and in-terquartile ranges (IQR). Significantly higher in stroke patients as compared to normal cases (P<0.0001). 10.1371/journal.pone.0072758.g002Figure 2 Correlation between serum cortisol levels and others predictors. (a) Correlation between the National Institutes of Health Stroke Scale (NIHSS) and serum cortisol levels. (b) Correlation between the serum glucose and cortisol levels. (c) Correlation between the infract volume and serum cortisol levels. Serum cortisol levels and outcome Serum cortisol levels in patients with a poor outcome were significantly greater than those in patients with a good outcome (643 [IQR, 456–786] vs 441 [IQR, 367–511] nmol/L; p<0.0001; Figure 3.). In univariate logistic regression analysis, we calculated the ORs of log-transformed cortisol levels as compared with the NIHSS score and other risk factors as presented in Table 2. With an unadjusted OR of 5.98 (95% CI, 3.63–10.52), cortisol had a strong association with functional outcome. After adjusting for all other significant outcome predictors, cortisol remained can be seen as an independent outcome predictor with an adjusted OR of 3.44 (95% CI, 2.58–6.23). In addition, age, glucose level, infarct volume, and the NIHSS score remained significant outcome predictors (Table 2). 10.1371/journal.pone.0072758.g003Figure 3 Serum cortisol levels in acute ischemic stroke patients with good and poor outcomes. Mann–Whitney U-test. All data are interquar-tile ranges (IQR). Significantly higher in poor as compared to good group (P<0.0001). 10.1371/journal.pone.0072758.t002Table 2 Univariate and multivariate Analysis. Parameter Univariate Analysis Multivariate Analysis ORa 95% CIa P ORa 95% CIa P Predictor: functional outcome Cortisol (increase per log unit)b 5.98 3.63–10.52 <0.0001 3.44 2.58–6.23 <0.0001 Age (increase per unit) 1.36 0.85–2.18 0.031 1.54 0.83–2.89 0.012 NIHSS (increase per unit) 1.23 1.06–1.45 <0.0001 1.30 1.14–1.47 <0.0001 Glucose (increase per unit) 1.19 1.04–1.36 0.012 1.10 0.92–1.31 0.030 CRP (increase per unit) 1.23 1.06–1.45 0.008 1.16 1.11–1.23 0.021 Infarct volume(increase per unit) 1.11 1.00–1.22 0.006 1.13 0.87–1.31 0.002 Predictor: death Cortisol (increase per log unit)b 10.32 5.68–18.45 <0.0001 4.21 1.89–9.24 <0.0001 Age (increase per unit) 1.42 0.99–1.87 0.023 1.34 1.08–1.99 0.043 NIHSS (increase per unit) 1.52 1.14–2.99 <0.0001 1.42 0.95–2.07 0.027 Glucose (increase per unit) 1.33 1.12–1.68 0.003 1.33 0.88–2.07 <0.0001 CRP (increase per unit) 1.29 1.08–1.65 0.011 1.12 0.98–1.56 0.016 Infarct volume(increase per unit) 1.19 1.02–1.54 0.007 1.06 1.04–1.09 <0.0001 a Note that the odds ratio corresponds to a unit increase in the explanatory variable. b This corresponds to an increase per unit of the log transformation of cortisol (thus, a log-transformed increase of 1 corresponds to a cortisol increase of 10 nmol/L). OR, odds ratio; CI, confidence interval; CRP, C-reactive protein; NIHSS, National Institutes of Health Stroke Scale. The area under the receiver operating characteristics (ROC) curve to predict outcome for cortisol with an AUC of 0.78 (0.71–0.85) was in the range of the NIHSS with an AUC of 0.81 (0.75–0.89) (see table 3). Cortisol had a higher prognostic accuracy as compared to glucose (AUC 0.55 (0.43–0.67), P<0.01), white blood count (WBC) (AUC 0.47 (0.39–0.58), P<0.001) and infarct volume AUC 0.69 (0.62–0.77), P = 0.01). Interesting, we found that combination of cortisol and NIHSS scores could improve the NIHSS scores (AUC of the combined model, 0.87; 95% CI, 0.82–0.92; p = 0.01). This improvement was stable in an internal 5-fold cross validation that resulted in an average AUC (standard error) of 0.81 (0.036) for the NIHSS and 0.87(0.028) for the combined model, corresponding to a difference of 0.06 (0.014). 10.1371/journal.pone.0072758.t003Table 3 Receiver operating characteristics curve analysis. Parameter Functional outcome at 90 days Mortality at 90 days AUC 95% confidence interval P AUC 95% confidence interval P Cortisol 0.78 0.71–0.85 0.81 0.73–0.79 NIHSS 0.81 0.75–0.89 0.32 0.85 0.78–0.91 0.16 Infarct volume 0.69 0.62–0.77 0.01 0.76 0.67–0.84 0.04 Age 0.61 0.53–0.69 <0.01 0.54 0.43–0.66 <0.01 Glucose 0.55 0.43–0.67 <0.01 0.64 0.51–0.72 0.02 CRP 0.54 0.44–0.68 <0.01 0.57 0.49–0.67 <0.01 WBC 0.47 0.39–0.58 <0.001 0.55 0.43–0.67 <0.01 Combined score⋇ 0.87 0.82–0.92 0.01 0.90 0.84–0.95 0.01 AUC, area under the curve; CRP, C-reactive protein; NIHSS, National Institutes of Health Stroke Scale; WBC, white blood cells. ⋇ including NIHSS and Cortisol. Cortisol levels in 34 patients who died were significantly greater as compared with patients who survived (682 [IQR, 511–878] vs 438 [IQR, 384–563] nmol/L; p<0.0001). After adjustment for other parameters, cortisol level remained an independent predictor for mortality with an OR of 4.21 (95% CI, 1.89–9.24; see Table 2). Receiver operating characteristic curves indicated the greatest discriminatory accuracies for cortisol level (AUC, 0.81; 95% CI, 0.73–0.89) and the NIHSS score (AUC, 0.85; 95% CI, 0.78–0.91). Combination of cortisol and NIHSS scores also could improve the NIHSS scores (AUC of the combined model, 0.90; 95% CI, 0.84–0.95; p = 0.01). Again, this improvement was stable in an internal 5-fold cross validation that resulted in an average AUC (standard error) of 0.85 (0.023) for the NIHSS and 0.90(0.027) for the combined model, corresponding to a difference of 0.05 (0.018). The time to death was analyzed by Kaplan–Meier survival curves based on cortisol quartiles. Patients in the lowest two quartile (cortisol <397 nmol/L and cortisol between 397 and 456 nmol/L) had a minimal risk for death, in contrast with patients with levels in the highest two quartiles (cortisol between 456 and 621 nmol/L and cortisol >621 nmol/L, respectively) (p<0.0001) (See the Figure 4.). 10.1371/journal.pone.0072758.g004Figure 4 Kaplan–Meier survival curves based on cortisol quartiles. Time to death was analysed by Kaplan–Meier curves based on cortisol quartiles. Discussion Acute ischemic stroke acts as a stressor and thus stimulates the HPA axis resulting in increased glucocorticoid levels [4]. In this study, we firstly assessed serum cortisol levels with regard to their accuracy to predict functional outcome and mortality in patients with acute ischemic stroke within 90 days in Chinese population. Our main finding is that cortisol can be seen as an independent short-term prognostic marker of functional outcome and death in Chinese patients with acute ischemic stroke even after correcting for possible confounding factors. Combined model (cortisol and NHISS score) can add significant additional predictive information to the clinical score of the NIHSS. We also found that that cortisol levels increased with infarct volume, neurological deficit (assessed by the NIHSS) and the clinical stroke syndrome. These results are in accordance with the results from other studies showing that hypercortisolemia was associated with older age, greater severity of neurological deficit, larger ischemic lesions on CT, worse prognoses (a greater disability and mortality) in stroke patients [8], [12], [23]. Age may influence serum cortisol [24]–[25]. We also found a modest positive correlation between levels of serum cortisol levels and age(r = 0.157, P = 0.021). Previous studies reported that fasting glucose level and insulin resistance increased with higher cortisol level [6]–[7].We also found that there was a positive correlation between levels of cortisol and glucose levels (r = 0.324, P<0.0001; Figure 3.). As expected, high levels of cortisol were associated with diabetes mellitus. The association may be confounded by obesity. It has been reported that cortisol correlated positively to inflammatory response after stroke, and it was suggested that cytokines modulate the cortisol response after acute stroke [11] by stimulating the HPA axis leading to increased levels of cortisol in the periphery [26]. Our findings were inconsistent with previous findings, and we did not find correlation between inflammatory response and serum cortisol levels. In our study, we found hypercortisolemia in 28.3% of patients with acute stroke. The frequency of hypercortisolemia in stroke patients has been reported in the range of 24%–38% [8], [12], [27]. This is very interesting because despite using different methods of assessment and the study population are not the same, a similar prevalence of hypercortisolemia was obtained, indicating a similar increase in adrenocortical output in stroke. Whether the high serum cortisol is just an epiphenomenon to stroke severity or independently contributes to prognosis remains uncertain. A severe stroke per se implicates a poor outcome. However, there are several other reasons explaining unfavorable outcome in patients with higher cortisol levels. It cannot be excluded that these HPA axis-related hormones, once released, secondarily reinforce damage of hypoxic brain tissue and thereby contribute to the poor outcomes. Firstly, increased exposure to cortisol contributes to increased fat accumulation in visceral depots [28]. Hypercortisolism itself may potentiate ischemic neuronal injury, especially in hippocampal neurons [29]–[30], and the corticosterone-synthesis inhibitor, metyrapone, was able to prevent ischemia-induced loss of synaptic function in the hippocampus of rats [31]. The hippocampus has an important role in the feedback regulation of the HPA axis. Dysfunction of hippocampal might result in false HPA axis feedback, which increase cortisol and causes a vicious circle [32]. Secondly, patients with stroke and high cortisol levels are more prone to suffer from adverse cardiac events, which may lead to higher mortality rates [32]–[33]. Finally, a bad prognosis after stroke is the development of infectious disease which is related to an immune dysregulation resulting from neuroendocrine disturbance after stroke [34]. High cortisol levels are more susceptible to infections [7]. The specific mechanism needs to be further studied in population-based larger cohort studies. Some limitations of this observational study merit consideration. First, without serial measurement of the circulating cortisol levels, this study yielded no data regarding when and how long cortisol is elevated in these patients. Second, the effects of circulating cortisol on long-term clinical outcome were not included in the study protocol, so these relationships were not examined beyond the 90-day clinical outcome. Third, we assessed all-cause mortality because classification of death in clinical practice can sometimes be difficult and unreliable [35]. Forth, this was only a preliminary study; these results need to be replicated in more prospective studies to determine whether or not they are stable and valid. If our findings are confirmed, clinicians assessing patients with acute stroke should consider measuring serum levels of cortisol routinely on admission. Conclusions Despite its inherent limitations, our study suggested that cortisol levels may reliably predict short-term prognosis at its onset in Chinese patient with acute ischemic stroke. Combined model (cortisol and NHISS score) can add significant additional predictive information to the clinical score of the NIHSS. We recommend that further studies should be carried out with respect to the mechanism between increased cortisol levels and poor outcome. If it is possible to elucidate this, the prognosis of these stroke patients might be improved. We are grateful to the Department of Neurology; the nurses, physicians, and patients who participated in our study; and the staff of the central laboratory of the Hospital. Both authors have contributed significantly, and both authors are in agreement with the content of the manuscript. ==== Refs References 1 Liu L , Wang D , Wong KS , Wang Y (2011 ) Stroke and stroke care in China: huge burden, significant workload, and a national priority . Stroke 42 : 3651 –3654 .22052510 2 Johnston SC , Mendis S , Mathers CD (2009 ) Global variation in stroke burden and mortality: estimates from monitoring, surveillance, and modeling . Lancet Neurol 8 : 345 –354 .19233730 3 McEwen BS (2007 ) Physiology and neurobiology of stress and adaptation: central role of the brain . Physiol Rev 87 : 873 –904 .17615391 4 Fassbender K , Schmidt R , Mossner R , Daffertshofer M , Hennerici M (1994 ) Pattern of activation of the hypothalamic–pituitary– adrenal axis in acute stroke. Relation to acute confusional state, extent of brain damage, and clinical outcome . Stroke 25 : 1105 –1108 .8202965 5 Fiorentino L , Saxbe D , Alessi CA , Woods DL , Martin JL (2012 ) Diurnal cortisol and functional outcomes in post-acute rehabilitation patients . J Gerontol A Biol Sci Med Sci 67 : 677 –682 .22219521 6 O'Neill PA , Davies I , Fullerton KJ , Bennett D (1991 ) Stress hormone and blood glucose response following acute stroke in the elderly . Stroke 22 : 842 –847 .1853403 7 Schoorlemmer RM , Peeters GM , van Schoor NM , Lips P (2009 ) Relationships between cortisol level, mortality and chronic diseases in older persons . Clin Endocrinol (Oxf) 71 : 779 –786 .19226268 8 Olsson T , Marklund N , Gustafson Y , Näsman B (1992 ) Abnormalities at different levels of the hypothalamic – pituitary – adrenocortical axis early after stroke . Stroke 23 : 1573 –1576 .1332219 9 Zetterling M , Engström BE , Hallberg L , Hillered L , Enblad P , et al (2011 ) Cortisol and adrenocorticotropic hormone dynamics in the acute phase of subarachnoid haemorrhage . Br J Neurosurg 25 : 684 –692 .22115015 10 Feibel JH , Hardy PM , Campell RG , Goldstein MN , Joynt RJ (1977 ) Prognostic value of the stress response following stroke . JAMA 238 : 1374 –1376 .578192 11 Christensen H , Boysen G , Johannesen HH (2004 ) Serum-cortisol reflects severity and mortality in acute stroke . J Neurol Sci 217 : 175 –180 .14706221 12 Slowik A , Turaj W , Pankiewicz J , Dziedzic T , Szermer P (2002 ) Hypercortisolemia in acute stroke is related to the inflammatory response . J Neurol Sc 196 : 27 –32 .11959152 13 Anne M , Juha K , Timo M , Mikko T , Olli V (2007 ) Neurohormonal activation in ischemic stroke: effects of acute phase disturbances on long-term mortality . Curr Neurovasc Res 4 : 170 –175 .17691970 14 Hatano S (1976 ) Experience from a multicentre stroke register: a preliminary report . Bull World Health Organ 54 : 541 –553 .1088404 15 Brott T , Adams HP Jr, Olinger CP , Marler JR , Barsanl WG , et al (1989 ) Measurements of acute cerebral infarction: A clinical examination scale . Stroke 20 : 864 –870 .2749846 16 Kasner SE , Chalela JA , Luciano JM , Cucchiara BL , Raps EC (1999 ) Reliability and validity of estimating the NIH stroke scale score from medical records . Stroke 30 : 1534 –1537 .10436096 17 Adams HP , Bendixen BH , Kappelle LJ , Biller J , Love BB (1993 ) Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment . Stroke 24 : 35 –41 .7678184 18 Bamford J , Sandercock P , Dennis M , Burn J , Warlow C (1991 ) Classification and natural history of clinically identifiable subtypes of cerebral infarction . Lancet 337 : 1521 –1526 .1675378 19 Bonita R , Beaglehole R (1988 ) Recovery of motor function after stroke . Stroke 19 : 1497 –1500 .3201508 20 Sims JR , Gharai LR , Schaefer PW , Vangel M , Rosenthal ES (2009 ) ABC/2 for rapid clinical estimate of infarct, perfusion, and mismatch volumes . Neurology 72 : 2104 –2110 .19528517 21 Ibrahimagić OC , Sinanović O , Cickusić A , Smajlović D (2005 ) Ischemic stroke as reaction to an acute stressful event . Med Arh 59 : 79 –82 .15875466 22 Mu DL , Wang DX , Li LH , Shan GJ , Li J (2010 ) High serum cortisol level is associated with increased risk of delirium after coronary artery bypass graft surgery: a prospective cohort study . Crit Care 14 : R238 .21192800 23 Murros K , Fogelholm R , Kettunen S , Vuorela AL (1993 ) Serum cortisol and outcome of ischemic brain infarction . J Neurol Sci 116 : 12 –17 .8509800 24 Parker CR Jr, Slayden SM , Azziz R , Crabbe SL , Hines GA (2000 ) Effects of aging on adrenal function in the human: responsiveness and sensitivity of adrenal androgens and cortisol to adrenocorticotropin in premenopausal and postmenopausal women . J Clin Endocrinol Metab 85 : 48 –54 .10634362 25 Wilkinson CW , Petrie EC , Murray SR , Colasurdo EA , Raskind MA (2001 ) Human glucocorticoid feedback inhibition is reduced in older individuals: evening study . J Clin Endocrinol Metab 86 : 545 –550 .11158007 26 Chrousos GP (1995 ) The hypothalamic– pituitary– adrenal axis and immune mediated inflammation . N Engl J Med 332 : 1351 –1362 .7715646 27 Aström M , Olsson T , Asplund K (1993 ) Different linkage of major depression to hypercortisolism early versus late after stroke: results from a 3-year longitudinal study . Stroke 24 : 52 –57 .8418550 28 Anagnostis P , Athyros VG , Tziomalos K , Karagiannis A , Mikhailidis DP (2009 ) The pathogenetic role of cortisol in the metabolic syndrome: a hypothesis . J Clin Endocrinol Metab 94 : 2692 –2701 .19470627 29 Sapolsky RM , Pulsinelli WA (1985 ) Glucocorticoids potentiate ischemic injury to neurons: therapeutic implications . Science 229 : 1397 –1400 .4035356 30 Fuchs E , Flügge G (1998 ) Stress, glucocorticoids and structural plasticity of the hippocampus . Neurosci Biobehav Rev 23 : 295 –300 .9884123 31 Krugers HJ , Maslam S , Korf J , Joëls M , Holsboer F (2000 ) The corticosterone synthesis inhibitor metyrapone prevents hypoxia/ischemia-induced loss of synaptic function in the rat hippocampus . Stroke 31 : 1162 –1172 .10797181 32 Katan M , Elkind MS (2011 ) Inflammatory and neuroendocrine biomarkers of prognosis after ischemic stroke . Expert Rev Neurother 11 : 225 –239 .21306210 33 Samuels MA (2007 ) The brain–heart connection . Circulation 116 : 77 –84 .17606855 34 Neidert S , Katan M , Schuetz P , Fluri F , Ernst A (2011 ) Anterior pituitary axis hormones and outcome in acute ischaemic stroke . J Intern Med 269 : 420 –432 .21205022 35 Potocki M , Breidthardt T , Mueller A (2010 ) Copeptin and risk stratification in patients with acute dyspnea . Critical Care 14 : R213 .21106053	24069157	PMC3771965	CC BY	2021-01-05 17:39:23	yes	PLoS One. 2013 Sep 12; 8(9):e72758
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24069144PONE-D-13-0411310.1371/journal.pone.0072333Research ArticlePolymorphism of 9p21.3 Locus Is Associated with 5-Year Survival in High-Risk Patients with Myocardial Infarction The 9p21.3 Locus and Mortality after STEMISzpakowicz Anna 1 Pepinski Witold 2 Waszkiewicz Ewa 1 Maciorkowska Dominika 3 Skawronska Małgorzata 2 Niemcunowicz-Janica Anna 2 Milewski Robert 4 Dobrzycki Sławomir 3 Musial Włodzimierz Jerzy 1 Kaminski Karol Adam 1 * 1 Department of Cardiology, Medical University of Bialystok, Bialystok, Poland 2 Department of Forensic Medicine, Medical University of Bialystok, Bialystok, Poland 3 Department of Invasive Cardiology, Medical University of Bialystok, Bialystok, Poland 4 Department of Statistics and Medical Informatics, Medical University of Bialystok, Bialystok, Poland Gong Yan Editor College of Pharmacy, University of Florida, United States of America * E-mail: fizklin@wp.plCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: AS WP ANJ SD WJM KAK. Performed the experiments: EW MS DM. Analyzed the data: AS WP RM KAK. Contributed reagents/materials/analysis tools: WP ANJ SD WJM KAK. Wrote the paper: AS. 2013 12 9 2013 17 4 2014 8 9 e7233325 1 2013 9 7 2013 © 2013 Szpakowicz et al2013Szpakowicz et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Objective The rs1333049, rs10757278 and rs4977574 are single nucleotide polymorphisms (SNPs) of chromosome 9p21 locus that are associated with prevalence of acute coronary syndromes (ACS). The rs1333049 SNP was also associated with cardiac outcome 6 months post ACS. No data concerning their association with long term prognosis after myocardial infarction is available. The aim of our study was to investigate the association of the 9p21.3 locus with 5-year overall mortality in patients with ST-elevation myocardial infarction (STEMI) treated invasively. Materials and Methods We performed a retrospective analysis of data collected prospectively in a registry of consecutive patients with STEMI treated with primary PCI. Genotyping was performed with a TaqMan method. The analyzed end-point was total 5-year mortality. Results The study group comprised 589 patients: 25.3% of females (n = 149), mean age 62.4±11.9 years, total 5-year mortality 16.6% (n = 98). When all the study group was analyzed, no significant differences in mortality were found between the genotypes. However, in high-risk patients (Grace risk score ≥155 points, n = 238), low-risk homozygotes had significantly better 5-year survival compared to other genotypes. The hazard ratio associated with high-risk genotype (high-risk homozygote or heterozygote) was: HR = 2.9 (95%CI 1.4–6.1) for the rs4977574 polymorphism, HR = 2.6 (1.25–5.3) for the rs1333049 one and HR = 2.35 (1.2–4.6) for the rs10757278 one (Cox proportional hazards model). Conclusions The 9p21.3 locus is associated with 5-year mortality in high-risk patients with STEMI. This finding, due to very high effect size, could potentially be applied into clinical practice, if appropriate methods are elaborated. This work was supported by National Science Center, Poland [N N 402 529139]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Several genome-wide association studies have shown a strong association between the chromosomal locus 9p21.3 and coronary artery disease (CAD) or myocardial infarction [1]–[8]. The results were further replicated in large-scale case-control studies [9]–[10]. The same locus was reported to give significant genomic signal for other diseases, like type 2 diabetes [11]–[14], aortic or intracranial aneurysms, peripheral artery disease or cancers [15]–[17]. There are several single nucleotide polymorphisms (SNPs) of the 9p21.3 locus associated with cardiovascular diseases, however, the functional link remains hypothetical. None of the SNPs is located within a protein coding region. The 9p21.3 locus contains only a sequence for an antisense RNA (ANRIL, CDKN2BAS). The nearby genes are coding cyclin-dependent kinases 2B and 2A (CDKN2A and CDKN2B) or methylthioadenosine phosphorylase (MTAP). The CDKN2B is potentially involved in pathogenesis of atherosclerosis, while it is a downstream target for transforming growth factor beta [18]–[19]. The problem is that SNPs influencing CDKN2B expression do not affect CAD risk and vice versa [20]. There is a very strong evidence for association between the 9p21.3 locus and myocardial infarction (MI). However, data regarding its influence on further prognosis is equivocal. In the GRACE registry that was performed in Europe (n = 3247, patients with all forms of an acute coronary syndrome, 6 months of follow-up), C allele of the rs1333049 polymorphism was independently associated with recurrent myocardial infarction or cardiac death [21]. No association with outcome was found in the population of the Post-Myocardial Infarction study (New Zealand, n = 816, median follow-up 9 years) or in Han Chinese patients with first ST-segment elevation myocardial infarction (STEMI, n = 520, median follow-up 29 months) [22]–[23]. The discrepancies were not related to type of treatment. Han Chinese patients underwent invasive procedure, participants from New Zealand received fibrinolytics and in GRACE registry patients were enrolled irrespective of applied strategy. There is a debate if genetic testing may become part of the risk assessment in MI patients. Some authors claim that the influence of particular polymorphisms on prognosis does not exceed 15–30%, thus making a whole genetic analysis useless for clinical risk assessment. Their opinions, however, are based on genome wide association studies that are especially biased to recognize markers with limited influence. This fact does not mean that particular polymorphisms would not be useful for special populations, where they could identify high risk patients. In the present study we aimed to investigate the association of the 9p21.3 locus with 5-year all-cause mortality of patients with myocardial infarction. We tested 3 previously described SNPs (rs4977574, rs1333049, rs10757278) as markers of this locus. The further goal of the study was selecting and characterizing patients who could have the greatest benefit from genotyping. Materials and Methods Ethics statement The study protocol was approved by Ethics Committee of Medical University of Bialystok. The study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Informed written consent has been obtained from the subjects. We performed a retrospective analysis of data collected prospectively in a single center. It comprised Caucasian patients with STEMI, inhabitants of North-Eastern Poland, who were hospitalized in years 2001–2005 and survived first 48 hours after admission. The 48 hour survival threshold was implemented because of the time from blood sampling to potential result of genetic testing. In this subpopulation it would be of no benefit, but the very early mortality might affect final results. No additional exclusion criteria were introduced. All the patients underwent coronary angiography within 12 hours from symptoms onset. STEMI was diagnosed based on rise in troponin I concentration or creatine kinase –MB fraction activity accompanied by chest pain history and new ECG abnormalities (lasting >20 minutes ST-segment elevation or left bundle branch block). The analyzed data included patients' history, physical examination on admission, routine laboratory tests, echocardiography, results of coronary angiography and invasive treatment. MDRD (Modification of Diet in Renal Disease) formula was used to estimate creatinine clearance. Grace risk score was calculated retrospectively, based on previously described method [24]. Appropriate number of points were given for: age, heart rate, systolic blood pressure, creatinine plasma concentration, Killip class and cardiac arrest (all parameters assessed on admission). All patients were also scored for ST-segment deviation and elevated cardiac markers. Next, all subjects were divided into high-risk group (≥155 points) and non-high-risk group, according to previously evaluated clinical cut-off level [25]. All the patients were treated according to contemporary guidelines. Blood samples were collected to EDTA tubes, treated with commercial DNA extraction kit (Blood Mini, A&A Biotechnology) and stored in −20 degrees Celsius. The SNPs (rs1333049, rs10757278, rs4977574) were assessed with a TaqMan SNP Genotyping Assay on the ABI 7500 real time PCR platform (Applied Biosystems), according to manufacturer's instructions. Ten percent of samples were genotyped twice (quality control requirements). The analyzed end-point was 5-year all-cause mortality. Data concerning survival was retrieved from the local population registry run by a Government Office, assuring the most complete follow-up possible. A statistical analysis was performed with STATISTICA 9.0 software. Distribution of variables was tested with Shapiro-Wilk test. Next, clinical parameters were compared between the genotypes with chi-2, t-Student or Mann-Whitney tests, as appropriate. Survival was compared with log-rank test. Univariate and multivariate analyses for 5-year survival were performed with Cox proportional hazards model. Variables with significant association with survival were included in a primary model of multivariate regression. In the case of the 3 SNPs, due to strong linkage between them, only the rs4977574 was included, because it showed the strongest effect, based on literature [2], [4], [5], [6]. The final model was selected in backward stepwise manner. Additionally, all SNPs in a univariate analysis as well as multivariate model were adjusted for severity of coronary artery disease (number of vessels with significant stenosis). Two-sided p value<0.05 was considered statistically significant. In the case of survival analysis (chi-2 and log-rank tests) multiple tests were performed, therefore p values were adjusted for Bonferroni correction (4 subgroup analyses). Due to very strong linkage between the 3 SNPs, we did not consider them for further multiple testing. The biostatistical parameters were calculated using ARLEQUIN v.3.0 software. The study was designed to have a statistical power of at least 80 percent to detect a 66% percent relative risk increase in 5-year mortality of high-risk homozygotes compared to other genotypes. Assuming 18% overall mortality rate and percentage of high-risk homozygotes around 25% [2], the target of events would be achieved in a group of 570 patients. Estimation of sample sizes or effect sizes in survival functions were performed with chi-square test. Results The registry comprised 609 patients. Nine of them were lost to follow-up (1.5%) and genotype could not be determined in 11 remaining cases due to poor sample quality (1.8%). No genotyping discrepancies were observed in the samples genotyped twice. The final study group consisted of 589 patients: 25.3% of females (n = 149), mean age 62.4±11.9 years, TIMI 3 obtained in 92% of patients (n = 542). Genotyping results are presented in table 1. The genotype frequency distributions showed no significant deviations from Hardy-Weinberg equilibrium (rs1333049 SNP: p = 0.12; rs10757278: p = 0.21; rs4977574: p = 0.17). The specific allele frequencies are comparable to previous reports (2, 4, 5). According to genetic localisation data, the SNPs rs1333049, rs10757278 and rs4977574 are closely linked. Consequently, pairwise comparison using the exact test disequilibrium analysis yielded departures from independence for all pairs of loci (p<0.0001, table 2). Therefore we chose the rs4977574 SNP with the strongest effect based on literature [2], [4], [5], [6] to present clinical characteristics of the study group (G allele carriers vs. AA homozygotes, table 3). No significant differences were observed between the genotypes. 10.1371/journal.pone.0072333.t001Table 1 Percentages of specific genotypes and associated mortality rates. Polymorphism (risk allele) rs1333049 (C) rs10757278 (G) rs4977574(G) The whole study group (n = 589) Genotype CC CG GG GG AG AA GG AG AA Percentage (n) 24.8 (146) 46.7 (275) 28.5 (168) 24.2 (143) 47.4 (279) 28.3 (167) 24.1 (142) 47.0 (277) 28.9 (170) 5-year mortality (n) 14.4 (21) 20.0 (55) 13.1 (22) 15.4 (22) 19.4 (54) 13.2 (22) 16.2 (23) 19.5 (54) 12.4 (21) Subgroup of high-risk patients (Grace risk score ≥155, n = 238) Genotype CC+CG GG GG+AG AA GG+AG AA Percentage (n) 72.7 (173) 27.3 (65) 71.4 (170) 28.6 (68) 72.7 (173) 27.3 (65) 5-year mortality (n) 31.8 (55)1 13.8 (9) 31.8 (54)2 14.7 (10) 32.4 (56)3 12.3 (8) Subgroup of patients with 3-vessel disease (n = 122) Genotype CC+CG GG GG+AG AA GG+AG AA Percentage (n) 66.39 (81) 33.6 (41) 69.7 (85) 30.3 (37) 65.6 (80) 34.4 (42) 5-year mortality (n) 29.6 (24)* 12.2 (5) 29.4 (25)* 10.8 (4) 31.2 (25)* 9.5 (4) 1 p = 0.01; 2 p = 0.029; 3 p = 0.008; * p>0.05 compared to homozygotes; all p values adjusted for Bonferroni correction. 10.1371/journal.pone.0072333.t002Table 2 Linkage disequilibrium of investigated SNPs. p SNP1 SNP2 D′ LD r2 <0.0001 rs1333049 rs10757278 0.88 0.21 0.77 <0.0001 rs1333049 rs4977574 0.83 0.2 0.68 <0.0001 rs10757278 rs4977574 0.89 0.22 0.78 10.1371/journal.pone.0072333.t003Table 3 Baseline characteristics of the study group based on rs4977574 genotype (risk allele- G). Characteristic Overall population N = 589 rs4977574 G-allele carriers N = 419 rs4977574 AA homozygotes N = 170 P Age (years) 62.4 (12.0) 63.0 (11.7) 60.9 (12.6) 0.43 Female gender (%) 25.3 (n = 149) 25.3 (n = 106) 25.3 (n = 43) 0.99 Body mass index (kg/m2) 24.7 (3.7) 24.5 (3.8) 25.0 (3.5) 0.6 Hypertension (%) 53.3 (n = 314) 54.1 (n = 227) 51.2 (n = 87) 0.5 Type 2 diabetes (%) 22.1 (n = 130) 23.4 (n = 98) 18.8 (n = 32) 0.22 Previous myocardial infarction (%) 10.9 (n = 64) 11.5 (n = 48) 9.4 (n = 16) 0.47 Systolic blood pressure (mmHg) 138.7 (28.3) 138.3 (29.8) 139.8 (24.3) 0.88 Heart rate (beats/min) 75.7 (17.8) 76.2 (18.1) 74.7 (16.9) 0.26 Killip class III or IV (%) 4.1 (n = 24) 4.0 (n = 17) 4.1 (n = 7) 0.97 ST-elevation in anterior leads 39.4 (n = 232) 40.1 (n = 168) 37.6 (n = 64) 0.58 TIMI flow grade 3 after procedure 92.0 (n = 542) 91.6 (n = 384) 92.9 (n = 158) 0.59 Stent implantation (%) 77.1 (n = 454) 76.6 (n = 321) 78.2 (n = 133) 0.67 No of vessels with significant stenosis 1.7 (0.8) 1.7 (0.8) 1.8 (0.8) 0.12 eGFR (ml/min/1.73 m2) 79.9 (23.3) 79.1 (24.1) 82.0 (21.2) 0.51 Haemoglobin on admission (mg/dl) 13.05 (1.6) 13.0 (1.6) 13.1 (1.7) 0.26 Ejection fraction (%) 45.9 (9.5) 45.2 (9.0) 45.9 (9.5) 0.26 Grace risk score 149 (35) 151 (35) 145 (34) 0.09 eGFR- estimated GFR, mean values with standard deviations are given, unless otherwise specified. A 5-year follow-up with a median of 1950 days (minimum 1824, maximum 3378 days) was performed. At the cut-off point of 1825 days (5 years) 98 patients died (16.6%). When all the study group was analyzed, no significant differences in mortality were found between the genotypes (table 1). Figure 1 presents Kaplan-Meier surviving curves for specific genotypes of rs4977574 polymorphism and 5-year mortality (p = 0.6 after adjustment for Bonferroni correction, log-rank test). 10.1371/journal.pone.0072333.g001Figure 1 The rs4977574 polymorphism and 5-year survival. No significant differences were observed between the genotypes. However, in the subgroup of high-risk patients (GRACE risk score ≥155, n = 238, 26.9% mortality [n = 64]), visual analysis of survival curves showed strikingly better survival of low-risk homozygotes compared to both other genotypes. The curves for heterozygotes and high-risk homozygotes had almost similar course and therefore were collapsed together for further analysis. Kaplan-Meier surviving curves for the subgroup of high-risk patients and rs4977574 polymorphism are shown in figure 2. The difference was statistically significant (p = 0.008 after adjustment for Bonferroni correction, log-rank test). No such trend was observed in low and medium-risk patients (p>0.99 after adjustment for Bonferroni correction, log-rank test, figure 3). Mortality rates for specific genotypes in the subgroup of high-risk patients are shown in table 1. 10.1371/journal.pone.0072333.g002Figure 2 The rs4977574 polymorphism and 5-year survival - subgroup of high-risk patients (Grace risk score ≥155). AA low-risk homozygotes had significantly higher probability of survival compared to other genotypes (p = 0.008 after adjustment for Bonferroni correction, log-rank test). 10.1371/journal.pone.0072333.g003Figure 3 The rs4977574 polymorphism and 5-year survival – medium and low-risk patients (Grace risk score <155). No significant differences were observed between the genotypes. Table 4 presents results of Cox proportional hazards model for 5-year survival in a subgroup of high-risk patients. The hazard ratio associated with high-risk genotype (high-risk homozygote or heterozygote) was: HR = 2.9 (95%CI 1.4–6.1) for the rs4977574 polymorphism, HR = 2.6 (1.25–5.3) for the rs1333049 one and HR = 2.35 (1.2–4.6) for the rs10757278 one. In a univariate analysis, apart from 3 investigated polymorphism, significant association was found in the case of Killip class, ejection fraction and type 2 diabetes. In multivariate regression the rs4977574 polymorphism remained related to mortality together with ejection fraction and type 2 diabetes. After adjustment for severity of coronary artery disease all p values were still below 0.05. 10.1371/journal.pone.0072333.t004Table 4 Univariate and multivariate analysis for 5-year mortality in a subgroup of high-risk patients (Grace risk score ≥155). Variable Hazard ratio 95% CI p Killip class 1.4 1.09–1.7 0.01 Ejection fraction (%) 0.95 0.93–0.98 0.0002 Type 2 diabetes 2.1 1.3–3.5 0.006 rs1333049 CC or CG genotype 2.6 (2.3) 1.25–5.3 (1.2–4.5) 0.004 (0.014) rs10757278 GG or AG genotype 2.35 (2.2) 1.2–4.6 (1.1–4.2) 0.006 (0.017) rs4977574 GG or AG genotype 2.9 (2.65) 1.4–6.1 (1.3–5.4) 0.004 (0.006) No of vessels with significant stenosis 0.98 0.9–1.06 0.66 Multivariate analysis Ejection fraction (%) 0.95 (0.96) 0.93–0.98 (0.94–0.99) 0.001 (0.01) Type 2 diabetes 1.9 (1.97) 1.1–3.2 (1.2–3.3) 0.014 (0.008) rs4977574 GG or AG genotype 2.7 (2.46) 1.3–5.7 (1.2–5.0) 0.009 (0.01) In the brackets values adjusted for severity of coronary artery disease (number of vessels with significant stenosis) are given. Next, similar trends between genotypes and survival were found in patients with 3-vessel disease (n = 122, Table 1). Additional analysis was done using Cox proportional hazard model that showed following hazard ratios associated with high-risk genotype: HR = 3.65 (95%CI 1.3–10.5) for the rs4977574 polymorphism, HR = 2.6 (1.002–6.9) for the rs1333049 one and HR = 3.0 (1.03–8.5) for the rs10757278 one. Discussion The 9p21.3 locus showed a significant association with 5-year survival of high-risk patients with STEMI (GRACE risk score ≥155 points or 3-vessel disease). This phenomenon is probably related to an increased number of events in the chosen subgroups. On the other hand, the 9p21.3 locus may influence atherosclerotic plaque development, stability, and, in this way, survival [15]. Such effect would be more pronounced in patients with advanced coronary atherosclerosis. It was already reported that 9p21 locus is associated with mortality in patients with multivessel coronary artery disease [26]. Another patomechanism that needs to be considered is sudden cardiac death due to probable arrhythmia that was shown in a prospective cohort [27]. Based on multivariate analysis, 9p21 locus polymorphism added prognostic information to previously established risk factors like ejection fraction and type 2 diabetes. This report supports results from the GRACE registry, that was also performed in European populations and showed association between the rs1333049 polymorphism and outcome within 6 months [21]. Our findings extend this effect to 5 years, however, they are limited to high-risk patients. Another difference is study population (patients with all forms of an acute coronary syndrome in the GRACE registry vs. the ones with STEMI). We observed remarkably high effect sizes for the association between high-risk genotypes and 5-year outcome. It makes these novel risk markers potentially more applicable in everyday practice. Furthermore, if only a particular subgroup of patients was to be genotyped, the method would be more cost-effective. The future interventional studies where additional therapeutic actions are applied in high-risk clinical and genetic profile would help translate this effect into clinical practice. The results from European registries are not in concordance with studies performed in other populations [22], [23]. The effect sizes of specific genotypes may be strongly related to genetic background as well as environmental factors. Therefore it is essential to validate results of genetic studies in regional populations. In Han Chinese patients with STEMI no differences were found between the 9p21 locus genotypes in 2-year event-free survival (cardiac death, non-fatal MI, recurrent angina or heart failure requiring hospitalization) [23]. Similarly, patients from Post-Myocardial Infarction Study (New Zealand, 9-year follow-up) had genotype-dependent variation neither in total mortality nor in hospital admissions due to reinfarctions or heart failure [22]. There is an Italian study that investigates closely linked polymorphism: rs1333040 [28]. Included participants had early-onset myocardial infarction (<45 years) and underwent coronary angiography without index event coronary revascularization. During 10-year follow-up the genotype significantly affected risk of coronary revascularization, but no influence on cardiac death or recurrent myocardial infarctions was observed. In general, the rate of events was low due to early age (5.1%, n = 77) and possibly therefore no effect on survival was shown. We have found no association between the 9p21.3 locus and either participants' age or severity of coronary artery disease, which is again consistent with the GRACE registry [21]. On the contrary, in the study from New Zealand high-risk homozygotes (rs1333049 SNP) were significantly younger, compared to other genotypes (60.6 vs. 62.8 years, p = 0.009) [22]. The report from the United States of America showed that the rs10757278 SNP influenced angiographic severity and progression of coronary artery disease [29]. Finally, in Chinese population the rs1333049 SNP contributed to severity of coronary artery disease, but only in diabetics [30]. The discrepancies might be again explained by different genetic effect sizes depending on a chosen population. It is surprising that no significant deviation from Hardy-Weinberg equilibrium was shown in our population. However, the study was not designed to prove the association between the 3 SNPs and myocardial infarction. Such analysis would require remarkably larger sample sizes. The number of patients investigated in this study was relatively low, but in our opinion it was large enough to search for associations of clinical importance. Very large study groups enable finding significant correlations of very small effect sizes that have no further meaning in everyday practice. The further limitation of the study is a retrospective type of analysis, however, the data was collected prospectively. Conclusions The 9p21.3 locus was associated with 5-year mortality in high-risk patients with STEMI. This finding, due to very high effect size, could potentially be applied into clinical practice. However, optimal set of risk genotypes and appropriate tools for this clinical setting are still to be identified and elaborated. ==== Refs References 1 Burton PR , Clayton DG , Cardon LR , Craddock N , Deloukas P , et al (2007 ) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls . Nature 447 : 661 –78 .17554300 2 Samani NJ , Erdmann J , Hall AS , Hengstenberg C , Mangino M , et al (2007 ) Genome-wide association analysis of coronary artery disease . N Engl J Med 357 : 443 –53 .17634449 3 Koch W , Türk S , Erl A , Hoppmann P , Pfeufer A , et al (2011 ) The chromosome 9p21 region and myocardial infarction in a European population . Atherosclerosis 217 : 220 –6 .21511257 4 Helgadottir A , Thorleifsson G , Manolescu A , Gretarsdottir S , Blondal T , et al (2007 ) A Common Variant on Chromosome 9p21 Affects the Risk of Myocardial Infarction . Science 316 : 1491 –93 .17478679 5 Kathiresan S , Voight BF , Purcell S , Musunuru K , Ardissino D , et al (2009 ) Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy-number variants . Nat Genet 41 : 334 –41 .19198609 6 O'Donnell CJ , Kavousi M , Smith AV , Kardia SL , Feitosa MF , et al (2011 ) Genome-wide association study for coronary artery calcification with follow-up in myocardial infarction . Circulation 124 : 2855 –64 .22144573 7 Takeuchi F , Yokota M , Yamamoto K , Nakashima E , Katsuya T , et al (2012 ) Genome-wide association study of coronary artery disease in the Japanese . Eur J Hum Genet 20 : 333 –40 .21971053 8 Lee JY , Lee BS , Shin DJ , Woo Park K , Shin YA , et al (2013 ) A genome-wide association study of a coronary artery disease risk variant . J Hum Genet 58 : 120 –6 .23364394 9 Butterworth AS , Braund PS , Farrall M , Hardwick RJ , Saleheen D , et al (2011 ) Large-scale gene-centric analysis identifies novel variants for coronary artery disease . PLoS Genet 7 : e1002260 .21966275 10 Scheffold T , Kullmann S , Huge A , Binner P , Ochs HR , et al (2011 ) Six sequence variants on chromosome 9p21.3 are associated with a positive family history of myocardial infarction: a multicenter registry . BMC Cardiovasc Disord 11 : 9 .21385355 11 Lyssenko V , Burtt NP , de Bakker PI , Chen H , Roix JJ , et al (2007 ) Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels . Science 316 : 1331 –6 .17463246 12 Zeggini E , Weedon MN , Lindgren CM , Frayling TM , Elliott KS , et al (2007 ) Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes . Science 316 : 1336 –41 .17463249 13 Scott LJ , Mohlke KL , Bonnycastle LL , Willer CJ , Li Y , et al (2007 ) A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants . Science 316 : 1341 –5 .17463248 14 Cugino D , Gianfagna F , Santimone I , de Gaetano G , Donati MB , et al (2012 ) Type 2 diabetes and polymorphisms on chromosome 9p21: A meta-analysis . Nutr Metab Cardiovasc Dis 22 : 619 –25 .21315566 15 Nambi V , Boerwinkle E , Lawson K , Brautbar A , Chambless L , et al (2012 ) The 9p21 genetic variant is additive to carotid intima media thickness and plaque in improving coronary heart disease risk prediction in white participants of the Atherosclerosis Risk in Communities (ARIC) Study . Atherosclerosis 222 : 135 –7 .22349088 16 Caldas C , Hahn SA , da Costa LT , Redston MS , Schutte M , et al (1994 ) Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma . Nat Genet 8 : 27 –32 .7726912 17 Cannon-Albright LA , Goldgar DE , Meyer LJ , Lewis CM , Anderson DE , et al (1992 ) Assignment of a locus for familial melanoma, MLM, to chromosome 9p13–p22 . Science 258 : 1148 –52 .1439824 18 Kalinina N , Agrotis A , Antropova Y , Ilyinskaya O , Smirnov V , et al (2004 ) Smad expression in human atherosclerotic lesions: evidence for impaired TGF-b/Smad signaling in smooth muscle cells of fibrofatty lesions. Arterioscler . Thromb Vasc Biol 24 : 1391n6 . 19 Schmid M , Sen M , Rosenbach MD , Carrera CJ , Friedman H , et al (2000 ) A methylthioadenosine phosphorylase (MTAP) fusion transcript identifies a new gene on chromosome 9p21 that is frequently deleted in cancer . Oncogene 19 : 5747 –54 .11126361 20 Zeller T , Wild P , Szymczak S , Rotival M , Schillert A , et al (2010 ) Genetics and beyond–the transcriptome of human monocytes and disease susceptibility . PLoS One 5 : e10693 .20502693 21 Buysschaert I , Carruthers KF , Dunbar DR , Peuteman G , Rietzschel E , et al (2010 ) A variant at chromosome 9p21 is associated with recurrent myocardial infarction and cardiac death after acute coronary syndrome: The GRACE Genetics Study . Eur Heart J 31 : 1132 –41 .20231156 22 Ellis KL , Pilbrow AP , Frampton CM , Doughty RN , Whalley GA , et al (2010 ) A common variant at chromosome 9P21.3 is associated with age of onset of coronary disease but not subsequent mortality . Circ Cardiovasc Genet 3 : 286 –93 .20400779 23 Peng WH , Lu L , Zhang Q , Zhang RY , Wang LJ , et al (2009 ) Chromosome 9p21 polymorphism is associated with myocardial infarction but not with clinical outcome in Han Chinese . Clin Chem Lab Med 47 : 917 –22 .19548844 24 Granger CB , Goldberg RJ , Dabbous O , Pieper KS , Eagle KA , et al (2003 ) Global Registry of Acute Coronary Events Investigators. Predictors of hospital mortality in the Global Registry of Acute Coronary Events . Arch Intern Med 163 : 2345 –2353 .14581255 25 Global Registry of Acute Coronary Events website. Available: http://www.outcomes-umassmed.org/GRACE/grace_risk_table.aspx. Accessed: 25 Jul 2013. 26 Gioli-Pereira L , Santos PC , Ferreira NE , Hueb WA , Krieger JE , et al (2012 ) Higher incidence of death in multi-vessel coronary artery disease patients associated with polymorphisms in chromosome 9p21 . BMC Cardiovasc Disord 12 : 61 .22856518 27 Newton-Cheh C , Cook NR , VanDenburgh M , Rimm EB , Ridker PM , et al (2009 ) A common variant at 9p21 is associated with sudden and arrhythmic cardiac death . Circulation 120 : 2062 –8 .19901189 28 Ardissino D , Berzuini C , Merlini PA , Mannuccio Mannucci P , Surti A , et al (2011 ) Investigators. Influence of 9p21.3 genetic variants on clinical and angiographic outcomes in early-onset myocardial infarction . J Am Coll Cardiol 58 : 426 –34 .21757122 29 Patel RS , Su S , Neeland IJ , Ahuja A , Veledar E , et al (2010 ) The chromosome 9p21 risk locus is associated with angiographic severity and progression of coronary artery disease . Eur Heart J 31 : 3017 –24 .20729229 30 Wang W , Peng WH , Lu L , Zhang RY , Zhang Q , et al (2011 ) Polymorphism on chromosome 9p21.3 contributes to early-onset and severity of coronary artery disease in non-diabetic and type 2 diabetic patients . Chin Med J 124 : 66 –71 .21362310	24069144	PMC3772090	CC BY	2021-01-05 17:47:15	yes	PLoS One. 2013 Sep 12; 8(9):e72333
==== Front Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2013/932391Review ArticlePreventative and Therapeutic Probiotic Use in Allergic Skin Conditions: Experimental and Clinical Findings Özdemir Öner 1 Göksu Erol Azize Yasemin 2 1Department of Pediatrics, Division of Allergy and Immunology, Research and Training Hospital of Sakarya University, Faculty of Medicine, Sakarya University, Adnan Menderes Caddesi, Sağlık Sokak No. 195, Adapazarı, 54100 Sakarya, Turkey2Department of Histology and Embryology, Faculty of Medicine, Afyon Kocatepe University, 03200 Afyonkarahisar, TurkeyÖner Özdemir: ozdemir_oner@hotmail.comAcademic Editor: Ibrahim Banat 2013 1 9 2013 2013 93239121 4 2013 18 7 2013 Copyright © 2013 Ö. Özdemir and A. Y. Göksu Erol.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Probiotics are ingested live microbes that can modify intestinal microbial populations in a way that benefits the host. The interest in probiotic preventative/therapeutic potential in allergic diseases stemmed from the fact that probiotics have been shown to improve intestinal dysbiosis and permeability and to reduce inflammatory cytokines in human and murine experimental models. Enhanced presence of probiotic bacteria in the intestinal microbiota is found to correlate with protection against allergy. Therefore, many studies have been recently designed to examine the efficacy of probiotics, but the literature on the allergic skin disorders is still very scarce. Here, our objective is to summarize and evaluate the available knowledge from randomized or nonrandomized controlled trials of probiotic use in allergic skin conditions. Clinical improvement especially in IgE-sensitized eczema and experimental models such as atopic dermatitis-like lesions (trinitrochlorobenzene and picryl chloride sensitizations) and allergic contact dermatitis (dinitrofluorobenzene sensitization) has been reported. Although there is a very promising evidence to recommend the addition of probiotics into foods, probiotics do not have a proven role in the prevention or the therapy of allergic skin disorders. Thus, being aware of possible measures, such as probiotics use, to prevent/heal atopic diseases is essential for the practicing allergy specialist. ==== Body 1. Background The interest in probiotic preventative/therapeutic potential in allergic disorders stemmed from the fact that probiotics have been shown to improve intestinal dysbiosis and permeability and to reduce inflammatory cytokines. Such effects would be desirable in treating allergic disorders including atopic dermatitis (AD). Therefore, several studies have been recently designed to examine the efficacy of probiotics in many allergic conditions, such as eczema and food allergies [1, 2]. Especially, the literature on the clinical probiotic use in other skin allergy reactions of human is very scarce. Therefore, this paper will mostly have to discuss the literature on the preventative and/or prophylactic role of probiotic use in AD. 1.1. Clinical and Experimental Essentials of Preventative and Therapeutic Probiotic Use in Allergic Skin Conditions Including the first publication in 1997, over 40 randomized, double-blind, and placebo-controlled clinical trials were conducted to study the effects of various probiotics on treatment and prevention of allergic diseases. In total, more than 3000 individuals (including those in placebo groups) have participated in these studies so far. In the first-time study done by Majamaa and Isolauri in 1997, the administration of Lactobacillus (Lctbs) rhamnosus GG (LGG) to highly selected patients (age < 2 years, challenge-proven cow's milk allergy, and mild-to-moderate eczema) significantly improved the total scoring of AD severity index (SCORAD) score [3]. Later, the Finnish study of Kalliomäki et al. was the first report to describe that the frequency of AD in neonates treated with LGG was half that of the placebo [4]. However, these results have been lately questioned by other trials, which reported no difference in the development and therapy of AD in neonates supplemented with LGG or other probiotics. Therefore, an allergy preventative or therapeutic effect of probiotics in AD and allergic skin conditions could not be consistently established. The aim of this paper is to characterize current knowledge of probiotic use in skin allergy reactions, including their preventative/therapeutic role in AD. As briefly mentioned above, there are good (animal) experimental and (human) clinical theoretical bases for using probiotics in the prevention and therapy of allergic skin conditions such as AD [5]. Germ-free animal models demonstrate that bacterial gut colonization is essential for maturation of immune function and induction of oral tolerance [6]. It has been proposed that a similar but a more subtle process may be occurring in human beings with progressively cleaner environments. Probiotic intestinal flora is arguably the most abundant source of early immune stimulation and contributes significantly to microbial burden in early life. A number of studies have suggested differences in the early colonization patterns of infants who go on to develop allergic diseases. These studies strongly suggest that the pattern of colonization in the first weeks of life may influence the patterns of allergic disease development [7, 8]. These notions have been supported by observations that gut flora can influence local and systemic immune responses. There has been speculation that intestinal flora may influence the maturing precursor cells that circulate through the gut before they home to other tissues. This may explain how probiotic species can influence systemic immune responses and immunoglobulin (Ig) A production in distal sites, such as the respiratory tract. Thus, certain probiotics seem to influence the gut's allergen-stimulated inflammatory response and provide a barrier effect against antigens that might otherwise ultimately lead to systemic allergic symptoms (such as eczema). Together with reported clinical effects in early allergic disease, this has logically led to a growing interest in the role of probiotics in allergy prevention [1, 2]. 1.2. Allergic Skin Conditions (Reactions) and Atopic Dermatitis (Eczema) The literature on probiotic use in allergic skin reactions mainly includes experiments in AD (human and animal), AD-like skin lesions, and allergic contact dermatitis in animal experiments. And AD can be accepted as a prototypic disease for skin allergy reactions. AD is the most common chronic skin allergy reaction in children and adults, with a prevalence of 10% to 20% in population. Geographic location affects the prevalence of this disease, with the highest prevalence in the USA and Europe [9]. Important factors in the susceptibility to develop AD include a genetic basis and environmental factors. Eczema refers to a chronic or relapsing itchy skin inflammation with typical lesions and locations. Eczema is called atopic if it is associated with IgE demonstrated by either positive skin prick tests or elevated antigen-specific IgE antibodies. The term atopy refers to a genetic predisposition to become sensitized and to mount an IgE response to allergens. AD has been linked to food hypersensitivity, especially milk and egg proteins. However, 40%–60% of children with AD may not develop IgE sensitization [10]. The term eczema has been recently proposed, but, for practical purposes, both AD and eczema will be used in this paper. There have been several proposed methods for classifying the severity of AD in various research studies mentioned in this paper, but only the SCORAD, established by the European Task Force on AD, has been validated for reproducibility and accuracy in assessing therapeutic response [9, 10]. The SCORAD combines objective measures, such as extent and severity of skin lesions, and subjective criteria, such as pruritus and sleep loss. Children with AD can be further classified as having mild, (≤25); moderate, (25–50); or severe, (≥50) disease based on their SCORAD scores. 1.3. What Are Probiotics? Year 2013 marks the 106 year since Metchnikoff suggested that the consumption of lactic acid bacteria (LAB) may benefit the human host's immune system [55]. However, not until the mid 1960s did the term probiotic become the trend. The term probiotics means “for life” and is defined by the World Health Organization and the Food and Agriculture Organization of the United Nations as “live microorganisms which, when administered in adequate amounts as part of food, confer a beneficial health effect by producing gut microflora on the host.” These probiotics are mainly represented by LAB [56]. Simply, probiotics are ingested live microbes that can modify intestinal microbial populations in a way that benefits the host. Probiotic intestinal flora contributes to microbial antigen exposure in early life and is one of the most abundant sources of early immune stimulation. Because allergic immune responses manifest early in life, there has been obvious interest in the potential benefits of modifying the gastrointestinal flora by using probiotic supplementation. However, the value of probiotics for primary prevention of these diseases is controversial [1, 2]. So far, there have been only several studies to address the role of probiotics in primary prevention of allergic skin conditions, with a reported suspicious reduction in the incidence of eczema. Since the role of probiotics in allergy prevention has remained controversial and there has been an urgent call for similar studies to address this further, this paper will try to highlight the role of probiotics in the therapy/prevention of allergic skin reactions and the future of this therapy. 2. Mechanisms of Probiotics' Effects in Allergic Skin Conditions Although the beneficial effects of probiotics on wide variety of atopic diseases have been suggested, little is known about how probiotics modulate the immune system, atopic disease development, and skin allergy reactions. Currently, only limited publications are available defining the effects of probiotics in murine or human models of AD and skin allergy reactions. Therefore, it is important to explore the effects of probiotics in these models [57]. In this section, experimental (animal) models and clinical studies showing mechanisms of probiotics' effects in skin allergy reactions and AD are being discussed [8, 58]. 2.1. Maturing Gut Barrier: Probiotic Regulation in Intestinal Epithelium and Upregulation of Host Immune Responses Recent data indicate that commensal intestinal microbiota represents a major modulator of intestinal homeostasis. Dysregulation of the symbiotic interaction between the intestinal microbiota and the mucosa may result in a pathological condition with potential clinical repercussions. For instance, it is shown that mice reared in germ-free conditions have underdeveloped immune systems and have no oral tolerance [6]. In contrast, pathogen-free mice are capable of reconstituting the bacterial flora with Bifidobacteria and tolerance development [59]. In addition to providing maturational signals for the gut-associated lymphoid tissue, probiotics balance the generation of pro- and anti-inflammatory cytokines in the gut. Some components of heat-treated LGG may have an ability to delay the onset and suppress the development of AD in NC/Nga mice, probably through a strong induction of IL-10 in intestinal lymphoid organs and systemic levels [14]. After probiotic consumption, decrease in fecal α-1 antitrypsin and serum TNF-α and changes in TGF-β and other cytokines point to downregulation of inflammatory mediators [18]. For instance, after a challenge study in infants allergic to cow's milk, fecal IgA levels were detected to be higher, and serum TNF-α levels were lower in the LGG-applied group compared with the placebo [32]. Similarly, another study by Kirjavainen et al. suggested that Bfdbm lactis Bb12 might modify gut microflora to alleviate early onset atopic eczema. And this modification was found to be compatible with reductions of serum TNF-α and fecal α-1-antitrypsin levels as well as an increase in fecal IgA level [60]. Moreover, probiotic bacteria may counteract the inflammatory process by stabilizing the gut microbial environment and the permeability barrier of the intestine, and by enhancing the degradation of enteral antigens and altering their immunogenicity [61]. This gut-stabilizing effect of probiotics could be explained by the improvement by probiotics of the immunological barrier of the intestine through intestinal IgA responses; see specifically [33, 62]. Oral treatment with probiotic Lctbs johnsonii NCC533 (La1) for a specific part of the weaning period was also shown to prevent the development of AD in model mice, NC/Nga, by modulating or accelerating the gut immune response with increased intestinal secretory IgA [63]. Consistent with these explanations, in children with food allergies, probiotics are shown to reverse increased intestinal permeability and to enhance frequently defective IgA responses [32, 64]. 2.2. Immunomodulation: Th1/Th2 Balance, IgE Production, and Cytokines In addition to maturing gut barrier, certain strains of Lactobacilli and Bifidobacteria modulate the production of cytokines by monocytes and lymphocytes and may divert the immune system in a regulatory or tolerant mode [59, 65]. Nonetheless, there are still some studies showing no significant effects of probiotics on either Th1 or Th2 cell responses to allergens. Although the cytokine stimulation profiles of different probiotic strains vary, the strains isolated from healthy infants mainly stimulate noninflammatory cytokines [66]. Therefore, it seems that changes in cytokine profiles induced by probiotics may be probiotic strain or site specific and dependent on the experimental system used. For instance, Lctbs reuteri induced proinflammatory and Th1 cytokines; Bfdbm bifidum/infantis and Lctbs lactis reduced Th2 cytokines [67]. Oral administration of LAB isolated from the traditional South Asian fermented milk “dahi” inhibits the development of AD in NC/Nga mice as well. Of the 41 strains tested from “dahi”, Lctbs delbrueckii subsp. lactis R-037 exhibited the greatest IL-12 induction, suggesting that it is a potent Th1 inducer [11]. Also, the antiallergic effects of one strain (T120) of LAB isolated from the Mongolian fermented milk using AD model mice (NC/Nga mice) were investigated. Strain T120 has already been identified as Enterococcus faecium, suppressed total IgE production, and induced IL-12 and IFN-γ production by splenocytes of NC/Nga mice. Furthermore, this strain enhanced the production of IL-10 by splenocytes, and activation of T regulatory (Treg) cells by strain T120 may inhibit atopic disease. In in vivo studies, intraperitoneal injection of strain T120 inhibited serum IgE elevation and AD symptoms in NC/Nga mice [12]. In another study, Lctbs plantarum strains from Kimchi were demonstrated to inhibit AD (house-dust mite-induced dermatitis) in NC/Nga mice. The three strains, CJLP55, CJLP133, and CJLP136, suppressed AD-like skin lesions and epidermal thickening. These same three strains decreased Th2 cytokines production such as IL-4 and IL-5 in lymph node cell cultures. The latter two, CJLP133 and CJLP136, increased IFN-γ secretion, while CJLP55 enhanced IL-10 production. These findings suggest that Lactobacilli isolated from Kimchi inhibit AD, probably by altering the balance of Th1/Th2 ratio or by inducing IL-10 production [68]. Similarly, Lctbs acidophilus strain L-55 suppressed the development of AD-like skin lesions induced by repeated application of TNCB in sensitized NC/Nga mice via a decrease in the serum total IgE level [69]. AD-like skin lesions were induced by sensitization to and repeated challenges with picrylchloride in the Th2-skewed NC/Nga mice strains. A new synbiotic, Lctbs casei subsp. casei together with dextran, reduces murine allergic reaction such as the development of AD-like skin lesions in NC/Nga mice. This synbiotic combination significantly decreased clinical skin severity scores induced by picryl chloride and total IgE levels in sera of NC/Nga mice [15]. Also, supplementation with KW3110 strain of LAB significantly attenuated the onset and exacerbation of AD-like skin lesions, accompanied by less mast cell infiltration and lower plasma IgE levels through its effects on IL-12 and IL-4 production in vitro [21]. Furthermore, oral administration of heat-killed Lctbs brevis SBC8803 ameliorates the development of dermatitis in AD model of NC/Nga mice. Eight-week-old male NC/Nga mice were sensitized by the topical application of picryl chloride to foot pads and shaved abdomens. Oral administration of Lctbs brevis SBC8803 significantly inhibited IgE production and ear swelling and suppressed the development of dermatitis in a dose-dependent manner. Immunosuppressive cytokines such as IL-10 and TGF-β production from Peyer's patch cells significantly increased in the treatment group, compared with the control group [22]. Consistently, oral supplementation with Lctbs rhamnosus CGMCC 1.3724 (LPR) in a study by Tanaka et al. has been demonstrated to prevent development of AD in NC/NgaTnd mice possibly by modulating local production of IFN-γ and plasma total IgE in skin biopsies, compared with untreated mice [19]. A decrease in the secretion of proinflammatory cytokines, IFN-γ, TNF-α, and IL-12, has been demonstrated. Consistently, in an experimental study, probiotic supplementation decreased the severity of allergic skin responses in allergen-sensitized pigs with a corresponding increase in IFN-γ expression [70]. Similarly, Pohjavuori et al. were able to demonstrate an increase of IFN-γ production in peripheral blood mononuclear cell in infants with AD treated with LGG instead of placebo [71]. Additionally, the improvement in AD severity in very young children with probiotic treatment was detected to be associated with significant increases in the capacity for Th1 IFN-γ responses and altered responses to skin and enteric flora. This effect was still evident 2 months after the supplementation was ceased [72]. Twelve human studies were included in a review, and 67% showed a positive association with TGF-β1 or TGF-β2 demonstrating protection against allergy-related outcomes in infancy and early childhood. High variability in concentrations of TGF-β was noted between and within studies, with some of it explained by maternal history of atopy or by consumption of probiotics. Human milk TGF-β appears to be essential in developing and maintaining appropriate immune responses in infants and may provide protection against adverse immunological outcomes, corroborating findings from experimental animal studies. In a study, the aim was to evaluate the effect of probiotic supplementation on the immunological composition of breast milk and colostrum in relation to sensitization and eczema in babies. Supplementation of probiotics during pregnancy was associated with low levels of TGF-β2 and slightly increased levels of IL-10 in colostrum. Infants receiving breast milk with low levels of TGF-β2 were less likely to become sensitized, and it was likely to find possibly less IgE-associated eczema in breast-fed infants during their first 2 years of life [44]. However, another trial by Boyle et al. showed that LGG treatment during pregnancy (prenatal) for the prevention of eczema was associated with decreased breast milk soluble CD14 and IgA levels, not TGF-β [47]. The difference between these studies depends on probiotic species, which may affect the immunological composition of breast milk. 2.3. Anti-Inflammatory Effects: Their Effects on Serum Inflammatory Parameters The anti-inflammatory effect of probiotics has been attributed to increased production of IL-10 by immune cells in the lamina propria, Peyer's patches, and the spleen of treated animals [66, 67, 73, 74]. Oral administration of LGG resulted in elevated IL-10 concentrations in atopic children, indicating that specific probiotics may have anti-inflammatory effects in vivo and may possibly enhance regulatory or tolerance-inducing mechanisms as well. In a review of the evidence from randomized controlled trial (RCTs) by Betsi et al. about probiotics for the treatment or prevention of AD, the results of 13 relevant randomized (placebo)-controlled trials were reviewed: 10 of which evaluated probiotics as treatment and 3 for prevention of AD. In four of those RCTs, clinical improvement was associated with a change in some inflammatory markers [75]. A study by Woo et al. evaluated the effect of Lctbs sakei supplementation in children with atopic eczema-dermatitis syndrome (AEDS). In this study, compared with placebo, probiotic administration was associated with lower pretreatment-adjusted serum levels of chemokines such as CCL17 and CCL27, which were significantly correlated with SCORAD total score [36]. Probiotic-induced chronic low-grade inflammation characterized by elevation of CRP, IgE, IgA, and IL-10 was shown in some studies, with the changes being typically observed in helminth infection-associated induction of regulatory mechanisms. The association of increased CRP with a decreased risk of eczema at 2 years of age in allergy-prone children supports the view that chronic, low-grade inflammation protects from eczema. The findings emphasize the role of chronic microbial exposure as an immune modulator protecting from allergy [40]. Primary administration of Lctbs johnsonii NCC533 (La1) in the weaning period suppressed the elevation of proinflammatory cytokines and CD86 gene expression levels in skin lesions of NC/Nga model mice. The suppression of proinflammatory cytokines such as IL-8/-12/-23 and CD86 expression by primary administration of La1 may significantly contribute to the inhibitory effects on the skin lesions like AD [76]. In a study by Rosenfeldt et al., 2 probiotic Lctbs strains (lyophilized Lctbs rhamnosus 19070-2 and Lctbs reuteri DSM 122460) were given in combination for 6 weeks to 1- to 13-year-old children with AD. During active treatment, serum eosinophil cationic protein (ECP) levels significantly decreased. A combination of Lctbs rhamnosus and Lctbs reuteri was beneficial in the management of AD, and the effect was more pronounced in atopic eczema patients [27]. Another study that was conducted by Brouwer et al. showed, during Lctbs species supplementation, that a moderate but significant reduction in soluble ECP levels was found. ECP, a cytotoxic protein released from activated eosinophils, has been used to monitor disease activity in AD. Thus, soluble ECP might be a more sensitive marker in acute exacerbations of the eczema than a marker of disease activity per se [52]. 2.4. Development of Tolerogenic Dendritic Cells Selected species of the Bfdbm genus were demonstrated to prime in vitro cultured neonatal dendritic cells (DCs) to polarize T cell responses and may, therefore, be used as candidates in primary prevention of allergic diseases. Bfdbm bifidum was found to be the most potent polarizer in in vitro-cultured DCs to drive Th1-cell responses involving increased IFN-γ-producing T-cells concomitant with reduction of IL-4-producing T-cells [77]. In addition, T-cells stimulated by Bfdbm bifidum matured DCs as producers of more IL-10 [78]. Moreover, Lctbs rhamnosus, a member of another genus of probiotic bacteria, modulates DCs functions to induce a novel form of T-cell hyporesponsiveness [79]. Lctbs reuteri/casei have been also shown to prime monocyte-derived DCs through the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) to drive the development of Treg cells [80]. These Treg cells produce increased levels of IL-10 and are capable of inhibiting the proliferation of bystander T-cells. This study suggests that the targeting of DC-SIGN by certain probiotic bacteria might explain their beneficial effect in the treatment of a number of inflammatory diseases, including AD. 2.5. Immunoregulation by T Regulatory (Treg) Cells Generation of CD4+/Foxp3+ Treg cells by probiotics administration suppresses immune and allergic disorders. Recently, two studies reported that oral administration of a certain probiotic strain could increase Foxp3+ Tregs [81]. It is known that the lower percentage of epidermal or dermal Foxp3+ cells in eczematous dermatitis might contribute to their pathogenesis [82]. The strain T120 of LAB was shown to be able to inhibit atopic disease in NC/Nga mice through enhanced production of IL-10 by splenocytes and activation of Treg cells [12]. In a recent study, a mixture of probiotics (Lctbs acidophilus, Lctbs casei, Lctbs reuteri, Bfdbm bifidum, and Streptococcus thermophilus) was identified, and it upregulates CD4+/Foxp3+ Treg cells. Administration of the probiotics mixture induced both T-cells and B-cells hyporesponsiveness and downregulated Th1, Th2, and Th17 cytokines [81, 83]. It also induced generation of CD4+/Foxp3+-Tregs from the CD4+/25 population and increased the suppressor activity of naturally occurring CD4+/25+-Tregs. Conversion of T cells into Foxp3+ Tregs is directly mediated by regulatory DCs that express high levels of IL-10 and TGF-β. In a murine AD model, treatment with this probiotic mixture significantly inhibited the clinical symptoms of AD progression by reducing IgE levels (total and specific IgE levels), infiltrated lymphocytes and granulocytes, and levels of AD-associated cytokines [81]. Lctbs casei treatment enhanced the frequency of FoxP3+-Tregs in the skin and increased the production of IL-10 by CD4+/25+-Tregs cells in skin-draining lymph nodes of hapten-sensitized mice. These data demonstrate that orally administered Lctbs casei efficiently alleviate T-cell-mediated skin inflammation without causing immune suppression, via mechanisms that include control of CD8+-effector T-cells and involve regulatory CD4+-T-cells. Lctbs casei may, thus, represent a probiotic of potential interest for immunomodulation of T-cell-mediated allergic skin diseases in human beings [25]. In sensitized BALB/c mice, skin inflammation was induced by topical allergen application. Escherichia coli Nissle 1917 was administered orally in a preventative manner. Oral Escherichia coli Nissle administration improved allergen-induced dermatitis dose dependently. In parallel, a reduction of epidermal thickness and infiltrating immune cells together with an enhanced number of Foxp3 (+) cells and a trend of increased IFN-γ, IL-10, and TGF-β expression levels was detected in eczematous skin. Our findings indicate that Escherichia coli Nissle alters the local allergen-induced immune response by increase of Foxp3 (+) cells and by favoring an immunoregulatory cytokine pattern [26]. 2.6. Lymphocyte Subpopulations Several studies reveal that the probiotics differently modulate peripheral blood immune parameters in healthy subjects and patients with AD. Gerasimov et al. conducted a study to assess the clinical efficacy and impact of Lctbs acidophilus and Bfdbm lactis with fructooligosaccharide on peripheral blood lymphocyte subsets in preschool children with moderate-to-severe AD. The percentage of CD4 and the percentage and absolute count of CD25 decreased; and the percentage and absolute count of CD8 increased significantly in the probiotic group at week 8, compared with placebo. They found a significant correlation between CD4 percentage, CD25 percentage, CD25 absolute count, and SCORAD values in the probiotic group at week 8. The administration of a probiotic mixture and fructooligosaccharide was correlated with significant clinical improvement in children with AD, with corresponding lymphocyte subpopulation changes in peripheral blood [46]. Also in other mice studies, contact hypersensitivity to the hapten 2,4-dinitrofluorobenzene, a model of allergic contact dermatitis mediated by CD8+-cytotoxic T-lymphocytes and controlled by CD4+-Treg cells, was studied. Daily oral administration of fermented milk containing Lctbs casei or Lctbs casei alone decreased skin inflammation by inhibiting the priming/expansion of hapten-specific IFN-γ-producing CD8+-effector T-cells. This study provides the first evidence that oral administration of Lctbs casei can reduce antigen-specific skin inflammation by controlling the size of the CD8+-effector pool [24]. Nevertheless, oral treatment with the probiotic bacteria Lctbs casei alone alleviated antigen-specific skin inflammation mediated by either protein-specific CD4+-T-cells or hapten-specific CD8+-T-cells in hapten-sensitized mice. In the model of CD8+-T-cell-mediated skin inflammation, reproducing allergic contact dermatitis in human beings, inhibition of skin inflammation was due to decreased CD8+-effector T-cells recruitment into the skin during the elicitation (i.e., symptomatic) phase of contact hypersensitivity [25]. 2.7. Toll-Like Receptor (TLR) Stimulation A number of experiments indicate that infectious agents can promote protection from ADs through mechanisms independent of their constitutive antigens, leading to stimulation of non-antigen-specific receptors such as TLRs. A family of pattern-recognition receptors such as TLRs on gut lymphoid and epithelial cells mediates innate immune responses to bacterial molecular patterns and, thereby, orchestrates acquired immunity. The transient protection offered by probiotics against IgE-associated allergic diseases is based on stimulation of TLRs, which produce mediators such as IL-6; these further induce IgA differentiation from naive B-cells. These events were shown to occur after probiotic administration to infants with eczema, as well as in infants who showed increased levels of serum CRP, IL-10, and IgE at the age of 6 months [40]. This probiotic-induced low-grade inflammation was characterized by elevation of CRP, IgE, IgA, and IL-10, with the changes being typically observed in helminth infection-associated induction of regulatory mechanisms. Moreover, the association of increased CRP with a decreased risk of eczema at 2 years of age in allergy-prone children supports the view that chronic, low-grade inflammation protects from eczema. The findings emphasize the role of chronic microbial exposure as an immune modulator protecting from allergy through activation of Treg cells. Consistently, LAB species such as Bfdbm bifidum/infantis and Lctbs salivarius were shown to be capable of activating TLR-2 [84]. In summary of the various effects of different probiotic strains in skin allergy reactions, local influences of probiotics potentially include reduction of gut permeability and systemic penetration of antigens, increased local IgA production, and alteration of local inflammation or tolerance induction. Some possible systemic effects consist of anti-inflammatory effects mediated by Toll-like receptors (TLRs), T-helper 1 (Th1) skewing of responses to allergens, and activation of tolerogenic dendritic cells (DCs), in addition to Treg cell production [1, 2, 85]. 3. Experimental (Animal Model) and Clinical (Human) Studies Showing the Role of Probiotics in the Prevention and Treatment of Allergic Skin Conditions The increased prevalence of allergic diseases is nowadays defined as an epidemic. AD is known as the earliest of these conditions, and it might act as an indicator for the development of IgE- or non-IgE-mediated allergic manifestations. Thus, being aware of possible measures, such as probiotic use, to prevent and/or heal atopic disease is essential for the practicing allergy specialist. Here, their role in the prevention/therapy of AD and allergic skin conditions under the recent literature gathered from Medline and Pubmed is discussed. 3.1. Experimental Studies Showing the Role of Probiotics in the Prevention/Treatment of Allergic Skin Conditions Over the several decades, animal models of AD and skin allergy reactions have received increasing attention. These models include NC/Nga mice, a hapten-induced mouse model, and transgenic and knockout mouse models. Although the pathogenesis of skin inflammation elicited in these models is not quite the same, it is pertinent to ask what these animal models really tell us about the pathogenesis and possible therapies for the disease. NC/Nga mice may yield information relevant to the dissection of the crucial components of the pathophysiology of skin allergy reactions and AD rather than the assessment of potentially therapeutic agents for their treatment. And this hapten-induced mouse model has been mostly used and created by repeated applications of 2,4,6-trinitrochlorobenzene (TNCB), that is, a simple and reproducible one. This model offers several advantages over others: by changing hapten and the mouse strain used, various types of chronic inflammation, probably reflecting heterogeneity in clinical presentation of skin allergy reactions and AD, can be induced. This model is also of enormous value in its high reproducibility as well as the ease of quantitative assessment by measuring ear thickness [57, 86]. Probiotic strains have been reported to have the ability to control allergic and inflammatory diseases. Here, some of the studies performed on experimental murine models of AD and AD-like lesions showing the role of probiotics will be discussed (the various effects of different probiotic strains, referred to in this paper, on AD, AD-like skin lesions, and allergic contact dermatitis in experimental (animal) studies are shown in Table 1 as well). 3.1.1. Murine Models of AD Induced by House-Dust Mite Sensitization Oral administration of Lctbs delbrueckii subsp. lactis R-037 isolated from the traditional South Asian fermented milk “dahi” inhibited the development of AD in NC/Nga mice [11]. In addition, the antiallergic effect of one strain (T120) of LAB isolated from the Mongolian fermented milk using AD model mice (NC/Nga mice) was investigated. And in in vivo studies, intraperitoneal injection of strain T120 subdued AD symptoms in NC/Nga mice [12]. In another study, Lctbs plantarum strains from Kimchi were investigated for their capacity to inhibit AD (house dust mite-induced dermatitis) in NC/Nga mouse. The three strains, CJLP55, CJLP133, and CJLP136, suppressed AD-like skin lesions and epidermal thickening [13, 23, 87]. Ingestion of heat-treated LGG was shown to prevent development of AD of NC/Nga mice in a study. Maternal and infant mice were fed with food containing or not containing heat-treated LGG during pregnancy and breastfeeding, and after weaning. Administration of food containing heat-treated LGG inhibited the onset and development of atopic skin lesions, accompanied by smaller numbers of mast cells and eosinophils in the affected skin sites [14, 21]. Moreover, a new synbiotic, Lctbs casei subsp. casei together with dextran reduced murine allergic reaction such as the development of AD-like skin lesions developed by Dermatophagoides pteronyssinus crude extract in NC/Nga mice. This combination significantly decreased clinical skin severity scores and total IgE levels in sera of NC/Nga mice [15]. Nevertheless, administration of LGG to puppies appeared to reduce immunologic indicators (allergen-specific IgE) of AD, although no significant decrease in clinical signs (dermatitis and pruritus) was detected. In this study, the efficacy of the probiotic LGG for the alleviation or prevention of clinical signs of AD in genetically predisposed dogs (2 adult Beagles with severe AD and 16 puppies) was evaluated. LGG was administered to the bitch during the second pregnancy and to the puppies of the second litter from 3 weeks to 6 months of age. Both litters were epicutaneously sensitized to Dermatophagoides farinae [16, 17]. In a recent study, the inhibitory effect of Bacillus subtilis on AD was studied too. The effects of continuous oral administration of Bacillus subtilis for 4 weeks on the development of AD induced by Dermatophagoides farinae body antigen in NC/Nga mice were evaluated using 4 groups of mice. Histopathological examination results revealed significant differences suggesting that continuous oral administration of Bacillus subtilis can be effective in alleviating the development of skin lesions induced by Dermatophagoides in NC/Nga mice [88]. 3.1.2. Murine Models of AD-Like Skin Lesions Induced by Trinitrochlorobenzene Sensitization In a study, Lctbs acidophilus strain L-55 suppressed the development of AD-like skin lesions induced by repeated applications of TNCB in sensitized NC/Nga mice. The increase of dermatitis score and ear swelling was also inhibited by strain L-55. Scratching behavior observed in the back and ears was inhibited by strain L-55 as well. Furthermore, strain L-55 also caused an inhibition of histological changes induced by repeated applications of TNCB [18, 89]. Oral treatment with probiotic Lctbs johnsonii NCC533 (La1) during the specific part of the weaning period prevented the development of AD in model mice, NC/Nga. In a similar study, La1 was also administered orally to the La1 group from 20 to 22 days after birth. After the induction of skin lesions in 6-week-old mice, the expression of genes supposedly involved in AD was evaluated. Gene expression of the proinflammatory cytokines such as IL-8, IL-12, and IL-23 was significantly enhanced in the lesional skin of the control group by the induction of the lesion, whereas gene expression of those in the La1 group was not elevated. Moreover, the La1 group showed a significantly lower gene expression of CD86 in Peyer's patches and mesenteric lymph nodes than the control group. The suppression of proinflammatory cytokines and CD86 expression by primary administration of La1 may significantly contribute to the inhibitory effect on the skin lesions [20, 90, 91]. Oral supplementation with Lctbs rhamnosus CGMCC 1.3724 (LPR) prevented development of AD in NC/NgaTnd mice possibly by modulating local production of IFN-γ in a study. Pregnant NC/NgaTnd mice were orally treated with the probiotic strain LPR, which was followed by treatment of pups until 12 weeks of age. LPR-treated mice exhibited significantly lower clinical symptoms of dermatitis and reduced scratching frequency, compared with untreated mice. The protective effect was also observed when mice started to be treated at weaning time (5 weeks of age) even with limited supplementation period of 2 weeks. However, treatment of mice with the probiotic starting 1 week after the onset of the disease (8 weeks of age) had limited effects [19]. 3.1.3. Murine Models of AD-Like Skin Lesions Induced by Picryl Chloride Sensitization AD-like skin lesions were induced by sensitization to and repeated challenges with picryl chloride in the Th2-skewed NC/Nga mouse strain. A new synbiotic, Lctbs casei subsp. casei together with dextran reduced murine allergic reaction such as the development of AD-like skin lesions in NC/Nga mice. This synbiotic combination significantly decreased clinical skin severity scores induced by picryl chloride, similar to dust mite sensitization, in NC/Nga mice [15]. Supplementation with KW3110 strain of LAB significantly attenuated the onset and exacerbation of AD-like skin lesions, accompanied by less mast cell infiltration [16]. Oral administration of heat-killed Lctbs brevis SBC8803 ameliorated the development of dermatitis in AD model NC/Nga mice. Eight-week-old male NC/Nga mice were sensitized by the topical application of picryl chloride to foot pads and shaved abdomens. These mice were boosted with picryl chloride by topical application onto the ears once a week for 9 weeks. The mice (n = 10 per group) were fed a diet containing 0%, 0.05%, or 0.5% of heat-killed Lctbs brevis from 2 weeks before the first sensitization to the end of the study. Oral administration of Lctbs brevis significantly inhibited ear swelling and suppressed the development of dermatitis in a dose-dependent manner [22]. 3.1.4. Murine Models of Allergic Contact Dermatitis Induced by Dinitrofluorobenzene Sensitization The aim of a few studies was to examine whether Lctbs casei could affect antigen-specific CD8+ -T-cell-mediated skin inflammation. In a study by Chapat et al., contact hypersensitivity to the hapten 2,4-dinitrofluorobenzene, a model of allergic contact dermatitis mediated by CD8+-cytotoxic T-lymphocytes and controlled by CD4+-Treg cells, was used. This study provided the first evidence that oral administration of Lctbs casei could reduce antigen-specific skin inflammation by controlling the size of the CD8+-effector pool [24]. Similarly, oral treatment with the probiotic bacteria Lctbs casei alone alleviated antigen-specific skin inflammation mediated by either protein-specific CD4+-T-cells or hapten-specific CD8+-T-cells in hapten-sensitized mice. In the model of CD8+-T-cell-mediated skin inflammation, which reproduces allergic contact dermatitis in human beings, inhibition of skin inflammation by Lctbs casei was due to attenuation of the recruitment of CD8+-effector T-cells into the skin during the elicitation (i.e., symptomatic) phase of contact hypersensitivity. These data demonstrate that orally administered Lctbs casei efficiently alleviate T-cell-mediated skin inflammation without causing immunosuppression [25]. In sensitized BALB/c mice, skin inflammation was induced by topical allergen application. Escherichia coli Nissle 1917 was administered orally in a preventative manner and it improved allergen-induced dermatitis dose dependently, consistent with a reduction of epidermal thickness that was detected in eczematous skin [26]. Lctbs sakei probio-65 that was isolated from Kimchi, a traditional Korean fermented food, was found to be effective in reducing allergic dermatitis in chemical allergen- (1-chloro-2,4-dinitrobenzene-) induced mice as well [68, 92]. 3.2. Clinical (Human) Studies Showing Probiotics' Effects in Allergic Skin Conditions including Eczema Mostly reported clinical (human) studies showing probiotics' effects in skin allergy reactions have been related to AD (eczema). Here, probiotics' effects in human AD are being discussed according to the IgE-sensitized (atopic) versus non-IgE-sensitized (nonatopic) eczema groups (the various effects of different probiotic strains, referred to in this paper, on allergic skin conditions including AD in clinical (human) studies are shown in Table 2). Is There Any Difference between IgE-Sensitized (Atopic) and Non-IgE-Sensitized (Nonatopic) Eczema Groups? A number of studies could only relate probiotic benefits to a certain subset of AD patients. In support of the efficacy of probiotics in IgE-sensitized children, some other studies also demonstrated comparable results as well. In brief, treatment with Lctbs rhamnosus for the first 2 years of life was associated with a significant reduction in the prevalence of any IgE-associated eczema by about a half [4]. Another study demonstrated that LGG alleviated atopic eczema/dermatitis syndrome symptoms in IgE-sensitized infants [18]. In food-sensitized atopic children, the efficacy of the probiotics such as Lctbs rhamnosus and Bifidobacterium (Bfdbm) lactis was demonstrated too [28]. This effect was more pronounced in patients with a positive skin prick test and increased IgE levels. Yet, some other studies failed to demonstrate that the severity and frequency of AD were decreased with the supplementation of probiotics, regardless of their IgE sensitization status. For instance, Boyle et al. and others could not show any effect even of LGG in infants with AD [47, 48]. A few meta-analyses also could not confirm that IgE sensitization was indeed a factor in determining the efficacy of probiotics in atopic children. However, the heterogeneity between studies may be attributable to probiotic strain-specific effects and other factors as well, meaning that some probiotic strains may still have a therapeutic role in eczema [1, 2]. 3.2.1. IgE-Sensitized (Atopic) Eczema Therapy and Prevention Recently was published one of the largest studies by Viljanen et al. to date that compared LGG or a probiotic mix (LGG, Lctbsrhamnosus LC705, Bfdbm breve Bb99, and Propionibacterium freudenreichii ssp. shermanii JS) with placebo. In that study, 230 Finnish children with AD were treated for 4 weeks with LGG, a mixture of four probiotic strains, or placebo. With supplementation of probiotics (LGG), Viljanen et al. found significant improvement on the SCORAD index only in “IgE-sensitize cow's milk-allergic infants of the AEDS.” Only in the subgroup of IgE-sensitized children did the LGG group show a greater reduction in SCORAD than the placebo group, but this effect could have been due to a higher baseline score in this subgroup. There was no difference between the groups at the end of the 4-week therapy, and 4 weeks after therapy was discontinued. Contrary to what would be expected, improvement was seen 4 weeks after discontinuation of therapy rather than during treatment [93]. Rosenfeldt et al. from Denmark in a study demonstrated that 2 lyophilized probiotic Lctbs strains (lyophilized Lctbs rhamnosus 19070-2 and Lctbs reuteri DSM 122460) were given in combination for 6 weeks to 1- to 13-year-old (mean age, 5.2 years) children with AD. This study used 2 different Lctbs species in older children. A combination of these was beneficial in the management of AD. Statistically significant improvement in SCORAD score was seen only in a subset of children with positive skin prick test results and elevated IgE levels [27]. Another study by Sistek et al. showed the efficacy of the probiotics Lctbs rhamnosus and Bfdbm lactis in food-sensitized children [28]. A study by a Finnish group used the same probiotic mixture with prebiotics. Kukkonen et al. in a trial using probiotic mix (Lctbs rhamnosus GG, Lctbs rhamnosus LC705, Bfdbm breve Bb99; and Propionibacterium freudenreichii ssp. shermanii JS) and prebiotic galactooligosaccharides demonstrated that the prevention of atopic eczema in high-risk Finnish infants is possible by modulating the infants' gut microbiota with probiotics and prebiotics. Probiotic treatment compared with placebo reduced IgE-associated (atopic) diseases. Probiotic treatment also reduced eczema and atopic eczema [29, 94]. In 2009, in a study by Kuitunen et al., 1223 Finnish mothers were randomized with infants at high risk for allergy to receive the same probiotic mixture (2 Lactobacilli, Bifidobacteria, and Propionibacteria) or placebo during the last month of pregnancy, and their infants were to receive it from birth until the age of 6 months. Infants also received a prebiotic galactooligosaccharide or placebo. At 5 years, the cumulative incidence of allergic diseases (eczema, food allergy, allergic rhinitis, and asthma) and IgE sensitization were evaluated. Frequencies of allergic and IgE-associated allergic disease and sensitization in the probiotic and placebo groups were similar. However, less IgE-associated allergic diseases occurred in cesarean-delivered children receiving probiotics. No allergy-preventative effect that extended to the age of 5 years was achieved with perinatal supplementation of probiotic bacteria to high-risk mothers and children. It conferred protection only to cesarean-delivered children [30]. Similarly, Abrahamsson et al. could not confirm a preventative effect of probiotics (Lctbs reuteri ATCC 55730) on infant eczema in a recently published study. However, they observed that the treated infants had less IgE-associated eczema at 2 years. Moreover, skin prick test reactivity was also less common in the treated group than in the placebo group, but this difference reached significance only for infants with allergic Swedish mothers [31]. In summary, all of these studies taken together demonstrate that probiotics might not be effective and/or therapeutic for all children with AD, but they offer benefits to a subset of IgE-sensitized children. 3.2.2. Non-IgE-Sensitized (Nonatopic) Eczema Therapy and Prevention Until now, several clinical studies have been published and have focused on the use of probiotics for therapy and primary prevention of atopic diseases. To date, the results of at least 15 prospective preventative studies with different Lctbs or Bfdbm strains (or mixture) in children at high risk for allergic diseases have been published. The first study in the literature by Isolauri et al. analyzed a benefit of LGG in mild AD disease in 1997. They observed 27 exclusively breastfed infants (median age, 4–6 months) with mild AD (median SCORAD score of 16), receiving extensively hydrolyzed whey formula with (LGG or Bfdbm strain) or without probiotics (placebo) for 8 weeks. They showed a reduction in the SCORAD by 15 points (from 16 to 1) for the LGG and by 16 points (from 16 to 0) for the Bfdbm arm, as compared with a reduction of 2–6 points (from 16 to 13–4) in the placebo arm. However, one month after therapy, SCORAD scores were comparable with those of placebo. Therefore, the probiotic effect was limited to acceleration of improvement in infants with mild disease [3]. The same investigators subsequently published 2 additional studies. One of these studies compared LGG with Bfdbm lactis Bb-12, both of which showed a significant improvement in SCORAD score over placebo. However, after 6 months, the median SCORAD score was zero in all groups, again suggesting that the effect is limited to rapid initiation of improvement [95]. The other study underlined the importance of viability for probiotic species. The use of heat-inactivated LGG resulted in adverse gastrointestinal symptoms with diarrhea, and study recruitment was halted. They concluded that supplementation of infant formulas with viable but not heat-inactivated LGG was found to be a potential approach for the management of atopic eczema and cow's milk allergy [96]. In an earlier study by Viljanen et al., probiotics have been suggested to be useful in children with AEDS. In 2010, a study by Woo et al. was performed to assess the clinical effect of Lctbs sakei supplementation in children with AEDS. In that study, children who aged 2 to 10 years with AEDS with a minimum SCORAD score of 25 were randomized to receive either daily Lctbs sakei KCTC 10755BP or daily placebo supplementation for 12 weeks. At week 12, SCORAD total scores adjusted by pretreatment values were lower after probiotic treatment than after placebo treatment. There was a 31% improvement in mean disease activity with probiotic use compared with a 13% improvement with placebo use. Therefore, significant differences in favor of probiotic treatment were also observed in proportions of patients achieving improvement of at least 30% and 50%. Interestingly, clinical improvement in this study was not just observed in the subgroup of IgE-sensitized children, contrary to the Viljanen et al. study, but it was also regardless of IgE sensitization [36]. Weston et al. from Australia published their experience with using Lctbs fermentum VRI-003 PCC for 8 weeks in 53 infants with AD. After 16 weeks, the probiotic group had significant reduction of SCORAD scores, while the placebo group did not. Lctbs fermentum caused a significant reduction in SCORAD scores. Although the change in SCORAD score from baseline in the probiotic group was significant, the difference between the probiotic and placebo groups did not reach significance by week 16 [37]. In a study by Hoang et al., they followed 14 cases of pediatric patients (ages of 8 months to 64 months) with a history of resistant eczema for a period of at least 6 months. All of these children received Lctbs rhamnosus cell lysate daily as an immunobiotic supplement. The results of this open-label nonrandomized clinical observation showed a substantial improvement in quality of life, skin symptoms, and day- and night-time irritation scores in children with the supplementation of Lctbs rhamnosus lysate. There were no intolerance or adverse reactions observed in these children. Lctbs rhamnosus cell lysate may, thus, be used as a safe and effective immunobiotic for the treatment and prevention of childhood eczema [38]. Bfdbm breve has been reported by Hattori et al. to improve cutaneous symptoms of AD patients. Fifteen children with AD who had Bfdbm-deficient microflora were selected for this study. Eight subjects in the Bifidobacteria-administered group were given oral administration of lyophilized Bfdbm breve M-16V strain. In the Bifidobacteria-administered group, the proportion of Bfdbm in the fecal microflora was increased, and the proportion of aerobic bacteria was decreased after 1 month of administration. Furthermore, significant improvement of allergic symptoms (in cutaneous symptoms and total allergic scores) was also observed in the Bifidobacteria-administered group. The tendency of allergic symptom improvement was remarkable compared with the control group; however, there was no correlation between changes in fecal microflora and allergic symptoms [39]. The Finnish study of Kalliomäki et al. was the first report to describe that the frequency of AD in the probiotic group was half that of the placebo. This hallmark study demonstrated that administration of LGG for 1 month before and 6 months after birth to their infants was associated with a significant reduction in the cumulative incidence of eczema during the first 7 years of life. The effect of probiotics on preventing AD has been demonstrated in infants of the Finnish pregnant mothers with a strong family history of eczema, allergic rhinitis, or asthma. The frequency of developing AD in the offspring was significantly reduced by 2, 4, and 7 years, by 50%, 44%, and 36%, respectively. But, there were no preventative effects on atopic sensitization and onset of respiratory allergic diseases [4]. Wickens et al. studied a differential effect of 2 probiotics in the prevention of eczema and atopy. Infants receiving Lctbs rhamnosus had a significantly reduced risk of eczema, compared with placebo, but this was not the case for B animalis subsp. lactis. In a double-blind, randomized placebo-controlled trial of infants at risk of allergic disease, pregnant women were randomized to take Lctbs rhamnosus HN001, Bfdbm animalis subsp. lactis strain HN019, or placebo daily from 35 weeks of gestation until 6 months if breastfeeding, and their infants were randomized to receive the same treatment from birth to 2 years (n : 474). Infants receiving Lctbs rhamnosus had a significantly reduced risk of eczema compared with placebo, but this was not the case for Bfdbm animalis subsp. lactis. There was no significant effect of Lctbs rhamnosus or Bfdbm animalis subsp. lactis on atopy. Lctbs rhamnosus (72%) was more likely than Bfdbm animalis subsp. lactis (22.6%) to be present in the feces at 3 months, although detection rates were similar by 24 months. The authors found out that supplementation with Lctbs rhamnosus, but not Bfdbm animalis subsp. lactis, substantially reduced the cumulative prevalence of eczema, but not atopy, by 2 years [34]. In a randomized double-blind study by Marschan et al., probiotic bacteria (Lctbs rhamnosus GG (ATCC 53103), Lctbs rhamnosus LC705, Bfdbm breve Bb99, and Propionibacterium freudenreichii ssp. Shermanii JS) or placebo had been given for 1 month before delivery to mothers and for 6 months to infants with a family history of allergy. Infants receiving probiotic bacteria had higher plasma levels of CRP, total IgA, total IgE, and IL-10 than infants in the placebo group. Increased plasma CRP level at the age of 6 months was associated with a decreased risk of eczema and with a decreased risk of allergic disease at the age of 2 years, when adjusted with probiotic use. The association of CRP with a decreased risk of eczema at 2 years of age in allergy-prone children supports the view that chronic, low-grade inflammation protects from eczema. Probiotic-induced low-grade inflammation was characterized by elevation of IgE, IgA, and IL-10, the changes typically observed in helminth infection-associated induction of regulatory mechanisms [40]. In the Panda study of Niers et al. administered was a mixture of probiotic bacteria (Bfdbm bifidum W23, Bfdbm lactis W52, and Lactococcus lactis W58; Ecologic Panda) for 6 weeks prenatally to mothers of high-risk children and to their offspring for the first 12 months of life. Although cumulative incidence of atopic eczema and IgE levels were similar in both treated and placebo groups, the parental reported eczema was significantly lower during the first 3 months of life in infants receiving probiotics. This particular combination of probiotic bacteria showed a preventative effect on the incidence of eczema in high-risk children, which seems to be sustained during the first 2 years of life. In addition to the previous studies, the preventative effect appeared to be established within the first 3 months of life in this study [41]. In a trial by Kim et al., 112 pregnant women with a family history of allergic diseases received a mixture of Bfdbm bifidum BGN4, Bfdbm lactis AD011, and Lctbs acidophilus AD031, starting at 4–8 weeks before delivery and continuing until 6 months after delivery. The cumulative incidence of eczema during the first 12 months was reduced significantly in the probiotic group; however, there was no difference in serum total IgE level or the sensitization against food allergens between the two groups. Prenatal and postnatal supplementation with a mixture of probiotics is an effective approach in preventing the development of eczema in infants at high risk of allergy during the first year of life [42]. In a randomized, double-blind trial by Dotterud et al., probiotics were given to pregnant women to prevent allergic diseases. In this study, children from a nonselected maternal population and women received probiotic milk or placebo from 36 weeks of gestation to 3 months postnatally during breastfeeding. The probiotic milk contained Lctbs rhamnosus GG, Lctbs acidophilus La-5, and Bfdbm animalis subsp. lactis Bb-12. At 2 years of age, all children were assessed for atopic sensitization, AD, asthma, and allergic rhinoconjunctivitis. Probiotics given to the nonselected mothers reduced the cumulative incidence of AD, but they had no effect on asthma or atopic sensitization [43]. Böttcher et al.'s study demonstrated that Lctbs reuteri supplementation during pregnancy is associated with reduced risk of sensitization during infancy. Swedish pregnant women were treated with Lctbs reuteri (n : 54) or placebo (n : 55) from gestational week 36 until delivery. The infants were followed prospectively for 2 years regarding development of eczema and sensitization as defined by a positive skin prick test and/or circulating allergen-specific IgE antibodies at 6, 12, and 24 months of age [44]. Of note, another recently published Swedish study demonstrated that administration of Lctbs casei F19 during weaning significantly reduced the incidence of eczema, indicating that proper timing of the probiotic intervention is a critical factor. That study also supports the notion that there is more than a single window of opportunity to manage allergic diseases. That study, moreover, evaluated the effects of feeding with Lctbs F19 during weaning period on the incidence of eczema and Th1/Th2 balance. In this intervention trial by West et al., infants were fed cereals with (n : 89) or without Lctbs F19 (n : 90) from 4 to 13 months of age. The cumulative incidences of eczema at 13 months 11% and 22% in the probiotic and placebo groups, respectively were (P : <0.05). At 13 months of age, the IFN-γ/IL-4 mRNA ratio was significantly higher in the probiotic group compared with the placebo group. The higher Th1/Th2 ratio in the probiotic group compared with the placebo group suggests enhancing effects of Lctbs F19 on the T-cell-mediated immune response. In contrast, there were no differences between groups in serum IgE concentrations. As a result, feeding Lctbs F19 during weaning could be an effective tool in the prevention of early manifestation of allergy such as eczema [35]. Oral administration of probiotic Escherichia coli after birth in the early postnatal period by Lodinova-Zadnikova et al. reduced frequency of serum-specific IgE allergies later in life (after 10 and 20 years) [45]. Gerasimov et al. conducted a study to assess the clinical efficacy and impact of Lctbs acidophilus DDS-1 and Bfdbm lactis UABLA-12 with fructooligosaccharide on peripheral blood lymphocyte subsets in preschool children with moderate-to-severe AD. In a randomized, double-blind, placebo-controlled, and prospective trial of 90 children aging 1–3 years with moderate-to-severe AD who were treated with a mixture of probiotics with fructooligosaccharide for 8 weeks versus placebo at the final visit, the percentage significant decrease in SCORAD was 33% in the probiotic group compared with 19% in the placebo group. Children receiving probiotics showed a greater decrease in the mean SCORAD score than did children from the placebo group at week 8. The administration of a probiotic mixture and fructooligosaccharide was associated with significant clinical improvement in children with AD, with corresponding lymphocyte subset changes in peripheral blood [46]. In brief, here, probiotics were more likely to be effective in treating moderately severe AD as well as mild atopic diseases. Although not every study result above was significant, the effect of probiotics did not seem to be greater just in the IgE-sensitized group than in the non-IgE-sensitized group. Nevertheless, there have been several reports in the literature showing no effect of probiotics, which are being discussed in the section below. 3.2.3. No Therapeutic or Preventative Effect of Probiotics in AD Regardless of IgE Sensitization It is striking that the proportion of children with AD and allergic sensitization such as in the study of Taylor [49] and Huurre et al. [97] was significantly higher in the probiotic group. In Taylor et al.'s trial, probiotic supplementation postnatally failed to reduce the risk of AD and increased the risk of allergen sensitization in high-risk children. Newborns of women with allergy (n : 231) received either Lctbs acidophilus (LAVRI-A1) or placebo daily for the first 6 months of life. Children were assessed for AD and other symptoms at 6 and 12 months and had allergen skin prick tests at 12 months of age. At 6 and 12 months, AD rates were similar in the probiotic and placebo groups. At 12 months, the rate of sensitization was significantly higher in the probiotic group. The presence of culturable Lactobacilli or Bfdbm in stools in the first month of life was not associated with the risk of subsequent sensitization or disease; however, the presence of Lctbs at 6 months of age was associated with increased risk of subsequent cow's milk sensitization. Early probiotic supplementation with Lctbs acidophilus did not reduce the risk of AD in high-risk infants and was associated with increased allergen sensitization in infants receiving supplements. There were 3 major differences between Taylor's study and the others. The type of probiotic product (Lctbs acidophilus), the supplementation period (1 year), and the timing of the introduction of the probiotic were different. Taylor et al. administered the probiotic supplement postnatally, while other studies administered probiotics before and after birth. Prenatal supplementation may prove to be crucial for the preventative benefit of probiotics in this disorder. The data from Taylor et al.'s study point in the same direction regarding allergic sensitization, also suggesting that the use of probiotics for primary prevention must be exercised with caution [49]. Similarly, a randomized, double-blind, placebo-controlled, and prospective trial by Kopp et al. of probiotics for primary prevention did show no clinical effects of LGG supplementation; 105 pregnant women from families with ≥1 member (mother, father, or child) with an atopic disease were randomly assigned to receive either the probiotic LGG or placebo. The supplementation period started 4 to 6 weeks before expected delivery, followed by a postnatal period of 6 months. The primary endpoint was the occurrence of AD at the age of 2 years. Secondary outcomes were severity of AD, recurrent episodes of wheezing bronchitis, and allergic sensitization at the age of 2 years. Notably, children with recurrent (≥5) episodes of wheezing bronchitis were more frequent in the LGG group (26%), as compared with the placebo group (9%). As a result, supplementation with LGG during pregnancy and early infancy neither reduced the incidence of AD nor altered the severity of AD in affected children but was associated with an increased rate of recurrent episodes of wheezing bronchitis. No difference was observed between both groups in total IgE concentrations or numbers of specific sensitization to inhalant allergens [50]. Furthermore, prenatal probiotic LGG treatment during pregnancy was not associated with reduced risk of eczema or IgE-associated eczema in a RCT by Boyle et al. [47, 48]. In a recent study, 250 pregnant women were recruited carrying infants at high risk of allergic disease to a RCT of probiotic supplementation (LGG) from 36 weeks of gestation until delivery. Grüber et al.'s study also did not show any effect of LGG in infants with AD regardless of their IgE sensitization status [51]. However, a study from the Netherlands by Brouwer et al. and another study from Germany by Fölster-Holst et al. showed no effect of LGG in infants with AD regardless of their IgE sensitization status. In a study conducted by Brouwer et al., after 4–6 weeks of baseline and double-blind and placebo-controlled challenges for diagnosis of cow's milk allergy, infants less than 5 months old with AD received a hydrolyzed whey-based formula as placebo (n : 17) or were supplemented with either Lctbs rhamnosus (n : 17) or LGG (n : 16) for 3 months. No statistically significant effects of probiotic supplementation on SCORAD, sensitization, inflammatory parameters, or cytokine production between groups were found. No clinical or immunological effects of the probiotic bacteria used in infants with AD were found [52]. A similar prospective study by Fölster-Holst et al. was performed to reassess the efficacy of orally administered LGG in infants with AD. In a randomized, double-blind, and placebo-controlled study, 54 infants aging 1–55 months with moderate-to-severe AD were randomized to receive LGG or placebo during an 8-week intervention phase. At the end of treatment, there were no significant differences between the groups with respect to clinical symptoms (SCORAD, pruritus, and sleep loss), immunological parameters, or health-related quality of life of the parents [53]. Additionally, Soh et al. in a clinical trial involving 253 infants with a family history of allergic disease utilized probiotic supplementation (Bfdbm longum + Lctbs rhamnosus) in the first 6 months of life in Asian infants at risk and evaluated the effects on eczema and atopic sensitization at the age of 1 year. Early life administration of a cow's milk formula supplemented with probiotics showed no effect on prevention of eczema or allergen sensitization in the first year of life in Asian infants at risk of allergic diseases [54]. A randomized, double-blind, and placebo-controlled study was conducted in 34 adult-type AD subjects who were treated with conventional topical corticosteroid and tacrolimus. In these kinds of patients, heat-killed Lctbs paracasei K71 (LAB diet) may have been shown to have some benefits as a complementary therapy for adult AD patients who were managed with the conventional treatment [98]. In a double-blind, placebo-controlled, and crossover study, Bfdbm animalis subsp. lactis LKM512 yogurt was given for 4 weeks to 10 adult AD patients (4 males + 6 females; average age: 22 years) who were diagnosed with moderate AD. Scores of itching and burning tended to improve to a greater extent by LKM512 yogurt consumption than by placebo consumption. LKM512 yogurt consumption may be effective against intractable adult-type AD [99]. LGG was the mostly used probiotic species in these studies. Firstly used by Kalliomäki et al. [4] with a success, however, other groups including Brouwer, Boyle, Kopp et al., Grüber et al., and Fölster-Holst et al. [47, 50–53] could not demonstrate any benefit in AD. For instance, Kopp et al. have shown that the probiotic LGG has no preventative effect on the development or the severity of AD at the age of 2 years in a German population of infants at high risk. Instead, there was a significantly higher risk of ≥5 episodes with wheezing bronchitis during the first 2 years in the LGG group, as compared with placebo. There were several methodological differences between these studies: Kopp et al. adapted the protocol of Kalliomäki et al. and continued to supplement LGG for 3 months after birth to the breastfeeding mothers and the following 3 months only to the neonates. This modification was made to achieve a more consistent probiotic delivery. Second, Finnish mothers received supplementation during the last 4 weeks of pregnancy, whereas pregnant women in this population commenced with LGG or placebo for 4 to 6 weeks. They extended the prenatal supplementation period, because a 4-week period is thought to be possibly too short for the suspection of the in utero effects of LGG supplementation. Also, a population in the study by Kopp et al. was being of higher risk compared with the Finnish population, which might account for the differing results. And more infants with older siblings were recruited compared with the Finnish study. Lastly, the Finnish and German populations are of different genetic backgrounds. In summary, there is unsatisfactory but fairly promising evidence to recommend the addition of probiotics to foods for prevention and treatment of AD [100]. Nonetheless, there is a large amount of conflicting data on the preventative/therapeutic effects of probiotics in especially human clinical trials of AD. Results from these trials, meta-analyses, and systematic reviews that combine results of studies from different types of probiotics to examine the effects in any disease should be interpreted with caution. One may quickly recognize the degree of heterogeneity among the different probiotic studies as well. Very few studies were similar in design. For instance, several different probiotic strains with different dosing regimens were used [101]. And some probiotic studies suggest short-term statistically significant improvement in SCORAD scores and no sustained benefit from continued ingestion. Consequently, subgroup analysis became critical in understanding the outcomes of the studies. Not all individuals in clinical trials receiving the probiotic agent benefited, but subsets of these patients, mainly those with moderate disease activity and IgE-associated disease (atopic eczema), seemed to have benefited the most. 4. Conclusion Currently, probiotics do not have a proven role in the prevention or therapy of allergic skin disorders. No single probiotic supplement or group of probiotic supplements has been yet demonstrated to efficiently affect the course of any allergic disease or manifestation. Therefore, probiotics cannot be recommended generally for primary prevention/therapy of allergic skin disorders [102]. If probiotics are used in patients with allergic skin disorders for any reasontherapy or prevention-cautionary approach ought to be taken. Abbreviations AD: Atopic dermatitis Lctbs: Lactobacillus LGG: Lctbs rhamnosus GG SCORAD: Severity scoring of atopic dermatitis Ig: Immunoglobulin LAB: Lactic acid bacteria TLR: Toll-like receptor Th1: T-helper 1 DC: Dendritic cell Treg: T regulatory TNCB: Trinitrochlorobenzene La1: Lctbs johnsonii NCC533 LPR: Lctbs rhamnosus CGMCC 1.3724 AEDS: Atopic eczema/dermatitis syndrome Bfdbm: Bifidobacterium RCTs: Randomized (placebo-) controlled trials. Table 1 The various effects of different probiotic strains, referred to in this paper, on allergic skin conditions including atopic dermatitis, atopic dermatitis-like skin lesions, and allergic contact dermatitis in experimental (animal) studies are shown. References Probiotic species Types of dermatitis in murine Outcomes Atopic dermatitis (AD) Watanabe et al. [11] Lctbs delbrueckii subsp. lactis Atopic dermatitis ↓ Hayashi et al. [12] Lactic acid bacteria Atopic dermatitis ↓ Won et al. [13] Lctbs plantarum House-dust mite-induced AD ↓ Sawada et al. [14] LGG Atopic dermatitis ↓ Ogawa et al. [15] Lctbs casei subsp. casei Atopic dermatitis ↓ Marsella et al. [16, 17] LGG Atopic dermatitis ↓ AD-like lesions (trinitrochlorobenzene sensitization) Viljanen et al. [18] Lctbs acidophilus Atopic dermatitis-like lesions ↓ Tanaka et al. [19, 20] Lctbs rhamnosus Atopic dermatitis-like lesions ↓ AD-like lesions (picrylchloride sensitization) Ogawa et al. [15] Lctbs casei subsp. casei Atopic dermatitis-like lesions ↓ Wakabayashi et al. [21] Lactic acid bacteria Atopic dermatitis-like lesions ↓ Segawa et al. [22] Lctbs brevis Atopic dermatitis-like lesions ↓ Allergic contact dermatitis (dinitrofluorobenzene sensitization) Park et al. [23] Lctbs sakei probio-65 (1-Chloro-2,4-dinitrobenzene)-induced allergic dermatitis ↓ Chapat et al. [24] Lctbs casei Allergic contact dermatitis ↓ Hacini-Rachinel et al. [25] Lctbs casei Allergic contact dermatitis ↓ Weise et al. [26] Escherichia coli Nissle 1917 Allergic contact dermatitis ↓ Lctbs: Lactobacillus; Bfdbm: bifidobacterium; LGG: Lactobacillus rhamnosus GG; ↓: decrease in symptoms or positive effect. Table 2 The various effects of different probiotic strains, referred to in this paper, in (human) clinical allergic skin conditions such as atopic and nonatopic eczema are shown. References Probiotic species Types of atopic dermatitis Outcomes Atopic (IgE-associated) Eczema Majamaa and Isolauri [3] LGG Food-sensitized eczema ↓ Viljanen et al. [18] LGG Atopic eczema/dermatitis syndrome ↓ Rosenfeldt et al. [27] Lctbs rhamnosus + Lctbs reuteri Atopic eczema ↓ Sistek et al. [28] Lctbs rhamnosus + Bfdbm lactis Eczema, food-sensitized atopy ↓ Kukkonen et al. and Kuitunen et al. [29, 30] Mix (LGG, Lctbs rhamnosus LC705, Bfdbm breve, and Propionibacterium) Atopic eczema ↓ Kuitunen et al. [30] Lctbs + Bfdbm + Propionibacteria IgE-associated allergy ↓ Abrahamsson et al. [31] Lctbs reuteri Atopic eczema ↓ Isolauri et al. [32, 33] Bfdbm or Lctbs Food (cow's milk) allergy ↓ Wickens et al. [34] Lctbs rhamnosus IgE-associated eczema ↓ Nonatopic Eczema Kalliomäki et al. [4] LGG Atopic dermatitis ↓ West et al. [35] Lctbs casei F19 Atopic dermatitis ↓ Woo et al. [36] Lctbs sakei Atopic dermatitis ↓ Weston et al. [37] Lctbs fermentum Atopic dermatitis ↓ Hoang et al. [38] Lctbs rhamnosus Atopic dermatitis ↓ Hattori et al. [39] Bfdbm breve Atopic dermatitis ↓ Wickens et al. [34] Lctbs rhamnosus, Bfdbm animalis (Bb-12) Atopic dermatitis ↓ Marschan et al. [40] Mix (LGG, Lctbs rhamnosus LC705, Bfdbm breve, and Propionibacterium) Atopic dermatitis ↓ Niers et al. [41] Bfdbm bifidum, Bfdbm lactis, and Lactococcus lactis Atopic dermatitis ↓ Kim et al. [42] Bfdbm bifidum, Bfdbm lactis, and Lctbs acidophilus Atopic dermatitis ↓ Dotterud et al. [43] LGG, Lctbs acidophilus, and Bfdbm animalis (Bb-12) Atopic dermatitis ↓ Böttcher et al. [44] Lctbs reuteri Atopic dermatitis (sensitization) ↓ Lodinova-Zadnikova et al. [45] Escherichia coli Atopic dermatitis (IgE allergies) ↓ Gerasimov et al. [46] Lctbs acidophilus and Bfdbm lactis Atopic dermatitis ↓ Eczema (atopic dermatitis) Boyle et al. [47, 48] LGG Atopic dermatitis ↔ Kuitunen et al. [30] Lctbs + Bfdbm + Propionibacteria Atopic dermatitis ↔ Taylor et al. [49] LGG or Lctbs acidophilus Atopic dermatitis ↔ Kopp et al. [50] LGG Atopic dermatitis ↔ Grüber et al. [51] LGG Atopic dermatitis ↔ Brouwer et al. [52] Lctbs rhamnosus Atopic dermatitis ↔ Fölster-Holst et al. [53] LGG Atopic dermatitis ↔ Soh et al. [54] Bfdbm longum + Lctbcs rhamnosus Eczema and atopic sensitization ↔ Bfdbm: Bifidobacterium; Lctbs: Lactobacillus; LGG: Lactobacillus rhamnosus GG; ↓: decrease in symptoms or positive effect, ↔: no change in symptoms or no effect. ==== Refs 1 Özdemir Ö Various effects of different probiotic strains in allergic disorders: an update from laboratory and clinical data Clinical and Experimental Immunology 2010 160 3 295 304 2-s2.0-77952181378 20345982 2 Özdemir Ö Any benefits of probiotics in allergic disorders? Allergy and Asthma Proceedings 2010 31 2 103 111 2-s2.0-77950822757 20406592 3 Majamaa H Isolauri E Probiotics: a novel approach in the management of food allergy Journal of Allergy and Clinical Immunology 1997 99 2 179 185 2-s2.0-0031041742 9042042 4 Kalliomäki M Salminen S Poussa T Isolauri E Probiotics during the first 7 years of life: a cumulative risk reduction of eczema in a randomized, placebo-controlled trial Journal of Allergy and Clinical Immunology 2007 119 4 1019 1021 2-s2.0-34047116237 17289135 5 Pelucchi C Chatenoud L Turati F Probiotics supplementation during pregnancy or infancy for the prevention of atopic dermatitis: a meta-analysis Epidemiology 2012 23 3 402 414 2-s2.0-84859630789 22441545 6 Cukrowska B Kozáková H Reháková Z Sinkora J Tlaskalová-Hogenová H Specific antibody and immunoglobulin responses after intestinal colonization of germ-free piglets with non-pathogenic Escherichia coli O86 Immunobiology 2001 204 4 425 433 11776397 7 Novak N Bieber T Leung DYM Immune mechanisms leading to atopic dermatitis Journal of Allergy and Clinical Immunology 2003 112 6 S128 S139 2-s2.0-0345800718 14657843 8 Walker WA Mechanisms of action of probiotics Clinical Infectious Diseases 2008 46 supplement 2 S87 S91 18181730 9 Darsow U Wollenberg A Simon D ETFAD/EADV eczema task force 2009 position paper on diagnosis and treatment of atopic dermatitis Journal of the European Academy of Dermatology and Venereology 2010 24 3 317 328 2-s2.0-76349105505 19732254 10 Boguniewicz M Leung DYM Pathophysiologic mechanisms in atopic dermatitis Seminars in Cutaneous Medicine and Surgery 2001 20 4 217 225 2-s2.0-0035211713 11770908 11 Watanabe T Hamada K Tategaki A Oral administration of lactic acid bacteria isolated from traditional South Asian fermented milk ‘dahi’ inhibits the development of atopic dermatitis in NC/Nga mice Journal of Nutritional Science and Vitaminology 2009 55 3 271 278 2-s2.0-70349308327 19602836 12 Hayashi A Kimura M Nakamura Y Yasui H Anti-atopic dermatitis effects and the mechanism of lactic acid bacteria isolated from Mongolian fermented milk Journal of Dairy Research 2009 76 2 158 164 2-s2.0-68349155776 19121229 13 Won TJ Kim B Lee Y Therapeutic potential of Lactobacillus plantarum CJLP133 for house-dust mite-induced dermatitis in NC/Nga mice Cellular Immunology 2012 277 1-2 49 57 22726349 14 Sawada J Morita H Tanaka A Salminen S He F Matsuda H Ingestion of heat-treated Lactobacillus rhamnosus GG prevents development of atopic dermatitis in NC/Nga mice Clinical and Experimental Allergy 2007 37 2 296 303 2-s2.0-33846458592 17250703 15 Ogawa T Hashikawa S Asai Y Sakamoto H Yasuda K Makimura Y A new synbiotic, Lactobacillus casei subsp. casei together with dextran, reduces murine and human allergic reaction FEMS Immunology and Medical Microbiology 2006 46 3 400 409 2-s2.0-33645078112 16553814 16 Marsella R Santoro D Ahrens K Early exposure to probiotics in a canine model of atopic dermatitis has long-term clinical and immunological effects Veterinary Immunology and Immunopathology 2012 146 2 185 189 2-s2.0-84859497831 22436376 17 Marsella R Evaluation of Lactobacillus rhamnosus strain GG for the prevention of atopic dermatitis in dogs The American Journal of Veterinary Research 2009 70 6 735 740 2-s2.0-67649484693 19496662 18 Viljanen M Kuitunen M Haahtela T Juntunen-Backman K Korpela R Savilahti E Probiotic effects on faecal inflammatory markers and on faecal IgA in food allergic atopic eczema/dermatitis syndrome infants Pediatric Allergy and Immunology 2005 16 1 65 71 2-s2.0-14644399194 15693914 19 Tanaka A Jung K Benyacoub J Oral supplementation with Lactobacillus rhamnosus CGMCC 1.3724 prevents development of atopic dermatitis in NC/NgaTnd mice possibly by modulating local production of IFN-γ Experimental Dermatology 2009 18 12 1022 1027 2-s2.0-70849099735 19555432 20 Tanaka A Fukushima Y Benyacoub J Blum S Matsuda H Prophylactic effect of oral administration of Lactobacillus johnsonii NCC533 (La1) during the weaning period on atopic dermatitis in NC/NgaTnd mice European Journal of Dermatology 2008 18 2 136 140 2-s2.0-42349101516 18424371 21 Wakabayashi H Nariai C Takemura F Nakao W Fujiwara D Dietary supplementation with lactic acid bacteria attenuates the development of atopic dermatitis-like skin lesions in NC/Nga mice in a strain-dependent manner International Archives of Allergy and Immunology 2008 145 2 141 151 2-s2.0-38349169577 17848807 22 Segawa S Hayashi A Nakakita Y Kaneda H Watari J Yasui H Oral administration of heat-killed Lactobacillus brevis SBC8803 ameliorates the development of dermatitis and inhibits immunoglobulin E production in atopic dermatitis model NC/Nga mice Biological and Pharmaceutical Bulletin 2008 31 5 884 889 2-s2.0-44349151001 18451512 23 Park CW Youn M Jung Y-M New functional probiotic Lactobacillus sakei probio 65 alleviates atopic symptoms in the mouse Journal of Medicinal Food 2008 11 3 405 412 2-s2.0-52149087768 18800885 24 Chapat L Chemin K Dubois B Bourdet-Sicard R Kaiserlian D Lactobacillus casei reduces CD8+ T cell-mediated skin inflammation European Journal of Immunology 2004 34 9 2520 2528 2-s2.0-4644331406 15307184 25 Hacini-Rachinel F Gheit H Le Luduec J-B Dif F Nancey S Kaiserlian D Oral probiotic control skin inflammation by acting on both effector and regulatory T cells PLoS ONE 2009 4 3 2-s2.0-63749121653 e4903 26 Weise C Zhu Y Ernst D Kühl AA Worm M Oral administration of Escherichia coli Nissle 1917 prevents allergen-induced dermatitis in mice Experimental Dermatology 2011 20 10 805 809 2-s2.0-80052966421 21740462 27 Rosenfeldt V Benfeldt E Nielsen SD Effect of probiotic Lactobacillus strains in children with atopic dermatitis Journal of Allergy and Clinical Immunology 2003 111 2 389 395 2-s2.0-0037327049 12589361 28 Sistek D Kelly R Wickens K Stanley T Fitzharris P Crane J Is the effect of probiotics on atopic dermatitis confined to food sensitized children? Clinical and Experimental Allergy 2006 36 5 629 633 2-s2.0-33646263600 16650048 29 Kukkonen K Savilahti E Haahtela T Probiotics and prebiotic galacto-oligosaccharides in the prevention of allergic diseases: a randomized, double-blind, placebo-controlled trial Journal of Allergy and Clinical Immunology 2007 119 1 192 198 2-s2.0-33845951414 17208601 30 Kuitunen M Kukkonen K Juntunen-Backman K Probiotics prevent IgE-associated allergy until age 5 years in cesarean-delivered children but not in the total cohort Journal of Allergy and Clinical Immunology 2009 123 2 335 341 2-s2.0-59449087653 19135235 31 Abrahamsson TR Jakobsson T Böttcher MF Probiotics in prevention of IgE-associated eczema: a double-blind, randomized, placebo-controlled trial Journal of Allergy and Clinical Immunology 2007 119 5 1174 1180 2-s2.0-34247502904 17349686 32 Isolauri E Studies on Lactobacillus GG in food hypersensitivity disorders Nutrition Today 1996 31 6 285 315 33 Isolauri E Sütas Y Kankaanpää P Arvilommi H Salminen S Probiotics: effects on immunity The American Journal of Clinical Nutrition 2001 73 2, supplement 444S 450S 11157355 34 Wickens K Black PN Stanley TV A differential effect of 2 probiotics in the prevention of eczema and atopy: a double-blind, randomized, placebo-controlled trial Journal of Allergy and Clinical Immunology 2008 122 4 788 794 2-s2.0-53049095486 18762327 35 West CE Hammarström M-L Hernell O Probiotics during weaning reduce the incidence of eczema Pediatric Allergy and Immunology 2009 20 5 430 437 2-s2.0-68349097604 19298231 36 Woo S-I Kim J-Y Lee Y-J Kim N-S Hahn Y-S Effect of Lactobacillus sakei supplementation in children with atopic eczema-dermatitis syndrome Annals of Allergy, Asthma and Immunology 2010 104 4 343 348 2-s2.0-77950011957 37 Weston S Halbert A Richmond P Prescott SL Effects of probiotics on atopic dermatitis: a randomised controlled trial Archives of Disease in Childhood 2005 90 9 892 897 2-s2.0-20444475470 15863468 38 Hoang BX Shaw G Pham P Levine SA Lactobacillus rhamnosus cell lysate in the management of resistant childhood atopic Eczema Inflammation and Allergy 2010 9 3 192 196 2-s2.0-77958462529 39 Hattori K Yamamoto A Sasai M Effects of administration of Bifidobacteria on fecal microflora and clinical symptoms in infants with atopic dermatitis Japanese Journal of Allergology 2003 52 1 20 30 2-s2.0-0037225805 12598719 40 Marschan E Kuitunen M Kukkonen K Probiotics in infancy induce protective immune profiles that are characteristic for chronic low-grade inflammation Clinical and Experimental Allergy 2008 38 4 611 618 2-s2.0-40949158856 18266878 41 Niers L Martín R Rijkers G The effects of selected probiotic strains on the development of eczema (the PandA study) Allergy 2009 64 9 1349 1358 2-s2.0-68949207372 19392993 42 Kim JY Kwon JH Ahn SH Effect of probiotic mix (Bifidobacterium bifidum, Bifidobacterium lactis , Lactobacillus acidophilus ) in the primary prevention of eczema: a double-blind, randomized, placebo-controlled trial Pediatric Allergy and Immunology 2010 21 2 e386 e393 2-s2.0-77950235521 19840300 43 Dotterud CK Storrø O Johnsen R Øien T Probiotics in pregnant women to prevent allergic disease: a randomized, double-blind trial British Journal of Dermatology 2010 163 3 616 623 2-s2.0-77955856378 20545688 44 Böttcher MF Abrahamsson TR Fredriksson M Jakobsson T Björkstén B Low breast milk TGF-β 2 is induced by Lactobacillus reuteri supplementation and associates with reduced risk of sensitization during infancy Pediatric Allergy and Immunology 2008 19 6 497 504 2-s2.0-49749117133 18221472 45 Lodinova-Zadnikova R Cukrowska B Tlaskalova-Hogenova H Oral administration of probiotic Escherichia coli after birth reduces frequency of allergies and repeated infections later in life (after 10 and 20 years) International Archives of Allergy and Immunology 2003 131 3 209 211 2-s2.0-0041663833 12876412 46 Gerasimov SV Vasjuta VV Myhovych OO Bondarchuk LI Probiotic supplement reduces Atopic Dermatitis in preschool children: a randomized, double-blind, placebo-controlled, clinical trial The American Journal of Clinical Dermatology 2010 11 5 351 361 2-s2.0-77954803014 20642296 47 Boyle RJ Ismail IH Kivivuori S Lactobacillus GG treatment during pregnancy for the prevention of eczema: a randomized controlled trial Allergy 2011 66 4 509 516 2-s2.0-79952021189 21121927 48 Boyle RJ Bath-Hextall FJ Leonardi-Bee J Murrell DF Tang ML-K Probiotics for the treatment of eczema: a systematic review Clinical and Experimental Allergy 2009 39 8 1117 1127 2-s2.0-67650436138 19573037 49 Taylor AL Dunstan JA Prescott SL Probiotic supplementation for the first 6 months of life fails to reduce the risk of atopic dermatitis and increases the risk of allergen sensitization in high-risk children: a randomized controlled trial Journal of Allergy and Clinical Immunology 2007 119 1 184 191 2-s2.0-33845931138 17208600 50 Kopp MV Hennemuth I Heinzmann A Urbanek R Randomized, double-blind, placebo-controlled trial of probiotics for primary prevention: no clinical effects of lactobacillus GG supplementation Pediatrics 2008 121 4 e850 e856 2-s2.0-40949124307 18332075 51 Grüber C Wendt M Sulser C Randomized, placebo-controlled trial of Lactobacillus rhamnosus GG as treatment of atopic dermatitis in infancy Allergy 2007 62 11 1270 1276 2-s2.0-35148888616 17919141 52 Brouwer ML Wolt-Plompen SAA Dubios AEJ No effects of probiotics on atopic dermatitis in infancy: a randomized placebo-controlled trial Clinical and Experimental Allergy 2006 36 7 899 906 2-s2.0-33745641869 16839405 53 Fölster-Holst R Müller F Schnopp N Prospective, randomized controlled trial on Lactobacillus rhamnosus in infants with moderate to severe atopic dermatitis British Journal of Dermatology 2006 155 6 1256 1261 2-s2.0-33750901306 17107398 54 Soh SE Aw M Gerez I Probiotic supplementation in the first 6 months of life in at risk Asian infants: effects on eczema and atopic sensitization at the age of 1 year Clinical and Experimental Allergy 2009 39 4 571 578 2-s2.0-62449251219 19134020 55 Metchnikoff E The Prolongation of Life: Optimistic Studies 1907 London, UK G. P. Putnam and Sons 56 Food and Agriculture Organization Report of Joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria FAO/WHO Report 10-1-2001 57 Shiohara T Hayakawa J Mizukawa Y Animal models for atopic dermatitis: are they relevant to human disease? Journal of Dermatological Science 2004 36 1 1 9 2-s2.0-5644282657 15488700 58 Bermudez-Brito M Plaza-Díaz J Muñoz-Quezada S Gómez-Llorente C Gil A Probiotic mechanisms of action Annals of Nutrition and Metabolism 2012 61 2 160 174 23037511 59 Sudo N Sawamura S-A Tanaka K Aiba Y Kubo C Koga Y The requirement of intestinal bacterial flora for the development of an IgE production system fully susceptible to oral tolerance induction Journal of Immunology 1997 159 4 1739 1745 2-s2.0-0031571230 60 Kirjavainen PV Arvola T Salminen SJ Isolauri E Aberrant composition of gut microbiota of allergic infants: a target of bifidobacterial therapy at weaning? Gut 2002 51 1 51 55 2-s2.0-0036298165 12077091 61 Pessi T Isolauri E Sütas Y Kankaanranta H Moilanen E Hurme M Suppression of T-cell activation by Lactobacillus rhamnosus GG-degraded bovine casein International Immunopharmacology 2001 1 2 211 218 2-s2.0-0035117985 11360922 62 Cebra JJ Influences of microbiota on intestinal immune system development The American Journal of Clinical Nutrition 1999 69 5 2-s2.0-0032935617 63 Inoue R Nishio A Fukushima Y Ushida K Oral treatment with probiotic Lactobacillus johnsonii NCC533 (La1) for a specific part of the weaning period prevents the development of atopic dermatitis induced after maturation in model mice, NC/Nga British Journal of Dermatology 2007 156 3 499 509 2-s2.0-33846952104 17300240 64 Kaila M Isolauri E Soppi E Virtanen E Laine S Arvilommi H Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain Pediatric Research 1992 32 2 141 144 2-s2.0-0026724964 1324462 65 Ruemmele FM Bier D Marteau P Clinical evidence for immunomodulatory effects of probiotic bacteria Journal of Pediatric Gastroenterology and Nutrition 2009 48 2 126 141 2-s2.0-66149146312 19179874 66 Christensen HR Frøkiær H Pestka JJ Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells Journal of Immunology 2002 168 1 171 178 2-s2.0-0036136282 67 Salminen SJ Gueimonde M Isolauri E Probiotics that modify disease risk Journal of Nutrition 2005 135 5 1294 1298 2-s2.0-18344387591 15867327 68 Won TJ Kim B Lim YT Oral administration of Lactobacillus strains from Kimchi inhibits atopic dermatitis in NC/Nga mice Journal of Applied Microbiology 2011 110 5 1195 1202 2-s2.0-79953752241 21338447 69 Sunada Y Nakamura S Kamei C Effect of Lactobacillus acidophilus strain L-55 on the development of atopic dermatitis-like skin lesions in NC/Nga mice International Immunopharmacology 2008 8 13-14 1761 1766 2-s2.0-55249100582 18790088 70 Thomas DJ Husmann RJ Villamar M Winship TR Buck RH Zuckermann FA Lactobacillus rhamnosus HN001 attenuates allergy development in a pig model PLoS ONE 2011 6 2 2-s2.0-79952215777 e16577 71 Pohjavuori E Viljanen M Korpela R Lactobacillus GG effect in increasing IFN-γ production in infants with cow’s milk allergy Journal of Allergy and Clinical Immunology 2004 114 1 131 136 2-s2.0-3442896194 15241356 72 Prescott SL Dunstan JA Hale J Clinical effects of probiotics are associated with increased interferon-γ responses in very young children with atopic dermatitis Clinical and Experimental Allergy 2005 35 12 1557 1564 2-s2.0-33646177970 16393321 73 Niers LEM Timmerman HM Rijkers GT Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines Clinical and Experimental Allergy 2005 35 11 1481 1489 2-s2.0-33644937046 16297146 74 Maassen CBM van Holten-Neelen C Balk F Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains Vaccine 2000 18 23 2613 2623 2-s2.0-0034701834 10775795 75 Betsi GI Papadavid E Falagas ME Probiotics for the treatment or prevention of atopic dermatitis: a review of the evidence from randomized controlled trials The American Journal of Clinical Dermatology 2008 9 2 93 103 2-s2.0-39549097953 18284263 76 Inoue R Otsuka M Nishio A Ushida K Primary administration of Lactobacillus johnsonii NCC533 in weaning period suppresses the elevation of proinflammatory cytokines and CD86 gene expressions in skin lesions in NC/Nga mice FEMS Immunology and Medical Microbiology 2007 50 1 67 76 2-s2.0-34248588146 17425659 77 Niers LEM Hoekstra MO Timmerman HM Selection of probiotic bacteria for prevention of allergic diseases: immunomodulation of neonatal dendritic cells Clinical and Experimental Immunology 2007 149 2 344 352 2-s2.0-34447300419 17521319 78 Hart AL Lammers K Brigidi P Modulation of human dendritic cell phenotype and function by probiotic bacteria Gut 2004 53 11 1602 1609 2-s2.0-7244226119 15479680 79 Braat H van den Brande J van Tol E Hommes D Peppelenbosch M van Deventer S Lactobacillus rhamnosus induces peripheral hyporesponsiveness in stimulated CD4+ T cells via modulation of dendritic cell function The American Journal of Clinical Nutrition 2004 80 6 1618 1625 2-s2.0-16544392923 15585777 80 Smits HH Engering A van der Kleij D Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin Journal of Allergy and Clinical Immunology 2005 115 6 1260 1267 2-s2.0-20444503737 15940144 81 Lavasani S Dzhambazov B Nouri M A novel probiotic mixture exerts a therapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10 producing regulatory T cells PLoS ONE 2010 5 2 2-s2.0-77749322389 e9009 82 Fujimura T Okuyama R Ito Y Aiba S Profiles of Foxp3+ regulatory T cells in eczematous dermatitis, psoriasis vulgaris and mycosis fungoides British Journal of Dermatology 2008 158 6 1256 1263 2-s2.0-43749092317 18363755 83 Kwon H-K Lee C-G So J-S Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders Proceedings of the National Academy of Sciences of the United States of America 2010 107 5 2159 2164 2-s2.0-76649140863 20080669 84 Hoarau C Lagaraine C Martin L Velge-Roussel F Lebranchu Y Supernatant of Bifidobacterium breve induces dendritic cell maturation, activation, and survival through a Toll-like receptor 2 pathway Journal of Allergy and Clinical Immunology 2006 117 3 696 702 2-s2.0-33644752602 16522473 85 Kalliomäki M Antoine J-M Herz U Rijkers GT Wells JM Mercenier A Guidance for substantiating the evidence for beneficial effects of probiotics: prevention and management of allergic diseases by probiotics Journal of Nutrition 2010 140 3 713 721 2-s2.0-77249173797 86 Jin H He R Oyoshi M Geha RS Animal models of atopic dermatitis The Journal of investigative dermatology 2009 129 1 31 40 2-s2.0-58149340599 19078986 87 Kim JY Park BK Park HJ Park YH Kim BO Pyo S Atopic dermatitis-mitigating effects of new Lactobacillus strain, Lactobacillus sakei probio 65 isolated from Kimchi Journal of Applied Microbiology 2013 115 2 517 526 23607518 88 Goto K Iwasawa D Kamimura Y Yasuda M Matsumura M Shimada T Clinical and histopathological evaluation of Dermatophagoides farinae-induced dermatitis in NC/Nga mice orally administered Bacillus subtilis Journal of Veterinary Medical Science 2011 73 5 649 654 2-s2.0-79958034947 21206175 89 Sunada Y Nakamura S Kamei C Effects of Lactobacillus acidophilus strain L-55 on experimental allergic rhinitis in BALB/c mice Biological and Pharmaceutical Bulletin 2007 30 11 2163 2166 2-s2.0-36048931210 17978493 90 Granato D Bergonzelli GE Pridmore RD Marvin L Rouvet M Corthésy-Theulaz IE Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins Infection and Immunity 2004 72 4 2160 2169 15039339 91 Guéniche A Benyacoub J Buetler TM Smola H Blum S Supplementation with oral probiotic bacteria maintains cutaneous immune homeostasis after UV exposure European Journal of Dermatology 2006 16 5 511 517 17101471 92 Won TJ Kim B Lim YT Oral administration of Lactobacillus strains from Kimchi inhibits atopic dermatitis in NC/Nga mice Journal of Applied Microbiology 2011 110 5 1195 1202 2-s2.0-79953752241 21338447 93 Viljanen M Savilahti E Haahtela T Probiotics in the treatment of atopic eczema/dermatitis syndrome in infants: a double-blind placebo-controlled trial Allergy 2005 60 4 494 500 2-s2.0-14744293190 15727582 94 Kukkonen K Kuitunen M Haahtela T Korpela R Poussa T Savilahti E High intestinal IgA associates with reduced risk of IgE-associated allergic diseases Pediatric Allergy and Immunology 2010 21 1 67 73 2-s2.0-76149143135 19566584 95 Isolauri E Rautava S Kalliomäki M Kirjavainen P Salminen S Role of probiotics in food hypersensitivity Current Opinion in Allergy and Clinical Immunology 2002 2 3 263 271 2-s2.0-0036275394 12045425 96 Kirjavainen PV Salminen SJ Isolauri E Probiotic bacteria in the management of atopic disease: underscoring the importance of viability Journal of Pediatric Gastroenterology and Nutrition 2003 36 2 223 227 2-s2.0-0037530080 12548058 97 Huurre A Laitinen K Rautava S Korkeamäki M Isolauri E Impact of maternal atopy and probiotic supplementation during pregnancy on infant sensitization: a double-blind placebo-controlled study Clinical and Experimental Allergy 2008 38 8 1342 1348 2-s2.0-47649113201 18477013 98 Moroi M Uchi S Nakamura K Beneficial effect of a diet containing heat-killed Lactobacillus paracasei K71 on adult type atopic dermatitis Journal of Dermatology 2011 38 2 131 139 2-s2.0-79251551091 21269308 99 Matsumoto M Aranami A Ishige A Watanabe K Benno Y LKM512 yogurt consumption improves the intestinal environment and induces the T-helper type 1 cytokine in adult patients with intractable atopic dermatitis Clinical and Experimental Allergy 2007 37 3 358 370 2-s2.0-33847795377 17359386 100 Prescott SL Björkstén B Probiotics for the prevention or treatment of allergic diseases Journal of Allergy and Clinical Immunology 2007 120 2 255 262 17544096 101 Fiocchi A Burks W Bahna SL Clinical use of probiotics in pediatric allergy (cuppa): a world allergy organization position paper World Allergy Organization Journal 2012 5 11 148 167 23282383 102 Salfeld P Kopp MV Probiotics cannot be generally recommended for primary prevention of atopic dermatitis Journal of Allergy and Clinical Immunology 2009 124 1 p. 170 2-s2.0-67649224661	24078929	PMC3773919	CC BY	2021-01-05 10:06:53	yes	Biomed Res Int. 2013 Sep 1; 2013:932391
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24069158PONE-D-13-1784810.1371/journal.pone.0072783Research ArticleTNF-α Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation TNF-α Induces cPLA2 ExpressionLee I-Ta 1 2 Lin Chih-Chung 1 Cheng Shin-Ei 2 Hsiao Li-Der 2 Hsiao Yu-Chun 2 Yang Chuen-Mao 2 * 1 Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan 2 Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan Liu Guangwei Editor Department of Immunology, China * E-mail: chuenmao@mail.cgu.edu.twCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: ITL CCL CMY. Performed the experiments: ITL SEC LDH YCH. Analyzed the data: HCT ITL LDH CMY. Contributed reagents/materials/analysis tools: ITL CCL SEC LDH YCH. Wrote the paper: ITL CCL CMY. 2013 19 9 2013 8 9 e727831 5 2013 11 7 2013 © 2013 Lee et al2013Lee et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Principal Findings We demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, p42, p38, JNK2, c-Jun, c-Fos, or ATF2. We showed that TNF-α markedly stimulated p42/p44 MAPK, p38 MAPK, and JNK1/2 phosphorylation which were attenuated by their respective inhibitors. In addition, TNF-α also stimulated c-Jun and ATF2 phosphorylation which were inhibited by pretreatment with SP600125 and SB202190, respectively, but not PD98059. Furthermore, TNF-α-induced cPLA2 promoter activity was abrogated by transfection with the point-mutated AP-1 cPLA2 construct. Finally, we showed that TNF-α time-dependently induced p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs. On the other hand, TNF-α induced in vivo binding of c-Jun, c-Fos, ATF2, and p300 to the cPLA2 promoter in these cells. In an in vivo study, we found that TNF-α induced leukocyte count in BAL fluid of mice and cPLA2 mRNA levels in lung tissues via MAPKs and AP-1. Significance Taken together, these results demonstrated that TNF-α-induced cPLA2 expression was mediated through p38 MAPK- and JNK1/2-dependent p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs. This work was supported by the Ministry of Education, Taiwan, grant numbers EMRPD1C0261 and EMRPD1C0271; National Science Council, Taiwan, grant numbers NSC101-2321-B-182-013, NSC101-2320-B-182-039-MY3, NSC99-2321-B182-003, and NSC98-2320-B-255-001-MY3; and Chang Gung Medical Research Foundation, grant numbers CMRPD180373, CMRPD1B0381, CMRPG391033, and CMRPG3B1091. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Lung inflammation is a pivotal event in the pathogenesis of chronic obstructive pulmonary disease (COPD) and asthma [1]. Several lipid mediators, such as eicosanoids generated from arachidonic acid (AA) have been identified in situ in airway secretion of asthmatics [2], [3]. The generation of eicosanoids is first initiated through the release of AA from membrane phospholipids hydrolyzed by the action of phospholipase A2 (PLA2) enzymes [4]. AA is further converted to prostaglandins (PGs), such as PGE2 by the constitutive enzyme cyclooxygenase (COX)-1 or the inducible COX-2 in various cell types [5], [6]. The PLA2 superfamily is composed of three main types of lipolytic enzymes, including secretory PLA2, the 85 kDa cytosolic group IV PLA2 (cPLA2), and a calcium-independent group VI PLA2 in mammalian cells [7]. cPLA2 is the only one that plays a key role in mediating agonist-induced AA release for eicosanoid production in various cell types [8]. It has been demonstrated that activation of the MAPKs, including p42/p44 MAPK, p38 MAPK, and JNK1/2, by pro-inflammatory stimuli leads to the phosphorylation of cPLA2 at Ser505 and Ser727 [9] with Ca2+/calmodulin kinase II-dependent phosphorylation of Ser515 associated with increased enzymatic activity [10]. cPLA2 has been shown to be implicated in acute lung injury induced by sepsis [11] and bronchial reactivity associated with anaphylaxis [12]. Furthermore, increased PGE2 synthesis is dependent on an increase in cPLA2 activity in various cell types [13], [14]. Elevated levels of pro-inflammatory cytokines, including TNF-α in the bronchoalveolar lavage fluid have been detected in allergic asthmatic patients. TNF-α exerts as a potent stimulus in inflammatory responses through up-regulation of target genes, such as cPLA2 in various cell types [15], [16]. The expression of cPLA2 induced by TNF-α may be integrated to the signaling networks that augment lung inflammation by enhancing PGE2 synthesis. Although cPLA2 has been shown to mediate inflammatory reactions, the detail mechanisms underlying TNF-α-induced cPLA2 expression and PGE2 synthesis in human lung epithelial cells (HPAEpiCs) were not completely understood. Several extracellular stimuli elicit a broad spectrum of biological responses through activation of MAPK cascades leading to phosphorylation of specific target proteins [17]. Moreover, we have demonstrated that TNF-α causes a rapid phosphorylation of p42/p44 MAPK or p38 MAPK and up-regulation of COX-2 in human airway smooth muscle cells [18]. In addition, JNK1/2, p42/p44 MAPK, and p38 MAPK have also been shown to be involved in lipopolysaccharide (LPS)-induced cPLA2 induction in canine tracheal smooth muscle cells [19]. On the other hand, we have also indicated that MAPKs and NF-κB were involved in TNF-α-induced PGE2 release in human airway smooth muscle cells [20]. Therefore, in this study, we investigated the roles of MAPKs in TNF-α-mediated cPLA2 expression and PGE2 synthesis in HPAEpiCs. AP-1 is a heterogeneous collection of dimeric transcription factors comprising Jun, Fos, and ATF subunits. Among AP-1 subunits, c-Jun is the most important transcriptional activator in inflammatory status [21]. AP-1 activity is regulated by multiple mechanisms, including phosphorylation by various MAPKs [22]. Among MAPKs, JNK1/2 predominantly plays an important role in TNF-α-induced AP-1 activity, which contributes to the induction of TNF-α-targeted genes [23]. Histone acetyltransferases (HATs), such as p300 and CREB-binding protein functioning as transcriptional co-activators and signal integrators have been proved to play a vital role in expression of inflammatory genes, such as cPLA2 or COX-2 [20], [24]. By this model, the activities of HATs must be tightly regulated in response to various stimuli, such as TNF-α, IL-1β, and bacterial toxins [25], [26]. It has been demonstrated that pulmonary inflammation, exacerbated asthma, and COPD induced by exposure to diesel exhaust particulate matter are related to the p300 activation and recruitment to the promoter region of COX-2 [27]. Thus, the role of p300 in TNF-α-mediated AP-1 activation leading to cPLA2 expression was also investigated in HPAEpiCs. In addressing these questions, experiments were performed to investigate the mechanisms underlying TNF-α-induced cPLA2 expression and PGE2 synthesis in HPAEpiCs. These findings suggested that in HPAEpiCs, TNF-α-induced cPLA2 expression associated with PGE2 release was, at least in part, mediated through JNK1/2- and p38 MAPK-dependent p300-AP-1 signaling pathway. These results demonstrated that MAPKs and AP-1 may be the critical components implicated in cPLA2 expression and PGE2 synthesis in TNF-α-challenged HPAEpiCs. Methods Materials Recombinant human TNF-α was from R&D System (Minneapolis, MN). Anti-cPLA2, anti-GAPDH, anti-TNFR1, anti-p42, anti-p38, anti-JNK2, anti-c-Jun, anti-c-Fos, anti-ATF2, and anti-p300 antibodies were from Santa Cruz (Santa Cruz, CA). Anti-phospho-p42/p44 MAPK, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were from Cell Signaling (Danver, MA). Actinomycin D (Act. D), cycloheximide (CHI), SP600125, PD98059, SB202190, AACOCF3, and Tanshinone IIA were from Biomol (Plymouth Meeting, PA). AH 6809, SC-19220, and GW627368X were from Cayman (Ann Arbor, MI). Other chemicals were from Sigma (St. Louis, MO). Cell culture Human pulmonary alveolar epithelial cells (HPAEpiCs, type II alveolar epithelial cells) were purchased from the ScienCell Research Lab. (San Diego, CA) and grown as previously described [28]. Western blot analysis Growth-arrested HPAEpiCs were incubated with thrombin at 37°C for the indicated time intervals. The cells were washed, scraped, collected, and centrifuged at 45000× g at 4°C for 1 h to yield the whole cell extract, as previously described [28]. Samples were denatured, subjected to SDS-PAGE using a 10% running gel, transferred to nitrocellulose membrane, incubated with an anti-cPLA2 antibody for 24 h, and then incubated with an anti-mouse horseradish peroxidase antibody for 1 h. The immunoreactive bands were detected by ECL reagents. Real-time PCR Total RNA was extracted using TRIzol reagent. mRNA was reverse-transcribed into cDNA and analyzed by real-time RT-PCR. Real-time PCR was performed using SYBR Green PCR reagents (Applied Biosystems, Branchburg, NJ) and primers specific for cPLA2 and GAPDH mRNAs. The levels of cPLA2 expression were determined by normalizing to GAPDH expression. Measurement of cPLA2 luciferase activity For construction of the cPLA2-luc plasmid, human cPLA2 promoter, a region spanning −2375 to +75 bp, was cloned into pGL3-basic vector (Promega, Madison, WI). cPLA2-luc activity was determined as previously described [28] using a luciferase assay system (Promega, Madison, WI). Firefly luciferase activities were standardized for β-gal activity. Measurement of PGE2 generation Cells were cultured in 6-well culture plates. After reaching confluence, cells were treated with TNF-α for the indicated times. After treatment, the medium were collected and stored at −80°C until being assayed. PGE2 was assayed using the PGE2 enzyme immunoassay kit (Cayman) according to the manufacturer's instructions. Transient transfection with siRNAs Human siRNAs of scrambled, TNFR1, p42, p38, JNK2, c-Jun, c-Fos, and ATF2 were from Sigma (St. Louis, MO). Transient transfection of siRNAs (100 nM) was performed using a Lipofectamine™ RNAiMAX reagent according to the manufacturer's instructions. Chromatin immunoprecipitation assay To detect the association of nuclear proteins with human cPLA2 promoter, chromatin immunoprecipitation (ChIP) analysis was conducted as previously described [20]. DNA immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody was purified. The DNA pellet was re-suspended in H2O and subjected to PCR amplification with the forward primer 5′-GAATTCAACCTGATTTCATTTTCTTCC-3′ and the reverse primer 5′-CTTCAGGCTCCTCAATGCCTCTAGCTTTCAG-3′, which were specifically designed from the cPLA2 promoter region. PCR products were analyzed on ethidium bromide-stained agarose gels (1%). Co-immunoprecipitation assay Cell lysates containing 1 mg of protein were incubated with 2 µg of an anti-p300 antibody at 4°C for 24 h, and then 10 µl of 50% protein A-agarose beads was added and mixed for 24 h at 4°C. The immunoprecipitates were collected and washed three times with a lysis buffer without Triton X-100. 5X Laemmli buffer was added and subjected to electrophoresis on SDS-PAGE, and then blotted using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. Animal care, ethics statement, and experimental procedures Male ICR mice aged 6–8 weeks were purchased from the National Laboratory Animal Centre (Taipei, Taiwan) and handled according to the guidelines of Animal Care Committee of Chang Gung University approved for this study (IACUC approval number: 12-048) and National Institutes of Health Guides for the Care and Use of Laboratory Animals. All treatments were performed under pentobarbital sodium anesthesia, and all efforts were made to minimize suffering. ICR mice were anesthetized with intraperieritoneal injection of 200 µl of pentobarbital sodium (5 mg/ml) and placed individually on a board in a near vertical position and the tongues were withdrawn with a lined forceps. TNF-α (0.125 mg/kg body weight) was placed posterior in the throat and aspirated into lungs. Control mice were administrated sterile 0.1% BSA. Mice regained consciousness after 15 min. Mice were i.p. given one dose of PD98059, SB202190, SP600125, Tanshinone IIA, AH 6809, SC-19220, GW627368X, or AACOCF3 (2 mg/kg) for 1 h prior to TNF-α treatment, and sacrificed after 24 h. Isolation of bronchoalveolar lavage (BAL) fluid Mice were injected with TNF-α at a dose of 0.75 mg/kg and sacrificed 24 h later. BAL fluid was performed through a tracheal cannula using 1 ml aliquots of ice-cold PBS medium. BAL fluid was centrifuged at 500× g at 4°C, and cell pellets were washed and re-suspended in PBS. Leukocyte count was determined by a hemocytometer. Analysis of data All data were estimated and made using a GraphPad Prism Program (GraphPad, San Diego, CA, USA). Data were expressed as the mean±S.E.M. and analyzed by one-way ANOVA followed with Tukey's post-hoc test. P<0.05 was considered significant. Results TNF-α induces cPLA2 expression and PGE2 release in HPAEpiCs To determine the effect of TNF-α on cPLA2 expression, cells were incubated with TNF-α for the indicated time intervals. As shown in Fig. 1A, TNF-α induced cPLA2 protein expression in a time-dependent manner with a maximal response within 16–24 h. Moreover, TNF-α also enhanced cPLA2 mRNA accumulation in a time-dependent manner with a maximal response within 4–6 h (Fig. 1B). On the other hand, TNF-α markedly induced cPLA2 promoter activity in these cells (Fig. 1B). cPLA2 is the major form of PLA2, which selectively hydrolyzes membrane phospholipids at the sn-2 position and is the rate-limiting enzyme in the release of AA [20]. AA is further converted to PGs (i.e. PGE2) by the constitutive enzyme COX-1 or by the inducible COX-2. In our previous study, up-regulation of COX-2 has been shown to induce PGE2 synthesis by TNF-α [18]. Therefore, the synthesis of PGE2 is a good index of AA release that is more sensitive than [3H]AA mobilization [18]. We further tested the effect of TNF-α on PGE2 synthesis as a parameter of cPLA2 activity. As shown in Fig. 1C, TNF-α induced a time-dependent increase in PGE2 synthesis. These results suggested that TNF-α induces cPLA2 expression associated with PGE2 generation in HPAEpiCs. 10.1371/journal.pone.0072783.g001Figure 1 TNF-α induces cPLA2 protein and mRNA expression. Cells were incubated with TNF-α for the indicated time intervals. (A) The protein levels of cPLA2 were determined by Western blot, (B) the mRNA levels of cPLA2 were determined by real-time PCR, and the promoter activity of cPLA2 was determined in the cell lysates. (C) Cells were incubated with TNF-α (30 ng/ml) for the indicated time intervals. The media were collected and analyzed for PGE2 release. Data are expressed as mean±S.E.M. of three independent experiments. P<0.05; # P<0.01, as compared with the cells exposed to vehicle alone. TNF-α induces cPLA2 expression via TNFR1 in HPAEpiCs To further determine whether TNF-α-induced cPLA2 expression required transcription or translation, cells were stimulated with TNF-α (30 ng/ml) in the presence of an inhibitor of transcriptional level, actinomycin D (Act. D) or translational level, cycloheximide (CHI) and cPLA2 protein expression was determined by Western blot. As shown in Figs. 2A and B, TNF-α-mediated cPLA2 protein expression and PGE2 release was abolished by either Act. D or CHI in a concentration-dependent manner, while cPLA2 mRNA levels were only attenuated by Act. D. Taken together, these findings demonstrated that the induction of cPLA2 expression by TNF-α depends on de novo protein synthesis in HPAEpiCs. Most of TNF-α actions are elicited through TNFR1 [29]. Thus, we investigated whether TNF-α induced cPLA2 expression via TNFR1 in these cells. As shown in Fig. 2C, transfection with TNFR1 siRNA markedly reduced TNFR1 protein expression, and then inhibited TNF-α-induced cPLA2 expression in HPAEpiCs. Therefore, TNFR1 mainly plays a key role in TNF-α-induced inflammatory responses. 10.1371/journal.pone.0072783.g002Figure 2 TNF-α induces cPLA2 expression via TNFR1 in HPAEpiCs. (A) Cells were pretreated with Act. D or CHI for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA2 were determined by Western blot. (B) Cells were pretreated with Act. D (1 µM) or CHI (1 µM) for 1 h, and then incubated with TNF-α for 6 h (for cPLA2 mRNA levels) or 24 h (for PGE2 release). cPLA2 mRNA levels were determined by real-time PCR. The media were collected and analyzed for PGE2 release. (C) Cells were transfected with scrambled or TNFR1 siRNA, and then incubated with TNF-α for 24 h. The protein expression of TNFR1 and cPLA2 were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P<0.01, as compared with the cells exposed to TNF-α alone. p42/p44 MAPK is involved in TNF-α-induced cPLA2 expression in HPAEpiCs Previous studies demonstrated that TNF-α could induce MAPKs activation in human airway smooth muscle cells [18], [20]. Thus, we further investigated whether TNF-α-induced cPLA2 expression was also mediated via p42/p44 MAPK in HPAEpiCs. As shown in Figs. 3A and B, pretreatment with PD98059 (an inhibitor of MEK1/2) attenuated TNF-α-induced cPLA2 protein and mRNA expression, and promoter activity. To further ensure that TNF-α-induced cPLA2 expression was mediated via p42/p44 MAPK, as shown in Fig. 3C, transfection with p42 siRNA significantly down-regulated p42 protein expression and subsequently led to a decrease of cPLA2 protein expression by TNF-α. Finally, we showed that TNF-α stimulated p42/p44 MAPK phosphorylation in a time-dependent manner, which was reduced by PD98059 during the period of observation (Fig. 3D). These data indicated that MEK1/2-p42/p44 MAPK cascade was involved in TNF-α-induced cPLA2 expression in HPAEpiCs. 10.1371/journal.pone.0072783.g003Figure 3 p42/p44 MAPK is involved in TNF-α-induced cPLA2 expression. (A) Cells were pretreated with PD98059 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA2 were determined by Western blot. (B) Cells were pretreated with PD98059 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p42 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p42 and cPLA2 were determined. (D) Cells were pretreated with or without PD98059 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p42/p44 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. P<0.05; # P<0.01, as compared with the cells exposed to TNF-α alone. TNF-α induces cPLA2 expression via p38 MAPK in HPAEpiCs cPLA2 expression induced by LPS has been shown to be mediated through p38 MAPK in canine airway smooth muscle cells [19]. To determine whether p38 MAPK was also involved in TNF-α-induced cPLA2 expression in HPAEpiCs, a p38 MAPK inhibitor, SB202190 was used. As shown in Figs. 4A and B, pretreatment with SB202190 inhibited TNF-α-induced cPLA2 protein and mRNA expression, and promoter activity. To further ensure that TNF-α-induced cPLA2 expression was mediated via p38 MAPK in these cells, as shown in Fig. 4C, transfection with p38 siRNA significantly down-regulated p38 MAPK protein expression and subsequently led to a decrease of cPLA2 protein expression by TNF-α. Finally, we showed that TNF-α stimulated p38 MAPK phosphorylation in a time-dependent manner, which was reduced by SB202190 during the period of observation (Fig. 4D). These data indicated that p38 MAPK cascade was involved in TNF-α-induced cPLA2 expression in HPAEpiCs. 10.1371/journal.pone.0072783.g004Figure 4 p38 MAPK is involved in TNF-α-induced cPLA2 expression. (A) Cells were pretreated with SB202190 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA2 were determined by Western blot. (B) Cells were pretreated with SB202190 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p38 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p38 and cPLA2 were determined. (D) Cells were pretreated with or without SB202190 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P<0.01, as compared with the cells exposed to TNF-α alone. TNF-α enhances cPLA2 expression via JNK1/2 in HPAEpiCs Expression of cPLA2 in lung epithelial cells and non-small cell lung cancer is mediated by Sp1 and c-Jun through JNK1/2 activation [30]. To characterize the role of JNK1/2 in TNF-α-induced cPLA2 expression in HPAEpiCs, a selective inhibitor of JNK1/2, SP600125, was used. As shown in Figs. 5A and B, pretreatment with SP600125 blocked TNF-α-induced cPLA2 protein and mRNA expression, and promoter activity. To further ensure that TNF-α-induced cPLA2 expression was mediated via JNK1/2 in HPAEpiCs, as shown in Fig. 5C, transfection with JNK2 siRNA significantly down-regulated JNK2 expression and subsequently led to a decrease of cPLA2 protein expression in response to TNF-α. Finally, we showed that TNF-α stimulated JNK1/2 phosphorylation in a time-dependent manner, which was reduced by SP600125 during the period of observation (Fig. 5D). These results suggested that JNK1/2 activation was required for TNF-α-induced cPLA2 expression in HPAEpiCs. 10.1371/journal.pone.0072783.g005Figure 5 JNK1/2 is involved in TNF-α-induced cPLA2 expression. (A) Cells were pretreated with SP600125 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA2 were determined by Western blot. (B) Cells were pretreated with SP600125 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or JNK2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of JNK2 and cPLA2 were determined. (D) Cells were pretreated with or without SP600125 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-JNK1/2 were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P<0.01, as compared with the cells exposed to TNF-α alone. AP-1 is involved in TNF-α-induced cPLA2 expression and PGE2 release in HPAEpiCs AP-1 is a transcription factor which is a heterodimeric protein composed of proteins belonging to the c-Fos, c-Jun, ATF, and JDP families [21], which regulates gene expression induced by various stimuli, including cytokines, growth factors, stress, and bacterial and viral infections [21]. To characterize the role of AP-1 in TNF-α-induced cPLA2 expression in HPAEpiCs, a selective inhibitor of AP-1, Tanshinone IIA, was used. As shown in Figs. 6A and B, pretreatment with Tanshinone IIA blocked TNF-α-induced cPLA2 protein and mRNA expression, and promoter activity. To further ensure that TNF-α-induced cPLA2 expression was mediated via AP-1 in HPAEpiCs, as shown in Fig. 6C, transfection with c-Jun or c-Fos siRNA significantly down-regulated c-Jun or c-Fos expression and subsequently led to a decrease of cPLA2 protein expression by TNF-α. To further confirm the role of AP-1 in TNF-α-mediated cPLA2 promoter induction, point-mutated AP-1 cPLA2 promoter construct was used. As shown in Fig. 6D, TNF-α-stimulated cPLA2 promoter activity was prominently lost in HPAEpiCs transfected with point-mutated AP-1 cPLA2 promoter. Finally, we found that pretreatment with PD98059, SB202190, SP600125, or Tanshinone IIA markedly reduced TNF-α-induced PGE2 release in these cells (Fig. 6E). Thus, these data suggested that TNF-α induces cPLA2 expression via an AP-1 signaling in HPAEpiCs. 10.1371/journal.pone.0072783.g006Figure 6 AP-1 is involved in TNF-α-induced cPLA2 expression. (A) Cells were pretreated with Tanshinone IIA (TSIIA) for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA2 were determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (TSIIA), and then incubated with TNF-α for 6 h. cPLA2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled, c-Jun, or c-Fos siRNA, and then incubated with TNF-α for 24 h. The protein levels of c-Jun, c-Fos, and cPLA2 were determined. (D) Cells were transfected with pGL3-empty, wild-type cPLA2 promoter, or AP-1-mutated cPLA2 promoter, and then incubated with TNF-α for 6 h. The promoter activity of cPLA2 was determined in the cell lysates. (E) Cells were pretreated with PD98059 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Tanshinone IIA (TSIIA; 10 µM) for 1 h, and then incubated with TNF-α for 24 h. The media were collected and analyzed for PGE2 release. Data are expressed as mean±S.E.M. of three independent experiments. # P<0.01, as compared with the cells exposed to TNF-α alone (A, B, and E). # P<0.01, as compared with cells transfected with wild-type cPLA2 promoter stimulated by TNF-α (D). TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation in HPAEpiCs ATF2 is a member of the ATF/cyclic AMP-responsive element binding protein family of transcription factors and implicated in inflammatory responses [31]. To ensure that TNF-α-induced cPLA2 expression was mediated via ATF2 in HPAEpiCs, as shown in Fig. 7A, transfection with ATF2 siRNA significantly down-regulated ATF2 expression and subsequently led to a decrease of cPLA2 protein expression by TNF-α. On the other hand, we demonstrated that TNF-α time-dependently induced c-Fos and c-Jun protein expression or c-Jun and ATF2 phosphorylation in these cells (Fig. 7B). We further investigated the relationship between MAPKs and AP-1 in TNF-α-stimulated HPAEpiCs. As shown in Fig. 7C, TNF-α-enhanced ATF2 phosphorylation was inhibited by SB202190, but not PD98059 and SP600125. However, c-Jun phosphorylation stimulated by TNF-α was inhibited by SP600125, but not PD98059 oand SB202190. Thus, we suggested that TNF-α-induced cPLA2 expression is mediated through AP-1 activation which is regulated by p38 MAPK and JNK1/2 but not p42/p44 MAPK in HPAEpiCs. 10.1371/journal.pone.0072783.g007Figure 7 TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P<0.01, as compared with the cells exposed to TNF-α alone. The transcriptional co-activator p300 displays an intrinsic HAT activity which participates in transcriptional activation through the destabilization of nucleosome structure. p300 is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways [20]. Moreover, co-immunoprecipitation study revealed that TNF-α-stimulated p300 directly associated with c-Fos, c-Jun, or ATF2 in a time-dependent manner with a maximal response within 30 min. Finally, the in vivo recruitment of p300, ATF2, c-Fos, and c-Jun to the cPLA2 promoter was assessed by a ChIP assay. In vivo binding of p300, ATF2, c-Fos, and c-Jun to the cPLA2 promoter occurred as early as 15 min and was sustained for 30 min following TNF-α stimulation (Fig. 7E). TNF-α induces leukocyte accumulation in BAL and cPLA2 mRNA expression in mice via MAPKs and AP-1 TNF-α has been shown to induce ROS generation via NADPH oxidase activation, which in turn initiates the activation of various signaling pathways, including PKCs, PI3K/Akt, and MAPKs or transcription factors, such as NF-κB and AP-1, and ultimately induces expression of cPLA2. Moreover, cPLA2 induction may trigger airway and pulmonary diseases, such as asthma and COPD [1]. To further confirm the effects of TNF-α on animal models, mice were (i.p.) injected with PD98059, SB202190, SP600125, or Tanshinone IIA, and then administrated by oropharyngeal route with TNF-α for 24 h. As shown in Fig. 8A, TNF-α markedly induced cPLA2 mRNA expression in lung tissues of mice, which was reduced by PD98059, SB202190, SP600125, or Tanshinone IIA. In addition, we also showed that PD98059, SB202190, SP600125, Tanshinone IIA, or AACOCF3 (an inhibitor of cPLA2) reduced TNF-α-induced leukocyte count in BAL fluid of mice (Fig. 8B). PGE2, one of the major PGs products, exerts its biological activities by binding to specific cell surface receptors, designated PGE2 receptors (EPs). To investigate whether PGE2 could induce leukocyte count in BAL fluid of mice, AH 6809 (an EP1 and EP2 receptor antagonist), SC-19220 (an EP1 receptor antagonist), or GW627368X (an EP4 receptor antagonist) was used. As shown in Fig. 8C, these three EP receptor antagonists reduced TNF-α-induced leukocyte count in BAL fluid of mice. These data suggested that TNF-α may promote leukocyte accumulation and lung inflammation via cPLA2-mediated PGE2 release to cause airway and pulmonary diseases, such as asthma and COPD. 10.1371/journal.pone.0072783.g008Figure 8 TNF-α induces leukocyte accumulation in BAL and cPLA2 mRNA expression in mice via MAPKs and AP-1. (A) Mice were i.p. given one dose of PD98059, SB202190, SP600125, or Tanshinone IIA (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. Lung tissues were homogenized to extract mRNA. The levels of cPLA2 mRNA were determined by real-time PCR. (B, C) Mice were i.p. given one dose of PD98059, SB202190, SP600125, Tanshinone IIA, AACOCF3, AH 6809, SC-19220, or GW627368X (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. (D) Schematic representation of the signaling pathways involved in the TNF-α-induced cPLA2 expression in HPAEpiCs. TNF-α-induced cPLA2 expression and PGE2 release are mediated through p38 MAPK- and JNK1/2-dependent p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs. Discussion Asthma and COPD are pulmonary disorders characterized by various degrees of inflammation and tissue remodeling. Up-regulation of cPLA2 expression by mesenchymal cells in several extra-pulmonary sites may play a key role in generation of PGE2, known as a biologically active lipid mediator implicated in inflammatory responses [32]. TNF-α has been confirmed to induce the late-phase airway hyperresponsiveness and inflammation mediated through activation of cPLA2 [33], but little is known about the intracellular signaling pathways leading to its expression. TNF-α has also been shown to activate MAPKs pathways in several cell types [18], [34]. In addition, AP-1 activity is regulated by multiple mechanisms, including phosphorylation by various MAPKs [22]. Among MAPKs, JNK1/2 predominantly plays an important role in TNF-α-induced AP-1 activity, which contributes to the induction of TNF-α-targeted genes [23]. However, in HPAEpiCs, whether TNF-α-induced cPLA2 expression was mediated through the activation of MAPKs and AP-1 was still unknown. In this study, TNF-α induced cPLA2 expression and PGE2 production which were attenuated by pretreatment with the inhibitors of MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), and AP-1 (Tanshinone IIA) or transfection with siRNAs of p42, p38, JNK2, c-Fos, c-Jun, ATF2, and TNFR1. Here, our results suggested that in HPAEpiCs, TNF-α-induced cPLA2 expression associated with PGE2 release was, at least in part, mediated through JNK1/2- and p38 MAPK-dependent p300-AP-1 signaling pathway. These results demonstrated that MAPKs and AP-1 may be the critical components implicated in cPLA2 expression and PGE2 synthesis in TNF-α-challenged HPAEpiCs. Accumulating evidence demonstrates that TNF-α may activate downstream protein kinases leading to the expression of inflammatory proteins [18], [29]. All known responses to TNF-α are triggered by binding to one of two distinct receptors, designated as TNFR1 and TNFR2 [29]. However, based on cell culture experiments and studies with receptor knockout mice, both the proinflammatory and the programmed cell death pathways that are activated by TNF-α, and associated with tissue injury, are largely mediated through TNFR1 [29], [35]. In contrast, TNFR2 has been shown to mediate signals that promote tissue repair and angiogenesis [36]. Indeed, in HPAEpiCs, we also showed that TNFR1 plays a key role in mediating TNF-α-induced inflammatory responses. Several extracellular stimuli elicit a broad spectrum of biological responses mediated through activation of MAPKs, including p42/p44 MAPK, p38 MAPK, and JNK1/2. Since TNF-α plays an important role in different cellular responses, the activation of these MAPKs is not necessarily restricted to TNF-α-induced cPLA2 expression. For example, activation of JNK1/2 and p42/p44 MAPK is required for up-regulation of cPLA2 in response to oncogenic Ras in normal epithelial cells [16]. In canine airway smooth muscle cells, up-regulation of cPLA2 by LPS is mediated through these MAPKs pathways [19]. Moreover, IL-1β induces expression of cPLA2 in human airway smooth muscle cells, which is regulated by p38 MAPK and JNK1/2, but not p42/p44 MAPK [37]. In the present study, our results demonstrated that activation of p42/p44 MAPK, p38 MAPK, or JNK1/2 was necessary for TNF-α-induced cPLA2 expression and PGE2 release in HPAEpiCs. These results were consistent with the reports indicating that activation of MAPKs plays a pivotal role in the expression of cPLA2 in various cell types [19], [20]. AP-1 is a dimeric transcription factor comprising proteins from several families whose common denominator is the possession of basic leucine zipper (bZIP) domains that are essential for dimerization and DNA binding. It has been well established that inflammatory responses following exposure to extracellular stimuli are highly dependent on activation of AP-1 which plays an important role in the expression of several target genes [22]. Our group has indicated that IL-1β could induce cPLA2 expression via p42/p44 MAPK- and JNK1/2-dependent AP-1 activation in RA synovial fibroblasts (RASFs) [38]. In addition, we also demonstrated that cigarette smoke extract (CSE) induces cPLA2 expression via MAPKs, AP-1, and NF-κB in human tracheal smooth muscle cells [39]. Here, in HPAEpiCs, we also found that TNF-α-induced cPLA2 expression and PGE2 release was decreased via AP-1 inhibition. On the other hand, we also demonstrated that TNF-α could enhance c-Jun and c-Fos protein expression, which may promote TNF-α-mediated induction of inflammatory genes. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through JNK1/2 [22]. The transcription factor ATF2 (also called CRE-BP1) binds to both AP-1 and CRE DNA response elements and is a member of the ATF/CREB family of leucine zipper proteins. Various forms of cellular stresses, including genotoxic agents, inflammatory cytokines, and UV irradiation, stimulate the transcriptional activity of ATF2. Cellular stresses activate ATF2 by phosphorylation of Thr69 and Thr71 [21], [22]. Moreover, in HPAEpiCs, TNF-α could stimulate c-Jun and ATF2 phosphorylation in a time-dependent manner. We further established that p38 MAPK, but not p42/p44 MAPK and JNK1/2 plays a key role in mediating TNF-α-induced ATF2 activation in these cells. However, TNF-α-induced c-Jun phosphorylation was regulated via JNK1/2 activation. Although p42/p44 MAPK was involved in TNF-α-induced cPLA2 expression, which was not mediated through activation of ATF2 and c-Jun in HPAEpiCs. In the future, we will investigate whether p42/p44 MAPK may regulate other transcription factors, such as NF-κB or Elk-1, leading to cPLA2 expression. In non-small cell lung cancer cells, cPLA2 gene expression can be regulated by various transcription factors, including Sp1 and c-Jun [30]. Both Sp1 and c-Jun have been reported to interact with co-activator, p300, one of HAT members [40], [41]. HATs, such as p300 and CREB binding protein (CBP) act as protein bridges, thereby connecting different transcriptional activators via protein-protein interactions to the basal transcriptional machinery, including transcription factor IIB (TFIIB), TATA-binding protein, and the RNA polymerase II complex [42]. They also function as a scaffolding protein which builds a multi-component transcriptional regulatory complex. Raised activity of intrinsic HAT may cause remodeling of chromatin structure by acetylation of the NH2 terminus of core nucleosomal histones [42]. Chromatin remodeling after p300/CBP associated with histone acetylation is believed to participate in active transcription of pro-inflammatory genes upon stimulation by various mediators. Here, we found that TNF-α time-dependently induced p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs. Finally, we also established that TNF-α markedly induced in vivo binding of p300, ATF2, c-Fos, and c-Jun to the cPLA2 promoter. Based on the observation from literatures and our findings, Fig. 8D reveals a model for the signaling mechanisms implicated in TNF-α-induced cPLA2 expression and PGE2 release in HPAEpiCs. Indeed, previous study showed that cigarette smoke extract (CSE) induced cPLA2 expression in airway smooth muscle cells via the NADPH oxidase-dependent p42/p44 MAPK and p38 MAPK/c-Fos and JNK1/2/c-Jun/p300 pathways [39]. In addition, our group also indicated that TNF-α induced cPLA2 expression through MAPKs, and then the activated MAPKs regulated the activity of p300 and acetylation of histone H4 and hence led to cPLA2 expression [20]. Chi et al. demonstrated that IL-1β induced cPLA2 expression via activation of p42/p44 MAPK and JNK1/2, which further stimulated AP-1 activation in rheumatoid arthritis synovial fibroblasts [38]. However, this study is the first to demonstrate that in HPAEpiCs, the mechanisms underlying TNF-α-mediated activation of MAPKs and AP-1 was required for the expression of cPLA2. Finally, association of p300, ATF2, c-Jun, and c-Fos led to cPLA2 gene transcription. The mechanisms by which TNF-α induced cPLA2 expression may be an important link in the pathogenesis of lung inflammatory diseases. Therefore, understanding the mechanisms underlying TNF-α-induced cPLA2 expression in HPAEpiCs is important to develop new therapeutic strategies. We thank Ms. Chi-Yin Lee for her technical assistance. ==== Refs References 1 Lee IT , Yang CM (2012 ) Role of NADPH oxidase/ROS in pro-inflammatory mediators-induced airway and pulmonary diseases . Biochem Pharmacol 84 : 581 –590 .22587816 2 Barnes PJ (1989 ) New concepts in the pathogenesis of bronchial hyperresponsiveness and asthma . J Allergy Clin Immunol 83 : 1013 –1026 .2659643 3 Henderson WR Jr, Tang LO , Chu SJ , Tsao SM , Chiang GK , et al (2002 ) A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model . Am J Respir Crit Care Med 165 : 108 –116 .11779739 4 Borsch-Haubold AG , Bartoli F , Asselin J , Dudler T , Kramer RM , et al (1998 ) Identification of the phosphorylation sites of cytosolic phospholipase A2 in agonist-stimulated human platelets and HeLa cells . J Biol Chem 273 : 4449 –4458 .9468497 5 Hsieh HL , Wu CY , Hwang TL , Yen MH , Parker P , et al (2006 ) BK-induced cytosolic phospholipase A2 expression via sequential PKC-δ, p42/p44 MAPK, and NF-κB activation in rat brain astrocytes . J Cell Physiol 206 : 246 –254 .15991247 6 Yang CM , Chien CS , Hsiao LD , Luo SF , Wang CC (2002 ) Interleukin-1β-induced cyclooxygenase-2 expression is mediated through activation of p42/44 and p38 MAPKS, and NF-κB pathways in canine tracheal smooth muscle cells . Cell Signal 14 : 899 –911 .12220616 7 Six DA , Dennis EA (2000 ) The expanding superfamily of phospholipase A2 enzymes: classification and characterization . Biochim Biophys Acta 1488 : 1 –19 .11080672 8 Leslie CC (1997 ) Properties and regulation of cytosolic phospholipase A2 . J Biol Chem 272 : 16709 –16712 .9201969 9 Lin LL , Wartmann M , Lin AY , Knopf JL , Seth A , et al (1993 ) cPLA2 is phosphorylated and activated by MAP kinase . Cell 72 : 269 –278 .8381049 10 Gijon MA , Spencer DM , Siddiqi AR , Bonventre JV , Leslie CC (2000 ) Cytosolic phospholipase A2 is required for macrophage arachidonic acid release by agonists that Do and Do not mobilize calcium. Novel role of mitogen-activated protein kinase pathways in cytosolic phospholipase A2 regulation . J Biol Chem 275 : 20146 –20156 .10867029 11 Nagase T , Uozumi N , Ishii S , Kume K , Izumi T , et al (2000 ) Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2 . Nat Immunol 1 : 42 –46 .10881173 12 Uozumi N , Kume K , Nagase T , Nakatani N , Ishii S , et al (1997 ) Role of cytosolic phospholipase A2 in allergic response and parturition . Nature 390 : 618 –622 .9403692 13 Dieter P , Kolada A , Kamionka S , Schadow A , Kaszkin M (2002 ) Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group IV cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2 . Cell Signal 14 : 199 –204 .11812647 14 Gilroy DW , Newson J , Sawmynaden P , Willoughby DA , Croxtall JD (2004 ) A novel role for phospholipase A2 isoforms in the checkpoint control of acute inflammation . FASEB J 18 : 489 –498 .15003994 15 Hulkower KI , Wertheimer SJ , Levin W , Coffey JW , Anderson CM , et al (1994 ) Interleukin-1β induces cytosolic phospholipase A2 and prostaglandin H synthase in rheumatoid synovial fibroblasts. Evidence for their roles in the production of prostaglandin E2 . Arthritis Rheum 37 : 653 –661 .8185692 16 Van Putten V , Refaat Z , Dessev C , Blaine S , Wick M , et al (2001 ) Induction of cytosolic phospholipase A2 by oncogenic Ras is mediated through the JNK and ERK pathways in rat epithelial cells . J Biol Chem 276 : 1226 –1232 .11042196 17 Marshall CJ (1994 ) MAP kinase kinase kinase, MAP kinase kinase and MAP kinase . Curr Opin Genet Dev 4 : 82 –89 .8193545 18 Lin CC , Hsiao LD , Chien CS , Lee CW , Hsieh JT , et al (2004 ) Tumor necrosis factor-α-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-κB . Cell Signal 16 : 597 –607 .14751545 19 Luo SF , Lin WN , Yang CM , Lee CW , Liao CH , et al (2006 ) Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-κB pathways . Cell Signal 18 : 1201 –1211 .16278065 20 Lee CW , Lin CC , Lee IT , Lee HC , Yang CM (2011 ) Activation and induction of cytosolic phospholipase A2 by TNF-α mediated through Nox2, MAPKs, NF-κB, and p300 in human tracheal smooth muscle cells . J Cell Physiol 226 : 2103 –2114 .21520062 21 Vesely PW , Staber PB , Hoefler G , Kenner L (2009 ) Translational regulation mechanisms of AP-1 proteins . Mutat Res 682 : 7 –12 .19167516 22 Shaulian E (2010 ) AP-1−The Jun proteins: Oncogenes or tumor suppressors in disguise? Cell Signal 22 : 894 –899 .20060892 23 Karin M , Gallagher E (2009 ) TNFR signaling: ubiquitin-conjugated TRAFfic signals control stop-and-go for MAPK signaling complexes . Immunol Rev 228 : 225 –240 .19290931 24 Joo M , Wright JG , Hu NN , Sadikot RT , Park GY , et al (2007 ) Yin Yang 1 enhances cyclooxygenase-2 gene expression in macrophages . Am J Physiol Lung Cell Mol Physiol 292 : L1219 –L1226 .17220375 25 Deng WG , Zhu Y , Wu KK (2004 ) Role of p300 and PCAF in regulating cyclooxygenase-2 promoter activation by inflammatory mediators . Blood 103 : 2135 –2142 .14630807 26 Lee CW , Lin CC , Lin WN , Liang KC , Luo SF , et al (2007 ) TNF-α induces MMP-9 expression via activation of Src/EGFR, PDGFR/PI3K/Akt cascade and promotion of NF-κB/p300 binding in human tracheal smooth muscle cells . Am J Physiol Lung Cell Mol Physiol 292 : L799 –L812 .17158602 27 Cao D , Bromberg PA , Samet JM (2007 ) COX-2 expression induced by diesel particles involves chromatin modification and degradation of HDAC1 . Am J Respir Cell Mol Biol 37 : 232 –239 .17395887 28 Lee IT , Wang SW , Lee CW , Chang CC , Lin CC , et al (2008 ) Lipoteichoic acid induces HO-1 expression via the TLR2/MyD88/c-Src/NADPH oxidase pathway and Nrf2 in human tracheal smooth muscle cells . J Immunol 181 : 5098 –5110 .18802114 29 Lee IT , Luo SF , Lee CW , Wang SW , Lin CC , et al (2009 ) Overexpression of HO-1 protects against TNF-α-mediated airway inflammation by down-regulation of TNFR1-dependent oxidative stress . Am J Pathol 175 : 519 –532 .19608869 30 Blaine SA , Wick M , Dessev C , Nemenoff RA (2001 ) Induction of cPLA2 in lung epithelial cells and non-small cell lung cancer is mediated by Sp1 and c-Jun . J Biol Chem 276 : 42737 –42743 .11559711 31 Lee SH , Bahn JH , Whitlock NC , Baek SJ (2010 ) Activating transcription factor 2 (ATF2) controls tolfenamic acid-induced ATF3 expression via MAP kinase pathways . Oncogene 29 : 5182 –5192 .20581861 32 Khanapure SP , Garvey DS , Janero DR , Letts LG (2007 ) Eicosanoids in inflammation: biosynthesis, pharmacology, and therapeutic frontiers . Curr Top Med Chem 7 : 311 –340 .17305573 33 Choi IW , Sun K , Kim YS , Ko HM , Im SY , et al (2005 ) TNF-α induces the late-phase airway hyperresponsiveness and airway inflammation through cytosolic phospholipase A2 activation . J Allergy Clin Immunol 116 : 537 –543 .16159621 34 Byeon HE , Um SH , Yim JH , Lee HK , Pyo S (2012 ) Ohioensin F suppresses TNF-α-induced adhesion molecule expression by inactivation of the MAPK, Akt and NF-κB pathways in vascular smooth muscle cells . Life Sci 90 : 396 –406 .22280832 35 van VC , Bukczynska PE , Puryer MA , Sadek CM , Shields BJ , et al (2005 ) Selective regulation of tumor necrosis factor-induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase . Nat Immunol 6 : 253 –260 .15696169 36 Bradley JR (2008 ) TNF-mediated inflammatory disease . J Pathol 214 : 149 –160 .18161752 37 Pascual RM , Carr EM , Seeds MC , Guo M , Panettieri RA Jr, et al (2006 ) Regulatory features of interleukin-1β-mediated prostaglandin E2 synthesis in airway smooth muscle . Am J Physiol Lung Cell Mol Physiol 290 : L501 –L508 .16299051 38 Chi PL , Chen YW , Hsiao LD , Chen YL , Yang CM (2012 ) Heme oxygenase 1 attenuates interleukin-1β-induced cytosolic phospholipase A2 expression via a decrease in NADPH oxidase/reactive oxygen species/activator protein 1 activation in rheumatoid arthritis synovial fibroblasts . Arthritis Rheum 64 : 2114 –2125 .22231145 39 Cheng SE , Luo SF , Jou MJ , Lin CC , Kou YR , et al (2009 ) Cigarette smoke extract induces cytosolic phospholipase A2 expression via NADPH oxidase, MAPKs, AP-1, and NF-κB in human tracheal smooth muscle cells . Free Radic Biol Med 46 : 948 –960 .19280714 40 Hung JJ , Wang YT , Chang WC (2006 ) Sp1 deacetylation induced by phorbol ester recruits p300 to activate 12(S)-lipoxygenase gene transcription . Mol Cell Biol 26 : 1770 –1785 .16478997 41 Wang YN , Chen YJ , Chang WC (2006 ) Activation of extracellular signal-regulated kinase signaling by epidermal growth factor mediates c-Jun activation and p300 recruitment in keratin 16 gene expression . Mol Pharmacol 69 : 85 –98 .16214953 42 Goodman RH , Smolik S (2000 ) CBP/p300 in cell growth, transformation, and development . Genes Dev 14 : 1553 –1577 .10887150	24069158	PMC3777958	CC BY	2021-01-05 17:39:57	yes	PLoS One. 2013 Sep 19; 8(9):e72783
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24098807PONE-D-13-2708910.1371/journal.pone.0076809Research ArticleGenome-Wide Analysis of the Dof Transcription Factor Gene Family Reveals Soybean-Specific Duplicable and Functional Characteristics Genome-Wide Analysis of Dof Gene Family in SoybeanGuo Yong Qiu Li-Juan * The National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI)/Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, P. R. China Liu Ji-Hong Editor Key Laboratory of Horticultural Plant Biology (MOE), China * E-mail: qiulijuan@caas.cnCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: YG LJQ. Performed the experiments: YG. Analyzed the data: YG LJQ. Contributed reagents/materials/analysis tools: YG. Wrote the manuscript: YG LJQ. 2013 30 9 2013 8 9 e768091 7 2013 30 8 2013 © 2013 Guo et al2013Guo et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The Dof domain protein family is a classic plant-specific zinc-finger transcription factor family involved in a variety of biological processes. There is great diversity in the number of Dof genes in different plants. However, there are only very limited reports on the characterization of Dof transcription factors in soybean (Glycine max). In the present study, 78 putative Dof genes were identified from the whole-genome sequence of soybean. The predicted GmDof genes were non-randomly distributed within and across 19 out of 20 chromosomes and 97.4% (38 pairs) were preferentially retained duplicate paralogous genes located in duplicated regions of the genome. Soybean-specific segmental duplications contributed significantly to the expansion of the soybean Dof gene family. These Dof proteins were phylogenetically clustered into nine distinct subgroups among which the gene structure and motif compositions were considerably conserved. Comparative phylogenetic analysis of these Dof proteins revealed four major groups, similar to those reported for Arabidopsis and rice. Most of the GmDofs showed specific expression patterns based on RNA-seq data analyses. The expression patterns of some duplicate genes were partially redundant while others showed functional diversity, suggesting the occurrence of sub-functionalization during subsequent evolution. Comprehensive expression profile analysis also provided insights into the soybean-specific functional divergence among members of the Dof gene family. Cis-regulatory element analysis of these GmDof genes suggested diverse functions associated with different processes. Taken together, our results provide useful information for the functional characterization of soybean Dof genes by combining phylogenetic analysis with global gene-expression profiling. This work was supported by the National Natural Science Foundation of China (31071446 and 31271753), the Fundamental Research Funds for ICS-CAAS (Grant to Y. G.), the State High-tech Research and Development Program (2013AA102602) and the National Transgenic Major Program (2013ZX08004-001 and 2013ZX08004-002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The transcriptional regulation of gene expression influences or controls many important cellular processes, such as signal transduction, morphogenesis, and environmental stress responses [1]. Transcription factors (TFs) are a group of proteins that control cellular processes by regulating the expression of downstream target genes [2]. Therefore, the identification and functional characterization of TFs is essential for the reconstruction of transcriptional regulatory networks [3]. In plants, ~60 families of TFs have been identified based on bioinformatics analysis and manual inspection [4,5]. The Arabidopsis genome codes for at least 1533 TFs, which account for about 5.9% of its estimated total number of genes [1]. As for soybean (Glycine max), ~12.2% of the 46,430 predicted protein-coding loci have been identified to encode 5,671 putative TFs [6]. The Dof (DNA binding with one finger) TF family belongs to a class of plant-specific TFs that are not found in other eukaryotes such as yeast, Caenorhabditis elegans, Drosophila , fish or humans [7]. Bioinformatics analysis predicts 36 Dof genes in the Arabidopsis genome and 30 in the rice genome [8], while 41 have been described in poplar [9], 31 in wheat [10], and 28 in sorghum [11]. Dof protein is characterized by an N-terminal Dof domain of 50-52 amino-acid residues structured as a Cys2/Cys2 (C2/C2) zinc finger that recognizes a cis-regulatory element containing the common core sequence 5’- (T/A)AAAG-3’ [12-14]. The Dof domain is bifunctional, mediating both DNA-protein and protein-protein interactions. Different Dof TFs may form homo- and/or hetero-dimeric complexes through the Dof domain in a given cell type and have various functions, acting as positive or negative regulators of their targets [15,16]. Other than the conserved Dof domain, diversified transcriptional regulation domains are also located at the C-terminal regions of Dof proteins. The conserved Dof domain might endow all Dof domain proteins with similar characteristics, while the diversified regions outside the Dof domain might be linked to the different functions of distinct Dof domain proteins [14]. Dof TFs are associated with many plant-specific physiological processes related to stress responses, photosynthesis, growth and development [17-27]. In Arabidopsis , some of the well-characterized Dof genes include DAG1 and DAG2 which are associated with seed germination [17,28], and CDF1, CDF2 and CDF3 which are involved in the photoperiodic control of flowering [19]. Some of the Dof TF genes (AtDof2.4, AtDof5.8 and AtDof5.6/HCA2) are reported to be expressed specifically in cells at an early stage of vascular tissue development [18,29]. In rice, OsDof3 is involved in gibberellins-regulated expression [30]. Maize Dof1 and Dof2 are activators of gene expression associated with carbohydrate metabolism, including the gene encoding phosphoenolpyruvate carboxylase [25,27]. In wheat, the Dof TF gene WPBF functions both during seed development and other growth and development processes [31]. A Dof gene, StDof1, which is expressed in epidermal fragments highly-enriched in guard cells, interacts in a sequence-specific manner with a KST1 promoter fragment containing the TAAAG motif in tomato [12]. Some Dof TF genes also take part in the stress and defense responses of plants. Previous study showed that the RNA expression levels of three Dof genes (OBP1, OBP2, and OBP3) increase following treatment with auxin, salicylic acid or cycloheximide, while the OBP proteins have similar in vitro DNA-binding properties and are able to interact with OBF4, a bZIP transcription factor [32]. In response to drought treatment, some TaDof genes are down-regulated and two of them (TaDof14 and TaDof15) are significantly upregulated, indicating that these genes may be involved in drought adaptation [10]. Although quite a few Dof TFs have been functionally characterized in the model plant Arabidopsis and others, the functions of most members of the Dof family remain unknown. Especially in soybean, the typical legume species, there are only very limited reports on the functional characterization of Dof TFs. Wang et al. (2006) identified 28 GmDof proteins with recognizable Dof domain from 39 putative unigenes for the Dof gene family after analysis of their Expressed Sequence Tags (ESTs) in soybean [33,34] and detailed study of two GmDof genes suggested they increased the content of total fatty-acids and lipids in transgenic Arabidopsis by upregulating genes that were associated with fatty-acid biosynthesis [34]. Completion of the soybean genome greatly facilitated the identification of gene families at the whole-genome level [6]. In the present study, a genome-wide identification of Dof domain TFs in soybean was performed and revealed an expanded Dof family with 78 members. Detailed analysis of the sequence phylogeny, genome organization, gene structure, conserved motifs, duplication status, expression profiling, and cis-elements was performed. It is noteworthy that nearly all of the GmDof genes (38 pairs) were preferentially-retained duplicates located in duplicated regions of the genome, indicating soybean-specific duplicable characteristics of the Dof gene family in this species. The putative soybean-specific functions of the predicted GmDof genes were investigated by analyzing the expression profiles using RNA-seq data and cis-regulatory elements associated with these genes in the promoter region. Our data provide a basis for the further evolutionary and functional characterization of the Dof gene family in soybean. Materials and Methods Database search and sequence retrieval The Dof sequences of Arabidopsis thaliana and Oryza sativa were downloaded from the Arabidopsis genome TAIR release 9.0 (http://www.arabidopsis.org/) and the rice genome annotation database (http://rice.plantbiology.msu.edu/, release 5.0). The amino-acid sequence of the Dof domain was used to search for potential Dof-domain homolog hits in the whole-genome sequence of G. max with BLASTP at the Phytozome database (http:/www.phytozome.net) [35]. All non-redundant hits with expected values <1E-5 were collected and compared with the Dof family in PlantTFDB (http://planttfdb.cbi.edu.cn/) [5] and LegumeTFDB (http://legumetfdb.psc.riken.jp/) [36]. As for the incorrectly-predicted genes, manual re-annotation was performed using the on-line web server GENSCAN (http://genes.mit.edu/GENSCAN.html) [37] and/or RT-PCR cloning. The re-annotated sequences were further manually analyzed to confirm the presence of the Dof domain using the InterProScan program (http://www.ebi.ac.uk/Tools/InterProScan/) [38]. Protein Alignment and Phylogenetic Analysis Multiple sequence alignments of the full-length deduced amino-acid sequences of Dof proteins were performed by Clustal X (version 1.83) [39]. The distribution of amino-acid residues at the corresponding positions in domain profiles for the conserved Dof domains of GmDofs were created using WebLogo [40]. Unrooted phylogenetic trees were constructed with MEGA 4.0 using the Neighbor-Joining (NJ) method and the bootstrap test carried out with 1000 iterations [41]. The pairwise gap deletion mode was used to ensure that the more divergent C-terminal domains could contribute to the topology of the NJ tree. Genomic structure and chromosomal location The Gene Structure Display Server program [42] was used to illustrate the exon/intron organization for individual Dof genes by comparison of the coding sequences with their corresponding genomic DNA sequences from Phytozome (http://www.phytozome.net/gmax). The chromosomal locations of soybean Dofs were mapped to the duplicated blocks using the CViT (Chromosome Visualization Tool) genome search and synteny viewer at the Legume Information System (http://comparative-legumes.org/) [43,44]. The deduced amino-acid sequences of all GmDofs were used to search against the soybean genome and the results were displayed using CViT. Calculation of Ks and Ka to date duplication events Clustal X (version 1.83) was used to make pairwise alignments of the paralogous nucleotide sequences [39]. Ks (synonymous substitution rate) and Ka (non-synonymous substitution rate) were estimated using the program DnaSp v5 [45]. The Ks values were then used to calculate the approximate date of duplication event (T = Ks/2λ), assuming a clock-like rate (λ) of synonymous substitution of 6.1×10−9 substitutions/synonymous site/year for soybean [6,46,47]. Identification of conserved motifs The deduced amino-acid sequences of the 78 GmDofs were analyzed by MEME (Multiple EM for Motif Elicitation) version 4.9.0 (http://meme.nbcr.net/meme/cgi-bin/meme.cgi) [48] for motif analysis. To identify conserved motifs in these sequences, selection of the maximum number of motifs was set to 30 with a minimum width of 6 and a maximum width of 200 amino-acids, while other factors were set at default values. Structural motif annotation was performed using the SMART (http://smart.embl-heidelberg.de) [49] and Pfam (http://pfam.sanger.ac.uk) databases [50]. Expression analysis of soybean Dof genes The genome-wide transcriptome data from seeds during several stages of development and throughout the soybean life cycle (obtained with high-throughput sequencing) were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov; accession numbers SRX062325–SRX062334). The transcript data were obtained from seeds at five stages of development (globular, heart, cotyledon, early-maturation, and dry seeds), vegetative tissue (leaves, roots, stems, and whole seedlings), and reproductive tissue (floral buds). All transcript data were analyzed with Cluster 3.0 [51] and the heat map was viewed in Java Treeview [52]. Cis-regulatory element analysis For promoter analysis, 1000-bp sequences upstream from the initiation codon of the putative GmDofs were retrieved. These sequences were then subjected to search in the PLACE database (http://www.dna.affrc.go.jp/PLACE/signalscan.html) [53] to identify cis-regulatory elements. Results and Discussion Identification of Dof-encoding gene family in soybean In order to identify the Dof gene family in the soybean genome, the amino-acid sequence of the conserved Dof domain was used to perform a BLAST search against the Glycine max v1.1 genome (http://www.phytozome.net). A total of 79 non-redundant Dof transcription factor-encoding genes were identified from the whole genome. The presence of the conserved Dof domain in the predicted GmDof protein was a typical feature for consideration as a member of the Dof TF family. To verify the reliability of our results, all of the putative Dof protein sequences were subjected to functional analysis by InterProScan. A typical zinc-finger Dof-type profile was found in all GmDof-encoding genes except for one, annotated as Glyma08g12230, which appears to be a pseudogene owing to a stop codon within the Dof domain. The 78 soybean Dof genes were numbered from GmDof01.1 to GmDof20.2 following the nomenclature proposed for Arabidopsis and according to their positions on different chromosomes. The identified GmDof genes encode peptides ranging from 147 to 555 amino-acids in length with an average of 335. The detailed information of the Dof family genes in soybean, including accession numbers and similarities to their Arabidopsis orthologs, as well as nucleotide and protein sequences, are listed in Table 1 and Additional Table S1. The Dof gene family in soybean is largest compared with the estimates for other plant species, which range from ~36 in Arabidopsis [13], ~30 in rice [8], ~28 in sorghum [11] and ~27 in Brachypodium distachyon [54]. The member of Dof genes in soybean is roughly 2.4-fold that in Arabidopsis , which is consistent with the ratio of 1.4-1.6 putative Populus homologs for each Arabidopsis gene, based on comparative genomics studies [9]. This ratio is almost consistent with that among all the putative protein coding genes of these three species, although the genome size of soybean (1,115 Mb) is almost 9.7 times that of Arabidopsis (115 Mb) and 2.3 times that of Populus (480 Mb) [6,55,56]. 10.1371/journal.pone.0076809.t001Table 1 Summary of Dof family members in soybean. Gene Symbol Gene Locus Gene Location Amino Acids Introns Score E-value GmDof01.1 Glyma01g02610 Gm01: 2137617-2139436 337 0 106.4 8.00E-24 GmDof01.2 Glyma01g05960 Gm01: 5750259-5754433 479 1 92.0 4.00E-20 GmDof01.3 Glyma01g38970 Gm01: 50951027-50952807 336 0 104.4 3.10E-23 GmDof02.1 Glyma02g06970 Gm02: 5595711-5596415 234 0 96.7 5.50E-21 GmDof02.2 Glyma02g10250 Gm02: 8123065-8125204 371 1 101.3 2.30E-22 GmDof02.3 Glyma02g12081 Gm02: 10302501-10306472 485 1 95.9 1.00E-20 GmDof02.4 Glyma02g35296 Gm02: 40034736-40035659 307 0 102.1 1.60E-22 GmDof03.1 Glyma03g01030 Gm03: 756237-758785 472 1 92.8 9.20E-20 GmDof03.2 Glyma03g41980 Gm03: 47319684-47321893 257 0 105.1 1.70E-23 GmDof04.1 Glyma04g31690 Gm04: 35880682-35882596 341 0 99.8 8.00E-22 GmDof04.2 Glyma04g33410 Gm04: 39029262-39032664 470 1 100.5 4.30E-22 GmDof04.3 Glyma04g35650 Gm04: 42048974-42051454 344 1 110.2 5.50E-25 GmDof04.4 Glyma04g41170 Gm04: 47030349-47032300 297 1 105.1 1.80E-23 GmDof04.5 Glyma04g41830 Gm04: 47667211-47668500 289 0 110.5 4.30E-25 GmDof05.1 Glyma05g00970 Gm05: 586599-589518 473 1 98.2 2.00E-21 GmDof05.2 Glyma05g02220 Gm05: 1636697-1639230 330 1 105.5 1.30E-23 GmDof05.3 Glyma05g07460 Gm05: 7516304-7518205 292 0 104.8 2.00E-23 GmDof05.4 Glyma05g29090 Gm05: 34760928-34763043 165 1 92.0 1.60E-19 GmDof06.1 Glyma06g12950 Gm06: 10094214-10095083 289 0 112.1 1.40E-25 GmDof06.2 Glyma06g13671 Gm06: 10805902-10807867 206 1 104.8 2.40E-23 GmDof06.3 Glyma06g19330 Gm06: 15557061-15559563 353 1 108.2 2.00E-24 GmDof06.4 Glyma06g20950 Gm06: 17335571-17338829 458 1 100.9 2.90E-22 GmDof06.5 Glyma06g22797 Gm06: 19579399-19580371 303 1 99.8 6.80E-22 GmDof07.1 Glyma07g01461 Gm07: 936400-938618 211 0 98.6 1.40E-21 GmDof07.2 Glyma07g05950 Gm07: 4649017-4651265 281 0 107.1 4.90E-24 GmDof07.3 Glyma07g31340 Gm07: 36361704-36363720 332 0 97.1 4.70E-21 GmDof07.4 Glyma07g31860 Gm07: 36820811-36821677 288 0 93.2 7.60E-20 GmDof07.5 Glyma07g31870 Gm07: 36829670-36831859 348 1 103.2 6.90E-23 GmDof07.6 Glyma07g35690 Gm07: 41004726-41008389 479 1 97.1 5.20E-21 GmDof08.1 Glyma08g20840 Gm08: 15829658-15831897 213 0 93.6 5.80E-20 GmDof08.2 Glyma08g24591 Gm08: 18749907-18753887 463 1 95.1 1.70E-20 GmDof08.3 Glyma08g37530 Gm08: 36252447-36254191 403 0 105.9 9.00E-24 GmDof08.4 Glyma08g47290 Gm08: 46169187-46171177 367 1 108.6 1.50E-24 GmDof09.1 Glyma09g33350 Gm09: 39841007-39842035 342 0 105.9 9.00E-24 GmDof09.2 Glyma09g37170 Gm09: 42705807-42709793 503 1 91.7 2.00E-19 GmDof10.1 Glyma10g10142 Gm10: 9742414-9743975 309 0 102.4 1.10E-22 GmDof10.2 Glyma10g31700 Gm10: 40190913-40205863 324 1 103.2 6.80E-23 GmDof11.1 Glyma11g06300 Gm11: 4474891-4476607 339 0 104.0 3.70E-23 GmDof11.2 Glyma11g14920 Gm11: 10654917-10656815 288 1 104.0 4.30E-23 GmDof11.3 Glyma11g15761 Gm11: 11423453-11425703 310 1 101.7 2.10E-22 GmDof12.1 Glyma12g06880 Gm12: 4679868-4681949 307 1 104.0 3.40E-23 GmDof12.2 Glyma12g07710 Gm12: 5322929-5325618 305 1 107.8 2.90E-24 GmDof13.1 Glyma13g05480 Gm13: 5801463-5804791 488 1 96.3 7.60E-21 GmDof13.2 Glyma13g24600 Gm13: 27964926-27967177 353 1 102.1 1.50E-22 GmDof13.3 Glyma13g24611 Gm13: 27973342-27974271 309 0 96.7 6.50E-21 GmDof13.4 Glyma13g25120 Gm13: 28389200-28391375 336 0 97.1 4.80E-21 GmDof13.5 Glyma13g30331 Gm13: 33007956-33010080 147 1 86.3 8.00E-18 GmDof13.6 Glyma13g31100 Gm13: 33571320-33573635 357 1 103.2 6.30E-23 GmDof13.7 Glyma13g31110 Gm13: 33583810-33584763 317 0 102.1 1.40E-22 GmDof13.8 Glyma13g31560 Gm13: 33969725-33970600 278 0 93.2 6.00E-20 GmDof13.9 Glyma13g40420 Gm13: 40913246-40915457 285 1 104.0 3.80E-23 GmDof13.10 Glyma13g41031 Gm13: 41429101-41431274 269 1 102.4 1.10E-22 GmDof13.11 Glyma13g42820 Gm13: 42682406-42684307 212 0 103.2 5.80E-23 GmDof15.1 Glyma15g02620 Gm15: 1777967-1779680 211 0 103.2 7.00E-23 GmDof15.2 Glyma15g04430 Gm15: 3099789-3101706 304 1 102.8 8.70E-23 GmDof15.3 Glyma15g04980 Gm15: 3568928-3571019 285 1 101.3 2.50E-22 GmDof15.4 Glyma15g07730 Gm15: 5453626-5455994 285 0 93.2 6.70E-20 GmDof15.5 Glyma15g08230 Gm15: 5800695-5803209 313 0 102.1 1.40E-22 GmDof15.6 Glyma15g08250 Gm15: 5817356-5819506 353 1 109.8 6.50E-25 GmDof15.7 Glyma15g08860 Gm15: 6264258-6266252 153 1 86.3 8.00E-18 GmDof15.8 Glyma15g29870 Gm15: 32718091-32721358 464 1 93.2 7.10E-20 GmDof16.1 Glyma16g02550 Gm16: 2119565-2121907 276 0 107.1 4.90E-24 GmDof16.2 Glyma16g26030 Gm16: 30193624-30194977 236 0 94.7 2.00E-20 GmDof17.1 Glyma17g08950 Gm17: 6612406-6614430 300 0 99.4 9.30E-22 GmDof17.2 Glyma17g09710 Gm17: 7203819-7206839 330 1 108.6 1.70E-24 GmDof17.3 Glyma17g10920 Gm17: 8207249-8210723 471 1 99.4 0.0 GmDof17.4 Glyma17g21540 Gm17: 20917544-20919496 352 0 105.5 1.30E-23 GmDof18.1 Glyma18g26870 Gm18: 30922106-30923215 369 0 104.4 2.90E-23 GmDof18.2 Glyma18g38560 Gm18: 46153747-46155733 363 1 102.8 9.20E-23 GmDof18.3 Glyma18g49520 Gm18: 58916821-58920915 501 1 95.1 1.70E-20 GmDof18.4 Glyma18g52661 Gm18: 61211505-61213733 363 1 102.4 1.20E-22 GmDof19.1 Glyma19g02710 Gm19: 2647356-2650816 385 1 97.1 4.90E-21 GmDof19.2 Glyma19g29610 Gm19: 37285687-37288840 483 1 90.9 3.00E-19 GmDof19.3 Glyma19g38660 Gm19: 45513027-45514071 271 0 104.0 4.00E-23 GmDof19.4 Glyma19g38750 Gm19: 45606704-45607516 270 0 99.4 8.40E-22 GmDof19.5 Glyma19g44670 Gm19: 50031772-50033750 252 0 102.8 7.40E-23 GmDof20.1 Glyma20g04600 Gm20: 4815565-4819043 482 1 95.5 1.20E-20 GmDof20.2 Glyma20g35910 Gm20: 44105729-44107846 300 1 103.2 5.70E-23 To investigate the features of the homologous domain sequences, and the frequency of the most prevalent amino-acids at each position within the soybean Dof domain, multiple-alignment analysis using the amino-acid sequences of the Dof domains from 78 GmDofs was performed. In general, the basic regions of the Dof domains had 52 basic residues. The distribution of amino-acid residues at the corresponding positions of the soybean Dof domains also revealed that it was very similar to that of Arabidopsis , as expected from the evolutionary distances among plants (Figure 1). The Dof domain of soybean revealed highly-conserved sequences and 26 out of 52 amino-acids were 100% conserved in all GmDof proteins, including four absolutely-conserved cysteine residues that presumably coordinate zinc ion. Other highly conserved residues in the soybean Dof domains were Pro-4, Arg-5, Ser-8, Thr-11, Lys-12, Phe-13, Cys-14, Tyr-15, Asn-17, Asn-18, Tyr-19, Gln-23, Pro-24, Arg-25, Arg-33, Trp-35, Thr-36, Gly-38, Gly-39, Arg-42, Gly-47 and Gly-49. These highly-conserved residues were also nearly identical to the Dof domain proteins of other plants such as sorghum and tomato [11,57]. Moreover, five other amino-acid residues showed variation in less than three sequences among all GmDofs. 10.1371/journal.pone.0076809.g001Figure 1 Dof domains are highly conserved across all Dof proteins in soybean. The sequence logos are based on alignments of all soybean Dof domains. Multiple alignment analysis of 78 typical soybean Dof domains was performed with ClustalW. The bit score indicates the information content for each position in the sequence. Asterisks indicate the conserved cysteine residues (Cys) in the Dof domain. Phylogenetic Relationships and Gene Structure of Soybean Dof Genes To examine the phylogenetic relationships among the Dof domain proteins in soybean, an unrooted tree was constructed from alignments of the full-length amino-acid sequences of all GmDof proteins (Figure 2A). The observed sequence similarity and phylogenetic tree topology allowed us to classify the soybean Dof gene family into nine subgroups (subgroups I-IX). Each subgroup had 4-19 members and the very high bootstrap value in each subgroup suggested a common origin for the Dof genes in each subgroup. Inspection of the phylogenetic tree topology revealed several pairs of Dof proteins with a high degree of homology in the terminal nodes of each subgroup, suggesting that they are putative paralogous pairs (Figure 2A). A total of 38 pairs of putative paralogous Dof proteins were identified, accounting for nearly the entire family (except for GmDof17.4 and GmDof05.4), with sequence identity ranging from 72% to 97% (see Additional Table S2 for details). So many putative paralogous Dof proteins supported the hypothesis that they evolved from a recent soybean genome duplication event [58]. 10.1371/journal.pone.0076809.g002Figure 2 Phylogenetic relationships and gene structure of soybean Dof genes. (A) The phylogenetic tree of soybean Dof proteins constructed from a complete alignment of 78 GmDof proteins using MEGA 4.0 by the neighbor-joining method with 1,000 bootstrap replicates. Percentage bootstrap scores >50% are indicated on the nodes. The nine major phylogenetic subgroups designated I to IX are indicated. (B) Exon/intron structures of Dof genes from soybean. Exons are represented by green boxes and introns by black lines. The sizes of exons and introns can be estimated using the scale below. It is well known that gene structural diversity is a possible mechanism for the evolution of multigene families. In order to gain further insight into the structural diversity of Dof genes, we compared the exon/intron organization in the coding sequences of individual Dof genes in soybean. A detailed illustration of the exon/intron structures is shown in Figure 2B. According to their predicted structures, 35 of the GmDof genes have no introns whereas 38 contain one intron generally placed up-stream of the Dof domain, except for five (GmDof10.2, GmDof20.2, GmDof13.5, GmDof15.7, and GmDof05.4) with a down-stream intron. These exon/intron structures are similar to those of Arabidopsis , rice, and other plants [8,11,54]. The most closely-related members in the same subgroup generally showed the same exon/intron pattern, with the position and length of the intron almost completely conserved within most subgroups (Figure 2). For instance, the Dof genes in subgroups II, IV, VII and VIII all lacked an intron, while all members of subgroups III and IX contained one intron. In contrast, the gene structure appeared to be more variable in subgroups I, V and VI, which had the largest numbers of exon/intron structural variants with striking distinctions. Chromosomal location and duplication of soybean Dof genes Genome chromosomal location analyses revealed that GmDofs were non-randomly distributed on 19 of the 20 chromosomes (Figure 3). Nearly all GmDof genes were distributed on the chromosome arms while none were on the heterochromatin regions around the centromeric repeats. Among these chromosomes, chromosome 13 contained the largest number of eleven Dof genes followed by eight on chromosome 15. In contrast, no Dof genes were found on chromosome 14 and only two occurred on six chromosomes (chromosome 03, 09, 10, 12, 16, and 20). Substantial clustering of Dof genes was evident on several chromosomes, especially on those with high densities of the genes. For example, GmDof07.4 and GmDof07.5 located in an 8.8-kb segment on chromosome 07, while GmDof15.5 and GmDof15.6 located within a 19-kb segment on chromosome 15. Similarly, four genes (GmDof13.2 and 13.3, and GmDof13.6 and 13.7) were arranged in two clusters in 10-kb and 13-kb segments on chromosome 13 respectively (Figure 3). 10.1371/journal.pone.0076809.g003Figure 3 Chromosomal locations, region duplications, and predicted clusters for soybean Dof genes. The schematic diagram of genome-wide chromosome organization and segmental duplication arising from the genome duplication event in soybean was derived from the CViT genome search and synteny viewer at the Legume Information System (http://comparative-legumes.org). Colored blocks to the left of each chromosome show duplications with chromosomes of the same color. For example, the gray blocks at the bottom of Gm10 correspond with regions on the brown Gm20, and vice versa. The chromosomal positions of all Dof genes in soybean were mapped on each chromosome. The locations of centromeric repeats are shown as black rectangles over the chromosomes. The chromosome numbers are indicated at the top of each bar and sizes of chromosomes are represented by the vertical scale. Segmental duplication, tandem duplication, and transposition events are the main causes of gene-family expansion. Two or more genes located on the same chromosome confirms a tandem duplication event, while gene duplication on different chromosomes is designated a segmental duplication event [59]. Previous studies revealed that the soybean genome has undergone at least two rounds of genome-wide duplication followed by multiple segmental duplication, tandem duplication, and transposition events such as retroposition and replicative transposition [58]. To detect a potential relationship between putative paralogous pairs of soybean Dofs and potential segmental duplications, the Dof genes were mapped to the duplicated blocks using the CViT genome search and synteny viewer at the Legume Information System (http://comparative-legumes.org/) [43,44]. The distributions of Dof genes relative to the corresponding duplicate genomic blocks are illustrated in Figure 3. Within the duplicated blocks associated with a duplication event, 22 out of 38 putative paralogous pairs were preferentially-retained duplicates that were located in a segmental duplication of a long fragment (>1 Mb), and 13 putative paralogous pairs were located in a segmental duplication of a short fragment (<1 Mb) (Table 2). Another two putative paralogous pairs lacked the corresponding duplicates and only one putative paralogous pair (GmDof19.3/19.4) was possibly due to tandem duplication in the same orientation. These results implied that segmental duplication was predominant for Dof gene evolution in soybean, and that tandem duplication was involved. This relationship between soybean Dofs and potential segmental duplications suggests that dynamic changes occurred following segmental duplication, leading to loss of some of the genes. 10.1371/journal.pone.0076809.t002Table 2 Duplicated Dof genes in soybean and the dates of the duplication blocks. Gene 1 Gene 2 Fragment Duplication Ka Ks Ka/Ks Date (Mya) GmDof07.3 GmDof13.4 Small 0.0313 0.1010 0.3099 8.28 GmDof07.5 GmDof13.2 Small 0.0662 0.1355 0.4886 11.11 GmDof13.6 GmDof15.6 Large 0.0556 0.0951 0.5846 7.80 GmDof07.4 GmDof13.3 Small 0.0916 0.1079 0.8489 8.84 GmDof13.7 GmDof15.5 Large 0.0441 0.1205 0.3660 9.88 GmDof02.2 GmDof18.4 Small 0.0498 0.0938 0.5309 7.69 GmDof13.10 GmDof15.2 Large 0.0555 0.1133 0.4898 9.29 GmDof08.3 GmDof18.1 None 0.1244 0.3315 0.3753 27.17 GmDof13.11 GmDof15.1 Large 0.0424 0.1295 0.3274 10.61 GmDof10.2 GmDof20.2 Large 0.0615 0.1561 0.3940 12.80 GmDof04.4 GmDof06.2 Large 0.0496 0.1395 0.3556 11.43 GmDof11.3 GmDof12.2 Small 0.0369 0.1188 0.3106 9.74 GmDof13.9 GmDof15.3 Large 0.0379 0.1148 0.3301 9.41 GmDof05.2 GmDof17.2 Large 0.0406 0.1156 0.3512 9.48 GmDof04.1 GmDof06.5 None 0.0811 0.2524 0.3213 20.69 GmDof04.5 GmDof06.1 Large 0.0807 0.2125 0.3798 17.42 GmDof02.4 GmDof10.1 Small 0.0410 0.1334 0.3073 10.93 GmDof03.1 GmDof19.2 Small 0.0503 0.1633 0.3080 13.39 GmDof08.2 GmDof15.8 Small 0.0901 0.1474 0.6113 12.08 GmDof07.6 GmDof20.1 Small 0.0458 0.1444 0.3172 11.84 GmDof05.1 GmDof17.3 Large 0.0448 0.0732 0.6120 6.00 GmDof13.1 GmDof19.1 Large 0.0633 0.1013 0.6249 8.30 In order to trace the dates of the duplication blocks, the DnaSP program was used to estimate the Ks and Ka distances, as well as the Ka/Ks ratios. The approximate dates of duplication events were calculated using Ks. Table 2 shows the results of analysis of segmental and tandem duplication blocks. The segmental duplications of the Dof genes in soybean originated from 6.0 Mya (million years ago, Ks = 0.0732) to 27.17 Mya (Ks = 0.2018), with the mean of 11.90 Mya (Ks = 0.1452); the Ks of tandem duplication of GmDof19.3 and GmDof19.4 was 0.0111, dating the duplication event at 0.91 Mya. Since the soybean genome underwent two polyploidy events at 13 and 58 Mya, all the segmental duplications of the GmDof genes occurred around 13 Mya when Glycine -specific duplication occurred in the soybean genome. The Ka/Ks ratios of 15 segmental duplication pairs and one tandem duplication pair were <0.3, while the ratios of the other 22 segmental duplication pairs were all >0.3, suggesting that significant functional divergence of some GmDof genes might have occurred after the duplication events. Phylogenetic analysis of the Dof gene family in soybean, Arabidopsis , and rice To investigate the molecular evolution and phylogenetic relationships among the Dof domain proteins in soybean, Arabidopsis , and rice, the 78 predicted GmDof proteins were subjected to multiple sequence alignment along with 36 Arabidopsis and 30 rice Dof proteins, and an unrooted phylogenetic tree was constructed using the NJ method, based on the alignment of all the Dof amino-acid sequences (Figure 4, Additional Table S3). The NJ tree showed that all the Dof family proteins from the three higher plants were divided into four Major Clusters of Orthologous Groups (MCOG A, B, C, and D) and nine well-supported clades (Figure 4), similar to previous reports [8,13]. Among these, group C constituted the largest clade, containing 47 members and accounting for 32.6% of the total Dof genes, and the other three groups contained 25 (Group A), 30 (Group B), and 42 (Group D) members, respectively. In general, the Dof members demonstrated an interspersed distribution in most subfamilies, indicating that the expansion of Dof genes occurred before the divergence of soybean, Arabidopsis , and rice. Based on the phylogenetic tree, several putative orthologs (GmDof06.3/AtDof5.6, OsDof-2/GmDof07.6 (GmDof09.2), AtDof1.6/OsDof-10, or AtDof2.4/OsDof-16/GmDof13.10 (GmDof15.2)) and paralogs (AtDof5.7/AtDof4.7, OsDof-13/OsDof-30, GmDof03.1/GmDof19.2) were also identified. 10.1371/journal.pone.0076809.g004Figure 4 Phylogenetic tree of all Dof domain containing proteins from soybean, Arabidopsis , and rice. The deduced full-length amino-acid sequences of 78 soybean, 36 Arabidopsis and 30 rice Dof genes were aligned by Clustal X 1.83 and the phylogenetic tree was constructed using MEGA 4.0 by the neighbor-joining method with 1,000 bootstrap replicates. Each Dof subgroup is indicated by a specific color. Moreover, since most of the Arabidopsis Dof genes with similar functions showed a tendency to fall into one subgroup, soybean Dof genes in the same subgroup may have similar functions. In subgroup A, eight soybean Dof genes clustered with the Arabidopsis Dof genes AtDof2.4, AtDof4.7, AtDof5.7 and AtDof3.6(OBP3) in subgroup B1, and these have been identified to be involved in tissue differentiation (vascular development, floral organ abscission, leaf blade polarity and growth regulation) [20,29,32,60,61]. About 19 GmDofs showed maximum similarity with AtDof5.5(CDF1), AtDof5.2(CDF2), AtDof3.3(CDF3), AtDof2.3(CDF4), AtDof1.10(CDF5), and AtDof1.5(COG1) of Arabidopsis representing subgroup D1, which are basically CDF (Cycling Dof Factor) proteins associated with the regulation of photoperiodic flowering time by repressing the CONSTANS gene [19,62]. Specifically, the Arabidopsis Dof proteins AtDof4.2, 4.3, 4.4 and 4.5 constitute the distinct subgroup C3 and OsDof-13, 24, 25, 30 constitute the distinct subgroup D3, similar to what has been reported in Arabidopsis and rice clusters C3 and D3 [8]. These sets of Dof genes might be exclusively present in Arabidopsis/rice as no apparent counterpart in soybean as well as other plants. Conserved motifs outside the Dof domain To reveal the diversification of Dof genes in soybean, putative motifs were predicted by the program MEME (Multiple Em for Motif Elicitation), and a total of 30 conserved motifs were found in all the 78 Dof proteins (Figure 5). Motif 1 was uniformly present in all the Dof proteins and represents the conserved Dof domain. Moreover, a number of common motifs were found in all soybean Dofs (the amino-acid consensus sequence of each motif is listed in Additional Table S4). As expected, most of the closely-related members in the phylogenetic tree had common motif compositions. For example, there were no conserved motifs outside the Dof domain in Subgroup I, while motifs 2, 3, 4, 5, 6, 7, 9, 10, 12, 17, and 22 appeared in nearly all the members of subgroup IX. In other subgroups, motifs 8 and 15 were specific to subgroup III, motifs 20 and 24 were specific to subgroup IV, motifs 18 and 29 were specific to subgroup V, motifs 11, 21, 19, 23, and 30 were specific to subgroup VI, motif 13 was specific to subgroup VII, and motifs 25, 26 and 27 were specific to subgroup VIII. These similarities in motif patterns might be related to similar functions of the Dof proteins within the same subgroup. 10.1371/journal.pone.0076809.g005Figure 5 Schematic distributions of the conserved motifs among defined gene clusters. Motifs were identified by means of MEME software using the deduced amino-acid sequences of the 78 GmDofs. The relative position of each identified motif in all Dof proteins is shown. Multilevel consensus sequences for the MEME defined motifs are listed in Table S4. Expression pattern of Dof genes in soybean Since high-throughput sequencing and gene expression analyses have been performed on many soybean tissues at various developmental stages, publicly-available RNA-Seq data is thought to be a useful resources for studying gene expression profiles. Distinct transcript abundance patterns were readily identifiable in the RNA-Seq dataset at NCBI. Nearly all Dof genes (except for three: GmDof02.4, GmDof13.1, and GmDof19.3) have sequence reads in at least one tissue, their universal expression also indicating the importance of Dof TFs. The expression profiles of the 75 Dof genes were analyzed as shown in Figure 6. Most of the Dof genes showed distinct tissue-specific expression patterns across the ten tissues examined. All of the GmDofs having expression profiles were clustered into nine groups based on their expression patterns. The genes in clusters A-I were mainly expressed in root/floral bud, root, root/globular embryo, floral bud/globular embryo, leaf/floral bud, floral bud, cotyledon/early-maturation embryo, heart/cotyledon embryo, and dry seed. 10.1371/journal.pone.0076809.g006Figure 6 Heatmap of expression profiles for soybean Dof genes across different tissues. The genome-wide transcriptome data of soybean were generated from the NCBI database (accession numbers SRX062325–SRX062334). The expression data were gene-wise normalized and hierarchically clustered. The relative expression level of a particular gene in each row was normalized against the mean value. The color scale below represents expression values, green indicating low levels and red indicating high levels of transcript abundance. The sources of the samples were as follows: SDLG (whole seedlings 6 days after imbibition), LEAF (leaves), ROOT (roots), STEM (stems), FBUD (floral buds), GLOB (globular-stage embryos), HRT (heart-stage embryos), COT (cotyledon-stage embryos), EM (early maturation stage embryos), and DRY (dry soybean seeds). Detailed analysis of the expression patterns of GmDofs showed that some of the genes clustered in the same subgroup of the phylogenetic tree (Figure 2) had similar expression patterns, also indicating the existence of redundancy among the Dof genes in these subgroups. For example, all of the GmDofs in subgroup VII were mainly expressed in floral buds while all of genes in subgroup V were mainly expressed in root and/or globular embryo. Most of the genes in subgroup IX had dominant expression patterns in floral buds and/or globular embryo. However, some Dof members in the same subgroups also had totally different expression patterns, even among paralogous genes with high identity of amino-acid sequences. In subgroup I of the phylogenetic tree (Figure 2), there were five kinds of expression patterns among all eight GmDof members. Three of four pairs of paralogous genes (GmDof07.3/13.4, GmDof07.5/13.2, and GmDof13.6/15.6) had different expression patterns and one pair (GmDof13.8/15.4) was mainly expressed in floral buds and globular embryo. The genes in the same subgroup with different expression pattern, especially paralogous genes, also revealed their functional diversity despite these Dof genes had highly similar amino-acid sequences. Cis-regulatory element analysis The transcription rate of a gene is determined by trans-acting TFs that bind to cis-regulatory elements in promoters, additional co-factors, and chromatin accessibility [63]. A common approach to identify functional cis-acting promoter elements is to discover over-represented motifs in co-expressed genes. It is assumed that promoter motifs conserved in clusters of co-expressed and functionally-related genes may be involved in mediating coordinated gene activity [64,65]. The promoter regions of the GmDof genes (1000-bp sequences upstream from the translational start site) were analyzed using the PLACE database to identify putative cis-elements. According to the PLACE results, many similar cis-acting regulatory DNA elements associated with root, leaf, flower, seed, nodulin, abiotic or biotic stress, and hormone (Additional Table S5) occurred in the promoter regions of the 78 GmDof genes. For example, cis-elements related to root-specific (ROOTMOTIFTAPOX1), leaf-specific (CACTFTPPCA1), and flower-specific (POLLEN1LELAT52) were present in all soybean GmDof promoters (Additional Table S5). Especially, all of the GmDof promoters contained Dof elements (DOFCOREZM) ranging from 4 to 37 copies, indicating the important role of Dof TFs in regulating themselves. Furthermore, the differences in common cis-elements across these promoter regions, including both number and distance from the start codon (Additional Table S5), indicated that the number of cis-elements and their distance from the start site affect the responsiveness of GmDofs to the environment and development. Conclusions Transcriptional regulation is an important mechanism underlying gene expression. The number, position and interaction between different cis-elements and the TFs at a given gene promoter determine the gene expression pattern. These TFs can be classified into gene families according to the presence of a particular DNA-binding domain. In this study, a comprehensive analysis was conducted and a multitude of Dof gene family members were identified in the soybean genome. Genome-wide analysis revealed the existence of 78 full-length Dof genes, and multiple sequence alignment of the GmDof proteins showed strong conservation of four cysteine residues and the other amino-acid residues in the Dof domains. Phylogenetic analysis revealed that all GmDofs were clustered into nine distinct subgroups. The exon/intron structure and motif composition of the Dofs were highly conserved in each subfamily, indicating their functional conservation. The Dof genes were non-randomly distributed within and across 19 chromosomes, and a high proportion of GmDofs were preferentially-retained duplicates located on duplicated blocks. Soybean-specific segmental duplications of the genome contributed significantly to the expansion of the soybean Dof gene family. The comparative phylogenetic analysis of soybean Dof proteins with Arabidopsis and rice Dof proteins revealed four Major Clusters of Orthologous Groups and nine well-supported clades. The global expression profile analysis provided insight into the soybean-specific functional divergence among members of the Dof gene family. A majority of GmDofs showed specific temporal and spatial expression patterns, based on RNA-seq data analyses. The expression patterns of duplicate genes were partially redundant or divergent. The cis-regulatory element analysis of the predicted Dof genes revealed differences in common cis-elements across these promoter regions including both their number and distance from the start codon. The results presented here provide information useful for the functional characterization of soybean gene families by combining phylogenetic analysis with global gene expression profiling. Supporting Information Table S1 Complete list of soybean Dof gene sequences identified in the present study. The list comprises 78 GmDof gene sequences. The amino-acid sequences were deduced from their corresponding coding sequences; the genomic DNA sequences were obtained from Phytozome. Most of the transcripts were based on the Glycine max v1.1 annotation and some were from v1.0. Some of the Dof genes were re-annotated based on GENESCAN, paralogous genes, and/or RT-PCR. (XLS) Click here for additional data file. Table S2 Pairwise identities between homologous pairs of Dof genes from soybean. Pairwise identities and sequence alignments of the 38 homologous pairs identified from the soybean Dof family. (XLS) Click here for additional data file. Table S3 List of Dof genes from A. thaliana and O. sativa used for phylogenetic analysis. The Dof sequences of A. thaliana and O. sativa were downloaded from Arabidopsis genome TAIR release 9.0 (http://www.Arabidopsis.org/) and those of O. sativa from the rice genome annotation database (http://rice.plantbiology.msu.edu/, release 5.0). The nomenclature is according to previous reports [8,13]. (XLS) Click here for additional data file. Table S4 Multilevel consensus sequences for the MEME-defined motifs found among different Dof proteins from soybean. Consensus amino-acid sequences obtained from analysis of the 78 soybean Dof proteins with MEME software. The motif numbers are equivalent to those described in Figure 5. Motif 1 corresponds to the Dof DNA-binding domain. (XLS) Click here for additional data file. Table S5 The cis-acting regulatory DNA elements of 78 GmDof promoters. The motifs of the soybean GmDof promoters were predicted by PLACE (http://www.dna.affrc.go.jp/PLACE/). The numbers show the occurrence frequency of the motifs in one promoter. The sequences were from the 1-kb sequence upstream of the ATG. (XLS) Click here for additional data file. The authors thank Prof. Iain C Bruce (Zhejiang University, China) for critical reading of the manuscript and the reviewers for their constructive comments on earlier versions of this manuscript. ==== Refs References 1 Riechmann JL , Heard J , Martin G , Reuber L , Jiang C et al. (2000 ) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes . Science 290 : 2105 -2110 . doi:10.1126/science.290.5499.2105. PubMed: 11118137.11118137 2 Qu L-J , Zhu Y-X (2006 ) Transcription factor families in Arabidopsis: major progress and outstanding issues for future research . Curr Opin Plant Biol 9 : 544 -549 . doi:10.1016/j.pbi.2006.07.005. PubMed: 16877030.16877030 3 Riaño-Pachón DM , Ruzicic S , Dreyer I , Mueller-Roeber B (2007 ) PlnTFDB: an integrative plant transcription factor database . BMC Bioinformatics 8 : 42 . doi:10.1186/1471-2105-8-42. PubMed: 17286856.17286856 4 Guo AY , Chen X , Gao G , Zhang H , Zhu QH et al. (2008 ) PlantTFDB: a comprehensive plant transcription factor database . Nucleic Acids Res 36 : D966 -D969 . PubMed: 17933783.17933783 5 Zhang H , Jin J , Tang L , Zhao Y , Gu X et al. (2011 ) PlantTFDB 2.0: update and improvement of the comprehensive plant transcription factor database . Nucleic Acids Res 39 : D1114 -D1117 .21097470 6 Schmutz J , Cannon SB , Schlueter J , Ma J , Mitros T et al. (2010 ) Genome sequence of the palaeopolyploid soybean . Nature 463 : 178 -183 . doi:10.1038/nature08670. PubMed: 20075913.20075913 7 Moreno-Risueno MA , Martínez M , Vicente-Carbajosa J , Carbonero P (2007 ) The family of DOF transcription factors: from green unicellular algae to vascular plants . Mol Genet Genomics 277 : 379 -390 . doi:10.1007/s00438-006-0186-9. PubMed: 17180359.17180359 8 Lijavetzky D , Carbonero P , Vicente-Carbajosa J (2003 ) Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families . BMC Evol Biol 3 : 17 . doi:10.1186/1471-2148-3-17. PubMed: 12877745.12877745 9 Yang X , Tuskan GA , Cheng MZ (2006 ) Divergence of the Dof gene families in poplar, Arabidopsis, and rice suggests multiple modes of gene evolution after duplication . Plant Physiol 142 : 820 -830 . doi:10.1104/pp.106.083642. PubMed: 16980566.16980566 10 Shaw LM , McIntyre CL , Gresshoff PM , Xue GP (2009 ) Members of the Dof transcription factor family in Triticum aestivum are associated with light-mediated gene regulation . Funct Integr Genomics 9 : 485 -498 . doi:10.1007/s10142-009-0130-2. PubMed: 19578911.19578911 11 Kushwaha H , Gupta S , Singh VK , Rastogi S , Yadav D (2011 ) Genome wide identification of Dof transcription factor gene family in sorghum and its comparative phylogenetic analysis with rice and Arabidopsis . Mol Biol Rep 38 : 5037 -5053 . doi:10.1007/s11033-010-0650-9. PubMed: 21161392.21161392 12 Plesch G , Ehrhardt T , Mueller-Roeber B (2001 ) Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression . Plant J 28 : 455 -464 . PubMed: 11737782.11737782 13 Yanagisawa S (2002 ) The Dof family of plant transcription factors . Trends Plant Sci 7 : 555 -560 . doi:10.1016/S1360-1385(02)02362-2. PubMed: 12475498.12475498 14 Yanagisawa S (2004 ) Dof domain proteins: plant-specific transcription factors associated with diverse phenomena unique to plants . Plant Cell Physiol 45 : 386 -391 . doi:10.1093/pcp/pch055. PubMed: 15111712.15111712 15 Krohn NM , Yanagisawa S , Grasser KD (2002 ) Specificity of the stimulatory interaction between chromosomal HMGB proteins and the transcription factor Dof2 and its negative regulation by protein kinase CK2-mediated phosphorylation . J Biol Chem 277 : 32438 -32444 . doi:10.1074/jbc.M203814200. PubMed: 12065590.12065590 16 Yanagisawa S (1997 ) Dof DNA-binding domains of plant transcription factors contribute to multiple protein-protein interactions . Eur J Biochem 250 : 403 -410 . doi:10.1111/j.1432-1033.1997.0403a.x. PubMed: 9428691.9428691 17 Gualberti G , Papi M , Bellucci L , Ricci I , Bouchez D et al. (2002 ) Mutations in the Dof zinc finger genes DAG2 and DAG1 influence with opposite effects the germination of Arabidopsis seeds . Plant Cell 14 : 1253 -1263 . doi:10.1105/tpc.010491. PubMed: 12084825.12084825 18 Guo Y , Qin G , Gu H , Qu LJ (2009 ): Dof5.6/HCA2, a Dof transcription factor gene, regulates interfascicular cambium formation and vascular tissue development in Arabidopsis . Plant Cell 21 : 3518 -3534 .19915089 19 Imaizumi T , Schultz TF , Harmon FG , Ho LA , Kay SA (2005 ) FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis . Science 309 : 293 -297 . doi:10.1126/science.1110586. PubMed: 16002617.16002617 20 Kim HS , Kim SJ , Abbasi N , Bressan RA , Yun DJ et al. (2010 ) The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis . Plant J 64 : 524 -535 . doi:10.1111/j.1365-313X.2010.04346.x. PubMed: 20807212.20807212 21 Negi J , Moriwaki K , Konishi M , Yokoyama R , Nakano T et al. (2013 ) A Dof transcription factor, SCAP1, is essential for the development of functional stomata in Arabidopsis . Curr Biol 23 : 479 –484 . doi:10.1016/j.cub.2013.04.037. PubMed: 23453954.23453954 22 Park DH , Lim PO , Kim JS , Cho DS , Hong SH et al. (2003 ) The Arabidopsis COG1 gene encodes a Dof domain transcription factor and negatively regulates phytochrome signaling . Plant J 34 : 161 -171 .12694592 23 Skirycz A , Radziejwoski A , Busch W , Hannah MA , Czeszejko J et al. (2008 ) The DOF transcription factor OBP1 is involved in cell cycle regulation in Arabidopsis thaliana . Plant J 56 : 779 -792 . doi:10.1111/j.1365-313X.2008.03641.x. PubMed: 18665917.18665917 24 Ward JM , Cufr CA , Denzel MA , Neff MM (2005 ) The Dof transcription factor OBP3 modulates phytochrome and cryptochrome signaling in Arabidopsis . Plant Cell 17 : 475 -485 . doi:10.1105/tpc.104.027722. PubMed: 15659636.15659636 25 Yanagisawa S (2000 ) Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize . Plant J 21 : 281 -288 . doi:10.1046/j.1365-313x.2000.00685.x. PubMed: 10758479.10758479 26 Yanagisawa S , Akiyama A , Kisaka H , Uchimiya H , Miwa T (2004 ) Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions . Proc Natl Acad Sci U S A 101 : 7833 -7838 . doi:10.1073/pnas.0402267101. PubMed: 15136740.15136740 27 Yanagisawa S , Sheen J (1998 ) Involvement of maize Dof zinc finger proteins in tissue-specific and light-regulated gene expression . Plant Cell 10 : 75 -89 . doi:10.2307/3870630. PubMed: 9477573.9477573 28 Papi M , Sabatini S , Bouchez D , Camilleri C , Costantino P et al. (2000 ) Identification and disruption of an Arabidopsis zinc finger gene controlling seed germination . Genes Dev 14 : 28 -33 . PubMed: 10640273.10640273 29 Konishi M , Yanagisawa S (2007 ) Sequential activation of two Dof transcription factor gene promoters during vascular development in Arabidopsis thaliana . Plant Physiol Biochem 45 : 623 -629 . doi:10.1016/j.plaphy.2007.05.001. PubMed: 17583520.17583520 30 Washio K (2003 ) Functional dissections between GAMYB and Dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy1A gene in the rice aleurone . Plant Physiol 133 : 850 -863 . doi:10.1104/pp.103.027334. PubMed: 14500792.14500792 31 Dong G , Ni Z , Yao Y , Nie X , Sun Q (2007 ) Wheat Dof transcription factor WPBF interacts with TaQM and activates transcription of an alpha-gliadin gene during wheat seed development . Plant Mol Biol 63 : 73 -84 . PubMed: 17021941.17021941 32 Kang HG , Singh KB (2000 ) Characterization of salicylic acid-responsive, Arabidopsis Dof domain proteins: overexpression of OBP3 leads to growth defects . Plant J 21 : 329 -339 . doi:10.1046/j.1365-313x.2000.00678.x. PubMed: 10758484.10758484 33 Tian AG , Wang J , Cui P , Han YJ , Xu H et al. (2004 ) Characterization of soybean genomic features by analysis of its expressed sequence tags . Theor Appl Genet 108 : 903 -913 . doi:10.1007/s00122-003-1499-2. PubMed: 14624337.14624337 34 Wang HW , Zhang B , Hao YJ , Huang J , Tian AG et al. (2007 ) The soybean Dof-type transcription factor genes, GmDof4 and GmDof11, enhance lipid content in the seeds of transgenic Arabidopsis plants . Plant J 52 : 716 -729 . doi:10.1111/j.1365-313X.2007.03268.x. PubMed: 17877700.17877700 35 Goodstein DM , Shu S , Howson R , Neupane R , Hayes RD et al. (2012 ) Phytozome: a comparative platform for green plant genomics . Nucleic Acids Res 40 : D1178 -D1186 . doi:10.1093/nar/gkr944. PubMed: 22110026.22110026 36 Mochida K , Yoshida T , Sakurai T , Yamaguchi-Shinozaki K , Shinozaki K et al. (2010 ) LegumeTFDB: an integrative database of Glycine max, Lotus japonicus and Medicago truncatula transcription factors . Bioinformatics 26 : 290 -291 . doi:10.1093/bioinformatics/btp645. PubMed: 19933159.19933159 37 Stormo GD (2000 ) Gene-finding approaches for eukaryotes . Genome Res 10 : 394 -397 . doi:10.1101/gr.10.4.394. PubMed: 10779479.10779479 38 Quevillon E , Silventoinen V , Pillai S , Harte N , Mulder N et al. (2005 ) InterProScan: protein domains identifier . Nucleic Acids Res 33 : W116 -W120 . doi:10.1093/nar/gni118. PubMed: 15980438.15980438 39 Thompson JD , Gibson TJ , Plewniak F , Jeanmougin F , Higgins DG (1997 ) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools . Nucleic Acids Res 25 : 4876 -4882 . doi:10.1093/nar/25.24.4876. PubMed: 9396791.9396791 40 Crooks GE , Hon G , Chandonia JM , Brenner SE (2004 ) WebLogo: a sequence logo generator . Genome Res 14 : 1188 -1190 . doi:10.1101/gr.849004. PubMed: 15173120.15173120 41 Tamura K , Dudley J , Nei M , Kumar S (2007 ) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0 . Mol Biol Evol 24 : 1596 -1599 . doi:10.1093/molbev/msm092. PubMed: 17488738.17488738 42 Guo AY , Zhu QH , Chen X , Luo JC (2007 ) GSDS: a gene structure display server . Yi Chuan 29 : 1023 -1026 . doi:10.1360/yc-007-1023. PubMed: 17681935.17681935 43 Cannon EK , Cannon SB (2011 ) Chromosome visualization tool: a whole genome viewer . Int J Plants Genomics , 2011 : 373875 . doi:10.1155/2011/373875. Article ID 22220167. 44 Cannon SB , Shoemaker RC (2012 ) Evolutionary and comparative analyses of the soybean genome . Breed Sci 61 : 437 -444 . doi:10.1270/jsbbs.61.437. PubMed: 23136483.23136483 45 Librado P , Rozas J (2009 ) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data . Bioinformatics 25 : 1451 -1452 . doi:10.1093/bioinformatics/btp187. PubMed: 19346325.19346325 46 Lavin M , Herendeen PS , Wojciechowski MF (2005 ) Evolutionary rates analysis of Leguminosae implicates a rapid diversification of lineages during the tertiary . Syst Biol 54 : 575 -594 . doi:10.1080/10635150590947131. PubMed: 16085576.16085576 47 Lynch M , Conery JS (2000 ) The evolutionary fate and consequences of duplicate genes . Science 290 : 1151 -1155 . doi:10.1126/science.290.5494.1151. PubMed: 11073452.11073452 48 Bailey TL , Williams N , Misleh C , Li WW (2006 ) MEME: discovering and analyzing DNA and protein sequence motifs . Nucleic Acids Res 34 : W369 -W373 . doi:10.1093/nar/gkl198. PubMed: 16845028.16845028 49 Letunic I , Copley RR , Schmidt S , Ciccarelli FD , Doerks T et al. (2004 ) SMART 4.0: towards genomic data integration . Nucleic Acids Res 32 : D142 -144 .14681379 50 Finn RD , Mistry J , Schuster-Böckler B , Griffiths-Jones S , Hollich V et al. (2006 ) Pfam: clans, web tools and services . Nucleic Acids Res 34 : D247 -D251 . doi:10.1093/nar/gkj149. PubMed: 16381856.16381856 51 de Hoon MJ , Imoto S , Nolan J , Miyano S (2004 ) Open source clustering software . Bioinformatics 20 : 1453 -1454 . doi:10.1093/bioinformatics/bth078. PubMed: 14871861.14871861 52 Page RD (1996 ) TreeView: an application to display phylogenetic trees on personal computers . Comput Appl Biosci 12 : 357 -358 . PubMed: 8902363.8902363 53 Higo K , Ugawa Y , Iwamoto M , Korenaga T (1999 ) Plant cis-acting regulatory DNA elements (PLACE) database . Nucleic Acids Res 27 : 297 -300 .9847208 54 Hernando-Amado S , González-Calle V , Carbonero P , Barrero-Sicilia C (2012 ) The family of DOF transcription factors in Brachypodium distachyon: phylogenetic comparison with rice and barley DOFs and expression profiling . BMC Plant Biol 12 : 202 . doi:10.1186/1471-2229-12-202. PubMed: 23126376.23126376 55 Tuskan GA , Difazio S , Jansson S , Bohlmann J , Grigoriev I et al. (2006 ) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray) . Science 313 : 1596 -1604 . doi:10.1126/science.1128691. PubMed: 16973872.16973872 56 AGI (2000 ) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana . Nature 408 : 796 -815 . doi:10.1038/35048692. PubMed: 11130711.11130711 57 Cai X , Zhang Y , Zhang C , Zhang T , Hu T et al. (2013 ) Genome-wide analysis of plant-specific Dof transcription factor family in tomato . J Integr Plant Biol 55 : 552 -566 . doi:10.1111/jipb.12043. PubMed: 23462305.23462305 58 Schlueter JA , Lin JY , Schlueter SD , Vasylenko-Sanders IF , Deshpande S et al. (2007 ) Gene duplication and paleopolyploidy in soybean and the implications for whole genome sequencing . BMC Genomics 8 : 330 . doi:10.1186/1471-2164-8-330. PubMed: 17880721.17880721 59 Liu Y , Jiang H , Chen W , Qian Y , Ma Q et al. (2011 ) Genome-wide analysis of the auxin response factor (ARF) gene family in maize (Zea mays) . Plant Growth Regul 63 : 225 -234 . doi:10.1007/s10725-010-9519-0. 60 Kang HG , Foley RC , Oñate-Sánchez L , Lin C , Singh KB (2003 ) Target genes for OBP3, a Dof transcription factor, include novel basic helix-loop-helix domain proteins inducible by salicylic acid . Plant J 35 : 362 -372 . doi:10.1046/j.1365-313X.2003.01812.x. PubMed: 12887587.12887587 61 Wei PC , Tan F , Gao XQ , Zhang XQ , Wang GQ et al. (2010 ) Overexpression of AtDOF4.7, an Arabidopsis DOF family transcription factor, induces floral organ abscission deficiency in Arabidopsis . Plant Physiol 153 : 1031 -1045 . doi:10.1104/pp.110.153247. PubMed: 20466844.20466844 62 Fornara F , Panigrahi KC , Gissot L , Sauerbrunn N , Rühl M et al. (2009 ) Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response . Dev Cell 17 : 75 -86 . doi:10.1016/j.devcel.2009.06.015. PubMed: 19619493.19619493 63 Wasserman WW , Sandelin A (2004 ) Applied bioinformatics for the identification of regulatory elements . Nat Rev Genet 5 : 276 -287 . doi:10.1038/nrg1315. PubMed: 15131651.15131651 64 Do JH , Choi DK (2008 ) Clustering approaches to identifying gene expression patterns from DNA microarray data . Mol Cells 25 : 279 -288 . PubMed: 18414008.18414008 65 Tavazoie S , Hughes JD , Campbell MJ , Cho RJ , Church GM (1999 ) Systematic determination of genetic network architecture . Nat Genet 22 : 281 -285 . doi:10.1038/10343. PubMed: 10391217.10391217	24098807	PMC3786956	CC BY	2021-01-05 17:47:15	yes	PLoS One. 2013 Sep 30; 8(9):e76809
==== Front Open BiolOpen BiolRSOBroyopenbioOpen Biology2046-2441The Royal Society 2402653710.1098/rsob.130102rsob1301021001129ResearchResearch ArticleMHF1–2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination Fission yeast Mhf1 and Mhf2Bhattacharjee Sonali †Osman Fekret Feeney Laura Lorenz Alexander ‡Bryer Claire Whitby Matthew C. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UKe-mail: matthew.whitby@bioch.ox.ac.uk† Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, NY 11724, USA. ‡ Present address: The Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. 9 2013 9 2013 3 9 13010223 6 2013 16 8 2013 2013© 2013 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.The histone-fold proteins Mhf1/CENP-S and Mhf2/CENP-X perform two important functions in vertebrate cells. First, they are components of the constitutive centromere-associated network, aiding kinetochore assembly and function. Second, they work with the FANCM DNA translocase to promote DNA repair. However, it has been unclear whether there is crosstalk between these roles. We show that Mhf1 and Mhf2 in fission yeast, as in vertebrates, serve a dual function, aiding DNA repair/recombination and localizing to centromeres to promote chromosome segregation. Importantly, these functions are distinct, with the former being dependent on their interaction with the FANCM orthologue Fml1 and the latter not. Together with Fml1, they play a second role in aiding chromosome segregation by processing sister chromatid junctions. However, a failure of this activity does not manifest dramatically increased levels of chromosome missegregation due to the Mus81–Eme1 endonuclease, which acts as a failsafe to resolve DNA junctions before the end of mitosis. anaphase bridgecentromereDNA helicaseDNA replicationhomologous recombinationcover-dateSeptember 2013 ==== Body 2. Introduction Homologous recombination (HR) is a fundamental process in chromosome biology, being deployed in various ways to facilitate the repair and tolerance of DNA lesions in which genetic information is lost or corrupted in both strands of the double helix (e.g. a DNA double-strand break; DSB). It also promotes genome duplication by enabling the restart of collapsed replication forks, and in most studied eukaryotes serves a crucial role during meiosis in establishing chiasmata that guide correct disjunction of the homologous chromosomes during the first meiotic division. A potential consequence of its action is the rearrangement of genetic material, which in the germline can have the desirable effect of driving genetic diversity, but in somatic cells can lead to the loss or alteration of gene function, which in turn can result in disease and death. Many proteins contribute to the correct functioning of HR; however, among these are a core cohort that is directly responsible for catalysing the key DNA transactions that occur [1]. Included here are nucleases and DNA helicases that often work hand in hand to generate a region of single-stranded DNA (ssDNA) with an exposed 3′-OH terminus onto which the central recombinase Rad51 can load supported by various mediator and accessory proteins. Once bound, Rad51 catalyses invasion of its DNA into an intact homologous duplex, forming a displacement (D) loop where the 3′ end of the invading strand can be used to prime DNA synthesis. The end stages of HR involve either the dissociation or cleavage of the D-loop or its maturation into one or two Holliday junctions (HJs) that similarly can be processed by a variety of DNA helicases/translocases and structure-specific nucleases to disengage the recombining DNA molecules, enabling them to segregate during mitosis/meiosis. One of the core components of the HR machinery is the FANCM DNA translocase [2–4]. In humans, FANCM is encoded by one of 15 genes in which mutations can cause the rare genetic disease Fanconi anaemia (FA), characterized by progressive bone marrow failure, developmental problems and cancer proneness. At a cellular level, deficiencies in FA proteins result in hypersentivity to DNA interstrand cross-linking (ICL) agents such as cisplatin, increased chromosomal abnormalities, increased DNA bridges during mitosis, and high rates of bi- and multinucleated cells that result from failed cytokinesis [5]. The products of the FA genes are part of a DNA repair network with eight members (FANCA, -B, -C, -E, -F, -G, -L and -M), forming the so-called FA core complex that monoubiquitinates FANCD2 and FANCI, which in turn are thought to direct subsequent repair events involving HR and translesion DNA synthesis [6]. FANCM's role here is to target the core complex to sites of stalled replication, which promotes the monoubiquitination reaction [2,7–9]. However, various lines of evidence also point to key roles for FANCM in HR that are independent of core complex activation and FANCD2/I monoubiquitination [4]. Not least among these are studies of the yeast orthologues of FANCM (Mph1 in Saccharomyces cerevisiae and Fml1 in Schizosaccharomyces pombe), which operate in environments that are devoid of most other FA proteins. FANCM, Mph1 and Fml1 are superfamily 2 DNA helicases/translocases, and in vitro can use the energy from hydrolysing ATP to drive fork remodelling, HJ branch migration and D-loop dissociation [10–15]. Based on the findings of cell biological and genetic experiments, it is thought that these activities are used to support at least two reactions related to HR in vivo, namely the reversal of stalled replication forks (which could be used to generate a substrate for HR to promote replication restart) and the processing of recombination intermediates that enables the recombining DNAs to disjoin. In the latter case, several studies have shown that FANCM and its orthologues can limit the formation of crossover (CO) recombinants that stem from the cleavage of D-loops/HJs by structure-specific nucleases such as Mus81–Eme1 [12–14,16–20]. COs are reciprocal exchanges of the chromosomal regions that flank the site at which HR has acted. In meiotic cells, they are necessary for the establishment of chiasmata, but in somatic cells they can result in deleterious genome rearrangements if the recombining DNAs are other than perfectly aligned sister chromatids. The ability of FANCM/Mph1/Fml1 to direct CO avoidance most probably relates to its D-loop dissociation activity, which negates the need for junction resolution by a nuclease, and, in the context of a DSB, drives repair via a sub-pathway of HR called synthesis-dependent strand annealing that generates only non-crossover (NCO) recombinants. Recently, it was found that FANCM interacts with a complex of two small histone-fold proteins named MHF1 and MHF2 (i.e. FANCM-associated Histone-Fold protein 1 and 2) [21,22]. The interaction occurs via a region just on the C-terminal side of FANCM's helicase domain, which docks onto a heterotetramer configuration of MHF1 and MHF2 that resembles the H3–H4 heterotetramer within histone octamers [23]. For brevity, we will refer to both the yeast and human form of this complex as MHF henceforth. Genetic studies in HeLa and/or chicken DT40 cells have shown that MHF functions alongside FANCM in promoting FANCD2 monoubiquitination following induction of ICLs, and suppressing spontaneous sister chromatid exchange (SCE), albeit in the latter case not to the same extent as FANCM [21,22]. At least in part, it appears to fulfil these roles by promoting the stability, chromatin association and substrate targeting of FANCM. In vitro purified MHF binds to double-stranded DNA (dsDNA) and enhances the fork reversal activity of FANCM [21,22]. Intriguingly, there is a synergistic increase in the DNA binding activity of the FANCM–MHF complex, resulting from the establishment of an additional DNA binding site, which is presumably important for substrate targeting [22,23]. Interestingly, MHF1 and MHF2 are also components of the constitutive-centromere-associated network (CCAN), going under the names of CENP-S and CENP-X, respectively [24,25]. Here, they interact with CENP-T and CENP-W to form a stable heterotetramer that can wrap DNA around itself in a manner that is thought to be analogous to the tetrasome formed by the histone H3–H4 heterotetramer [25]. CENP-T interacts directly with the Ndc80 complex of the outer kinetochore, which in turn attaches to the microtubules of the mitotic spindle [26–28]. In this way, CENP-T-W-S-X is thought to form a point of anchorage for the kinetochore at the centromere that is additional to that formed by the interaction of Ndc80 to CENP-A-containing nucleosomes via Mis12 and CENP-C. DT40 cells deficient in MHF exhibit noticeable defects in kinetochore architecture, including reduced localization of Ndc80 to the outer kinetochore and an increase in the intrakinetochore distance between CENP-T and Ndc80, and depletion of MHF2 in HeLa cells results in numerous mitotic defects, including a high proportion of misaligned chromosomes at the metaphase plate [24]. To gain a greater understanding of MHF's roles in DNA recombination and repair, and how this relates to its function at the centromere, we have conducted a genetic and biochemical analysis of S. pombe MHF. We show that MHF's DNA repair/recombination role is distinct from its centromeric role, with the former depending largely on its physical interaction with Fml1 and the latter being independent of Fml1. We also reveal that MHF is recruited to DNA bridges and trailing segments of DNA during mitosis in a Fml1-dependent manner. Impairment of Fml1's catalytic activity or interaction with MHF increases the frequency of mitotic DNA bridges, but relatively few of these lead to gross chromosome missegregation, seemingly due to processing by the Mus81–Eme1 endonuclease. Our data indicate that unresolved recombination intermediates often persist into mitosis and are processed by Fml1–MHF or Mus81–Eme1 even as late as anaphase/telophase. 3. Results 3.1. Mhf1 and Mhf2 localize to centromeres, and are needed for correct chromosome segregation during meiosis In contrast to fml1, deletion of either mhf1 or mhf2 in fission yeast results in a marked reduction in growth and viability indicating that MHF performs a critical function that does not require Fml1 (figure 1a). Epifluorescence microscopy of strains expressing GFP-tagged forms of Mhf1 and Mhf2 show that they co-localize with the CCAN component Mis6 (CENP-I), confirming that Mhf1 and Mhf2 are centromeric proteins in fission yeast (figure 1b; electronic supplementary material, figure S1). Analysis of meiotic chromosome segregation using strains in which chromosome 2 is marked with an array of lacO sequences bound by the LacI repressor protein fused to GFP showed that deletion of mhf2 results in a high proportion of meiosis I and II segregation defects (figure 1c,d). By contrast, a fml1Δ mutant exhibits almost wild-type levels of accuracy for meiotic chromosome segregation (figure 1d). Altogether these data are consistent with the notions that MHF plays an important role at the centromere in establishing proper kinetochore function, which is needed for faithful chromosome segregation, and that this function is independent of its involvement with Fml1 in recombination. Figure 1. MHF functions at the centromere and promotes viability and chromosome segregation independently of Fml1. (a) Spot assay showing the relative growth of strains MCW1221, MCW2080, MCW4639 and MCW4777 on YES agar after 3 days at 30°C. (b) Example cells from a culture of MCW5846 showing the co-localization of Mhf1-GFP with Mis6-mCherry. (c) Schematic of the meiotic chromosome segregation assay. (d) Meiotic chromosome segregation in asci from wild-type (AY167-1D × FO652), fml1Δ (MCW4172 × MCW6196) and mhf2Δ (MCW4777 × MCW5113) homozygous crosses. 3.2. MHF interacts with the C-terminal region of Fml1 Human MHF binds to a region on the C-terminal side of FANCM's helicase domain [22,23]. To see whether the same is true for the fission yeast orthologues, we established an in vitro assay for determining their interaction using purified MHF (figure 2; electronic supplementary material, figure S2A) and Fml1 fused to maltose binding protein (MBP). Essentially, MBP-Fml1 bound to amylose resin was tested for its ability to retain MHF on the resin, with detection of the complex on a Western blot using an antibody against a His-tag fused to Mhf2. As expected, full-length Fml1, which is 834 amino acids (figure 2a), retained MHF on the resin, whereas MBP or resin alone did not (figure 2b). We next tested various fragments of Fml1 for their ability to bind MHF; by this approach, we narrowed down the point of interaction to a region between amino acids, 620 and 700 (figure 2a,b; electronic supplementary material, figure S2B,C). Further division of this region established that amino acids 650–690 encompassed the site of interaction (figure 2c), and that amino acids 670–690 are sufficient to retain MHF on the resin, but only under conditions of limited washing (figure 2d). To identify key residues needed for the interaction, we chose to mutate the tyrosine and arginines in the 670–690 region to alanine as such changes have been shown to frequently disturb protein–protein interactions in other cases [29] (figure 2e). Three combinations of mutations were tested, and whereas changing arginines 683, 686 and 687 to alanine had no effect on the ability of the 650–690 amino acid fragment to interact with MHF, changing tyrosine 672 together with arginines 674 and 678 to alanine totally abolished binding both in the context of the 650–690 amino acid fragment and the full-length protein (figure 2f,g). Figure 2. MHF interacts with Fml1. (a) Schematic of Fml1 and the various truncated forms of it used in (b) and (c). The position of the MBP fusion, DNA helicase motifs (blue bars), and region encompassing the site of MHF interaction (red box) are shown. The numbers refer to amino acid positions. (b–d) Western blots showing the amount of His-tagged Mhf2 retained on amylose resin that has been pre-incubated with the indicated MBP-Fml1 fragment (the numbers refer to amino acid positions). (e) Amino acids 670–690 in Fml1 with the mutations tested in (f) indicated. (f) Western blot showing the retention of His-tagged Mhf2 on amylose resin pre-incubated with MBP-Fml1650–690 or its mutant derivatives 1–3. (g) Western blot showing that full-length Fml1AAA fused to MBP fails to retain HisMhf2 on amylose resin. (h) SDS-PAGE analysis of gel filtration fractions 32–38 from the purification of the Fml1576–725–MHF complex. The gel was stained with Coomassie blue. (i) EMSA comparing the ability of MHF and the Fml1576–725–MHF complex to bind linear dsDNA. The amounts of protein are 49 nM (lanes b and f), 98 nM (lanes c and g), 490 nM (lanes d and h) and 980 nM (lanes e and i). 3.3. MHF's ability to bind dsDNA appears to be enhanced by its interaction with Fml1's C-terminal domain It has recently been shown that the interaction between human MHF and FANCM generates a DNA binding site, which results in a synergistic increase in DNA binding of the protein complex [23]. To see whether the same is true for S. pombe MHF and Fml1, we co-expressed Fml1's C-terminal domain (residues 576–725) with Mhf1 and His-tagged Mhf2 in Escherichia coli and purified the complex by nickel affinity and gel filtration chromatography (figure 2h). We then compared the DNA binding ability of this complex to that of the MHF complex using an electrophoretic mobility shift assay (EMSA; figure 2i). The MHF complex without Fml1576–725 binds a 50 bp linear dsDNA to form a single retarded band at relatively high protein concentrations (greater than 1.48 µM; electronic supplementary material, figure S2D). By contrast, much lower concentrations of the MHF–Fml1576–725 complex (less than or equal to 490 nM) can achieve the same amount of DNA binding, consistent with the idea that the interaction between MHF and Fml1 forges an additional DNA binding site (figure 2i, lanes h and i). However, a caveat to this experiment is that we were unable to purify Fml1576–725 to homogeneity and therefore are uncertain whether or not this region of Fml1 binds DNA in its own right. 3.4. Fml1's ability to interact with MHF is important but not essential for its role in DNA repair We have previously shown that Fml1 is needed for the repair/tolerance of DNA damage induced by the alkylating agent methyl methanesulfonate (MMS) and the DNA ICL agent cisplatin [12,14]. To see whether MHF also plays a role here alongside Fml1, we compared the MMS and cisplatin sensitivities of fml1Δ, mhf1Δ and mhf2Δ single and double mutant strains (figure 3a). As described earlier, both mhf1Δ and mhf2Δ mutants exhibit poor growth, and our data here show that this is not worsened when they are combined together or with fml1Δ. The poor growth hampers the comparison of genotoxin sensitivities, but nevertheless it is clear that both mhf1Δ and mhf2Δ exhibit similar levels of hypersensitivity to MMS and cisplatin, which is not further enhanced when combined with fml1Δ. This epistatic relationship indicates that MHF and Fml1 function in the same pathway for the repair/tolerance of ICLs and MMS-induced damage. Figure 3. MHF functions with Fml1 in the same DNA damage tolerance/repair pathway. (a) Spot assays comparing the growth and genotoxin sensitivities after 5 days of growth at 30°C for (a) strains MCW1221, MCW2080, MCW4639, MCW4777, MCW5127, MCW5790 and MCW5126, and (b) strains MCW1221, MCW2080, MCW5895 and MCW4405. To see how important the interaction between Fml1 and MHF is for their ability to promote DNA repair, we compared the MMS and cisplatin sensitivities of a fml1Δ strain with one in which Y672, R674 and R678 in fml1 were mutated to alanine and a natMX4 marker inserted adjacent to its 3′ untranslated region (fml1AAA::natMX4; figure 3b). Interestingly, the fml1AAA mutant, although hypersensitive to MMS and cisplatin, is not as sensitive as a fml1Δ mutant. However, it exhibits the same sensitivity as a strain containing a truncated form of Fml1, in which the entire C-terminal domain from amino acid 604–834 is deleted (fml1ΔC1–603). Importantly, the hypersensitivity of both fml1AAA and fml1ΔC1–603 mutants is not due to an altered level of Fml1 protein (see electronic supplementary material, figure S3) nor to the presence of the linked natMX4 marker in these strains [12]. Together these data indicate that the critical role of Fml1's C-terminal domain is to mediate the interaction with MHF, and that without this Fml1 is still able to promote DNA repair, albeit at a reduced efficiency. 3.5. MHF functions with Fml1 to promote non-crossover recombination Fml1 plays a major role in promoting NCO recombination both in mitotic and meiotic cells, and at least in the latter case MHF is also involved [19]. To further investigate the involvement of MHF in promoting NCO recombination, we used a plasmid gap repair assay, in which a plasmid containing a double-stranded gap within a copy of ade6 is repaired by HR with a mutant copy (ade6-M26) on chromosome III, resulting in integration of the plasmid into the chromosome (CO) or recircularization of the plasmid (NCO) (figure 4a) [14]. In a wild-type strain, approximately 75% of the repaired plasmids contain a fully restored copy of ade6+ resulting from a gene conversion (GC) event and only 10% of these are COs (figure 4b; electronic supplementary material, figure S4). A comparison of fml1Δ, mhf1Δ and mhf2Δ single and double mutants showed that they all exhibit wild-type levels of GC and gap repair, but, unlike wild-type, approximately 35% of ade+ recombinants are COs (figure 4b; electronic supplementary material, figure S4). The similarity in the percentage of COs in both single and double mutant strains indicates that MHF plays an essential role in the Fml1-dependent pathway of NCO recombination in mitotic cells. Interestingly, the fml1AAA mutant exhibits only approximately 20% COs among ade+ recombinants (figure 4b), which suggests that MHF may not need to interact with Fml1 in order to provide at least some assistance in promoting NCO recombination in mitotic cells. Figure 4. MHF promotes NCO recombination in both mitotic and meiotic cells. (a) Schematic of the plasmid gap repair assay. The filled circle indicates the position of the M26 mutation. (b) Percentage of Ade+ transformants that are COs in strains MCW1193, MCW2096, MCW5345, MCW5346, MCW5790, MCW5983, MCW4893 and MCW6001. Values are means from three experiments ±s.d. (c) Schematic of the meiotic recombination assay showing the different types of CO and NCO recombinants that can be associated with an ade6+ convertant. The filled circles indicate the position of the 3083 and L469 mutations. (d) Percentage of CO associated with GC events at ade6-3083 from wild-type and mutant crosses (see electronic supplementary material, table S1). Values are means from at least six crosses ±s.d. Statistical significance in comparison with wild-type and fml1+::natMX4 is p < 0.01. MHF supports Fml1 in directing NCO recombination during meiosis [19]. To see whether the interaction between these proteins is important for this, we compared the percentage of COs associated with GC at the ade6-3083 meiotic recombination hotspot in wild-type, fml1Δ and fml1AAA strains (figure 4c,d; electronic supplementary material, table S1). As seen previously, there is a small but significant increase in the percentage of COs associated with GC in a fml1Δ mutant compared with wild-type. A similar increase is also seen in the fml1AAA mutant, indicating that the interaction between Fml1 and MHF is important for directing NCO recombination during meiosis. 3.6. MHF functions with Fml1 to promote gene conversion at blocked replication forks Replication fork stalling at the RTS1 protein–DNA barrier induces Rad51-dependent recombination, which can give rise to both GC (conversion-types) and deletions (deletion-types) between flanking ade6− heteroalleles (figure 5a) [30]. Fml1 plays a role here in promoting GC, probably by catalysing the reversal of the stalled fork, and recent work has implicated Mhf2 in assisting it in this function [12,14,22]. To confirm that MHF works with Fml1 in promoting GC at the RTS1 barrier, we compared the frequency of ade+ deletion- and conversion-types in fml1Δ, mhf1Δ and mhf2Δ single and double mutant strains, and in the fml1AAA mutant (figure 5b,c). Consistent with published data, absence of fml1 has no effect on the frequency of deletion-types, but reduces conversion-types by approximately sevenfold. Both mhf1Δ and mhf2Δ single mutants likewise show a reduction (approx. threefold) in conversion-type frequency, but interestingly exhibit a small increase in deletion-types. Importantly, both fml1Δ mhf1Δ and fml1Δ mhf2Δ double mutants exhibit essentially the same deletion-type and conversion-type frequency as a fml1Δ single mutant. Similar to what was seen in the plasmid gap repair assay, the fml1AAA mutant exhibits a more modest effect on recombination than fml1Δ, with a reduction in conversion-types of twofold when compared with a fml1+::natMX4 strain, which exhibits slightly higher recombinant frequencies than a wild-type without the natMX4 marker. Altogether these data indicate that MHF promotes Fml1-dependent GC at stalled replication forks, and can do so at a reduced level even when unable to interact with Fml1's C-terminal domain. In the absence of MHF, Fml1 retains some ability to act but at a much reduced efficiency. Moreover, without MHF Fml1 may act in an aberrant fashion to promote the formation of deletion-types. Figure 5. MHF functions with Fml1 to promote RTS1-induced direct repeat recombination. (a) Schematic of the recombination substrate on chromosome III, including the two classes of Ade+ recombinant product. (b) Deletion-type frequencies and (c) conversion-type frequencies in strains MCW4713, MCW3060, MCW4770, MCW5217, MCW5218, MCW5220, MCW4797 and MCW5932. All values are mean±s.d. Statistical significance in comparison with wild-type and fml1+::natMX4 is p < 0.01 and **p < 0.001. 3.7. MHF localizes to non-centromeric sites in a Fml1-dependent fashion As shown in figure 1b, Mhf1-GFP co-localizes with the centromeric protein Mis6, consistent with it being a component of the CCAN. However, unlike Mis6, it also forms a speckling of fluorescence throughout the rest of the nucleus, which might represent its localization to sites across the genome where Fml1 is actively engaged in DNA repair and recombination. To investigate this, we assessed whether Mhf1-GFP localization is affected by deletion of fml1 (figure 6; electronic supplementary material, figure S5). In a fml1Δ strain, Mhf1-GFP centromeric localization appears unaltered. By contrast, its wider distribution throughout the nucleus is lost or greatly diminished in almost all cells, and this is also true in a fml1AAA strain. This loss in general nuclear fluorescence is not due to a change in the amount of Mhf1-GFP, which is the same in both wild-type and fml1 mutant strains (see electronic supplementary material, figure S6). To see whether the non-centromeric localization of MHF is dependent on Fml1's catalytic activity, we assessed Mhf1-GFP fluorescence in a fml1D196N strain, which contains a mutation in Fml1's helicase motif II that destroys its ATPase activity (and therefore its ability to unwind and branch migrate DNA junctions) without affecting its DNA binding [12]. Similar to the wild-type strain, Mhf1-GFP localized to both centromeric and non-centromeric sites throughout the nuclei of fml1D196N mutant cells (figure 6; electronic supplementary material, figure S5). Altogether these data indicate that MHF is recruited to and/or retained at non-centromeric chromosomal sites through its interaction with Fml1. Figure 6. Nuclear localization of Mhf1-GFP in wild-type and fml1 mutant cells. The strains are MCW5846, MCW5963, MCW6152 and MCW6132. 3.8. Fml1 limits mitotic bridge and tail formation In human cells, the FA pathway plays a role in limiting the occurrence of DNA bridges connecting segregating sister chromatids during mitosis [31]. To see whether Fml1 plays a similar role in S. pombe, we analysed binucleate cells from asynchronously growing cultures of wild-type and fml1Δ strains, using DAPI to stain the DNA (figure 7a,b). Only approximately 3% of wild-type binucleate cells exhibit a DNA bridge between the two masses of segregating DNA (figure 7c). By contrast, approximately 25% of fml1Δ binucleates exhibit a DNA bridge, albeit some of these are discontinuous and therefore perhaps better described as DNA tails with a small gap at or near the midpoint between the main DNA masses (figure 7b,c). The bridges and tails in the fml1Δ strain are also on average longer than those seen in wild-type cells (figure 7d). A similar frequency and length of bridges and tails was also seen among fml1D196N binucleates, whereas in a fml1AAA mutant their frequency is less, albeit still fivefold more than in a wild-type (figure 7c,d). Altogether these data indicate that Fml1's catalytic activity and interaction with MHF are needed for the efficient and timely resolution of DNA connections between sister chromatids. Figure 7. Mitotic DNA bridges in wild-type and fml1 mutant cells. (a) Examples of wild-type cells undergoing mitosis. (b) Examples of fml1Δ mutant cells exhibiting a mitotic DNA bridge (top row) and tails (bottom row). (c) Frequency of mitotic DNA bridges/tails and (d) length of mitotic DNA bridges in strains MCW1221, MCW2080, MCW4778 and MCW5895. In all cases, values are mean±s.d. (e) Examples of Mhf1-GFP co-localizing with mitotic DNA bridges/tails in wild-type (MCW5846) and fml1D196N mutant cells (MCW6132). 3.9. MHF localizes to mitotic bridges and tails in a Fml1-dependent fashion In human cells, immunostaining for FANCM has revealed that it forms bridges between segregating DNA in telophase, suggesting that it plays a role during this late stage of mitosis to resolve persistent connections between sister chromatids [31]. Similarly, Mhf1-GFP localizes to more than 90% of the mitotic DNA bridges or tails detected in wild-type cells by DAPI staining (figure 7e; electronic supplementary material, figure S7). This localization is far more striking in fml1D196N cells, which exhibit a greater frequency and length of bridges and tails than wild-type (figure 7e; electronic supplementary material, figure S7). In a few cases, we also observed Mhf1-GFP localizing to the region between the segregating DNA masses when no DNA bridge or tail was detected by DAPI staining (see electronic supplementary material, figure S7). Importantly, the localization of Mhf1-GFP to mitotic DNA bridges and tails is lost or greatly diminished in both fml1Δ and fml1AAA mutants (see electronic supplementary material, figure S7). Altogether these data indicate that MHF is recruited to and/or retained at mitotic DNA bridges and tails through its interaction with Fml1. The fact that these bridges are occasionally detected in wild-type cells suggests that Fml1 together with MHF can act as late as mitosis to resolve connections between sister chromatids. 3.10. Mus81–Eme1 functions as a failsafe for resolving sister chromatid connections in the absence of Fml1 Even though the frequency of mitotic DNA bridges and tails increases in a fml1Δ mutant, this does not lead to a correspondingly high frequency of aberrant chromosome segregation among cells that have laid down a division septum (figure 8a,b). This suggests that DNA bridges are resolved prior to cytokinesis. A prime candidate for resolving DNA junctions between sister chromatids is the Mus81–Eme1 endonuclease, whose orthologue in budding yeast is activated during G2 and M phase by CDK- and Polo-like kinase-dependent phosphorylation of Mms4 (the orthologue of Eme1) [32,33]. Consistent with Mus81–Eme1 playing an important role in resolving sister chromatid junctions, a high frequency (approx. 40%) of mus81Δ binucleate cells exhibit mitotic DNA bridges, tails and lagging chromosomes (see electronic supplementary material, figure S8). Moreover, unlike in a fml1Δ mutant, there is a similarly high number of septated cells with aberrant chromosomal segregation, including ‘cut’ (where DNA spans the division septum), missegregation and failed segregation phenotypes (figure 8a,b). A mus81Δ fml1Δ double mutant exhibits a marked reduction in growth and viability compared with either single mutant [14], and this correlates with a range of abnormal cell and nuclear morphologies, including many cells in which the DNA appears to be fragmented, possibly as a consequence of aberrant chromosome segregation or as part of an apoptotic response. Among septated cells, the frequency of aberrant chromosome segregation is much higher than in either single mutant, although it should be noted that the nuclear fragmentation, which is prevalent in the double mutant, complicates this analysis (figure 8b). Altogether these data indicate that Mus81–Eme1 is able to process the majority of DNA junctions that would normally be dealt with by Fml1. Figure 8. Partial suppression of aberrant chromosome segregation and genotoxin sensitivity in a fml1Δ mus81Δ double mutant by deleting rad51. (a) Examples of aberrant chromosome segregation in septated mus81Δ (MCW1779) and mus81Δ fml1Δ (MCW2428) mutant cells. Arrowheads indicate the division septum. (b) Frequency of aberrant chromosome segregation among septated cells in strains MCW1221, MCW2080, MCW1088, MCW1779, MCW2428 and MCW4490. Values are based on the analysis of approximately 300 septated cells from three independent asynchronously growing cultures. (c) Spot assay comparing the growth and genotoxin sensitivity of strains MCW3792, MCW1235, MCW2428 and MCW4490. Plates were photographed after 5 days growth at 30°C. In vitro Mus81–Eme1 and Fml1 can process a similar spectrum of DNA junctions, including model replication forks and recombination intermediates such as D-loops and HJs [4,34]. The frequency of aberrant chromosome segregation in a fml1Δ mus81Δ double mutant is reduced almost twofold by deleting rad51 (figure 8b), and rad51 deletion also partially suppresses the hypersensitivity of a fml1Δ mus81Δ double mutant to ultraviolet (UV) light and MMS, which are agents that induce HR (figure 8c). These data suggest that much of the impaired chromosome segregation in a fml1Δ mus81Δ mutant is due to unresolved recombination intermediates that presumably impede sister chromatid separation. The fact that rad51 deletion does not improve the growth and viability of a fml1Δ mus81Δ mutant more fully is probably due to Mus81 also having a role in a Rad51-independent DNA repair pathway [35]. 4. Discussion Recent studies have established that vertebrate MHF functions as a component of the CCAN as well as an accessory factor for FANCM; however, it has been unclear whether these functions are entirely distinct or overlap. In HeLa cells, transiently transfected GFP-FANCM localizes to centromeres in an MHF1-dependent fashion, suggesting that it plays a role there in humans [23]. However, we have shown that, at least in fission yeast, MHF's function at the centromere is distinct from its role in supporting Fml1 in DNA repair and recombination. This conclusion is based on our observations that Mhf1-GFP localizes to centromeres in a Fml1-independent manner, and that, unlike a fml1Δ mutant, mhf1/2Δ mutants exhibit poor viability and high rates of meiotic chromosome missegregation. Although these data do not exclude the possibility that Fml1 functions at centromeres together with MHF, they do indicate that any role it might play there is non-essential. Similar to human MHF, fission yeast MHF binds linear dsDNA but not ssDNA (see electronic supplementary material, figure S2; S.B. & M.C.W. 2013, unpublished data), whereas the helicase domains of both FANCM and Fml1 bind ssDNA and branched dsDNA structures but not linear dsDNA [4,22]. The Fml1–MHF complex is therefore endowed with the ability to bind all of the constituent parts of a stalled replication fork or D-loop, forming multiple protein–DNA contacts that presumably enable efficient targeting of these substrates in vivo. Moreover, the interaction between MHF and FANCM/Fml1 appears to generate an additional DNA binding site, which further enhances this ability of the complex [23] (figure 2i). Unsurprisingly, MHF is needed for the localization of FANCM to chromatin and its efficient recruitment to DNA ICLs [22,23]. Our data indicate that this dependency is likely to be reciprocal as Mhf1-GFP exhibits reduced or no localization to non-centromeric chromatin when unable to interact with Fml1 (because Fml1 is either deleted or mutated in its MHF interaction domain). Interdependence between both components of the FANCM/Fml1–MHF complex for localizing to non-centromeric chromatin would accord with its synergistic increase in DNA binding in vitro [22,23]. MHF supports Fml1 in promoting NCO meiotic recombination and Rad51-dependent recombination at blocked replication forks [19,22]. We have shown here that it is also essential for Fml1's role in CO avoidance during mitotic DSB repair, and works together with Fml1 in promoting the tolerance/repair of both MMS and cisplatin-induced DNA damage. However, at least for promoting RTS1-induced direct repeat recombination, it is evident that Fml1 retains some ability to act without MHF. This is also true in chicken DT40 cells where deletion of FANCM results in a bigger increase in SCE than deletion of MHF1 [22]. More surprisingly, the interaction between Fml1 and MHF, at least mediated by Fml1's C-terminal domain, is not essential for MHF to make a contribution to Fml1-mediated DNA repair and recombination. Of course, we cannot be certain that the Y672A, R674A and R678A mutations generated in our study totally prevent Fml1 from interacting with MHF in vivo, which could occur additionally via unknown intermediary proteins and/or as a consequence of post-translational modification. Replication forks blocked at RTS1 are restarted by a recombination-dependent process [36], and therefore the reduction in RTS1-induced GC in both fml1Δ and mhf1/2Δ mutants probably reflects the fact that Fml1–MHF is a key component of the replication restart machinery. Indeed FANCM's ATPase activity has been shown to be important for stabilizing and restarting stalled replication forks in human cells [37]. Fml1's ability to catalyse fork reversal, which is probably enhanced by MHF, would generate a substrate for the recruitment of other recombination proteins, leading ultimately to the assembly of a Rad51-DNA filament, which catalyses the key step of strand invasion that reprimes DNA synthesis [12,14]. Without a fully functional Fml1–MHF complex replication restart would be impaired, and this could result in unreplicated regions of the genome persisting into mitosis, leading to an increase in mitotic DNA bridges (as observed in fml1Δ, fml1D196N and fml1AAA mutants). A similar scenario has been proposed to explain the increase in ultrafine anaphase bridges (UFBs) detected by immunostaining for the BLM DNA helicase in FANCM-depleted cells [31]. However, a failure to resolve recombination intermediates (i.e. D-loops and HJs) between sister chromatids could also account for the occurrence of mitotic DNA bridges in the fml1 mutants. The possibility that some UFBs in human cells are caused by unresolved recombination intermediates has been largely discounted because UFB occurrence increases upon deletion of RAD51 [38]. However, in S. pombe, deletion of Rad51 and its key mediator Rad52, which together are responsible for all recombination-dependent replication restart (RDR) [36], results in a relatively small increase in the frequency of mitotic DNA bridges compared with a fml1Δ mutant (L.F. & M.C.W. 2013, unpublished data). Therefore, a failure to promote RDR cannot account for all of the mitotic DNA bridges that are observed when Fml1 is absent or impaired. A failure of FANCM to prevent/resolve UFBs correlates with an increase in multinucleated cells, which are thought to arise as a consequence of cytokinesis failure due to DNA being trapped in the cleavage furrow [31]. In S. pombe, septation proceeds even when DNA spans the division plane, resulting in cut phenotypes and uneven distribution of DNA between daughter cells [39,40]. Interestingly, very few of the mitotic DNA bridges in a fml1Δ mutant give rise to a cut or chromosome missegregation phenotype, indicating that alternative pathways of restarting stalled replication forks and processing recombination intermediates are able to act during mitosis to achieve sister chromatid separation prior to septation. At least one of these pathways appears to depend on Mus81–Eme1 since a mus81Δ fml1Δ double mutant exhibits a synergistic increase in chromosome missegregation and cell inviability. Importantly, these phenotypes are partially suppressed by deleting rad51, suggesting that some of the aberrant chromosome segregation is due to unresolved recombination intermediates. Mus81–Eme1's ability to act late in the cell cycle to resolve recombination intermediates accords with the finding in budding yeast that Mus81–Mms4 nucleolytic activity is activated in G2 and M phase [32,33]. Intriguingly, while Mus81–Eme1 appears to be able to resolve most of the recombination intermediates that accumulate in a fml1Δ mutant, the reverse is not true, as the majority of mitotic DNA bridges in a mus81Δ mutant seemingly give rise to cut and chromosome missegregation phenotypes. Single HJs formed during the repair of broken replication forks may account for these bridges, as neither Fml1 nor RecQ helicase-dependent double HJ dissolution would be able to substitute effectively for Mus81–Eme1 in resolving them productively [34]. One of the intriguing observations in our study is the speckled localization of Mhf1-GFP at non-centromeric sites throughout the nucleus, which depends on MHF's interaction with Fml1 but not on Fml1's ATPase activity. This pattern of localization is observed in essentially all cells within an asynchronously growing population, and presumably represents binding of Fml1–MHF or Fml1-dependent deposition of MHF to multiple genomic sites. Given MHF's potential to associate with other histone-fold proteins [22,25], it is possible that it forms distinct regions of chromatin at sites where replication forks have been perturbed or recombination enacted. Such chromatin could persist until its displacement in the following S-phase. Enrichment of Mhf1-GFP on mitotic DNA bridges may therefore represent either the redeployment of Fml1–MHF to replication/recombination intermediates in M-phase or its retention at these sites following earlier recruitment in S- or G2-phase. The former is certainly possible because in human cells FANCM has been shown to localize to UFBs in telophase [31]. Regardless of when it is recruited, Mhf1-GFP's presence on bridges suggests that Fml1–MHF is able to act during the late stages of the cell cycle to promote sister chromatid segregation. MHF is a member of a growing list of CCAN proteins that also function in DNA repair/recombination [41]. It has been speculated that recombination might play a role in proper centromere function [42], and if true this would provide a link between these seemingly disparate processes. However, at least in the case of MHF, its recombination function, which is rooted in its interaction with Fml1, is distinct from its key centromeric role. Nevertheless, it is intriguing to note that both its functions share a common aim in promoting chromosome segregation. Defining the evolutionary origin of this dual role presents an interesting challenge for future research. 5. Material and methods 5.1. Schizosaccharomyces pombe strains and plasmids Schizosaccharomyces pombe strains are listed in electronic supplementary material, table S2. The mhf1Δ::kanMX6 and mhf2Δ::natMX4 strains were made by gene targeting using derivatives of pFA6a-kanMX6 [43] (pMW871) and pAG25 [44] (pMW872), respectively. The initial gene deletion was made in a diploid S. pombe strain from which haploid segregants were obtained. The mhf1::GFP-kanMX6 strain was made by gene targeting directly in a haploid S. pombe strain using a derivative of pFA6a-GFP(S65 T)-kanMX6 [43] (pCB1). Similarly, fml1AAA::natMX4, fml1ΔC1–603::natMX4, fml1+::13Myc-natMX4, fml1AAA::13Myc-natMX4 and fml1ΔC1–603::13Myc-natMX4 strains were made by gene targeting in a haploid S. pombe strain using derivatives of pAG25 (pJBB79, pJBB9, pJBB28, pJBB81 and pJBB7, respectively). The plasmids for expressing full-length or fragments of Fml1 fused to MBP are all derivatives of pMAL-c2x (New England BioLabs) with a BamHI–XbaI insert encoding the stated portion of Fml1. The plasmid for co-expressing Mhf1 and His-tagged Mhf2 (pMW891) was made by first cloning the cDNA for mhf2 as an NdeI–BamHI fragment into pET14b to make pMW884. A BglII–SalI fragment containing the T7 promoter and mhf1 cDNA from the pT7-7 derivative pMW889 was then cloned into these sites in pMW884. The plasmid for co-expressing Mhf1, His-tagged Mhf2 and Fml1576–725 (pCB6) was made by amplifying the T7 promoter and fml1576–725 fragment from the pT7-7 derivative pCB5 and cloning this as a NheI fragment into pMW891. All plasmids were verified by DNA sequencing. 5.2. Media and genetic methods Media and genetic methods followed standard protocols [45]. The complete and minimal media were yeast extract with supplements (YES) and Edinburgh minimal medium plus 3.7 mg ml−1 sodium glutamate (EMMG), plus appropriate amino acids (0.25 mg ml−1), respectively. Sporulation of crosses was performed on malt extract agar (MEA). Low adenine media (YELA) was supplemented with 0.01 mg ml−1 adenine. Ade+ recombinants were selected on YES lacking adenine and supplemented with 0.2 mg ml−1 guanine to prevent uptake of residual adenine. 5.3. Spot assays Exponentially growing cells from liquid cultures were harvested, washed and resuspended in water at a density of 1×107–1×103 cellsml−1. Aliquots (10 µl) of the cell suspensions were spotted onto YES agar plates containing genotoxins as indicated. For UV, plates were irradiated using a Stratalinker (Stratagene). Plates were photographed after 3–5 days growth at 30°C as indicated. 5.4. Microscopy Cells from an exponentially growing culture in YES were harvested and fixed with 70% ethanol for subsequent microscopy. The fixed cells were stained with DAPI and analysed using an Olympus BX50 epifluorescence microscope equipped with the appropriate filter sets to detect blue, green and red fluorescence (Chroma Technology, VT). Black and white images were acquired with a CoolSNAP HQ2 CCD camera (Photometrics, AZ) controlled by MetaMorph software (v. 7.7.3.0, Molecular Devices, CA). Images were pseudo-coloured and overlayed using Photoshop CS5 (v. 12.0, Adobe Systems, CA). 5.5. Recombination assays The direct repeat recombination, plasmid gap repair and meiotic recombination assays have been described previously [14,30,46,47]. Two sample t-tests were used to determine the statistical significance of differences in recombination values between strains unless otherwise stated. 5.6. Protein expression and purification A 1 l culture of E. coli BL21 (DE3) CodonPlus-RIL cells (Stratagene) transformed with pMW891 was grown with aeration at 25°C in LB broth containing 50 μg ml–1 ampicillin and 20 μg ml−1 chloramphenicol to an A600 of 0.5. Mhf1-HisMhf2 was induced by adding IPTG to a final concentration of 0.5 mM, following which the cells were grown with aeration at 25°C for a further 5 h. The cells were then harvested by centrifugation, resuspended in 20 ml Buffer H (50 mM potassium phosphate, pH 8.0, 0.3 M NaCl, 10% glycerol) and frozen at −80°C until needed. The frozen cells were defrosted and mixed with 1% Triton X-100, 10 mM β-mercaptoethanol and EDTA-free protease inhibitor cocktail (Roche) before passage through a French pressure cell at 19 000 p.s.i. All subsequent steps were performed at 4°C. The lysates were cleared by centrifugation at 19 000g for 50 min, and the supernatant was loaded directly onto a 1 ml nickel-nitrilotriacetic acid Superflow column (Qiagen) that had been pre-equilibrated with Buffer H. The column was then washed with 60 ml of Buffer H plus 20 mM imidazole before eluting bound Mhf1-HisMhf2 with Buffer H plus 100 mM imidazole into three 1 ml fractions. The second 1 ml fraction contained the peak of Mhf1-HisMhf2 and was loaded directly onto a HiLoad 16/60 Superdex 200 gel filtration column (Amersham Biosciences), which was then developed with 120 ml of Buffer A (50 mM Tris–HCl, pH8.0, 1 mM EDTA, 1 mM DTT, 10% glycerol) plus 0.3 M NaCl. Fractions of 2 ml were collected, and the peak fractions containing Mhf1-HisMhf2 (fractions 36–42) were pooled, diluted with an equal volume of Buffer A and loaded onto a 1 ml Hi-Trap Heparin column (GE Healthcare). The column was then washed with 5 ml of Buffer A plus 0.1 M NaCl before eluting bound protein with an 18 ml linear gradient from 0.1 to 1.0 M NaCl. The peak of Mhf1-HisMhf2 eluted between 0.41 and 0.43 M NaCl, and these fractions were pooled and stored as aliquots at −80°C. The purification of Fml1576–725-Mhf1-HisMhf2 followed the same protocol as for Mhf1-HisMhf2 except the expression plasmid was pCB6 and the final heparin step was omitted. The peak of Fml1576–725-Mhf1-HisMhf2 eluted from the gel filtration column in fractions 34–36, and these fractions were pooled and stored as aliquots at −80°C. Both full-length Fml1 and fragments of it were expressed as fusion proteins with MBP from the appropriate pMAL-c2x derivative in BL21 (DE3) CodonPlus-RIL cells. Cells were grown as 100 ml cultures at 25°C with aeration in LB broth containing 50 μg ml−1 ampicillin and 20 μg ml−1 chloramphenicol. At a cell density corresponding to an A600 of 0.6, IPTG was added to a final concentration of 250 μM and incubation continued for a further 12 h at 18°C. Cells were then harvested by centrifugation and resuspended in 10 ml of Buffer M (20 mM Tris–HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 10% glycerol). DTT (1 mM), Triton X-100 (1%) and PMSF (4 mM) were added to the sample before it was lysed by passage through a French pressure cell at 19 000 p.s.i. All subsequent steps were performed at 4°C. Cell debris was removed by centrifugation at 19 000g and the cleared lysate was then loaded onto a 0.5 ml amylose column (New England Biolabs) pre-equilibrated with Buffer M, which was then washed with 12 ml of Buffer M before eluting bound protein with Buffer M plus 10 mM maltose. Protein samples were pooled and stored as aliquots at −80°C. In all cases, protein amounts were estimated using a Bio-Rad protein assay kit with bovine serum albumin as the standard. 5.7. Protein–protein interaction assay Full-length Fml1 and fragments of Fml1 fused to MBP were immobilized on 100 μl amylose resin by incubating them for 2 h at 4°C on a rotating wheel. After removing unbound protein by washing with Buffer M, purified Mhf1-HisMhf2 was added and the mixture incubated for 3 h at 4°C on a rotating wheel. Unbound protein was removed by three consecutive 20 min washes with 0.5 ml Buffer M before eluting bound protein with Buffer M plus 10 mM maltose. Samples were then analysed for the presence of Mhf1-HisMhf2 by Western blotting using anti-polyhistidine antibody (Sigma). 5.8. DNA substrates The 32P-labelled linear dsDNA substrate was made by annealing oligonucleotides 2 and 44 (5′-CAACGTCATAGACGATTACATTGCTAGGACATCTTTGCCCACGTTGACCC-3′) as described by Whitby & Dixon [48]. 5.9. DNA binding assays Reaction mixtures (20 µl) contained 1.1 nM 32P-labelled linear duplex DNA in buffer (25 mM Tris–HCl, pH 8.0, 1 mM DTT, 100 µg ml−1 bovine serum albumin, 6% glycerol) plus protein as indicated. Reactions were incubated on ice for 15 min and then loaded onto a 4% native polyacrylamide gel in low-ionic-strength buffer (6.7 mM Tris–HCl, pH 8.0, 3.3 mM sodium acetate, 2 mM EDTA) that had been pre-cooled at 4°C. Gels were run for 1 h and 45 min at 160 V with continuous buffer recirculation. Gels were then dried on 3 MM Whatman paper and analysed by Phosphor Imaging using a Fuji FLA3000 and Image Gauge software. Supplementary Material Bhattacharjee et al Supplementary Material Acknowledgements We thank Zsofi Novak for constructing strain MCW4490, Elizabeth Murray for help with the mitotic DNA bridge analysis, and Stefania Castagnetti and Mitsuhiro Yanagida for supplying strains. Funding statement This work was supported by a grant (090767/Z/09/Z) from the Wellcome Trust. S.B. was supported by a K. Pathak Clarendon Scholarship from Exeter College, University of Oxford. ==== Refs References 1 Heyer WD Ehmsen KT Liu J 2010 Regulation of homologous recombination in eukaryotes . Annu. Rev. Genet. 44 , 113 –139 (doi:10.1146/annurev-genet-051710-150955)20690856 2 Meetei AR 2005 A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M . Nat. Genet. 37 , 958 –963 (doi:10.1038/ng1626)16116422 3 Mosedale G Niedzwiedz W Alpi A Perrina F Pereira-Leal JB Johnson M Langevin F Pace P Patel KJ 2005 The vertebrate Hef ortholog is a component of the Fanconi anemia tumor-suppressor pathway . Nat. Struct. Mol. Biol. 12 , 763 –771 (doi:10.1038/nsmb981)16116434 4 Whitby MC 2010 The FANCM family of DNA helicases/translocases . DNA Repair 9 , 224 –236 (doi:10.1016/j.dnarep.2009.12.012)20117061 5 Kee Y D'Andrea AD 2010 Expanded roles of the Fanconi anemia pathway in preserving genomic stability . Genes Dev. 24 , 1680 –1694 (doi:10.1101/gad.1955310)20713514 6 Wang W 2007 Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins . Nat. Rev. Genet. 8 , 735 –748 (doi:10.1038/nrg2159)17768402 7 Ciccia A 2007 Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM . Mol. Cell 25 , 331 –343 (doi:10.1016/j.molcel.2007.01.003)17289582 8 Kim JM Kee Y Gurtan A D'Andrea AD 2008 Cell cycle-dependent chromatin loading of the Fanconi anemia core complex by FANCM/FAAP24 . Blood 111 , 5215 –5222 (doi:10.1182/blood-2007-09-113092)18174376 9 Singh TR 2009 Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M . Blood 114 , 174 –180 (doi:10.1182/blood-2009-02-207811)19423727 10 Gari K Decaillet C Delannoy M Wu L Constantinou A 2008 Remodeling of DNA replication structures by the branch point translocase FANCM . Proc. Natl Acad. Sci. USA 105 , 16 107 –16 112 (doi:10.1073/pnas.0804777105) 11 Gari K Decaillet C Stasiak AZ Stasiak A Constantinou A 2008 The Fanconi anemia protein FANCM can promote branch migration of Holliday junctions and replication forks . Mol. Cell 29 , 141 –148 (doi:10.1016/j.molcel.2007.11.032)18206976 12 Nandi S Whitby MC 2012 The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair . Nucleic Acids Res. 40 , 9584 –9595 (doi:10.1093/nar/gks715)22844101 13 Prakash R 2009 Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination . Genes Dev. 23 , 67 –79 (doi:10.1101/gad.1737809)19136626 14 Sun W Nandi S Osman F Ahn JS Jakovleska J Lorenz A Whitby MC 2008 The fission yeast FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during double-strand break repair . Mol. Cell 32 , 118 –128 (doi:10.1016/j.molcel.2008.08.024)18851838 15 Zheng XF Prakash R Saro D Longerich S Niu H Sung P 2011 Processing of DNA structures via DNA unwinding and branch migration by the S. cerevisiae Mph1 protein . DNA Repair (Amst) 10 , 1034 –1043 (doi:10.1016/j.dnarep.2011.08.002)21880555 16 Bakker ST 2009 FANCM-deficient mice reveal unique features of Fanconi anemia complementation group M . Hum. Mol. Genet. 18 , 3484 –3495 (doi:10.1093/hmg/ddp297)19561169 17 Rosado IV Niedzwiedz W Alpi AF Patel KJ 2009 The Walker B motif in avian FANCM is required to limit sister chromatid exchanges but is dispensable for DNA crosslink repair . Nucleic Acids Res. 37 , 4360 –4370 (doi:10.1093/nar/gkp365)19465393 18 Deans AJ West SC 2009 FANCM connects the genome instability disorders Bloom's syndrome and Fanconi anemia . Mol. Cell 36 , 943 –953 (doi:10.1016/j.molcel.2009.12.006)20064461 19 Lorenz A Osman F Sun W Nandi S Steinacher R Whitby MC 2012 The fission yeast FANCM ortholog directs non-crossover recombination during meiosis . Science 336 , 1585 –1588 (doi:10.1126/science.1220111)22723423 20 Crismani W Girard C Froger N Pradillo M Santos JL Chelysheva L Copenhaver GP Horlow C Mercier R 2012 FANCM limits meiotic crossovers . Science 336 , 1588 –1590 (doi:10.1126/science.1220381)22723424 21 Singh TR 2010 MHF1-MHF2, a histone-fold-containing protein complex, participates in the Fanconi anemia pathway via FANCM . Mol. Cell 37 , 879 –886 (doi:10.1016/j.molcel.2010.01.036)20347429 22 Yan Z 2010 A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability . Mol. Cell 37 , 865 –878 (doi:10.1016/j.molcel.2010.01.039)20347428 23 Tao Y Jin C Li X Qi S Chu L Niu L Yao X Teng M 2012 The structure of the FANCM-MHF complex reveals physical features for functional assembly . Nat. Commun. 3 , 782 (doi:10.1038/ncomms1779)22510687 24 Amano M Suzuki A Hori T Backer C Okawa K Cheeseman IM Fukagawa T 2009 The CENP-S complex is essential for the stable assembly of outer kinetochore structure . J. Cell Biol. 186 , 173 –182 (doi:10.1083/jcb.200903100)19620631 25 Nishino T Takeuchi K Gascoigne KE Suzuki A Hori T Oyama T Morikawa K Cheeseman IM Fukagawa T 2012 CENP-T-W-S-X forms a unique centromeric chromatin structure with a histone-like fold . Cell 148 , 487 –501 (doi:10.1016/j.cell.2011.11.061)22304917 26 Malvezzi F Litos G Schleiffer A Heuck A Mechtler K Clausen T Westermann S 2013 A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors . EMBO J. 32 , 409 –423 (doi:10.1038/emboj.2012.356)23334295 27 Nishino T Rago F Hori T Tomii K Cheeseman IM Fukagawa T 2013 CENP-T provides a structural platform for outer kinetochore assembly . EMBO J. 32 , 424 –436 (doi:10.1038/emboj.2012.348)23334297 28 Schleiffer A Maier M Litos G Lampert F Hornung P Mechtler K Westermann S 2012 CENP-T proteins are conserved centromere receptors of the Ndc80 complex . Nat. Cell Biol. 14 , 604 –613 (doi:10.1038/ncb2493)22561346 29 Moreira IS Fernandes PA Ramos MJ 2007 Hot spots: a review of the protein–protein interface determinant amino-acid residues . Proteins 68 , 803 –812 (doi:10.1002/prot.21396)17546660 30 Ahn JS Osman F Whitby MC 2005 Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast . EMBO J. 24 , 2011 –2023 (doi:10.1038/sj.emboj.7600670)15889146 31 Vinciguerra P Godinho SA Parmar K Pellman D D'Andrea AD 2010 Cytokinesis failure occurs in Fanconi anemia pathway-deficient murine and human bone marrow hematopoietic cells . J. Clin. Invest. 120 , 3834 –3842 (doi:10.1172/JCI43391)20921626 32 Matos J Blanco MG Maslen S Skehel JM West SC 2011 Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis . Cell 147 , 158 –172 (doi:10.1016/j.cell.2011.08.032)21962513 33 Gallo-Fernandez M Saugar I Ortiz-Bazan MA Vazquez MV Tercero JA 2012 Cell cycle-dependent regulation of the nuclease activity of Mus81–Eme1/Mms4 . Nucleic Acids Res. 40 , 8325 –8335 (doi:10.1093/nar/gks599)22730299 34 Osman F Whitby MC 2007 Exploring the roles of Mus81–Eme1/Mms4 at perturbed replication forks . DNA Repair 6 , 1004 –1017 (doi:10.1016/j.dnarep.2007.02.019)17409028 35 Doe CL Osman F Dixon J Whitby MC 2004 DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51 . Nucleic Acids Res. 32 , 5570 –5581 (doi:10.1093/nar/gkh853)15486206 36 Lambert S Mizuno K Blaisonneau J Martineau S Chanet R Freon K Murray JM Carr AM Baldacci G 2010 Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange . Mol. Cell 39 , 346 –359 (doi:10.1016/j.molcel.2010.07.015)20705238 37 Blackford AN Schwab RA Nieminuszczy J Deans AJ West SC Niedzwiedz W 2012 The DNA translocase activity of FANCM protects stalled replication forks . Hum. Mol. Genet. 21 , 2005 –2016 (doi:10.1093/hmg/dds013)22279085 38 Chan KL Palmai-Pallag T Ying S Hickson ID 2009 Replication stress induces sister-chromatid bridging at fragile site loci in mitosis . Nat. Cell Biol. 11 , 753 –760 (doi:10.1038/ncb1882)19465922 39 Uemura T Yanagida M 1984 Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization . EMBO J. 3 , 1737 –1744 6090122 40 Sofueva S Osman F Lorenz A Steinacher R Castagnetti S Ledesma J Whitby MC 2011 Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier . Nucleic Acids Res. 39 , 6568 –6584 (doi:10.1093/nar/gkr340)21576223 41 Zeitlin SG Baker NM Chapados BR Soutoglou E Wang JY Berns MW Cleveland DW 2009 Double-strand DNA breaks recruit the centromeric histone CENP-A . Proc. Natl Acad. Sci. USA 106 , 15 762 –15 767 (doi:10.1073/pnas.0908233106) 42 McFarlane RJ Humphrey TC 2010 A role for recombination in centromere function . Trends Genet. 26 , 209 –213 (doi:10.1016/j.tig.2010.02.005)20382440 43 Bahler J Wu JQ Longtine MS Shah NG McKenzie A 3rdSteever AB Wach A Philippsen P Pringle JR 1998 Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe . Yeast 14 , 943 –951 (doi:10.1002/(SICI)1097-0061(199807)14:10<943::AID-YEA292>3.0.CO;2-Y)9717240 44 Goldstein AL McCusker JH 1999 Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae . Yeast 15 , 1541 –1553 (doi:10.1002/(SICI)1097-0061(199910)15:14<1541::AID-YEA476>3.0.CO;2-K)10514571 45 Moreno S Klar A Nurse P 1991 Molecular genetic analysis of fission yeast Schizosaccharomyces pombe . Methods Enzymol. 194 , 795 –823 (doi:10.1016/0076-6879(91)94059-L)2005825 46 Osman F Whitby MC 2009 Monitoring homologous recombination following replication fork perturbation in the fission yeast Schizosaccharomyces pombe . Methods Mol. Biol. 521 , 535 –552 (doi:10.1007/978-1-60327-815-7_31)19563128 47 Osman F Dixon J Doe CL Whitby MC 2003 Generating crossovers by resolution of nicked Holliday junctions: a role for Mus81–Eme1 in meiosis . Mol. Cell 12 , 761 –774 (doi:10.1016/j.dnarep.2007.02.019)14527420 48 Whitby MC Dixon J 1998 Substrate specificity of the SpCCE1 holliday junction resolvase of Schizosaccharomyces pombe . J. Biol. Chem. 273 , 35 063 –35 073 (doi:10.1074/jbc.273.52.35063)	24026537	PMC3787749	CC BY	2021-01-04 23:14:51	yes	Open Biol. 2013 Sep; 3(9):130102
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24098415PONE-D-13-2700210.1371/journal.pone.0075994Research ArticleA Novel Alkaloid from Marine-Derived Actinomycete Streptomyces xinghaiensis with Broad-Spectrum Antibacterial and Cytotoxic Activities Novel Alkaloid with Biological ActivitiesJiao Wence 1 Zhang Fenghua 2 Zhao Xinqing 1 * Hu Jiehan 3 Suh Joo-Won 4 1 School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China 2 First Affiliated Hospital of Dalian Medical University, Dalian, China 3 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 4 Division of Bioscience and Bioinformatics, Myongji University, Yongin, Korea Virolle Marie-Joelle Editor University Paris South, France * E-mail: xqzhao@dlut.edu.cnCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: XZ WJ. Performed the experiments: WJ. Analyzed the data: JH WJ XZ. Contributed reagents/materials/analysis tools: XZ JS FZ. Wrote the paper: WJ XZ. 2013 1 10 2013 8 10 e7599429 6 2013 19 8 2013 © 2013 Jiao et al2013Jiao et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Due to the increasing emergence of drug-resistant bacteria and tumor cell lines, novel antibiotics with antibacterial and cytotoxic activities are urgently needed. Marine actinobacteria are rich sources of novel antibiotics, and here we report the discovery of a novel alkaloid, xinghaiamine A, from a marine-derived actinomycete Streptomyces xinghaiensis NRRL B24674T. Xinghaiamine A was purified from the fermentation broth, and its structure was elucidated based on extensive spectroscopic analysis, including 1D and 2D NMR spectrum as well as mass spectrometry. Xinghaiamine A was identified to be a novel alkaloid with highly symmetric structure on the basis of sulfoxide functional group, and sulfoxide containing compound has so far never been reported in microorganisms. Biological assays revealed that xinghaiamine A exhibited broad-spectrum antibacterial activities to both Gram-negative persistent hospital pathogens (e.g. Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli) and Gram-positive ones, which include Staphylococcus aureus and Bacillus subtilis. In addition, xinghaiamine A also exhibited potent cytotoxic activity to human cancer cell lines of MCF-7 and U-937 with the IC50 of 0.6 and 0.5 µM, respectively. This work was supported by the Next-Generation BioGreen 21 program of the rural development Administration, Republic of Korea (No. PJ0080932011). The authors also acknowledge the financial support by the state key laboratory open program from Key Laboratory of Marine Bio-resources Sustainable Utilization (No. LMB111002), and the state key laboratory open program from Key Laboratory of Bioreactor Engineering, East China University of Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The increasing prevalence of infections caused by multi-drug-resistant (MDR) bacterial pathogens has aroused worldwide concern. In addition to methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa strains which have caused serious healthcare-associated and community-onset infections [1], [2], Acinetobacter baumannii has received increasing attention recently as a persistent hospital pathogens due to the rapidly increasing number of infections among compromised and injured patients and the global spread of strains with resistance to multiple antibiotics [3], [4]. However, studies on novel antibiotics to treat drug resistant A. baumannii are still very limited [5]. Therefore, there is an urgent need to seek for new antibiotics for clinical infections caused by the MDR bacterial pathogens. Marine-derived actinomycetes are rich sources of novel secondary metabolites which harbour unique structures and have diverse biological activities such as antimicrobial, antitumor and immunosuppressive activities [6]–[8]. The obligate marine genera Salinispora and Marinispora have been characterized [9], [10], and structurally unique and biologically active secondary metabolites have been isolated, such as salinosporamide A with excellent cytotoxicity from S. tropica CNB-392 and marinomycins A with strong antimicrobial and cytotoxic activities from Marinispora sp. CNQ-140 [11]–[13]. Marine-derived streptomycetes are also widely studied as novel antibiotic producers, where interesting compounds with antibacterial activities and anticancer activities were reported to be isolated [14], [15]. In our previous studies, a marine-derived actinobacterium Streptomyces xinghaiensis was identified to be a new species, which was proved to exhibit broad-spectrum antibacterial activities [16]. Herein, we report the isolation, structure elucidation, and biological activities of a new compound with promising activities against various bacterial pathogens, including the two notorious opportunistic pathogens A. baumannii and P. aeruginosa. To our knowledge, the sulfoxide functional group-containing compound has so far never been observed in microorganisms. Materials and Methods Ethics statement The clinical methicillin-resistant S. aureus (5301, 5438 and 5885) and A. baumannii used as test strains were isolated from the sputum samples of patients and provided by the First Affiliated Hospital of Dalian Medical University. The study and protocols using the bacterial strains from patients were approved by the Ethics Committee of First Affiliated Hospital of Dalian Medical University, China. The Ethics Committee of First Affiliated Hospital of Dalian Medical University waived the need for a written informed consent. Microbial strains and culture media S. xinghaiensis was preserved in our lab as a glycerol stock at −80°C and also at China General Microbiological Culture Collection centre (CGMCC) with accession number of CGMCC 2251. S. aureus (CGMCC 1.89), B. subtilis (CGMCC 1.73), E. coli (CGMCC 1.797), P. aeruginosa (CGMCC 1.2031) and C. albicans (CGMCC 2.538) were used as test strains. The clinical isolates including strains S. aureus (5301, 5438 and 5885) and A. baumannii used as test strains were isolated from the sputum samples of patients, and the procedure used for strain isolation was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University. The Ethics Committee of First Affiliated Hospital of Dalian Medical University waived the need for a written informed consent. Bacteria and yeast strain were maintained on Luria Bertani (LB, tryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/l) and Yeast Extract Peptone Dextrose (YPD, yeast extract 5 g/l, peptone 10 g/l, glucose 20 g/l) slants at 4°C, respectively. The solid medium were prepared by adding 1.5% agar into the liquid media. S. xinghaiensis was activated in Tryptic Soytone Broth (TSB, BD Difco™) seed medium and the production medium was optimized based on the medium described by Wang et al [17], and was prepared as follows: soluble starch 20 g/l, soybean powder 25 g/l, (NH4)2SO4 2 g/l, NaCl 2 g/l, K2HPO4 0.5 g/l and CaCO3 5 g/l. The medium was prepared with distilled water, and the pH was adjusted to 7.0 prior to sterilization. Cell lines Four human cancer cell lines for cytotoxicity assays were purchased from the Committee of Type Culture Collection of the Chinese Academy of Sciences (CTCCCAS, Shanghai, China). The accession numbers of human breast cancer cells line MCF-7, human live cancer cell line SMMC-7721, human acute myelogenous leukemia cell line U-937 and human small cell lung cancer cell line NCI-H1688 are TCHu 74, TCHu 52, TCHu159 and TCHu154, respectively. All the cell lines were maitained in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum and cultured at 37°C in humidified air containing 5% CO2. Culture conditions of S. xinghaiensis The fresh culture of S. xinghaiensis from the TSB agar plate was inoculated into 250 ml shaker flask with 50 ml TSB medium and cultivated at 30°C at 200 rpm for two days. The seed cultures were then inoculated into the production medium (200 ml medium in 500 ml shake flasks) with an inoculation rate of 10% and cultured at 30°C at 200 rpm for 9 days. Three percent resin HP-20 (w/v) was added into the production medium at the 3rd day after inoculation to improve the production of antibiotics. General experimental procedures Optical rotations were determined with a JASCO P-1020 digital polarimeter. The ultraviolet (UV) data were obtained on a HP8453 spectrophotometer. Thermo Nicolet spectrometer was used for scanning IR spectroscopy. Electrospray ionization (ESI) spectra in both positive and negative ion modes were measured on a HP1100 LC-MS spectrometer. High-resolution-electrospray-ionization mass spectra (HR-ESIMS) were performed on a Q-TOF Micro Mass Spectrometer. 1H NMR, 13C NMR, DEPT135, HSQC and HMBC spectra were recorded on a Bruker Advance DPX400 spectrometer (400 and 100 MHz for 1H and 13C NMR, respectively) using tetramethylsilane (TMS) as an internal standard. Elemental analysis was performed on Vario EL ΙΙΙ (Elementar, Germany). Extraction and isolation of antibacterial compounds The fermentation broth (4 L) including both mycelia and resins was directly mixed with two volumes of methanol (MeOH) and was shaken at 150 rpm for 120 min to extract the bioactive compounds. Then the mixture was centrifuged at 5000 rpm for 10 min, after which the supernatant was concentrated under reduced pressure to obtainan aqueous solution. The aqueous solution was subsequently extracted three times with two volumes of ethyl acetate (EtOAC) and concentrated under reduced pressure to give a crude extract (10 g). The crude extract was firstly analyzed using a HPLC system (Waters, USA) equipped with a Waters Symmetry C18 column (4.6 mm×250 mm, 5 µm). The mobile phase consisted of H2O/acetonitrile (ACN) with 0.1% TFA added to both solvents and a gradient elution step was applied as follows: 0–15 min, 5–100% ACN; 15–25 min, 100% ACN. The flow rate was 0.85 ml/min and the elution was monitored by the UV absorption at 254 nm. The crude extract was then subjected to column chromatography (CC) over silica gel (300–400 mesh, Qingdao Marine Chemical Factory, Qingdao, China), and eluted with stepwise gradients of CH2Cl2, EtOAC and MeOH (100% CH2Cl2, CH2Cl2/EtOAc 2∶1, CH2Cl2/EtOAc 1∶1, CH2Cl2/EtOAc 1∶2, 100% EtOAc, EtOAc/MeOH 2∶1, EtOAc/MeOH 1∶1, EtOAc/MeOH 1∶2, and finally 100% MeOH, v/v), respectively, to obtain nine fractions (Fr.01-Fr.09). Bioassay-guided fractionation of the crude extract revealed that the antibacterial compounds against S. aureus existed in fractions Fr.04, Fr.06, Fr.07 and Fr.09 (174.3, 250.3, 186.5 and 145.7 mg, respectively), which were then fractionated by flash C18 column chromatography eluting with 30%, 60%, 80% and 100% MeOH/water mixtures to give several sub-fractions. The sub-fractions (Fr.04-2, Fr.06-3, Fr.07-2 and Fr.09-4) containing antibacterial compounds were then further purified by semi-preparative reversed-phase HPLC equipped with C18 column (Waters Symmetry Prep™ C18 7.8 mm×300 mm, 7 µm, 3 ml/min) to give compound E (named later as xinghaiamine A), D1, D2, D3, C1, C2, B1 and B2 (For detailed isolation procedure, see Fig. S1). Xinghaiamine A Brown, viscous oil; [α]D 20+10.8° (c 0.245, MeOH); UV (MeOH) λmax (log ε) 232.5 (3.88), 295.3 (1.15), 320.6 (0.08) nm; IR (MeOH) λmax 2924, 2852, 1725, 1656, 1561, 1465, 1382, 1280, 1233, 1082 cm−1; 1H and 13C NMR, see Table 1; positive ESIMS showed a [M+Na]+ peak at m/z 747.5 and a [M-H]+ peak at m/z 723.2; HR-ESIMS m/z 747.3390 ([M+Na]+, calcd 747.3385 for C50H48N2OSNa). 10.1371/journal.pone.0075994.t001Table 1 NMR spectroscopic data (CD3OD) of xinghaiamine Aa Position δC, type δH (J in Hz) 1H-1H cosy HMBC 1 107.7 CH 6.46, s 3 3, 5, 9 2 143.2 C 3 108.3 CH 6.76, m 1 1, 5, 11 4 138.3 C 5 120.4 C 6 134.5 C 7 120.0 CH 8.17, m 9 5, 9, 12, 19 8 140.2 C 9 116.1 CH 8.15, s 7 1, 5, 7, 19 10 122.2 C 11 120.7 CH 7.28, m 12 3, 5, 6 12 125.8 CH 7.28, m 11 4, 5, 7 13 27.2 CH2 1.80, m 14 15, 21, 23, 24 14 27.5 CH2 1.92, m 13, 15 22, 23 15 53.4 CH 3.09, t (8) 14, 23 2, 13, 18, 22 16 51.8 CH2 2.85, t (8) 17 2, 18 17 22.3 CH2 1.49, m 16 19, 21, 23 18 37.1 C 19 37.1 C 20 28.8 CH2 1.29, s 8, 18, 22, 25 21 61.2 C 22 34.3 C 23 24.8 CH 2.12, d (12) 15 13, 14, 19, 21 24 20.9 CH3 1.33, s 13, 21, 23 25 17.3 CH3 1.23, s 8, 18, 20 a Data was recorded in CD3OD, 400 MHz for 1H-NMR and 100 MHz for 13C-NMR. The signals were assigned in combination with 1H-1H COSY, HSQC and HMBC. Biological activity assays Antimicrobial activities of xinghaiamine A against S. aureus (CGMCC 1.89), B. subtilis (CGMCC 1.73), E. coli (CGMCC 1.797), P. aeruginosa (CGMCC 1.2031), C. albicans (CGMCC 2.538) and methicillin-resistant S. aureus (MRSA 5301, 5438 and 5885) as well as A. baumannii were investigated. Xinghaiamine A was dissolved in MeOH to test the MIC (minimal inhibitory concentration, defined as the lowest concentration of xinghaiamine A inhibiting visible growth of test strains) values, which were achieved using the method as described previously [18]. Tetracycline and vancomycin were employed as positive controls for the model strains and MRSA isolates respectively and MeOH was employed as the negative control. The in vitro cytotoxic activities of xinghaiamine A against MCF-7, SMMC-7721, U-937 and NCI-H1688 were evaluated using the MTT (Methyl-Thiazol-Tetrozolium) method [19]. Briefly, 200 µL of cell suspension was inoculated to 96-well plates with a final concentration of 105 cells/ml and cultured for 24 h. After that, 50 µl of xinghaiamine A dissolved in DMSO adjusted to various concentrations was added to each well. After the exposure to xinghaiamine A for 48 h, 20 µL of 5 mg/ml MTT solution was added to each well, and the plates were incubated for 4 h at 37°C. Then, 150 µl of DMSO was added in each well. The absorbance caused by formazan crystallization was recorded at 550 nm using scanning mutiwell spectrophotometer. The measurements were repeated for three times, and average value was obtained. The cell viability was calculated using the following formula: cell viability (%) = (OD550 nm of the group treated with xinghaiamine A/OD550 nm of the untreated group) ×100%. Cisplatin and DMSO were employed as positive and negative control, respectively. Results Analysis of fermentation crude extract The crude extract was analyzed by HPLC and it was found that compounds with retention time at 11.18, 11.32, 11.62, 12.16, 13.36, 13.62, 13.92 and 16.64 min may belong to the same family due to the similar UV absorption (Fig. 1), which we successively named B1, B2, C1, C2, D1, D2, D3 and E, temporarily. To evaluate the antibacterial activity of these compounds, a pre-bioassay was performed by collecting samples every minute and employing S. aureus as an indicator after about 20 mg crude extract was subjected to the analytical HPLC. The results indicated that the antibacterial activities of S. xinghaiensis were mainly due to this compound family, especially compound E, which we named xinghaiamine A and it exhibited antibacterial activity against S. aureus and was more easily purified. Thus we subsequently focused on this compound in the large scale isolation and purification. 10.1371/journal.pone.0075994.g001Figure 1 Production of a family of compounds with unique UV spectra by S. xinghaiensis. 1a , HPLC profile of fermentation crude extract produced by S. xinghaiensis. The elution was monitored at 254 nm at a flow rate of 0.85 ml/min. 1b, UV absorption of the compound family. Compound E was named as xinghaiamine A. Structural analysis of xinghaiamine A Xinghaiamine A was isolated as brown viscous oil. The positive- and negative-ion mode ESIMS analysis of xinghaiamine A (m/z 747.5 [M+Na]+; m/z 723.2 [M-H]+) indicated a molecular weight of 724 Da (Fig. S2). The positive-ion mode HR-ESIMS data of xinghaiamine A exhibited a [M+Na]+ molecular ion peak at m/z 747.3390 (calculated 747.3385) consistent with the molecular formula of C50H48N2OS, requiring 27 degrees of unsaturation (Fig. S3). The characteristic absorption bands at 232, 295, and 320 nm of UV spectra demonstrated that a conjugated naphthalene ring chromophore (maxima at 218, 261, and 331 nm) was involved in xinghaiamine A. The IR absorption band at 1082 cm−1 suggested the presence of sulfoxide functional group (Fig. S4), which was also supported by a sulfur content of 4.3% in the elemental analysis result (Fig. S10). The 13C NMR spectrum (Table 1) in combination with the DEPT135 experiments and 2D NMR spectra possessed 25 carbon signals, including two methyls, five methylenes, eight methines, and ten quaternary carbons, which revealed a highly symmetric molecular structure of xinghaiamine A on the basis of sulfoxide moiety. All of the protons were assigned to carbons by HSQC experiments. For each part, an interpretation of 13C NMR and DEPT spectrum data showed the aromatic part has twelve carbons, including six methines and six quaternary carbons, accounting for ten out of fourteen degrees of unsaturation. Further interpretation of 1H NMR spectrum of xinghaiamine A accounts for six aromatic protons at δ6.46 (s), 6.76 (m), 7.29 (m), 7.27 (m), 8.15 (s), and 8.17 (m). Analysis of NMR with both COSY (H1/H3, H7/H9, H11/H12) and HMBC spectrum (H1 to C3, H1 to C5, H1 to C9, H3 to C1, H3 to C5, H3 to C11, H7 to C5, H7 to C9, H7 to C12, H7 to C19, H9 to C1, H9 to C5, H9 to C7, H9 to C19, H11 to C3, H11 to C5, H11 to C6, H12 to C4, H11 to C5, H12 to C7) showed correlations which illustrated that these proton and carbon signals were components of a naphthalene ring substituted at the position of C-2, C-8 and C-4, C-6 (conjugate ring), respectively. The remaining aliphatic fragment requiring the other five sites of unsaturation was deduced to have five or four rings by sharing a ring with the aromatic portion. An interpretation of 13C NMR and DEPT spectroscopic data showed that the aliphatic part had thirteen carbons and eighteen protons, indicative of two methyls, five methylenes, two methines, and four quaternary carbons (Table 1). The δC 37.1 (C-19,-C) and δC 140.2 (C-8, -C) were established to be at the junction of the aromatic and aliphatic fragment by the HMBC correlations from H-20 to C-8, H-25 to C-8, H-7 to C-19 as well as H-9 to C-19. Also, the HMBC correlations from H-15 and H-16 to C-2 proved that C-2 was substituted with N. Obviously, the proton signals at δH 3.09 (C-15, -CH) and δH 2.85 (C-16, -CH2) were connected to N with a relative low magnetic field. The 1H-1H COSY cross peaks of H-13/H-14, H-14/H-15 and H-16/H-17 suggested that δH 1.80 (C-13, -CH2) and δH 3.09 (C-15, -CH) were linked to δH 1.92 (C-14, -CH2), δH 1.49 (C-17 -CH2) was linked to δ2.85 (C-16, -CH2), respectively. δC 61.2 (C-21,-C) was established to be linked with the sulfoxide moiety due to the unique lowest chemical shift in aliphatic fragment, which was also consistent with the characteristic of sulfoxide functional group. The structure of other aliphatic fragment was established on the basis of 1H-1H cosy (H15/H23) and HMBC correlations of (See Table 1, Fig. 2, 3 and Fig. S5, S6, S7, S8, S9). 10.1371/journal.pone.0075994.g002Figure 2 Proposed chemical structure of xinghaiamine A. 10.1371/journal.pone.0075994.g003Figure 3 Selected 1H-1H COSY (bold lines) and HMBC (red arrows) correlations of xinghaiamine A. Antimicrobial tests Xinghaiamine A showed broad-spectrum antibacterial activities against several test strains. It exhibited superior antibacterial activity to S. aureus, B. subtilis, E. coli, and A. baumanii with the MIC values of 0.69, 0.35 0.17, 2.76 and 11.04 µM, respectively, which were much lower than those of tetracycline. However, the inhibition of xinghaiamine A to P. aeruginosa was not as good as that of tetracycline. Xinghaiamine A also showed considerable activities to the clinical MRSA isolates with MIC values of 2.76 and 5.52 µM, albeit not so good as the powerful antibiotic vancomycin. The inhibition to the pathogenic bacteria and clinical MRSA isolates of xinghaiamine A demonstrated that it has the potential to be an effective antibiotic to deal with the multi-drug resistant pathogens, especially S. aureus and A. baumanii. No obvious antifungal activity to C. albicans of xinghaiamine A was found when it was tested at concentrations up to 176.64 µM (Table 2). 10.1371/journal.pone.0075994.t002Table 2 Antimicrobial activities of xinghaiamine A (MIC, µM) against the test strains. MIC (µM)a xinghaiamine Ab tetracyclinec vancomycinc S. aureus 0.69 4.50 −d B. subtilis 0.35 2.25 − E. coli 0.17 1.13 − A. baumanii 2.76 9.0 − P. aeruginosa 11.04 4.50 − C. albicans >176.64 − − MRSA 5301 5.52 − 0.35 MRSA 5438 2.76 − 0.18 MRSA 5885 5.52 − 0.70 a MIC represented the lowest compound concentration apparently inhibiting microorganism growth. b Xinghaiamine A was dissolved in MeOH for MIC test and MeOH was used as the negative control. c Tetracycline and vancomycin were employed as positive controls for the pathogenic bacteria (S. aureus, B. subtilis, E. coli, A. baumanii and P. aeruginosa) and clinical MRSA isolates (5301, 5438 and 5885), respectively. d “−” indicated that the positive control was not measured for the test strains. In vitro cytotoxicity assays In vitro cytotoxicity assays of xinghaiamine A against four human cancer cell lines revealed that xinghaiamine A exhibited considerable broad-spectrum anti-proliferative activities (Table 3). Of the four cell lines, superior activity of xinghaiamine A against U-937 was observed, with the minimum IC50 value of 0.5 µM. Good cytotoxic activities of xinghaiamine A were also observed agaist MCF-7 and NCI-H1688, with the IC50 values much lower than that of the control. In contrast, comparable activity against SMMC-7721 of xinghaiamina A and cisplatin was observed (Table 3). 10.1371/journal.pone.0075994.t003Table 3 In vitro cytotoxicity of xinghaiamine A (IC50 µM) against four human cancer cell lines. Cytotoxicity (IC50,µM) MCF-7 SMMC-7721 U-937 NCI-H1688 Xinghaiamine Aa 0.6 6.3 0.5 2.2 Cisplatinb 4.2 8.5 13.5 14.6 a Xinghaiamine A was dissolved in DMSO for the IC50 test and DMSO was used as the negative control. b Cisplatin was employed as positive control for the human tumor cell lines. Discussion The sulfoxide containing natural products are very limited in nature, and the current naturally occurred sulfoxide compounds are commonly peptide derivatives from terrestrial plants including Brussels sprouts (S-methyl-cysteine sulfoxide) [20], and Allium siculums (S-alk(en)yl-L-cysteine sulfoxide, S-n-butyl-cysteine sulfoxide) [21]-[23], as well as rare marine secondary metabolites from marine invertebrates (marine sponge Pseudoceratina purpurea, ascidian Polycitor sp., etc.), which include psammaplin N, eudistomin K, lehualides J, varacins B and D, eudistomidin E and aplisulfamines [24]–[29]. However, so far no sulfoxide antibiotic has been reported to be produced by microorganisms. The sulfoxide moiety presented in xinghaiamine A is unprecedented in metabolites from marine actinomycete. Sulfoxide compounds have broad-spectrum of biological activities, including excellent antimicrobial, pesticidic and antitumor activities, and chemical synthesis of sulfoxide compounds also has aroused the interests of researchers [30]. The discovery of xinghaiamine A as the first sulfoxide compound from marine Streptomyces sp. also promotes the idea that the sulfoxide compounds from marine invertebrates may also have a microbial origin. Xinghaiamine A exhibited broad-spectrum antibacterial activities against various tested strains, including A. baumanii, which remains one of the major multiply resistant bacterial pathogens for serious healthcare-associated and community-onset infections. The isolation of xinghaiamine A seems to provide powerful potential to combat the emergence of multi-drug-resistant microbial pathogens. In addition, compared with cisplatin, xinghaiamine A also displayed promising cytotoxic activities against a series of human cancer cell lines. Recently, the rapid development of resistance to multiple drugs in tumor chemotherapy has urged for the searching for novel drugs and the results above revealed that xinghaiamine A could be a potential clinically useful antitumor drug to combat with the increasing multi-drug resistant cancer cell lines, and the current study provided basis for further develop this novel compound for anticancer therapy. Supporting Information Figure S1 Flow chart of isolation and purification of antibacterial compounds from S. xinghaiensis . (TIF) Click here for additional data file. Figure S2 ESI-MS spectrum of xinghaiamine A, (a) positive-ion mode (b) negative-ion mode. (TIF) Click here for additional data file. Figure S3 Positive ion mode of HR-ESIMS spectrum of xinghaiamine A. (TIF) Click here for additional data file. Figure S4 IR spectrum of xinghaiamine A. (TIF) Click here for additional data file. Figure S5 13C NMR and DEPT135 spectrum of xinghaiamine A. Compound was dissolved in CH3OD and data was recorded on a Bruker Advance DPX400 spectrometer of 400 MHz for 13C NMR using TMS as internal standard. (TIF) Click here for additional data file. Figure S6 1H NMR spectrum of xinghaiamine A. Compound was dissolved in CH3OD and data was recorded on a Bruker Advance DPX400 spectrometer of 100 MHz for 1H NMR using TMS as internal standard. (TIF) Click here for additional data file. Figure S7 1H-1H COSY spectrum of xinghaiamine A. (TIF) Click here for additional data file. Figure S8 HSQC spectrum of xinghaiamine A. (TIF) Click here for additional data file. Figure S9 HMBC spectrum of xinghaiamine A. (TIF) Click here for additional data file. Figure S10 Elementary analysis of xinghaiamine A. The experiments were performed twice which were described as E1 and E2, respectively. (TIF) Click here for additional data file. The authors are grateful to Professor Hans-Pieter Fiedler in University of Tuebingen, Germany for identification of xinghaiamine A family compounds as the possible novel compounds. The authors also acknowledge the helpful discussions with Dr. Shuangjun Lin in Shanghai Jiao Tong University, China for structure elucidation. ==== Refs References 1 de Kraker MEA , Wolkewitz M , Davey PG , Grundmann H (2011 ) Clinical impact of antimicrobial resistance in European hospitals: excess mortality and length of hospital stay related to methicillin-resistant Staphylococcus aureus bloodstream infections . Antimicrob Agents Chemother 55 : 1598 –1605 .21220533 2 Xu Z , Fang X , Wood TK , Huang ZJ (2013 ) A systems-level approach for investigating Pseudomonas aeruginosa biofilm formation . PLoS ONE 8 : e57050 doi:10.1371/journal.pone.0057050 23451140 3 Peleg AY , Seifert H , Paterson DL (2008 ) Acinetobacter baumannii: emergence of a successful pathogen . Clin Microbiol Rev 21 : 538 –582 .18625687 4 McConnell MJ , Actis L , Pachón J (2013 ) Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models . FEMS Microbiol Rev 37 : 130 –155 .22568581 5 Lee KH , Kim KW , Rhee KH (2010 ) Identification of Streptomyces sp. KH29, which produces an antibiotic substance processing an inhibitory activity against multidrug-resistant Acinetobacter baumannii . J Microbiol Biotechnol 20 : 1672 –1676 .21193822 6 Manivasagan P , Venkatesan J , Sivakumar K , Kim SK (2013 ) Marine actinobacterial metabolites: Current status and future perspectives . Microbiol Res 168 : 311 –332 .23480961 7 Zotchev SB (2012 ) Marine actinomycetes as an emerging resource for the drug development pipelines . J Biotechnol 158 : 168 –175 .21683100 8 Blunt JW , Copp BR , Keyzers RA , Munro MHG , Prinsep MR (2013 ) Marine natural products . Nat Prod Rep 30 : 237 –323 .23263727 9 Maldonado LA , Fenical W , Jensen PR , Kauffman CA , Mincer TJ , et al (2005 ) Salinispora arenicola gen. nov., sp. nov. and Salinispora tropica sp. nov., obligate marine actinomycetes belonging to the family Micromonosporaceae . Int J Syst Evol Microbiol 55 : 1759 –1766 .16166663 10 Jensen PR , Mincer TJ , Williams PG , Fenical W (2005 ) Marine actinomycete diversity and natural product discovery . Antonie Van Leeuwenhoek 87 : 43 –48 .15726290 11 Feling RH , Buchanan GO , Mincer TJ , Kauffman CA , Jensen PR , et al (2003 ) Salinosporamide A: A Highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus Salinospora . Angew Chem Int Edit 42 : 355 –357 . 12 Williams PG , Buchanan GO , Feling RH , Kauffman CA , Jensen PR , et al (2005 ) New cytotoxic salinosporamides from the marine actinomycete Salinispora tropica . J Org Chem 70 : 6196 –6203 .16050677 13 Kwon HC , Kauffman CA , Jensen PR , Fenical W (2006 ) Marinomycins A–D, antitumor-antibiotics of a new structure class from a marine actinomycete of the recently discovered genus “Marinispora” . J Am Chem Soc 128 : 1622 –1632 .16448135 14 Raju R , Piggott AM , Barrientos D , Leticia X , Khalil Z , et al (2010 ) Heronapyrroles A–C: farnesylated 2-nitropyrroles from an Australian marine derived Streptomyces sp . Org Lett 12 : 5158 –5161 .20949971 15 Kondratyuk TP , Park EJ , Yu R , van Breemen RB , Asolkar RN , et al (2012 ) Novel marine phenazines as potential cancer chemopreventive and anti-inflammatory agents . Mar Drugs 10 : 451 –464 .22412812 16 Zhao XQ , Li WJ , Jiao WC , Li Y , Yuan WJ , et al (2009 ) Streptomyces xinghaiensis sp. nov., isolated from marine sediment . Int J Syst Evol Microbiol 59 : 2870 –2874 .19628610 17 Wang F , Kong R , Liu B , Jing Zhao , Rongguo Qiu , et al (2012 ) Functional characterization of the genes tauO, tauK, and tauI in the biosynthesis of tautomycetin . J Microbiol 50 : 770 –776 .23124744 18 Wang J , Galgoci A , Kodali S , Herath KB , Jayasuriya H , et al (2003 ) Discovery of a small molecule that inhibits cell division by blocking FtsZ, a novel therapeutic target of antibiotics . J Biol Chem 278 : 44424 –44428 .12952956 19 Burres NS , Clement JJ (1989 ) Antitumor activity and mechanism of action of the novel marine natural products mycalamide-A and -B and onnamide . Cancer Res 49 : 2935 –2940 .2720652 20 Marks HS , Hilson JA , Leichtweis HC , Stoewsand GS (1992 ) S-methylcysteine sulfoxide in Brassica vegetables and formation of methyl methanethiosulfinate from brussels sprouts . J Agric Food Chem 40 : 2098 –2101 . 21 Kubec R , Kim S , McKeon DM , Musah RA (2002 ) Isolation of S-n-butylcysteine sulfoxide and six n-butyl-containing thiosulfinates from Allium siculum . J Nat Prod 65 : 960 –964 .12141853 22 Rose P , Whiteman M , Moore PK , Zhu YZ (2005 ) Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium: the chemistry of potential therapeutic agents . Nat Prod Rep 22 : 351 –368 .16010345 23 Hornickova J , Velisek J , Ovesna J , Stavelikova H (2009 ) Distribution of S-alk(en)yl-L-cysteine sulfoxides in garlic (Allium sativum L.) . Czech J Food Sci 27 : 232 –235 . 24 Graham SK , Lambert LK , Pierens GK , Hooper JNA , Garson MJ (2010 ) Psammaplin metabolites new and old: an NMR study involving chiral sulfur chemistry . Aust J Chem 63 : 867 –872 . 25 Lake RJ , Brennan MM , Blunt JW , Munro MHG , Pannell LK (1988 ) Eudistomin K sulfoxide – an antiviral sulfoxide from the New Zealand ascidian Ritterella sigillinoides . Tetrahedron Lett 29 : 2255 –2256 . 26 Barber JM , Quek NCH , Leahy DC , Miller JH , Bellows DS , et al (2011 ) Lehualides E−K, cytotoxic metabolites from the Tongan marine sponge Plakortis sp . J Nat Prod 74 : 809 –815 .21351759 27 Makarieva TN , Stonik VA , Dmitrenok AS , Grebnev BB , Isakov VV , et al (1995 ) Varacin and three new marine antimicrobial polysulfides from the far-eastern ascidian Polycitor sp . J Nat Prod 58 : 254 –258 .7769392 28 Murata O , Shigemori H , Ishibashi M , Sugama K , Hayashi K , et al (1991 ) Eudistomidins E and F, new β-carboline alkaloids from the okinawan marine tunicate Eudistoma glaucus . Tetrahedron Lett 32 : 3539 –3542 . 29 Aiello A , Fattorusso E , Imperatore C , Luciano P , Menna M , et al (2012 ) Aplisulfamines, new sulfoxide-containing metabolites from an Aplidium tunicate: absolute stereochemistry at chiral sulfur and carbon atoms assigned through an original combination of spectroscopic and computational methods . Mar Drugs 10 : 51 –63 .22363220 30 Prilezhaeva EN (2000 ) Sulfones and sulfoxides in the total synthesis of biologically active natural compounds . Russian Chemical Reviews 69 : 367 –408 .	24098415	PMC3787992	CC BY	2021-01-05 17:12:12	yes	PLoS One. 2013 Oct 1; 8(10):e75994
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24098407PONE-D-13-0864210.1371/journal.pone.0075928Research ArticleBiodegradation of the Allelopathic Chemical m-Tyrosine by Bacillus aquimaris SSC5 Involves the Homogentisate Central Pathway Biodegradation of m-TyrosineKhan Fazlurrahman Kumari Munesh Cameotra Swaranjit Singh * Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Chandigarh, India van Schaik Willem Editor University Medical Center Utrecht, The Netherlands * E-mail: ssc@imtech.res.inCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SSC FK. Performed the experiments: FK MK. Analyzed the data: SSC FK. Wrote the paper: SSC FK. 2013 1 10 2013 9 7 2014 8 10 e7592827 2 2013 23 8 2013 © 2013 Khan et al2013Khan et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. m-Tyrosine is an amino acid analogue, exuded from the roots of fescue grasses, which acts as a potent allelopathic and a broad spectrum herbicidal chemical. Although the production and toxic effects of m-tyrosine are known, its microbial degradation has not been documented yet. A soil microcosm study showed efficient degradation of m-tyrosine by the inhabitant microorganisms. A bacterial strain designated SSC5, that was able to utilize m-tyrosine as the sole source of carbon, nitrogen, and energy, was isolated from the soil microcosm and was characterized as Bacillus aquimaris. Analytical methods such as HPLC, GC-MS, and 1H-NMR performed on the resting cell samples identified the formation of 3-hydroxyphenylpyruvate (3-OH-PPA), 3-hydroxyphenylacetate (3-OH-PhAc), and homogentisate (HMG) as major intermediates in the m-tyrosine degradation pathway. Enzymatic assays carried out on cell-free lysates of m-tyrosine-induced cells confirmed transamination reaction as the first step of m-tyrosine degradation. The intermediate 3-OH-PhAc thus obtained was further funneled into the HMG central pathway as revealed by a hydroxylase enzyme assay. Subsequent degradation of HMG occurred by ring cleavage catalyzed by the enzyme homogentisate 1, 2-dioxygenase. This study has significant implications in terms of understanding the environmental fate of m-tyrosine as well as regulation of its phytotoxic effect by soil microorganisms. The authors have no support or funding to report. ==== Body Introduction m-Tyrosine is a non-proteinogenic amino acid analogue with phytotoxic properties that is exuded from the roots of fine fescue grasses to grant a competitive advantage over other plants [1], [2], [3], [4]. m-Tyrosine also arises from the oxidation of L-phenylalanine by hydroxyl radicals and peroxynitrite [5], [6]. The phytotoxic activity of m-tyrosine is due to its inhibitory effects on the root development of competing plants [1]. Similarly, m-tyrosine also inhibits growth of prokaryotic cells, Escherichia coli and Bacillus sp. [7], [8], [9]. Previous studies showed that the cytotoxic effects of m-tyrosine is the result of its misincorporation into cellular protein in place of phenylalanine [10], [11], [12], [13]. The accumulation of m-tyrosine in mammalian tissues is used as an indicator for determining the oxidative stress and aging process [14], [15]. Several bacteria, e. g. Streptomyces spp., use m-tyrosine as a precursor for the synthesis of antibiotics such as mureidomycins, pacidamycins, and napsamycins [16]. Plant roots exude various allelochemicals to compete with other plants; however, soil microorganisms residing nearby the roots influence their phytotoxic effects either by co-metabolic transformation or utilizing them as sole carbon, nitrogen, and energy sources [17], [18], [19]. Thus, soil microorganisms can either enhance or decrease the phytotoxic properties of allelochemicals which interferes with defense properties of m-tyrosine producing plants. In some cases, the microbial degradation products of the allelopathic chemicals pose potential phytotoxic effects, while some of the microbially transformed products of allelochemicals decrease their level of phytotoxic effect as compared to the original compounds [3], [18], [19], [20], [21], [22], [23], [24], [25]. Several studies reported microbial degradation of previously characterized allelochemicals such as sorgoleone, juglone, benzoxazinoids, and other flavonoids [26], [27], [28], [29], while there is no information available on the degradation of m-tyrosine by means of bacterial isolates. The present study shows the isolation and characterization of an aerobic bacterium from a soil microcosms set up for the degradation of m-tyrosine. The strain Bacillus aquimaris SSC5 isolated from a soil microcosm utilizes m-tyrosine as the sole source of carbon, nitrogen, and energy. The resting cell studies and enzyme assays confirm the formation of 3-OH-PPA, 3-OH-PhAc, and HMG as the major metabolic intermediates. Based on the above studies we conclude that the degradation of m-tyrosine occurs via the homogentisate central pathway in Bacillus aquimaris SSC5. Materials and Methods Chemicals and growth media Analytical grade m-tyrosine (>98% purity), 3-hydroxyphenylacetic acid (>99% purity), and homogentisate (98% purity) were purchased from Sigma-Aldrich (St, Louis, MO, USA). Minimal salt medium (MSM) used in the present study was prepared as described earlier [30], with a minor modification i.e. absence of nitrogen source [(NH4)2SO4]. Nutrient agar (NA) and nutrient broth (NB) both at one-quarter strength (1/4th) were used for bacterial growth and culture maintenance. m-Tyrosine degradation in soil microcosms The rhizosphere soil samples of fine fescue grasses (Festuca rubra ssp. commutata) were collected from the lawn of the Institute of Microbial Technology, Chandigarh, India. The soil pH, moisture, total organic carbon, and total nitrogen were 6.4, 5.2%, 3.2%, and 4.7%, respectively. Before their use in inoculating the microcosms, the soil samples were sieved through a 2 mm mesh for the removal of stones and debris. Soil microcosms studies were carried out as described previously with minor modifications [31]. Subsequently, 10 g soil sample was suspended in 50 ml MSM in a 250-ml Erlenmeyer flask supplemented with 200 µM m-tyrosine. The above microcosms were also supplemented with or without (i) 10 mM glucose and 5 mM succinate as the sole carbon source, or (ii) 0.8 g l−1 NH4Cl as the sole nitrogen source, or (iii) 10 mM glucose, 5 mM succinate and 0.8 g l−1 NH4Cl as the sole carbon and nitrogen source. A control was also set up containing 10 g autoclaved soil in 50 ml MSM and m-tyrosine (200 µM). Further, these flasks were incubated at 30°C under shaking condition at 150 rpm. Samples were withdrawn from the test and control flasks at regular intervals and analyzed for the disappearance of m-tyrosine. All experiments were carried out in triplicate. Isolation and identification of m-tyrosine degrading bacteria The soil slurries that mediated complete degradation of m-tyrosine were serially diluted and spread plated on 1/4th-diluted nutrient agar (1/4-NA) and a selective media (MSM-agar supplemented with 500 µM m-tyrosine). Following seven days of incubation, the bacterial colonies that appeared on the agar were selected for characterization and were tested for m-tyrosine degradation. Initial screening was performed by inoculating selected bacterial colonies in 10 ml McCartney vials containing 5 ml carbon-free MSM supplemented with different concentrations (50–500 µM) of m-tyrosine. Bacterial growth, along with the measurement of the amount of ammonia released and depletion of substrate were used as an indicator for positive degrading activity as described earlier for 4-nitroaniline and 2-chloro-4-nitroaniline degradation studies [32], [33]. Further, the efficient m-tyrosine degrading strain was identified using polyphasic taxonomy and 16S rRNA gene sequencing as described earlier [34]. The 16S rRNA gene sequence (1375 bp) of strain SSC5 was compared to those of the type strains of Bacillus using the BLAST search function of EzTaxon server version 2.1 (www.eztaxon.org). Growth studies and degradation of m-tyrosine by strain SSC5 The growth studies of strain SSC5 were performed in 50 ml carbon-free MSM supplemented with different concentrations (50–500 µM) of m-tyrosine by inoculating (1%, v/v) overnight seed culture grown in 1/4-NB. Cultures were incubated on a rotary shaker at 200 rpm at 30°C. After every 8 hours, cultures were withdrawn and optical cell density was monitored at 600 nm using Lambda EZ 201 UV-visible spectrophotometer (Perkin-Elmer Inc, USA). Bacterial growth was also monitored by measuring the total protein of the cultures with the Pierce BCA protein assay kit (Thermo Scientific, USA) according to the procedure as described earlier [32], [33]. The culture fluid samples were centrifuged at 8,000× g for 10 min to obtain cell-free supernatants which were used for the analysis of the amount of ammonia released, depletion of m-tyrosine, and identification of intermediates using the method described later. An un-inoculated flask and a flask that was inoculated with heat-killed cells of strain SSC5 were used as abiotic and negative controls respectively. Another control experiment was also conducted by inoculating strain SSC5 in carbon-free MSM only. Resting cell studies In order to identify the metabolic intermediates of m-tyrosine catabolic pathway in strain SSC5, a resting cell study was carried out according to the method described elsewhere [35]. A volume of 1.6 liter of 1/4-NB supplemented with m-tyrosine (300 µM) was inoculated with an overnight 1/4-NB grown seed culture (6%, v/v) of strain SSC5 and was incubated at 30°C under shaking at 200 rpm up to 24 hours. When the optical density (OD600) of the culture reached 1.3–1.4, the induced cells were harvested by centrifugation at 8,000× g at room temperature for 10 min, washed twice with sodium phosphate buffer (20 mM, pH 7.2) and suspended in 100 ml of carbon-free MSM. This suspension was divided into four aliquots of 25 ml each. First two aliquots with heat killed cells (by incubating in boiling water for 30 min) and without heat killed cells not supplemented with m-tyrosine were used as negative controls, respectively. The heat killed cells supplemented with m-tyrosine in third aliquot was used as another control. The fourth aliquot supplemented with 300 µM m-tyrosine was used as test. Similarly, the resting cell studies on 3-OH-PhAc and HMG were also carried out with the m-tyrosine grown cells of strain SSC5. Each flask was incubated at 30°C with shaking at 200 rpm. Samples (2.0 ml) were withdrawn from both control and experimental flasks at regular intervals of 2 hours and were analyzed for the amount of ammonia released, followed by High Performance Liquid Chromatography (HPLC), Gas-Chromatography Mass-Spectroscopy (GC-MS) and 1H-nuclear magnetic resonance (1H-NMR) spectra analysis (methods described later). Enzyme assays with cell-free lysates m-Tyrosine-induced cells of strain SSC5 were harvested by centrifugation, washed twice and suspended in the sodium phosphate buffer (20 mM, pH 7.2). The cell suspensions lysed by passages through a French pressure cell (20,000 lb/in2) were centrifuged at 12,000 rpm for 30 min at 4°C and supernatant was separated to obtain cell-free extract. The cell-free ectract was used for determing activities of transaminase, hydroxylase, and homogentisate 1, 2-dioxygenase, respectively as described below. Protein content of the cell-free extracts was determined with the Pierce BCA protein assay kit (Thermo Scientific, USA). Transaminase enzyme assay The enzyme assay, for the transamination reaction from the cell-free lysates of strain SSC5, was carried out according to the method described earlier with minor modifications [36], [37]. One ml reaction volume contained sodium phosphate buffer (20 mM, pH 7.0), 100 µM α-ketoglutarate (α-KG), 25 µM pyridoxal phosphate, cell-free lysate (0.5 mg ml−1 of total protein), and 100 µM m-tyrosine. Transamination reaction was also carried out by taking the cell-free lysates prepared from the glucose grown cells. The transamination reactions were initiated by adding 100 µM m-tyrosine to the reaction mixture and incubated at 30°C. Enzyme activity was calculated based on substrate depletion as determined by HPLC. The reactions that lacked either α-KG or cell-free lysates prepared from cells of strain SSC5 grown with m-tyrosine or glucose were used as controls. Hydroxylase enzyme assay It has been reported that the formation of homogentisate (HMG) from 3-hydroxyphenylacetate (3-OH-PhAc) occurs by the action of 3-hydroxyphenylacetate 6-hydroxylase [38], [39]. The hydroxylase assay was carried out as described previously with minor modifications [40]. One ml reaction volume contained sodium phosphate buffer (20 mM, pH 7.0), 100 µM 3-OH-PhAc, 200 µM NADH, 50 µM FAD, and cell-free lysate (0.5 mg ml−1 of total protein). The hydroxylase reaction was initiated by adding 100 µM 3-OH-PhAc to the reaction mixture and incubated at 25°C. The control reactions lacking either substrate or cell-free lysate were also used during the above reaction. Substrate depletion and product formation was quantified by HPLC. The enzyme specific activity was determined based on the substrate depletion in the reaction mixture. Homogentisate 1, 2-dioxygenase enzyme assay The homogentisate 1, 2-dioxygenase activity in the cell-free lysate was determined spectrophotometrically by measuring the formation of maleylacetoacetate (MA) at 330 nm as described previously [41], [42]. The specific activity was calculated with the help of molar extinction coefficient of MA, 13,500/M/cm [43]. One ml reaction volume contained sodium phosphate buffer (20 mM, pH 7.0), 50 µM FeSO4, 2 mM ascorbate, cell-free lysate (0.5 mg ml−1 of total protein), and 100 µM HMG. Similarly, another reaction was carried out with addition of cell-free lysate prepared either from the glucose or 3-OH-PhAc grown cells. The reactions were initiated by adding 100 µM HMG to the mixture and incubated at 30°C. The enzyme activity was determined spectrophotometrically by measuring the absorbance at a wavelength of 330 nm at a time interval of 1 min using Lambda EZ 201 UV-visible spectrophotometer (Perkin-Elmer Inc, Massachusetts, USA). Reactions without cell-free lysate was set up as negative control. Analytical methods Ammonia concentration was monitored by a colorimetric method using the ‘Ammonia Estimation Kit’ (Sigma Aldrich, USA) according to the manufacturers' recommendations. The brown color pigment formation in the media was quantified by measuring the optical density at 400 nm in spectrophotometer as previously reported [44]. Similarly, the quantitative measurement of m-tyrosine degradation and identification of metabolic intermediates were analyzed by HPLC as per the method described earlier with minor modifications [3]. Samples (2.0 ml) from soil microcosms were withdrawn at regular time intervals and centrifuged for 10 min at 5,000 rpm. The clear supernatants were filtered with 0.2 µm membrane filters (Millipore Inc. USA) and analyzed by HPLC. Similarly, the cell-free aqueous culture supernatants collected from growth studies, resting cell studies, and enzyme assays were also filtered and analyzed by HPLC. The HPLC used for the sample analysis was a Waters HPLC system (Waters, USA) equipped with a UV detector and a RP-C18/Lichrospher 5-µM column. The mobile phase and the flow rate were the same as described by Kaur et al. [3]. The peaks of eluents were monitored at 280 nm. m-Tyrosine as well as its metabolic intermediates were quantified using calibration curves made with authentic standards. Further, the identified and unidentified metabolic intermediates detected from the samples of resting cell studies were also analyzed by GC-MS and 1H-NMR. For the GC-MS analysis, samples were prepared by mixing an equal volume of ethyl acetate to the cell-free aqueous culture and liquid-liquid extraction was performed by layer separation sequentially at neutral and acidic pH. The extracted organic phase was pooled and dried under nitrogen flow using RotaVapor II (BUCHI, Switzerland). The derivatization of metabolic intermediates in the sample was performed by using bis(trimethylsilyl)-trifluoroacetamide as previously described [45]. The derivatized samples were analyzed by GC-MS using QP2010S (Shimadzu Scientific Instruments, USA) with temperature program and other parameters used for GC analysis as reported previously [46]. 1H-NMR spectra of putatative intermediates were recorded using Bruker Avance DRX-300 spectrometer (Bruker, Germany) according to the method described earlier [47]. The dried extracted sample was dissolved in deuterated chloroform (CDCl3) in 5-mm NMR tubes and the spectra was recorded at 300 MHz. The chemical shift (δ) is given in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard. Nucleotide sequence A total of 1375 base pairs of the 16S rRNA gene of strain SSC5 were sequenced. This sequence has been deposited to GenBank with accession no. KC607748. Results Degradation of m-tyrosine in soil microcosms Samples collected from the soil microcosms were analyzed by HPLC in order to determine the degradation kinetics of m-tyrosine. Degradation of m-tyrosine in carbon-amended soil microcosms was initiated with a lag phase of ∼2 days, and thereafter the rate of degradation increased and was completed within 6 days of incubation (Figure 1). In contrast, degradation of m-tyrosine in the unamended soil microcosms was slower (Figure 1). The rate of m-tyrosine degradation was much slower in microcosms amended with only nitrogen (NH4Cl) or with a combination of nitrogen and carbon when compared to the above carbon-amended or unamended set ups (Figure 1). This difference in the rate of m-tyrosine degradation in soil microcosms was likely because the microorganisms utilized more the more favorable nitrogen source NH4Cl. No transformation of m-tyrosine was observed in the sterile soil (Figure 1). The above results clearly indicate that the soil microorganims utilized m-tyrosine as sole source of carbon, nitrogen, and energy 10.1371/journal.pone.0075928.g001Figure 1 Biotransformation of m-tyrosine in soil microcosms under aerobic conditions. (▪), carbon-amended; (○), unamended; (Δ), nitrogen-amended; (•), carbon and nitrogen-amended; (□), sterile. Values are presented as arithmetic mean of data obtained from experiments carried out in triplicate; error bars represent standard deviation. Isolation and characterization of m-tyrosine degrading bacteria Fifteen morphologically different colonies were isolated from the soil microcosms. These isolates were further screened for their ability to utilize m-tyrosine as the sole source of carbon, nitrogen, and energy . Among these isolates, one strain designated SSC5 was found to be an efficient m-tyrosine degrader and therefore was further studied to characterize its m-tyrosine degradation pathway. Strain SSC5 was found to be Gram variable, endospore forming, aerobic, catalase positive, and oxidase negative. The colonies were pale orange-yellow in color, slightly raised with irregular edges observed after overnight incubation on NA at 30°C. The optimal growth temperature and pH were 30–37°C and 6.0–7.0, respectively. This strain produced acid from D-fructose, D-glucose, maltose, D-ribose, sucrose, and D-trehalose. The strain efficiently hydrolyzed casein, starch and Tween 80; however, it was not capable of hydrolyzing aesculin, hypoxanthine, xanthine, and tyrosine. The above data identifies strain SSC5 as closely related to Bacillus aquimaris TF-12 [48]. Strain SSC5 utilized acetate, glucose, and succinate as sole carbon sources. The partial 16S rRNA gene sequence (1375 bp) of strain SSC5 showed 99% sequence similarity to that of Bacillus aquimaris strain TF-12. Thus, based on the morphological and physiological characteristics and phylogenetic analysis, strain SSC5 was identified as Bacillus aquimaris SSC5. Growth and degradation of m-tyrosine by strain SSC5 A growth study of strain SSC5 was carried out using different concentrations of m-tyrosine ranging from 50 to 500 µM. Growth of strain SSC5 was completely abolished at an m-tyrosine concentration of 400 µM , whereas 300 µM of m-tyrosine was found to be optimal for its growth. Strain SSC5 experienced a lag phase of ∼10 hours during its growth in carbon-free MSM supplemented with 300 µM m-tyrosine (Figure 2). Following lag phase, SSC5 grew exponentially, as determined by the increase of total cell protein up to the value of 9.6 µg ml−1. The increase in growth was accompanied by a decrease in the concentration of m-tyrosine. The complete depletion of m-tyrosine was observed at 48 hours of incubation (Figure 2). Although, no metabolic intermediates were identified during the growth study, a slight accumulation of ammonia (84 µM) was observed (Figure 2). This non-stoichiometric release of ammonia suggested that m-tyrosine might possibly be utilized as the sole nitrogen source for the growth of strain SSC5. The above results are in close agreement with the results reported earlier on the degradation of 3-nitrotyrosine by Burkholderia sp. strain JS165 and Variovorax paradoxus JS171 [36]. Growth yield of strain SSC5 on m-tyrosine was found to be 0.35 g of cells/g of m-tyrosine. The rate of m-tyrosine degradation by strain SSC5 during the growth studies was calculated to be 8.6 nmol min−1 mg of protein−1. Strain SSC5 was unable to grow in MSM in the absence of m-tyrosine as well as in MSM supplemented with L-tyrosine. Thus, the above results indicated that m-tyrosine is being utilized as the sole source of carbon, nitrogen, and energy by strain SSC5. 10.1371/journal.pone.0075928.g002Figure 2 Growth of Bacillus aquimaris SSC5 on m-tyrosine as the sole carbon, nitrogen, and energy source. (•), m-tyrosine; (▴), total protein; (○), NH4 +. Values are presented as arithmetic mean of data obtained from experiments carried out in triplicate; error bars represent standard deviation. Elucidation of catabolic pathway for m-tyrosine degradation by strain SSC5 During the growth study, no putative metabolic intermediates of m-tyrosine degradation were identified; therefore, samples from resting cell studies and enzyme assays were analyzed for the identification of intermediates. Previous studies on the bacterial degradation of L-tyrosine have shown that the first step of degradation is the transamination reaction which results in the formation of 4-hydroxyphenylpyruvate (4-OH-PPA), which subsequently undergoes degradation either by the homogentisate (HMG) or the homoprotocatechuate (HPC) central pathway [38], [49]. HPLC analysis of the samples from the resting cell study using m-tyrosine induced cells showed the presence of three additional compounds, eluting at 4.82, 6.36, and 8.10 min, respectively (Figure 3A). Peaks with the retention time (Rt) of 6.36 and 8.10 min, corresponded to the authentic standards of 3-hydroxyphenylacetate (3-OH-PhAc) and HMG, respectively. However, the peak with an Rt value of 4.82 min was not identified. In the subsequent GC-MS analysis, these compounds were eluted at 10.94 (3-OH-PhAc), 12.22 (unknown intermediate), and 15.85 min (HMG), respectively (Figure 3B). The unknown intermediate (Rt, 12.22 min; m/z 324) was identified as 3-hydroxyphenylpyruvate (3-OH-PPA) on the basis of mass fragmentation pattern in GC-MS analysis (Figure 4). The identities of 3-OH-PhAc (Rt, 10.94 min; m/z 296) and HMG (Rt, 15.85 min; m/z 384) intermediates were also confirmed by mass fragmentation pattern analysis (Figure 4). The formation of 3-OH-PPA as a m-tyrosine degradation pathway intermediate was confirmed by 1H-NMR analysis. The 1H-NMR analysis showed appearance of two broadened singlets of hydroxyl proton, one from carboxylic acid (COOH) and another from aromatic hydroxyl (C-3-OH) with the chemical shift values of 5.0 and 11.0 ppm, respectively (Figure 5). Similarly, two singlets appeared with the chemical shift values of 4.78 and 6.53 ppm showing the presence of methylene proton (CH2) and benzene proton (C-2-H) (Figure 5). Three doublet of doublets appeared at 6.54, 6.62, and 6.97 ppm which corresponded with the chemical shift values of benzene protons at position C-4, C-5, and C-6, respectively (Figure 5). The above 1H-NMR results confirmed the formation of 3-OH-PPA as the first metabolic intermediate of m-tyrosine degradation. 10.1371/journal.pone.0075928.g003Figure 3 Representative HPLC and GC-MS chromatograms of intermediates identified during the degradation of m-tyrosine by resting cells of Bacillus aquimaris SSC5. (A) HPLC chromatograms of intermediates identified at different time intervals. (B) GC-MS chromatograms of TMS-derivatized authentic standards and the identified intermediates. Peaks at Rt values correspond to: 5.62 min = m-tyrosine with m/z 325; 10.94 min = 3-OH-PhAc with m/z 296; 12.22 min = 3-OH-PPA with m/z 324; 15.85 min = HMG with m/z 384. 10.1371/journal.pone.0075928.g004Figure 4 Mass fragmentation patterns of m-tyrosine intermediates including 3-OH-PPA, 3-OH-PhAc, and HMG analyzed by GC-MS. 10.1371/journal.pone.0075928.g005Figure 5 1H-NMR analysis of the 3-hydroxyphenylpyruvate (3-OH-PPA) intermediate indentified during the degradation of m-tyrosine by resting cells of Bacillus aquimaris SSC5. m-Tyrosine-induced cells of strain SSC5 completely degraded m-tyrosine within 8 hours of incubation with the appearance and disappearance of intermediates namely 3-OH-PPA, 3-OH-PhAc, and HMG (Figure 6A). Thus, results from the resting cell study established that the degradation of m-tyrosine occurs with the formation of 3-OH-PPA and 3-OH-PhAc as intermediates, which are subsequently transformed into HMG. The induced cells of strain SSC5 also degraded 3-OH-PhAc via the formation of HMG as an intermediate and the complete degradation of 3-OH-PhAc occurred after 12 hours of incubation (Figure 6B). Similarly, HMG was degraded completely by the same cells after 8 hours of incubation (Figure 6C). Boiled cells were used as a control for the experiment related to HMG degradation and a brown color was observed in the medium, whereas there was no such color formation in the test samples (Figure 6C). The above results suggested that the dead (heat-inactivated) cells could not transform HMG, leading to the accumulation of HMG in the culture medium. Upon chemical oxidation and polymerization of HMG, pyomelanin pigments were formed that turned the color of the medium brown [44], [50]. This phenomenon has been described previously by Mendez et al. [42]. Previous reports also showed that the mutational inactivation of pathway gene(s) responsible for the catabolism of HMG, results in the formation of brown pigments due to accumulation of HMG and its chemical oxidation [38], [51], [52]. Our results provide evidence that the degradation of m-tyrosine occurs via the central pathway of HMG degradation. 10.1371/journal.pone.0075928.g006Figure 6 Degradation kinetics of m-tyrosine, 3-hydroxyphenylacetate, and homogentisate by m-tyrosine-induced cells of strain SSC5. (A) m-Tyrosine degradation kinetics. (•), m-tyrosine; (○), 3-OH-PPA; (Δ), 3-OH-PhAc; (▴), HMG; (▪), m-tyrosine in abiotic control. (B) 3-Hydroxyphenylacetate degradation kinetics. (•), 3-OH-PhAc; (▪), HMG. (C) Homogentisate degradation kinetics. (•), HMG; (○), brown color (as measured by the OD400) in control. Values are presented as arithmetic mean of data obtained from experiments carried out in triplicate; error bars represent standard deviation. m-Tyrosine transaminase activity Previous studies have shown that the first step of L-tyrosine degradation occurs by transamination reaction with the formation of 4-OH-PPA [38]. Thus, the identification of the 3-OH-PPA intermediate during the resting cell study leads us to posit that the first step of m-tyrosine degradation is also initiated with a transamination reaction. The transaminase enzyme assay from the cell-free lysates prepared from the m-tyrosine grown cells showed the accumulation of 3-OH-PPA (data not shown). Since the accumulation of 3-OH-PPA could not be quantified by HPLC due to the non availability of authentic standard, the enzyme activity was calculated based on the quantitative determination of m-tyrosine disappearance. The enzyme assay further supported the results of resting cells study and confirmed that the first step of m-tyrosine degradation occurs by transamination reaction in strain SSC5. The specific activity for the transaminase enzyme was found to be 4.9±0.17 nmol min−1 mg of protein−1. No transamination activity was observed in the control reactions that lacked either α-KG or cell-free lysates prepared from cells of strain SSC5 grown on m-tyrosine or glucose. 3-Hydroxyphenylacetate-6-hydroxylase activity Adding an additional hydroxyl group (-OH) on C-6 of the 3-OH-PhAc ring would result in the formation of HMG [38], [39]. Therefore, identification of HMG intermediate during the resting cell study clearly indicates the involvement of hydroxylation reaction in the conversion of 3-OH-PhAc to HMG. Previous studies have demonstrated that the formation of HMG from 3-OH-PhAc occurs by the action of 3-hydroxyphenylacetate-6-hydroxylase [38], [39]. Enzyme assay using the cell-free lysates prepared from m-tyrosine grown cells of strain SSC5 showed hydroxylase activity on 3-OH-PhAc with the formation of HMG as a product. The specific activity of hydroxylase enzyme was found to be 3.20±0.13 nmol min−1 mg of protein−1. The above experiment provided clear evidence on the involvement of a NADH-dependent hydroxylation reaction in the third step of m-tyrosine degradation by strain SSC5. Homogentisate 1, 2-dioxygenase activity To validate the funneling of 3-OH-PhAc into the HMG central pathway (i.e. the fourth step of m-tyrosine degradation), an enzyme assay for homogentisate 1, 2-dioxygenase was carried out using the cell-free lysate prepared from the m-tyrosine and 3-OH-PhAc grown cells, respectively. The cell-free lysates prepared from 3-OH-PhAc grown cells showed slightly higher 1, 2-dioxygenase activity as compared to the lysates prepared from the cells grown in presence of m-tyrosine (Figure 7). The specific enzyme activity was found to be 28.57±0.32 nmol min−1 mg of protein−1 when grown on 3-OH-PhAc and 26.32±0.15 nmol min−1 mg of protein−1 when grown on m-tyrosine. 10.1371/journal.pone.0075928.g007Figure 7 Homogentisate (HMG) 1, 2-dioxygenase activity from the cell-free lysates prepared from m-tyrosine-induced cells of strain SSC5. HMG 1, 2-dioxygenase activity was measured by maleylacetoacetate (MA) formation at 330 nm absorbance from cell-free lysates in spectrophotometer. (•), m-tyrosine grown cells; (○), 3-OH-PhAc grown cells; (▪), glucose grown cells. It is known that the homogentisate 1, 2-dioxygenase enzyme transforms HMG to maleylacetoacetate (MA) as a result of ring cleavage of HMG [38], [42]. The cell-free lysates prepared from the cells grown with glucose did not show any dioxygenase activity. Similarly, no dioxygenase activity was observed in the control reactions that lacked cell-free lysates. The above results proved that the homogentisate 1, 2-dioxygenase involved in the homogentisate central pathway, is inducible in nature in the presence of the parent substrate as well as the respective intermediates. Our findings are in close agreement with the results of Mandez et al. [42], who reported 1, 2-dioxygenase activity in the cell-free lysates prepared from 3-OH-PhAc grown cells of Burkholderia xenovorans LB400. Discussion Allelopathy is an important mechanism for the competitive advantage of several plants by exudation of various phytotoxic molecules from their roots [1], [53], [54]. The phytotoxic levels of allelochemicals are primarly influenced by process such as their sorption on soil particles and their chemical decomposition. However, soil microbes can also affect the outcome of allelopathic interaction between plants by degrading the released allelochemicals [3], [23], [24], [55]. Kaur et al. [3], have also reported that the outcome of allelopathic interactions of m-tyrosine in sterilized soil with a particular species was significantly diminished when non-sterile soil was used. Our microcosm studies showed that the soil microorganisms rapidly degrade m-tyrosine in carbon-amended as well as unamended soil (Figure 1). This study showed that soil microbes influence the phytotoxic properties of m-tyrosine either by utilizing it as sole carbon, and nitrogen source or transforming it into nontoxic products. Strain SSC5 isolated from the soil microcosms was able to utilize m-tyrosine as the sole carbon, nitrogen, and energy source under aerobic conditions. The degradation pathway of m-tyrosine in this bacterium has been proposed based on the metabolites identified during the growth and resting cell studies and the enzyme assays (Figure 8). Previous studies have shown that the degradation of L-tyrosine in eukaryotes and prokaryotes occurs by a common peripheral pathway i.e. formation of 4-OH-PPA through the transamination reaction, which funnels into either the HMG or the HPC central pathway [38], [42], [52], [56], [57], [58]. The initial step of m-tyrosine degradation occurs with the removal of amino substituent by the action of transaminase activity as determined in crude cell lysates, which is closely similar to the results of Nishino and Spain [36]. They identified 4-hydroxy-3-nitrophenylacetate instead of 4-hydroxy-3-nitrophenylpyruvate from 3-nitrotyrosine by the transamination reaction due to the non-availability of a standard. The GC-MS and 1H-NMR analysis of the samples collected from the resting cell study and the transaminase enzyme assay confirmed the formation of 3-OH-PPA as the first metabolic intermediate. Earlier reports have shown that the second step of L-tyrosine degradation is the transformation of 4-OH-PPA into the HMG intermediate with the removal of CO2 by the action of 4-hydroxyphenylpyruvate dioxygenase [38], [59]. However, with the identification of the 3-OH-PhAc intermediate in the resting cell study it is presumed that the second degradation step involves the conversion of 3-OH-PPA to 3-OH-PhAc, possibly via a coupled decarboxylation-mooxygenation reaction unlike 4-hydroxyphenylpyruvate dioxygenase which mediates a decarboxylation coupled to a dioxygenation. The decarboxylase enzyme assay from the crude cell lysates could not be carried out due to the non-availability of a 3-OH-PPA standard. During the resting cell study on 3-OH-PhAc, m-tyrosine-induced cells transformed 3-OH-PhAc into HMG and this observation was also supported by the NADH-dependent hydroxylase enzyme assay. The results of the hydroxylase activity assay are in close agreement with the results reported earlier [40], [42]. 10.1371/journal.pone.0075928.g008Figure 8 Proposed metabolic pathway for aerobic degradation of m-tyrosine by Bacillus aquimaris SSC5. Based on the data from our study, 3-OH-PPA, 3-OH-PhAc, and HMG are identified as the metabolic intermediates during degradation of m-tyrosine. The homogentisate is not the central bacterial catabolic pathway for the degradation of phenylacetic acid and phenylalanine, but it is employed by bacteria for the degradation of hydroxylated phenylacetic acid derivatives and, in rare cases, for phenylalanine [38], [42], [60], [61]. The homogentisate central pathway involves three successive catabolic enzymes i.e. homogentisate 1, 2-dioxygenase, isomerase, and hydrolase, respectively. Homogentisate 1, 2-dioxygenase cleaves the HMG ring and forms MA that is subsequently isomerized by the isomerase enzyme into fumarylacetoacetate (FA) [38]. Further, FA is transformed into fumarate and acetoacetate by the action of a hydrolase [38]. Homogentisate 1, 2-dioxygenase enzyme assays with the cell-free lysates prepared from m-tyrosine grown cells showed the conversion of HMG to MA. These data are in close agreement with the results reported earlier [38], [42]. Based on the above studies, it is proposed that the degradation of m-tyrosine by strain SSC5 occurs through the HMG central pathway via the formation of 3-OH-PPA and 3-OH-PhAc as major intermediates. The m-tyrosine degradation pathway proposed by us is similar to the pathway of L-tyrosine degradation reported earlier in a few bacterial isolates [45], [58], [62], [63]. Conclusion This is the first report on metabolism of m-tyrosine by Bacillus aquimaris SSC5, isolated from soil microcosms. The strain SSC5 utilizes m-tyrosine as the sole carbon, nitrogen, and energy source under aerobic conditions. The degradation of m-tyrosine occurs with the formation of 3-OH-PPA, 3-OH-PhAc, and HMG as intermediates. The initial step of m-tyrosine degradation occurs by the transamination of the amino substituent with the formation of 3-OH-PPA as the first metabolite. Subsequent degradation occurs with the formation of 3-OH-PhAc and HMG as intermediates. Finally, the HMG produced, degrades to MA catalyzed by homogentisate 1, 2-dioxygenase. The present study has significant implications in terms of understanding the environmental fate of m-tyrosine and also the masking effect of soil microorganisms over its allelopathic effect. Further studies are needed to establish the regulation and biochemistry of non-proteinogenic amino acid metabolic pathway in Bacillus aquimaris SSC5. We thank the reviewers and the editor for their helpful suggestions to improve our paper. FK thanks DBT for the Research Associateship and MK acknowledges her research fellowship from UGC, India. This is the IMTECH communication number 16/2013. ==== Refs References 1 Bertin C , Weston LA , Huang T , Jander G , Owens T , et al (2007 ) Grass roots chemistry: meta-tyrosine, an herbicidal nonprotein amino acid . Proc Natl Acad Sci U S A 104 : 16964 –16969 .17940026 2 Duke SO (2007 ) The emergence of grass root chemical ecology . Proc Natl Acad Sci U S A 104 : 16729 –16730 .17942678 3 Kaur H , Kaur R , Kaur S , Baldwin IT , Inderjit (2009 ) Taking ecological function seriously: soil microbial communities can obviate allelopathic effects of released metabolites . PLoS One 4 : e4700 .19277112 4 Huang T , Rehak L , Jander G (2012 ) meta-Tyrosine in Festuca rubra ssp. commutata (Chewings fescue) is synthesized by hydroxylation of phenylalanine . Phytochemistry 75 : 60 –66 .22192329 5 Huggins TG , Wells-Knecht MC , Detorie NA , Baynes JW , Thorpe SR (1993 ) Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation . J Biol Chem 268 : 12341 –12347 .8509373 6 van der Vliet A , O'Neill CA , Halliwell B , Cross CE , Kaur H (1994 ) Aromatic hydroxylation and nitration of phenylalanine and tyrosine by peroxynitrite. Evidence for hydroxyl radical production from peroxynitrite . FEBS Lett 339 : 89 –92 .8313984 7 Smith LC , Ravel JM , Lax SR , Shive W (1964 ) The effects of phenylalanine and tyrosine analogs on the synthesis and activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetases . Arch Biochem Biophys 105 : 424 –430 .14186748 8 Aronson JN , Wermus GR (1965 ) Effects of m-tyrosine on growth and sporulation of Bacillus species . J Bacteriol 90 : 38 –46 .16562040 9 Aronson JN , Bowe PA , Swafford J (1967 ) Mesosomes in m-tyrosine-inhibited Bacillus thuringiensis . J Bacteriol 93 : 1174 –1176 .6025420 10 Inderjit, Callaway RM , Vivanco JM (2006 ) Can plant biochemistry contribute to understanding of invasion ecology? Trends Plant Sci 11 : 574 –580 .17092763 11 Haney RL , Brinton WF , Evans E (2008 ) Soil CO2 respiration: Comparison of chemical titration, CO2 IRGA analysis and the Solvita gel system . Renew Agr Food Syst 23 : 171 –176 . 12 Anderson JPE (1982 ) Soil respiration. Methods of soil snalysis. Part 2 . Chemical and microbiological properties Agronomy Monograph 831 –871 . 13 Gurer-Orhan H , Ercal N , Mare S , Pennathur S , Orhan H , et al (2006 ) Misincorporation of free m-tyrosine into cellular proteins: a potential cytotoxic mechanism for oxidized amino acids . Biochem J 395 : 277 –284 .16363993 14 Molnar GA , Nemes V , Biro Z , Ludany A , Wagner Z , et al (2005 ) Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase . Free Radic Res 39 : 1359 –1366 .16298866 15 Matayatsuk C , Poljak A , Bustamante S , Smythe GA , Kalpravidh RW , et al (2007 ) Quantitative determination of ortho- and meta-tyrosine as biomarkers of protein oxidative damage in beta-thalassemia . Redox Rep 12 : 219 –228 .17925094 16 Winn M , Goss RJ , Kimura K , Bugg TD (2010 ) Antimicrobial nucleoside antibiotics targeting cell wall assembly: recent advances in structure-function studies and nucleoside biosynthesis . Nat Prod Rep 27 : 279 –304 .20111805 17 Levy E, Carmeli S (1994) Biological control of plant pathogens by antibiotic-producing bacteria. Allelopathy: American Chemical Society. pp. 300–309. 18 Lambers H, Colmer T, Inderjit(2005) Soil microorganisms: An important determinant of allelopathic activity. Root Physiology: from Gene to Function: Springer Netherlands. pp. 227–236. 19 Inderjit (1996 ) Plant phenolics in allelopathy . Bot Rev 62 : 186 –202 . 20 Blum U , Shafer SR (1988 ) Microbial populations and phenolic acids in soil . Soil Biol Biochem 20 : 793 –800 . 21 Nair M , Whitenack C , Putnam A (1990 ) 2,2′-OXO-1,1′-azobenzene a microbially transformed allelochemical from 2,3-benzoxazolinone: I . J Chem Ecol 16 : 353 –364 .24263495 22 Blum U (1998 ) Effects of microbial utilization of phenolic acids and their phenolic acid breakdown products on allelopathic Interactions . J Chem Ecol 24 : 685 –708 . 23 Inderjit, Pollock JL , Callaway RM , Holben W (2008 ) Phytotoxic effects of (+/−)-catechin in vitro, in soil, and in the field . PLoS One 3 : e2536 .18648546 24 Perry L , Thelen G , Ridenour W , Callaway R , Paschke M , et al (2007 ) Concentrations of the allelochemical (+/−)-catechin in centaurea maculosa soils . J Chem Ecol 33 : 2337 –2344 .18030533 25 Ehlers BK (2011 ) Soil microorganisms alleviate the allelochemical effects of a thyme monoterpene on the performance of an associated grass species . PLoS One 6 : e26321 .22125596 26 Macias FA , Oliveros-Bastidas A , Marin D , Castellano D , Simonet AM , et al (2005 ) Degradation studies on benzoxazinoids. Soil degradation dynamics of (2R)-2-O-beta-D-glucopyranosyl-4-hydroxy-(2H)- 1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and its degradation products, phytotoxic allelochemicals from Gramineae . J Agri Food Chem 53 : 554 –561 . 27 Rettenmaier H , Kupas U , Lingens F (1983 ) Degradation of juglone by Pseudomonas putida J 1 . FEMS Microbiol Lett 19 : 193 –195 . 28 Schmidt SK (1988 ) Degradation of juglone by soil bacteria . J Chem Ecol 14 : 1561 –1571 .24276429 29 Shaw LJ , Morris P , Hooker JE (2006 ) Perception and modification of plant flavonoid signals by rhizosphere microorganisms . Environ Microbiol 8 : 1867 –1880 .17014487 30 Fazlurrahman , Fazlurrahman, Batra M , Pandey J , Suri CR , Jain RK (2009 ) Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil . Lett Appl Microbiol 49 : 721 –729 .19818008 31 Perreault NN , Halasz A , Manno D , Thiboutot S , Ampleman G , et al (2012 ) Aerobic mineralization of nitroguanidine by Variovorax strain VC1 isolated from soil . Environ Sci Technol 46 : 6035 –6040 .22563908 32 Khan F , Pandey J , Vikram S , Pal D , Cameotra SS (2013 ) Aerobic degradation of 4-nitroaniline (4-NA) via novel degradation intermediates by Rhodococcus sp. strain FK48 . J Hazard Mater 254–255C : 72 –78 . 33 Khan F , Pal D , Vikram S , Cameotra SS (2013 ) Metabolism of 2-chloro-4-nitroaniline via novel aerobic degradation pathway by Rhodococcus sp. strain MB-P1 . PLoS One 8 : e62178 .23614030 34 Kaur I , Kaur C , Khan F , Mayilraj S (2012 ) Flavobacterium rakeshii sp. nov., isolated from marine sediment, and emended description of Flavobacterium beibuense Fu et al. 2011 . Int J Syst Evol Microbiol 62 : 2897 –2902 .22247214 35 Shettigar M , Pearce S , Pandey R , Khan F , Dorrian SJ , et al (2012 ) Cloning of a novel 6-chloronicotinic acid chlorohydrolase from the newly isolated 6-chloronicotinic acid mineralizing Bradyrhizobiaceae strain SG-6C . PLoS One 7 : e51162 .23226482 36 Nishino SF , Spain JC (2006 ) Biodegradation of 3-nitrotyrosine by Burkholderia sp. strain JS165 and Variovorax paradoxus JS171 . Appl Environ Microbiol 72 : 1040 –1044 .16461647 37 Kittell BL , Helinski DR , Ditta GS (1989 ) Aromatic aminotransferase activity and indoleacetic acid production in Rhizobium meliloti . J Bacteriol 171 : 5458 –5466 .2551887 38 Arias-Barrau E OE , Luengo JM , Fernandez C , Galan B , et al (2004 ) The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida . J Bacteriol 186 : 5062 –5077 .15262943 39 Arias-Barrau E , Sandoval A , Naharro G , Olivera ER , Luengo JM (2005 ) A two-component hydroxylase involved in the assimilation of 3-hydroxyphenyl acetate in Pseudomonas putida . J Biol Chem 280 : 26435 –26447 .15866873 40 Van Berkel WJH , Van Den Tweel WJJ (1991 ) Purification and characteriation of 3-hydroxyphenylacetate 6-hydroxylase: a novel FAD-dependent monooxygenase from a Flavobacterium species . Eur J Biochem 201 : 585 –592 .1935954 41 Fernandez-Canon JMPM (1997 ) Spectrophotometric determination of homogentisate using Aspergillus nidulans homogentisate dioxygenase . Anal Biochem 245 : 218 –221 .9056215 42 Mendez V , Agullo L , Gonzalez M , Seeger M (2011 ) The homogentisate and homoprotocatechuate central pathways are involved in 3- and 4-hydroxyphenylacetate degradation by Burkholderia xenovorans LB400 . PLoS One 6 : e17583 .21423751 43 Seegmiller JE , Zannoni VG , Laster L , La Du BN (1961 ) An enzymatic spectrophotometric method for the determination of homogentisic acid in plasma and urine . J Biol Chem 236 : 774 –777 .13749617 44 Carreira A , Ferreira LM , Loureiro V (2001 ) Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid . Appl Environ Microbiol 67 : 3463 –3468 .11472920 45 Pometto AL (1985 ) L-Phenylalanine and L-tyrosine catabolism by selected Streptomyces species . Appl Environ Microbiol 49 : 727 –729 .3994376 46 Deutsch JC (1997 ) Determination of p-hydroxyphenylpyruvate, p-hydroxyphenyllactate and tyrosine in normal human plasma by gas chromatography-mass spectrometry isotope-dilution assay . J Chromatogr B Biomed Sci Appl 690 : 1 –6 .9106023 47 Prakash D , Kumar R , Jain RK , Tiwary BN (2011 ) Novel pathway for the degradation of 2-chloro-4-nitrobenzoic acid by Acinetobacter sp. strain RKJ12 . Appl Environ Microbiol 77 : 6606 –6613 .21803909 48 Yoon JH , Kim IG , Kang KH , Oh TK , Park YH (2003 ) Bacillus marisflavi sp. nov. and Bacillus aquimaris sp. nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea . Int J Syst Evol Microbiol 53 : 1297 –1303 .13130010 49 Arunachalam U , Massey V , Vaidyanathan CS (1992 ) p-Hydroxyphenylacetate-3-hydroxylase. A two-protein component enzyme . J Biol Chem 267 : 25848 –25855 .1464599 50 Yabuuchi E , Ohyama A (1972 ) Characterization of “pyomelanin”-producing strains of Pseudomonas aeruginosa . Int JSyst Bacteriol 22 : 53 –64 . 51 Rodriguez-Rojas A , Mena A , Martin S , Borrell N , Oliver A , et al (2009 ) Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection . Microbiology 155 : 1050 –1057 .19332807 52 Schmaler-Ripcke J , Sugareva V , Gebhardt P , Winkler R , Kniemeyer O , et al (2009 ) Production of pyomelanin, a second type of melanin, via the tyrosine degradation pathway in Aspergillus fumigatus . Appl Environ Microbiol 75 : 493 –503 .19028908 53 Bais HP , Vepachedu R , Gilroy S , Callaway RM , Vivanco JM (2003 ) Allelopathy and exotic plant invasion: from molecules and genes to species interactions . Science 301 : 1377 –1380 .12958360 54 Weston LA , Duke SO (2003 ) Weed and crop allelopathy . Crit Rev Plant Sci 22 : 367 –389 . 55 Inderjit (2001 ) Soil: environmental effect on allelochemical activity . Agron J 93 : 79 –84 . 56 Sparnins VL , Chapman PJ (1976 ) Catabolism of L-tyrosine by the homoprotocatechuate pathway in gram-positive bacteria . J Bacteriol 127 : 362 –366 .931949 57 Lindstedt S , Odelhog B , Rundgren M (1977 ) Purification and some properties of 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P. J. 874 . Biochemistry 16 : 3369 –3377 .889802 58 Denoya CD , Skinner DD , Morgenstern MR (1994 ) A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli . J Bacteriol 176 : 5312 –5319 .8071207 59 Serre L , Sailland A , Sy D , Boudec P , Rolland A , et al (1999 ) Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway . Structure 7 : 977 –988 .10467142 60 Olivera ER , Reglero A , Martinez-Blanco H , Fernandez-Medarde A , Moreno MA , et al (1994 ) Catabolism of aromatics in Pseudomonas putida U. Formal evidence that phenylacetic acid and 4-hydroxyphenylacetic acid are catabolized by two unrelated pathways . Eur J Biochem 221 : 375 –381 .8168524 61 Teufel R , Mascaraque V , Ismail W , Voss M , Perera J , et al (2010 ) Bacterial phenylalanine and phenylacetate catabolic pathway revealed . Proc Natl Acad Sci U S A 107 : 14390 –14395 .20660314 62 Blakley ER (1972 ) Microbial conversion of p-hydroxyphenylacetic acid to homogentisic acid . Can J Microbiol 18 : 1247 –1255 .4403325 63 Boer L , Harder W , Dijkhuizen L (1988 ) Phenylalanine and tyrosine metabolism in the facultative methylotroph Nocardia sp. 239 . Arch Microbiol 149 : 459 –465 .	24098407	PMC3788032	CC BY	2021-01-05 17:47:15	yes	PLoS One. 2013 Oct 1; 8(10):e75928
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24098600PONE-D-13-2805310.1371/journal.pone.0078521Research ArticleComparative Immunogenicity of HIV-1 gp160, gp140 and gp120 Expressed by Live Attenuated Newcastle Disease Virus Vector Immune Response Induced by NDV Expressed HIV gp140Khattar Sunil K. 1 Samal Sweety 1 LaBranche Celia C. 2 Montefiori David C. 2 Collins Peter L. 3 Samal Siba K. 1 * 1 Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America 2 Division of Surgical Sciences, Duke University, Durham, North Carolina, United States of America 3 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, United States of America Tyagi Anil Kumar Editor University of Delhi, India * E-mail: ssamal@umd.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SKK SKS CCL DCM. Performed the experiments: SKK SS CCL. Analyzed the data: SKK SS CCL. Contributed reagents/materials/analysis tools: SKK SKS CCL DCM. Wrote the manuscript: SKK CCL DCM PLC SKS. 2013 1 10 2013 8 10 e785218 7 2013 19 9 2013 2013This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.The development of a vaccine against human immunodeficiency virus-1 (HIV-1) capable of inducing broad humoral and cellular responses at both the systemic and mucosal levels will be critical for combating the global AIDS epidemic. We previously demonstrated the ability of Newcastle disease virus (NDV) as a vaccine vector to express oligomeric Env protein gp160 and induce potent humoral and mucosal immune responses. In the present study, we used NDV vaccine strain LaSota as a vector to compare the biochemical and immunogenic properties of vector-expressed gp160, gp120, and two versions of gp140 (a derivative of gp160 made by deleting the transmembrane and cytoplasmic domains), namely: gp140L, which contained the complete membrane-proximal external region (MPER), and gp140S, which lacks the distal half of MPER. We show that, similar to gp160, NDV-expressed gp140S and gp120, but not gp140L, formed higher-order oligomers that retained recognition by conformationally sensitive monoclonal antibodies. Immunization of guinea pigs by the intranasal route with rLaSota/gp140S resulted in significantly greater systemic and mucosal antibody responses compared to the other recombinants. Immunization with rLaSota/140S, rLaSota/140L rLaSota/120 resulted in mixed Th1/Th2 immune responses as compared to Th1-biased immune responses induced by rLaSota/160. Importantly, rLaSota/gp140S induced neutralizing antibody responses to homologous HIV-1 strain BaL.26 and laboratory adapted HIV-1 strain MN.3 that were stronger than those elicited by the other NDV recombinants. Additionally, rLaSota/gp140S induced greater CD4+ and CD8+ T-cell responses in mice. These studies illustrate that rLaSota/gp140S is a promising vaccine candidate to elicit potent mucosal, humoral and cellular immune responses to the HIV-1 Env protein. This research was supported by NIH R21 grant AI-093198 awarded to S.K.S and by the NIAID Primate Central Immunology Laboratory Contract HHSN27201100016C awarded to D.C.M. P.L.C. was supported by the NIAID, NIH Intramural Research Program. The views expressed herein neither necessarily reflect the official policies of the Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The HIV-1 envelope (Env) glycoprotein is the major viral neutralization antigen and its efficacy in protection against HIV-1 has been demonstrated in animal models [1-3]. . The HIV-1 Env also is a target for cell mediated immune responses that can contribute to protection [4,5]. Env is synthesized as a 160-kDa precursor gp160 that is processed by furin or related host cellular proteases into its soluble attachment subunit gp120 and transmembrane subunit gp41 [6,7]. gp120 and gp41 are organized on virions as trimeric spikes with three gp120 proteins non-covalently associated with three gp41 subunits [8]. The viral envelope initiates infection by contact through a gp120-CD4 interaction. This interaction also stabilizes the structure of a coreceptor binding site on gp120 that engages one of two coreceptors (CCR5 or CXCR4) [9]. The viral spike possesses a number of characteristics that subvert humoral immunity, including heavy glycosylation, conformational flexibility, and sequence variability in immunodominant domains. Therefore, significant efforts have been made to design and construct either purified Env glycoprotein immunogens or vaccine vectors that present Env glycoprotein as functional trimeric complexes, thereby preferentially exposing relevant neutralizing determinants to the immune system [10,11]. Various forms of Env glycoprotein have been evaluated as vaccine immunogens to protect against HIV-1. Env vaccines consisting of gp120 subunit proteins or peptide fragments thereof have shown a lack of protective efficacy in clinical trials [12]. Soluble forms, called gp140, which contains the membrane proximal external region (MPER) but lacks the transmembrane and cytoplasmic domains have been designed and are cleaved in the same fashion as gp160, resulting in gp120 subunits along with a thermodynamically favored 6-helix bundle formed by gp41 moiety [13-17]. Various strategies have been used to produce stable trimers of gp140 [11,18,19]. Several replication-competent and non-replication-competent viral vector have been used to express oligomeric Env immunogens and to stimulate immune responses against HIV-1 [10]. Although immunogenicity studies with these trimers have thus far showed some improvements in breadth and potency of neutralization when compared with monomeric gp120, adequate protection against diverse primary HIV-1 isolates has not been achieved. Newcastle disease virus (NDV) is a member of the genus Avulavirus in the family Paramyxoviridae. The genome of NDV is a single-stranded, negative-sense RNA of 15 kb that contains six genes in the order 3′-N-P-M-F-HN-L-5′. [20]. NDV has several properties that are useful as a vaccine vector in humans. NDV is an avian virus that is attenuated in humans due to a natural host range restriction [21]. NDV shares only a low level of amino acid sequence identity with, and is antigenically distinct from, common animal and human pathogens, and thus vaccination should not be affected by preexisting immunity. NDV has been used to express protective antigens of several human pathogens in non-human primate models [21-24]. Further, the ability of NDV to express HIV-1 Gag [25,26] and gp160 [27] antigens and to generate Gag-and Env-specific immune response in mice and guinea pigs, respectively has been demonstrated. In the present study, we constructed NDV vectors expressing several engineered derivatives of gp160, including two forms of gp140. These were evaluated for neutralizing immune responses in guinea pigs and T-cell responses in mice. This showed that engineering gp140 containing MPER resulted in substantial increases in the induction of HIV-1-specific serum and mucosal antibodies and CD4+ and CD8+ T lymphocytes. Materials and Methods Ethical Statement Female Hartley guinea pigs (aged 5-6 weeks) and female BALB/c mice (aged 5 weeks) were obtained from Charles River Laboratories, Wilmington, MA and National Cancer Institute, Bethesda, MD, respectively. The study was done in AAALAC-approved animal facility and under the authority of Institutional Animal Care and Use Committee (IACUC) of University of Maryland, College Park, MD. All the guinea pigs used in this study were housed in isolator cages in our Bio Safety Level-2+ facility and all the mice used in this study were kept under specific pathogen-free conditions in Individually Ventilated Cages (IVCs) in our Bio Safety Level-2+ facility. All the animals were cared for in strict accordance with established guidelines, and the experimental procedures were performed with approval from IACUC. The intranasal inoculation and bleeding in guinea pigs was performed after injecting Ketamine and Xylazine anesthesia. The intranasal inoculation and bleeding in mice was performed after anaesthetizing the mice by inhalation of isofluorane in specialized chambers. All the efforts are made to minimize sufferings in both guinea pigs and mice. All the experiments where 9-day old embryonated chicken eggs were used ended on or before day 13. Before collecting allantoic fluid from the eggs, the embryos were sacrificed by incubating the eggs at 4°C in a refrigerator for 2 hour. Cells, viruses, antibodies and protein HEp-2, DF1, Vero and TZM-bl cells were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). 293T/17 cells were grown and maintained in Opti-MEM I reduced serum medium containing 10% FBS. Recombinant and wild-type NDV strains were grown in 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs. The modified vaccinia virus strain Ankara expressing the T7 RNA polymerase was grown in primary chicken embryo fibroblast cells. Purified recombinant HIV-1 BaL gp120 protein was obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP). A pool of HIV gp120 monoclonal antibodies was kindly provided by Dr. Anthony DeVico, University of Maryland School of Medicine, UMB, Baltimore, MD. Construction of recombinant NDVs expressing HIV-1 gp140 and gp120 and gp160 A 2598-nucleotide (nt) cDNA encoding human-codon-optimized HIV-1 glycoprotein gp160 (852 amino acids [aa]) derived from the CCR5-tropic clade B strain BaL1 was modified by PCR to add NDV transcription signals and flanking PmeI sites and was inserted at the unique PmeI site between P and M genes in a cloned cDNA of the full-length antigenome of the lentogenic NDV vaccine strain LaSota (Figure 1) [27,28]. Additional constructs were made encoding two different versions of gp140, namely gp140L (2082 nt, 679 aa) and gp140S (2034 nt, 663 aa), as well as gp120 (1560 nt, 506 aa), and similarly were inserted at the PmeI site between the P and M genes (Figure 1). Recombinant viruses (designated rLaSota/gp160, rLaSota/gp140L, rLaSota/gp140S and rLaSota/gp120) were recovered as described previously [28] and were plaque purified and grown in 9-day-old embryonated SPF chicken eggs [29,30]. 10.1371/journal.pone.0078521.g001Figure 1 Gene map of recombinant NDV LaSota (rLaSota) and structures of gp160, gp120, gp140L, and gp140S. The proteins are shown as unprocessed primary translation products annotated to show the locations of the furin cleavage site, the membrane-proximal external region (MPER), the transmembrane (TM) and cytoplasmic (CT) domains, and the total aa lengths. The cDNAs encoding these proteins were modified by PCR to add NDV gene-start (GS) and gene-end (GE) transcription signals, an intergenic (IG) nucleotide, and flanking PmeI sites, and were inserted individually between the NDV P and M genes. Sequence flanking the Env ORFs is shown in positive-sense, and PmeI sites used in the construction are shown in italics. NDV genes (N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuraminidase protein; L, large polymerase protein) are shown as open boxes. Expression of HIV-1 gp140, gp120 and gp160 Env proteins in cells infected with recombinant virus The expression of gp140, gp120 and gp160 by rLaSota/gp140L and rLaSota/gp140S, rLaSota/gp120 and rLaSota/gp160 was examined by Western blot analysis. Briefly, DF1 cells were infected with rLasota/gp140L, rLaSota/gp140S, rLaSota/gp120, rLaSota/gp160 and rLaSota at a multiplicity of infection (MOI) of 0.01 PFU. The cells were harvested at 48 h post-infection and lysed using radioimmunoprecipition buffer. The cell culture medium supernatants were collected at 48 h post-infection and were concentrated 10x by passing through Amicon filters. The cell lysate and cell culture medium supernatants were analyzed by Western blotting using a 1:10 dilution of a pool of gp120-specific monoclonal antibodies. Western blots were scanned and densitometric analysis of gp120 protein bands were performed by using the Photoshop program. The expression of gp140, 120 and 160 by the recombinant viruses was further examined in Vero cells by immunofluorescence assay. Briefly, confluent monolayers of Vero cells on 4 well Lab-Tek chamber slides were infected with the recombinant viruses at an MOI of 0.1. After 24 h, the infected cells were washed with PBS and either fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 min for detection of total antigen. After further washing with PBS, the cells were incubated for 30 min with 3% normal goat serum to block nonspecific binding sites and incubated for 1 h with 1:10 dilution of a pool of gp120-specific monoclonal antibodies. The cells were rinsed with PBS and incubated with a 1:1000 dilution of Alexa Fluor 488 conjugated goat anti-mouse immunoglobulin G antibody (Invitrogen, Carlsbad, CA) for 45 min. The cells were washed with PBS and analyzed with a fluorescent microscope. Analysis of HIV-1 gp160, gp140 and gp120 protein oligomers The oligomeric state of gp140, gp120 and gp160 expressed by the NDV recombinants was analyzed by cross linking infected cell lysates followed by Western blotting. Briefly, lysates of rLaSota/gp140L, rLaSota/gp140S, rLasota/gp120, rLaSota/gp160 and rLaSota infected DF1 cells in PBS were incubated at room temperature for 30 min with a final concentration of 1 mM Dithiobis (succinimidyl propionate) [DSP; Pierce], a thiol-cleavable, amine-reactive and membrane-permeable crosslinker. After crosslinking, samples were prepared in Laemmli sample buffer (100 mM Tris, pH 6.8, 2% SDS, 15% glycerol) with or without 5% β- mercaptoethanol and boiled for 5 min to make reduced and non-reduced samples, respectively. SDS-PAGE and Western blot analysis were performed as described before. Pathogenicity of rNDVs in embryonated chicken eggs The pathogenicity of rLasota/gp140L, rLasota/gp140S, rLasota/gp120, rLasota/gp160 and rLaSota in chickens was evaluated by an internationally established in vivo test: the mean death time (MDT) test in 9-day-old SPF embryonated chicken eggs. The MDT test was performed by a standard procedure [31]. Briefly, a series of 10-fold dilutions of fresh allantoic fluid from eggs infected with the test virus were made in sterile PBS, and 0.1 ml of each dilution was inoculated into the allantoic cavity of each of five eggs. The eggs were incubated at 37°C and examined four times daily for 7 days. The time that each embryo was first observed dead was recorded. The highest dilution that killed all embryos was considered the minimum lethal dose. The MDT was recorded as the time (in h) for the minimum lethal dose to kill the embryos. The MDT has been used to classify NDV strains as velogenic (MDT < 60 h), mesogenic (MDT 60 to 90 h), and lentogenic (MDT > 90 h). Growth characteristics of rNDVs in DF1 cells To determine multicycle growth kinetics of rLaSota/gp140L, rLaSota/gp140S, rLaSota/gp120, rLaSota/gp160 and rLaSota, DF1 cells in duplicate wells of six-well plates were infected with each virus at an MOI of 0.01 PFU. After 1 h of adsorption, the cells were washed with DMEM and then incubated with DMEM containing 5% FBS and 5% allantoic fluid. The cell culture supernatant samples were collected and replaced with an equal volume of fresh medium at 8 h intervals until 64 h post-infection. The titers of virus in the samples were quantified by plaque assay in DF1 cells. Guinea pig immunizations Female Hartley guinea pigs weighing approximately 375 gm each were obtained from Charles River Laboratories, Wilmington, MA. A total of 27 guinea pigs were divided into four groups of 6 animals each that received rLaSota/gp160, rLaSota/gp140L, rLaSota/gp140S, or rLaSota/gp120, and a control group of 3 animals that received the empty rLaSota vector. Animals were immunized by intranasal (i.n.) route on days 0 and 14 with 300 µl (150 µl in each nostril) of allantoic fluid containing 106 PFU/ml of the indicated virus. All animals were sacrificed 76 days after the second boost (i.e. 90 days following the first immunization). Blood was collected on day 0 (pre-bleed) and on days 7, 14, 21, 28, 35, 42, 56, 70 and 90. Sera were prepared and stored at -70°C. Vaginal washes and fecal samples were collected in parallel with the blood samples. To collect vaginal washes, animal feeding needles (Fisher Scientific) were used to flush 100 µl of PBS containing protease inhibitor cocktail (Sigma) four to six times into vaginal cavity. Vaginal washes were spun at 10,000 rpm for 15 min to remove cellular debris and supernatants were collected and stored at -70°C. Fecal sample were collected in PBS containing antibiotics, vortexed and incubated at 37 °C for 20 min and spun at 4,000 rpm for 10 min. Supernatants were collected and stored at -70°C Measuring gp120-specific total IgG, IgG1, IgG2a and IgA antibodies in sera, vaginal washes and fecal samples by ELISA HIV-1 Env-specific antibody titers were determined by isotype-specific ELISA. Ninety-six-well Maxisorp ELISA plates (Nunc, Denmark), coated overnight with 100 µl/well of 1 µg/ml purified recombinant HIV-1 BaL gp120 protein in sodium caronate/bicarbonate buffer (pH 9.8), were blocked first with 3% skimmed milk in water for 30 sec and then with 2% sucrose in water for 30 sec. Plates were dried for 2 h at 37°C. Serial dilutions of sera or vaginal washes or fecal samples from immunized guinea pigs were prepared in dilution buffer (Synbiotics Carporation, San Diego, CA), added to the plates, and incubated for 2 h at room temperature. The plates were washed three times with plate washing solution (Synbiotics Carporation) and incubated for 1 h with a 1:1,000 dilution of an isotype-specific secondary antibody; namely, horseradish peroxidase (HRP)-conjugated goat anti-guinea pig IgG (KPL, Gaithersburg, MD), goat anti-guinea pig IgG1, goat anti-guinea pig IgG2a (Novus Biologicals, Littleton, CO), or sheep anti-guinea pig IgA (Immunology Consultants Laboratory, Newberg, OR). The plates were washed three times and developed with ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) peroxidase substrate solution (Synbiotics Corporation), stopped by the addition of peroxidase stop solution, and analyzed at 405 nm with ELx800 ELISA plate reader (BioTek, VT). ELISA endpoint titers were defined as the highest reciprocal serum dilution at which the mean OD value of duplicate wells were >2-fold above the mean OD value plus 2 SD of serum or vaginal wash or fecal sample from negative control animals. Commercial NDV ELISA kits (Synbiotics Corporation) was used to detect antibodies against the NDV antigens. Detection of Env-specific IFN-γ producing cells by intracellular cytokine staining (ICS) Six-week-old female BALB/c mice (Charles River Laboratories, Wilmington, Massachusetts) in groups of six animals each were immunized by the i.n. route on days 0 and 14 with the indicated virus in 50 µl (25 µl in each nostril) of allantoic fluid containing 105 PFU/ml. Splenocytes were collected on day 56 and stimulated for 12 h with either 10 µg/ml of HIV-1 consensus subtype B 10-mer overlapping Env peptide pools (AIDS Research and Reference Reagent Program) or medium alone. Cells were incubated for 6 h with 10 µg/ml brefeldin A (Sigma). After blocking Fcγ receptors (rat anti-mouse CD16/32; BD Biosciences), cells were stained with Alexa Fluor 488-conjugated anti-mouseCD3ε and APC-Cy™7 conjugated anti-CD4 and per CP-Cy TM5 conjugated anti-mouse CD8 for 30 min at 4°C. The cells were fixed, permeabilized (Cytofix/Cytoperm Plus, BD Biosciences) and stained with PE anti–IFN-γ mAbs for 30 min at 4°C (BD Biosciences). Cells were analyzed by flow cytometry and Flowjo software was used for data analysis. The frequencies of cells positive for IFN-γ+ and CD4 or CD8 were determined. Data are representative of three experiments where spleens from two mice were pooled in each experiment. Neutralization assays Neutralizing antibody activity was measured in 96-well culture plates by using Tat-regulated Luc reporter gene expression to quantify reductions in virus infection in either TZM-bl or A3R5.7 cells. Assays in TZM-bl cells were performed with HIV Env-pseudotyped viruses as described previously [32]. TZM-bl cells were used for neutralization of clade B tier 1 HIV-1 strains BaL.26 and MN.3 and heterologous clade B tier 2 HIV-1 strains RHPA4259.7 and TRO.11. Briefly, neutralization assays were performed with serial dilutions of heat-inactivated (56°C, 1 hr) samples. Serum samples were diluted over a range of 1:20 to 1:43740 in cell culture medium and were pre-incubated with virus (~150,000 relative light unit equivalents) for 1 hr at 37 °C before addition of cells. Following 48 hr incubation, cells were lysed and luciferase activity was determined using a microtiter plate luminometer and BriteLite Plus Reagent (PerkinElmer). The A3R5.7 cell line (A3.01/R5.7) is a derivative of the CEM human lymphoblastoid cell line that naturally expresses CD4 and CXCR4 [33] and was engineered to express CCR5 [34]. The A3R5 assay was performed with clade B tier 2 HIV-1 strains SC22.3C2.LucR. T2A.ecto and REJO. LucR. T2A.ecto essentially as previously described [35]. As with the TZM-bl assay, diluted samples were incubated with virus (~50,000 RLU equivalents) for 1 hr at 37 °C prior to adding cells. After incubating for 4 days, a defined portion of the cell suspension was transferred to 96-well white solid plates (Costar) for measurement of luminescence using the ViviRen Live Cell Substrate as described by the supplier (Promega). For both the TZM-bl and A3R5 assays, neutralization titers are the sample dilution at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. For each animal in this study, pre- and post-immune serum samples were assayed side-by-side. As is often the case with guinea pig sera, a low-level background signal was present in many pre-immune serum samples; the background has been subtracted from the neutralization titers presented. Statistical analysis Statistical analysis of serological responses was performed by unpaired t test (two-tailed) with the use of Prism 5.0 (Graph Pad Software Inc., SanDiego, CA) with a significance level of P <0.05. Results Generation of rNDVs expressing HIV-1 gp160, gp140L, gp140S, and gp120 A recombinant version of the avirulent NDV vaccine strain LaSota was used to construct four viruses expressing different forms of the Env glycoprotein of HIV-1 strain BaL.1, namely: (i) the full-length, 852-aa gp160 protein (virus rLaSota/gp160); (ii) a 679-aa gp140 protein (gp140L, virus rLaSota/gp140L) that contained the complete 30-aa MPER at its C-terminus and terminated with the sequence WYIKI immediately adjacent to the transmembrane domain; (iii) a shorter, 663-aa gp140 protein (gp140S, virus rLaSota/gp140S) that contained a partial, 14-aa long MPER at its C-terminus and terminated with the sequence DKWAS; and (iv) a 560-aa gp120 protein (virus rLaSota/gp120) that terminated with the cleavage site sequence REKR (Figure 1). The recombinant viruses were recovered and propagated in embryonated chicken eggs with peak titers of 108 to 109 HA units and in DF1 cells with peak titers 107 to 108 pfu/ml. To determine the stability of the gp160, gp140 and gp120 genes in rLaSota vector, the recovered viruses were passaged 10 times in embryonated chicken eggs and the sequences were confirmed. Expression of HIV-1 gp140 and gp120 and gp160 Env proteins and analysis of oligomers To investigate the expression of the various forms of Env, DF1 cells were infected with rLaSota/160, rLaSota/gp140L, rLaSota/gp140S, and rLaSota/gp120, and cell lysates and culture medium supernatants were harvested, subjected to gel electrophoresis, and analyzed by Western blot using a pool of gp120-specific monoclonal antibodies. Analysis of cell lystates revealed the presence of precursor protein (either 160 kDa or 140 kDa) as well as the 120 kDa protein derived by cleavage of gp140 or gp160 or expressed directly by rLaSota/gp120 (Figure 2A). Densitometric analysis of Western blot showed that level of gp140 and gp120 proteins expressed by rLaSota/gp140S was 2-fold higher compared to rLaSota/gp140L (Figure 2A). Analysis of medium supernatants showed that gp120 was secreted from cells infected with rLaSota/gp140L, rLaSota/gp140S, and rLaSota/gp120, whereas gp120 expressed by rLaSota/gp160 remained cell-associated (Figure 2B). Densitometric analysis of gp120 protein band present in Western blot revealed that the level of gp120 secreted by rLaSota/gp140S was 3-fold higher compared to rLaSota/gp140L. The intracellular expression of Env protein in response to the NDV vectors also was analyzed in Vero cells by indirect immunofluorescence using a pool of gp120-specific monoclonal antibodies (Figure 3). This showed that the distribution of Env protein expressed by rLaSota/gp140L, rLaSota/gp140S, and rLaSota/gp120 was quite different compared to rLaSota/gp160, which is not surprising since the substantial secretion of gp120 observed with the first three viruses would involve the secretory pathway, whereas, in contrast, Env protein expressed by rLaSota/gp160 remained cell-associated and would accumulate on the plasma membrane. 10.1371/journal.pone.0078521.g002Figure 2 Detection of NDV-expressed Env proteins present in infected-cell lysates (A) and the cell culture medium (B). DF1 cells were infected with indicated viruses at an MOI of 0.01 PFU. After 48 h, the cell culture medium supernatants and cells were collected and processed. (A) The cell lysates were prepared from cells and subjected to SDS-PAGE under reducing conditions. (B) The cell culture medium supernatants were concentrated 10x by passing through Amicon filters and subjected to SDS-PAGE under reducing conditions. The gels were analyzed by Western blotting using a pool of gp120-specific monoclonal antibodies. The positions of HIV-1 gp160, gp140 precursor gp120 are indicated by arrows in the right margin. Molecular masses of marker proteins (in kilodaltons) are shown in the left margin. 10.1371/journal.pone.0078521.g003Figure 3 Immunofluorescence of Env protein expressed in Vero cells infected with rLaSota/gp160 (panel a), rLaSota/gp140L (panel b), rLaSota/gp140S (panel c), rLaSota/gp120 (panel d) and rLaSota (panel e) at an MOI of 0.1 PFU. Twenty-four h post-infection, the infected cells were fixed with paraformadehyde and permeabilized with Triton X-100 for detection of total antigen inside the cell. The cells were probed with a pool of gp120-specific monoclonal antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies, and analyzed by immunofluorescence. The cells were visualized under Nikon Eclipse TE fluorescent microscope. Arrows indicate areas of positive immunofluorescence. The oligomeric state of the Env proteins in DF1 cells infected with rLaSota/gp140L, rLaSota/gp140S, rLaSota/gp120, and rLaSota/gp160 was investigated by preparing infected cell lysates and subjecting them to cross-linking with the thiol-cleavable cross-linker DSP, followed by SDS-PAGE under reducing and non-reducing conditions and immunoblotting with gp120-specific monoclonal antibodies (Figure 4). Cross-linked Env protein expressed by rLaSota/gp160 migrated under non-reducing conditions as a broad oligomeric band of molecular mass greater than 188 kDa, as we previously described [27]. Under non-reducing conditions, cross-linked Env protein expressed by rLasota/gp140S migrated as a diffuse band of higher molecular weight, corresponding to higher order oligomeric forms. Unexpectedly, very little Env protein oligomers could be detected in rLasota/gp140L-infected cells. The bulk of cross-linked gp120 expressed by rLaSota gp120 migrated under non-reducing conditions as higher order oligomers. These data suggest that rLaSota/gp140S and rLaSota/gp120, similar to rLaSota/gp160, support the expression of one predominant oligomeric species of molecular mass greater than 188 kDa, probably representing dimers and or trimers. 10.1371/journal.pone.0078521.g004Figure 4 Oligomeric status of NDV-expressed Env proteins. DF1 cells were infected with indicated viruses at an MOI of 0.01 PFU. After 48 h, the cells were collected and cross-linked with DSP and subjected to SDS-PAGE under reducing (+R) or non-reducing (-R) conditions. The gels were analyzed by Western blotting using a pool of gp120-specific monoclonal antibodies. The positions of HIV-1 gp160, gp140 precursor gp120 are indicated by arrows in the right margin. Molecular masses of marker proteins (in kilodaltons) are shown in the left margin. Biological characterization of rNDVs expressing gp140, 120 and 160 proteins The multicycle growth kinetics of rLaSota/gp140L, rLaSota/gp140S rLaSota/gp120, rLaSota/gp160 in DF1 cells showed that the replication of these recombinants was essentially indistinguishable from that of parental rLaSota virus (Figure 5). The pathogenicity of these recombinants and their parental rLaSota virus was evaluated in 9-day-old embryonated chicken eggs by the MDT (mean death time) test. The values of MDT for rLaSota, rLaSota/gp140L, rLaSota/gp140S, rLaSota/gp120 and rLaSota/gp160 were 105 h, 107 h, 107 h, 106 h and 109 h, respectively. This showed that the insertion of the foreign gene conferred a marginal amount of attenuation to the NDV vector. 10.1371/journal.pone.0078521.g005Figure 5 Comparison of multicycle growth kinetics of rLaSota/gp160 (panel A), rLaSota/gp140L (panel B), rLaSota/gp140S (panel C), rLaSota/gp120 (panel D) viruses, each compared with the empty vector rLaSota virus. DF1 cells were infected with each virus at an MOI of 0.01 and cell culture media supernatant aliquots were harvested at 8 h intervals until 64 h post-infection. The virus titers in the aliquots were determined by plaque assay in DF1 cells. Values are averages from three independent experiments. Infection of guinea pigs To evaluate the immunogenicity of Env protein expressed by the various NDV recombinants, we immunized outbred female Hartley guinea pigs (n=6 for groups immunized with rLaSota/gp140L, rLaSota/gp140S, rLaSota/gp120 and rLaSota/gp160 and n=3 for the empty vector rLaSota control) on days 0 and 14 (Figure 6). Each animal received a dose of 300 µl (150 µl in each nostril) of allantoic fluid containing 106 PFU/ml of recombinant virus. We chose the i.n. route because it elicited the highest immune responses in our previous study with rNDV expressing gp160 protein [27]. The animals did not show any overt clinical signs of infection or any loss of body weight throughout the study. However, three animals each from the rLaSota/gp160 and rLaSota/gp140L groups and one animal from the rLaSota/gp120 group died due to physical injury (bone fracture) during captivity that was unrelated to the immunizations. Post-mortem analysis of different tissues such as lungs, trachea, spleen and brain revealed no lesions and no virus was isolated from these tissues. 10.1371/journal.pone.0078521.g006Figure 6 Immunization schedules. A. Guinea pig immunization. Twenty seven guinea pigs were divided into 5 groups (n=6 for rLaSota/gp160, rLaSota/gp140L, rLaSota/gp140S, rLaSota/gp120 groups; n=3 for rLaSota group). Animals in each group were immunized with two doses of each recombinant virus on days 0 and 14 by i.n. route of administration. Each dose consisted of 300 µl (150 µl in each nostril) of allantoic fluid containing 106 PFU/ml of virus. Blood, vaginal washes and fecal samples were collected on days 0, 7, 14, 21, 28, 42, 56, 70 and 90. All animals were sacrificed on day 90. B. Mice immunization. Thirty mice were divided in to 5 groups (n=6/group). Animals in each group were immunized with two doses of virus on days 0 and 14 by the i.n. route of administration. Each dose consisted of 50 µl (25 µl in each nostril) of allantoic fluid containing 105 PFU/ml of virus. All animals were sacrificed on day 56 and splenocytes were collected. Humoral immune responses The induction of NDV-specific serum antibodies was measured on days 28 and 56 using an NDV-specific ELISA (Figure 7A). All four animal groups exhibited high levels of NDV-specific IgG antibodies on these days, suggesting that each of the viruses replicated to same extent in the immunized animals. 10.1371/journal.pone.0078521.g007Figure 7 NDV-specific total IgG (panel A), and HIV-1 gp120-specific total IgG (panel B), IgG1 (panel C) and IgG2 (panel D) responses in guinea pig sera. The guinea pigs were immunized with the indicated rNDVs by the i.n. route. (A) The guinea pig sera were analyzed for NDV-specific antibodies by commercial NDV ELISA kits (Synbiotics Corporation). Mean ELISA end-point titers of NDV-specific serum antibodies on days 28 and 56 are shown. (B-D) The guine pig sera were analyzed by HIV-1 gp120 specific total IgG, IgG1 and IgG2 antibodies by isotype-specific ELISA with purified gp120. Mean ELISA end-point titers of gp120-binding serum antibodies of the indicated isotype on days 0, 7, 14, 21, 28, 42, 56, 70 and 90 are shown. Antibodies specific to gp120 were not detected in any animal on any day in the control rLaSota group. The graph shows the geometric mean value ± SEM for 3 animals in rLaSota, rLaSota/gp160 and rLaSota/gp140L groups, 6 animals in rLaSota/gp140S group and 5 animals in rLaSota/gp120 group. Arrows indicate time of rNDV immunizations on days 0 and 14. Statistical differences between the groups were calculated by unpaired t test (two-tailed). 1* indicates statistically significant differences (P<0.05) of rLaSota/gp140S vs. rLaSota/gp160, rLaSota/gp140L and rLaSota/gp120 groups. 2* indicates statistically significant differences (P<0.05) of rLaSota/gp140S vs. rLaSota/gp160 and rLaSota/gp140L groups. The induction of HIV-1 Env-specific serum antibodies was measured on days 7, 14, 21, 28, 42, 56, 70 and 90. Total serum IgG specific to BaL.1 gp120 was measured at each time point by ELISA (Figure 7B). Responses were detected on day 21 following the initial immunization in all of the groups. The boost on day 14 was followed by increased immune responses in all the groups. The highest gp120-spectific total IgG titer was observed with the rLaSota/gp140S group, followed by the rLasota/gp160 and rLaSota/gp120 groups, and the lowest titer was observed with rLaSota/140L. On day 42, rLaSota/gp140S group showed significantly higher titer compared to all the other groups (P<0.0001 for gp140S versus gp160 and gp40L groups, P=0.0008 for gp140S versus gp120 group) and on day 56, it showed significantly higher titer compared to rLaSota/gp160 and rLaSota/gp140L groups (P<0.05). In addition, serum IgG1 and IgG2a responses specific to BaL.1 gp120 were measured by isotype-specific ELISA (Figure 7C and D). Responses were detected on day 21 after the first immunization in all of the groups, and responses peaked by day 42. The IgG1 response was strongest in rLaSota/gp140S group followed by rLaSota/gp120, rLaSota/gp140L and rLaSota/gp160 groups. On day 42, rLaSota/gp140S group showed significantly higher titer compared to all the other groups (P<0.05), whereas on days 28, 56, 70 and 90, the IgG1 titer induced by rLaSota/gp140S was significantly higher compared to rLaSota/gp160 and rLaSota/gp140L (P<0.05). The IgG2 response also was strongest in rLaSota/gp140S group followed by rLaSota/gp120, rLaSota/gp160, and rLaSota/gp140L. On days 28 and 42, the IgG2 response induced by rLaSota/gp140S was significantly higher compared to rLaSota/gp160 and rLaSota/gp140L (P<0.05). We also calculated the ratio of serum IgG1:IgG2a to assess the Th1/Th2 balance. The ratio for rLaSota/gp160 was 1:3 on day 28 and 1:8 on day 90, indicative of a Th1-biased response. In contrast, the ratios for rLaSota/gp140L, rLaSota/gp140S, and rLaSota/gp120 varied from 1:0.7 to 1:1.4 on days 28 to 90 post-immunization and thus showed mixed Th1/Th2 responses. In addition, we assayed gp120-specific serum IgA in all the groups, but all animals at all-time points were negative. Mucosal immune responses Vaginal washes were collected from each animal at each time point and evaluated by ELISA using BaL.1 gp120-coated plates (Figure 8). In all the groups, the titer of total IgG peaked on day 42, decreased by day 56-70, and surprisingly peaked again on day 90 (Figure 8A). The response was greatest in the rLaSota/gp140S group followed by rLaSota/gp120, rLaSota/gp160, and rLaSota/gp140L. On day 56, the response induced by rLaSota/gp140S was significantly higher compared to rLaSota/gp160 and rLaSota/gp140L (P<0.05). We assayed the total IgG response in fecal samples in all of the groups but unable to detect any titer. We also analyzed the IgG1 and IgG2a responses in vaginal washes in all the groups (Figure 8B and C). Similar to total vaginal IgG response, the IgG1 and IgG2a responses peaked on day 42, decreased on day 56-70 and increased on day 90. The IgG1and IgG2a responses were strongest with rLaSota/gp140S followed by rLaSota/gp20, rLaSota/gp160, and rLaSota/gp140L. On day 42, the IgG1 response induced in rLaSota/gp140S group was significantly higher compared to rLaSota/gp160 rLaSota/gp140L groups (P<0.05), whereas on day 56, it was significantly higher in this group compared to all the other groups (P<0.05). Also the IgG2a response induced on day 56 in rLaSota/gp140S group was significantly higher compared to rLaSota/gp160 and rLaSota/gp140L groups (P<0.05). The vaginal IgG1:IgG2a ratio varied from 1: 3 to 1:7 at different time points for the rLaSota/gp160 group, indicative of a Th1-biased response. In the other groups, the vaginal Th1:Th2a ratio varied at different time points from 1:0.7 to 1:4.5, 1:0.6 to 1:2.3 and 1:1 to 1:4 for rLaSota/gp140L, rLaSota/gp140S, and rLaSota/gp120, indicative of a mixed Th1/Th2a response. BaL.1 gp120-specific IgA responses in vaginal washes also were measured (Figure 8D). A low titer was detected in the all the groups. The titer peaked on day 42, dropped on day 56 and increased slightly on day 90. The strongest response was detected in the rLaSota/gp140S group, followed by rLaSota/gp160, rLaSota/gp120, and rLaSota/gp140L. 10.1371/journal.pone.0078521.g008Figure 8 HIV-1 gp120-specific total IgG (panel A), IgG1 (panel B), IgG2a (panel C) and IgA (panel D) antibodies in vaginal washes collected from guinea pigs, detected by isotype-specific ELISA with purified gp120. The guinea pigs were immunized with the indicated rNDVs by the i.n. route. Mean ELISA end-point titers of gp120-binding vaginal wash antibodies of the indicated isotype on days 0, 7, 14, 21, 28, 42, 56, 70 and 90 are shown. Antibodies specific to gp120 were not detected in any animal on any day in control rLaSota group. The graph shows the geometric mean value ± SEM for 3 animals in rLaSota, rLaSota/gp160 and rLaSota/gp140L groups, 6 animals in rLaSota/gp140S group and 5 animals in rLaSota/gp120 group. Arrows indicate time of rNDV immunizations on days 0 and 14. Statistical differences between the groups were calculated by unpaired t test (two-tailed). 1* indicates statistically significant differences (P<0.05) of rLaSota/gp140S vs. rLaSota/gp160, rLaSota/gp140L and rLaSota/gp120 groups. 2* indicates statistically significant differences (P<0.05) of rLaSota/gp140S vs. rLaSota/gp160 and rLaSota/gp140L groups. Neutralizing antibody (NAb) responses Sera from days 28, 56, 70 and 90 from animals immunized with the rNDVs expressing gp140, gp120 and gp160 were evaluated in the TZM bl assay (which assays HIV-1 infection by measuring Tat-regulated luciferase expression in an indicator cell line) for the ability to neutralize homologous clade B tier 1 HIV-1 strains BaL.26 and MN.3 and heterologous clade B tier 2 HIV-1 strains RHPA4259.7 and TRO.11. NAb activity (expressed as IC50 value) against HIV-1 strain Bal.26 was detected in sera from all of the animals immunized with NDV expressing the various Env-derived proteins, with the highest titer observed with rLaSota/gp140S followed by rLaSota/gp120, rLaSota/gp160, and rLaSota/gp140L (Figure 9A). On days 28, 56, 70 and 90, the mean IC50 titers induced by rLaSota/gp140S was higher than rLaSota/gp160 and rLaSota/gp140L but it was significantly higher particularly on days 28 and 70 (P<0.05). Compared to rLaSota/gp120 group, NAb activity induced in rLaSota/gp140S group was also higher on all the days but it was significantly higher on day 28 (P = 0.002). A comparison of mean IC50 titres against HIV-1 strain MN.3 among all the groups on days 28 and 56 showed a significantly stronger NAb responses in rLaSota/gp140S group (P<0.05) [Figure 9B]. On day 70, NAb response was also significantly stronger in rLaSota/gp140S group compared to rLaSota/gp160 and rLaSota/gp140L groups. These data indicated that NAb response induced by rLaSota/gp140S against homologous clade B tier 1 HIV-1 strains BaL.26 and MN.3 was significantly stronger compared to other recombinant viruses early on but the differences diminished over time particularly for BaL.26 strain. Further, analysis of sera of day 56 of all the groups indicated a weak neutralization response against the clade B tier 2 viruses RHPA4259.7 and TRO.11 in TZM bl assay (Figure 9C). We then used more sensitive A3R5 assay to look for neutralization of Clade B tier 2 HIV-1 strains SC22.3C2.LucR. T2A.ecto and REJO. LucR. T2A.ecto. As shown in Figure 9C, NAb activity induced against SC22.3C2.LucR. T2A.ecto by rLaSota/gp140S was higher compared to other groups and was significantly greater compared to rLaSota/gp140L and rLaSota/gp120 groups (P<0.05). No response was observed against REJO. LucR. T2A.ecto virus (data not shown). 10.1371/journal.pone.0078521.g009Figure 9 Virus neutralizing antibody activity (50%-inhibitory-concentration [IC50] titers) against homologous HIV-1 clade B tier 1 strains (BaL.26 and MN.3) and heterologous clade B tier 2 strains (RHPA4257.7, TRO.11, and SC22.3C2.LucR. T2A.ecto) in sera from guinea pigs immunized with the indicated rNDVs. (A) Guinea pig sera obtained on days 28, 56, 70 and 90 were tested against BaL.26 pseudovirus by the TZM-bl assay (B) Guinea pig sera obtained on days 28, 56, 70 and 90 were tested against MN.3 pseudovirus by the TZM-bl assay (C) Guinea pig sera obtained on day 56 were tested against RHPA4257.7 and TRO.11 by the TZM-bl assay, and against SC22.3C2.LucR. T2A.ecto by the A3R5 assay. Horizontal bars indicate the geometric mean titer. Pre-immune sera were used to establish baseline-neutralizing activity in each individual guinea pig, and these values were subtracted from the values shown. The neutralizing antibody activity against each virus in sera obtained from guinea pigs immunized with rLaSota virus was <20. Statistical differences between the groups were calculated by unpaired t test (two-tailed) and shown by the numbers underneath the horizontal line. * indicates statistically significant differences (P<0.05) between groups. Cellular Immune Responses The ability of rNDVs to stimulate cellular immune responses against HIV-1 Env was evaluated in female BALB/c mice (n=6/group). This model was used because of the availability of immunological reagents. Mice were inoculated i.n. on days 0 and 14 with the various rNDVs expressing gp140S, gp140L, gp120 and gp160, and on day 56 splenocytes were isolated (Figure 6B). The splenocytes were stimulated in vitro with Env peptides, and processed for intracellular cytokine staining of IFN-γ and staining for CD4 or CD8 (Figure 10). A significant number of IFN-γ-producing CD4+ T cells and CD8+ T cells were detected, and were higher in the mice that received rLaSota/gp140S and rLaSota/gp120 than for rLaSota/gp160 and rLaSota/gp140L. Mice immunized with rLaSota/gp140S and rLaSota/gp120 demonstrated 9- and 5- fold increases in IFN-γ+CD4+ T cells compared to rLaSota/gp160 (Figure 10A). The number of IFN-γ+CD8+ T cells was higher with rLaSota/gp120, rLaSota/gp140S and rLaSota/gp140L compared to rLaSota/gp160 (Figure 10B) Collectively, these results demonstrate that administration of rNDV expressing gp140S can induce robust CD4+ and CD8+ T cell responses in addition to humoral and mucosal immune responses. 10.1371/journal.pone.0078521.g010Figure 10 HIV-1 Env-specific CD4+ (panel A) and CD8+ (panel B) T cell response. Mice in groups of 6 were immunized with 105 PFU/ml of the indicated rNDV by the i.n. route on days 0 and 14. On day 56, splenocytes were isolated, stimulated with a pool of overlapping Env peptides, and processed for intracellular cytokine staining for IFN-γ and CD4 and CD8. Discussion Recently, the modest success of the recent phase III RV144 trial, employing a prime-boost vaccine regime with a recombinant canarypox vector and purified recombinant gp120, emphasized that Env-based immunogens are important HIV-1 vaccine candidates [36]. It is important to further explore the immunogenicity of different structural variants of the Env glycoprotein. In addition, the optimal means to present Env antigen to the immune system requires continued investigation, particularly presentation by live viral vectors. Different Env-based immunogens including gp120 and gp140 have been shown to induce variable humoral and CTL responses in animal models. The results of clinical trials indicated that soluble gp120 elicits antibodies of narrow neutralization specificities that are unable to neutralize primary isolates [37-39]. A critical deficiency of gp120 vaccines is the absence of epitopes that are present in relatively conserved gp41 regions such as MPER. Therefore, gp160 was considered to be a better immunogen than gp120. A previous study showed that gp160 is cytotoxic but that truncation of its C-terminus to 140 kDa, by removal of the transmembrane and cytoplasmic domains, could remove its cytotoxicity [40]. Thereafter, more attention has been focused on generation of versions such as gp140 because it contains the entire ectodomain of HIV-1 Env, has the potential to form trimers indicative of an intact authentic conformation, and can be secreted from expressing cells. To stabilize recombinant gp140 trimers and to elicit a better immune response against HIV-1, various strategies have been employed, such as engineering a cleavage-deficient form produced by deletion of the furin cleavage site, or introduction of disulfide bonds to covalently link gp120 and gp41, or incorporation of trimerization motifs into the gp41 ectodomain [11]. In the present study, we used a live, replication-competent NDV vector to express and compare several forms of Env. Each Env ORF was engineered to be under the control of NDV transcription signals in the NDV genome and to be expressed as a separate mRNA. This strategy would provide for de novo synthesis of correctly folded and processed trimers in vivo. We retained the furin cleavage site on the premise that native cleavage might be important to the correct conformation. We generated two forms of 140, namely gp140L and gp140S that contained complete or partial MPER, respectively, and compared these to gp120 and gp160. The NDV vector was based on the LaSota strain, which is a naturally-occurring avirulent strain that is commonly used as a live NDV vaccine in chickens and thus poses no agricultural concerns. Initially, we investigated whether rNDV could efficiently express correctly folded gp140 and gp120 molecules and whether these molecules could be cleaved, assemble to form oligomers, and be secreted. NDV-expressed gp160, gp140L, and gp140S were shown to be cleaved, although cleavage of gp140L was somewhat less efficient. NDV-expressed gp120 and gp140S were efficiently secreted, whereas secretion of gp140L was much less efficient, and gp160 was not secreted consistent with its membrane-bound status. Chemical cross-linking and gel electrophoresis showed that gp160, gp140S, and gp120 efficiently formed conformationally intact higher-order oligomers, whereas gp140L did not. The low level of expression, cleavage, oligomer formation, and secretion by gp140L suggested that either this truncation mutant might have a defect in folding or the protein produced by this mutant could be unstable and rapidly degraded. Previously, we have shown the potential of rNDV as a vaccine vector to deliver HIV-1 gp160 protein by the i.n. route in guinea pigs [27]. In the present study, we immunized guinea pigs with two doses of the various NDV constructs by the i.n. route and evaluated the resulting serum and mucosal antibodies for the ability to bind to purified gp120 in ELISA and to neutralize homologous and heterologous strains of HIV-1. Our results showed that the rLaSota/gp140S group exhibited higher humoral and mucosal immune responses compared with the other groups. In addition, the immune responses elicited by rLaSota/140S and rLaSota/120 groups were qualitatively different in terms of antibody isotypes compared to responses elicited by rLaSota/160 group. Whereas, similar to our previous studies, rLaSota/160 induced a Th1-biased response (lower IgG1:IgG2a ratio), rLaSota/gp140S and rLaSota/120 induced mixed Th1/Th2 responses (higher IgG1:IgG2a ratio). Further, the neutralization responses elicited against the homologous strain BaL.26 and the laboratory adapted heterologous strain MN.3 differed among the various constructs and showed the following rank order: rLaSota/140S>rLaSota120>rLaSota160 >rLaSota140L. The lower IgG1 and neutralizing antibody responses for rLaSota/gp160 compared to rLaSota/gp140S and rLaSota/gp120 could be due to several reason: i) the transmembrane and cytoplasmic domains present in gp160 might influence the conformation of gp120, ii) some non-neutralization epitopes might be present on these domains, and the immune responses generated against these epitopes might create hindrance to neutralizing antibodies, iii) previous studies indicated that the cytoplasmic domain of gp41 contains some down regulatory sequences that could affect cell surface expression of gp160 [41,42], and (iv) the ability of gp140S and gp120 to be both cell-associated and secreted may provide increased immunogenicity. The finding that the immune responses elicited by NDV-expressed gp140S was superior to that of gp120 and gp160 supports the concept that trimeric gp140 envelope glycoprotein in a soluble form is a more efficient Env immunogen [43-46]. In the present study, we found that immunization of guinea pigs and mice with rLaSota/gp140L resulted in humoral, mucosal and cellular immune responses that are lower than those induced by rLaSota/gp140S. The gp41 sequence of rLaSota/gp140L is 16 aa longer and contains the complete 30-aa long MPER compared to shorter MPER sequence of gp41 present in rLaSota/gp140S. The lower level of immunogenicity induced by rLaSota/gp140L compared to rLaSota/gp140S could be due to a lower level of gp140L expression, inefficient cleavage, oligomerization or secretion. It is also possible that the presence of complete C-terminal MPER region in gp140L exerted immune suppression due to mimicry of this region to the self-protein cardiolipin [47]. It was shown earlier that the MPER region is weakly immunogenic when it was presented to the immune system on particulate Hepatititis B surface antigen particles [48]. However, recently analysis of human monoclonal antibody 10E8, isolated from an HIV-1 infected individual with high neutralization titers, demonstrated that the breadth of neutralizing antibody response of this antibody is mediated by recognition of MPER [49]. The differences in the immunogenicity between rLaSota/gp140L and rLaSota/gp140S warrant further investigation. We have demonstrated that rNDV expressing HIV Env protein is able to induce a potent humoral immune response against HIV [27]. However, several studies including recently concluded RV144 trial in Thailand suggested that in addition to Env specific antibodies, CD4+ T cell response are important for protection against HIV-1 [36,50]. Studies in Elite controller and in macaque models indicated that in addition to CD4+ T cells, CD8+ T cells appear to play a key role in control of viral replication and level of set point viral load [51-53] Little information is available regarding the induction of T cell responses against foreign proteins expressed by NDV-based vectors. In this study we evaluated rNDV expressing HIV Env proteins to induce HIV-1 specific CD4+ and CD8+ T cells in mice. Inoculation of mice with rNDV expressing different forms of HIV Env resulted in both CD4+ and CD8+ T cell immune responses against HIV Env. rLaSota/gp140S and rLaSota/gp120 produced higher T cell responses compared to other recombinants. These results have indicated that NDV is promising vector for inducing T cell responses against HIV Env. A major limitation of virus-based vectors is interference by anti-vector antibodies [54,55]. As noted, NDV has the advantage of being antigenically distinct from other common human pathogens, and thus its use should be unaffected by antibodies commonly present in the human population. Vector-specific antibodies also arise following the first vaccine administration, and these may interfere with a subsequent application of the same vaccine [56]. However, in the present study we found that NDV vectors can elevate the level of humoral and mucosal responses to the transgene following a booster vaccination. We think that NDV can re-infect because its low level of replication does not allow for the induction of solid immunity after only one inoculation. It also is possible that NDV is unusually immunogenic as it is a potent inducer of interferon and dendritic cell maturation and so even a low level of replication is very immunogenic. There was a decrease in IgG titer in vaginal washes on day 56 or 70 and surprisingly, it increased again on day 90. A possible explanation for this increase could be the production of long lived plasma cells from bone marrow [57]. In summary, we evaluated the potential of NDV strain LaSota as a vaccine vector for expressing different forms of the HIV-1 Env protein. In particular, we compared the humoral and mucosal immune responses in guinea pigs and cellular immune responses in mice induced by rNDVs expressing gp140S, gp140L, gp120 and gp160. Our results showed that rNDV is a promising vector that can induce mucosal, humoral and cellular immune responses against HIV-1 Env proteins. We evaluated two forms of gp140 expressed by rNDV and, surprisingly, found that deletion of part of MPER resulted in increased immunogenicity. The immune responses elicited by gp140S oligomers expressed by rNDV were several fold higher compared to those induced by rNDVs expressing gp120 and gp160 oligomers. Further, the ability of rLaSota/gp140S to induce neutralizing antibody responses against homologous BaL.26 and laboratory adapted MN.3 was superior to rLaSota/gp120 and rLaSota/gp160. We conclude that rNDV expressing soluble gp140 oligomers lacking the complete MPER produces strong mucosal, humoral and cellular immune responses that warrants further investigation in nonhuman primates. We thank Yunsheng Wang, Daniel Rockemann, and other lab members for their technical assistance and help. We thank Girmay Gebreluul and Yonas Araya for their help with handling of guinea pigs. ==== Refs References 1 Baba TW , Liska V , Hofmann-Lehmann R , Vlasak J , Xu W et al. (2000 ) Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection . Nat Med 6 : 200 -206 . doi:10.1038/72309. PubMed: 10655110.10655110 2 Mascola JR , Stiegler G , VanCott TC , Katinger H , Carpenter CB et al. (2000 ) Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies . Nat Med 6 : 207 -210 . doi:10.1038/72318. PubMed: 10655111.10655111 3 Balazs AB , Chen J , Hong CM , Rao DS , Yang L et al. (2012 ) Antibody-based protection against HIV infection by vectored immunoprophylaxis . Nature 481 : 81 -84 . doi:10.1038/nature10660. PubMed: 22139420. 4 Barouch DH , Liu J , Peter L , Abbink P , Iampietro MJ et al. (2013 ) Characterization of Humoral and Cellular Immune Responses Elicited by a Recombinant Adenovirus Serotype 26 HIV-1 Env Vaccine in Healthy Adults (IPCAVD 001) . J Infect Dis 207 : 248 -256 . doi:10.1093/infdis/jis671. PubMed: 23125443.23125443 5 de Souza MS , Ratto-Kim S , Chuenarom W , Schuetz A , Chantakulkij S et al. (2012 ) The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope . J Immunol 188 : 5166 -5176 . doi:10.4049/jimmunol.1102756. PubMed: 22529301.22529301 6 Franzusoff A , Volpe AM , Josse D , Pichuantes S , Wolf JR (1995 ) Biochemical and genetic definition of the cellular protease required for HIV-1 gp160 processing . J Biol Chem 270 : 3154 -3159 . doi:10.1074/jbc.270.7.3154. PubMed: 7852398.7852398 7 Decroly E , Vandenbranden M , Ruysschaert JM , Cogniaux J , Jacob GS et al. (1994 ) The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-1 SU) and gp41 (HIV-I TM) . J Biol Chem 269 : 12240 -12247 . PubMed: 8163529.8163529 8 Helseth E , Olshevsky U , Furman C , Sodroski J (1991 ) Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein . J Virol 65 : 2119 -2123 . PubMed: 2002555.2002555 9 Pierson TC , Doms RW (2003 ) HIV-1 entry and its inhibition . Curr Top Microbiol Immunol 281 : 1 -27 . doi:10.1007/978-3-642-19012-4_1. PubMed: 12932074.12932074 10 Robert-Guroff M (2007 ) Replicating and non-replicating viral vectors for vaccine development . Curr Opin Biotechnol 18 : 546 -556 . doi:10.1016/j.copbio.2007.10.010. PubMed: 18063357.18063357 11 Phogat S , Wyatt R (2007 ) Rational modifications of HIV-1 envelope glycoproteins for immunogen design . Curr Pharm Des 13 : 213 -227 . doi:10.2174/138161207779313632. PubMed: 17269929.17269929 12 Pitisuttithum P , Gilbert P , Gurwith M , Heyward W , Martin M et al. (2006 ) Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand . J Infect Dis 194 : 1661 -1671 . doi:10.1086/508748. PubMed: 17109337.17109337 13 Burton DR , Desrosiers RC , Doms RW , Koff WC , Kwong PD et al. (2004 ) HIV vaccine design and the neutralizing antibody problem . Nat Immunol 5 : 233 -236 . doi:10.1038/ni0304-233. PubMed: 14985706.14985706 14 Schülke N , Vesanen MS , Sanders RW , Zhu P , Lu M et al. (2002 ) Oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein . J Virol 76 : 7760 -7776 . doi:10.1128/JVI.76.15.7760-7776.2002. PubMed: 12097589.12097589 15 Srivastava IK , Stamatatos L , Kan E , Vajdy M , Lian Y et al. (2003 ) Purification, characterization, and immunogenicity of a soluble trimeric envelope protein containing a partial deletion of the V2 loop derived from SF162, an R5-tropic human immunodeficiency virus type 1 isolate . J Virol 77 : 11244 -11259 . doi:10.1128/JVI.77.20.11244-11259.2003. PubMed: 14512572.14512572 16 Yang X , Lee J , Mahony EM , Kwong PD , Wyatt R et al. (2002 ) Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin . J Virol 76 : 4634 -4642 . doi:10.1128/JVI.76.9.4634-4642.2002. PubMed: 11932429.11932429 17 Zhang PF , Cham F , Dong M , Choudhary A , Bouma P et al. (2007 ) Extensively cross-reactive anti-HIV-1 neutralizing antibodies induced by gp140 immunization . Proc Natl Acad Sci U S A 104 : 10193 -10198 . doi:10.1073/pnas.0608635104. PubMed: 17540729.17540729 18 Binley JM , Sanders RW , Clas B , Schuelke N , Master A et al. (2000 ) A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure . J Virol 74 : 627 -643 . doi:10.1128/JVI.74.2.627-643.2000. PubMed: 10623724.10623724 19 Du SX , Idiart RJ , Mariano EB , Chen H , Jiang P et al. (2009 ) Effect of trimerization motifs on quaternary structure, antigenicity, and immunogenicity of a noncleavable HIV-1 gp140 envelope glycoprotein . Virology 395 : 33 -44 . doi:10.1016/j.virol.2009.07.042. PubMed: 19815247.19815247 20 Krishnamurthy S , Samal SK (1998 ) Nucleotide sequences of the trailer, nucleocapsid protein gene and intergenic regions of Newcastle disease virus strain Beaudette C and completion of the entire genome sequence . J Gen Virol 79 (Pt 10): 2419 -2424 . PubMed: 9780047.9780047 21 Bukreyev A , Huang Z , Yang L , Elankumaran S , St Claire M et al. (2005 ) Recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates . J Virol 79 : 13275 -13284 . doi:10.1128/JVI.79.21.13275-13284.2005. PubMed: 16227250.16227250 22 DiNapoli JM , Kotelkin A , Yang L , Elankumaran S , Murphy BR et al. (2007 ) Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens . Proc Natl Acad Sci U S A 104 : 9788 -9793 . doi:10.1073/pnas.0703584104. PubMed: 17535926.17535926 23 DiNapoli JM , Yang L , Samal SK , Murphy BR , Collins PL et al. (2010 ) Respiratory tract immunization of non-human primates with a Newcastle disease virus-vectored vaccine candidate against Ebola virus elicits a neutralizing antibody response . Vaccine 29 : 17 -25 . doi:10.1016/j.vaccine.2010.10.024. PubMed: 21034822.21034822 24 DiNapoli JM , Nayak B , Yang L , Finneyfrock BW , Cook A et al. (2010 ) Newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of H5N1 highly pathogenic avian influenza virus protect against virus challenge in monkeys . J Virol 84 : 1489 -1503 . doi:10.1128/JVI.01946-09. PubMed: 19923177.19923177 25 Carnero E , Li W , Borderia AV , Moltedo B , Moran T et al. (2009 ) Optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses . J Virol 83 : 584 -597 . doi:10.1128/JVI.01443-08. PubMed: 19004953.19004953 26 Maamary J , Array F , Gao Q , García-Sastre A , Steinman RM et al. (2011 ) Newcastle disease virus expressing a dendritic cell-targeted HIV gag protein induces a potent gag-specific immune response in mice . J Virol 85 : 2235 -2246 . doi:10.1128/JVI.02036-10. PubMed: 21159873.21159873 27 Khattar SK , Samal S , Devico AL , Collins PL , Samal SK (2011 ) Newcastle disease virus expressing human immunodeficiency virus type 1 envelope glycoprotein induces strong mucosal and serum antibody responses in Guinea pigs . J Virol 85 : 10529 -10541 . PubMed: 21849467.21849467 28 Huang Z , Krishnamurthy S , Panda A , Samal SK (2001 ) High-level expression of a foreign gene from the most 3'-proximal locus of a recombinant Newcastle disease virus . J Gen Virol 82 : 1729 -1736 . PubMed: 11413385.11413385 29 Huang Z , Krishnamurthy S , Panda A , Samal SK (2003 ) Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist . J Virol 77 : 8676 -8685 . doi:10.1128/JVI.77.16.8676-8685.2003. PubMed: 12885886.12885886 30 Krishnamurthy S , Huang Z , Samal SK (2000 ) Recovery of a virulent strain of newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation . Virology 278 : 168 -182 . doi:10.1006/viro.2000.0618. PubMed: 11112492.11112492 31 Alexander DJ (1997 ) Newcastle disease and other avian Paramyxoviridae infection . Ames, IA : Iowa State University Press . 32 Li M , Gao F , Mascola JR , Stamatatos L , Polonis VR et al. (2005 ) Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies . J Virol 79 : 10108 -10125 . doi:10.1128/JVI.79.16.10108-10125.2005. PubMed: 16051804.16051804 33 Folks T , Benn S , Rabson A , Theodore T , Hoggan MD et al. (1985 ) Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus . Proc Natl Acad Sci U S A 82 : 4539 -4543 . doi:10.1073/pnas.82.13.4539. PubMed: 2989831.2989831 34 Kim JH , Pitisuttithum P , Kamboonruang C , Chuenchitra T , Mascola J et al. (2003 ) Specific antibody responses to vaccination with bivalent CM235/SF2 gp120: detection of homologous and heterologous neutralizing antibody to subtype E (CRF01.AE) HIV type 1. AIDS Res Hum Retroviruses 19: 807-816 35 Montefiori DC , Karnasuta C , Huang Y , Ahmed H , Gilbert P et al. (2012 ) Magnitude and breadth of the neutralizing antibody response in the RV144 and Vax003 HIV-1 vaccine efficacy trials . J Infect Dis 206 : 431 -441 . doi:10.1093/infdis/jis367. PubMed: 22634875.22634875 36 Rerks-Ngarm S , Pitisuttithum P , Nitayaphan S , Kaewkungwal J , Chiu J et al. (2009 ) Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand . N Engl J Med 361 : 2209 -2220 . doi:10.1056/NEJMoa0908492. PubMed: 19843557.19843557 37 Graham BS , McElrath MJ , Connor RI , Schwartz DH , Gorse GJ et al. (1998 ) Analysis of intercurrent human immunodeficiency virus type 1 infections in phase I and II trials of candidate AIDS vaccines. AIDS Vaccine Evaluation Group, and the Correlates of HIV Immune Protection Group . J Infect Dis 177 : 310 -319 . doi:10.1086/514209. PubMed: 9466516.9466516 38 Mascola JR , Snyder SW , Weislow OS , Belay SM , Belshe RB et al. (1996 ) Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group . J Infect Dis 173 : 340 -348 . doi:10.1093/infdis/173.2.340. PubMed: 8568294.8568294 39 Matthews TJ (1994 ) Dilemma of neutralization resistance of HIV-1 field isolates and vaccine development . AIDS Res Hum Retrovir 10 : 631 -632 . doi:10.1089/aid.1994.10.631. PubMed: 8074926.8074926 40 Chakrabarti BK , Kong WP , Wu BY , Yang ZY , Friborg J et al. (2002 ) Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization . J Virol 76 : 5357 -5368 . doi:10.1128/JVI.76.11.5357-5368.2002. PubMed: 11991964.11991964 41 Bültmann A , Muranyi W , Seed B , Haas J (2001 ) Identification of two sequences in the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein that inhibit cell surface expression . J Virol 75 : 5263 -5276 . doi:10.1128/JVI.75.11.5263-5276.2001. PubMed: 11333908.11333908 42 Bu Z , Ye L , Vzorov A , Taylor D , Compans RW et al. (2004 ) Enhancement of immunogenicity of an HIV Env DNA vaccine by mutation of the Tyr-based endocytosis motif in the cytoplasmic domain . Virology 328 : 62 -73 . doi:10.1016/j.virol.2004.06.041. PubMed: 15380359.15380359 43 Beddows S , Franti M , Dey AK , Kirschner M , Iyer SP et al. (2007 ) A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120 . Virology 360 : 329 -340 . doi:10.1016/j.virol.2006.10.032. PubMed: 17126869.17126869 44 Yang X , Wyatt R , Sodroski J (2001 ) Improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers . J Virol 75 : 1165 -1171 . doi:10.1128/JVI.75.3.1165-1171.2001. PubMed: 11152489.11152489 45 Bower JF , Yang X , Sodroski J , Ross TM (2004 ) Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d . J Virol 78 : 4710 -4719 . doi:10.1128/JVI.78.9.4710-4719.2004. PubMed: 15078953.15078953 46 Kovacs JM , Nkolola JP , Peng H , Cheung A , Perry J et al. (2012 ) HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120 . Proc Natl Acad Sci U S A 109 : 12111 -12116 . doi:10.1073/pnas.1204533109. PubMed: 22773820.22773820 47 Haynes BF , Fleming J , St Clair EW , Katinger H , Stiegler G et al. (2005 ) Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies . Science 308 : 1906 -1908 . doi:10.1126/science.1111781. PubMed: 15860590.15860590 48 Phogat S , Svehla K , Tang M , Spadaccini A , Muller J et al. (2008 ) Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles . Virology 373 : 72 -84 . doi:10.1016/j.virol.2007.11.005. PubMed: 18155743.18155743 49 Huang J , Ofek G , Laub L , Louder MK , Doria-Rose NA et al. (2012 ) Broad and potent neutralization of HIV-1 by a gp41-specific human antibody . Nature 491 : 406 -412 . doi:10.1038/nature11544. PubMed: 23151583.23151583 50 Porichis F , Kaufmann DE (2011 ) HIV-specific CD4 T cells and immune control of viral replication . Curr Opin HIV AIDS 6 : 174 -180 . doi:10.1097/COH.0b013e3283454058. PubMed: 21502921.21502921 51 Blankson JN (2010 ) Control of HIV-1 replication in elite suppressors . Discov Med 9 : 261 -266 . PubMed: 20350494.20350494 52 Demers KR , Reuter MA , Betts MR (2013 ) CD8(+) T-cell effector function and transcriptional regulation during HIV pathogenesis . Immunol Rev 254 : 190 -206 . doi:10.1111/imr.12069. PubMed: 23772621.23772621 53 Picker LJ , Hansen SG , Lifson JD (2012 ) New paradigms for HIV/AIDS vaccine development . Annu Rev Med 63 : 95 -111 . doi:10.1146/annurev-med-042010-085643. PubMed: 21942424.21942424 54 Kostense S , Koudstaal W , Sprangers M , Weverling GJ , Penders G et al. (2004 ) Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector . AIDS 18 : 1213 -1216 . doi:10.1097/00002030-200405210-00019. PubMed: 15166541.15166541 55 Cooney EL , Collier AC , Greenberg PD , Coombs RW , Zarling J et al. (1991 ) Safety of and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein . Lancet 337 : 567 -572 . doi:10.1016/0140-6736(91)91636-9. PubMed: 1671940.1671940 56 Papp Z , Babiuk LA , Baca-Estrada ME (1999 ) The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1 . Vaccine 17 : 933 -943 . doi:10.1016/S0264-410X(98)00279-5. PubMed: 10067700.10067700 57 Pulendran B , Ahmed R (2011 ) Immunological mechanisms of vaccination . Nat Immunol 12 : 509 -517 . PubMed: 21739679.21739679	24098600	PMC3788131	CC0	2021-01-05 17:12:14	yes	PLoS One. 2013 Oct 1; 8(10):e78521
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24116023PONE-D-13-2023210.1371/journal.pone.0075046Research ArticleAerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil Biodegradation of N-Methyl-4-NitroanilineKhan Fazlurrahman Vyas Bhawna Pal Deepika Cameotra Swaranjit Singh * Environmental Biotechnology and Microbial Biochemistry Laboratory, Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh, India van Raaij Mark J. Editor Centro Nacional de Biotecnologia - CSIC, Spain * E-mail: ssc@imtech.res.inCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: FK SSC. Performed the experiments: FK BV DP. Analyzed the data: FK SSC. Wrote the paper: FK SSC. 2013 8 10 2013 9 7 2014 8 10 e7504616 5 2013 8 8 2013 © 2013 Khan et al2013Khan et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH2 substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway. FK acknowledges the financial support from the Research Associateship from the Department of Biotechnology (DBT), India. BV and DP acknowledge for their research fellowship awards from DST and UGC India, respectively. This is the IMTECH communication number 50/2013. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction N-Methyl-4-nitroaniline (C7H8N2O2, MNA) is a nitroaromatic compound, used as an additive for lowering the melting temperature of highly energetic compounds, such as 2, 4-dinitroanisole (DNAN), that helps in easier synthesis of insensitive explosives [1], [2], [3]. During the process of manufacturing, packaging, and handling of insensitive explosives, it may get released into the environment through various waste streams [2]. Presence of electron withdrawing nitro group (−NO2) in the benzene ring results in acute toxicity to human and other life forms [2], [4]. As a result of toxicity and recalcitrant nature of nitroaromatic compounds, public concern has prompted the need to develop clean-up technologies for restoration of environments. Microbial degradation/or transformation could be used as an effective mechanism for the elimination of nitroaromatic compounds from the environment. Microbial degradation of nitroaromatic compounds can take place in both aerobic and anaerobic conditions, however, due to electron withdrawing effect of nitro group, only nitro group is reduced sequentially into aryl nitroso (Ar-NO), aryl hydroxylamine (Ar-NHOH), and aryl amine (Ar-NH2) without any aromatic ring cleavage [5], [6]. These reductive transformations have been shown to be slow and the reductively transformed products are known to be carcinogenic [7], [8], [9]. The oxidative degradation of recalcitrant compounds under aerobic conditions is expected to be one of the most effective mechanisms for rapid and complete mineralization, which occurs by non-specific enzymes [10], [11], [12]. There are few reports on biological transformation of MNA in anaerobic fluidized-bed bioreactors, where MNA was reductively transformed into 4-methoxy-phenylenediamine that gets accumulated as dead end product followed by dimer formation [1], [2]. Till date there is no information available on aerobic transformation/or degradation of MNA by means of bacterial isolates. Elucidation of catabolic pathways involved in degradation of xenobiotic compounds is critical for both ‘basic understanding’ and ‘development of remediation technologies’ [13], [14], [15]. Therefore, studies on isolation of MNA degrading aerobic bacteria and elucidation of corresponding metabolic pathways are of great ecological significance. In this communication we report isolation of a bacterial strain Pseudomonas sp. strain FK357, isolated from soil that utilizes MNA as the sole source of carbon, nitrogen, and energy. The degradation of MNA by strain FK357 occurs via the formation of 4-NA, 4-AP, and BT as the major metabolic intermediates. We propose that strain FK357 could be used as a model system for studying the molecular and biochemical mechanism of aerobic degradation of MNA as well as for the development of MNA bioremediation technology. Materials and Methods Chemicals and Growth Media N-Methyl-4-nitroaniline (MNA), 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and benzenetriol (BT) were purchased from Sigma-Aldrich (St, Louis, MO, USA). Minimal salt medium (MSM) used in the present study was prepared as described earlier [16] with slight modification i.e. absence of nitrogen source [(NH4)2SO4]. Stock solution (10 mM) of MNA prepared in HPLC grade methanol was added to an empty Erlenmeyer flask to obtain the working concentrations. Further, the residual methanol in the flask was evaporated under stream of air to leave the dry crystal of MNA in the bottom of flask. Nutrient agar (NA) and nutrient broth (NB) both at one-quarter strength (1/4th) were used as rich media for bacterial growth and culture maintenance. Soil Microcosms Study The non-polluted soil sample was collected from the lawn of Institute of Microbial Technology, Chandigarh, India. The pH of the soil was 5.9, and it consisted of 8.2% moisture, 3. 5% total organic carbon and 1.7% total nitrogen content, respectively. The soil samples were sieved through 2 mm mess to remove stones and debris. Soil microcosms experiment was carried out as described previously with slight modification [17]. Subsequently, 5 gm soil sample was suspended in 50 ml MSM in 250 ml Erlenmeyer flask supplemented with 100 µM MNA. Further, the above microcosms were each supplemented with or without (i) 10 mM glucose and 5 mM succinate as the sole carbon source, or (ii) 0.8 g L−1 NH4Cl as the sole nitrogen source, or (iii) 10 mM glucose, 5 mM succinate and 0.8 g L−1 NH4Cl as the sole carbon and nitrogen source, respectively. A control was also set up containing 5 gm autoclaved soil in 50 ml MSM and MNA (100 µM). All the above flasks were incubated in dark at 30°C under shaking condition (150 rpm). At regular time intervals, samples were withdrawn from the test and control flasks and analyzed for the disappearance of MNA. All the experiments were carried out in triplicates. Isolation and Characterization of MNA Degrading Bacteria Upon complete depletion of MNA in the soil microcosms, the serially diluted soil slurries was spread plated on selective medium (MSM-agar containing 700 µM MNA) and incubated at 30°C for one week. The bacterial colonies appeared on the plate were selected for characterization and were tested for MNA degradation. The selected bacterial isolates were used for primary screening by inoculating in 10 ml vials containing 3 ml carbon-free MSM supplemented with different concentrations (50–700 µM) of MNA. The positive bacterial isolates were selected on the basis of increase in growth accompanied by substrate depletion and release of ammonia (NH4 +) and nitrite (NO2 −) ions as measured colorimetrically at wavelength of 340 and 540 nm, respectively. Purity of the positive isolates was checked by streaking on 1/4-NA plates. Further, the efficient MNA degrading isolate was identified using polyphasic taxonomy and 16S rRNA gene sequencing [17]. The 16S rRNA gene sequence (1431 base pairs) of strain FK357 was compared to those of the type strains of Pseudomonas using the BLAST search. Growth Studies and Degradation of MNA by Strain FK357 The growth studies of strain FK357 and degradation of MNA were performed in 50 ml carbon-free MSM supplemented with varying concentrations of MNA ranging from 50 to 700 µM by inoculating (1%, v/v) seed culture grown in 1/4-NB. The flasks were incubated at 30°C under shaking condition of 200 rpm. At every 8 hours, culture was withdrawn and optical cell density at 600 nm (OD600 nm) was measured using Lambda EZ 201 UV-visible spectrophotometer (Perkin-Elmer Inc, USA). The bacterial growth was also monitored by measuring the total protein of the culture with the help of Pierce BCA protein assay kit (Thermo Scientific, USA). Culture fluid samples (2.0 ml) were centrifuged at 8,000 × g for 10 min to obtain cell-free supernatants which were used for analysis of the amount of NH4 + and NO2 − released. Subsequently, the above supernatants were also used for the quantitative determination of MNA disappearance and identification of metabolic intermediates by HPLC. Non-inoculated and inoculated flasks with heat killed cells of strain FK357 were used as abiotic and negative controls, respectively. Another control experiment was also conducted by inoculating strain FK357 in carbon-free MSM (not supplemented with MNA). Resting Cell Studies Resting cell studies on MNA degradation were carried out according to the method described elsewhere [18]. The overnight 1/4-NB grown seed culture (6%, v/v) of strain FK357 was inoculated into 1.0 L of 1/4-NB supplemented with MNA (150 µM) and incubated at 30°C under shaking at 200 rpm up to 24 hours. Similarly, to obtain un-induced cells, the strain FK357 was grown in 1/4-NB only. When the optical cell density of the culture reached to OD600 ranged between 1.2–1.5, the cells (induced and un-induced) were harvested by centrifugation at 8,000×g at 4°C for 10 min, washed twice with phosphate buffer (20 mM, pH 7.2) and suspended in 100 ml carbon-free MSM. In 25 ml cell suspension 150 µM each of MNA and 4-NA were supplemented separately. Similarly, the degradation of MNA and 4-NA by un-induced cells were also carried out by suspending the un-induced cells in 25 ml MSM supplemented with 150 µM each of MNA and 4-NA, separately. Non-inoculated and inoculated flasks with heat killed cells were used as abiotic and negative controls, respectively. Each flask was incubated at 30°C with shaking at 150 rpm. Samples (2.0 ml) were withdrawn from both control and experimental flasks at regular time intervals of 2 hours and were analyzed for the amount of NO2 −, and NH4 + released, followed by High Performance Liquid Chromatography (HPLC) and Gas-Chromatography Mass-Spectroscopy (GC-MS) analysis (methods described later). Enzyme Assays with Cell-free Lysates MNA-induced cells of strain FK357 were harvested by centrifugation and washed twice with phosphate buffer (20 mM, pH 7.2) and re-suspended in phosphate buffer. The cell suspensions lysed by passages through a French pressure cell (20,000 lb/in2) were centrifuged at 12,000 rpm for 30 min at 4°C and supernatant was separated to obtain cell-free enzyme extract, which was subsequently used for the enzyme assays. Protein content within cell-free extracts was determined with Pierce BCA protein assay kit (Thermo Scientific, USA). The cell-free extract was used for determining activities of N-demethylase, monooxygenase, and 1, 2, 4-benzenetriol 1, 2-dioxygenase, respectively as described below. Enzyme assay for N-demethylase Demethylation reaction i.e. removal of N-methyl group from MNA in cell-free lysates was carried out according to the method of Summers et al. [19]. The reaction was carried out in a total volume of 10 ml phosphate buffer (50 mM, pH 7.5) containing cell-free protein (2.0 mg ml−1) 150 µM NADH, 25 mM Fe (NH4)2(SO4)2·6 H2O and 150 µM MNA. The enzymatic reaction was initiated by addition of 150 µM MNA and incubated at 30°C under shaking (200 rpm). The control reactions lacking either substrate or cell-free protein were also used during the above reaction. The N-demethylase activity was determined by measuring time dependent depletion of substrate and formation of N-demethylated product by HPLC. The formation of formaldehyde (HCHO) in the demethylation reaction was quantitatively determined by using an assay based on the Hantzsch reaction [20]. Briefly, in 0.5 ml of culture supernatant equal volume of reagent B (2 M ammonium acetate, 0.05 M acetic acid and 0.02 M acetylacetone) was added and incubated at 58°C for 5 min. Presence of HCHO in the sample was determined colorimetrically by calculating the absorbance at 412 nm as the HCHO adduct diacetyldihydrolutidine. Enzyme assay for monooxygenase The monooxygenase mediated conversion of 4-NA into 4-AP was carried out in cell-free lysates prepared from MNA grown cells of strain FK357 as described previously [21]. The reaction was carried out in a total volume of 10 ml phosphate buffer (20 mM, pH 7.0) containing cell-free protein (2.0 mg ml−1) 200 µM NADPH, 150 µM FMN. The reaction was initiated by addition of 100 µM 4-NA and incubated at 28°C. The control reactions lacking either substrate or cell-free protein or cofactors (NADPH or FMN) were also used during the above reaction. The monooxygenase activity was determined by measuring time dependent depletion of substrate and formation of product by HPLC. Similarly, quantitative determination of NO2 − ions released in the above reaction was also estimated colorimetrically. Aniline dioxygenase assay The dioxygenation reaction involved in the conversion of 4-AP to BT by strain FK357 was measured with an oxygen electrode (YSI, Ohio, USA), according to the method as described previously [22], [23]. MNA-induced and un-induced cells (as described in the resting cell study) were harvested by centrifugation at 7,500×g at 4°C for 15 min. Pellets were washed twice with phosphate buffer (20 mM, pH 7.2) and re-suspended in the same buffer. This suspension was used for the assay of dioxygenase by measuring oxygen uptake at 30°C. The reaction was carried out in 1.85 ml volume air-saturated phosphate buffer (20 mM, pH 7.2) containing substrates (70 µM), and cells (0.25 mg of protein). 1, 2, 4-Benzenetriol 1, 2-dioxygenase enzyme assay It is known that the degradation of terminal intermediate 1, 2, 4-benzenetriol (BT) starts by the action of 1, 2, 4-benzenetriol 1, 2-dioxygenase with the formation of lower pathway intermediate i.e. maleylacetate (MA) [24]. 1, 2, 4-Benzenetriol 1, 2-dioxygenase activity was determined spectrophotometrically using Lambda EZ 201 UV-visible spectrophotometer (Perkin-Elmer Inc, Massachusetts, USA) [21]. The reaction mixture consisted of 50 mM sodium phosphate buffer (pH 7.0), 10 mM Fe (NH4)2(SO4)2·6 H2O, and 0.5 mg soluble protein of cell-free lysates, in the total reaction volume of 1.0 ml. Reaction was initiated by the addition of 70 µM of BT to the reaction mixture. Enzyme activity was monitored with wavelength scan over a range of 220–360 nm at an interval of 1 min. Analytical Methods The release of nitrite ions (NO2 −) was monitored with a colorimetric method using N-(1-naphthyl) ethylene-diamine-dihydrochloride and sulfanilic acid reagent as described earlier [25]. The NO2 − assay was carried out by mixing equal volume (0.1 ml) of reagent A [0.1% (w/v) sulfanilic acid in 30% (v/v) acetic acid] and reagent B [0.1% (w/v) naphthylethylenediamine in 30% acetic acid] to 0.1 ml culture supernatant. Presence of NO2 − in the sample was indicated by the appearance of purple colour and quantified by calculating the absorbance at 540 nm. Ammonia (NH4 +) concentrations were also monitored with a colorimetric method using ‘Ammonia Estimation Kit’ (Sigma Aldrich, USA) according to the manufacturers’ recommendation. Standard plots generated with known concentrations of (NH4)2SO4 and NaNO2 were used to determine the concentrations of NH4 + and NO2 − ions. The quantitative determination of MNA and its metabolic intermediates were analyzed by HPLC [21]. The samples collected from growth, resting cell studies and enzyme assays were analyzed by HPLC using Waters-HPLC 2489 model (Waters, USA) equipped with a UV detector and a C-18 reverse phase column at 30°C [21]. The mobile phase used for the separation of substrate and intermediates consisted of acetonitrile: water (30∶70, v/v) under isocratic condition with the flow rate of 1.0 ml min−1. The samples were injected with a constant injection volume of 20 µl. The peaks of eluents were monitored with UV detector over a wavelength scan of 220–290 nm. For the GC-MS analysis, samples were prepared by mixing equal volume of ethyl acetate to the cell-free aqueous culture and liquid-liquid extraction performed by layer separation sequentially at neutral and acidic pH. Extracted organic phase was pooled, dried under nitrogen flow using RotaVapor II (BUCHI, Switzerland). The samples were analyzed by GC-MS using QP2010S (Shimadzu Scientific Instruments, USA) as reported earlier [6]. Briefly, the chromatographic separation of substrate and intermediates was carried out with constant temperature of injector, oven, and detector at 280, 200, and 250°C, respectively. Positive molecular ion mass spectra were scanned in mass/charge (m/z) range of 0–160. Identity of different metabolic intermediates was ascertained by comparison of mass fragmentation pattern and subsequent mass spectral database match from the National Institute of Standards and Technology library (NIST). Nucleotide Sequence A total of 1431 base-pairs were sequenced for 16S rRNA gene of strain FK357; which has been deposited to GenBank under the accession no. KF011496. Results Soil Microcosms Study The rate of MNA degradation was slightly higher in nitrogen (NH4Cl) amended soil microcosms as compared to unamended soil microcosms (Figure 1). The higher rate of MNA degradation in nitrogen-amended soil microcosms, suggests the availability of most favorable nitrogen source from NH4Cl, which makes it easier for soil microorganisms to utilize N-methyl group as the carbon source. However, in case of soil microcosms amended with a combination of carbon and nitrogen, the rate of MNA degradation was completely inhibited. The inhibition of MNA degradation in the above soil microcosms was likely because the soil inhabitant microorganisms utilized more favorable carbon (succinate and glucose) and nitrogen (NH4Cl) source (Figure 1). There was no transformation of MNA in the sterile soil (Figure 1). 10.1371/journal.pone.0075046.g001Figure 1 Biotransformation of MNA in soil microcosms under aerobic conditions. (•), nitrogen-amended; (○), unamended; (▴), carbon-amended; (□), carbon and nitrogen-amended; (▪), sterile. Values are presented as arithmetic mean of data obtained from experiments carried out in triplicate; error bars represent standard deviation. Isolation and Characterization of N-methyl-4-nitroaniline Degrading Bacteria After 15 days of incubation, when complete degradation of MNA occurred, the serially diluted soil slurries from microcosms was spread plated on MNA selective plates. Total 35 morphologically different isolates were selected and further screened for the degradation of MNA. Among these isolates, strain FK357 was found to be an efficient MNA degrader. The strain FK357 was selected for the identification and study of MNA degradation along with elucidation of metabolic pathways. Strain FK357 was identified as a facultative anaerobe, Gram-negative, non-spore forming, non-motile, catalase and oxidase positive, rod-shaped bacterium. It showed optimal growth on nutrient rich medium (NA, NB) at 30°C; however it was also able to grow at 37°C. Strain FK357 was found negative for H2S production. This strain showed acid production from D-fructose, D-glucose, maltose, D-mannose, D-ribose, trehalose, and L-alanine, whereas, negative on glycerol, D-lactose, D-galactose, D-cellobiose, D-raffinose, and L-serine. Strain FK357 also utilizes acetate and succinate as sole carbon sources. The partial 16S rRNA gene sequence (1431 base pair) of strain FK357 showed 99% sequence similarity to that of several Pseudomonas putida strains. Thus, based on the biochemical tests, morphological and physiological characteristics, and phylogenetic analysis, strain FK357 was identified as Pseudomonas sp. strain FK357. The strain Pseudomonas sp. strain FK357 will be made available to other researchers upon reasonable request. Growth Study and Degradation of N-methyl-4-nitroaniline by Strain FK357 A growth study of strain FK357 was carried out using different concentrations of MNA ranging from 50 to 700 µM. Growth of strain FK357 was completely abolished at an MNA concentration of 500 µM, whereas 250 µM of MNA was found to be optimal for its growth with subsequent degradation of the parent compound (Figure 2A and B). Strain FK357 grew best on 250 µM concentration of MNA at 30°C and pH 7.2. Degradation of MNA during the growth study occurred with the stoichiometric production of nitrite ions and lesser amount of NH4 + (Figure 3). The complete depletion of MNA was observed at 96 hours of incubation (Figure 3). Depletion of MNA was accompanied with an increase in growth of strain FK357 as determined by measuring the total cell proteins up to the value of 10.64 µg ml−1. A transient accumulation of 4-NA occurred in the media during the degradation of MNA (Figure 3).We were unable to detect formaldehyde (HCHO) during the growth study, while transient accumulation of 4-NA, suggested that the first step of MNA degradation starts via the release of –CH3 group in the form of HCHO, followed by the removal of –NO2 and –NH2 groups, respectively. In growth media the accumulation of released NO2 − was stoichiometrically up to the value of 249.56±0.17 µM, whereas, a slight accumulation of NH4 + (51.06 µM) was observed. Thus, non-stoichiometric release of NH4 + suggested that it might possibly be utilized as a preferential nitrogen source for the growth of strain FK357. The above results are in close agreement with the results reported earlier on the degradation of amino or nitro containing aromatic compounds by different genera of bacteria including Pseudomonas [21], [26], [27], [28], [29], [30], [31]. The growth yield of strain FK357 on MNA was found to be 0.55 g of cells/g of MNA. The rate of MNA degradation by strain FK357 was calculated to be 3.26±0.08 nmol MNA min−1 mg of protein−1. Strain FK357 was unable to grow in MSM in the absence of MNA. The above results indicated that the strain FK357 utilizes MNA as the sole source of carbon, nitrogen, and energy. Growth of strain FK357 on 4-NA was also checked. Strain FK357 also utilizes 4-NA and the degradation of 4-NA occurred via stoichiometric accumulation of NO2 − and slight accumulation of NH4 + in the media (data not shown). The growth yield of strain on 4-NA and the rate of 4-NA degradation came out to be 0.43 g of cells/g of 4-NA and 2.73±0.13 nmol 4-NA min−1 mg of protein−1, respectively. In growth study, we identified only 4-NA as an intermediate; further metabolic intermediates were not identified. Based on the accumulation of NO2 − and NH4 + in the growth media, it could be proposed that the subsequent degradation of 4-NA intermediate may initiate either direct ‘oxidative deamination’ of aromatic nucleus or oxidative denitration with the formation of 4-nitrophenol or 4-aminophenol as the putative metabolic product, respectively. 10.1371/journal.pone.0075046.g002Figure 2 Growth characteristics and degradation kinetics of Pseudomonas sp. strain FK357 in carbon-free MSM supplemented with different concentrations of MNA. (•), 50 µM; (▴), 150 µM, (Δ), 250 µM; (○), 350 µM; (▪), 500 µM; (□), 700 µM. (A) Growth of strain FK357 on different concentrations of MNA. (B) Degradation of MNA at different concentrations by strain FK357. 10.1371/journal.pone.0075046.g003Figure 3 Growth of Pseudomonas sp. strain FK357 on MNA as the sole carbon, nitrogen, and energy source. (•), MNA; (▴), 4-NA; (Δ), NO2 − ; (▪), total protein; (○), NH4 +. Values are presented as arithmetic mean of data obtained from experiments carried out in triplicate; error bars represent standard deviation. Resting Cell Studies HPLC analysis of the samples from resting cell study, showed the formation of 4-NA, 4-AP, and BT intermediates as confirmed by comparison with authentic standards. The identified intermediates by HPLC were also confirmed by GC-MS analysis. The mass-fragmentation patterns of the above intermediates viz; 4-NA, 4-AP, and BT were matched with the known authentic standards (Figure 4). Based on the above identified intermediates, we hypothesized that the degradation of MNA occurs via the formation of 4-NA metabolite as a result of demethylation of N–CH3 group, followed by oxidative removal of –NO2 group with the formation of 4-AP. Subsequent degradation of 4-AP may occur by oxidative deamination of –NH2 group with the formation of BT as a terminal intermediate. 10.1371/journal.pone.0075046.g004Figure 4 Mass-fragmentation patterns of the metabolites identified by GC-MS analysis during the degradation of MNA by resting cells of Pseudomonas sp. strain FK357. (A) Mass-fragmentation patterns of 4-NA (B) Mass-fragmentation patterns of 4-AP, and (C) Mass-fragmentation patterns of BT. The resting cell study showed that the MNA-induced cells eliminate the lag phase for the depletion of MNA as observed in case of un-induced cells (Figure 5A). Complete degradation of MNA by the induced cells occurred within 6 hours of incubation with the rate of 0.35±0.07 nmol MNA min−1 mg of protein−1, whereas, un-induced cells degraded MNA after 15 hours of incubation with the rate of 0.17±0.12 nmol MNA min−1 mg of protein−1. The above results showed that the rate of MNA degradation by induced cells was two times higher compared to the rate of degradation by un-induced cells. The degradation of MNA occurs with the appearance followed by disappearance of 4-NA, 4-AP, and BT intermediates (Figure 5B). The cells pre-exposed with MNA also degraded 4-NA with the formation 4-AP and BT as intermediates and the complete degradation of 4-NA occurred after 10 hours of incubation (data not shown). 10.1371/journal.pone.0075046.g005Figure 5 Kinetics of MNA degradation by MNA-induced and un-induced cells of strain FK357 and identification of MNA degradation intermediates. (A) Kinetics of MNA degradation. (•), MNA; (○), MNA; (▪), MNA in abiotic control. (B) Identification of MNA degradation intermediates. (○), 4-NA; (▴), 4-AP; (Δ), BT. Values are presented as arithmetic mean of data obtained from experiments carried out in triplicate; error bars represent standard deviation. N-Demethylase Activity (Removal of -CH3) NADH-dependent enzyme assay in the crude cell extract prepared from MNA grown cells catalyzed stoichiometric transformation of MNA to 4-NA (Figure 6A). The conversion of MNA to 4-NA occurred by N-demethylation reaction with the removal of –CH3 group as determined by stoichiometric formation of HCHO (Figure 6A). The specific activity of N-demethylation reaction was found to be 12.33±0.15 nmol min−1 mg of protein−1. The identification of 4-NA product from MNA in the enzyme assay confirmed that the first step of MNA degradation is an N-demethylation reaction. 10.1371/journal.pone.0075046.g006Figure 6 Stoichiometric N-demethylation of MNA and monooxygenation of 4-NA by cell-free lysates prepared from MNA grown cells of strain FK357. (A) N-demethylation of MNA to 4-NA along with concomitant production of HCHO. (•), MNA; (▾), 4-NA; (○), HCHO. (B) Monooxygenation of 4-NA to 4-AP with stoichiometric release of NO2 −. (•), 4-NA; (▾), 4-AP; (○), NO2 −. Values are presented as arithmetic mean of data obtained from experiments carried out in triplicate; error bars represent standard deviation. Monooxygenase Activity (Removal of -NO2) The subsequent enzymatic transformation of 4-NA, which was identified as the first metabolite, was also carried out in the above crude cell extract. In the presence of NADPH and FMN cofactor, the crude cell extract showed stoichiometric transformation of 4-NA to 4-AP along with stoichiometric production of NO2 − (Figure 6B). The specific activity for the conversion of 4-NA to 4-AP was found to be 3.55±0.08 nmol min−1 mg of protein−1. No monooxygenase activity was observed in the control reactions that lacked either NADPH or FMN or cell-free protein. The above results showed that the second step of MNA degradation is a flavin-dependent monooxygenation reaction. Oxidative Deaminase Activity (Removal of –NH2) The rate of oxygen consumption by 4-AP, 4-chlorophenol (4-CP), and aniline in MNA grown cells of strain FK357 was higher as compared to the MNA and 4-NA (Table 1). However, negligible oxygen consumption was observed by 4-AP, 4-CP and aniline, when 1/4-NB grown cells were incubated with these substrates suggesting the inducible nature of enzyme. In contrast, the lesser amount of oxygen consumption by MNA and 4-NA in 1/4-NB grown cells demonstrated that the pathway enzymes involved in the initiation of MNA degradation have moderate constitutive activities. The inducible nature of enzyme involved in the oxidative deamination of –NH2 from aminophenols and anilines are in close agreement with the earlier reports [22], [23], [32], [33]. The stoichiometric release of NH4 + and formation of BT, chlorocatechol and catechol products from 4-AP, 4-CP and aniline suggested that the deamination of –NH2 group is a dioxygenation reaction and also it is inducible in nature. 10.1371/journal.pone.0075046.t001Table 1 Oxygen uptake by MNA-grown and 1/4-NB grown cellsa. Substrates Oxygen uptake (nmol O2/min/mg of protein) by cells grown in: MNA 1/4-NB N-Methyl-4-nitroaniline 65.22±0.7 33.19±0.1 4-Nitroaniline 79.50±1.2 28.70±1.1 4-Aminophenol 184.74±0.5 0.27±0.7 4-Chloroaminophenol 173.34±1.3 0.78±0.4 Aniline 190.10±2.1 0.50±0.1 a The reaction was carried in 1.85 ml volume air-saturated phosphate buffer (20 mM, pH 7.2) containing substrates (70 µM), and cells (0.25 mg of protein). Data represents means of at least three separate experiments. 1, 2, 4-Benzenetriol 1, 2-dioxygenase Activity Furthermore, 1, 2, 4-benzenetriol 1, 4-dioxygenase (BtD) activity on BT by crude cell extracts was carried out spectrophotometrically by monitoring time dependent wave length scan over a range of 200–500 nm. During the incubation of BT in crude cell extracts, the substrate peak with proximal absorbance at 283 nm gradually decreased, giving rise to a new peak with maximal absorbance at 243 nm (Figure 7). The absorbance at 243 nm is the characteristic of maleylacetate (MA). The specific activity for the BtD was found to be 2.43±0.30 nmol min−1 mg of protein−1. 10.1371/journal.pone.0075046.g007Figure 7 Formation of the maleylacetate (MA) from benzenetriol (BT) by crude cell extracts of Pseudomonas sp. strain FK357. Sample and reference cuvette contained phosphate buffer (50 mM, pH 7.0), 10 mM Fe (NH4)2(SO4)2.6 H2O, and 0.5 mg ml−1 protein in a 1.0-ml mixture. The reaction was initiated by addition of 70 µM BT and the spectra were recorded every 1 minute after the addition of BT. The arrows indicate the directions of spectral changes. Discussion Structurally MNA is an analogue to 4-nitroanisole, which contains O-methyl group (O-CH3) and a nitro group (−NO2), whereas MNA contains an N-methyl group (N-CH3) and a nitro group (−NO2). The mineralization of 4-nitroanisole has been earlier reported by Rhodococcus strain AS2 and AS3 [34]. The degradation of 4-nitroanisole starts with O-demethylation of O-CH3 with the formation of 4-nitrophenol, which subsequently get degraded as a result of ring cleavage via the formation of 4-nitrocatechol intermediate [34]. Soil microcosms study demonstrated the possibility of MNA degradation in aerobic conditions. Nitrogen (NH4Cl) amended soil microcosms showed the higher rate of MNA degradation as compared to that of unamended soil microcosms. The above result indicates that soil microbes utilizes N–CH3 group from MNA as the preferential single carbon source i.e. the easiest and first to be used, in the presence of most favourable nitrogen source (NH4Cl). Soil microcosms study suggested that microorganisms utilized MNA as sole source of carbon, nitrogen, and energy. Based on the identified intermediates during the growth study, resting cell study and enzyme assays, we proposed a novel MNA degradation pathway in Pseudomonas sp. strain FK357 (Figure 8). In this pathway, conversion of MNA into 4-NA is expected to be catalysed by a putative demethylase enzyme capable of removing N-CH3 via oxidative pathway. O-demethylation reactions are widely distributed among microorganisms capable of degrading aromatic compounds containing O-CH3 group, the removal of N-CH3 group from aromatic compounds has been shown only in few reports. Pseudomonas putida CBB5 has been isolated and characterized for N-demethylation reaction occurring in degradation pathway of several purine alkaloids [19], [35]. We were unable to quantify HCHO during the growth study of strain FK357, however, it was later quantified in resting cell study. Enzyme assay carried out for the N-demethylation reaction in crude extracts on MNA showed the formation of 4-NA along with stoichiometric production of HCHO. Interestingly, growth yield of strain FK357 on MNA was slightly higher as compared to its growth yield on 4-NA, the possible reason for which could be the availability of extra single carbon source i. e. N-CH3 from MNA ring. Earlier, it has been reported that the methyl group released either from N-methyl or O-methyl containing compounds oxidized to HCHO [19], [35], [36], [37], [38], [39], [40], [41]. There are various bacterial strains which have been characterized to utilize HCHO as the sole carbon source [20], [35], [42], [43], [44]. In the presence of nitrogen source, the strain FK357 also utilized HCHO as the sole carbon source (data not shown). Summers et al. [19] characterized N-demethylase (Ndm) gene from Pseudomonas putida CBB5, which showed the broad substrate activity on several purine alkaloids. Thus, based on the above study, we proposed the involvement of a similar bacterial N-demethylation reaction as the first step of MNA degradation. Subsequent degradation of MNA presumably initiated as a result of removal of substituted –NO2 or –NH2 group from the benzene ring. The mechanism for the removal of –NO2 and –NH2 groups from benzene ring poses an interesting question and challenge to the microbial systems for the preferential removal of these groups. Usually there are two reaction mechanisms for the removal of –NO2 group, one is oxidative and another is reductive [5]. In the oxidative reaction, the removal of –NO2 group as nitrite ions (NO2 −) takes place by hydroxylation, while −NO2 group get sequentially reduced to nitroso (−NO), hydroxylamine (−NHOH), and amino (−NH2) group in the reductive reaction [6], [45], [46]. Another reaction mechanism involves dioxygenation which removes –NO2 group by adding two hydroxyl groups simultaneously, one at the nitro-substituted position and the other at the adjacent position [5], [47]. Removal of –NO2 group by dioxygenation reaction has been reported earlier in the degradation pathway of nitrobenzene by Comamonas sp. strain JS765 [5], [48]. The dioxygenation of –NO2 group from nitrobenzene resulted in the formation of 1, 2-cis-dihydrodiol product [47], [49]. Similarly, the removal of –NH2 group from the benzene ring by dioxygenation reaction has also been reported for the degradation of anilines, diphenylamine, and chloroanilines [22], [23], [32], [50]. Aniline dioxygenase is one of the most commonly characterized enzyme reported for the removal of –NH2 group during the aniline and chloroaniline degradation pathways [22], [23], [27], [33], [50], [51]. 10.1371/journal.pone.0075046.g008Figure 8 Proposed metabolic pathway for aerobic degradation of MNA by Pseudomonas sp. strain FK357. The crude cell extracts showed conversion of 4-NA into 4-AP with stoichiometric production of NO2 − and the removal of –NO2 from 4-NA occurs via a hydroxylation reaction catalyzed by flavin-dependent monooxygenase (Figure 6A). This reaction is very common for aerobic microbial degradation of nitro or chloro containing aromatic compounds such as 2-chloro-4-nitrophenol, 2-chloro-4-nitroaniline, 4-nitroaniline, 4-nitrophenol, 2, 4-dichlorophenol, and 4-chlorophenol, respectively [6], [21], [33], [52], [53]. Oxygen uptake studies showed the conversion of 4-AP into BT product with stoichiometric release of NH4 +, suggesting that a dioxygenase enzyme catalyses the reaction by introducing two oxygen atoms on the benzene ring. Similar reports has also been shown in case of aniline degradation by strain Pseudomonas putida mt-2, Pseudomonas acidovorans CA28, Delftia sp. AN3, and Delftia tsuruhatensis AD9 which degrades aniline via the formation of catechol as the first metabolic product [22], [23], [29], [54]. Moraxella sp. strain G is known to mineralize 4-chloroaniline by dioxygenation reaction with the formation of chlorocatechol as a metabolic product along with stoichiometric release of NH4 + [55]. Furthermore, the crude cell extracts prepared from MNA grown cells also transforms BT to MA, suggesting the involvement of a ring cleavage enzyme i.e. 1, 2, 4- benzenetriol 1, 2-dioxygenase. Formation of MA from BT is commonly reported as a ring cleavage step in 4-nitrophenol degradation pathway [56], [57], [58]. Based on the above identified metabolites, the degradation pathway has been elucidated, showing N-demethylation of N-CH3 group as the first reaction followed by monooxygenase mediated removal of –NO2 (Figure 8). Further degradation of 4-AP occurs by the dioxygenation reaction resulting in formation of BT which subsequently gets ring cleaved by 1, 2, 4-benzenetriol 1, 2-dioxygenase enzyme. Conclusion We report isolation and characterization of bacterium Pseudomonas sp. strain FK357, which is the first naturally occurring aerobic bacterium capable of utilizing MNA as the sole source of carbon, nitrogen, and energy. Degradation of MNA by strain FK357 occurs via the formation of 4-NA, 4-AP, and BT as major intermediates (Figure 8). Strain FK357 could also be used as an important model system for studies on biochemical and molecular evolution of microbial degradation of MNA. The molecular components involved in the MNA degradation by strain FK357 are yet to be characterized. The future work has been proposed to carry out entire genome sequencing, annotation or genomic library construction of strain FK357 for the characterization of gene(s)/gene cluster involved in this important metabolic pathway. ==== Refs References 1 Platten WE 3rd, Bailey D , Suidan MT , Maloney SW (2010 ) Biological transformation pathways of 2,4-dinitro anisole and N-methyl paranitro aniline in anaerobic fluidized-bed bioreactors . Chemosphere 81 : 1131 –1136 .20855103 2 Arnett CM , Rodriguez G , Maloney SW (2009 ) Analysis of bacterial community diversity in anaerobic fluidized bed bioreactors treating 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) using 16S rRNA gene clone libraries . Microbes Environ 24 : 72 –75 .21566358 3 Agrawal JP (1998 ) Recent trends in high-energy materials . Prog Energ Combust 24 : 1 –30 . 4 Robidoux PY , Svendsen C , Caumartin J , Hawari J , Ampleman G , et al (2000 ) Chronic toxicity of energetic compounds in soil determined using the earthworm (Eisenia andrei) reproduction test . Environ Toxicol Chem 19 : 1764 –1773 . 5 Nishino SF , Spain JC (1995 ) Oxidative pathway for the biodegradation of nitrobenzene by Comamonas sp. strain JS765 . Appl Environ Microbiol 61 : 2308 –2313 .16535050 6 Ghosh A , Khurana M , Chauhan A , Takeo M , Chakraborti AK , et al (2010 ) Degradation of 4-nitrophenol, 2-chloro-4-nitrophenol, and 2,4-dinitrophenol by Rhodococcus imtechensis strain RKJ300 . Environ Sci Technol 44 : 1069 –1077 .20050667 7 Saupe A (1999 ) High-rate biodegradation of 3- and 4-nitroaniline . Chemosphere 39 : 2325 –2346 .10576103 8 Purohit V , Basu AK (2000 ) Mutagenicity of nitroaromatic compounds . Chem Res Toxicol 13 : 673 –692 .10956054 9 Padda RS , Wang C , Hughes JB , Kutty R , Bennett GN (2003 ) Mutagenicity of nitroaromatic degradation compounds . Environ Toxicol Chem 22 : 2293 –2297 .14551991 10 Das N , Chandran P (2011 ) Microbial degradation of petroleum hydrocarbon contaminants: an overview . Biotechnol Res Int 2011 : 941810 .21350672 11 O’Neill C , Lopez A , Esteves S , Hawkes FR , Hawkes DL , et al (2000 ) Azo-dye degradation in an anaerobic-aerobic treatment system operating on simulated textile effluent . Appl Microbiol Biotechnol 53 : 249 –254 .10709990 12 Zissi U , Lyberatos G (1996 ) Azo-dye biodegradation under anoxic conditions . Water Sci Technol 34 : 495 –500 . 13 Alexander M , Loehr RC (1992 ) Bioremediation review . Science 258 : 874 . 14 Singh S , Kang SH , Mulchandani A , Chen W (2008 ) Bioremediation: environmental clean-up through pathway engineering . Curr Opin Biotechnol 19 : 437 –444 .18760355 15 Soccol CR , Vandenberghe LP , Woiciechowski AL , Thomaz-Soccol V , Correia CT , et al (2003 ) Bioremediation: an important alternative for soil and industrial wastes clean-up . Indian J Exp Biol 41 : 1030 –1045 .15242296 16 Fazlurrahman, Batra M , Pandey J , Suri CR , Jain RK (2009 ) Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil . Lett Appl Microbiol 49 : 721 –729 .19818008 17 Perreault NN , Halasz A , Manno D , Thiboutot S , Ampleman G , et al (2012 ) Aerobic mineralization of nitroguanidine by Variovorax strain VC1 isolated from soil . Environ Sci Technol 46 : 6035 –6040 .22563908 18 Shettigar M , Pearce S , Pandey R , Khan F , Dorrian SJ , et al (2012 ) Cloning of a novel 6-chloronicotinic acid chlorohydrolase from the newly isolated 6-chloronicotinic acid mineralizing Bradyrhizobiaceae strain SG-6C . PLoS One 7 : e51162 .23226482 19 Summers RM , Louie TM , Yu CL , Subramanian M (2011 ) Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source . Microbiology 157 : 583 –592 .20966097 20 Chongcharoen R , Smith TJ , Flint KP , Dalton H (2005 ) Adaptation and acclimatization to formaldehyde in methylotrophs capable of high-concentration formaldehyde detoxification . Microbiology 151 : 2615 –2622 .16079340 21 Khan F , Pandey J , Vikram S , Pal D , Cameotra SS (2013 ) Aerobic degradation of 4-nitroaniline (4-NA) via novel degradation intermediates by Rhodococcus sp. strain FK48 . J Hazard Mater 254–255C : 72 –78 . 22 Liu Z , Yang H , Huang Z , Zhou P , Liu SJ (2002 ) Degradation of aniline by newly isolated, extremely aniline-tolerant Delftia sp. AN3 . Appl Microbiol Biotechnol 58 : 679 –682 .11956754 23 Liang Q , Takeo M , Chen M , Zhang W , Xu Y , et al (2005 ) Chromosome-encoded gene cluster for the metabolic pathway that converts aniline to TCA-cycle intermediates in Delftia tsuruhatensis AD9 . Microbiology 151 : 3435 –3446 .16207925 24 Kadiyala V , Spain JC (1998 ) A two-component monooxygenase catalyzes both the hydroxylation of p-nitrophenol and the oxidative release of nitrite from 4-nitrocatechol in Bacillus sphaericus JS905 . Appl Environ Microbiol 64 : 2479 –2484 .9647818 25 White GF , Snape JR , Nicklin S (1996 ) Biodegradation of glycerol trinitrate and pentaerythritol tetranitrate by Agrobacterium radiobacter . Appl Environ Microbiol 62 : 637 –642 .16535244 26 Qu Y , Spain JC (2011 ) Molecular and biochemical characterization of the 5-nitroanthranilic acid degradation pathway in Bradyrhizobium sp. strain JS329 . J Bacteriol 193 : 3057 –3063 .21498645 27 Fukumori F , Saint CP (1997 ) Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDN1) . J Bacteriol 179 : 399 –408 .8990291 28 Zhao JS , Ward OP (2001 ) Substrate selectivity of a 3-nitrophenol-induced metabolic system in Pseudomonas putida 2NP8 transforming nitroaromatic compounds into ammonia under aerobic conditions . Appl Environ Microbiol 67 : 1388 –1391 .11229938 29 McClure NC , Venables WA (1986 ) Adaptation of Pseudomonas putida mt-2 to growth on aromatic amines . J Gen Microbiol 132 : 2209 –2218 .3794647 30 Nishino SF , Spain JC (1993 ) Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes . Appl Environ Microbiol 59 : 2520 –2525 .8368838 31 Qu Y , Spain JC (2010 ) Biodegradation of 5-nitroanthranilic acid by Bradyrhizobium sp. strain JS329 . Appl Environ Microbiol 76 : 1417 –1422 .20081004 32 Shin KA , Spain JC (2009 ) Pathway and evolutionary implications of diphenylamine biodegradation by Burkholderia sp. strain JS667 . Appl Environ Microbiol 75 : 2694 –2704 .19251893 33 Khan F , Pal D , Vikram S , Cameotra SS (2013 ) Metabolism of 2-chloro-4-nitroaniline via novel aerobic degradation pathway by Rhodococcus sp. strain MB-P1 . PLoS One 8 : e62178 .23614030 34 Schafer A , Harms H , Zehnder AJ (1996 ) Biodegradation of 4-nitroanisole by two Rhodococcus spp . Biodegradation 7 : 249 –255 .8782395 35 Summers RM , Louie TM , Yu CL , Gakhar L , Louie KC , et al (2012 ) Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids . J Bacteriol 194 : 2041 –2049 .22328667 36 Blecher R , Lingens F (1977 ) The metabolism of caffeine by a Pseudomonas putida strain . Hoppe Seylers Z Physiol Chem 358 : 807 –817 .561017 37 Woolfolk CA (1975 ) Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen . J Bacteriol 123 : 1088 –1106 .1158847 38 Providenti MA , O’Brien JM , Ruff J , Cook AM , Lambert IB (2006 ) Metabolism of isovanillate, vanillate, and veratrate by Comamonas testosteroni strain BR6020 . J Bacteriol 188 : 3862 –3869 .16707678 39 Hibi M , Sonoki T , Mori H (2005 ) Functional coupling between vanillate-O-demethylase and formaldehyde detoxification pathway . FEMS Microbiol Lett 253 : 237 –242 .16242864 40 Cartwright NJ , Smith AR (1967 ) Bacterial attack on phenolic ethers: An enzyme system demethylating vanillic acid . Biochem J 102 : 826 –841 .16742500 41 Asano Y , Komeda T , Yamada H (1993 ) Microbial production of theobromine from caffeine . Biosci Biotechnol Biochem 57 : 1286 –1289 . 42 Adroer N , Casas C , de Mas C , Sola C (1990 ) Mechanism of formaldehyde biodegradation by Pseudomonas putida . Appl Microbiol Biotechnol 33 : 217 –220 .1366532 43 Marx CJ , Miller JA , Chistoserdova L , Lidstrom ME (2004 ) Multiple formaldehyde oxidation/detoxification pathways in Burkholderia fungorum LB400 . J Bacteriol 186 : 2173 –2178 .15028703 44 Roca A , Rodriguez-Herva JJ , Duque E , Ramos JL (2008 ) Physiological responses of Pseudomonas putida to formaldehyde during detoxification . Microb Biotechnol 1 : 158 –169 .21261833 45 Beunink J , Rehm HJ (1990 ) Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system . Appl Microbiol Biotechnol 34 : 108 –115 .1366971 46 Katsivela E , Wray V , Pieper DH , Wittich RM (1999 ) Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1 . Appl Environ Microbiol 65 : 1405 –1412 .10103229 47 Nolan LC , O’Connor KE (2008 ) Dioxygenase- and monooxygenase-catalysed synthesis of cis-dihydrodiols, catechols, epoxides and other oxygenated products . Biotechnol Lett 30 : 1879 –1891 .18612597 48 Lessner DJ , Johnson GR , Parales RE , Spain JC , Gibson DT (2002 ) Molecular characterization and substrate specificity of nitrobenzene dioxygenase from Comamonas sp. strain JS765 . Appl Environ Microbiol 68 : 634 –641 .11823201 49 Boyd DR , Bugg TD (2006 ) Arene cis-dihydrodiol formation: from biology to application . Org Biomol Chem 4 : 181 –192 .16391757 50 Urata M , Uchida E , Nojiri H , Omori T , Obo R , et al (2004 ) Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment . Biosci Biotechnol Biochem 68 : 2457 –2465 .15618615 51 Travkin VM , Solyanikova IP , Rietjens IM , Vervoort J , van Berkel WJ , et al (2003 ) Degradation of 3,4-dichloro- and 3,4-difluoroaniline by Pseudomonas fluorescens 26-K . J Environ Sci Health B 38 : 121 –132 .12617551 52 Perry LL , Zylstra GJ (2007 ) Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase . J Bacteriol 189 : 7563 –7572 .17720792 53 Gisi MR , Xun L (2003 ) Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100 . J Bacteriol 185 : 2786 –2792 .12700257 54 Loidl M , Hinteregger C , Ditzelmuller G , Ferschl A , Streichsbier F (1990 ) Degradation of aniline and monochlorinated anilines by soil-born Pseudomonas acidovorans strains . Arch Microbiol 155 : 56 –61 . 55 Zeyer J , Wasserfallen A , Timmis KN (1985 ) Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway . Appl Environ Microbiol 50 : 447 –453 .4051488 56 Jain RK , Dreisbach JH , Spain JC (1994 ) Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp . Appl Environ Microbiol 60 : 3030 –3032 .8085840 57 Chauhan A , Pandey G , Sharma NK , Paul D , Pandey J , et al (2010 ) p-Nitrophenol degradation via 4-nitrocatechol in Burkholderia sp. SJ98 and cloning of some of the lower pathway genes . Environ Sci Technol 44 : 3435 –3441 .20359211 58 Wei M , Zhang JJ , Liu H , Zhou NY (2010 ) para-Nitrophenol 4-monooxygenase and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-nitrocatechol in Pseudomonas sp. strain WBC-3 . Biodegradation 21 : 915 –921 .20361240	24116023	PMC3792944	CC BY	2021-01-05 17:47:15	yes	PLoS One. 2013 Oct 8; 8(10):e75046
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24130713PONE-D-13-2340910.1371/journal.pone.0075456Research ArticleEvaluation of the Replication, Pathogenicity, and Immunogenicity of Avian Paramyxovirus (APMV) Serotypes 2, 3, 4, 5, 7, and 9 in Rhesus Macaques Replication of APMVs in Rhesus MacaquesKhattar Sunil K. 1 Nayak Baibaswata 1 Kim Shin-Hee 1 Xiao Sa 1 Samal Sweety 1 Paldurai Anandan 1 Buchholz Ursula J. 2 Collins Peter L. 2 Samal Siba K. 1 * 1 Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America 2 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, United States of America Schnell Matthias Johannes Editor Thomas Jefferson University, United States of America * E-mail: ssamal@umd.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SKK UJB PLC SKS. Performed the experiments: SKK BN SHK SX SS AP UJB. Analyzed the data: SKK BN UJB PLC SKS. Contributed reagents/materials/analysis tools: PLC SKS. Wrote the paper: SKK UJB PLC SKS. 2013 10 10 2013 8 10 e754565 6 2013 15 8 2013 2013This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.Avian paramyxoviruses (APMV) serotypes 1–9 are frequently isolated from domestic and wild birds worldwide. APMV-1 (also called Newcastle disease virus, NDV) is attenuated in non-human primates and is being developed as a candidate human vaccine vector. The vector potential of the other serotypes was unknown. In the present study, we evaluated nine different biologically- or recombinantly-derived APMV strains for the ability to replicate and cause disease in rhesus macaque model. Five of the viruses were: biologically-derived wild type (wt) APMV-2, -3, -5, -7 and -9. Another virus was a recombinant (r) version of wt APMV-4. The remaining three viruses were versions of wt rAPMV-2, -4 and -7 in which the F cleavage site had been modified to be multi-basic. Rhesus macaques were inoculated intranasally and intratracheally and monitored for clinical disease, virus shedding from the upper and lower respiratory tract, and seroconversion. Virus shedding was not detected for wt APMV-5. Very limited shedding was detected for wt rAPMV-4 and modified rAPMV-4, and only in a subset of animals. Shedding by the other viruses was detected in every infected animal, and usually from both the upper and lower respiratory tract. In particular, shedding over a number of days in every animal was observed for modified rAPMV-2, wt APMV-7, and modified rAPMV-7. Modification of the F protein cleavage site appeared to increase shedding by wt rAPMV-2 and marginally by wt rAPMV-4. All APMVs except wt APMV-5 induced a virus-specific serum antibody response in all infected animals. None of the animals exhibited any clinical disease signs. These results indicate that APMVs 2, 3, 4, 7, and 9 are competent to infect non-human primates, but are moderately-to-highly restricted, depending on the serotype. This suggests that they are not likely to significantly infect primates in nature, and represent promising attenuated candidates for vector development. This research was supported by National Institute of Allergy and Infectious Diseases (NIAID) contract number N01A060009 (85% support) and NIAID, National Institutes of Health (NIH) Intramural Research Program (15% support). PLC and UJB were supported by the Intramural Research Program of the NIAID, NIH. The views expressed herein do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the United States Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction The avian paramyxoviruses (APMVs) are isolated from wild and domestic birds all over the world. The APMVs have been divided into nine serotypes (APMV 1 to 9) based on hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays [1]. More recently, viruses representing potential APMV serotypes 10 and 11 were isolated from Rockhopper Penguins [2] and common teal [3], respectively. APMV-1, which includes all strains of Newcastle disease virus (NDV), has been extensively characterized because virulent NDV strains cause severe disease in chickens. NDV strains are divided into three pathotypes based on their virulence in chickens: highly virulent (velogenic) strains cause severe respiratory and neurologic diseases; moderately virulent (mesogenic) strains cause milder disease, and nonpathogenic (lentogenic) strains cause inapparent infection. Mesogenic and lentogenic strains of NDV are highly restricted for replication and highly attenuated in non-avian species including primates, and are being developed as vaccine vectors for animal and human pathogens. Previous studies have shown that APMV serotypes 2-9 replicate not only in avian species, but also in mice and hamsters [4], [5]. However, their ability to replicate and possibly cause disease in primates, and their potential as human vaccine vectors, were unknown. The APMVs belong to family Paramyxoviridae, a large and diverse family that includes viruses from a wide variety of mammalian, avian, reptilian, and fish species around the world [6]. Some members of the family are responsible for major human and animal diseases, while others cause inapparent infections. Paramyxoviruses are pleomorphic and enveloped and contain a non-segmented, negative-sense, single-stranded RNA genome of 13–19 kb. On the basis of virus structure, genome organization and sequence relatedness, the family Paramyxoviridae is divided in to two subfamilies: Paramyxovirinae and Pneumovirinae [6]. The subfamily Paramyxovirinae is divided into five genera: Respirovirus (including Sendai virus and human parainfluenza virus types 1 and 3), Rubulavirus (including parainfluenza virus 5 [previously known as simian virus type 5], mumps virus, and human parainfluenza virus types 2 and 4), Morbillivirus (including measles and canine distemper viruses), Henipavirus (comprising Hendra and Nipah viruses), and Avulavirus (comprising the APMVs). Subfamily Pneumovirinae contains two genera, Pneumovirus (including human and bovine respiratory syncytial viruses) and Metapneumovirus (comprising human metapneumovirus and the avian metapneumoviruses) [6], [7] Although a lot of information is available for APMV-1, much less is known about the molecular biology, pathogenicity and host range of the other APMV serotypes. As an initial step towards their characterization, we have recently determined the complete genome sequences of APMV-2 to -9 [8]–[15] and we have developed reverse genetics systems for APMV-2, -3, -4 and -7 [16]–[19]. However, the biological characteristics and pathogenicity of APMV-2 to -9 remain poorly understood. APMV-2 has been associated with severe respiratory disease, reduced egg production and infertility in turkeys [20], [21]. APMV-3 has been associated with encephalitis and high mortality in caged birds, respiratory disease in turkeys and stunted growth in young chickens [22], [23]. APMV-4 strains have been isolated from chickens, ducks and geese [19]. APMV-5 causes disease in budgerigars that is characterized by depression, dyspnoea, diarrhea and high mortality [24]. APMV-6 and -7 cause mild respiratory disease in turkeys and are associated with a drop in egg production [25], [26]. APMV-8 and -9, isolated from ducks, waterfowl, and other wild birds, did not produce any clinical signs of viral infection in chickens [27], [28] In the last 10 years, reverse genetic techniques have made it possible to engineer NDV as a potential vaccine vector for both human and animal uses [29]–[39]. NDV vectors expressing a number of foreign antigens have been evaluated not only in avian hosts, but also in murine and nonhuman primate models [29]–[33], [40]–[42]. Several strains of NDV have been shown to be highly restricted for replication in these mammalian models, indicating that they are highly attenuated due to a strong host range restriction and represent promising vaccine vectors. However, NDV strains are highly related antigenically, and therefore the use of NDV vectors for multiple purposes would be compromised by the induction of vector-specific immunity. This limitation might be overcome by using other APMV serotypes that are antigenically distinct from NDV as alternative vaccine vectors. In addition, it is possible that one or more of the other APMV serotypes might have other advantageous properties that cannot be predicted, such as increased immunogenicity compared to NDV. Furthermore, the possibility exists that one or more of the other APMV serotypes might be pathogenic in certain non-avian hosts including primates. For example, an APMV-2-like virus was previously recovered from a cynomolgus monkey with respiratory disease [43], and an APMV-3-like virus was recovered from pigs [44]. Therefore, evaluation of APMV-2 to -9 in non-avian species was warranted. We recently showed that APMV-2 to -9 are competent to infect and replicate to low-to-moderate titers in mice and hamsters [37], [38]. However, rodents are uncertain predictors of performance in other species such as humans because it is possible, and indeed likely, that there will be differences in the level of host range restriction between rodents and other species including primates. In the present study, we sought to evaluate the replication and pathogenicity of APMV-2, -3, -4, -5, -7 and -9 in rhesus macaques as a surrogate for humans. The viruses included biologically- and recombinantly-derived wt viruses as well as several recombinant viruses in which the F protein cleavage site had been modified to be multi-basic and to contain the optimal furin protease cleavage site motif RX(R/K)R↓ (signature R and K residues underlined). This was done because the presence of a furin motif in the F protein cleavage site typically facilitates cleavage and is a major determinant of virulence for NDV strains [45], [46], although this paradigm is uncertain for the other APMV serotypes [16], [18], [19]. The present study showed that, except for APMV-5, all of the APMVs under evaluation replicated at varying levels in rhesus macaques without inducing any apparent clinical disease signs. Thus, APMV serotypes 2, 3, 4, 7, and 9 are infectious, replication-competent and attenuated in non-human primates. In future work, the reverse genetics system for APMV-2, -4, and -7 will be used for the development of APMV vectored vaccines. Materials and Methods Ethical Statement Adult rhesus macaques (Macaca mullatta) were obtained from the NIAID breeding colony located on Morgan Island, SC. This colony is AAALAC international accredited and OLAW assured. All animals are transported in accordance with USDA guidelines and permits. The non-human primate experiments were performed at a site approved by the Association for Assessment and Accreditation of Laboratory Animal Care International, with a protocol approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases. Since this study involved infectious agents, rhesus macaques were housed individually in microisolator cages for the duration of the study. This was necessary to prevent transmission of respiratory viruses to other non-human primates in the room and to animal handlers. All monkeys were able see other monkeys of the same species. Monkeys were on an environmental enrichment program (additional objects for manipulation, perches, food enrichment). Animals were observed at least two times a day by animal care staff for any illnesses or abnormalities. No animals were sacrificed for this study. All the experiments where 9-day old embryonated chicken eggs were used ended on or before day 13. Before collecting allantoic fluid from the eggs, the embryos were sacrificed by incubating the eggs at 4°C in a refrigerator for 2 hour. Cells and viruses Chicken embryo fibroblast (DF-1) and African green monkey kidney (Vero) cell lines, obtained from the American Type Culture Collection (ATCC, Manassas, VA), were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS. Nine APMV strains were used in this study (see Table 1), all of which had been constructed in previous work: (i) biologically-derived wt APMV-2 strain chicken/Yucaipa/California/56 [8]; (ii) a recombinant derivative of wt APMV-2, called rAPMV-2 (type 1 Africa), in which the natural cleavage site was replaced by the cleavage site RRRRR↓F that is present in a APMV type 1 strain isolated in Africa [16]; (iii) biologically-derived wt APMV-3 strain parakeet/Netherland/449/75 [9]; (iv) recombinant wt APMV-4 strain duck/Hong Kong/D3/75 [10]; (v) a derivative of rAPMV-4, called rAPMV-4/Fc-BC, in which its natural cleavage site was replaced by the cleavage site RRQKR↓F that is present in mesogenic NDV strain Baudette C [19]; (vi) biologically-derived wt APMV-5 strain budgerigar/Kunitachi/74 [11]; (vii) biologically-derived wt APMV-7 strain dove/Tennessee/4/75 [13]; (viii) a recombinant derivative of wt APMV-7, called rAPMV-7/Fcs-5B in which its natural cleavage site was replaced by the cleavage site RRKKR↓FI present in a velogenic NDV strain Nigeria/95, and which includes an amino acid substitution in the +2 position relative to the cleavage site [18]; and (ix) biologically-derived wt APMV-9 strain duck/New York/22/78 [15]. All the APMV serotypes except serotype 5 were grown in the allantoic cavity of 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs. APMV-5 was grown in Vero cells. The allantoic fluids from infected eggs were collected 72 h post-inoculation and virus titers were determined by hemagglutination (HA) assay. Further, the titers of APMVs were determined by plaque assay on DF-1 cells for APMV-2, -3, -4 and -9 and on Vero cells for APMV-5 and -7. The samples were inoculated in triplicate onto 24-well plates of DF-1 or Vero cells at 80% confluency and virus titer was determined by either plaque assay or immunostaining with N specific antibodies. 10.1371/journal.pone.0075456.t001Table 1 APMV strains, F protein cleavage sites, and protease dependence. APMV strain F protein cleavage site† Furin motif Exogenous protease required in vitro APMV-2 DKPASR FVG No No rAPMV-2 (type 1 Africa)* DRRRRR FVG* Yes No APMV-3 ARPRGR LFG No Yes APMV-4 ADIQPR FIG No No APVM-4/Fc-BC* ARRQKR FIG* Yes No APMV-5 GKRKKR FVG Yes No APMV-7 TLPSSR FAG No No APMV-7/Fcs-5B* TRRKKR FIG* Yes No APMV-9 RIREGR IFG No Yes * The F protein cleavage site was modified to contain the preferred furin motif. Amino acid substitutions are underlined. † The gap between amino acids in F protein cleavage site indicates position of cleavage. Infection of rhesus macaques Adult rhesus macaques (Macaca mulatta) were obtained from Morgan Island. The animals were confirmed to be seronegative for APMV serotypes by hemagglutination inhibition (HI) and plaque reduction assays. The rhesus macaques (4 animals per virus) were infected by the combined intranasal and intratracheal routes using a 1 ml inoculum per site containing 106.0 PFU per mL of the indicated virus, as described previously [47]. Nasal washes (6 ml/animal) and fecal swabs (4 ml/animal) were collected daily on days 0–10, 12, and 21 in phosphate buffered saline (PBS). Bronchoalveolar lavages (BAL; 10 ml/animal) were collected on days 2, 4, 6 and 8 in PBS. Tracheal lavages (3 ml/animal) were collected on days 10, 12 and 21 in PBS. All samples were snap-frozen on dry ice and stored at -80° C until analyzed. Serum samples were obtained on days 0, 21 and 28. Clinical observations were performed daily for 28 days after the inoculation. Virus detection and quantification The nasal wash, BAL, tracheal lavage and fecal samples from each monkey were clarified by centrifugation, and supernatants were collected. The clarified samples were diluted in serial 10-fold dilutions in PBS. The dilutions 100, 101, 102, 103 and 104 (0.1 ml each) were inoculated in triplicate into the allantoic cavity of 9-day-old embryonated chicken eggs. Eggs were incubated at 37°C for 4 days. Allantoic fluid was collected from each egg and the presence of virus was detected by hemagglutination (HA) test with 0.5% chicken RBC. The 50 percent egg infectious dose (EID50) virus titer (expressed as log10 EID50 per mL) was determined by the method of Reed and Muench [48]. Replication of APMV-5 in rhesus monkeys was determined by titration in Vero cells, followed by neuramindase (NA) assay as described by Huang et al., 2004 [49]. Serological analysis The serum antibody levels to the specific APMV serotype used for immunization were evaluated pre- and post-immunization by hemagglutination inhibition (HI) assay, virus neutralization (VN) assay, and Western blot analysis, except for the serum antibody response to APMV-5, which was evaluated by VN and by neuraminidase inhibition (NAI) assay. For the HI assay, 25 µl of each serum sample was first treated with 50 µl of receptor destroying enzyme II (catalog number YCC 340–122; Accurate Chemical and Scientific, Westbury NY) at a 1∶3 ratio (vol/vol) at 37°C overnight. Then, 25 µl of 5% sodium citrate was added and incubation was continued at 56°C for 30 min. Each serum sample was allowed to cool to room temperature and 100 µl of packed chicken RBCs were added. After incubation at 4°C for 30 min, samples were centrifuged at 1000 × g for 10 min. Supernatants were used for HI assay. For the HI assay, twofold serial dilutions of treated sera (50 µl) were prepared, and each dilution was combined with 4 HA units of a particular live and homologous APMV serotype. Following 1 h of incubation, 50 µl of 1% chicken RBC was added and incubated for 30 min at room temperature, and HA was scored as the mean reciprocal log2 (± standard errors of the mean) of the highest serum dilution causing complete inhibition of four HA units of the indicated APMV. In case of APMV-5, antibody titers were measured by NAI assay. For the NAI assay, APMV-5 strain budgerigar/Kunitachi/74 was used as the source of NA. The NA activity of APMV-5 was measured by a modified fluorometric assay [49]. Briefly, serial twofold dilutions of serum samples were prepared in 20 µl volumes of enzyme buffer (33 mM 2-N-morpholino ethanesulfonic acid [MES], pH 6.5, and 4 mM calcium chloride) in a 96-well plate. 20 µl APMV-5, diluted in enzyme buffer to a constant NA amount (an optical density at 450 nm [OD450] of 100,000), was added as source of NA, and incubated for 1 h at room temperature. Ten microliters of 12.5% (vol/vol) dimethyl sulfoxide was added to each well of a fluorometric assay plate (black 96-well plates; Microfluor, Franklin, MA). Ten microliters of each serum and virus mixture was transferred in duplicate to the assay plate. 10 µl of diluted virus or enzyme buffer alone were used as positive and negative controls, respectively. The reaction was initiated by the addition of 30 µl of substrate mix [1 volume of 330 mM MES, pH 6.4; 3 volumes of 10 mM calcium chloride; and 2 volumes of 0.5 mM 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUN) (Sigma)] to give a final concentration of 100 µM MUN in the assay. The reaction mixture was incubated at 37°C for 15 min with shaking, and the reaction was terminated by the addition of 150 µl of termination buffer (0.014 M sodium hydroxide in 83% [vol/vol] ethanol). The extent of the reaction was quantified by fluorometry at an excitation wavelength of 360 nm and an emission wavelength of 450 nm using the Victor3 multilabel plate reader (PerkinElmer). Readings from the substrate blanks were subtracted from the virus sample readings. The average background-corrected NA activity was calculated from 12 independent wells. The neuraminidase inhibition (NAI) titer for each sample was reported as the reciprocal log2 of the highest serum dilution resulting in a 50% or greater reduction in input NA activity. Neutralizing antibody titers in post-immunization sera collected on day 28 from rhesus macaques were measured in a microneutralization assay. Briefly, 2-fold serial dilutions of 100 µl of heat-inactivated monkey serum samples were mixed with 100 TCID50 of the homologous APMV serotype and incubated at 37°C for 1 h. Following incubation, a 50 µl volume of the virus-serum mixture from each serum dilution was added to approximately 150 µl of medium in quadruplicate wells of DF-1 (for APMV-2, -3, -4 and -9) or Vero (for APMV-5 and -7) cells in 96-well plates. The plates were then incubated at 37°C for 72 h and were scored for virus replication based on HA activity of the supernatant. The titer was defined as the the mean reciprocal log2 of the highest serum dilution resulting in complete neutralization of infection in 50% of the wells. Sera from infected rhesus monkeys were further assayed for APMV-specific antibodies by Western blotting. Partially purified APMV (5 µg per lane) were subjected to 10% SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. Membrane strips were incubated with a 1∶500 dilution of day 0 and day 28 serum samples from each individual monkey from each group followed by incubation with a 1∶5,000 dilution of HRP-conjugated goat anti-human IgG (Fab specific). The hemagglutinin (HN), fusion (F0 and F1) nucleocapsid (NP), phophoprotein (P) and matrix (M) proteins specific to a particular APMV serotype were detected by a chemiluminescence assay. Results Infection of macaques with APMVs and absence of clinical disease Adult rhesus macaques in groups of four were inoculated by the combined intranasal and intratracheal routes with 106 PFU per site of nine APMV strains representing six serotypes, namely serotypes 2, 3, 4, 5, 7, and 9 (Table 1 and Materials and Methods). Five strains were biologically derived wt viruses: APMV-2, -3, -4, -5, -7, and -9. Another virus was rAPMV-4, a recombinant derivative of wt APMV-4. The remaining three viruses, called rAPMV-2 (type 1 Africa), rAPMV-4/Fcs-BC, and rAPMV-7/Fcs-5B, were versions of wt rAPMV-2, -4, and -7, respectively, in which the naturally-occurring non-multi-basic F cleavage site was modified into a multi-basic site containing the optimal furin motif (Table 1). The animals were observed daily from days 0–10 and on days 12, 21 and 28 for any clinical signs of illness or distress. None of the animals exhibited any clinical signs of infection such as weight loss, increased body temperature, fever, or signs of respiratory distress, and no deaths were recorded. Thus, none of these APMV strains and cleavage site mutants was overtly pathogenic in rhesus macaques. Replication of APMV strains in the upper and lower respiratory tract of rhesus macaques To evaluate replication of the APMVs in the upper respiratory tract of rhesus macaques, nasal washes were collected from days 0 to 10 and on days 12 and 21. Virus titers were determined by titration in embryonated chicken eggs, with the exception of APMV-5, which does not grow in chicken eggs. Thus, serial dilutions were prepared from the nasal washes and inoculated into the allantoic cavity of embryonated 9-day-old chicken eggs, and 4 dpi the allantoic fluid was collected and assayed for the presence of virus by HA assay. Virus titers were expressed as log10 EID50 per mL (Table 2). Replication of APMV-5 was determined by a limiting dilution assay on Vero cells in which virus-infected wells were detected by NA activity. 10.1371/journal.pone.0075456.t002Table 2 Shedding of the indicated APMV strains from the upper respiratory tract of rhesus macaques.* Virus Animal No. Nasal wash virus titer (log10 EID50) on indicated days † 0 1 2 3 4 5 6 7 wt APMV-2 1 – 0.8 – – – – – – 2 – 0.8 – – – 1.8 – – 3 – 1.5 – 0.8 – – – 1.3 4 – 1.5 – – – 1.5 – – Mean ‡ – 1.1 0.4 0.5 0.4 1.0 0.4 0.6 rAPMV-2 (type1 Africa) 1 – – – – – 0.8 – – 2 – – – – – – – – 3 – 0.8 – 0.8 – 0.8 – 1.3 4 – 1.5 1.5 2.5 1.5 0.8 1.3 – Mean – 0.8 0.7 1.0 0.7 0.7 0.6 0.6 wt APMV-3 1 – – – – – – – – 2 – – – 1.4 2.3 1.8 – 1.3 3 – – – – – 0.8 – – 4 – 0.8 – – – – – Mean – 0.5 0.4 0.6 0.9 0.8 0.4 0.6 wt rAPMV-4 1 – – – – – – – – 2 – – – – – – – – 3 – – – – – – – – 4 – – – – – – – – Mean – 0.4 0.4 0.4 0.4 0.4 0.4 0.4 rAPMV-4/Fc-Bc 1 – – – – – – – – 2 – – – – – – – – 3 – – – – – – – – 4 – – – – – – – – Mean – 0.4 0.4 0.4 0.4 0.4 0.4 0.4 wt APMV-5 § 1 – – – – – – – – 2 – – – – – – – – 3 – – – – – – – – 4 – – – – – – – – Mean – – – – – – – – wt APMV-7 1 – 0.8 – – – 1.8 1.5 – 2 – 1.5 – – 2.3 2.3 0.8 – 3 – 1.5 – – – 1.5 0.8 – 4 – 0.8 – 0.8 0.8 – – – Mean – 1.1 0.4 0.5 1.0 1.5 0.9 0.4 rAPMV-7/Fcs-5B 1 – 1.3 – – – – – – 2 – 0.8 – – – – – – 3 – – – – – – – – 4 – – – – – – – – Mean – 0.7 0.4 0.4 0.4 0.4 0.4 0.4 wt APMV-9 1 – 0.8 – – – – – – 2 – 1.3 – – – – – – 3 – – 1.3 0.8 – – – 1.3 4 – 1.3 1.5 1.5 2.6 1.3 1.3 – Mean – 0.9 0.9 0.8 0.9 0.6 0.6 0.6 * Rhesus macaques in groups of four were inoculated simultaneously by the intranasal and intratracheal routes with a 1-ml inoculum per site containing 6.0 log10 PFU per ml of the indicated virus on day 0. Nasal washes were performed daily from day 0 to day 10 and on day 12. † The 50 percent egg infectious dose (EID50) virus titer (expressed as log10 EID50 per mL), determined as described in Materials and Methods. Limit of detection was 0.8 log10 per mL. For calculation of daily means, a value of 0.4 was used for samples with no detectable virus. ‡ For calculation of daily means, a value of 0.4 log10 per mL was used for samples with no detectable virus. § Shedding of wt APMV-5 was measured by limiting dilution assay of nasal wash fluid on Vero cells, followed by virus detection using a neuraminidase assay. Virus shedding from the upper respiratory tract was undetectable for 3 strains, namely wt rAPMV-4, rAPMV-4/Fc-BC, and wt APMV-5. For the other strains, low levels of shedding were detected in most of the animals. Specifically, wt APMV-2 was detected in nasal washes from all 4 animals on day 1, from 1 animal on day 3, 2 animals on day 5 and from 1 animal on day 7. The mean daily titers ranged from 0.8 to 1.1 log10 EID50 per mL, and the highest mean daily titer was detected on day 1. Replication of the recombinant version of APMV-2 with multi-basic F cleavage site derived from serotype 1 (Africa) was detected in 3 of 4 animals: 1 animal shed low titers of virus consecutively from days 1 to 6, another shed virus on 1 day, and another shed virus on 4 days. Shedding of wt APMV-3 was detected in 3 of 4 animals: 1 animal shed on days 3, 4, 5, and 7, while 2 animals shed detectable virus only on single days. Wt APMV-7 was isolated from all inoculated animals on day 1 and from either 1, 2 or 3 animals on days 3 to 6, whereas rAPMV-7/Fcs-5B was isolated only on day 1 from 2 animals and was not detected any of the animals on other days. Wt APMV-9 was detected on a single day (day 1) in 2 animals, while 2 animals shed over several days. To evaluate the replication of APMVs in the lower respiratory tract of the inoculated animals, BALs were collected on days 2, 4, 6 and 8, and tracheal lavages were collected on days 10 and 12. All samples were processed and titrated as described above. Virus was detected only in the BAL samples (Table 3). Each strain except for wt APMV-5 replicated detectably in the lower respiratory tract of rhesus macaques. Specifically, wt APMV-2 was detected in all 4 animals on day 2, and in 1 or 2 animals on days 4 and 6, respectively (Table 3). Replication of rAPMV-2 (type 1 Africa) seemed to be more robust: virus shedding was detected from day 2 to day 6 in all of the animals, with mean daily titers ranging from 1.5 to 2.5 log10 EID50 per mL. Shedding of wt APMV-3 was detected in all inoculated animals until day 4, and in 2 animals until day 6, with mean daily titers of 0.9 to 2.2. Interestingly, while wt rAPMV-4 had not been detected in nasal washes from any animal, as described above (Table 2), this virus was detected on day 2 in BALs of 2 animals (Table 3). Similarly, the F cleavage mutant rAPMV-4/Fc-BC had not been detected in nasal wash specimens (Table 2), it was detected on days 2 and/or 4 in BALs of 3 of 4 inoculated animals. Whereas wt rAPMV-2 was detected in BALs only on day 2, the cleavage site mutant derivative was detected on days 2 and 4. Consistent shedding of wt APMV-7 and rAPMV-7/Fcs-5B was detected in all of the animals from days 2 to 6. Mean daily titers for the 2 viruses were similar and ranged from 1.2 to 3 log10 EID50 per mL. Thus, while it appeared that the cleavage site mutation Fcs-5B had a negative effect on replication of APMV7 in the upper respiratory tract (Table 2), it did not affect replication of APMV-7 in the lower respiratory tract (Table 3). Both versions of APMV-7 were detected more readily from the lower versus the upper respiratory tract. Wt APMV-9 was detected on day 2 in 3 of the 4 inoculated animals, and in 1 animal each on day 4 and 6. 10.1371/journal.pone.0075456.t003Table 3 Detection of the indicated APMV strains from the lower respiratory tract of rhesus macaques.* Virus Animal No. Virus titer in BAL (log10 EID50) on indicated days † Sum of daily titers ‡ 2 4 6 8 wt APMV-2 1 2.3 0.8 – – 3.9 2 1.5 – 1.8 – 4.1 3 2.5 – – – 3.7 4 2.3 1.5 – – 4.6 Mean § 2.1 0.8 0.7 0.4 4.1 rAPMV-2 (type1 Africa) 1 2.5 2.5 2.5 – 7.9 2 2.5 1.5 1.8 – 6.2 3 2.5 2.3 0.8 – 6.0 4 2.5 1.5 0.8 – 5.2 Mean 2.5 1.9 1.5 0.4 6.3 wt APMV-3 1 2.5 1.8 1.5 – 6.2 2 1.5 1.5 – – 3.8 3 2.3 2.3 1.3 – 6.3 4 2.5 1.5 – – 4.8 Mean 2.2 1.8 0.9 0.4 5.3 wt rAPMV-4 1 1.8 – – – 3.0 2 0.8 – – – 2.0 3 – – – – 1.6 4 – – – – 1.6 Mean 0.8 0.4 0.4 0.4 2.0 rAPMV-4/Fc-Bc 1 – 1.5 – – 2.7 2 – – – – 1.6 3 1.3 – – – 2.5 4 1.5 0.8 – – 3.1 Mean 0.9 0.8 0.4 0.4 2.5 wt APMV-5 ¶ 1 – – – – – 2 – – – – – 3 – – – – – 4 – – – – – Mean – – – – – wt APMV-7 1 3.5 2.3 1.4 – 7.6 2 2.8 1.5 1.5 – 6.2 3 2.8 1.5 1.3 – 6.0 4 2.8 1.8 0.8 – 5.8 Mean 3.0 1.8 1.2 0.4 6.4 rAPMV-7/Fcs-5B 1 3.5 1.8 2.3 – 8.0 2 2.5 3.5 0.8 – 7.2 3 3.5 1.8 2.5 – 8.2 4 2.5 1.5 0.8 – 5.2 Mean 3.0 2.1 1.6 0.4 7.1 wt APMV-9 1 – 1.3 – – 2.5 2 2.3 – – – 3.5 3 1.5 – – – 2.7 4 0.8 – 0.8 – 2.4 Mean 1.2 0.6 0.5 0.4 2.8 * This is a continuation of the experiment described in Table? 2. BAL was performed on days 2, 4, 6, and 8. † The 50 percent egg infectious dose (EID50) virus titer (expressed as log10 EID50 per mL) was determined as described in materials and methods. Limit of detection was 0.8 log10 per mL. ‡ The sum of daily titers (area under the curve) was used to determine magnitude of virus shedding. A value of 0.4 was used for samples with no detectable virus. § For calculation of daily means, a value of 0.4 log10 per mL was used for samples with no detectable virus. ¶ Shedding of wt APMV-5 was determined by limiting dilution assay of BAL fluid on Vero cells, followed by virus detection using a neuraminidase assay. For an overall comparison of virus replication in the lower respiratory tract, the sum of the daily BAL titers from days 2 to 8 was calculated for each animal, and the mean sum of daily titers of each virus was calculated (Table 3). This identified wt rAPMV-4, rAPMV-4/Fc-BC, and wt AMPV-9 as the viruses with the lowest level of replication in the lower respiratory tract [in the order: wt rAPMV-4 (2.0 log10) < rAPMV/4Fc-BC (2.5 log10) < wt APMV-9 (2.8 log10)]. Wt APMV-2 and wt APMV-3 replicated to slightly higher overall levels [wt APMV-2 (4.1 log10) < wt APMV3 (5.3 log10)], while rAPMV-2 (type 1 Africa), wt APMV-7, and rAPMV7/Fcs-5B were identified as the viruses with the highest mean sum of daily titers [rAPMV-2 (type 1 Africa) (6.3 log10) < wt APMV-7 (6.4 log10) < rAPMV7/Fcs-5B (7.1 log10)]. Interestingly, the mean sum of daily titers of rAPVM-2 with F cleavage site from type 1 Africa was significantly higher than that of wt rAPMV2, showing that the replacement of the F cleavage site by that of type 1 Africa significantly increased the replication of APMV-2 in the lower respiratory tract (One Way Anova with Tukey post-hoc analysis). In summary, APMV of serotypes 2, 3, 7 and 9, and derivatives thereof with F cleavage site mutations replicated in rhesus macaques at a low-to-moderate level over a period of days. rAPMV-2 (type 1 Africa) and wt APMV7 showed the most consistent replication in both the upper and lower respiratory tract of rhesus macaques. Replication of APMV serotype 4 was undetectable in the upper respiratory tract; in the lower respiratory tract, a low level of shedding, close to the level of detection, was detected. No virus shedding was detectable from the upper or lower respiratory tract for any of the viruses after day 7 post-infection, indicating that replication of all of these viruses was self-limiting. Furthermore, there was no detectable spread of the any of these APMVs to the gastrointestinal tract (not shown). Only one of the viruses tested, namely wt APMV-5, could not be detected nasal washes or BAL from any of the inoculated animals. APMV serotype-specific serum antibody responses Serum samples of the rhesus macaques infected with the various APMV strains were collected on days 0, 21 and 28 dpi. Serotype-specific serum antibody titers were determined by a modified HI assay using chicken erythrocytes, except in the case of APMV-5, in which case titers were determined by an NI assay in Vero cells (Table 4). All the animals were confirmed to be seronegative for APMV-specific antibodies on day 0. Each of APMV serotypes induced a serum antibody response. However, the magnitude of the response varied. On day 21, the highest mean HI antibody titers were observed in rhesus macaques immunized wt APMV-2 (7.00 log2 ± 1.4). Moderate mean HI antibody titers were observed with wt APMV-3 (5.8±0.5), wt APMV-7 (5.3±0.5), rAPMV-7/Fcs-5B (4.5±0.6) and wt APMV-9 (4.50±0.6), and the lowest mean HI antibody titers were observed with rAPMV-2 (type 1 Africa) (3.3±0.5) and wt APMV-5 (2.5±1.7). In case of wt rAPMV-4 and rAPMV-4/Fc-BC, only two of the four animals had serum HI antibody responses. The mean HI antibody titer values determined on day 28 were similar to day 21 values. 10.1371/journal.pone.0075456.t004Table 4 Serum antibody responses in rhesus macaques infected with the indicated APMV.* Virus Animal No. HI antibody titer † (mean reciprocal log2 ± standard error) Neutralizing antibody titer ‡ (mean reciprocal log2 ± standard error Day Day 21 28 28 wt APMV-2 1 7 7 2 2 8 7 3 3 5 5 2 4 8 7 5 Mean 7.0±1.4 6.5±1.0 3.0±1.4 rAPMV-2 (type1 Africa) 1 3 3 0 2 3 3 0 3 3 3 0 4 4 4 0 Mean 3.3±0.5 3.3±0.5 0 wt APMV-3 1 6 6 7 2 6 6 7 3 6 6 5 4 5 5 6 Mean 5.8±0.5 5.8±0.5 6.3±1.0 wt rAPMV-4 1 3 4 7 2 0 0 6 3 0 0 5 4 4 4 6 Mean 1.8±2.1 2.0±2.3 6.0±0.8 rAPMV-4/Fc-Bc 1 0 0 4 2 4 4 6 3 0 0 4 4 3 3 5 Mean 1.8±2.1 1.8±2.1 4.8±1.0 wt APMV-5 § 1 3 3 3 2 3 3 3 3 0 0 0 4 4 4 0 Mean 2.5±1.7 2.5±1.7 1.5±1.7 wt APMV-7 1 5 5 3 2 5 5 3 3 6 7 6 4 5 6 5 Mean 5.3±0.5 5.8±1.0 4.3±1.5 rAPMV-7/Fcs-5B 1 4 4 2 2 5 5 4 3 5 5 3 4 4 4 2 Mean 4.5±0.6 4.5±0.6 2.8±1.0 wt APMV-9 1 5 6 10 2 5 5 9 3 4 4 9 4 4 5 10 Mean 4.5±0.6 5.0±0.8 9.5±0.6 * This is continuation of the experiment described in Tables? 2 and 3. Serum samples were collected before inoculation on day 0, and after inoculation on days 21 and 28. † The hemagglutination inhibition (HI) titer is expressed as the mean reciprocal log2 (± standard errors of the mean) of the highest serum dilution causing complete inhibition of four HA units of the indicated APMV, except for APMV-5, which was evaluated by neuraminidase inhibition (NI) assay. All the values are averages from three independent experiments. The mean serum HI titers in all the day 0 serum samples were 0. ‡ The serum neutralizing antibody titers were expressed as mean reciprocal log2 of the serum dilution resulting in complete neutralization of infection in 50% of the wells (mean ± standard errors of the mean). The limit of detection is 0. The mean neutralizing antibody titers in all the day 0 serum samples were 0. § The antibody response to APMV-5 was evaluated by NI assay as described in Materials and Methods. The NI titer is expressed as the mean reciprocal log2 (± standard errors of the mean) of the highest serum dilution causing neuraminidase inhibition. The ability of the serum samples collected on day 28 to neutralize the respective APMV serotype was assessed by a micro-neutralization assay (Table 4). The antisera from the monkeys immunized with wt APMV-9 had the highest mean neutralizing antibody titer of log2 9.5±0.6. The next highest titer was observed in monkeys infected with wt rAPMV-3 (6.3±1.0). The neutralizing antibody titer in monkeys infected with other serotypes decreased in the order: wt rAPMV-4 (6.0±0.8) > rAPMV-4/Fc-BC (4.8±1.0) > wt APMV-7 (4.3±1.5) > wt APMV-2 (3.0±1.4) > rAPMV-7/Fcs-5B (2.8±1.0). Surprisingly, serum neutralizing antibodies were not detected in the case of rAPMV-2 (type 1 Africa). In this study, no direct correlation was found between HI titer and neutralizing titer. In addition, we examined the serum samples collected on day 28 from rhesus monkeys for reactivity against different APMV proteins in purified virions from egg allantoic fluids by Western blotting assay (Fig. 1). We did not examine APMV-5 because infectious virus was not recovered from any of the animals, and thus infectivity is minimal or negative. We probed the Western blots with pre-immune and immune serum samples from all the four monkeys infected with each of the other APMVs. Every animal reacted to the respective NP, F1, P and M proteins with varying degrees of intensities when 5 µg of each APMV virus preparation were run on the gels and blotted to nitrocellulose. For the HN and F0 proteins, reactive bands were recognized to varying extents or not at all by sera from individual animals, which is consistent with the known drastic changes in immune reactivity of paramyxovirus HN and F proteins when denatured [50]. All pre-immune serum samples were found negative. 10.1371/journal.pone.0075456.g001Figure 1 Analysis of the reactivity of sera from rhesus macaques collected following infection with the indicated APMVs, evaluated by Western blotting against purified virus of the homologous APMV strain. Virus representing each of the indicated APMV strains was purified from sucrose gradient centrifugation from allantoic fluid from infected eggs, and was denatured and reduced and subjected to 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. The membranes were cut into strips and incubated with 1∶500 dilutions of day 0 and 28 sera from each of the monkeys in each group. The positions of the HN, F0, NP, F1, P and M proteins are indicated. The positions and sizes (kDa) of size markers are indicated to the left. Discussion APMVs are frequently isolated from wide variety of avian species around the world and have been grouped into nine established serotypes, although two additional serotypes have been provisionally identified (Introduction). Among the nine established serotypes, APMV-1 (NDV) is the most extensively characterized due to its importance as a major pathogen of poultry. APMV serotypes 2 to 9 are frequently isolated from both domestic and wild birds, but have been largely uncharacterized until recently. NDV has been shown to infect not only avian species but also non-avian species, although its replication is restricted in non-avian hosts [29], [30]. NDV is being developed as promising viral vaccine vector for delivery of a number of antigens of animal and human pathogens [51]. However, the potential of APMV-2 to -9 as vaccine vectors for humans was not known. Previously, we demonstrated the replication of APMV-2 to -9 in hamsters and mice [4], [5]. However, low phylogenetic and anatomic relatedness between the rodent models and humans necessitate the use of more relevant models for assessment of replication and pathogenicity of APMV-2 to -9. Therefore, the goal of this study was to evaluate the replication and pathogenicity of a number of non-NDV APMV serotypes in non-human primates. Rhesus macaques were evaluated for permissiveness to infection, clinical disease, magnitude of replication, shedding and induction of antibodies. Rhesus macaques have been used commonly as a non-human primate model to study replication and pathogenicity of various viruses, to screen attenuation phenotypes of live virus vaccines, and to evaluate immune responses of virus vaccine candidates. The safety, immunogenicity, and protective efficacy of NDV has also been demonstrated in this model [29]–[33], [51]. In this study, we chose to evaluate nine different APMV strains. Six of these were wt viruses representing six different serotypes, specifically biologically-derived wt APMV -2, -3, -5, -7, and -9, as well as a recombinant version of wt APMV-4. We chose wt APMV-2, -5, -7 and wt rAPMV-4 because we previously showed that replication of these viruses in cell culture does not require, and is not enhanced by, exogenous protease supplementation [8], [11], [13], [19]. Since this property is known to correlate with pantropic replication and virulence by NDV in chickens, it was possible that it might confer increased replication of these non-NDV APMVs in the rhesus macaques, although we note that this NDV paradigm had not been consistent for these non-NDV APMVs in chickens [16], [18], [19]. Wt APMV-3 was selected because this virus replicates to high titer in cell culture and a reverse genetics system has been developed for this serotype [9], [17]. Wt APMV-9 was selected because this virus replicates well in nasal turbinate and lungs of mice and hamsters [4], [5]. The other three viruses evaluated in this study were recombinant versions of APMV-2, -4, and -7 in which the naturally-occurring F protein cleavage sites were replaced by multi-basic cleavage sites containing the optimal furin motif. Specifically, rAPMV-2 (type 1 Africa) contained the multi-basic cleavage site RRRRR↓F that is present in a virulent African NDV strain; rAPMV-4/Fc-BC contained the cleavage site RRQKR↓F derived from the mesogenic NDV strain Baudette C; and rAPMV-7/Fcs-5B contained the cleavage site RRKKR↓F derived from the velogenic NDV strain Nigeria/95. As noted, in NDV, multi-basic cleavage sites generally are associated with greater virulence in chickens. Rhesus macaques infected with APMV-2, -3, -4, -5, -7 and -9 showed no signs of any disease. APMV-2, -3, -7, and -9 replicated to low titers in upper respiratory tract as evidenced by low level of shedding of these viruses in nasal washes of animals from day 1 to 7. APMV-4 and -5 were found negative for shedding from upper respiratory tract. Similarly, we previously showed that rhesus macaques or African green monkeys that were infected by the combined intranasal and intratracheal routes with NDV strain BC were mostly negative for viral shedding [29]. Together, these results indicate that APMVs are highly restricted in replication in the upper respiratory tract of monkeys. However, analysis of BAL samples in the present study revealed replication of all these APMVs except wt APMV-5 in lower respiratory tract until day 6 post infection, although at different levels. The magnitude of shedding of wt APMV-2, rAPMV-2 (type 1 Africa), wt APMV-3, wt APMV-7, and rAPMV-7/Fcs-5B was moderate, while in case of wt APMV-9, wt rAPMV-4, and rAPMV-4/Fc-BC it was low. In contrast, a very low level of shedding (as determined by plaque assay of tracheal lavages or samples of lung tissues) was detected from lower respiratory tract of AGMs infected with NDV in earlier studies [29]. This indicates that, compared to NDV, wt and recombinant APMV-2 and 7, and wt APMV-3 replicate more efficiently in the lower respiratory tract in non-human primates. This apparent increased replication may provide an advantageous increase in immunogenicity for an expressed foreign antigen. Despite the somewhat greater replication of certain APMVs in the respiratory tract, analysis of fecal samples of rhesus macaques infected with all these APMVs revealed no shedding, suggesting a lack of spread to the gastrointestinal tract. These observations suggest that like NDV, APMV-2, -3, -4,-7 and -9 are highly restricted and highly attenuated in non-human primates. For a number of the APMV strains, replication in the upper respiratory tract appeared to be less efficient compared to the lower respiratory tract. One possible explanation is that there might be a lower density of APMV receptors in the upper respiratory tract of non-human primates compared to lower respiratory tract, and hence a lower level of replication. A second possible factor is that birds have a higher body temperature compared to primates. Therefore, both the upper and lower respiratory tract of primates may be suboptimal for APMV replication, and this effect would be more pronounced in the upper respiratory tract due to its relatively lower temperature. The low level of shedding of APMVs from upper respiratory tract of non-human primates predicts that shedding also should be reduced from humans, which would limit release into the environment and would be a desirable feature for a vaccine vector. We previously showed that, whereas wt APMV-2, -4, and -7 did not form syncytia or plaques in cell culture, despite their independence from added protease, in each case the introduction of the respective multi-basic site resulted in a gain-in-function conferring the ability to form syncytia and plaques [16], [18], [19]. The introduction of the multi-basic sites also increased virus replication in vitro - by 10-fold [16], [18], [19]in the case of rAPMV-2 (type 1 Africa) and rAPMV-7/Fcs-5B, and by 500- to 5000-fold in the case of rAPMV-4/Fc-BC. However, there was no observed difference in replication or pathogenicity in chickens [16], [18], [19]. For the purpose of developing APMV vaccine vectors for possible human use, these mutations had at least two potential advantages. First, the increased growth in vitro would facilitate vaccine manufacture. Second, the cleavage site mutations might increase vector replication in primates, in which case a modest increase in replication might increase the immunogenicity of an expressed foreign antigen while retaining the attenuation phenotype. In the present study, the introduction of the multi-basic sites were associated with an apparent increase in replication in the case of rAPMV-2 (type 1 Africa), and possibly a marginal increase in the case of rAPMV-4/Fc-BC, and an apparent decrease in replication in the case of rAPMV-7/Fcs-5B. With respect to the absence of clinical disease, the mutants remained as attenuated as their wt parents. However, a larger study would be required to confirm these preliminary observations. A single inoculation via combined intranasal and intratracheal routes in rhesus macaques with wt APMV-2, wt APMV-3, wt APMV-7, rAPMV-7/Fcs-5B and wt APMV-9 induced substantial HI antibody titer, whereas serum HI antibody responses induced by rAPMV-2 (type 1 Africa), wt rAPMV-4, rAPMV-4/Fc-BC, and wt APMV-5 was low. The HI titers correlated either completely or partially with the level of shedding from the lower respiratory tract except in the case of wt APMV-5, for which no shedding was observed but a low level of NI titer was produced. To date, APMV-5 has only been isolated from budgerigars, which are thought to be its natural host [24]. These observations indicated that this virus probably is strongly host restricted. In this study, no direct correlation was found between HI titer and neutralization titer. In the cases of wt APMV-3, rAPMV-4, rAPMV-4/Fc-BC, and wt APMV-9, the neutralization titers were higher compared to the HI titers, whereas the converse was true for the remaining APMVs. Since HI titers reflect antibodies specific for the attachment site of HN whereas neutralizing antibodies reflect broader antibody responses against antigenic sites throughout the HN and F proteins, the instances of relatively higher neutralizing titers might reflect a disproportionately greater contribution from additional epitopes in the HN and F proteins. The serum HI antibody titers induced in rhesus monkeys by wt APMV-2, wt APMV-3, wt APMV-7, rAPMV-7/Fcs-5B, and wt APMV-9 was either higher or equal to the HI antibody titers induced by mesogenic NDV strain BC in African green monkeys in previous studies [52]. These serotypes are thus candidates for further development as vaccines for human use. In the cases of wt rAPMV-4, rAPMV-4/Fc-BC, and wt APMV-5 (where not all of the monkeys seroconverted) and rAPMV-2 (type 1 Africa), the HI antibody titers were lower than that induced by NDV in a previous study [52], suggesting that these serotypes are less promising. One incongruity in the present study is the low titers of HI antibodies, and lack of detectable neutralizing antibodies, induced by rAPMV-2 (type 1 Africa) despite the relatively robust replication of this vector and compared to the greater immunogenicity of its wt APMV-2 parent despite the lower replication of the parent. In summary, we have evaluated the replication and pathogenicity of APMV serotypes-2, -3, -4, -5, -7, and -9 in non-human primates. These respiratory viruses are highly restricted for replication in respiratory tract of rhesus macaques. We have also demonstrated a substantial level of immunogenicity for wt APMV-2, -3 -7 and -9 and recombinant wt APMV-4, APMV-4/Fc-BC, and APMV-7/Fcs-5B in this non-human primate model. As these APMVs are serologically distinct from NDV and from each other, they may be developed as vectors and used in heterologous prime-boost combinations to induce robust systemic and mucosal immune responses against foreign antigens. We thank Daniel Rockemann for his excellent technical assistance and help. ==== Refs References 1 Alexander DJ (2003) Avian paramyxoviruses 2–9;. editor. Ames: Iowa state University Press. 2 Miller PJ , Afonso CL , Spackman E , Scott MA , Pedersen JC , et al (2010 ) Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands . J Virol 84 : 11496 –11504 .20702635 3 Briand FX, Henry A, Brown P, Massin P, Jestin V (2013) Complete genome sequence of a newcastle disease virus strain belonging to a recently identified genotype. Genome Announc 1. 4 Khattar SK , Kumar S , Xiao S , Collins PL , Samal SK (2011 ) Experimental infection of mice with avian paramyxovirus serotypes 1 to 9 . PLoS One 6 : e16776 .21347313 5 Samuel AS , Subbiah M , Shive H , Collins PL , Samal SK (2011 ) Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9 . Vet Res 42 : 38 .21345199 6 Lamb R PJ (2007) Paramyxoviridae: the viruses and their replication. Philadelphia: Lippincott Williams & Wilkins. 7 Rima B AD, Billeter MA, Collins PL, Kingsbury DW (1995) Family Paramyxoviridae. Vienna: Springer-Verlag. 8 Subbiah M , Xiao S , Collins PL , Samal SK (2008 ) Complete sequence of the genome of avian paramyxovirus type 2 (strain Yucaipa) and comparison with other paramyxoviruses . Virus Res 137 : 40 –48 .18603323 9 Kumar S , Nayak B , Collins PL , Samal SK (2008 ) Complete genome sequence of avian paramyxovirus type 3 reveals an unusually long trailer region . Virus Res 137 : 189 –197 .18691616 10 Nayak B , Kumar S , Collins PL , Samal SK (2008 ) Molecular characterization and complete genome sequence of avian paramyxovirus type 4 prototype strain duck/Hong Kong/D3/75 . Virol J 5 : 124 .18937854 11 Samuel AS , Paldurai A , Kumar S , Collins PL , Samal SK (2010 ) Complete genome sequence of avian paramyxovirus (APMV) serotype 5 completes the analysis of nine APMV serotypes and reveals the longest APMV genome . PLoS One 5 : e9269 .20174645 12 Xiao S , Subbiah M , Kumar S , De Nardi R , Terregino C , et al (2010 ) Complete genome sequences of avian paramyxovirus serotype 6 prototype strain Hong Kong and a recent novel strain from Italy: evidence for the existence of subgroups within the serotype . Virus Res 150 : 61 –72 .20206652 13 Xiao S , Paldurai A , Nayak B , Subbiah M , Collins PL , et al (2009 ) Complete genome sequence of avian paramyxovirus type 7 (strain Tennessee) and comparison with other paramyxoviruses . Virus Res 145 : 80 –91 .19540277 14 Paldurai A , Subbiah M , Kumar S , Collins PL , Samal SK (2009 ) Complete genome sequences of avian paramyxovirus type 8 strains goose/Delaware/1053/76 and pintail/Wakuya/20/78 . Virus Res 142 : 144 –153 .19341613 15 Samuel AS , Kumar S , Madhuri S , Collins PL , Samal SK (2009 ) Complete sequence of the genome of avian paramyxovirus type 9 and comparison with other paramyxoviruses . Virus Res 142 : 10 –18 .19185593 16 Subbiah M , Khattar SK , Collins PL , Samal SK (2011 ) Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 2 increase cleavability and syncytium formation but do not increase viral virulence in chickens . J Virol 85 : 5394 –5405 .21450835 17 Kumar S , Nayak B , Collins PL , Samal SK (2011 ) Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3 vector in chickens . J Virol 85 : 6521 –6534 .21525340 18 Xiao S , Khattar SK , Subbiah M , Collins PL , Samal SK (2012 ) Mutation of the f-protein cleavage site of avian paramyxovirus type 7 results in furin cleavage, fusion promotion, and increased replication in vitro but not increased replication, tissue tropism, or virulence in chickens . J Virol 86 : 3828 –3838 .22258248 19 Kim SH , Xiao S , Shive H , Collins PL , Samal SK (2013 ) Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 4 confer increased replication and syncytium formation in vitro but not increased replication and pathogenicity in chickens and ducks . PLoS One 8 : e50598 .23341874 20 Bankowski RA , Almquist J , Dombrucki J (1981 ) Effect of paramyxovirus yucaipa on fertility, hatchability, and poult yield of turkeys . Avian Dis 25 : 517 –520 .7259687 21 Lipkind MA , Weisman Y , Shihmanter E , Shoham D , Aronovici A (1979 ) The isolation of yucaipa-like paramyxoviruses from epizootics of a respiratory disease in turkey poultry farms in Israel . Vet Rec 105 : 577 –578 .532076 22 Alexander DJ , Pattison M , Macpherson I (1983 ) Avian paramyxoviruses of PMV-3 serotype in British turkeys . Avian Pathol 12 : 469 –482 .18766806 23 Tumova B , Robinson JH , Easterday BC (1979 ) A hitherto unreported paramyxovirus of turkeys . Res Vet Sci 27 : 135 –140 .523797 24 Nerome K , Nakayama M , Ishida M , Fukumi H (1978 ) Isolation of a new avian paramyxovirus from budgerigar (Melopsittacus undulatus) . J Gen Virol 38 : 293 –301 .627875 25 Chang PC , Hsieh ML , Shien JH , Graham DA , Lee MS , et al (2001 ) Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks . J Gen Virol 82 : 2157 –2168 .11514725 26 Saif YM , Mohan R , Ward L , Senne DA , Panigrahy B , et al (1997 ) Natural and experimental infection of turkeys with avian paramyxovirus-7 . Avian Dis 41 : 326 –329 .9201395 27 Yamane N , Arikawa J , Odagiri T , Ishida N (1982 ) Characterization of avian paramyxoviruses isolated from feral ducks in northern Japan: the presence of three distinct viruses in nature . Microbiol Immunol 26 : 557 –568 .7132788 28 Capua I , De Nardi R , Beato MS , Terregino C , Scremin M , et al (2004 ) Isolation of an avian paramyxovirus type 9 from migratory waterfowl in Italy . Vet Rec 155 : 156 . 29 Bukreyev A , Huang Z , Yang L , Elankumaran S , St Claire M , et al (2005 ) Recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates . J Virol 79 : 13275 –13284 .16227250 30 DiNapoli JM , Kotelkin A , Yang L , Elankumaran S , Murphy BR , et al (2007 ) Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens . Proc Natl Acad Sci U S A 104 : 9788 –9793 .17535926 31 DiNapoli JM , Yang L , Suguitan A Jr, Elankumaran S , Dorward DW , et al (2007 ) Immunization of primates with a Newcastle disease virus-vectored vaccine via the respiratory tract induces a high titer of serum neutralizing antibodies against highly pathogenic avian influenza virus . J Virol 81 : 11560 –11568 .17715243 32 DiNapoli JM , Nayak B , Yang L , Finneyfrock BW , Cook A , et al (2010 ) Newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of H5N1 highly pathogenic avian influenza virus protect against virus challenge in monkeys . J Virol 84 : 1489 –1503 .19923177 33 DiNapoli JM , Yang L , Samal SK , Murphy BR , Collins PL , et al (2010 ) Respiratory tract immunization of non-human primates with a Newcastle disease virus-vectored vaccine candidate against Ebola virus elicits a neutralizing antibody response . Vaccine 29 : 17 –25 .21034822 34 Huang Z , Elankumaran S , Yunus AS , Samal SK (2004 ) A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV . J Virol 78 : 10054 –10063 .15331738 35 Khattar SK , Collins PL , Samal SK (2010 ) Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1 . Vaccine 28 : 3159 –3170 .20189484 36 Khattar SK , Samal S , Devico AL , Collins PL , Samal SK (2012 ) Newcastle disease virus expressing human immunodeficiency virus type 1 envelope glycoprotein induces strong mucosal and serum antibody responses in Guinea pigs . J Virol 85 : 10529 –10541 . 37 Nayak B , Rout SN , Kumar S , Khalil MS , Fouda MM , et al (2009 ) Immunization of chickens with Newcastle disease virus expressing H5 hemagglutinin protects against highly pathogenic H5N1 avian influenza viruses . PLoS One 4 : e6509 .19654873 38 Nayak B , Kumar S , DiNapoli JM , Paldurai A , Perez DR , et al (2010 ) Contributions of the avian influenza virus HA, NA, and M2 surface proteins to the induction of neutralizing antibodies and protective immunity . J Virol 84 : 2408 –2420 .20032181 39 Xiao S , Kumar M , Yang X , Akkoyunlu M , Collins PL , et al (2011 ) A host-restricted viral vector for antigen-specific immunization against Lyme disease pathogen . Vaccine 29 : 5294 –5303 .21600949 40 Nakaya T , Cros J , Park MS , Nakaya Y , Zheng H , et al (2001 ) Recombinant Newcastle disease virus as a vaccine vector . J Virol 75 : 11868 –11873 .11689668 41 Martinez-Sobrido L , Gitiban N , Fernandez-Sesma A , Cros J , Mertz SE , et al (2006 ) Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector . J Virol 80 : 1130 –1139 .16414990 42 Carnero E , Li W , Borderia AV , Moltedo B , Moran T , et al (2009 ) Optimization of human immunodeficiency virus gag expression by newcastle disease virus vectors for the induction of potent immune responses . J Virol 83 : 584 –597 .19004953 43 Kusagawa S , Komada H , Mao X , Kawano M , Nishikawa F , et al (1993 ) Antigenic and molecular properties of Murayama virus isolated from cynomolgus monkeys: the virus is closely related to avian paramyxovirus type 2 . Virology 194 : 828 –832 .8503187 44 Lipkind M , Shoham D , Shihmanter E (1986 ) Isolation of a paramyxovirus from pigs in Israel and its antigenic relationships with avian paramyxoviruses . J Gen Virol 67 (Pt 3) : 427 –439 . 45 Peeters BP , de Leeuw OS , Koch G , Gielkens AL (1999 ) Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence . J Virol 73 : 5001 –5009 .10233962 46 Panda A , Huang Z , Elankumaran S , Rockemann DD , Samal SK (2004 ) Role of fusion protein cleavage site in the virulence of Newcastle disease virus . Microb Pathog 36 : 1 –10 .14643634 47 Bukreyev A , Lamirande EW , Buchholz UJ , Vogel LN , Elkins WR , et al (2004 ) Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS . Lancet 363 : 2122 –2127 .15220033 48 Reed LJ MH (1938 ) A simple method of estimation of 50% end points . Am J Hyg (Lond) 27 : 493 –497 . 49 Huang Z , Panda A , Elankumaran S , Govindarajan D , Rockemann DD , et al (2004 ) The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence . J Virol 78 : 4176 –4184 .15047833 50 Mottet G , Portner A , Roux L (1986 ) Drastic immunoreactivity changes between the immature and mature forms of the Sendai virus HN and F0 glycoproteins . J Virol 59 : 132 –141 .2423701 51 Bukreyev A , Collins PL (2008 ) Newcastle disease virus as a vaccine vector for humans . Curr Opin Mol Ther 10 : 46 –55 .18228181 52 DiNapoli JM , Ward JM , Cheng L , Yang L , Elankumaran S , et al (2009 ) Delivery to the lower respiratory tract is required for effective immunization with Newcastle disease virus-vectored vaccines intended for humans . Vaccine 27 : 1530 –1539 .19168110	24130713	PMC3794941	CC0	2021-01-05 17:12:46	yes	PLoS One. 2013 Oct 10; 8(10):e75456
==== Front Exp Ther MedExp Ther MedETMExperimental and Therapeutic Medicine1792-09811792-1015D.A. Spandidos 10.3892/etm.2013.1265etm-06-04-0899ArticlesRosuvastatin suppresses platelet-derived growth factor-BB-induced vascular smooth muscle cell proliferation and migration via the MAPK signaling pathway GAN JIANTING 1LI PING 1WANG ZHENGDONG 1CHEN JIAN 1LIANG XIANGWEN 1LIU MING 1XIE WENCHAO 1YIN RUIXING 2HUANG FENG 21 Department of Cardiology, Sixth Affiliated Hospital of Guangxi Medical University, Yulin, Guangxi 537000;2 Department of Cardiology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. ChinaCorrespondence to: Professor Ping Li, Department of Cardiology, Sixth Affiliated Hospital of Guangxi Medical University, 497 Education Road, Yulin, Guangxi 537000, P.R. China, E-mail: guangxiliping2012@163.com10 2013 20 8 2013 20 8 2013 6 4 899 903 24 4 2013 08 8 2013 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.An imbalance in the proliferation and migration of vascular smooth muscle cells (VSMCs) is significant in the onset and progression of vascular diseases, including arteriosclerosis and restenosis subsequent to vein grafting or coronary intervention. Rosuvastatin, a selective inhibitor of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, has pharmacological properties including the ability to reduce low-density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein-cholesterol (VLDL-C) levels, slow atherosclerosis progression and improve coronary heart disease outcomes. However, little is known concerning the molecular mechanism by which rosuvastatin affects vascular cell dynamics. In this study, we studied the inhibitory role of rosuvastatin on platelet-derived growth factor-BB (PDGF-BB)-induced VSMC proliferation and migration, as well as the molecular mechanisms involved. MTT data showed that rosuvastatin markedly inhibited the proliferation of PDGF-BB-induced VSMCs in a time-dependent manner. VSMCs are able to dedifferentiate into a proliferative phenotype in response to PDGF-BB stimulation; however, rosuvastatin effectively attenuated this phenotype switching. Moreover, we also showed that rosuvastatin significantly suppressed PDGF-BB-induced VSMC migration, which may be a result of its inhibitory effect on the protein expression of matrix metalloproteinase-2 (MMP2) and MMP9. Investigation into the molecular mechanisms involved revealed that rosuvastatin inhibited the mitogen-activated protein kinase (MAPK) signaling pathway by downregulating extracellular signal-regulated kinase (ERK) and p38 MAPK, although the phosphorylation level of c-Jun N-terminal kinase (c-JNK) was not altered following rosuvastatin treatment. In conclusion, the present study showed that rosuvastatin suppressed PDGF-BB-induced VSMC proliferation and migration, indicating that rosuvastatin has the potential to become a promising therapeutic agent for the treatment of atherosclerosis and restenosis. rosuvastatinvascular smooth muscle cellplatelet-derived growth factorproliferationmigrationmitogen-activated protein kinase ==== Body Introduction The unbalanced proliferation of vascular smooth muscle cells (VSMCs) acts as a critical factor in the initiation and progression of vascular diseases, such as restenosis and arteriosclerosis, subsequent to coronary intervention or vein grafting (1,2). Therefore, antiproliferative agents for VSMCs may serve as effective strategies for attenuating proliferative vascular diseases, as well as for reducing the incidence of cardiovascular complications, including bypass graft failure and in-stent restenosis (3,4). It has been well established that during the repair of vascular injury, multiple cytokines and growth factors are released that stimulate vascular cell proliferation (5–7). For example, following angioplasty, the upregulated production of platelet-derived growth factor (PDGF) initiates proliferation-related signaling pathways to stimulate VSMC proliferation in response to vascular injury (8,9). As a result, developing effective agents to suppress the PDGF-induced abnormal proliferation of vascular cells shows promise for improving the efficacy of cardiovascular surgery. Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of 3-hydroxy-3-methylglutaryl CoA to mevalonate, a precursor of cholesterol (10). As a result, HMG-CoA reductase inhibitors, such as statins, may be utilized for lowering cholesterol. Among all statins, rosuvastatin is a selective HMG-CoA reductase inhibitor, the main action site of which is the liver, the target organ for lowering cholesterol (11). Rosuvastatin increases the number of hepatic cell surface receptors for low-density lipoprotein-cholesterol (LDL-C), promotes the absorption and catabolism of LDL-C and inhibits the synthesis of very low-density lipoprotein-cholesterol (VLDL-C), thereby reducing total VLDL-C and LDL-C levels. Moreover, rosuvastatin is also able to reduce plasma triglycerides and increase high-density lipoprotein-cholesterol (HDL-C) levels (12). It has been shown that rosuvastatin is able to slow atherosclerosis progression and improve coronary heart disease outcomes (11); however, the molecular mechanism behind the action of rosuvastatin on vascular cell dynamics has not been fully elucidated. Therefore, this study aimed to investigate whether rosuvastatin was able to inhibit PDGF-BB-stimulated VSMC proliferation and migration, as well as the associated molecular mechanism. Materials and methods Materials and agents Rosuvastatin was obtained from AstraZeneca (London, UK). Recombinant mouse PDGF-BB was purchased from Supbio Company (Changsha, China). DMSO and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA), while antibodies for smooth muscle-α-actin (SMA), smoothelin, desmin, phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), ERK, phospho-p38, p38, phospho-c-Jun N-terminal kinase (JNK), JNK, matrix metalloproteinase-2 (MMP2), MMP9 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Cell culture VSMCs were isolated from the thoracic aortas of Sprague Dawley rats, and cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS). VSMCs of passage five were used in this study. MTT assay VSMCs were cultured to 70% confluence and serum-starved for 24 h. In the experimental group, cells were treated with rosuvastatin (10 μM) and PDGF-BB (20 ng/ml) for 6, 12, 24 and 48 h. In the control group, cells were cultured without any treatment. In the negative control (NC) group, cells were treated only with PDGF-BB (20 ng/ml) for 6, 12, 24 and 48 h. Following treatment, an MTT assay was used to examine the viability of the cells in all groups. Cells were plated at a density of 104/well, and incubated at 37°C with 5% CO2 for 3 h, subsequent to adding MTT (Promega, Madison, WI, USA) to the medium at a final concentration of 0.5 μg/ml. The medium was then removed and 100 μl DMSO was added. The plate was gently rotated on an orbital shaker for 10 min to completely dissolve the precipitation. A microplate reader (Bio-Rad, Hercules, CA, USA) was used to determine the absorbance at 570 nm. Cell migration assay For all groups, migration was measured in 24-well Transwell chambers (Chemicon, Temecula, CA, USA). In the control group, cells were cultured without any treatment. In the NC group, cells were cultured following the addition of PDGF-BB (20 ng/ml). In the experimental group, cells were cultured with rosuvastatin (10 μM) and PDGF-BB (20 ng/ml). Subsequent to 24 h incubation at 37°C with 5% CO2, the migrated cells were stained and counted. Western blot analysis In the control group, the cells were cultured without any treatment. In the NC group, cells were cultured following the addition of PDGF-BB (20 ng/ml) for 48 h. In the experimental group, cells were cultured with rosuvastatin (10 μM) and PDGF-BB (20 ng/ml) for 48 h. Cold radio-immunoprecipitation assay (RIPA) lysis buffer was used to solubilize the cells. Protein was separated with 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked in 5% non-fat dried milk in phosphate-buffered saline (PBS) overnight, prior to being incubated with specific primary antibodies (Santa Cruz Biotechnology, Inc.) for 3 h. Primary antibodies for SMA, smoothelin, desmin, p-ERK1/2, ERK, phospho-p38, p38, phospho-c-JNK, JNK, MMP2, MMP9 and GAPDH were used. All antibodies were mouse monoclonal antibodies bought from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Following incubation with rabbit anti-mouse secondary antibody (Santa Cruz Biotechnology, Inc.), immune complexes were detected using an enhanced chemiluminescence (ECL) Western Blotting Substrate kit (Biovision, San Francisco, CA, USA). Statistical analysis Data are expressed as the mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA). All analyses were performed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Results Inhibitory effect of rosuvastatin on the proliferation of PDGF-BB-stimulated VSMCs The effect of rosuvastatin on the proliferation of PDGF-stimulated VSMCs was studied using an MTT assay. As shown in Fig. 1, the cell proliferation rate in the experimental group was significantly reduced in a time-dependent manner when compared with that in the NC group, indicating that rosuvastatin had an inhibitory effect on the cell proliferation of the PDGF-BB-induced VSMCs. Inhibitory effect of rosuvastatin on the PDGF-BB-induced phenotype switching of the VSMCs VSMCs are able to dedifferentiate into a proliferative phenotype in response to vascular injury. Under such conditions, the protein expression of the smooth muscle markers SMA, smoothelin and desmin are decreased. Therefore, we tested whether rosuvastatin was able to regulate the phenotype switching of PDGF-BB-stimulated VSMCs. VSMCs were stimulated with PDGF-BB (20 ng/ml) for 48 h in the presence and absence of 10 μM rosuvastatin. Western blotting data showed that PDGF-BB stimulation reduced the SMA protein expression, indicating the dedifferentiation of the VSMCs into a proliferative phenotype (Fig. 2). However, 10 μM rosuvastatin attenuated this effect, suggesting that rosuvastatin inhibits the switch of PDGF-BB-stimulated VSMCs into a proliferative phenotype. Inhibitory effect of rosuvastatin on the PDGF-BB-stimulated migration of VSMCs We further determined the effect of rosuvastatin on the migration ability of PDGF-BB-stimulated VSMCs. VSMCs were stimulated with PDGF-BB (20 ng/ml) for 48 h in the presence/absence of 10 μM rosuvastatin. As demonstrated in Fig. 3A, PDGF-BB stimulation markedly enhanced the migration of VSMCs when compared with that in the control group without any treatment. However, rosuvastatin significantly inhibited the migration of PDGF-BB-stimulated VSMCs. Western blotting results showed that the protein expression of MMP2 and MMP9 was notably suppressed with rosuvastatin treatment (Fig. 3B). Inhibitory effect of rosuvastatin on the mitogen-activated protein kinase (MAPK) signaling pathway activated by PDGF-BB in VSMCs It has been demonstrated that the MAPK signaling pathway is important in VSMC proliferation in response to PDGF-BB stimulation. Thus, we determined the activity of the MAPK signaling pathway in PDGF-BB-stimulated VSMCs with or without the treatment of rosuvastatin for 48 h. As shown in Fig. 4, western blotting data demonstrated that the phospho-ERK1/2 and phospho-p38 MAPK protein levels in the PDGF-BB-stimulated VSMCs treated with rosuvastatin were significantly lower than those in the PDGF-BB-stimulated VSMCs without rosuvastatin treatment, although the phosphorylation level of c-JNK was not affected. These results indicated that it was likely that rosuvastatin suppressed the proliferation of PDGF-BB-stimulated VSMCs by downregulating the activity of the MAPK signaling pathway. Discussion Rosuvastatin is a selective HMG-CoA reductase inhibitor that has multiple biological activities, which include inhibiting HMG-CoA reductase activity, increasing LDL receptor levels and inhibiting VLDL-C synthesis. As a result, rosuvastatin has been commonly used as an anti-hyperlipidemic therapy. Recently, accumulating evidence has shown that rosuvastatin exhibits anti-arteriosclerotic activity (13). However, the molecular mechanisms of rosuvastatin underlying its actions in vascular diseases, including restenosis and arteriosclerosis, have not been fully elucidated. Vascular injury leads to the marked upregulation of VSMC proliferation and migration, which further results in neointima formation. In the present study, to the best of our knowledge, we showed for the first time that rosuvastatin effectively suppressed PDGF-BB-stimulated VSMC proliferation and migration in vitro, and that these effects may partly be attributed to the downregulation of the activity of the MAPK signaling pathway, as well as the decreased protein expression of MMP2 and MMP9. These data indicate that rosuvastatin may be beneficial in the protection against the neointima formation associated with restenosis and arteriosclerosis subsequent to vein grafting or coronary intervention. It has been demonstrated that vascular injury may affect VSMC plasticity and lead to the dedifferentiation of VSMCs into a proliferative phenotype (14). Our study showed that PDGF-BB treatment inhibited VSMC proliferation, as well as the protein expression of VSMC markers (smooth muscle markers SMA, smoothelin and desmin), indicating that VSMCs dedifferentiated into a proliferative phenotype. However, rosuvastatin effectively attenuated these alterations, suggesting that rosuvastatin is able to inhibit PDGF-BB-induced VSMC proliferation. The migration of VSMCs is crucial in the repair of vascular injury, i.e., the development of restenosis and atherosclerotic lesions subsequent to by-pass graft or angioplasty (15), and PDGF-BB has been revealed to have the ability to induce VSMC migration via multiple mechanisms (16–18). In this study, we showed that rosuvastatin effectively inhibited PDGF-BB-induced VSMC migration, accompanied by the decreased protein expression of MMP2 and MMP9. MMP2 and MMP9 are critical enzymes participating in extracellular matrix (ECM) remodeling, as well as cell proliferation and invasion, and are important in cardiovascular diseases (19–22). Thus, we hypothesize that the inhibitory effect of rosuvastatin on PDGF-BB-induced VSMC migration may be partly attributed to its inhibitory effect on the expression of MMP2 and MMP9. Since the expression levels of MMP2 and MMP9 have been demonstrated to be regulated by the MAPK signaling pathway, which also regulates cell proliferation (23,24), we further determined the phosphorylation levels of three MAPKs in PDGF-BB-stimulated VSMCs with or without rosuvastatin treatment. Although data concerning the phosphorylation of c-JNK revealed no difference, irrespective of rosuvastatin treatment, the phosphorylation levels of ERK1/2 and p38 were significantly upregulated in PDGF-BB-stimulated VSMCs, while rosuvastatin treatment effectively attenuated these effects. This suggests that rosuvastatin had an inhibitory effect on the PDGF-BB-induced MAPK activation in VSMCs. Several other studies have demonstrated that the MAPK signaling pathway participates in the PDGF-BB-induced VSMC proliferation and migration (25,26). Zhao et al (25) showed that ERK nuclear translocation was involved in the PDGF-BB-stimulated migration of VSMCs, while Zhu et al (26) observed that the phosphorylation of ERK1/2 and p38 was markedly induced following PDGF-BB treatment in VSMCs, which was consistent with our results. In conclusion, the present study showed for the first time, to the best of our knowledge, that rosuvastatin inhibited PDGF-BB-induced VSMC proliferation and migration, which are critical in neointimal hyperplasia. Moreover, these protective effects were shown to be associated with the cell cycle arrest, the downregulated activity of the MAPK signaling pathway, as well as reductions in the protein expression levels of MMP2 and MMP9. This study indicated that rosuvastatin showed promising effects for preventing the neointima formation associated with arteriosclerosis and restenosis subsequent to vein grafting or coronary intervention. This study was supported by the Guangxi Science and Technology Department Supporting Project (no. Gui Ke Gong 1140003A-50). Figure 1. Rosuvastatin inhibited the proliferation of platelet-derived growth factor-BB (PDGF-BB)-stimulated vascular smooth muscle cells (VSMCs). Con, VSMCs were cultured without any treatment; NC, VSMCs were treated only with PDGF-BB (20 ng/ml) for 6, 12, 24 and 48 h; rosuvastatin, VSMCs were treated with rosuvastatin (10 μM) and PDGF-BB (20 ng/ml) for 6, 12, 24 and 48 h. An MTT assay was used to examine the viability of cells in all groups. OD, optical density. Figure 2. Rosuvastatin inhibited the platelet-derived growth factor-BB (PDGF-BB)-induced phenotype switching of vascular smooth muscle cells (VSMCs). Con, VSMCs were cultured without any treatment; NC, VSMCs were treated only with PDGF-BB (20 ng/ml) for 48 h; rosuvastatin, VSMCs were treated with rosuvastatin (10 μM) and PDGF-BB (20 ng/ml) for 48 h. The protein expression levels of the smooth muscle markers smooth muscle-α-actin (SMA), smoothelin and desmin were determined. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Figure 3. Rosuvastatin suppressed the platelet-derived growth factor-BB (PDGF-BB)-stimulated migration of vascular smooth muscle cells (VSMCs). Con, VSMCs were cultured without any treatment; NC, VSMCs were treated only with PDGF-BB (20 ng/ml) for 48 h; rosuvastatin, VSMCs were treated with rosuvastatin (10 μM) and PDGF-BB (20 ng/ml) for 48 h. (A) Transwell assay was used to determine the migration of VSMCs. (B) Protein expression of matrix metalloproteinase 2 (MMP2) and 9 (MMP9) was determined by western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. **Significantly different from the NC group (P<0.01). Figure 4. Rosuvastatin inhibited the mitogen-activated protein kinase (MAPK) signaling pathway activated by platelet-derived growth factor-BB (PDGF-BB) in vascular smooth muscle cells (VSMCs). Con, VSMCs were cultured without any treatment; NC, VSMCs were treated only with PDGF-BB (20 ng/ml) for 48 h; rosuvastatin, VSMCs were treated with rosuvastatin (10 μM) and PDGF-BB (20 ng/ml) for 48 h. Western blot analysis was used to determine the protein expression of phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), ERK, phospho-p38, p38, phospho-c-Jun N-terminal kinase (JNK) and JNK. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. ==== Refs References 1. Doran AC Meller N McNamara CA Role of smooth muscle cells in the initiation and early progression of atherosclerosis Arterioscler Thromb Vasc Biol 28 812 819 2008 18276911 2. Hao H Gabbiani G Bochaton-Piallat ML Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development Arterioscler Thromb Vasc Biol 23 1510 1520 2003 12907463 3. Obata JE Nakamura T Kitta Y In-stent restenosis is inhibited in a bare metal stent implanted distal to a sirolimus-eluting stent to treat a long de novo coronary lesion with small distal vessel diameter Catheter Cardiovasc Interv 2 4 2013 (Epub ahead of print). 4. Mazurova VV Sukhorukov OE Zakharova OV Comparing moderately late results of the application of stents coated with a medicinal antiproliferative agent for the treatment of patients with various forms of coronary heart disease: their efficacy and safety Klin Med (Mosk) 90 30 35 2012 (In Russian). 22997717 5. Babapulle MN Eisenberg MJ Coated stents for the prevention of restenosis: part I Circulation 106 2734 2740 2002 12438301 6. Zhao Y Liu YX Xie SL Deng BQ Wang JF Nie RQ Increased expression of granulocyte colony-stimulating factor mediates mesenchymal stem cells recruitment after vascular injury Chin Med J (Engl) 124 4286 4292 2011 22340401 7. Niida T Isoda K Kitagaki M IκBNS regulates inter-leukin-6 production and inhibits neointimal formation after vascular injury in mice Cardiovasc Res 93 371 379 2012 22135163 8. Park ES Kang SI Yoo KD Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway Exp Cell Res 319 982 991 2013 23328306 9. Sun L Zhao R Zhang L Salvianolic acid A inhibits PDGF-BB induced vascular smooth muscle cell migration and proliferation while does not constrain endothelial cell proliferation and nitric oxide biosynthesis Molecules 17 3333 3347 2012 22418933 10. Sohda T Iwata K Hirano G 3-Hydroxyl-3-methylglut aryl-coenzyme A reductase is up regulated in hepatocellular carcinoma associated with paraneoplastic hypercholesterolemia Med Mol Morphol 4 3 2013 11. DiNicolantonio JJ Lavie CJ Serebruany VL O’Keefe JH Statin wars: the heavyweight match - atorvastatin versus rosuvastatin for the treatment of atherosclerosis, heart failure, and chronic kidney disease Postgrad Med 125 7 16 2013 23391667 12. Abel T Fehér J Role of rosuvastatin in current lipid-lowering therapy Orv Hetil 151 1424 1428 2010 (In Hungarian). 20719717 13. Waters DD Exploring new indications for statins beyond atherosclerosis: Successes and setbacks J Cardiol 55 155 162 2010 20206067 14. Zhu L Hao Y Guan H Cui C Tian S Yang D Wang X Zhang S Wang L Jiang H Effect of sinomenine on vascular smooth muscle cell dedifferentiation and neointima formation after vascular injury in mice Mol Cell Biochem 373 53 62 2013 23065380 15. Iida M Tanabe K Matsushima-Nishiwaki R Kozawa O Iida H Adenosine monophosphate-activated protein kinase regulates platelet-derived growth factor-BB-induced vascular smooth muscle cell migration Arch Biochem Biophys 530 83 92 2013 23298764 16. Li P Liu Y Yi B MicroRNA-638 is highly expressed in human vascular smooth muscle cells and inhibits PDGF-BB-induced cell proliferation and migration through targeting orphan nuclear receptor NOR1 Cardiovasc Res 99 185 193 2013 23554459 17. Park ES Lee KP Jung SH Compound K, an intestinal metabolite of ginsenosides, inhibits PDGF-BB-induced VSMC proliferation and migration through G1 arrest and attenuates neointimal hyperplasia after arterial injury Atherosclerosis 228 53 60 2013 23473423 18. Yi N Chen SY Ma A Tunicamycin inhibits PDGF-BB-induced proliferation and migration of vascular smooth muscle cells through induction of HO-1 Anat Rec (Hoboken) 295 1462 1472 2012 22821808 19. Gao J Ding F Liu Q Yao Y Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9 Mol Cell Biochem 376 21 32 2013 23232575 20. Mishra A Srivastava A Mittal T Garg N Mittal B Association of matrix metalloproteinases (MMP2, MMP7 and MMP9) genetic variants with left ventricular dysfunction in coronary artery disease patients Clin Chim Acta 413 1668 1674 2012 22664146 21. Deatrick KB Luke CE Elfline MA The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling J Vasc Surg 3 9 2013 (Epub ahead of print). 22. Halade GV Jin YF Lindsey ML Matrix metalloproteinase (MMP)-9: a proximal biomarker for cardiac remodeling and a distal biomarker for inflammation Pharmacol Ther 139 32 40 2013 23562601 23. Yang JL Lin JH Weng SW Crude extract of Euphorbia formosana inhibits the migration and invasion of DU145 human prostate cancer cells: The role of matrix metalloproteinase-2/9 inhibition via the MAPK signaling pathway Mol Med Rep 7 1403 1408 2013 23525212 24. Santarpia L Lippman SM El-Naggar AK Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy Expert Opin Ther Targets 16 103 119 2012 22239440 25. Zhao Y Lv M Lin H ROCK1 induces ERK nuclear translocation in PDGF-BB-stimulated migration of rat vascular smooth muscle cells IUBMB Life 64 194 202 2012 22215561 26. Zhu L Guan H Cui C Gastrodin inhibits cell proliferation in vascular smooth muscle cells and attenuates neointima formation in vivo Int J Mol Med 30 1034 1040 2012 22922870	24137286	PMC3797300	CC BY	2021-01-04 23:17:10	yes	Exp Ther Med. 2013 Oct 20; 6(4):899-903
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24204775PONE-D-13-1566110.1371/journal.pone.0077228Research ArticleProtein Tyrosine Phosphatase 1B and Insulin Resistance: Role of Endoplasmic Reticulum Stress/Reactive Oxygen Species/Nuclear Factor Kappa B Axis PTP1B and Insulin ResistancePanzhinskiy Evgeniy Ren Jun Nair Sreejayan * School of Pharmacy & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, Wyoming, United States of America Ushio-Fukai Masuko Editor University of Illinois at Chicago, United States of America * E-mail: sreejay@uwyo.eduCompeting Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: EP JR SN. Performed the experiments: EP. Analyzed the data: EP. Contributed reagents/materials/analysis tools: EP SN. Wrote the paper: EP JR SN. 2013 18 10 2013 8 10 e7722816 4 2013 1 9 2013 © 2013 Panzhinskiy et al2013Panzhinskiy et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [3H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH2-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition. This work was supported in part by grants from NIH P20RR016474 (SN, JR). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Obesity has reached epidemic proportions worldwide and is associated with an increased risk of disability and morbidity [1]. Obesity is a major risk factor for the development of stroke, congestive heart failure, myocardial infarction, atherosclerosis, sleep apnea, fatty liver disease, dementia, and cancer [2], [3]. Insulin resistance is a hallmark of obesity-associated metabolic syndrome and type 2 diabetes mellitus. It is characterized by impairment in glucose-uptake by insulin sensitive tissues [4]. Insulin, a key hormone regulating metabolism of glucose and lipids, is produced by pancreatic islet beta-cells and exerts its biological effects by binding and activating the insulin receptor (IR) in insulin sensitive tissues (muscle, liver, adipose). Activated insulin receptor phosphorylates the downstream docking protein insulin receptor substrate 1 (IRS-1), which subsequently, through the activation of the phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B (PKB) pathway leads to the translocation of glucose transporter type 4 (GLUT4) vesicles to the cell surface, leading to cellular glucose uptake [8]. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin signaling transduction [5]. PTP1B is able to interact with IR and IRS-1 to hydrolyze tyrosine phosphorylation induced by insulin action, causing an impairment of glucose uptake [6]. Global knock out of PTP1B in mice exhibit a phenotype with low adiposity, elevated insulin sensitivity and increased energy expenditure [7], [8]. Insulin resistant conditions, such as those seen with high-fat diet feeding, leptin deficiency, hyperglycemia or age-induced impairment in insulin signaling, are associated with increased expression of PTP1B in insulin-sensitive tissues [9]–[11]. Inhibition of PTP1B also improves palmitate-induced insulin resistance in cultured myotubes [12]. Nieto-Vasquez and colleagues demonstrated that immortalized PTP1B deficient myocytes had increased insulin-dependent glucose uptake and were protected against TNF-α-induced insulin resistance [13]. In addition, whole-body PTP1B-deficient mice were protected against TNF-α-induced insulin resistance owing to enhanced insulin sensitivity in skeletal muscle tissue. Delibegovic and colleagues showed that mice with muscle-specific deletion of PTP1B had improved glucose uptake and insulin signaling in skeletal muscle after high-fat diet feeding [14]. The endoplasmic reticulum (ER) is the cellular organelle responsible for multiple functions including protein and lipid biosynthesis, folding of newly synthesized peptides, modification of secreted proteins and detoxification of xenobiotics. Changes in nutrients and energy status in pathological conditions such as obesity overwhelm the capacity of ER leading to the accumulation of misfolded/unfolded proteins, a condition termed as endoplasmic reticulum stress (ER stress) [15]. In response to ER stress, the molecular chaperone glucose-regulated protein 78 (GRP78) results in its dissociates from the three ER-localized transmembrane signal transducers: inositol-requiring enzyme (IRE1), double-stranded RNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6), which results in the mobilization of adaptive cell signaling events of the unfolded protein response (UPR) [16]. Accumulating evidence suggests that chronic activation of ER stress plays a pivotal role in pathophysiology of obesity, insulin resistance and type 2 diabetes [17], [18]. While the role of ER stress in pathogenesis of obesity associated insulin resistance and inflammation has been widely studied in pancreatic beta-cells, hepatocytes, and adipocytes [19]–[22], knowledge regarding ER stress activation in skeletal muscle are rather limited and controversial. Previous studies have shown that UPR can be activated by high-fat diet-feeding in mice and in cultured myotubes treated with palmitic acid and other well-known ER stress inducers [23]. Studies in cultured cells from muscle biopsies from diabetic subjects have demonstrated that UPR is significantly elevated in in these cells following palmitic acid treatment [24]. On the other hand, more recent work from Deldicque and colleagues showed an absence of UPR in human skeletal muscle, despite increase in body mass, subcutaneous fat deposits, and intramyocellular lipid content after 6 weeks of fat-rich diet [25]. Given the importance of skeletal muscle in regulating whole body glucose homeostasis, the role of ER stress in muscle insulin resistance and the underlying mechanism assumes importance. PTP1B is tethered to the ER membrane, via a hydrophobic proline-reach region of 35-amino acid residues at the C-terminus [26]. Since PTP1B resides in the ER, it has been proposed that its function might affect development of UPR. Indeed, studies have shown that PTP1B deficiency protects against high-fat diet-induced ER stress in liver [27], [28]. Furthermore, PTP1B upregulates UPR by potentiating IRE-1α-mediated signaling pathways [29]. Our recent findings suggest that ER stress impairs glucose uptake in cultured myotubes by upregulating PTP1B, although the mechanism by which it does so is unclear [30]. Several pathways have been implicated in the regulation of PTP1B expression, including activation of UPR-associated ATF6 [31], binding of transcription factor YB-1 to enhancer region of Ptp1b promoter [32], and induction of NFκB [11]. However the exact mechanism by which ER stress can induce the ER stress remains largely unknown. The present study was therefore aimed at evaluating the role of PTP1B in high-fat diet-induced ER stress in skeletal muscle in vivo. We hypothesized that PTP1B deletion will augment UPR in skeletal muscle of high-fat diet fed mice. To this end, we assessed systemic glucose homeostasis and protein markers of ER stress, insulin signaling, and autophagy pathway in skeletal muscle and in cultured myotubes. Materials and Methods Ethics statement All the animal experimental procedures described in this study were approved by the University of Wyoming's Animal Use and Care Committee (Laramie, WY). Materials 2-Deoxy-[3H]-D-Glucose-1, propylene glycol and hematoxylin were from Sigma (St Lois, MO), tauroursodeoxycholic acid (TUDCA) and tunicamycin were from Calbiochem (Darmstadt, Germany); PTP1B siRNA sequences, non-target siRNA sequences and DharmaFECT transfection reagent were from Thermo Scientific (Rockford, IL); antibodies against-GRP78, -CHOP, -GAPDH, -phospho-eIF2α, -phospho-JNK1/2, eIF2α, JNK1/2, -phospho-Akt, Akt, Beclin-1, p62, NFκB p65, lamin A, NCK1, LC-3B, ATG5, ATG7 and LumiGLO reagent were from Cell Signaling Technology (Boston, MA); antibodies against PTP1B were from Millipore (Billerica, MA, USA); antibodies against ATG5 were from Abgent (San Diego, CA). Anti-rabbit IgG antibody were from Sigma-Aldrich (St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and horse serum were from Invitrogen (Carlsbad, CA). Animals Mice were maintained with access to food and water ad libitum, and were housed in the School of Pharmacy Animal Facility at the University of Wyoming at constant humidity and temperature with a light/dark cycle of 12 hours. C57BL6 (C57) mice were obtained from Jackson Laboratory (Bar Harbor, ME). PTP1B whole body knockout mice (PTP1BKO) were kindly provided by Dr. Michel L. Tremblay (McGill Cancer Center, Quebec, Canada). In brief, five week-old adult female C57 or PTP1BKO mice were randomly assigned to receive either low-fat (10 kcal% fat, 20 kcal% protein, 70 kcal% carbohydrate; Catalogue # D12450B, Research Diets, New Brunswick, NJ) or high-fat (45 kcal% fat, 20 kcal% protein, 35 kcal% carbohydrate; Catalogue # D12451, Research Diets, New Brunswick, NJ) diets for a period of 3 or 20 weeks. Some high-fat diet-fed C57 mice also received ad libitum drinking water supplemented with 40 mM NAC, as described before [33], [34]. At the end of 3- or 20-week treatment, overnight fasted mice were sacrificed and internal organs, epididymal fat pads, and blood samples were collected. Tibia length was measured as a marker of body size growth, since in extreme obesity conditions body weight is not a reliable normalization factor due to excessive fat accumulation. Gastrocnemius muscle and liver tissues were homogenized in RIPA lysis buffer (Upstate, Lake Placid, NY) using a PowerGen Homogenizer 125 (Fisher Scientific, Hampton, NH) and sonicated using Sonic Dismembrator 100 (Fisher Scientific, Hampton, NH). The homogenates were then centrifuged at 14000 g for 15 min and soluble fraction was used for protein expression analysis by Western Blot as described below. Intraperitoneal -glucose and –insulin tolerance tests After 20 weeks of high-fat diet-feeding, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. For IPGTT mice were fasted overnight for 12 h, and received intraperitoneally injection of D-glucose (2g kg−1). For IPITT the mice received intraperitoneal injection of insulin (0.5 U kg−1). Glucose concentration in a drop of blood obtained from tail-clipping was measured using Accu-Chek Advantage glucometer (Roche, Manheim, Germany) at 0, 30, 60, 90, and 120 min time points following the glucose challenge. Area under the curve (AUC) for each individual time curve was calculated using GraphPad Prism 5.04 software (GraphPad, Sowtware, La Jolla, CA). Oil Red O staining Fresh frozen liver sections (8 µm) were fixed with in ice cold 10% formalin, washed with water and placed in propylene glycol. Samples were stained in 0.5% Oil Red O solution in propylene glycol for 10 minutes at 60°C. After differentiating in 85% propylene glycol for 5 minutes, slides were rinsed in water and counter stained with hematoxylin. Following mounting in VectMount AQ (Vector Laboratories, Bulingame, CA) liver sections were observed under the microscope. Glucose uptake assay Ex vivo [3H]-2-deoxy-glucose-uptake assay was performed as previously described [35]. Briefly, gastrocnemius muscles were dissected out from mice, incubated in Krebs–Ringer phosphate HEPES buffer (KRPH buffer; 10 mM phosphate buffer, pH 7.4, 1 mM MgSO4, 1 mMCaCl2, 136 mM NaCl, 4.7 mM KCl, 10 mM HEPES (pH 7.6)) for 30 min in an atmosphere containing 5% CO2 and then incubated for 30 min at 37°C in the presence of 2-deoxy-[3H]-glucose (0.2 µCi) and 100 nmol/l insulin. At the end of the incubation period, the muscles were washed three times with ice-cold PBS. The muscle strips were freeze-dried, weighed, lysed in PBS containing 0.2 mol/l NaOH. Glucose uptake was assessed by scintillation counting using Beckman LS5000TD liquid scintillation system (Beckman Coulter, Pasadena, CA). The counts were adjusted by the muscle weight. Triglyceride and cholesterol quantification Total liver triglycerides and cholesterol levels were determined by Triglyceride Assay Kit and Cholesterol Quantitation Colorimetric/Fluorometric Kit (BioVision, Milpitas, CA) following the manufacturer's protocol. Colorimetric assays were measured at 570 nm wavelength on SpectraMax 190 spectrophotometer (Molecular devices, Sunnyvale, CA). PTP1B activity assay Hydrolyzing activity of PTP1B in gastrocnemius muscle lysates was determined using PTP Assay Kit 2 (Millipore, Billerica, MA) per manufacturer protocol. Cell culture and differentiation Mouse muscle myoblasts cell line (C2C12) was obtained from American Type Culture Collection (Rockville, MD), was maintained in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin–streptomycin under a humidified atmosphere of 5% CO2 in air. After reaching a confluence of the cells, the culture medium was substituted with DMEM containing 2% horse serum to initiate myogenic differentiation [36]. After differentiation myotubes were serum-free starved for 24 h and then were subjected to various treatment described below. Treatment of cells Endoplasmic reticulum stress was induced in quiescent C2C12 myotubes by treating them with tunicamycin (0.1 µg/ml) or palmitic acid (0.8 mmol/l) for 24 hours. Palmitic acid was prepared by conjugating it with bovine serum albumin as previously reported [37]. For some experiments cells were co-treated with 1 mmol/l tauroursodeoxycholic acid (TUDCA), 10 mmol/l N-acetylcysteine (NAC) or 100 μmol/l pyrrolidine dithiocarbamate (PDTC). Western blot analysis Cells were lysed in RIPA lysis buffer followed by sonication and centrifugation at 14000 g for 15 min. An aliquot of 50 µg of lysates in Laemmli sample buffer (BioRad, Hercules, CA) containing 5% 2-mercaptoethanol were heated at 95°C for 5 min and separated using 10% or 12% (for proteins with MW below 30) SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes, and incubated in the primary antibody against specific proteins overnight at 4°C. Following treatment with anti-rabbit IgG HRP-linked antibody protein bands were detected and quantified by enhanced chemiluminescence autoradiography by molecular imager Gel Doc XR + System (Bio Rad, Hercules, CA). All protein levels were normalized to GAPDH levels; phospho-eIF2α, -JNK, and -Akt were normalized to corresponding total protein levels. Average values for control (untreated) group were used for normalization between different blots, when acquired ratios for controls were substantially different among the blots. Measurement of intracellular reactive oxygen species (ROS) C2C12 cells were plated in 96-well cell culture plates and treated according to experimental design. At the end of the experiment cells were washed with PBS buffer pH 7.4 (137 mmol/l NaCl, 2.7 mmol/l KCl, 10 mmol/l Na2HPO4, 1.8 mmol/l KH2PO4) and incubated with 5 μmol/l dihydroethidium (DHE; Invitrogen, Carlsbad, CA) in PBS for 30 min at 37°C under a humidified atmosphere of 5% CO2 in air. Cells were then washed 3 times with PBS and fluorescence was then measured at excitation/emission wavelength of 488/590 using SpectraMax Gemini XS plate reader (Molecular Devices, Sunnyvale, California). Confocal microscopy C2C12 cultured myoblasts grown in chamber slides or 10 µM fresh frozen sections of gastrocnemius muscles were incubated with 5 μmol/l DHE in PBS for 30 min at 37°C under a humidified atmosphere of 5% CO2 in air. Cells or tissue were then washed 3 times with PBS. Following nuclear staining with DAPI (Invitrogen, Carlsbad, CA), the cells or tissue were observed under Zeiss LSM 710 confocal microscope. DHE fluorescence intensity was quantified with ImageJ. Subcellular fractionation Nuclear and cytosolic extracts were prepared as described in [38]. Briefly, C2C12 cells were lysed in ice-cold buffer containing 4 mmol/l HEPES, pH 7.4; 320 mmol/l sucrose; 1 mmol/l dithiothreitol; 10 mmol/l MgCl2; 5 mmol/l KCl dithiothreitol; 0.1% Triton X-100; 5 mmol/l NaF; and 2 mmol/l NaVO3. Protein content was measured as described above, and concentration for each sample was accordingly adjusted. An aliquot was collected and suspended with Laemmli sample buffer to serve as a whole cell extract. The remaining homogenate was spun down at 2000 g for 3 min at 4°C. The supernatant was spun down again at 2000 g for 10 min at 4°C, soluble fraction mixed with sample buffer and used as a cytosolic fraction. The remaining pellet was washed 3 times with lysis buffer, resuspended in sample buffer at a volume equal to the final volume of supernatant, and used as a nuclear fraction. Separation of nuclear and cytosolic fractions was verified by Western blotting for the cytosolic GAPDH and the nuclear protein lamin A. Transfection of cells with PTP1B siRNA A total of 25 nmol/l of PTP1B siRNA or the same amount of non-target siRNA were transfected for 48 hours into C2C12 myoblasts using DharmaFECT® transfection reagent per the manufacturer's instructions. Statistical analysis Data were expressed as mean ± SEM. Statistical analysis was performed with analysis of variance (ANOVA) followed by Newman-Keuls post hoc test using GraphPad Prism 5.04 software. A p-value less than 0.05 were considered to be statistically significant. Results PTP1B knockout mice are protected against high-fat diet-induced gain weight, glucose intolerance and hepatic steatosis As anticipated C57 mice fed with a high-fat diet developed severe obesity as by demonstrated by an in body weight gain, epididymal fat pads and relative (normalized to tibia length) weight of heart, liver, and kidney compared with mice kept on the normal diet (Table 1). In contrast, high-fat diet-fed, PTP1B deleted mice had lower body weight gain, adiposity and degree of heart and liver hypertrophy. PTP1B knockout did not affect body composition of mice that received a normal diet (Table 1). High-fat diet feeding also induced hyperglycemia in C57 mice, which was abolished in PTP1B knockout mice (Table 1). In mice receiving the normal diet PTP1B resulted in a small but insignificant decrease in fasting blood glucose levels. 10.1371/journal.pone.0077228.t001Table 1 Morphometry and fasting glucose of C57BL/6 and PTP1BKO female mice fed with a ND or a HFD for a 20 week period. Parameter C57BL/6 ND PTP1BKO ND C57BL/6 HFD PTP1BKO HFD Body weight (BW) (g) 26.73±1.00 25.26±0.65 41.50±4.00* 28.23±1.84 †,‡ Tibia length (TL) (mm) 17.5±0.4 17.6±0.2 18.3±0.1 18.3±0.1 Epididymal fat pad (g) 0.43±0.06 0.42±0.10 3.13±0.57* 1.03±0.33‡ Heart weight (HW) (mg) 116±4 107±4 146±6* 123±6‡ HW/TL 7.73±0.17 8.02±0.42 10.76±0.37* 9.55±0.45†,‡ Liver weight (LW) (g) 1.20±0.09 1.12±0.09 1.50±0.03* 1.11±0.1‡ LW/TL 0.068±0.006 0.063±0.004 0.083±0.002* 0.061±0.005‡ Kidney weigh (KW) (mg) 253±8 238±6 297±11* 280±20 KW/TL 14.5±0.7 13.5±0.5 16.2±0.6 15.4±1.2 Fasting blood glucose (mg/dL) 80±8 71±13 111±14* 98±3† Values are mean ± SEM, n = 5–6 mice per group, * p<0.05 vs. C57BL/6 ND group, † p<0.05 vs. PTP1BKO ND, ‡ p<0.05 vs. C57BL/6 HFD group. To further study the effect of PTP1B deletion the diet-induced obesity model we performed IPGTT and IPITT to access glucose tolerance and insulin sensitivity respectively. As shown in Fig. 1a–d PTP1B knockout marginally improved whole-body blood glucose disposal in mice that received a normal diet in both the IPGTT and IPITT. High-fat feeding resulted in the severe glucose intolerance (Fig. 1a) and insulin resistance (Fig. 1b), characterized by increased area under the post-challenge blood glucose curves. Conversely, deletion of PTP1B reversed the effect of HFD feeding on blood-glucose disposal, as indicated by lower AUCs (area under the curve) compared to that of the C57 mice. 10.1371/journal.pone.0077228.g001Figure 1 PTP1B knockout mice are protected against obesity-induced glucose intolerance and hepatic steatosis. Female C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks. At the end of the experiment IPGTT (a) and IPITT (b) were performed. Area under the curve (AUC) for each individual curve of IPGTT (c) and IPITT (d) was calculated (n = 6). Liver fat content was analyzed with (e) Oil Red O staining (n = 6), (f) triglyceride assay kit (n = 6), and (g) cholesterol quantitation colorimetric kit (n = 6). Red staining – fat droplets, blue – DAPI stained nuclei. Scale bars, 100±µm. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice. Since the liver weight was the most striking morphological difference observed between PTP1BKO and C57 mice (Table 1), we compared the degree of hepatic steatosis between the groups. Oil Red O staining revealed accumulation of excessive fat droplets in livers of high-fat diet-fed C57 mice, compared to those that received the normal diet. In contrast, livers of PTP1BKO mice subjected to high-fat diet did not show any signs of steatosis (Fig. 1e). No difference was observed in the hepatic content between the C57 and PTP1BKO mice that received a normal diet. Consistent with these findings, both hepatic triglycerides and total cholesterol levels were elevated in livers of C57 mice in response to high-fat feeding (Fig. 1f, g). In contrast, deletion of PTP1B protected the liver from high-fat diet induced accumulation of triglycerides and cholesterol. PTP1B deletion improves glucose uptake and insulin signaling in skeletal muscle of high-fat diet-fed mice Previous studies suggested that the skeletal muscle is a main site of the peripheral action of PTP1B in regulating whole body glucose homeostasis [8], [14], [39]. Thus to access the cellular consequence of the PTP1B deletion, we measured insulin-stimulated glucose uptake in ex-vivo skeletal muscle tissues from C57 and PTP1B KO mice fed with normal or high-fat diet. As expected, high-fat diet resulted in skeletal muscle insulin resistance as evidenced by a ∼3-fold decrease in muscle glucose uptake compared to that seen in the muscle samples from mice that received a normal diet (Fig. 2a). Muscle samples from PTP1B knockout mice subjected to a normal diet had a slightly higher insulin-stimulated muscle glucose uptake compared to similarly fed C57 mice. More importantly, the muscle samples from the high-fat diet fed PTP1B deleted mice exhibited a ∼2 fold increase in muscle glucose uptake compared to the muscle samples obtained from C57 mice that received the high-fat diet (Fig. 2a). 10.1371/journal.pone.0077228.g002Figure 2 PTP1B deletion improves diet-induced insulin resistance and glucose uptake in skeletal muscle in mice. (a) [3H]-2-deoxy-glucose-uptake assay (n = 6); (b) representative Western blots and (c) densitometric analysis (n = 6) of Akt phosphorylation in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks and challenged with insulin for 30 minutes. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice. To understand the mechanism involved in this process, we evaluated insulin signaling pathway by monitoring insulin-stimulated levels of phosphorylation of Akt, a key downstream molecule in the insulin signaling pathway. As anticipated, in C57 mice insulin challenge resulted in a significant increase of Akt phosphorylation levels with no difference in basal Akt levels (Fig. 2 b–c). PTPKO mice showed a small but insignificant increase in insulin-stimulated Akt phosphorylation under normal-diet conditions. However in high-fat diet-fed mice PTP1B deletion significantly increased levels of Akt phosphorylation in response to insulin stimulation, indicating central role of PTP1B in regulation of insulin signaling in skeletal muscle of obese mice (Fig. 2 b–c). High-fat diet-associated elevated ER stress in skeletal muscle is attenuated by PTP1B deletion High-fat diet-fed mice exhibited significantly higher PTP1B protein levels in gastrocnemius muscle compared with age- and sex-matched lean control mice (Fig. 3 a, b), which was consistent with our previous studies [30]. Activity of PTP1B was also elevated in high-fat diet-fed mice compared to normal diet-fed mice (Fig. 3c). Increased levels of PTP1B protein in obese mice were accompanied by elevated ER stress levels, as indicated by increased expression of GRP78 protein and phosphorylation of eIF2α and JNK2 proteins (Fig. 3 a, d–e). However PTP1B deletion resulted in a near complete inhibition of high-fat diet-induced expression of GRP78, phospho-eIF2α and phospho-JNK2 (Fig. 3 a, d–e), indicating a permissive role of PTP1B in development of high-fat diet-induced ER stress. These ER stress marker were unaltered in PTP1BKO mice that received the normal diet. Furthermore, neither high-fat diet feeding nor PTP1B deletion altered total eIF2α and JNK2 expression. 10.1371/journal.pone.0077228.g003Figure 3 The effect of PTP1B deletion on obesity-induced ER stress in skeletal muscle. Representative Western blots (a), PTP1B activity assay (b) and densitometric analysis (n = 6) of PTP1B (c), GRP78 (d), phospho- and total-eIF2α (p-eIF2α and t-eIF2α, respectively) (e), and phospho- and total-JNK2 (p-JNK and t-JNK, respectively) (f) protein levels in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice. Effect of PTP1B knockout on autophagy and NCK1 in high-fat diet-fed mice Accumulating body of recent data indicates that ER stress is also a potent trigger of autophagy through IRE1 or PERK pathways [40]. To examine the potential impact of PTP1B deficiency on autophagic degradation pathway, the autophagy markers ATG5, ATG7, Beclin-1 and autophagy cargo adapter p62 were evaluated in skeletal muscle from C57 and PTP1BKO mice following -normal or –high-fat diet feeding. We did not observe any effect of diet or genetic background on the protein expression levels of ATG5, ATG7, and Beclin-1 (Fig. 4 a–d). However, Western blot analysis revealed a significant increase in p62 expression in skeletal muscle of high-fat diet-fed mice, indicating partial inhibition of autophagy pathway (Fig. 4 a, e). Surprisingly, normal diet-fed mice lacking PTP1B had lower expression of p62 in skeletal muscle. High-fat diet feeding resulted in an increase in the protein levels of p62, although they were significantly lower than that observed in the C57 mice subjected to high-fat feeding (Fig. 4 a, e). 10.1371/journal.pone.0077228.g004Figure 4 The effect of PTP1B deletion on autophagic flux and NCK1 expression in skeletal muscle of obese mice. Representative Western blots (a) and densitometric analysis (n = 6) of ATG5 (b), ATG7 (c), Beclin-1 (d), p62 (e), and NCK1 (f) protein levels in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice, ‡ p<0.05 compared with PTP1BKO ND mice. It has been previously shown that deletion of non-catalytic region of tyrosine kinase 1 (NCK1) adaptor protein attenuates ER stress signaling and improves insulin signaling in liver of obese mice [41]. We therefore assessed the effect of high-fat diet on NCK1 protein expression levels in skeletal muscle of C57 and PTP1BKO mice. Interestingly, skeletal muscles from PTP1B knockout mice had significantly lower levels of NCK1 under both normal and high-fat diet conditions (Fig. 4a, f), although high-fat diet feeding by itself did not alter the expression levels of NCK1. Effect of PTP1B knockout on ER stress, autophagy and NCK1 in mice received short term high-fat diet feeding The observed effects of PTP1B deletion upon expression of ER stress markers, p62 and NCK1 could possibly result from leaner phenotype of PTP1B knockout mice under high-fat diet feeding. Thus to rule out this possibility we compared C57 and PTP1BKO mice after just 3 weeks of high-fat diet feeding, when there is still no significant difference in body weight between genotypes (20.4±0.4 and 19.8±0.2 respectively). Even shorter period of high-fat diet feeding resulted in elevated expression of PTP1B and ER stress markers (GRP78, phospho-eIF2α and phosphor-JNK2) (Fig. 5 a–e). In contrast, mice lacking PTP1B did not show increase in expression of PTP1B, GRP78, phospho-eIF2α and phosphor-JNK2 (Fig. 5 a–e), confirming our previous findings. We failed to observe any increase in protein levels of autophagy marker p62, but consistently with our earlier results its expression along with NCK1, was decreased in PTP1B knockout mice (Fig. 5 a, f–g). 10.1371/journal.pone.0077228.g005Figure 5 The effect of PTP1B deletion on short term high-fat diet-induced ER stress in skeletal muscle. Representative Western blots (a) and densitometric analysis (n = 6) of PTP1B (b), GRP78 (c), phospho- and total-eIF2α (p-eIF2α and t-eIF2α, respectively) (d), and phospho- and total-JNK2 (p-JNK and t-JNK, respectively) (e), p62 (f), and NCK1 (g) protein levels in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 3 weeks. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice. Tunicamycin induced expression of PTP1B is dependent on reactive oxygen species In an effort to understand the potential cross-talk between ER stress and PTP1B in inducing skeletal muscle insulin resistance we used cultured C2C12 myotubes that were rendered insulin resistant by treatment with the ER stress inducer tunicamycin. Previously we had shown that ER stress caused insulin resistance in cultured myotubes by increasing expression of PTP1B protein [30]. Because prolonged ER stress leads to accumulation of intracellular reactive oxygen species (ROS) and oxidative stress [42], we were interested in knowing whether ER-stresses induced PTP1B expression is dependent on ROS production. As expected tunicamycin treatment induced the production of intracellular ROS in cultured myotubes by ∼2.5 fold, as detected both by confocal microscopy (Fig. 6a) and spectrophotometric measurements (Fig. 6b). Consistently with our previous results, tunicamycin treatment led to increased expression of ER stress markers GRP78 and phospho-eIF2, and concomitant increase in PTP1B protein levels (Fig. 7a–d). To test if accumulation of intracellular ROS is essential for tunicamycin-induced expression of ER stress and PTP1B, we used the ROS scavenger N-acetylcysteine (NAC). NAC was able to block tunicamycin-induced production of ROS (Fig. 6a, b), which completely inhibited tunicamycin-induced expression of PTP1B, without significantly altering ER stress (Fig. 7a–d). These data suggest that ROS production is required for tunicamycin-induced expression of PTP1B in myotubes, but not for UPR activation. To ascertain that ER stress can cause ROS accumulation upon tunicamycin treatment we used chemical chaperone tauroursodeoxycholic acid (TUDCA), which has been shown to be able to alleviate ER stress [43]. Treatment with TUDCA indeed alleviated ER stress in tunicamycin-treated myotubes, as evidenced by decreased expression of GRP78 and phosphorylation of eIF2α (Fig. 7e–g) which significantly lowered intracellular ROS levels in cultured myotubes (Fig. 6a, b) suggesting that tunicamycin-induced ER stress results in ROS generation. 10.1371/journal.pone.0077228.g006Figure 6 The effect of NAC and TUDCA on tunicamycin (TM)-induced ROS production in cultured myotubes. Representative confocal microscopy pictures (a) and spectrophotometric quantification (n = 6) (b) of intracellular ROS production in C2C12 myotubes treated with tunicamycin in presence of 10 mmol/l NAC or 1 mmol/l TUDCA for 24 hours. ROS detection dye DHE is shown as red, the nucleus is stained with DAPI (blue), with purple suggests colocalization. Scale bars, 50 µm. 10.1371/journal.pone.0077228.g007Figure 7 The role of ROS in tunicamycin (TM)-induced PTP1B protein expression in C2C12 myotubes. (a–d) Representative Western blots (a) and densitometric analysis (n = 6) of PTP1B (b), GRP78 (c), and phospho- and total-eIF2α (p-eIF2α and t-eIF2α, respectively) (d) protein levels in C2C12 myotubes treated with tunicamycin in presence of 10 mmol/l NAC for 24 hours. (e–g) Representative Western blots (e) and densitometric analysis (n = 6) of GRP78 (f), and p-eIF2α and t-eIF2α (g) protein levels in C2C12 myotubes treated with tunicamycin in presence of 1 mmol/l TUDCA for 24 hours. * p<0.05 compared with non-treated control cells, † p<0.05 compared with tunicamycin-treated cells. NFκB mediates tunicamycin-induced PTP1B expression in cultured myotubes ROS activate the transcription of a variety of genes via activation of the transcription factor NFκB [44]. Interestingly, a putative cis-regulatory element for NFκB binding site has been previously identified within the Ptp1b promoter [45]. Additionally the p65 subunit of NFκB has been shown to bind and activate Ptp1b promoter in response to TNFα treatment in a liver of mice [11]. Therefore, we examined the possible involvement of NFκB signaling pathway in ER stress-induced expression of PTP1B. When activated, the inhibitor of κB (IκB) kinase phosphorylates IκB regulatory domain, leading to its degradation, which releases the p65 subunit of NFκB leading to the translocation of NFκB to the nucleus and subsequent transcription of the target genes [46]. To detect activation of NFκB signaling we separated the cytosolic and nuclear fractions, and determined p65 subunit content in each of them. Tunicamycin treatment resulted in the activation of the NFκB pathway in cultured C2C12 myotubes as indicated by an increase in levels of p65 in the nuclear fraction with a concomitant decrease of p65 in the cytosolic fraction (Fig. 8a–c). Inhibition of the NFκB signaling pathway using the pharmacological inhibitor pyrrolidine dithiocarbamate (PDTC) blocked the tunicamycin-induced nuclear translocation of p65, without affecting basal NFκB levels (Fig. 8a–c). Interestingly, PDTC treatment also prevented tunicamycin-induced expression of PTP1B in cultured myotubes, suggesting that the activation of NFκB pathway mediates expression of PTP1B under ER stress conditions (Fig. 8a, d). 10.1371/journal.pone.0077228.g008Figure 8 The role of NFκB in tunicamycin (TM)-induced PTP1B expression in myotubes. Representative Western blots (a) and densitometric analysis (n = 6) of nuclear NFκB (Nucl. NFκB) (b), cytosolic NFκB (Cyt. NFκB) (c), and PTP1B (d) protein expression in C2C12 myotubes treated with tunicamycin in presence of 10 mmol/l NAC, 100 μmol/l PDTC or 1 mmol/l TUDCA for 24 hours * p<0.05 compared with non-treated control cells, † p<0.05 compared with tunicamycin-treated cells. To ascertain that NFκB signaling is activated in response to ER stress-induced UPR, we performed the experiment in the presence of TUDCA. TUDCA had no effect on NFκB under basal condition, but was able to prevent the nuclear translocation of p65 in response to tunicamycin challenge (Fig. 8a–c). Furthermore, TUDCA effectively attenuated the expression of PTP1B protein to near-basal levels in tunicamycin-induced, but not control cells (Fig. 8a, d). These results indicate that ER stress by itself is necessary and sufficient to induce PTP1B expression through the activation of NFκB. Furthermore, myotubes co-treated with ROS scavenger NAC and tunicamycin failed to activate NFκB and nuclear translocation of p65, while NAC did not alter NFκB signaling under basal condition (Fig. 8a–c). More interesting, prevention of intracellular ROS production with NAC completely abolished tunicamycin-induced expression of PTP1B protein, indicating that ER stress causes activation of the NFκB via the generation of ROS (Fig. 8a, d). To confirm that activation of NFκB pathway was caused by the induction of ER stress rather than specific effects of tunicamycin, we also used palmitic acid a physiologically relevant inducer of ER stress. Indeed palmitic acid, a well established ER stress inducer in C2C12 cells [30], was able to induce activation of NFκB pathway, as shown by p65 nuclear translocation (Fig. S1a–c). Also co-treatment of C2C12 cells with PTP1B siRNA had no effect on tunicamycin-induced activation of NFκB pathway (Fig. S1d–g), indicating that ER stress-ROS-NFκB signaling axis functions upstream of PTP1B. ROS-NFκB axis mediates high-fat diet-induced PTP1B expression in skeletal muscle To confirm our in vitro findings regarding the role of ROS-NFκB axis in regulation of PTP1B expression in vivo, we used short term (3 weeks) high-fat diet feeding mice model. To evaluate the role of ROS, mice fed with high-fat diet received the antioxidant NAC supplement in drinking water for the duration of the experiment. High-fat diet feeding resulted in increase of intramuscular ROS production, which was abrogated by NAC supplementation (Fig. 9 a–b). We also observed NFκB activation in skeletal muscle after 3 weeks of high-fat diet feeding, as indicated by p65 nuclear translocation (Fig. 9c–e). Protein expression of PTP1B was also concomitantly increased in high-fat diet-fed mice (Fig. 9c, f). Additionally, NAC supplementation was able to prevent activation of p62, thus decreasing PTP1B expression. 10.1371/journal.pone.0077228.g009Figure 9 The role of ROS and NFκB in high-fat diet-induced PTP1B expression in skeletal muscle. Gastrocnemius muscles were isolated from C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) in a presence or absence of NAC supplementation diets for 3 weeks. (a–b) Representative confocal microscopy pictures (a) and quantification (n = 6) (b) of intracellular ROS production. ROS detection dye DHE is shown as red, the nucleus is stained with DAPI (blue), with purple suggests colocalization. Scale bars, 50 µm. (c–f) Representative Western blots (c) and densitometric analysis (n = 6) of nuclear NFκB (Nucl. NFκB) (d), cytosolic NFκB (Cyt. NFκB) (e), and PTP1B (f) protein expression. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice. Discussion Obesity-induced ER stress has been proposed as a common pathway linking body weight gain and insulin resistance [17], [18]. PTP1B, which serves a negative regulator of insulin signaling, is tightly linked to ER function and takes part in the cross-talk between itself and ER stress [26]. Given that skeletal muscle is a major site of the peripheral actions of PTP1B in regulating glucose homeostasis [13], [39], in our study we focused on molecular causes and consequences of this cross talk. We first employed a high-fat diet model of obesity in mice lacking PTP1B. Consistent with previous studies we found improved systemic insulin sensitivity, decreased body weight gain, and lower adiposity in high-fat diet-fed mice lacking PTP1B (Fig. 1a–d, Table 1.) [7], [8]. However, in contrast to previous observations [7] in our model we found a significant decrease in heart and liver weight in the PTP1B knockout mice subjected to a high-fat diet compared to C57 mice. These discrepancies may be explained by differences in a genetic background of mice, experimental diet used, duration of treatment, and the fact that we used female mice for our study whereas the previous study used male mice. The gender effect could also be an issue as we saw a slightly greater improvement in insulin sensitivity in female mice compared to male mice under high-fat diet conditions. However, in our pilot studies we did not observe any striking differences between expression levels of ER-stress markers and PTP1B protein in skeletal muscle of female mice compared to those from male mice (data not shown). We also found improved glucose uptake and insulin signaling in skeletal muscle of PTP1B whole body knockout mice (Fig. 2). These observations were consistent with the previous study done using mice with muscle-specific deletion of PTP1B [14]. We found that knockdown of PTP1B resulted in a drastic decrease in high-fat diet induced accumulation of fat droplet, triglycerides and cholesterol in the liver, indicating a role of PTP1B in hepatic fat metabolism (Fig. 1e–g). This observation is consistent with findings from Shimizu and colleagues, who showed that PTP1B activates hepatic lipogenesis, by regulating gene expression of sterol regulatory element-binding protein-1 (SREBP-1) via enhancement of protein phosphatase 2A (PP2A) activity [47]. We found that ER stress markers were elevated in the skeletal muscle of mice fed with high-fat diet for long (Fig. 3) or short (Fig. 5a–d) term, which was consistent with previous observations made by us and other investigators [23], [30]. Expression of PTP1B protein was also elevated in the skeletal muscle of mice in both high-fat diet-feeding models (Fig. 3, Fig. 5a–d). Our present findings in conjunction with our previous studies strongly suggest that the induction of expression of PTP1B in skeletal muscle under high-fat diet feeding conditions is mediated by the activation of ER stress. Also, here for the first time we report that PTP1B deletion protects against high-fat diet-induced ER stress in skeletal muscle (Fig. 3, Fig. 5a–d). Our findings were similar to results of Delibegovic and colleagues, who reported that liver specific deletion of PTP1B attenuated hepatic ER stress in a dietary model [48]. We also assessed autophagy as a process linked to ER stress [40]. We failed to observe any effect of high-fat diet feeding on expression of autophagy markers such as Beclin-1, LC-3B (data not shown), ATG5 and ATG7, which was consistent with previous studies [49]. However, we found significant increase in the expression of p62 protein in skeletal muscle of mice fed with high-fat diet for 20 weeks, but not for 3 weeks, indicating an impaired autophagic flux in chronic obesity model (Fig. 4a, e; Fig. 5a, f). Similar observations have been made in the heart tissue of mice fed with a high-fat diet [50]. Interestingly, PTP1B deletion decreased the amount of p62 in skeletal muscle independent of diet, thus improving autophagy (Fig. 4a, e; Fig. 5a, f). To our knowledge, this is the first report of the effect of PTP1B or lack thereof on the process of autophagy. In effort to explain impaired activation of UPR in high-fat diet-fed PTP1B knockout mice we evaluated the expression levels of adaptor protein NCK1, which can modulate activation of the UPR in a complex with protein tyrosine phosphatase [51]. Intriguingly, mice lacking PTP1B had lower levels of skeletal muscle NCK1, a protein which is necessary for the induction of ER stress signaling and development of insulin resistance in obese mice [41] (Fig. 4a, f; Fig. 5a, g). This observation may explain, at least in part, the protective effect of PTP1B deletion against diet-induced ER stress in skeletal muscle tissue. From the current study using in vivo high-fat diet feeding model, it seems that PTP1B induction promotes UPR, since the absence of PTP1B results in impaired activation of UPR. Thus there is an indication of a feed-back loop between PTP1B induction and promotion of ER stress, as we showed that ER stress increase PTP1B in vitro and PTP1B can modify UPR in vivo. NCK1 is a perfect candidate molecule linking PTP1B and UPR, because it is required for activation of the certain branches of UPR [51]. We observed a decreased expression of NCK1 protein in PTP1B knockout mice (Fig. 4a, f; Fig. 5a, g), which might negatively affect feed-back loop between PTP1B and ER stress and can possibly explain the mitigated UPR response in high-fat diet fed knockout mice. Interestingly, our previous study using acute in vivo induction of ER stress with tunicamycin, also revealed impaired phosphorylation of eIF2α and JNK in mice lacking PTP1B, confirming idea of a positive feed-back loop [30]. As a second part of our study we investigated the potential mechanisms by which ER stress activation leads to the induction of PTP1B expression. Using C2C12 cultured myotubes we first evaluated the role ER stress-dependent ROS production in induction of PTP1B expression. We found that ER stress driven intracellular ROS production is necessary for the induction of PTP1B expression in cultured myotubes (Fig. 6). These results were somewhat surprising, because PTP1B undergoes reversible inhibition through oxidation of active-site cysteine residues [52]; and its phosphatase activity has been shown to be inactivated by ROS in systemic sclerosis dermal fibroblasts [53]. However the effect of ROS on the expression levels of PTP1B has not been studied in details before. Our data shows that ER stress-mediated production of ROS as novel regulation of PTP1B expression (Fig. 7). Since ROS are capable of activating NFκB signaling pathway, mainly through the classical IKK-dependent pathway [44], [54], we hypothesized that the ER stress-induced ROS leads to the induction of NFκB pathway. It also has been shown that Ptp1b promoter has NFκB binding site and that PTP1B expression is induced by inflammation through activation of NFκB pathway [11], [45]. So we further investigated NFκB as a possible candidate responsible for ER stress-induced PTP1B expression. We found that tunicamycin-induced ER stress increases the amount of nuclear NFκB p65 subunit and it is required for induction of PTP1B expression (Fig. 8). Furthermore ER-stress dependent p65 translocation was dependent on intracellular ROS production, indicating that ROS may be functioning upstream causing NFκB activation under obese conditions (Fig. 8). Our observations that induction of PTP1B expression is mediated by increased NFκB p65 in the nuclear fraction, is consistent with a recent study showing simultaneous increase in hypothalamic PTP1B expression and activation of the NFκB signaling in aged rats [55]. Another study using an aging-associated obesity model also showed concomitant increase in expression of PTP1B and inflammatory pathway in liver and muscle [56]. We were able to confirm our in vitro findings using short term high-fat diet feeding model and antioxidant supplementation. Our data indicated activation of ROS-NFκB axis in skeletal muscle of mice after 3 weeks of high-fat feeding, was prevented by NAC supplementation (Fig. 9). Thus we demonstrated a direct link between ER stress-mediated activation of NFκB signaling and increased PTP1B expression under high-fat diet conditions. Based on our current findings and previous work by Zabolotny [11] it appears that both inflammation and ER stress additively contribute do the induction of NFκB signaling pathway, leading to increased PTP1B expression and insulin resistance in obesity. Although it is likely that the proteins identified in these studies are predominantly form skeletal muscle, as we did not perfuse the tissue prior to isolating the gastrocnemius muscle we cannot rule out the contribution of extraneous tissues such vascular cells. Collectively, our data demonstrate that under high-fat diet feeding conditions PTP1B plays a critical role in the development of ER stress in skeletal mus. High-fat diet feeding leads to activation of UPR in skeletal muscle, which may leads to the insulin resistance in the tissue. On the other hand, PTP1B deletion results in attenuation of ER stress in the skeletal muscle of high-fat diet-fed mice. Our in vitro findings indicate that ER stress-induced ROS production is required for NFκB-dependent activation of PTP1B expression. Taken together, it is likely that high-fat diet-associated ER stress induces PTP1B expression by activating ROS- NFκB axis, resulting in insulin resistance. Supporting Information Figure S1 (TIF) Click here for additional data file. ==== Refs References 1 Ahima RS (2011 ) Digging deeper into obesity . The Journal of Clinical Investigation 121 : 2076 –2079 .21633174 2 Hotamisligil GS (2006 ) Inflammation and metabolic disorders . Nature 444 : 860 –867 .17167474 3 Orgel E, Mittelman S (2012) The links between insulin resistance, diabetes, and cancer. Current Diabetes Reports: Current Science Inc. 1–10. 4 Shoelson SE , Lee J , Goldfine AB (2006 ) Inflammation and insulin resistance . The Journal of Clinical Investigation 116 : 1793 –1801 .16823477 5 Yip S-C , Saha S , Chernoff J (2010 ) PTP1B: a double agent in metabolism and oncogenesis . Trends in Biochemical Sciences 35 : 442 –449 .20381358 6 Panzhinskiy E , Ren J , Nair S (2013 ) Pharmacological inhibition of protein tyrosine phosphatase 1B: a promising strategy for the treatment of obesity and type 2 diabetes mellitus . Current Medicinal Chemistry 20 : 2609 –2625 .23627940 7 Klaman LD , Boss O , Peroni OD , Kim JK , Martino JL , et al (2000 ) Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice . Molecular and Cellular Biology 20 : 5479 –5489 .10891488 8 Elchebly M , Payette P , Michaliszyn E , Cromlish W , Collins S , et al (1999 ) Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene . Science 283 : 1544 –1548 .10066179 9 Pauli J , Ropelle ER , Cintra DE , De Souza CT , da Silva ASR , et al (2010 ) Acute exercise reverses aged-induced impairments in insulin signaling in rodent skeletal muscle . Mechanisms of Ageing and Development 131 : 323 –329 .20307567 10 Gonzalez-Rodriguez A , Gutierrez JAM , Sanz-Gonzalez S , Ros M , Burks DJ , et al (2010 ) Inhibition of PTP1B restores IRS1-mediated hepatic insulin signaling in IRS2-deficient mice . Diabetes 59 : 588 –599 .20028942 11 Zabolotny JM , Kim Y-B , Welsh LA , Kershaw EE , Neel BG , et al (2008 ) Protein-tyrosine phosphatase 1B expression is induced by inflammation in vivo . Journal of Biological Chemistry 283 : 14230 –14241 .18281274 12 Zhao M , Zhang ZF , Ding Y , Wang JB , Li Y (2012 ) Astragalus polysaccharide improves palmitate-induced insulin resistance by inhibiting PTP1B and NF-kappaB in C2C12 myotubes . Molecules 17 : 7083 –7092 .22728372 13 Nieto-Vazquez I , Fernández-Veledo S , de Alvaro C , Rondinone CM , Valverde AM , et al (2007 ) Protein-tyrosine phosphatase 1B-deficient myocytes show increased insulin sensitivity and protection against tumor necrosis factor-α-induced insulin resistance . Diabetes 56 : 404 –413 .17259385 14 Delibegovic M , Bence KK , Mody N , Hong E-G , Ko HJ , et al (2007 ) Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B . Molecular and Cellular Biology 27 : 7727 –7734 .17724080 15 Back SH , Kaufman RJ (2012 ) Endoplasmic reticulum stress and type 2 diabetes . Annual Review of Biochemistry 81 : 767 –793 . 16 Malhotra JD , Kaufman RJ (2007 ) Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxid Redox Signal 9 : 2277 –2293 .17979528 17 Özcan U , Cao Q , Yilmaz E , Lee AH , Iwakoshi NN , et al (2004 ) Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes . Science 306 : 457 –461 .15486293 18 Hotamisligil GS (2005 ) Role of endoplasmic reticulum stress and c-Jun NH2-terminal kinase pathways in inflammation and origin of obesity and diabetes . Diabetes 54 : S73 –S78 .16306344 19 Fu S , Watkins SM , Hotamisligil GS (2012 ) The role of endoplasmic reticulum in hepatic lipid homeostasis and stress signaling . Cell Metabolism 15 : 623 –634 .22560215 20 Pagliassotti MJ (2012 ) Endoplasmic reticulum stress in nonalcoholic fatty liver disease . Annual Review of Nutrition 32 : 17 –33 . 21 Scheuner D , Kaufman RJ (2008 ) The unfolded protein response: a pathway that links insulin demand with β-cell failure and diabetes . Endocrine Reviews 29 : 317 –333 .18436705 22 Gregor MF , Hotamisligil GkS (2007 ) Thematic review series: adipocyte biology. Adipocyte stress: the endoplasmic reticulum and metabolic disease . Journal of Lipid Research 48 : 1905 –1914 .17699733 23 Deldicque L , Cani PD , Philp A , Raymackers J-M , Meakin PJ , et al (2010 ) The unfolded protein response is activated in skeletal muscle by high-fat feeding: potential role in the downregulation of protein synthesis . American Journal of Physiology – Endocrinology And Metabolism 299 : E695 –E705 .20501874 24 Peter A , Weigert C , Staiger H , Machicao F , Schick F , et al (2009 ) Individual stearoyl-CoA desaturase 1 expression modulates endoplasmic reticulum stress and inflammation in human myotubes and is associated with skeletal muscle lipid storage and insulin sensitivity in vivo . Diabetes 58 : 1757 –1765 .19478146 25 Deldicque L , Van Proeyen K , Francaux M , Hespel P (2011 ) The unfolded protein response in human skeletal muscle is not involved in the onset of glucose tolerance impairment induced by a fat-rich diet . European Journal of Applied Physiology and Occupational Physiology 111 : 1553 –1558 .21188411 26 Popov D (2012 ) Endoplasmic reticulum stress and the on site function of resident PTP1B . Biochemical and Biophysical Research Communications 422 : 535 –538 .22609202 27 Delibegovic M , Zimmer D , Kauffman C , Rak K , Hong E–G , et al (2009 ) Liver-specific deletion of protein-tyrosine phosphatase 1B (PTP1B) improves metabolic syndrome and attenuates diet-induced endoplasmic reticulum stress . Diabetes 58 : 590 –599 .19074988 28 Agouni A , Mody N , Owen C , Czopek A , Zimmer D , et al (2011 ) Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically induced endoplasmic reticulum stress . Biochemical Journal 438 : 369 –378 .21605081 29 Gu F , Nguyên DT , Stuible M , Dubé N , Tremblay ML , et al (2004 ) Protein-tyrosine phosphatase 1B potentiates IRE1 signaling during endoplasmic reticulum stress . Journal of Biological Chemistry 279 : 49689 –49693 .15465829 30 Panzhinskiy E , Hua Y , Culver B , Ren J , Nair S (2012 ) Endoplasmic reticulum stress upregulates protein tyrosine phosphatase 1B and impairs glucose uptake in cultured myotubes . Diabetologia 56 : 598 –607 .23178931 31 Wang N , Zhang D , Mao X , Zou F , Jin H , et al (2009 ) Astragalus polysaccharides decreased the expression of PTP1B through relieving ER stress induced activation of ATF6 in a rat model of type 2 diabetes . Molecular and Cellular Endocrinology 307 : 89 –98 .19524131 32 Fukada T , Tonks NK (2003 ) Identification of YB-1 as a regulator of PTP1B expression: implications for regulation of insulin and cytokine signaling . EMBO Journal 22 : 479 –493 .12554649 33 Clark J , Clore EL , Zheng K , Adame A , Masliah E , et al (2010 ) Oral N-acetyl-cysteine attenuates loss of dopaminergic terminals in α-synuclein overexpressing mice . PLoS ONE 5 : e12333 .20808797 34 Novelli ELB , Santos, Assalin HB , Souza G , Rocha K , et al (2009 ) N-acetylcysteine in high-sucrose diet-induced obesity: Energy expenditure and metabolic shifting for cardiac health . Pharmacological Research 59 : 74 –79 .18996201 35 Kandadi MR , Rajanna PK , Unnikrishnan MK , Boddu SP , Hua Y , et al (2010 ) 2-(3,4-Dihydro-2H-pyrrolium-1-yl)-3oxoindan-1-olate (DHPO), a novel, synthetic small molecule that alleviates insulin resistance and lipid abnormalities . Biochem Pharmacol 79 : 623 –631 .19769946 36 Nedachi T , Kanzaki M (2006 ) Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes . Am J Physiol Endocrinol Metab 291 : E817 –828 .16735448 37 Wang XL , Zhang L , Youker K , Zhang MX , Wang J , et al (2006 ) Free fatty acids inhibit insulin signaling-stimulated endothelial nitric oxide synthase activation through upregulating PTEN or inhibiting Akt kinase . Diabetes 55 : 2301 –2310 .16873694 38 Battiprolu PK , Hojayev B , Jiang N , Wang ZV , Luo X , et al (2012 ) Metabolic stress-induced activation of FoxO1 triggers diabetic cardiomyopathy in mice . The Journal of Clinical Investigation 122 : 1109 –1118 .22326951 39 Zabolotny JM , Haj FG , Kim Y-B , Kim H-J , Shulman GI , et al (2004 ) Transgenic overexpression of protein-tyrosine phosphatase 1B in muscle causes insulin resistance, but overexpression with leukocyte antigen-related phosphatase does not additively impair insulin action . Journal of Biological Chemistry 279 : 24844 –24851 .15031294 40 Hoyer-Hansen M , Jaattela M (2007 ) Connecting endoplasmic reticulum stress to autophagy by unfolded protein response\ and calcium . Cell Death and Differentitation 14 : 1576 –1582 . 41 Latreille M , Laberge M-K , Bourret Gv , Yamani L , Larose L (2010 ) Deletion of Nck1 attenuates hepatic ER stress signaling and improves glucose tolerance and insulin signaling in liver of obese mice . American Journal of Physiology – Endocrinology And Metabolism 300 : E423 –E434 .20587749 42 Gregersen N, Bross P (2010) Protein misfolding and cellular stress: an overview. In: Bross P, Gregersen N, editors. Protein Misfolding and Cellular Stress in Disease and Aging: Humana Press. pp. 3–23. 43 Ceylan-Isik AF , Sreejayan N , Ren J (2011 ) Endoplasmic reticulum chaperon tauroursodeoxycholic acid alleviates obesity-induced myocardial contractile dysfunction . Journal of Molecular and Cellular Cardiology 50 : 107 –116 .21035453 44 Silveira LR , Fiamoncini J , Hirabara SM , Procópio J , Cambiaghi TD , et al (2008 ) Updating the effects of fatty acids on skeletal muscle . Journal of Cellular Physiology 217 : 1 –12 .18543263 45 MohammadTaghvaei N , Taheripak G , Taghikhani M , Meshkani R (2012 ) Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor kB (NF-kB) activation . Cellular Signalling 24 : 1964 –1970 .22580159 46 Brasier A (2006 ) The NF-κB regulatory network . Cardiovascular Toxicology 6 : 111 –130 .17303919 47 Shimizu S , Ugi S , Maegawa H , Egawa K , Nishio Y , et al (2003 ) Protein-tyrosine phosphatase 1B as new activator for hepatic lipogenesis via sterol regulatory element-binding protein-1 gene expression . Journal of Biological Chemistry 278 : 43095 –43101 .12941932 48 Delibegovic M , Zimmer D , Kauffman C , Rak K , Hong EG , et al (2009 ) Liver-specific deletion of protein-tyrosine phosphatase 1B (PTP1B) improves metabolic syndrome and attenuates diet-induced endoplasmic reticulum stress . Diabetes 58 : 590 –599 .19074988 49 Turpin SM , Ryall JG , Southgate R , Darby I , Hevener AL , et al (2009 ) Examination of ‘lipotoxicity’ in skeletal muscle of high-fat fed and ob/ob mice . The Journal of Physiology 587 : 1593 –1605 .19204053 50 Guo R, Zhang Y, Turdi S, Ren J (2013) Adiponectin knockout accentuates high fat diet-induced obesity and cardiac dysfunction: Role of autophagy. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease: In press. 51 Latreille M , Larose L (2006 ) Nck in a complex containing the catalytic subunit of protein phosphatase 1 regulates eukaryotic initiation factor 2α signaling and cell survival to endoplasmic reticulum stress . Journal of Biological Chemistry 281 : 26633 –26644 .16835242 52 Chiarugi P , Cirri P (2003 ) Redox regulation of protein tyrosine phosphatases during receptor tyrosine kinase signal transduction . Trends in Biochemical Sciences 28 : 509 –514 .13678963 53 Tsou P-S , Talia NN , Pinney AJ , Kendzicky A , Piera-Velazquez S , et al (2012 ) Effect of oxidative stress on protein tyrosine phosphatase 1B in scleroderma dermal fibroblasts . Arthritis & Rheumatism 64 : 1978 –1989 .22161819 54 Gloire G , Legrand-Poels S , Piette J (2006 ) NF-kappaB activation by reactive oxygen species: fifteen years later . Biochemical Pharmacology 72 : 1493 –1505 .16723122 55 García-San Frutos M , Teresa F-A , Carrascosa JM , Horrillo D , Barrús MT , et al (2012 ) Involvement of protein tyrosine phosphatases and inflammation in hypothalamic insulin resistance associated with ageing: Effect of caloric restriction . Mechanisms of Ageing and Development 133 : 489 –497 .22733037 56 González-Rodríguez Á , Más-Gutierrez JA , Mirasierra M , Fernandez-Pérez A , Lee YJ , et al (2012 ) Essential role of protein tyrosine phosphatase 1B in obesity-induced inflammation and peripheral insulin resistance during aging . Aging Cell 11 : 284 –296 .22221695	24204775	PMC3799617	CC BY	2021-01-05 17:12:58	yes	PLoS One. 2013 Oct 18; 8(10):e77228
==== Front Case Rep Obstet GynecolCase Rep Obstet GynecolCRIM.OBGYNCase Reports in Obstetrics and Gynecology2090-66842090-6692Hindawi Publishing Corporation 10.1155/2013/602407Case ReportIntravenous Leiomyoma with Extension to the Heart: A Case Report and Review of the Literature Demirkiran Fuat 1 2 Sal Veysel 1 Kaya Umit 2 Alhan Cem 3 Tokgozoglu Nedim 1 1Division of Gynecologic Oncology, Department of Gynecology and Obstetrics, Cerrahpasa Medical Faculty, Istanbul University, 34303 Istanbul, Turkey2Obstetrics and Gynecology Department, Acıbadem Kadıkoy Hospital, Istanbul, Turkey3Cardiovascular Surgery Department, Acıbadem Kadıkoy Hospital, Istanbul, TurkeyVeysel Sal: veyselsal@yahoo.comAcademic Editors: K. Dafopoulos, M. Furuhashi, K. Nasu, and B. Piura 2013 29 9 2013 2013 6024078 7 2013 11 9 2013 Copyright © 2013 Fuat Demirkiran et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Intravenous leiomyomatosis with cardiac extension is an extremely rare uterine tumor. We report here a case of intravenous leiomyoma extending to the right atrium, diagnosed in a patient having leiomyoma. Case Presentation. A 39-year-old woman with no symptoms and a past medical history of two myomectomy operations (7 and 3 years previously) was admitted to our clinic for routine control. We detected a uterine fibroid of 8 centimeters and 4 small solid masses of 1-2 centimeters near the uterus and ovaries at vaginal ultrasonography. Computed tomography (CT) was performed to investigate the abdominal cavity. It revealed a mass originating from the left common iliac vein, which invaded the inferior vena cava (IVC) and extended to the right atrium in addition to the uterine fibroids and pelvic masses. The operation was performed with a combined team of gynecologists and cardiac surgeons and a one-stage operation was accomplished. The postoperative course was uneventful. Conclusion. Abdominal CT is a useful imaging technique for the diagnosis of unusual pathology in a patient with uterine fibroid having suspicious pelvic masses. Also, when a right atrial mass is identified in a female with a prior history of hysterectomy because of leiomyoma or in whom there is a uterine myoma, then intravenous leiomyomatosis should be considered. ==== Body 1. Introduction Intravenous leiomyomatosis (IVL) is a histologically benign, rare smooth muscle tumor derived from either a uterine venous wall or uterine leiomyoma [1]. Although this tumor is usually confined to the pelvic cavity, it sometimes extends into the cardiac cavity. There is no strong relationship between the extent of cardiac involvement and clinical manifestations resulting in the misdiagnosis of the tumor [2–4]. Birch-Hirschfeld [5] first presented a case of IVL in 1896, and Durck first presented a case of intracardiac extension of IVL in 1907; cases of intracardiac extension account for about 10% [6, 7]. To date, less than 300 cases have been reported in the English literature. In 2011 Guo et al. published the largest cohort of patients at a single centre, including a total of 10 patients treated over a 10-year period [8]. We present an unusual case of intravenous leiomyoma arising from the left common iliac vein and extending to the inferior vena cava and right atrium with extensive intracaval attachment. We include a brief review of the literature. 2. Case Report A 39-year-old woman with no symptoms and a past medical history of two myomectomy operations (7 and 3 years previously) was admitted to our clinic for routine control. She had 2 normal deliveries. We detected a uterine fibroid of 8 centimeters and 4 small solid masses of 1-2 centimeters near the uterus and ovaries at vaginal ultrasonography. Computed tomography (CT) was performed to investigate the abdominal cavity. It revealed a mass originating from the left common iliac vein, which invaded the inferior vena cava (IVC) and extended to the right atrium in addition to the uterine fibroids and pelvic masses (Figure 1(a)). Tumor markers were normal, CA125: 13 U/mL (0–35), CA19.9: 6 U/mL (0–37), and CA15.3: 12 U/mL (4.5–29). Transthoracic echocardiogram showed a large and mobile irregular mass in the right atrium projecting through the IVC with no pedicle visible. An urgent operation was planned due to the risk of a sudden cardiac event. The operation was performed with a combined team of gynecologists and cardiac surgeons, and a one-stage operation was accomplished. The surgical approach was made through median sternotomy and laparotomy. The abdomen was explored via a median laparotomy, and a large soft intraligamentary fibroid was seen in the left site of the uterus. There are small retroperitoneal solid masses of different sizes. Also a solid mass were easily palpated within the left common iliac vein and the inferior vena cava. At first, hysterectomy and bilateral salpingo-oophorectomy was performed and all pelvic masses removed out easily. Then, external circulation by cardiopulmonary bypass was instituted to permit cardiac access. Following the institution of deep hypothermia and total circulatory arrest, intracardiac and intravenous masses were removed out by cardiac surgeons (Figure 2). The postoperative course was uneventful. The patient was discharged on the fourth postoperative day. Pathological examination of all the masses revealed to be composed of benign smooth muscle cells with fibrous tissue consistent with leiomyoma. There were no sarcomatous changes. Followup of the patient in the 4th month revealed an excellent patency of vascular structures and no recurrence of tumor at the control computed tomography scan (Figure 1(b)). 3. Discussion IVL is a condition that only affects women, the majority of whom have undergone a previous hysterectomy due to uterine leiomyoma. A review of the cases reported in the literature demonstrates that 64% of the women had undergone a previous hysterectomy, with a range of 6 months to 20 years before presentation with the intravenous portion of the tumor [9]. Our patient underwent two myomectomy operations (7 and 3 years previously). Two theories have been proposed to explain the origin of IVL [10, 11]. One suggests that the neoplasm arises from the vascular wall, and the other implicates vascular invasion of the myometrial veins by the leiomyoma. However, the exact etiology of the vascular invasion by the tumor remains unknown. Since it is a very rare condition, IVL is usually diagnosed after an operation for myoma uteri. In the present case, we were preoperatively able to make a diagnosis with CT. Because of pelvic masses seen on ultrasonographic examination, abdominal cavity was evaluated by CT. The patient had no cardiac symptoms and was in a good general condition. So, in that case, intravenous mass was detected accidentally. Abdominal CT or MRI are not routinely suggested before the operation for uterine fibroid. Usually, right atrial mass is detected firstly in patients with cardiac symptoms. Then IVL is diagnosed by echocardiography, CT, and MRI [12]. In most cases, cardiac catheterization and a contrast study of the inferior vena cava can be adopted to plan the operative strategy [13]. A successful therapy for IVL is dependent on the total surgical excision of the tumor. In the first total resection, reported in 1982 by Ariza et al., there was a delayed laparotomy after resection of the intracardiac portion of the tumor [14]. Recently, one-stage complete resection of the tumors has been performed using circulatory arrest with deep hypothermia for the patients. Incomplete resection results in regrowth or recurrence of the tumor. In our case, we have performed a one-stage operation, which was accomplished with a combined team of gynecologists and cardiac surgeons. 4. Conclusion Abdominal CT is a useful imaging technique for the diagnosis of unusual pathology in a patient with uterine fibroid having suspicious pelvic masses. Also, when a right atrial mass is detected in a female with a prior history of hysterectomy because of leiomyoma or in whom there is a uterine myoma, then IVL should be considered. Figure 1 (a) Preoperative computed tomography scan reveals intracaval filling defects extending between the left iliac vein and the right atrium. (b) Postoperative computed tomography scan in the 4th month of followup reveals excellent patency of vascular structures and no recurrence of tumor. Figure 2 Gross specimen of the tumor removed from the right atrium and inferior vena cava (bottom), uterine and ovarian component (upper side). ==== Refs 1 Clement PB Intravenous leiomyomatosis of the uterus Pathology Annual 1988 23 153 183 2-s2.0-0024197932 3060811 2 Reynen K Köckeritz U Strasser RH Metastases to the heart Annals of Oncology 2004 15 3 375 381 2-s2.0-1842607619 14998838 3 Kullo IJ Oh JK Keeney GL Khandheria BK Seward JB Intracardiac leiomyomatosis: echocardiographic features Chest 1999 115 2 587 591 2-s2.0-0032961537 10027468 4 Yu L Shi E Gu T Xiu Z Fang Q Wang C Intravenous leiomyomatosis with intracardiac extension: a report of two cases Journal of Cardiac Surgery 2011 26 1 56 60 2-s2.0-78751550613 21073522 5 Birch-Hirschfeld FV Lehrbuch der Pathologischen Anatomie 1896 5th edition Leipzig, Germany FCW Vogel 6 Baca López FM Martínez-Enriquez A Castrejón-Aivar FJ Ruanova-León D Yánez-Gutiérrez L Echocardiographic study of an intravenous leiomyoma: case report and review of the literature Echocardiography 2003 20 8 723 725 2-s2.0-10744232286 14641377 7 Durck H Ueber ien Kontinvierlich durch die entere Holhlvene in das Herz vorwachsendes: Fibromyom des uterus Munchen Med Wehnschr 1907 54 p. 1154 8 Guo X Zhang C Fang L Echocardiographic characteristics of intravenous leiomyomatosis with intracardiac extension: a single-institution experience Echocardiography 2011 28 9 934 940 2-s2.0-80053610646 21854425 9 Harris LM Karakousis CP Intravenous leiomyomatosis with cardiac extension: tumor thrombectomy through an abdominal approach Journal of Vascular Surgery 2000 31 5 1046 1051 2-s2.0-0034039002 10805899 10 Norris HJ Parmley T Mesenchymal tumors of the uterus. V. Intravenous leiomyomatosis. A clinical and pathologic study of 14 cases Cancer 1975 36 6 2164 2178 2-s2.0-0016693863 1203870 11 Tierney WM Ehrlich CE Bailey JC Intravenous leiomyomatosis of the uterus with extension into the heart American Journal of Medicine 1980 69 3 471 475 2-s2.0-0018832983 7416191 12 Kang L-Q Zhang B Liu B-G Liu F-H Diagnosis of intravenous leiomyomatosis extending to heart with emphasis on magnetic resonance imaging Chinese Medical Journal 2012 125 1 33 37 2-s2.0-84855849477 22340462 13 Hayasaka K Tanaka Y Fujii M Himi K Negishi N Intravenous leiomyomatosis Journal of Computer Assisted Tomography 2000 24 1 83 85 2-s2.0-0033815259 10667665 14 Ariza A Cerra C Hahn IS Shaw RK Rigney B Intravascular leiomyomatosis of the uterus. A case report Connecticut Medicine 1982 46 12 700 703 2-s2.0-0020347199 7151431	24191207	PMC3804365	CC BY	2021-01-05 10:37:08	yes	Case Rep Obstet Gynecol. 2013 Sep 29; 2013:602407
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24167561PONE-D-13-2626310.1371/journal.pone.0077131Research ArticleHistones-Mediated Lymphocyte Apoptosis during Sepsis Is Dependent on p38 Phosphorylation and Mitochondrial Permeability Transition Histones Induce Lymphocyte Apoptosis during SepsisLiu Zhan-Guo 1 Ni Shu-Yuan 2 Chen Gui-Ming 1 Cai Jing 1 Guo Zhen-Hui 3 Chang Ping 1 * Li Yu-Sheng 4 * 1 Department of ICU, Southern Medical University, Zhujiang Hospital, Guangzhou, China 2 Department of ICU, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 3 Guangdong Provincial Key Laboratory of Geriatric Infection and Organ Function Support, Department of Medical Intensive Care Unit, General Hospital of Guangzhou Military Command, Guangzhou, China 4 Department of Pathophysiology, Southern Medical University, Guangzhou, China Caldwell Charles C. Editor University of Cincinnati, United States of America * E-mail: changp963@163.com (PC); tobyaron@163.com (YSL)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: ZL YL PC SN. Analyzed the data: YL PC ZL SN GC JC ZG. Wrote the paper: YL ZL PC SN. 2013 22 10 2013 8 10 e7713125 6 2013 28 8 2013 © 2013 Liu et al2013Liu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Lymphocyte apoptosis is one reason for immunoparalysis seen in sepsis, although the triggers are unknown. We hypothesized that molecules in plasma, which are up-regulated during sepsis, may be responsible for this. In this study, peripheral lymphocyte apoptosis caused by extracellular histones was confirmed both in mouse and human primary lymphocytes, in which histones induced lymphocyte apoptosis dose-dependently and time-dependently. To identify which intracellular signal pathways were activated, phosphorylation of various mitogen-activated protein kinases (MAPKs) were evaluated during this process, and p38 inhibitor (SB203580) was used to confirm the role of p38 in lymphocyte apoptosis induced by histones. To investigate the mitochondrial injury during these processes, we analyzed Bcl2 degradation and Rhodamine 123 to assess mitochondrial-membrane stability, via cyclosporin A as an inhibitor for mitochondrial permeability transition (MPT). Then, caspase 3 activation was also checked by western-blotting. We found that p38 phosphorylation, mitochondrial injury and caspase 3 activation occurred dose-dependently in histones-mediated lymphocyte apoptosis. We also observed that p38 inhibitor SB203580 decreased lymphocyte apoptotic ratio by 49% (P<0.05), and inhibition of MPT protected lymphocytes from apoptosis. Furthermore, to investigate whether histones are responsible for lymphocyte apoptosis, various concentrations of histone H4 neutralization antibodies were co-cultured with human primary lymphocytes and plasma from cecal ligation and puncture (CLP) mice or sham mice. The results showed that H4 neutralization antibody dose-dependently blocked lymphocyte apoptosis caused by septic plasma in vitro. These data demonstrate for the first time that extracellular histones, especially H4, play a vital role in lymphocyte apoptosis during sepsis which is dependent on p38 phosphorylation and mitochondrial permeability transition. Neutralizing H4 can inhibit lymphocyte apoptosis, indicating that it could be a potential target in clinical interventions for sepsis associated immunoparalysis. This work was supported by grants from the National Natural Science Foundation for Youth of China (81101451 and 81100008), the Natural Science Foundation of Guangdong Province (S2011010003106) and the Guangdong Medical Science and Technology Research Fund (B2011208). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Sepsis causes long-term immunosuppression or immunoparalysis, leading to multiple organ failure (MOF) and possibly death [1]. Although sepsis has been recognized as one of the top causes of mortality worldwide, its incidence is continuing to rise dramatically, with approximately 1,400 deaths/day worldwide [2]. Severe sepsis or septic shock is one of the leading causes of admissions to intensive care units. However, there is no specific treatment currently available due to limited understanding of the underlying mechanism behind sepsis [3]. Recently, bundle therapy has been used with barely satisfactory effect, and the costs are high. Hence, further research into the mechanism of sepsis is urgently needed. It is increasingly being recognized that lymphocyte apoptosis is a vital process in the pathogenesis of sepsis [4], and it is one mechanism of immunosuppression during sepsis, not only because it reduces the number of these critical immune effecter cells [5], but also because of the immunoparalysis caused by apoptotic cells [6]. Moreover, apoptosis of lymphocytes during early stage of sepsis is the major reason for death from this condition [7]. In addition, a reduction in lymphocyte apoptosis is associated with improvement in survival rate in the cecal ligation and puncture (CLP) mouse model [8]. Therefore, understanding the mechanism of lymphocyte apoptosis is crucial for developing effective anti-sepsis therapies [9], [10]. It has been shown that mitogen-activated protein kinases (MAPKs) are involved in the regulation of lymphocyte apoptosis [11], [12]. Furthermore, p38 inhibition is useful for inhibiting lymphoid immunesuppression [13] and improving survival [14] in sepsis. Meanwhile, lymphocyte apoptosis is also mediated by mitochondrial injury [4], [5], [11], [15], resulting in caspase 3 activation [5]. In addition, over-expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), which is an anti-apoptosis protein that acts through stabilizing mitochondrial membrane, protects lymphocytes from apoptosis caused by sepsis [16]–[18]. Therefore, the function of components of the MAPK signaling pathway, especially p38, and mitochondrial injury in lymphocyte apoptosis during sepsis are investigated in the present study. Increases in extracellular histones in the blood of patients with sepsis are associated with prognosis and mortality. Esmon and colleagues reported that levels of extracellular histones were increased in the sera of baboons challenged with E. coli and samples collected from patients with sepsis [19], [20]. In addition, histone H4 neutralization antibody has been shown to have a protective effect in various mouse models of sepsis [19], [20]. Furthermore, extracellular histone H4 has been identified as a major antimicrobial component, which induces the death of microbes in the human body [21]. Histones also cause death of endothelial cells during sepsis [20] and induce apoptosis of renal tubular epithelial cells [22]. Based on the above results, we hypothesized that increased levels of extracellular histones are the direct reason for apoptosis of peripheral lymphocytes during sepsis, which results in an irreversible immune dysfunction. These effects may occur through MAPK phosphorylation (especially p38), mitochondrial injury and caspase 3 activation. To confirm this hypothesis, we tested the effect of histones on lymphocytes, and found that histones could lead to lymphocyte apoptosis dose-dependently and time-dependently through p38 phosphorylation, mitochondrial injury and caspase 3 activation. The present study appears to be the first report recognizing a relationship between lymphocyte apoptosis and histone release during sepsis, and addressing the mechanism by which histones induce lymphocyte apoptosis. These results not only add to the understanding of sepsis, but also provide a potential target for anti-immunoparalysis therapies in sepsis. Methods Reagents Unless otherwise stated, all the reagents used in this study were purchased from Sigma (St. Louis, MO, USA). Animal Model All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. Eighteen male mice (8 to 12 weeks old) were randomly separated into three groups (Normal, Sham and CLP). The CLP sepsis mouse model was established following the published protocol [23]. Sham-operated mice underwent operation without ligation and puncture. Un-operated mice were used as the normal group. Plasma or peripheral lymphocytes were harvested 6 h after surgery. Blood of each mouse was too little to separate enough number of lymphocytes for flow-cytometry analysis, so we mixed the lymphocytes of six mice of one group together. Also, we mixed the plasma of the six mice in one group to do the western blotting. And the experiment was repeated three times. Human Subjects Ethical approval was given by the Committee on the Ethics of Experiments of Southern Medical University and all participants provided written informed consent. Peripheral venous blood was taken from three healthy volunteers aged between 20 and 30 years old for each experiment, and was collected into vacuum tubes containing dried lithium heparin. Lymphocytes were separated immediately after collection. Separation and Stimulation of Lymphocytes Lymphocytes were separated from heparinized whole blood using a lymphocyte separation medium (MP Biomedicals, Santa Ana, CA, USA) in accordance with the manufacturer’s instructions. Separated lymphocytes were cultured at a concentration of 1×106/ml in a 96-well plate at 37°C with 5% CO2, and were treated with various concentrations (0, 50, 100, 200 µg/ml) of histones (VWR International, Radnor, PA, USA) for a set time (2 h), or were treated with a set concentration (100 µg/ml) of histones for various time durations (0, 2 and 3 h). After incubation, the lymphocytes were collected for analysis of apoptosis, p38 phosphorylation, mitochondrial injury and caspase 3 activation. Inhibitor of p38 activation (25 or 10 µmol/L SB203580) or dimethyl sulfoxide (DMSO) was incubated together with 100 µg/ml histones for 2 h, and then the peripheral lymphocyte apoptotic ratios were tested. Inhibitor of mitochondrial permeability transition, cyclosporin A (CSA) (25 ng/ml or 50 ng/ml), was pre-incubated with human peripheral lymphocytes for 12 h, and then the cells were treated with 50 µg/ml histones for 3 h. Finally, the peripheral lymphocyte apoptotic ratios were tested. To assess extracellular histone H4 neutralization, 20 µl of plasma from CLP mice (which contained histones) or sham mice was co-incubated with various concentrations (0, 10, 25 µg/ml) of H4 neutralization antibody (Cell Signaling Technology, Danvers, MA, USA) for 20 minutes at room temperature. Then plasma from each group was added to the supernatant of isolated human lymphocytes for 2 h, which was followed by flow-cytometry analysis. Animal Treatment Twelve male mice (8 to 12 weeks old) were randomly separated into two groups. The mice were injected with phosphate-buffered saline (PBS) or histones (60 mg/kg weight) through the caudal vein. Whole blood was taken 6 h after injection and lymphocytes were separated for apoptotic ratio analysis. Flow-cytometry Analysis For detection of apoptosis by flow-cytometry analysis, treated lymphocytes were stained with Annexin V and propidium iodide (PI) (BD, Franklin Lakes, NJ, USA) following the manufacturer’s instructions. Detection of Mitochondrial Injury Rhodamine 123 (Rho123) was used for mitochondrial injury detection in accordance with the manufacturer’s handbook. Immunoblotting Using our previously published protocol [24], we used primary antibodies against histone H4, p38, phosphorylated p38 (p-p38), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Bcl2 (Cell Signaling Technology, Danvers, MA, USA) followed by incubation with secondary antibodies. Statistical Analysis Data were analyzed using SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). All data are presented as means ± SD. The differences between experimental and control groups were assessed by the two-tailed unpaired Student’s t-test and p<0.05 was considered significant. Results Levels of Extracellular Histone H4 and Peripheral Apoptotic Lymphocytes Increase during the Early Phase of Sepsis in Mice The sepsis mouse model was established by CLP. Whole blood was collected 6 h later, and the histone H4 level in plasma was tested by western blotting. The result showed that levels of histone H4 in mouse plasma were significantly increased compared with normal or sham group (Fig. 1A). Meanwhile, the peripheral lymphocyte apoptotic ratio (13.11±0.90%) was increased after 6 h in the CLP mice compared with that of normal or sham mice (2.99±0.67%) (Fig. 1B). 10.1371/journal.pone.0077131.g001Figure 1 Lymphocyte apoptotic ratio was increased by extracellular histones in CLP mouse model. A. The levels of plasmic histone H4 of normal, sham or CLP mice were detected by western blotting 6± SD (n = 3). p<0.05, as compared with normal group. ‡ p<0.05, as compared with Sham group. C. Histones were injected into mice at the dose of 60 mg/kg weight. Lymphocytes were separated from whole blood 6 h after injection for apoptosis analysis by flow-cytometry. Values are presented as means ± SD (n = 6). p<0.05, as compared with PBS group. Intravenous Injection of Histones Induces Peripheral Lymphocyte Apoptosis in Mice To test if increased extracellular histone H4 and peripheral apoptotic lymphocytes are interdependent events during sepsis, we injected histones into the caudal vein of anesthetized mice. The peripheral lymphocyte apoptotic ratio was increased by 11.59±0.99% compared with that of the PBS group 6 h after injection (Fig. 1C). Histones are Associated with the Apoptosis of Human Peripheral Lymphocytes in Dose-dependent and Time-dependent Manner The in-vivo mouse experiment showed that extracellular histones, which were increasingly released during sepsis, could induce apoptosis of peripheral lymphocytes. In order to confirm the effect of extracellular histones and expound the mechanisms by which histones induce lymphocyte apoptosis in patients with sepsis, we cultured isolated human lymphocytes with histones in vitro. As shown in Fig. 2A, the various concentrations (0, 50, 100, 200 µg/ml) of extracellular histones led to peripheral lymphocyte apoptosis in a dose-dependent manner after 2 h treatment (15.64±2.44%, 77.98±2.90%, 93.61±2.86%, 94.30±3.31%, respectively). Additionally, this dose dependency was mainly correlated with an early apoptotic ratio (7.85±0.53%, 58.14±10.68%, 84.27±6.71%, 85.22±4.72%, respectively). The late apoptotic ratio made no statistically significant difference. Extracellular histones also led to peripheral lymphocyte apoptosis in a time-dependent manner of apoptotic status, but not of a significant ratio. The apoptotic peripheral lymphocytes were largely found to be in early apoptosis (89.54±2.02%) after 2 h of stimulation, but majority found in late apoptosis (91.80±1.54%) after 3 h. The total apoptotic ratios of the two groups were the same (Fig. 2B). Because of this, the early and late apoptotic ratio is not given for other experiments when the composition was the same. 10.1371/journal.pone.0077131.g002Figure 2 Histones induced human lymphocyte apoptosis dose-dependently and time-dependently. Human lymphocytes were cultured with histones of various concentrations (0, 50, 100, 200 µg/ml) or 100 µg/ml histones for various time durations (0, 2, 3 h). Lymphocyes were harvested and apoptotic ratio was detected by flow-cytometry. A. Dose-dependent manner. Values are presented as means ± SD (n = 3). P<0.05, as compared with 0 µg/ml. ‡P<0.05, as compared with 50 µg/ml. B. Time-dependent manner. Values are presented as means ± SD (n = 3). P<0.05, as compared with 0 µg/ml group. Human Peripheral Lymphocyte Apoptosis Associated with Extracellular Histones is p38 Phosphorylation-dependent To confirm which MAPK signal pathway is necessary for peripheral lymphocyte apoptosis induced by histones, the phosphorylation of p38, ERK and JNK were assessed. The data showed that histones only enhanced the phosphorylation of p38 after 2 h of stimulation (Fig. 3A). The results of ERK and JNK phosphorylation were not shown. The p38 pathways in lymphocytes was blocked by SB203580 after 2 h of incubation. The results showed that SB203580 was able to significantly decrease the peripheral lymphocyte apoptotic ratio (22.21±2.79% vs. 43.83±4.70%) (Fig. 3C). 10.1371/journal.pone.0077131.g003Figure 3 Inhibition of p38 phosphorylation blocked lymphocyte apoptosis induced by histones. A. Western blotting results of P38 phosphorylation. Lymphocytes were harvested after histones treatment for 2± SD (n = 3). ‡P<0.05, as compared with 0 µg/ml group. C. Human lymphocytes were exposed to 100 µg/ml histones with DMSO, 10 and 25 µmol/L SB203580. Lymphocyes were harvested 2 h after treatment for apoptosis detection by flow-cytometry. Values are presented as means ± SD (n = 3). P<0.05, as compared with control. Mitochondrial Injury is a Key Mechanism Underlying Histones-mediated Apoptosis in Lymphocytes Mitochondrial injury plays an important role in the development of lymphocyte apoptosis. Esmon found that histones could cause mitochondrial injury in endothelial cells after injection of histones to mice [20]. Thus, we proposed that mitochondrial injury might be a vital process during histone associated peripheral lymphocyte apoptosis. To confirm this hypothesis, we compared mitochondrial injury in lymphocytes cultured with various concentrations of histones using Rho123 (a dye that reflects the stability of the mitochondrial membrane). Our results showed that histones led to mitochondrial injury dose-dependently (Fig. 4A). And inhibition of mitochondrial permeability transition by CSA could decrease the peripheral lymphocyte apoptosis in a dose dependent manner (Fig. 4D). 10.1371/journal.pone.0077131.g004Figure 4 Mitochondrial injury is a key mechanism to induce histones-mediated apoptosis in lymphocytes. A. Human lymphocytes were cultured with various concentrations (0, 50, 100, 200 µg/ml) of histones. Lymphocyes were harvested 2 h after treatment and mitochondrial injury was detected by flow-cytometry. M5 represent the percentage of lymphocytes without mitochondrial injury. Values are presented as means ± SD (n = 3). P<0.05, as compared with 0 µg/ml group. B. Western blotting results of Bcl2. Lymphocytes were harvested after histones treatment for 2 h. Equal protein aliquots of cell lysate were examined by immunoblotting with antibodies against GAPDH or Bcl2. GAPDH was used to verify equal gel loading and transblot efficiencies. C. Bar graph of relative Bcl2 intensity. Values are presented as means ± SD (n = 3). ‡P<0.05, as compared with 0 µg/ml group. D. Inhibition of mitochondrial permeability transition by CSA (25 and 50 ng/ml) can decrease the peripheral lymphocyte apoptosis. alues are presented as means ± SD (n = 3). ▾ P<0.05, as compared with CSA 0 ng/ml His 50 µg/ml group. The expression level of Bcl2, a type of anti-apoptosis protein, is another important marker for mitochondrial-membrane stability. We found that the Bcl2 expression level was downregulated after histones stimulation (Fig. 4B). Extracellular Histones Activate Caspase 3 during Human Peripheral Lymphocyte Apoptosis In addition to the mitochondrial pathway, any proapoptotic pathway will eventually trigger caspase 3 activation. Thus, we compared the activation of caspase 3 between three groups treated with various concentrations of histones (0, 50, 100 µg/ml). Using gray-value comparison, we found that histones can cause caspase 3 activation dose-dependently (Fig. 5). 10.1371/journal.pone.0077131.g005Figure 5 Histones induced caspase 3 activation in a dose-dependent manner. A. Isolated human lymphocytes were exposed to various concentrations (0, 50, 100 µg/ml) of histones. Lymphocyes were harvested 1.5 h after treatment and casepase 3 activation was detected by western blotting. Celeaved caspase 3 represents the activation of caspase 3. GAPDH was used to verify equal gel loading and transblot efficiencies. B. Bar graph of relative activated caspase 3 intensity. Values are presented as means ± SD (n = 3). P<0.05, as compared with 0 µg/ml. ‡P<0.05, as compared with 50 µg/ml group. Extracellular Histone H4 Neutralization Dose-dependently Reduces Human Peripheral Lymphocyte Apoptosis We were interested in assessing whether it is possible to inhibit peripheral lymphocyte apoptosis through extracellular histone neutralization, and whether histones are the only reason for lymphocyte apoptosis. In order to standardize our experiments, we co-cultured plasma from CLP mice (containing histones) or sham mice and isolated human lymphocytes with various concentrations of H4 neutralization antibody (0, 10, 25 µg/ml) and control IgG (25 µg/ml). As shown in Fig. 6, the CLP mouse plasma increased the peripheral lymphocyte apoptotic ratio compared with the sham mouse plasma (73.18±2.44% vs. 44.32±5.52%). In addition, the H4 neutralization antibody reduced the peripheral lymphocyte apoptotic ratio in a dose-dependent manner (55.68±2.60% and 35.29±1.34% respectively vs. 73.18±2.44%). Unexpectedly, we found that 25 µg/ml of H4 neutralization antibodies obviously inhibited the peripheral lymphocyte apoptotic ratio caused by the sepsis plasma (35.29±1.34% vs. 44.32±5.52%) (Fig. 6). The histone H4 level in CLP mouse plasma was checked by western blotting (data not shown). 10.1371/journal.pone.0077131.g006Figure 6 Extracellular histone H4 neutralization antibody blocked human peripheral lymphocyte apoptosis induced by plasma of sepsis mouse model dose-dependently. Isolated human lymphocytes were exposed to 20 µl plasma of sham or CLP mice with various concentrations of H4 neutralization antibody (0, 10, 25 µg/ml) and control IgG (25 µg/ml). Lymphocytes were harvested at 2 h and apoptotic ratio was detected by Flow-cytometry. Values are presented as means ± SD (n = 3). P<0.05, as compared with sham. ‡P<0.05, as compared with CLP group. ▾ P<0.05, as compared with CLP+Anti-H4 10 µg/ml group. Discussion Previous data studies have shown that extensive lymphocyte apoptosis occurred in a number of organs [25]–[28], especially in the circulatory system [5], [15], during sepsis. It has also been shown that the peripheral lymphocyte apoptotic ratio increases in the CLP mouse model, which was confirmed by the current study (Fig. 1). However, the triggers of lymphocyte apoptosis during sepsis are yet unknown. Tumor necrosis factor (TNF)-α, which is increased in patients with sepsis [29], can induce apoptosis during sepsis in certain types of cells through a death receptor. However, anti-TNF-α antibodies are unable to block lymphocyte apoptosis in the septic mouse model [30]. Thus, TNF-α couldn’t be the reason for the peripheral lymphocyte apoptosis that occurs during sepsis. In the current study, we first found that extracellular histones were associated with peripheral lymphocyte apoptosis in both mouse (Fig. 1B) and human primary lymphocytes (Fig. 2) in a dose-dependent and time-dependent manner, indicating that extracellular histones are the triggers of this apoptosis. We observed that not only did the extracellular histone H4 neutralization antibody have a dose-dependent effect on inhibition of peripheral lymphocyte apoptosis, but that the appropriate dose could effectively suppress the apoptosis (Fig. 6). These results indicate that extracellular histone H4 is the dominant stimulant for sepsis-related peripheral lymphocyte apoptosis. Absolute lymphocyte count is decreased not only in sepsis, but also in critically ill patients without sepsis; however, depressed absolute lymphocyte count in sepsis is not the same as that in critically ill patients without sepsis, who can experience a return to normal values quickly. In sepsis, lymphocyte count decreases persistently throughout the course of the disease, which leads to irreversible immunosuppression or immunoparalysis, resulting in serious complications. Identification of extracellular histones inducing peripheral lymphocyte apoptosis presents a model of immunosuppression formation and maintenance, which is like a vicious positive feedback circle (Fig. 7). Histones are released into blood both by the damaged cells in burned, traumatized or surgically operated tissue, and by the dying neutrophils that migrate in increasing numbers into the injured area. Extracellular histones, especially H4, initially induce peripheral lymphocyte apoptosis, as this condition occurs earlier than other types of immunocytes in sepsis [8], [31]. The apoptotic lymphocytes then become the new sources of histones, and these increase the level of extracellular histones in blood. The dramatic rise in extracellular histones activates platelets through toll-like receptor 4 (TLR4) [32], and this may activate neutrophil extracellular traps (NETs) [33], which are another source of histones [19]. In this condition, increased number of neutrophils migrating into the injured area during sepsis is not helpful for patient recovery [8], which has been demonstrated by previous research [34]. In the meantime, professional phagocytes in blood, such as neutrophils, macrophages or certain dendritic cells (DCs), engulf the apoptotic lymphocytes, and this also results in immunosuppression through different pathways [35]–[37]. Consequently, even if the pathogenic factor is removed, extracellular histones can still be released into blood from the apoptotic peripheral lymphocytes and NETs. This results in further failure of lymphocytes, as well as of other immunocytes, such as neutrophils, macrophages or DCs, in which dysfunction occurs either through the phagocytosis of apoptotic cells. Therefore, once infection, burn or trauma occurs to a certain extent, various types of immunocytes are unable to act normally, and the immunosuppressive condition of sepsis maintains itself by a vicious positive feedback circle, unless the extracellular histones and the apoptotic cells are eliminated. This model presents us one of potential mechanisms of immunoparalysis status during sepsis. 10.1371/journal.pone.0077131.g007Figure 7 The immunosuppressive condition of sepsis maintains itself by a vicious positive feedback circle caused by extracellular histones. The importance of our work lies not only in the identification of extracellular histones as the trigger of peripheral lymphocyte apoptosis, but also indicates possible anti-immunoparalysis targets in sepsis. SB203580, which is a p38 phosphorylation blocker, decreased the peripheral lymphocyte apoptotic ratio from 43.83±4.70% to 22.21±2.79% (Fig. 3C). This indicates that inhibition of p38 phosphorylation is a potential candidate for anti-immunoparalysis therapies through blocking of peripheral lymphocyte apoptosis, as demonstrated by previous research [13], [14]. Inhibition of mitochondrial permeability has a protective function in the septic mouse model [38], and the mechanism may be involved in inhibiting the lymphocyte apoptosis, as shown in the current study. Extracellular histones lead to peripheral lymphocyte apoptosis through mitochondrial injury and inhibition of mitochondrial permeability transition by CSA (Fig. 4). Finally, we found that H4 neutralization antibodies effectively blocked the peripheral lymphocyte apoptosis caused by septic plasma (Fig. 6). This indicates that anti-H4 therapies might be a potential clinical intervention to fight immunoparalysis status of sepsis, which is currently a challenge. Recently, we are carring out in-vivo studies are addressing the effects of anti-H4 therapies on peripheral lymphocyte apoptosis and the functions of other immunocytes during sepsis. ==== Refs References 1 Stearns-Kurosawa DJ , Osuchowski MF , Valentine C , Kurosawa S , Remick DG (2011 ) The pathogenesis of sepsis . Annu Rev Pathol 6 : 19 –48 .20887193 2 Russell JA (2006 ) Management of sepsis . N Engl J Med 355 : 1699 –1713 .17050894 3 Vincent JL , Sakr Y , Sprung CL , Ranieri VM , Reinhart K , et al (2006 ) Sepsis in European intensive care units: results of the SOAP study . Crit Care Med 34 : 344 –353 .16424713 4 Lang JD , Matute-Bello G (2009 ) Lymphocytes, apoptosis and sepsis: making the jump from mice to humans . Crit Care 13 : 109 .19216722 5 Hotchkiss RS , Osmon SB , Chang KC , Wagner TH , Coopersmith CM , et al (2005 ) Accelerated lymphocyte death in sepsis occurs by both the death receptor and mitochondrial pathways . J Immunol 174 : 5110 –5118 .15814742 6 Hotchkiss RS , Chang KC , Grayson MH , Tinsley KW , Dunne BS , et al (2003 ) Adoptive transfer of apoptotic splenocytes worsens survival, whereas adoptive transfer of necrotic splenocytes improves survival in sepsis . Proc Natl Acad Sci U S A 100 : 6724 –6729 .12736377 7 Atmatzidis S , Koutelidakis IM , Chatzimavroudis G , Kotsaki A , Louis K , et al (2011 ) Detrimental effect of apoptosis of lymphocytes at an early time point of experimental abdominal sepsis . BMC Infect Dis 11 : 321 .22099496 8 Muenzer JT , Davis CG , Chang K , Schmidt RE , Dunne WM , et al (2010 ) Characterization and modulation of the immunosuppressive phase of sepsis . Infect Immun 78 : 1582 –1592 .20100863 9 Hotchkiss RS , Tinsley KW , Swanson PE , Chang KC , Cobb JP , et al (1999 ) Prevention of lymphocyte cell death in sepsis improves survival in mice . Proc Natl Acad Sci U S A 96 : 14541 –14546 .10588741 10 Hotchkiss RS , Coopersmith CM , Karl IE (2005 ) Prevention of lymphocyte apoptosis–a potential treatment of sepsis? Clin Infect Dis 41 Suppl 7S465 –469 .16237649 11 Torcia M , De Chiara G , Nencioni L , Ammendola S , Labardi D , et al (2001 ) Nerve growth factor inhibits apoptosis in memory B lymphocytes via inactivation of p38 MAPK, prevention of Bcl-2 phosphorylation, and cytochrome c release . J Biol Chem 276 : 39027 –39036 .11495898 12 Gendron S , Couture J , Aoudjit F (2003 ) Integrin alpha2beta1 inhibits Fas-mediated apoptosis in T lymphocytes by protein phosphatase 2A-dependent activation of the MAPK/ERK pathway . J Biol Chem 278 : 48633 –48643 .13679375 13 Song GY , Chung CS , Chaudry IH , Ayala A (2001 ) MAPK p38 antagonism as a novel method of inhibiting lymphoid immune suppression in polymicrobial sepsis . Am J Physiol Cell Physiol 281 : C662 –669 .11443065 14 O’Sullivan AW , Wang JH , Redmond HP (2009 ) NF-kappaB and p38 MAPK inhibition improve survival in endotoxin shock and in a cecal ligation and puncture model of sepsis in combination with antibiotic therapy . J Surg Res 152 : 46 –53 .19027920 15 Chang KC , Unsinger J , Davis CG , Schwulst SJ , Muenzer JT , et al (2007 ) Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis . FASEB J 21 : 708 –719 .17307841 16 Hotchkiss RS , Swanson PE , Knudson CM , Chang KC , Cobb JP , et al (1999 ) Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis . J Immunol 162 : 4148 –4156 .10201940 17 Oberholzer C , Oberholzer A , Bahjat FR , Minter RM , Tannahill CL , et al (2001 ) Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis . Proc Natl Acad Sci U S A 98 : 11503 –11508 .11553765 18 Iwata A , Stevenson VM , Minard A , Tasch M , Tupper J , et al (2003 ) Over-expression of Bcl-2 provides protection in septic mice by a trans effect . J Immunol 171 : 3136 –3141 .12960340 19 Chaput C , Zychlinsky A (2009 ) Sepsis: the dark side of histones . Nat Med 15 : 1245 –1246 .19893552 20 Xu J , Zhang X , Pelayo R , Monestier M , Ammollo CT , et al (2009 ) Extracellular histones are major mediators of death in sepsis . Nat Med 15 : 1318 –1321 .19855397 21 Lee DY , Huang CM , Nakatsuji T , Thiboutot D , Kang SA , et al (2009 ) Histone H4 is a major component of the antimicrobial action of human sebocytes . J Invest Dermatol 129 : 2489 –2496 .19536143 22 Allam R , Scherbaum CR , Darisipudi MN , Mulay SR , Hagele H , et al (2012 ) Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4 . J Am Soc Nephrol 23 : 1375 –1388 .22677551 23 Rittirsch D , Huber-Lang MS , Flierl MA , Ward PA (2009 ) Immunodesign of experimental sepsis by cecal ligation and puncture . Nat Protoc 4 : 31 –36 .19131954 24 Li Y , Dinwiddie DL , Harrod KS , Jiang Y , Kim KC (2010 ) Anti-inflammatory effect of MUC1 during respiratory syncytial virus infection of lung epithelial cells in vitro . Am J Physiol Lung Cell Mol Physiol 298 : L558 –563 .20081068 25 Ayala A , Herdon CD , Lehman DL , DeMaso CM , Ayala CA , et al (1995 ) The induction of accelerated thymic programmed cell death during polymicrobial sepsis: control by corticosteroids but not tumor necrosis factor . Shock 3 : 259 –267 .7600193 26 Hotchkiss RS , Swanson PE , Freeman BD , Tinsley KW , Cobb JP , et al (1999 ) Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction . Crit Care Med 27 : 1230 –1251 .10446814 27 Hotchkiss RS , Tinsley KW , Swanson PE , Schmieg RE Jr, Hui JJ , et al (2001 ) Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+ T lymphocytes in humans . J Immunol 166 : 6952 –6963 .11359857 28 Coopersmith CM , Stromberg PE , Dunne WM , Davis CG , Amiot DM 2nd, et al (2002 ) Inhibition of intestinal epithelial apoptosis and survival in a murine model of pneumonia-induced sepsis . JAMA 287 : 1716 –1721 .11926897 29 Deitch EA (1998 ) Animal models of sepsis and shock: a review and lessons learned . Shock 9 : 1 –11 . 30 Hiramatsu M , Hotchkiss RS , Karl IE , Buchman TG (1997 ) Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway . Shock 7 : 247 –253 .9110409 31 Le Tulzo Y , Pangault C , Gacouin A , Guilloux V , Tribut O , et al (2002 ) Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome . Shock 18 : 487 –494 .12462554 32 Semeraro F , Ammollo CT , Morrissey JH , Dale GL , Friese P , et al (2011 ) Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4 . Blood 118 : 1952 –1961 .21673343 33 Clark SR , Ma AC , Tavener SA , McDonald B , Goodarzi Z , et al (2007 ) Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood . Nat Med 13 : 463 –469 .17384648 34 Alves-Filho JC , de Freitas A , Spiller F , Souto FO , Cunha FQ (2008 ) The role of neutrophils in severe sepsis . Shock 30 Suppl 13 –9 . 35 Fadok VA , Bratton DL , Konowal A , Freed PW , Westcott JY , et al (1998 ) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF . J Clin Invest 101 : 890 –898 .9466984 36 Ren G , Su J , Zhao X , Zhang L , Zhang J , et al (2008 ) Apoptotic cells induce immunosuppression through dendritic cells: critical roles of IFN-gamma and nitric oxide . J Immunol 181 : 3277 –3284 .18713999 37 Esmann L , Idel C , Sarkar A , Hellberg L , Behnen M , et al (2010 ) Phagocytosis of apoptotic cells by neutrophil granulocytes: diminished proinflammatory neutrophil functions in the presence of apoptotic cells . J Immunol 184 : 391 –400 .19949068 38 Larche J , Lancel S , Hassoun SM , Favory R , Decoster B , et al (2006 ) Inhibition of mitochondrial permeability transition prevents sepsis-induced myocardial dysfunction and mortality . J Am Coll Cardiol 48 : 377 –385 .16843190	24167561	PMC3805602	CC BY	2021-01-05 17:13:26	yes	PLoS One. 2013 Oct 22; 8(10):e77131
==== Front ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XHindawi Publishing Corporation 10.1155/2013/421569Research ArticleEpidemiological Pattern of Newly Diagnosed Children with Type 1 Diabetes Mellitus, Taif, Saudi Arabia Kamal Alanani Naglaa Mohamed 1 Alsulaimani Adnan Amin 2 1Pediatric Department, Faculty of Medicine, Cairo University, Cairo, Egypt2Pediatric Department, Faculty of Medicine, Taif University, Saudi ArabiaNaglaa Mohamed Kamal Alanani: nagla.kamal@kasralainy.edu.egAcademic Editors: K. Gillespie and L. Lowes 2013 9 10 2013 2013 4215691 8 2013 25 8 2013 Copyright © 2013 N. M. Kamal Alanani and A. A. Alsulaimani.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction and Aim. Type-1-diabetes mellitus (T1DM) is the most commonly diagnosed type of DM in children and adolescents. We aim to identify the epidemiological profile, risk factors, clinical features, and factors related to delayed diagnosis or mismanagement in children with newly diagnosed T1DM in Taif, Saudi Arabia. Patients and Methods. Ninety-nine newly diagnosed patients were included in the study along with 110 healthy controls. Patients were classified into 3 groups (I: >2 years, II: 2–>6 years, and III: 6–12 years). Both patients and controls were tested for C-peptide, TSH, and autoantibodies associated with DM and those attacking the thyroid gland. Results. Diabetic ketoacidosis was present in 79.8%. Delayed and missed diagnoses were recorded in 45.5%, with significant correlation to age and district of origin. Severity at presentation showed significant correlation with age and cow's milk feeding. Group I, those with misdiagnosis or positive DM related autoantibodies, had more severe presentations. The correlation of C-peptide and TSH levels in patients and controls was significant for C-peptide and nonsignificant for TSH. Conclusion. Misdiagnosis and mismanagement are common and account for more severe presentation, especially in young children >2 years. Early introduction of cow's milk appears to be a risk factor for the development of T1DM. ==== Body 1. Introduction Type 1 diabetes mellitus (T1DM) is the most commonly diagnosed type of DM in children and adolescents. It presents usually with symptomatic hyperglycemia and imparts the immediate need for exogenous insulin replacement [1]. The presentation of T1DM is either as classic new onset DM (most common), silent DM, or diabetic ketoacidosis (DKA) which constitutes 20%–40% of cases [2]. The classic new onset T1DM patients present with polyuria, polydipsia, polyphagia, weight loss, and lethargy, while those with silent T1DM are typically diagnosed by families or physicians with high index of suspicion. Children who present with DKA also present with dehydration, vomiting, altered mental status, and rapid deep respiration (Kussmaul's breathing) [3]. Because DKA is a potentially preventable acute complication of DM and a predominant cause of mortality in children with diabetes, early recognition and prompt treatment should substantially reduce mortality in children with T1DM [4]. Increased public awareness of early symptoms of diabetes is needed to reduce the incidence and severity of DKA. In addition, greater medical alertness to the possibility of T1DM in a young child should be stressed [5]. The aim of the present study is to identify the main epidemiological patterns, clinical features, and risk factors for newly diagnosed children with T1DM in Taif district, Saudi Arabia. We aimed also at identification of the risk factors related to delayed diagnosis or mismanagement of newly diagnosed patients. 2. Patients and Methods 2.1. Patients We carried a prospective study during the period from March 2011 to March 2013 on ninety-nine Saudi children with newly diagnosed T1DM aged from birth to twelve years old from those attending the Pediatric Endocrinology outpatient clinics, the Pediatric Emergency units and the Pediatric inpatients wards, Taif region Hospitals, Saudi Arabia. One hundred and ten healthy children of matched age and sex from those attending the Well Child clinics served as controls. Written informed consents were obtained from all patients' and controls' parents for contribution into the current study. The study was approved by the research and ethical committees of the contributing hospitals. 2.2. Methods Patients were classified according to age into 3 groups ((I): <2 years, (II): 2–<6 years, and (III): 6–12 years). All patients were subjected to the following. Full history taking: age, district of origin, mode of presentation, clinical symptoms preceding diagnosis, duration before diagnosis, delayed or misdiagnosis, associated endocrinopathies or autoimmune diseases at onset, nutritional history at infancy (breastfeeding, cow's milk feeding whether formulas, yogurt, or other milk derivatives), family history including consanguinity, and positive family history of diabetes, other endocrinopathies or autoimmune diseases. Thorough clinical examination: anthropometric measurements obtained following the recommendations of the international biological program [6] and plotted on the Saudi children growth charts [7], thorough general and body systems examination [8]. Classification: DKA categorization was carried according to the American Diabetes Association into three stages of severity [9]. Mild: blood pH mildly decreased to between 7.25 and 7.30 (normal 7.35–7.45); serum bicarbonate decreased to 15–18 mmol/L (normal above 20); the patient is alert. Moderate: pH 7.00–7.25, bicarbonate 10–15, mild drowsiness may be present. Severe: pH below 7.00, bicarbonate below 10, stupor or coma may occur. Laboratory investigations: all patients were tested at initial presentation for blood glucose, urine acetone, blood gases, serum electrolytes, kidney functions, thyroid stimulating hormone (TSH), autoantibodies associated with DM (insulin autoantibodies (IAA) and glutamic acid decarboxylase antibodies (GADA), islet cells antibodies (ICA)), and autoantibodies to the thyroid gland. After stabilization and an overnight fasting (>6–8 hours), patients were tested for fasting blood sugar and fasting C-peptide. All controls were tested for fasting blood sugar, fasting C-peptide, TSH, and autoantibodies associated with DM and those against thyroid gland. 3. Statistical Methods Data management and analysis were performed using Statistical Analysis Systems, SAS versus 8.02. Numerical data were summarized using means and standard deviations or medians and ranges. Categorical data were summarized as percentages. Differences between two groups with respect to numeric variables were tested using the Student's t-test or Mann-Whitney, for small sample size. A one-way analysis of variance for small sample size, Kruskall-Wallis test, was performed to test differences between more than two groups. Chi-square test was used to compare groups with respect to categorical data or Fisher's exact test, for small sample size [10]. All P values are two sided. P values < 0.05 were considered significant. 4. Results Ninety-nine children, 52 males (52.5%) and 47 females (47.5%), with newly diagnosed T1DM were included in the current study. The mean age at diagnosis was 6.8 years ±4.6 (range 0.39–12 years) in males and 6.6 ± 4.2 years (range 0.19–12 years) in females. Patients were classified according to age into 3 groups ((I): <2 years, (II): 2–<6 years, and (III): 6–12 years). The demographic data of the studied population are shown in Table 1. Sixty percent of patients were coming from rural areas and 40% from urban areas. They were distributed more or less similarly among the three age groups with P > 0.05 (Table 1). The main symptoms preceding the diagnosis are illustrated in Figure 1 where polyuria, polydipsia, and weight loss (the classic triad of DM) had the highest percentage followed by nocturia and nocturnal enuresis then abdominal pain follows. Disturbed conscious level was present in an appreciable percentage (46.5%) being more frequent in those <2 yrs (60.9%) compared to 40.9% and 42.6% in group II and III, respectively. The distribution and correlation of the main symptoms experienced by the patients prior to diagnosis with the studied age groups are shown in Table 2 where polyphagia was found to be more frequent than decreased appetite and refusal of feeding (Figure 1), with no age differences. Abdominal pain was found more frequent as the age gets older. Around 14.3% of those below 2 years had colics and refusal of feeding, while 40.9% and 74% of both groups II and III, respectively, had abdominal pain with a statistically significant higher frequency in the 6–12 years age group (P < 0.001). Nocturia and nocturnal enuresis were found to be more frequent in group III with P < 0.001. Most patients presented with moderate to severe DKA (51.5%), followed by mild DKA in 28.3%. Only 20.2% presented with hyperglycemia without ketoacidosis. The correlation between the mode of presentation and age is shown in Table 1, where those <2 years presented more severe. The majority of the cases with abdominal pain (84.6%) presented with DKA at diagnosis, being moderate to severe in 2/3. All cases complaining of vomiting at diagnosis were in DKA, again being moderate to severe in 2/3. The triad of abdominal pain, vomiting, and dehydration were evident in all cases presenting with moderate to severe DKA (Table 3). The duration of symptoms before diagnosis varied greatly in such way that the standard deviation was found to be greater than the mean itself, accordingly we preferred the median which was found to be 14 days (range 1 to 180 days), as shown in Table 1. There was a significant correlation between the duration of symptoms and the districts of origin with higher duration of symptoms among those coming from rural areas, P value = 0.028 (Figure 2). Twelve patients had prolonged duration of symptoms before diagnosis. Eight patients had 60 days duration; 5 of them presented to us with moderate to severe DKA, and the other 3 presented with mild DKA. Two patients (4 months and 1.6 years) presented after 90 days duration of symptoms with hyperglycemia and mild DKA, respectively. They were misdiagnosed as gastroenteritis (GE) and weaning problems, respectively. One child, 11 years, had 140 days duration of symptoms. He presented with moderate to severe DKA and was misdiagnosed as GE. The child with the longest duration of symptoms, 180 days, presented with hyperglycemia only and had delayed diagnosis due to atypical symptoms (headache, nausea, and abdominal pain). The severity of presentation at the time of diagnosis among the different age groups is presented in Table 1, which shows that 20 children (20.2%) presented with hyperglycemia without ketosis, 28 (28.3%) with mild DKA, and 51 (51.5%) with moderate to severe DKA. Group I and III presented more severe with moderate to severe DKA (P < 0.05) (Table 1). High percentage of the studied population (45.5%) were misdiagnosed at their initial presentation. They were misdiagnosed either as upper (25%) or lower (37.5%) respiratory tract infection, or gastroenteritis (37.5%). Misdiagnosis was found to be statistically higher in group I as compared to the other 2 groups (69.6% versus 36.4 and 38.9%, P < 0.05) (Table 1) with a higher percentage of misdiagnosis among those living in rural areas (Figure 3). Regarding family history (FH) of DM, it was found that positive FH of type-2-DM (T2DM) only was more common in the three age groups as compared to FH of T1DM only or to both T1DM and T2DM (Table 1). Analysis of the type of feeding in the studied population revealed that only 19 patients were absolutely breastfed while 80 patients were cow's milk fed (Table 4). The median duration of breastfeeding in the study population was 1.5 years, range from zero (no breastfeeding) to 2 years. The duration of breastfeeding in the absolutely breastfed group was 0.68–2 years. Among them, 17 (89.5%) had positive FH of diabetes; 9 with T2DM; and 8 with both T1DM and T2DM but among them only 4 had the positive FH of DM in a first degree relative. Early introduction of cow's milk was classified according to the time of introduction into 3 subclasses: <3 months (28, 28.3%), 3–6 months (24, 24.2%), and >6 months (28, 28.3%). The relation between type of feeding and the age at presentation is illustrated in Figure 4 where the mean age of breastfed group was 6.8 years (range 0.1–12 yrs). The mean age for the 3 subgroups of cow's milk feeding (<3 months, 3–6 months, and >6 months) was 5.7 yrs (range 0.3–12 yrs), 6.5 yrs (range 0.9–12 yrs), and 7.6 yrs (range 0.6–12 yrs), respectively. Those who were cow's milk fed presented at earlier age, P value < 0.05. Implications of different factors on the severity of diabetes presentation are studied in Figures 3 and 4. Figure 3, highlights that small children (<2 years), those with misdiagnosis, or positive antibodies to insulin and islet cells had more severe presentations as most of them presented with moderate to severe DKA. These antibodies were detected with statistically higher percentage in patients with moderate to severe DKA as compared to other groups, (P < 0.001). Table 4 showed that 80.8% of patients were cow's milk fed which could raise the issue of early cow's milk feeding as a risk factor for T1DM. It also showed a significant correlation between early introduction of cow's milk and the severity of presentation. Regarding family history, it was statistically insignificant in the three groups (Table 4), yet children having positive FH for DM had lower frequency of misdiagnosis as compared to those with negative FH. Twenty-four patients had FH of autoimmune disease, goiter in 20 and collagen vascular disease in 4. Table 5 displays the correlation of C-peptide and TSH levels in patients and controls which was significant for C-peptide and nonsignificant for TSH. However, there was statistical variation in TSH levels and thyroid antibodies in the 3 studied patients groups with tendency to be higher in group III (Table 1). Regarding consanguinity, all patients had positive consanguinity that is why the role of consanguinity cannot be judged. 5. Discussion The current study included 99 children with newly diagnosed diabetes who were classified according to age into 3 age groups ((I): <2 years, (II): 2–<6 years and (III): 6–12 years) with higher incidence of disease in the older age group which is in accordance with several authors [11–14] while the younger age groups showed a rising incidence (45.5% in <6 years) which was in agreement with Harjutsalo et al. [15] and Patterson et al., [16], who expected in their studies doubling of the incidence of T1DM over the next 10 years. The EURODIAB reported a very wide range of incidence rates within Europe with the greatest increase in incidence rate among the young age groups [16]. In the present study, 60% of patients came from rural areas and 40% from urban areas with a ratio of 1.4 : 1. In agreement with other studies [17], the classic triad of DM were the most common symptoms preceding diagnosis in all age groups indicating that simple increased awareness of the necessity to consider diabetes in presence of this triad should improve recognition of diabetes with no delay in all age groups even infants. Abdominal pain was more frequent as the age gets older specially the 6–12 years age group when the child can more reliably relay his symptoms. Increasing the awareness of the public and health care professionals about these symptoms as possible features of diabetes and the necessity of routine urine or blood glucose analysis when these symptoms are encountered can decrease the frequency and severity of DKA. Same applies to nocturia and nocturnal enuresis, which are considered reliable symptoms in those above 2 years. Roche et al. (2005) agreed with us in that respect [18]. Disturbed conscious level was present in an appreciable percentage signifying more severe presentation at time of diagnosis. It was more frequent in those below 2 yrs which relates to the delay in diagnosis and the severity of diabetes in this young age group. Infants below 2 years of age showed the highest frequency of moderate to severe DKA (73.9%). This finding is in agreement with several studies [5, 19–23]. Neu et al. [19] and Szypowska and Skórka [23] suggested that children under 2 yrs of age remain the most prone to DKA due to delay in diagnosis as well as more aggressive β-cell destruction which is in agreement with the current study as seen from the highest incidence of misdiagnosis (Figure 2). The median duration of symptoms before diagnosis was 14 days (range 1–180 days) which was significantly higher in rural compared to urban areas; Du Prel et al. [24] suggested that the risk for T1DM is higher in children living in socially deprived and less densely populated areas. In addition, our study attributed the significantly increased severity of symptoms at presentation also to misdiagnosis (Figure 2). Misdiagnosis was found in 45.5% of the included patients, with around 25% being misdiagnosed as upper respiratory tract infections, 37.5% as lower respiratory tract infections, and 37.5% as gastroenteritis. Misdiagnosis was found to be the cause of long duration and severe presentations with moderate to severe DKA. In Saudi Arabia, Rotavirus is considered the most common cause of childhood gastroenteritis [25, 26]; on the other hand, Honeyman et al. 2000 [27] reported that serologically defined Rotavirus infection is significantly associated with an increase in ICA levels, then with IAA and GADA. This could explain the high levels of these autoantibodies in the present study especially in cases with moderate to severe DKA. A consensus statement from American Diabetes Association precisely points out delayed diagnosis as one of the main causes of DKA development in many children with newly diagnosed T1DM [28]. Regarding severity at presentation, it ranged from hyperglycemia to severe DKA. The frequencies of the different grades of severity were comparable between urban and rural areas. No significant difference was found in the duration of symptoms neither in relation to age nor to the degree of severity at presentation. This was similar to Olak-Białoń et al. [29], who found no correlation between duration of symptoms and both the severity of DKA and age of children. In the present study, positive family history of T2DM was more frequent than type 1 or both, in the 3 age groups, which was in agreement with Barone et al. [30]. Unfortunately, the presence of positive FH did not improve recognition of diagnosis and did not decrease duration of symptoms nor severity at presentation in our cohort which was in contrary to Blanc et al. [31] who found significantly less frequency of DKA in children with positive FH of T1DM. This might point to the low awareness and sometimes to denial and high resistance of the families with a positive FH of DM to admit the fact that other family members are prone to the development of DM. This issue is a real actual obstacle which we faced in many patients which was very evident up to the stage that some parents of children who presented with DKA even the severe grade were highly reluctant to admit that their children had T1DM and a false fixed believe that this might be a transient event which will resolve. Another great obstacle which might be very unique is the great believe of parents in traditional herbal medicines and their false thinking that this type of medicine can treat their children without the use of insulin. All these issues highlight the great need to emphasize and stress on health education and awareness about DM especially for members of families with positive FH. A multidisciplinary approach with involvement of physicians, dietitians, nutritionists, health educators, diabetic educators, social workers, and psychiatrists is highly recommended. An interesting finding in the current study which reflects the lack of awareness about healthy life style behaviors is the very low percentage of exclusively breastfed infants (19.2%) compared to 80.8% with cow's milk feeding which was introduced as early as <3 months age. Correlation of type of feeding with age at presentation revealed an earlier median age of around 4 years in children with early cow's milk introduction (<3 months) compared to an older median age of 7 years and 8 years in exclusively breastfed and late cow's milk introduction (>6 months), respectively. These findings may support the suggestions for the protective role of breastfeeding on one hand and the early cow's milk introduction as a risk factor for development of T1DM on the other hand. A review paper “Milk and Diabetes” [32] listed several studies which suggest that cow's milk products are associated with the onset of T1DM, and several other studies which show no correlation. The literature is still equivocal about the relationship of cow's milk products and the risk of type 1 diabetes, particularly the relationship between the time of their introduction to an infant and the onset of diabetes. Another review article [33] discussed this unsettled issue with several studies relating the age of introduction of dairy products to the risk of T1DM in particular when formula is introduced before 4 months of age, while other studies were not supporting this association. The same article added more evidence that cow's milk proteins trigger T1DM where antibodies to bovine beta-lactoglobulin were detected in the serum of children with diabetes while individuals without diabetes did not have this antibody [33]. According to a recent review article, most studies suggest that the early introduction of complex foreign proteins may be a risk factor for beta-cell autoimmunity, and a pilot intervention trial has implied that weaning to a highly hydrolyzed formula may decrease the risk of beta-cell autoimmunity [34]. Achenbach et al. 2005 [35] reported that children who develop autoantibodies within the first 2 years of life are those who most often develop multiple islet autoantibodies and progress to T1DM in childhood. This was very similar to the current study which revealed that most of cases below 2 years developed ICA, IAA, and GADA. Achenbach added that children who develop autoantibodies above 2 years have a slower progression to multiple antibodies and T1DM [35]. Kordonouri et al., 2002 [36], agreed with us in reporting significant prevalence of thyroid antibody titers with older age (Table 1). They added that TSH levels were higher in patients with thyroid autoimmunity [36]. Accordingly, the American Diabetes Association [37] and several authors [38, 39] recommended annual screening for thyroid disease in all T1DM subjects with TSH measurement; this procedure is considered the most sensitive way to identify patients with thyroid dysfunction, as autoantibodies may persist for many years without thyroid dysfunction. Fasting C-peptide was done in all patients after stabilization at their initial presentation which ranged from 0.25 to 3.8 ng/mL (mean 1.07 and median 0.87 ng/mL) that was in agreement with Levitt Katz et al. [40] who reported fasting C-peptide levels 0.38 ± 0.37 ng/mL with 83% sensitivity in distinguishing T1DM from T2DM. 6. Conclusions The classic triad (polyuria, polydipsia, and weight loss) is the commonest presenting symptom of diabetes in children. Misdiagnosis and mismanagement are common and account for more severe presentation among newly diagnosed children with diabetes, with those below 2 years of age being the most vulnerable group to such problem. Early introduction of cow's milk feeding appears to be a risk factor for the development of T1DM but more studies with larger population are warranted to validate these results. Increasing the awareness of the public especially in rural areas about the following: the great benefit of absolute breastfeeding in the 1st 6 months of their babies' life; the possibility of occurrence of DM in young children especially those with positive FH of DM and the better outcome when diagnosed early. A multidisciplinary approach with involvement of physicians, dietitians, nutritionists, health educators, diabetic educators, social workers, and psychiatrists is highly recommended. Conflict of Interests The authors declare that they have no conflict of interests. Acknowledgment The authors thank Dr. Khaled El-Saban, Professor of Nuclear Medicine, for his great help in the current study. Figure 1 Common symptoms preceding presentation and common signs at presentation in the studied group. Figure 2 Duration of symptoms in relation to district in the studied group. Figure 3 Risk stratification of severity of presentation of type I diabetes mellitus in the studied patients. Note that analysis of the severity of presentation in relation to the district of origin and age groups was statistically nonsignificant for those from urban areas (χ2 = 1.25, P > 0.05), but highly significant statistically for those from rural areas (χ2 = 16.4 and P = 0.0025), indicating that children living in rural areas had more severe presentation (moderate to severe DKA). Figure 4 Relation between type of feeding and age at presentation in the studied patients. Table 1 Demographic data of the studied patients. Point of comparison <2 years 2–<6 years 6–12 years Total P value No. % No. % No. % No. % Number 23 23.2 22 22.2 54 54.4 99 100 — District Urban 10 43.5 7 31.8 23 42.6 40 40 P > 0.05 Rural 13 56.5 15 68.2 31 57.4 59 60 Symptoms duration (X ± SD) 21 ± 17.6 22 ± 22.5 27 ± 33.6 P > 0.05 Median 10 16 14 Severity of presentation Hyperglycemia without DKA 4 17.4 5 22.7 11 20.4 20 20.2 P = 0.04 Mild DKA 2 8.7 10 45.5 16 29.6 28 28.3 Moderate to severe DKA 17 73.9 7 31.8 27 50 51 51.5 Misdiagnosis 16 69.6 8 36.4 21 38.9 45 45.5 P < 0.05 FH T1DM 1 4.7 0 0 2 3.7 3 3 P > 0.05 T2DM 15 71.5 13 59.1 29 53.7 57 57.6 P < 0.05 T1DM and T2DM 3 14.2 5 22.7 10 18.5 18 18.2 P > 0.05 Thyroid autoantibodies 2 7 7 31.8 18 33.3 27 27.3 P < 0.03 TSH 0.87 ± 0.07 2.99 ± 0.7 3.28 ± 1.9 P < 0.05 DKA: diabetic ketoacidosis, FH: family history, T1DM: type 1 diabetes mellitus, T2DM: type 1 diabetes mellitus, TSH: thyroid stimulating hormone. Table 2 Distribution and correlation of the main clinical symptoms preceding diagnosis and the different patients age groups. Symptoms Age groups (years) Total number P value <2 2–<6 6–12 Polyuria and Polydipsia 20 22 51 93 NS 87% 100.0% 94.4% 93.9% Weight loss 15 21 52 88 NS 65.2% 95.5% 96.3% 88.9% Polyphagia 5 5 12 22 NS 21.7% 22.7% 22.2% 22.2% Refusal of feeding 3 1 4 8 NS 14.3% 4.5% 7.4% 8.1% Abdominal pain 3 9 40 52 P < 0.001 13% 40.9% 74.1% 52.5% Vomiting 11 7 23 41 P = 0.393 47.8% 31.8% 42.6% 41.4% Dehydration 7 2 11 20 NS 30.4% 9.1% 20.4% 20.2% Disturbed conscious level 14 9 23 46 P = 0.284 60.9% 40.9% 42.6% 46.5% Nocturia and nocturnal enuresis 8 18 48 74 P < 0.001 34.7% 81.8% 88.9% 74.7% Infection 4 5 6 15 NS 17.4% 22.7% 11.1% 15.2% NS: nonsignificant value, *highly significant. N.B: the classic triad; polyuria, polydipsia, and weight loss; are the dominant presenting symptoms in the 3 age groups followed by vomiting and disturbed conscious level in those <2 years, nocturia and nocturnal enuresis in 2–<6 years age group, and abdominal pain, nocturia, and nocturnal enuresis in the 6–12 years age group. Table 3 Frequency of diabetic ketoacidosis among the different clinical symptoms and signs. Symptoms/signs Mode of presentations P value Mild DKA Moderate to severe DKA Total number of patients with DKA Total number of children with symptom Classic triad 0 8 8 8 P < 0.05 0.0 100% 100% 100% Dehydration 0 17 17 20 P < 0.05* 0.0 100% 85% 100% Vomiting 7 32 39 41 P < 0.001** 18% 82.1% 95.1% 100% Abdominal pain 13 31 44 52 P < 0.01* 29.5% 70.5% 84.6% 100% DKA: diabetic ketoacidosis, significant, highly significant. N.B: most children with DKA whatever the degree of severity presented with abdominal pain and vomiting. Dehydration was present in 100% of those with moderate to severe DKA. Table 4 Implication of different risk factors on the severity of presentation. Points of comparison Hyperglycemia Mild DKA Moderate-severe DKA Total P value No. % No. % No. % No. % 20 20.2 28 28.3 51 51.5 99 100 District Urban 11 27.5 10 25 19 47.5 40 40 P > 0.05 Rural 9 15.3 18 30.5 32 54.2 59 60 Symptom duration 12 ± 7.6 14 ± 9.5 15 ± 8.6 — P > 0.05 Type of feeding Breastfeeding 5 26.3 2 10.5 12 63.2 19 19.2 P < 0.05 Cow's milk 16 20.0 27 33.7 37 46.3 80 80.8 <3 months 6 21.4 7 25.0 15 53.6 28 28.3 3–6 months 2 8.3 13 54.2 9 37.5 24 24.2 >6 months 8 28.6 7 25 13 46.4 28 28.3 Positive family history of DM 17 85 23 82.1 38 74.5 78 78.8 P > 0.05 No.: number, DM: diabetes mellitus, DKA: diabetic ketoacidosis. Table 5 Correlation of C-peptide and TSH levels to patients and controls. Point of comparison Controls Patients P-value No. Mean Std. deviation Median Min. Max. No. Mean Std. deviation Median Min. Max. Fasting C-peptide (ng/mL) 110 1.28 0.38 1.30 0.80 1.80 99 1.07 0.66 0.87 0.25 3.80 P < 0.001* TSH μIU/mL 110 3.11 1.60 3.60 0.80 5.30 99 2.38 1.71 2.65 0.80 6.20 0.058 No.: number; Std.: standard; Min.: minimum; Max.: maximum; **highly significant. ==== Refs 1 Haller MJ Atkinson MA Schatz D Type 1 diabetes mellitus: etiology, presentation, and management Pediatric Clinics of North America 2005 52 6 1553 1578 2-s2.0-27844463189 16301083 2 Van Belle TL Coppieters KT Von Herrath MG Type 1 diabetes: etiology, immunology, and therapeutic strategies Physiological Reviews 2011 91 1 79 118 2-s2.0-78751662679 21248163 3 Craig ME Hattersley A Donaghue KC Definition, epidemiology and classification of diabetes in children and adolescents Pediatric Diabetes 2009 10 supplement 12 3 12 2-s2.0-70349563930 4 Scibilia J Finegold D Dorman J Why do children with diabetics die? Acta Endocrinologica 1986 113 279 326 333 2-s2.0-0022464328 5 Mallare JT Cordice CC Ryan BA Carey DE Kreitzer PM Frank GR Identifying risk factors for the development of diabetic ketoacidosis in new onset type 1 diabetes mellitus Clinical Pediatrics 2003 42 7 591 597 2-s2.0-0141653890 14552517 6 Hiernaux J Tanner JM Growth and physical studies Human Biology: A Guide To Field Methods 1969 Oxford, UK Blackwell Scientific Publications 7 El-Mouzan MI Al-Herbish AS Al-Salloum AA Qurachi MM Al-Omar AA Growth charts for Saudi children and adolescents Saudi Medical Journal 2007 28 10 1555 1568 2-s2.0-35948943479 17914520 8 Yosipovitch G DeVore A Dawn A Obesity and the skin: skin physiology and skin manifestations of obesity Journal of the American Academy of Dermatology 2007 56 6 901 916 2-s2.0-34248169203 17504714 9 Kitabchi AE Umpierrez GE Murphy MB Kreisberg RA Hyperglycemic crises in adult patients with diabetes: a consensus statement from the American Diabetes Association Diabetes Care 2006 29 12 2739 2748 2-s2.0-33845474117 17130218 10 Dawson B Trapp GT Basic and Clinical Biostatistics 2001 3rd edition Norwalk, Conn, USA Lange medical book, Appleton and Lange 11 Said Al Magamsi M Said Habib H Clinical presentation of childhood type 1 diabetes mellitus in the Al-Madina region of Saudi Arabia Pediatric Diabetes 2004 5 2 95 98 2-s2.0-3142773511 15189495 12 Evertsen J Alemzadeh R Wang X Increasing incidence of pediatric type 1 diabetes mellitus in Southeastern Wisconsin: relationship with body weight at diagnosis PLoS ONE 2009 4 9 2-s2.0-70149122372 e6873 13 Mayer-Davis EJ Beyer J Bell RA Diabetes in African American youth Diabetes Care 2009 32 2 S112 S122 2-s2.0-65449151930 19246576 14 Xin Y Yang M Chen XJ Tong YJ Zhang LH Clinical features at the onset of childhood type 1 diabetes mellitus in Shenyang, China Journal of Paediatrics and Child Health 2010 46 4 171 175 2-s2.0-77951832762 20546479 15 Harjutsalo V Sjöberg L Tuomilehto J Time trends in the incidence of type 1 diabetes in Finnish children: a cohort study The Lancet 2008 371 9626 1777 1782 2-s2.0-43849096855 16 Patterson CC Dahlquist GG Gyürüs E Green A Soltész G Incidence trends for childhood type 1 diabetes in Europe during 1989-2003 and predicted new cases 2005-20: a multicentre prospective registration study The Lancet 2009 373 9680 2027 2033 2-s2.0-67049159857 17 Ismail NA Kasem OM Abou-El-Asrar M El-Samahy MH Epidemiology and management of type 1 diabetes mellitus at the ain shams university pediatric hospital Journal of the Egyptian Public Health Association 2008 83 1-2 107 32 18992206 18 Roche EF Menon A Gill D Hoey H Clinical presentation of type 1 diabetes Pediatric Diabetes 2005 6 2 75 78 2-s2.0-21444453760 15963033 19 Neu A Willasch A Ehehalt S Hub R Ranke MB Ketoacidosis at onset of type 1 diabetes mellitus in children: frequency and clinical presentation Pediatric Diabetes 2003 4 2 77 81 14655263 20 Bui H To T Stein R Fung K Daneman D Is diabetic ketoacidosis at disease onset a result of missed diagnosis? Journal of Pediatrics 2010 156 3 472 477 2-s2.0-77149157258 19962155 21 Pawłowicz M Birkholz D Niedźwiecki M Balcerska A Difficulties or mistakes in diagnosing type 1 diabetes in children?: demographic factors influencing delayed diagnosis Pediatric Diabetes 2009 10 8 542 549 2-s2.0-77449146845 19496971 22 Abdul-Rasoul M Al-Mahdi M Al-Qattan H Ketoacidosis at presentation of type 1 diabetes in children in Kuwait: frequency and clinical characteristics Pediatric Diabetes 2010 11 5 351 356 2-s2.0-77955248009 19821943 23 Szypowska A Skórka A The risk factors of ketoacidosis in children with newly diagnosed type 1 diabetes mellitus Pediatric Diabetes 2011 12 4 302 306 2-s2.0-79957551199 21129138 24 Du Prel J-B Icks A Grabert M Holl RW Giani G Rosenbauer J Socioeconomic conditions and type 1 diabetes in childhood in North Rhine-Westphalia, Germany Diabetologia 2007 50 4 720 728 2-s2.0-33847660377 17294165 25 ElAssouli SM Banjar ZM Mohammed KA Milaat WA ElAssouli M-Z Genetic and antigenic analysis of human rotavirus prevalent in Al-Taif, Saudi Arabia Journal of Tropical Pediatrics 1996 42 4 211 219 2-s2.0-0029788996 8816033 26 Kheyami AM Areeshi MY Dove W Nakagomi O Cunliffe NA Hart CA Characterization of rotavirus strains detected among children and adults with acute gastroenteritis in Gizan, Saudi Arabia Saudi Medical Journal 2008 29 1 90 93 2-s2.0-40949107810 18176680 27 Honeyman MC Coulson BS Stone NL Association between rotavirus infection and pancreatic islet autoimmunity in children at risk of developing type 1 diabetes Diabetes 2000 49 8 1319 1324 2-s2.0-0033853920 10923632 28 Wolfsdorf J Glaser N Sperling MA Diabetic ketoacidosis in infants, children, and adolescents: a consensus statement from the American Diabetes Association Diabetes Care 2006 29 5 1150 1159 2-s2.0-33746363863 16644656 29 Olak-Białoń B Deja G Jarosz-Chobot P Buczkowska EO The occurrence and analysis of chosen risk factors of DKA among children with new onset of DMT1 Pediatric Endocrinology, Diabetes and Metabolism 2007 13 2 85 90 2-s2.0-34547235094 30 Barone B Rodacki M Zajdenverg L Family history of type 2 diabetes is increased in patients with type 1 diabetes Diabetes Research and Clinical Practice 2008 82 1 e1 e4 2-s2.0-52949152923 31 Blanc N Lucidarme N Tubiana-Rufi N Factors associated to ketoacidosis at diagnosis of type 1 diabetes in children Archives de Pediatrie 2003 10 4 320 325 2-s2.0-0038452557 12818752 32 Schrezenmeir J Jagla A Milk and diabetes Journal of the American College of Nutrition 2000 19 176S 190S 2-s2.0-0034027582 10759142 33 Goldfarb MF Relation of time of introduction of cow milk protein to an infant and risk of type-1 diabetes mellitus Journal of Proteome Research 2008 7 5 2165 2167 2-s2.0-48749120884 18410136 34 Knip M Virtanen SM Åkerblom HK Infant feeding and the risk of type 1 diabetes The American Journal of Clinical Nutrition 2010 9 15 1506S 1513S 35 Achenbach P Bonifacio E Koczwara K Ziegler A-G Natural history of type 1 diabetes Diabetes 2005 54 supplement 2 S25 S31 2-s2.0-33644761987 16306336 36 Kordonouri O Klinghammer A Lang EB Grüters-Kieslich A Grabert M Holl RW Thyroid autoimmunity in children and adolescents with type 1 diabetes: a multicenter survey Diabetes Care 2002 25 8 1346 1350 2-s2.0-0036673890 12145233 37 Silverstein J Klingensmith G Copeland K Care of children and adolescents with type 1 diabetes: a statement of the American Diabetes Association Diabetes Care 2005 28 1 186 212 2-s2.0-14644408638 15616254 38 Hansen D Bennedbaek FN Hoier-Madsen M Hegedus L Jacobsen BB A prospective study of thyroid dysfunction, morphology and autoimmunity in young patients with type 1 diabetes European Journal of Endocrinology 2003 148 2 245 251 12590645 39 Hoffman RP Kordonouri O Hartmann R Holl RW Thyroid stimulating hormone screening is more sensitive for detecting thyroid abnormalities in children and adolescents with type 1 diabetes Diabetes Care 2003 26 1 255 256 2-s2.0-0043172388 12502703 40 Levitt Katz LE Jawad AF Ganesh J Abraham M Murphy K Lipman TH Fasting c-peptide and insulin-like growth factor-binding protein-1 levels help to distinguish childhood type 1 and type 2 diabetes at diagnosis Pediatric Diabetes 2007 8 2 53 59 2-s2.0-34247381130 17448127	24222737	PMC3809599	CC BY	2021-01-05 11:54:56	yes	ScientificWorldJournal. 2013 Oct 9; 2013:421569
==== Front Oncol LettOncol LettOLOncology Letters1792-10741792-1082D.A. Spandidos 10.3892/ol.2013.1586ol-06-05-1253ArticlesClinical significance of the induction of macrophage differentiation by the costimulatory molecule B7-H3 in human non-small cell lung cancer SUN JING 1MAO YONG 2ZHANG YANG-QIN 1GUO YUN-DI 1MU CHUAN-YONG 3FU FENG-QING 3ZHANG XUE-GUANG 31 Institute of Medical Biotechnology, Suzhou Health College, Suzhou, Jiangsu 215009, P.R. China2 Department of Gastroenterology, The Fourth Affiliated Hospital of Soochow University, Wuxi, Jiangsu 214062, P.R. China3 Clinical Immunology Laboratory, The First Affiliated Hospital of Suzhou University, Suzhou, Jiangsu 215007, P.R. ChinaCorrespondence to: Professor Xue-Guang Zhang, Clinical Immunology Laboratory, The First Affiliated Hospital of Suzhou University, 708 Renmin Road, Suzhou, Jiangsu 215007, P.R. China, E-mail: xueguangzh@yahoo.com.cn11 2013 13 9 2013 13 9 2013 6 5 1253 1260 25 12 2012 05 6 2013 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.B7-H3, a member of the B7 family of molecules, is expressed in certain types of human cancer and is important in tumor development and progression. Although several studies have reported that the expression of B7-H3 is correlated with poor outcomes in patients with cancer, its exact role in cancer remains unknown. In the present study, the expression levels of B7-H3 in the pathological specimens of 105 patients treated for non-small cell lung cancer (NSCLC) were examined by immunohistochemistry. A high expression level of B7-H3 was observed in 46.9% of the 105 NSCLC tissue specimens. These patients demonstrated a more advanced tumor grade and a shorter survival time. In addition, we also examined the levels of tumor-associated macrophages (TAMs) in NSCLC tissues and observed that the levels were positively correlated with the expression of B7-H3, and that higher levels of macrophages were associated with lower levels of infiltrating T cells and a shorter survival time. These results demonstrated that TAMs are important in the evasion of tumor immune surveillance in NSCLC. Furthermore, through knockdown of B7-H3 by RNA interference, we observed that soluble B7-H3 was capable of inducing macrophages to express higher levels of macrophage mannose receptor (MMR) and lower levels of human leukocyte antigen (HLA)-DR, as well as higher levels of interleukin-10 (IL-10) and lower levels of IL-1β in vitro. These observations are characteristic of an anti-inflammatory/reparatory (alternative/M2) phenotype. Therefore, our data suggests that B7-H3 proteins are involved in the progression of NSCLC by inducing the development of monocytes into anti-inflammatory cells. non-small cell lung cancerB7-H3tumor-associated macrophage ==== Body Introduction Primary lung cancer is the leading cause of cancer-related mortality worldwide and has a poor prognosis compared with other types of cancer (1). Approximately 80% of all lung cancer cases are histologically classified as non-small cell lung cancer (NSCLC) (2). The overall 5-year survival rate of NSCLC is 10–15% (3), despite the availability of numerous treatment strategies, including conventional surgery, chemotherapy, radiotherapy, immunotherapy or a combination of these therapies (4). The pathogenesis of NSCLC involves multiple pathways and may result from numerous factors, such as smoking, bacterial infection and environmental contamination. Several studies have demonstrated that the overall survival and recurrence rates of lung cancer are associated with the type of local immune response (5). Macrophages are the primary immune cell type that infiltrates solid tumors, contributing ≤50% of the tumor cell mass (6). Consequently, these tumor-associated macrophages (TAMs) are important in determining the clinical outcome of disease. A number of studies have demonstrated that depending on the type of micro-environmental stimulation, TAMs are capable of exhibiting a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype (7–9). The M1 phenotype is known to suppress tumor growth, while the M2 phenotype promotes tumor growth. M1 macrophages are induced by lipopolysaccharide (LPS)/interferon-γ (IFN-γ) and are able to propagate the T helper 1 (Th1) response of T cells. Therefore, M1 macrophages are considered to be pro-inflammatory, which is reflected by their expression of interleukin-1β (IL-1β), IL-6, IL-8 or tumor necrosis factor-α (TNF-α). M2 macrophages are induced by IL-4 (M2a), immune complexes (M2b) or IL-13/IL-10 (M2c), and are mainly responsible for propagating the Th2 response of T cells. Thus, M2 macrophages are considered as anti-inflammatory, which is reflected by their expression of IL-10, CD36, scavenger receptor-A or the mannose receptor. Macrophages are critical in tumor progression; however, the mechanism by which the M2 phenotype is induced and promotes tumor angiogenesis is unclear. B7-H3, a member of the B7 family, was cloned from a dendritic cell (DC) cDNA library in 2001 (10) and is expressed at low levels in several normal lymphoid and peripheral tissues (11). However, several studies have revealed that B7-H3 expression is elevated in numerous types of cancer, including lung cancer (12,13), prostate cancer (14,15), ovarian carcinoma (16), colorectal carcinoma (17), pancreatic cancer (18,19) and gastric cancer (20). These studies demonstrated a variety of conflicting functions for B7-H3; however, the majority of clinical data revealed a positive correlation between B7-H3 expression and tumor size, progression and prognosis. We hypothesize that the conflicting findings among different studies may be due to the existence of two isoforms of B7-H3: One of which is 4IgB7-H3, possessing four Ig-like domains in the extracellular domain; and the other of which is 2IgB7-H3, with two Ig-like domains in the extracellular domain due to alternative splicing (21,22). However, the expression patterns of each isoform in tumors and the mechanism by which the isoforms of B7-H3 affect cancer progression remain unknown. In the present study, the pathological specimens of 105 patients treated for NSCLC were evaluated by immunohistochemistry to characterize the expression levels of B7-H3 in human NSCLC tissues. A negative correlation between the expression levels of B7-H3 and the survival time of patients was observed. In addition, we identified an inverse correlation between B7-H3 expression and the levels of tumor-infiltrating macrophages, suggesting that B7-H3 is important in the suppression of tumor immune surveillance. Furthermore, we used in vitro assays to investigate the role of B7-H3 in regulating macrophage differentiation into M2 macrophages and its involvement in tumor progression. Taken together, our study establishes an important role of B7-H3 in cancer progression and suggests that the expression of B7-H3 may be involved in suppressing cancer immune surveillance. Materials and methods Patients Tissues from 105 patients who underwent surgery for lung cancer between January and December, 2005 at the Department of Thoracic Surgery, The First Affiliated Hospital of Suzhou University (Suzhou, China) and The Fourth Affiliated Hospital of Soochow University (Wuxi, China) were used in the study. None of the patients received pre-operative chemotherapy or radiotherapy. The paraffin blocks of the tumor tissues were obtained from the archival collections of the Department of Pathology, The First Affiliated Hospital of Suzhou University and The Fourth Affiliated Hospital of Soochow University, and all 105 specimens were diagnosed with NSCLC by hematoxylin and eosin (H&E) staining. The patients’ pathological reports were reviewed and their clinical parameters are shown in Table I. This study was approved by the ethics committee of the First Affiliated Hospital of Suzhou University. Written informed consent was obtained from the patients. Cell culture and antibodies The A549, H1299 and H460 lung cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and the SPCA-1 lung cancer cell line was purchased from the Shanghai Cell Biology Institute of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured at 37ºC in a humidified incubator supplemented with 5% CO2. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation from the peripheral blood of healthy donors (Suzhou Central Blood Bank, Suzhou, China). Mouse anti-human B7-H3 monoclonal antibody (clone no. 4H7) was established and characterized at the Clinical Immunology Laboratory, The First Affiliated Hospital of Suzhou University). Mouse anti-human CD3 monoclonal antibody (clone no. SP7), mouse anti-human CD68 monoclonal antibody (clone no. KP1) and horseradish peroxidase (HRP)-labeled goat anti-mouse/rabbit secondary antibody were purchased from Maixin Bio, Ltd. (Fuzhou, China). Macrophage mannose receptor (MMR; CD206) antibody labeled with FITC was purchased from BioLegend (San Diego, CA, USA) and human leukocyte antigen (HLA)-DR labeled with phycoerythrin was purchased from Immunotech (Marseille, France). RT-PCR RNA was extracted from the NSCLC cell lines and tissues using TRIzol reagent (Takara Bio, Inc., Shiga, Japan) and converted into cDNA with an oligo(dT) primer using the PrimeScript First Strand cDNA Synthesis kit (Takara Biomedical Technology, Dalian, China). The sequence of the sense primer used in the PCR was 5′-CACTGTGGTTCTGCCTCA-3′ and the sequence of the antisense primer was 5′-GCTGTCTTGGAGCCTTCT-3′. Both types of primers were synthesized by Invitrogen Life Technologies (Carlsbad, CA, USA). Western blotting for B7-H3 Western blot analysis was performed using standard protocols. NSCLC cell lines and tissues were acquired and lysed with RIPA lysis buffer (Beyotime, Nantong, China). The lysates were then run in reducing buffer on Tris-glycine 4–20% acrylamide gradient gels (Novex; Invitrogen Life Technologies) and transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). Mouse anti-human B7-H3 antibodies were used at a concentration of 1 μg/ml for 2 h. The membranes were washed three times with wash buffer prior to incubation with goat anti-mouse-HRP conjugate (Molecular Probes, Eugene, OR, USA) at 0.2 μg/ml for 1 h. The membranes were then developed by enhanced chemiluminescence (Amersham Biosciences, Pittsburg, PA, USA). Immunohistochemistry Immunohistochemistry was performed using the Maixin-Bio™ method according to the manufacturer’s instructions. Briefly, 3-μm-thick consecutive sections were cut by a microtome, dewaxed in xylene and rehydrated through a series of graded ethanol solutions. Antigens were retrieved by heating the tissue sections at 100ºC for 30 min in citrate solution or EDTA solution when required. The sections were cooled down and immersed in 0.3% H2O2 solution for 20 min to block endogenous peroxidase activity, then rinsed in phosphate-buffered saline (PBS) for 5 min, blocked with 5% BSA at room temperature for 20 min, and incubated with primary antibodies against CD3, CD68 or B7-H3 (final concentration in use, 10 μg/ml) at 4ºC overnight. Negative controls were performed by replacing the specific primary antibody with PBS. Following three washes with PBS, the sections were incubated with secondary antibodies for 30 min at room temperature. Diaminobenzene was used as the chromogen and hematoxylin was used as the nuclear counterstain. The sections were dehydrated, cleared and mounted. Evaluation of B7-H3 immunohistochemical staining Two independent observers who were blinded to the clinicopathological parameters of the patients examined the immunohistochemically stained sections. The sections were considered as positive when the tumor cells demonstrated cytoplasmic or membranous B7-H3 immunostaining. The B7-H3 immunostaining intensities were scored according to the following scale: Grade 0, negative; grade 1, weak positive; grade 2, moderate positive and grade 3, strong positive. The negative grade represented no tumor cells demonstrating positive immunostaining. For the analysis, the B7-H3 immunostaining intensities were classified as follows: The sections scored as grades 0 and 1 were defined as the low expression group and the sections scored as grades 2 and 3 were defined as the high expression group. Evaluation of infiltrating T lymphocytes and macrophages in NSCLC tissues Tumor infiltrating T lymphocytes and macrophages in the tumor nest were determined according to the immunostaining intensities of CD3 and CD68. Firstly, the infiltrating T lymphocytes and macrophages in the tumor stroma were examined at low magnification (×40) and categorized according to the densities as follows: Grade 0, scanty; grade 1, moderate infiltration; grade 2, abundant infiltration and grade 3, massive infiltration. The group containing grade 0 and 1 densities was defined as the low infiltration group and the group containing grade 2 and 3 densities was defined as the high infiltration group. The results from each of the five areas were averaged and used in the statistical analysis. Small interfering RNA (siRNA) experiments Three stealth RNA interference (RNAi) and two control RNAi sequences were obtained from algorithms (Invitrogen Life Technologies) based against the sequence accession no. AJ583695 from GenBank. Stealth RNAi was pooled in equimolar amounts and used at a working concentration of 10 μM in order to knockdown B7-H3 expression. Control RNAi was also pooled in equimolar amounts and used at a working concentration of 10 μM. The SPCA-1 NSCLC cell line was transfected with RNAi using Lipofectamine™ 2000 (Invitrogen Life Technologies) and replated for 48 h prior to use. As an additional control, SPCA-1 cells were mock transfected without the use of RNAi. To evaluate the effect of transfection, real-time PCR was performed to analyze the levels of B7-H3 expression and the primer sequences used were as follows: 5′-GGCTGTCTGTCTGTCTCATTG-3′ and 5′-TCCATCATCTTCTTTGCTGTCA-3′ (Invitrogen Life Technologies). PBMCs were isolated by Ficoll-Hypaque gradient centrifugation from the peripheral blood of healthy donors (Suzhou Central Blood Bank). The CD14 Positive Selection kit (Stem Cell Therapeutics, Toronto, Ontario, Canada) was used to isolate human monocytes from PBMCs and the purity of the monocytes was >95%, as identified by anti-CD14 staining. Cell suspensions were added to flat-bottom 6-well plates at a density of 106 cells/well and the plates were incubated at 37ºC and 5% CO2, with SPCA-1 cells (2.5×105), which had been transfected with B7-H3-specific RNAi. To ensure there was cytokine secretion from tumor cells in the culture, the supernatant collected from the SPCA-1 cells was added and cultured for 7 days. The soluble B7-H3 in the supernatant does not interfere with the experimental results since the level of soluble B7-H3 in the SPCA-1 supernatant is extremely low. After 3 days, the cells were collected and incubated with MMR or HLA-DR for 30 min at 4ºC and washed. At the same time, the supernatants were harvested and frozen at −80ºC until assayed for the secretion of IL-10 and IL-1β (BD Biosciences, Franklin Lakes, NJ, USA). Statistical analysis All data are expressed as the mean ± standard deviation. Statistical analysis was performed using the Student’s t-test and analysis of variance. Correlations were evaluated by the Pearson’s correlation test. P<0.05 was considered to indicate a statistically significant result. Results B7-H3 expression in NSCLC tissues and cell lines In order to examine the B7-H3 expression levels in NSCLC tissues and cells, RT-PCR and western blotting were performed. We detected two unambiguous bands in the NSCLC tissues and cell lines. One predominant band at ~1500 bp represented the 4IgB7-H3 isoform and a minor band at ~800 bp represented the 2IgB7-H3 isoform (Fig. 1A). These data supported the results of several other studies conducted in normal tissues and cells (23,24). The expression of B7-H3 was regulated following RNA transcription (25) and the B7-H3 protein was only expressed in a minority of tissues and cells, including activated lymphocytes and tumor cells. To determine the protein expression levels of B7-H3, western blot analysis was performed and we observed that the two isoforms of B7-H3 were expressed equally in tumor tissues and cell lines (Fig. 1B). The band at 110 kDa represented the 4IgB7-H3 isoform and the band at ~65 kDa represented the 2IgB7-H3 isoform. We additionally characterized the expression of B7-H3 in resected specimens from 105 NSCLC patients by immunohistochemistry. Our results demonstrated that the intensity of B7-H3 expression varied and the immunolocalization of the B7-H3 molecule was predominantly in the membrane and cytoplasm of the tumor cells. We observed 58 cases of low B7-H3 expression, including 14 cases of grade 0 (Fig. 2A) and 44 cases of grade 1 (Fig. 2B). The other 47 cases were of high B7-H3 expression, including 33 cases of grade 2 (Fig. 2C) and 14 cases of grade 3 (Fig. 2D). Correlation of B7-H3 expression with the clinical parameters of patients We analyzed B7-H3 expression in NSCLC tissues and observed that its expression had a positive correlation with a patient’s tumor grade (P=0.048), suggesting that B7-H3 is involved in cancer progression. However, the B7-H3 expression levels did not correlate with other clinicopathological parameters, including gender, age, tumor location, tumor size, nodal metastasis and distant metastasis (Table I). We additionally examined whether there was an association between survival time and B7-H3 expression in tumor cells. The cumulative survival time was calculated using the Kaplan-Meier method and analyzed using the log-rank test. This analysis demonstrated that carcinoma patients with high B7-H3 expression exhibited significantly shorter survival times (P=0.0230; Fig. 3A). Infiltrating macrophages in NSCLC tissues and their correlation with B7-H3 expression, survival time and infiltrating T lymphocytes Following the examination of the immunohistochemically stained sections, a mass of macrophages that had infiltrated NSCLC tissues was detected. To elucidate the underlying mechanisms participating in the effect of TAMs in NSCLC, we performed an immunohistochemical examination to detect the number of infiltrating macrophages labeled with CD68 and analyzed its correlation with survival time. The results demonstrated that a shorter survival time was associated with a higher number of infiltrating macrophages (Fig. 3B). To assess the signals by which B7-H3 regulates macrophage differentiation, we additionally examined whether there was an association between B7-H3 expression and TAMs. The infiltration of macrophages had a significant correlation with the expression of B7-H3. Tumor cells that expressed higher levels of B7-H3 exhibited higher levels of macrophage infiltration (Table II). In order to assess whether the effect of TAMs was tumor-suppressive or tumor-facilitative, we examined the number of tumor-infiltrating T lymphocytes and its correlation with the number of TAMs in the specimens of 105 NSCLC patients by CD3 immunostaining. We observed that the level of T-cell infiltration in the tumor nest and stroma was correlated with nodal metastasis (P=0.042; Table I). Function of B7-H3 in macrophage differentiation To assess the role of B7-H3 in inducing the differentiation of macrophages, RNAi technology was used to knockdown B7-H3 expression in SPCA-1 cell lines prior to coculture with monocytes. Pools of specific or control RNAi were transfected into SPCA-1 cells and screened for effective inhibition of B7-H3. Effective and specific knockdown of B7-H3 persisted for at least 8 days following transfection and the highest rate of transfection was observed at 48 h. Monocytes isolated from PBMCs were cocultured with SPCA-1 cells that had been transfected with pooled B7-H3 RNAi or pooled control RNAi, or with mock-transfected SPCA-1 (no RNAi) cells. After 3 days of coculture with B7-H3 knockdown SPCA-1 cells, the monocytes were harvested to assay the expression of MMR and HLA-DR. The monocytes cocultured with siB7-H3-treated SPCA-1 cells expressed lower MMR and higher HLA-DR levels compared with those cocultured with control RNAi-treated SPCA-1 cells (Fig. 4B and C). The production of IL-1β and IL-10 by macrophages was measured by enzyme-linked immunosorbent assay (ELISA). The monocytes cocultured with siB7-H3-treated SPCA-1 cells demonstrated increased levels of IL-1β cytokine production compared with those cocultured with SPCA-1 cells transfected with control RNAi (Fig. 5A). By contrast, the secretion of IL-10 by monocytes cocultured with siB7-H3-treated SPCA-1 cells was reduced compared with that of monocytes cocultured with SPCA-1 cells transfected with control RNAi FLS (Fig. 5B). Discussion Although B7-H3 is a costimulatory molecule that is expressed in T cells, natural killer cells and antigen-presenting cells (11), its expression is not limited to immune cells. The B7-H3 protein is also expressed in osteoblasts (26), fibroblasts, fibroblast-like synoviocytes (24) and epithelial cells (27). This broad expression pattern most likely suggests that B7-H3 possesses more diverse immunological and nonimmunological functions, particularly in peripheral tissues. B7-H3 has been demonstrated to be highly expressed in a variety of different types of human cancer (28), including prostate cancer (15), gastric cancer (29), ovarian cancer (16), colorectal cancer (17) and urothelial cell carcinoma (30). The results of in vitro and in vivo studies strongly suggest a possible involvement of B7-H3 in the T-cell responses involved in the regulation of antitumor immunity (31,32), while the majority of other studies have proposed opposite functions for B7-H3. These studies have identified that higher levels of tumor B7-H3 expression were correlated with a more advanced tumor grade, more common lymph node metastasis, advanced pathological stage, shorter survival time and higher incidence of recurrence (33–35). In the present study, we demonstrated that B7-H3 was highly expressed in lung cancer cells. Increased B7-H3 expression was detected in 86.6% of the 105 NSCLC specimens examined. B7-H3 expression was significantly correlated with the patients’ survival time; higher levels of B7-H3 expression were associated with a shorter survival time. Based on previous studies and the results of the present study, we concluded that B7-H3 expression may play a critical physiological and pathological role in the oncogenesis and development of NSCLC; however, its exact role remains unclear. In the present study, we demonstrated that B7-H3 expression was positively associated with the levels of infiltrating macrophages and the number of macrophages had a negative correlation with the patients’ survival time. These data suggest that the macrophages that infiltrate the tumor tissues may be important in promoting tumor progression (7). TAMs exhibit a continuum of phenotypes ranging from pro-inflammatory (M1-like) to anti-inflammatory (M2-like), and these phenotypes vary in their effects on tumor cells. Furthermore, TAMs are considered as a polarized population of M2 macrophages, particularly when the tumor begins to invade, vascularize and develop (36,37). The classification of polarized macrophages, as either the M1 or M2 phenotype, is mainly based on the differential secretion of cytokines. The M1 phenotype secretes IL-12 and TNF-α, and the M2 phenotype secretes IL-10. Our present study demonstrated that the B7-H3 signal pathway significantly increased the levels of IL-10 secretion and MMR expression, consequently switching the macrophage phenotype from M1 to M2. We have previously demonstrated that the soluble form of B7-H3 is released from monocytes, DCs and activated T cells, and is able to be detected using the ELISA assay established in the Clinical Immunology Laboratory, The First Affiliated Hospital of Suzhou University (38). In addition, we previously identified that the levels of soluble B7-H3 molecules in serum from patients with NSCLC or colorectal carcinoma were significantly higher compared with those in patients with other pulmonary diseases or healthy volunteers (17,39). A high level of circulating B7-H3 in patients was demonstrated to be correlated with node metastasis, distant metastasis and clinical stage. It was suggested that B7-H3 may provide a promising serum biomarker to improve NSCLC diagnosis and prognostic assessment (39). In the present study, we identified that soluble B7-H3 was only produced from the 2IgB7-H3 isoform, while the 4IgB7-H3 isoform was only expressed in the membrane (22). Thus, 2IgB7-H3 is highly expressed in tumor tissues as well as in tumor cell lines. However, the underlying mechanism of soluble B7-H3 in serum has yet to be elucidated. We hypothesize that soluble B7-H3 may function as a chemotactic factor, attracting monocytes in the peripheral blood to migrate to tumor tissues, inducing the development of macrophages, thereby promoting tumor oncogenesis and development. In conclusion, our present study indicated that the costimulatory molecule B7-H3 is important in NSCLC progression. The significantly elevated levels of soluble B7-H3 were stimulated by highly expressed 2IgB7-H3 on tumor cells. The B7-H3 signaling pathway may be involved in switching macrophages to the M2 phenotype and the negative regulation of the T lymphocyte-mediated immune response. Future studies should focus on examining the precise mechanism by which B7-H3 expression is regulated in the tumor environment. Overall knowledge of the clinical implications of this and of potential therapeutic interventions targeting the B7-H3 signaling pathway in NSCLC warrant further investigation. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (nos. 31100626 and 30972718) and the National Natural Science Foundation of Jiangsu Province (BK2011320). Figure 1 Expression of B7-H3 in lung cancer cell lines and tumor tissues. (A) PCR analysis of the A549, H460, H1299 and SPCA-1 lung cancer cell lines and tumor tissues using a B7-H3-specific primer. PCR analysis was performed in over six cases of non-small cell lung cancer. The product of ~1500 bp corresponded with the 4IgB7-H3 molecule, whereas the product of 800 bp corresponded with the 2IgB7-H3 gene. (B) Identification of B7-H3 isoforms in the lung cancer cell lines and tumor tissues. The membrane proteins extracted from the A549, H460, H1299 and SPCA-1 cell lines, and tumor tissues were detected by western blotting using a B7-H3 antibody. A band of ~110 kDa represented the 4IgB7-H3 protein, while a protein of ~65 kDa represented the 2IgB7-H3 protein. Figure 2 B7-H3 immunostaining in non-small cell lung cancer tissues. (A) Negative, (B) weak positive, (C) moderate positive and (D) strong positive. Magnification, ×400. Diaminobenzene was used as the chromogen and hematoxylin was used as the nuclear counterstain. Figure 3 Kaplan-Meier survival analysis of the correlation between survival time and the expression of B7-H3 or the number of infiltrating macrophages labeled with CD68 in 36 patients with non-small cell lung cancer. Figure 4 B7-H3 may inhibit the differentiation of monocytes into TAMs in the SPCA-1 cell line. (A) SPCA-1 cell lines highly express B7-H3. (B) Monocytes may express higher levels of MMR and lower levels of HLA-DR when cocultured with the SPCA-1 cell line. (C) Monocytes were cocultured with SPCA-1 cells transfected with pooled B7-H3-specific RNAi. SPCA-1 cells lose the ability to induce the differentiation of monocytes when transfected with B7-H3-specific RNAi. TAMs, tumor-associated macrophages; MMR, macrophage mannose receptor; HLA, human leukocyte antigen. Figure 5 Monocytes were harvested following 3 days of coculture with SPCA-1 cells. The cytokine levels in the supernatants were measured by enzyme-linked immunosorbent assay. Error bars represent 95% confidence intervals. The two-tailed t-test analysis was used to compare cytokine production from monocytes cultured with SPCA-1 or SPCA-1 (RNAi). (A) IL-10 and (B) IL-1β production. The data are representative of three experiments with pooled B7-H3 RNAi or pooled control RNAi (control). IL, interleukin; RNAi, RNA interference. Table I Correlation between clinical parameters, B7-H3 expression and T-cell infiltration. B7-H3 expression Macrophage infiltration T-cell infiltration Clinical parameter Cases Low High P-valuea Low High P-value Low High P-value Gender Male 77 43 (42.5)b 34 (34.4) 0.835 39 (43.2) 38 (32.7) 0.094 36 (38.9) 41 (38.1) 0.206 Female 28 15 (15.5) 13 (12.5) 20 (15.7) 8 (12.3) 17 (14.1) 11 (13.9) Age (years) ≤60 51 28 (28.2) 23 (22.8) 0.946 29 (28.7) 22 (22.3) 0.893 27 (25.7) 24 (25.2) 0.624 >60 54 30 (29.8) 24 (24.1) 30 (30.3) 24 (23.7) 26 (27.3) 28 (26.7) Histological subtype Adenocarcinoma 45 26 (24.9) 19 (20.1) 0.720 24 (28.2) 21 (26.8) 0.023e 22 (22.7) 23 (22.3) 0.869 Squamous cell carcinoma 47 24 (26.0) 23 (21.0) 31 (24.1) 16 (22.9) 25 (23.7) 22 (23.3) Large cell carcinoma 13 8 (7.2) 5 (5.8) 4 (6.37) 9 (6.3) 6 (6.6) 7 (6.4) Tumor size (cm) ≤4 54 32 (29.8) 22 (24.2) 0.393 32 (30.3) 22 (22.7) 0.514 25 (27.3) 29 (26.7) 0.378 >4 51 26 (28.2) 25 (22.8) 27 (28.7) 24 (22.3) 28 (25.7) 23 (25.2) Tumor statusc pT1 11 8 (7.6) 3 (2.8) 0.048e 7 (7.4) 4 (5.6) 0.302 4 (6.6) 7 (6.4) 0.618 pT2 45 30 (28.6) 15 (14.3) 28 (25.2) 17 (20.8) 22 (24.7) 23 (24.3) pT3 31 13 (12.4) 18 (17.1) 13 (15.3) 18 (11.7) 18 (13.6) 13 (13.4) pT4 18 7 (6.7) 11 (10.5) 11 (9.1) 7 (2.9) 10 (8.1) 8 (7.9) Nodal metastasis Without 47 26 (26.0) 21 (21.0) 0.988 29 (26.3) 18 (20.7) 0.305 19 (24.1) 28 (22.8) 0.042e With 58 32 (32.0) 26 (26.0) 30 (31.8) 28 (25.2) 35 (29.8) 23 (28.1) Distant metastasisd Without 101 56 (55.8) 45 (45.2) 0.765 59 (56.2) 42 (42.8) 0.072 50 (51.0) 51 (50.0) 0.624 With 4 2 (2.21) 2 (1.79) 0 (2.8) 4 (2.2) 3 (2.02) 1 (1.98) a P-values were calculated using the Pearson’s χ2 test or the χ2 test for trend. b Numbers inside the parentheses represent the percentage of patients. c The stage was determined by pathological (p) examination. T1, tumor ≤3 cm diameter, surrounded by lung or visceral pleura, without invasion and more proximal than the lobar bronchus. T2, tumor >3 cm, however ≤7 cm, or a tumor with any of the following features: Involvement of the main bronchus >2 cm distal to the carina; invasion of the visceral pleura; association with atelectasis or obstructive pneumonitis that extends to the hilar region, however, does not involve the entire lung. T3, tumor >7 cm diameter or with any of the following features: Direct invasion of the chest wall, diaphragm, phrenic nerve, mediastinal pleura, parietal pericardium or main bronchus <2 cm from the carina without involvement of the carina; atelectasis or obstructive pneumonitis of the entire lung or separate tumor nodules in the same lobe. T4, tumor of any size that invades the mediastinum, heart, great vessels, trachea, recurrent laryngeal nerve, esophagus, vertebral body and carina, or with separate tumor nodules in a different ipsilateral lobe. d These patients demonstrated distant metastasis, and presented with metastais of the brain or bone. e Signifies P<0.05. Table II Correlation between the levels of infiltrating macrophages and B7-H3 expression or T lymphocytes in NSCLC tissues. Infiltrating macrophages in NSCLC tissues Group Low (n) Ratio (%) High (n) Ratio (%) χ2 value P-value B7-H3 expression 59 56.19 46 43.80 4.5788 0.0324a Low expression 38 36.19 20 19.04 High expression 21 20.00 26 24.76 Infiltrating T lymphocytes 59 56.19 46 43.81 4.9567 0.0260a Low infiltrating 36 34.29 18 17.14 High infiltrating 23 21.90 28 26.67 Total 105 105 a Signifies P<0.05. NSCLC, non-small cell lung cancer; n, number of cases. ==== Refs References 1 Patrick DJ Fitzgerald SD Sesterhenn IA Davis CJ Kiupel M Classification of canine urinary bladder urothelial tumours based on the World Health Organization/International Society of Urological Pathology consensus classification J Comp Pathol 135 190 199 2006 17054974 2 Gansler T Ganz PA Grant M Sixty years of CA: a cancer journal for clinicians CA Cancer J Clin 60 345 350 2011 21075954 3 Ginsberg RJ Resection of non-small cell lung cancer: how much and by what route Chest 112 Suppl 4 S203 S205 1997 4 Payne DG The role of radiotherapy in bronchopulmonary cancer Rev Mal Respir 10 401 422 1993 (In French) 8256027 5 Derniame S Vignaud JM Faure GC Béné MC Alteration of the immunological synapse in lung cancer: a microenvironmental approach Clin Exp Immunol 154 48 55 2008 18761663 6 Faget J Biota C Bachelot T Early detection of tumor cells by innate immune cells leads to T(reg) recruitment through CCL22 production by tumor cells Cancer Res 71 6143 6152 2011 21852386 7 Mantovani A Bottazzi B Colotta F Sozzani S Ruco L The origin and function of tumor-associated macrophages Immunol Today 13 265 270 1992 1388654 8 Mantovani A Allavena P Sica A Tumour-associated macrophages as a prototypic type II polarised phagocyte population: role in tumour progression Eur J Cancer 40 1660 1667 2004 15251154 9 Mantovani A Sozzani S Locati M Infiltration of tumours by macrophages and dendritic cells: tumour-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes Novartis Found Symp 256 137 145 2004 15027487 10 Chapoval AI Ni J Lau JS B7-H3: a costimulatory molecule for T cell activation and IFN-gamma production Nat Immunol 2 269 274 2001 11224528 11 Sun M Richards S Prasad DV Mai XM Rudensky A Dong C Characterization of mouse and human B7-H3 genes J Immunol 168 6294 6297 2002 12055244 12 Sun Y Liu J Gao P Wang Y Liu C Expression of Ig-like transcript 4 inhibitory receptor in human non-small cell lung cancer Chest 134 783 788 2008 18625675 13 Sun Y Wang Y Zhao J B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143 151 2006 16782226 14 Chavin G Sheinin Y Crispen PL Expression of immunosuppresive B7-H3 ligand by hormone-treated prostate cancer tumors and metastases Clin Cancer Res 15 2174 2180 2009 19276267 15 Lehmann BD Paine MS Brooks AM Senescence-associated exosome release from human prostate cancer cells Cancer Res 68 7864 7871 2008 18829542 16 Zang X Sullivan PS Soslow RA Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104 1112 2010 20495537 17 Sun J Chen LJ Zhang GB Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163 1171 2010 20333377 18 Yamato I Sho M Nomi T Clinical importance of B7-H3 expression in human pancreatic cancer Br J Cancer 101 1709 1716 2009 19844235 19 Loos M Hedderich DM Ottenhausen M Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 2009 20035626 20 Wu CP Jiang JT Tan M Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457 459 2006 16489649 21 Ling V Wu PW Spaulding V Duplication of primate and rodent B7-H3 immunoglobulin V- and C-like domains: divergent history of functional redundancy and exon loss Genomics 82 365 377 2003 12906861 22 Sun J Fu F Gu W Origination of new immunological functions in the costimulatory molecule B7-H3: the role of exon duplication in evolution of the immune system PLoS One 6 e24751 2011 21931843 23 Zhou YH Chen YJ Ma ZY 4IgB7-H3 is the major isoform expressed on immunocytes as well as malignant cells Tissue Antigens 70 96 104 2007 17610414 24 Tran CN Thacker SG Louie DM Interactions of T cells with fibroblast-like synoviocytes: role of the B7 family costimulatory ligand B7-H3 J Immunol 180 2989 2998 2008 18292521 25 Xu H Cheung IY Guo HF Cheung NK MicroRNA miR-29 modulates expression of immunoinhibitory molecule B7-H3: potential implications for immune based therapy of human solid tumors Cancer Res 69 6275 6281 2009 19584290 26 Suh WK Wang SX Jheon AH The immune regulatory protein B7-H3 promotes osteoblast differentiation and bone mineralization Proc Natl Acad Sci USA 101 12969 12973 2004 15317945 27 Saatian B Yu XY Lane AP Expression of genes for B7-H3 and other T cell ligands by nasal epithelial cells during differentiation and activation Am J Physiol Lung Cell Mol Physiol 287 L217 L225 2004 15047568 28 Chen YW Tekle C Fodstad O The immunoregulatory protein human B7H3 is a tumor-associated antigen that regulates tumor cell migration and invasion Curr Cancer Drug Targets 8 404 413 2008 18690846 29 Arigami T Uenosono Y Hirata M Yanagita S Ishigami S Natsugoe S B7-H3 expression in gastric cancer: a novel molecular blood marker for detecting circulating tumor cells Cancer Sci 102 1019 1024 2011 21251161 30 Boorjian SA Sheinin Y Crispen PL T-cell coregulatory molecule expression in urothelial cell carcinoma: clinicopathologic correlations and association with survival Clin Cancer Res 14 4800 4808 2008 18676751 31 Kobori H Hashiguchi M Piao J Kato M Ritprajak P Azuma M Enhancement of effector CD8+ T-cell function by tumour-associated B7-H3 and modulation of its counter-receptor triggering receptor expressed on myeloid cell-like transcript 2 at tumour sites Immunology 130 363 373 2010 20141543 32 Loos M Hedderich DM Friess H Kleeff J B7-h3 and its role in antitumor immunity Clin Dev Immunol 2010 683875 2010 21127709 33 Crispen PL Sheinin Y Roth TJ Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150 5157 2008 18694993 34 Hofmeyer KA Ray A Zang X The contrasting role of B7-H3 Proc Natl Acad Sci USA 105 10277 10278 2008 18650376 35 Katayama A Takahara M Kishibe K Expression of B7-H3 in hypopharyngeal squamous cell carcinoma as a predictive indicator for tumor metastasis and prognosis Int J Oncol 38 1219 1226 2011 21344157 36 Mantovani A Sozzani S Locati M Allavena P Sica A Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes Trends Immunol 23 549 555 2002 12401408 37 Murdoch C Muthana M Coffelt SB Lewis CE The role of myeloid cells in the promotion of tumour angiogenesis Nat Rev Cancer 8 618 631 2008 18633355 38 Zhang G Hou J Shi J Yu G Lu B Zhang X Soluble CD276 (B7-H3) is released from monocytes, dendritic cells and activated T cells and is detectable in normal human serum Immunology 123 538 546 2008 18194267 39 Zhang G Xu Y Lu X Diagnosis value of serum B7-H3 expression in non-small cell lung cancer Lung Cancer 66 245 249 2009 19269710	24179504	PMC3813612	CC BY	2021-01-04 23:20:30	yes	Oncol Lett. 2013 Nov 13; 6(5):1253-1260
==== Front Oncol LettOncol LettOLOncology Letters1792-10741792-1082D.A. Spandidos 10.3892/ol.2013.1546ol-06-05-1351ArticlesInhibition of δ-opioid receptors induces brain glioma cell apoptosis through the mitochondrial and protein kinase C pathways ZHOU LIXIANG 1GUO XUDONG 2CHEN MO 3FU SHUANGLIN 1ZHOU JINGBIN 2REN GANG 2YANG ZIRONG 2FAN WENHAI 21 Department of Neurosurgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China2 Department of Neurosurgery, The Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning 116001, P.R. China3 Department of Neurosurgery, People’s Hospital of Jilin Province, Changchun, Jilin 130000, P.R. ChinaCorrespondence to: Dr Wenhai Fan, Department of Neurosurgery, The Affiliated Zhongshan Hospital of Dalian University, 6 Jiefang Street, Zhongshan, Dalian, Liaoning 116001, P.R. China, E-mail: wenhaif123@163.com11 2013 26 8 2013 26 8 2013 6 5 1351 1357 08 2 2013 01 8 2013 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.Brain glioma is a malignant tumor with a high incidence rate and poor prognosis that has become a focus of studies of central nervous system diseases. Previous studies have suggested that δ-opioid receptors may affect the proliferation and apoptosis of numerous types of tumor cells. However, to date, their precise mechanism(s) of action have not been elucidated. The present study aimed to investigate the effects of inhibiting δ-opioid receptors in brain glioma cell proliferation and apoptosis and their relevant molecular mechanisms. Various doses of naltrindole were supplied to treat brain glioma cells using the MTT method to assess the proliferation index. Flow cytometry was used to investigate the changes in cell apoptosis and mitochondrial membrane potential. The expression levels of Bax, Bcl-2, Bcl-xL, cytochrome c, caspase-9, caspase-3 and protein kinase C (PKC) were measured using western blotting. Naltrindole was observed to inhibit brain glioma cell proliferation and promote apoptosis in a dose- and time-dependent manner. Furthermore, the addition of naltrindole lead to changes in the brain glioma cell membrane potential and regulated Bax translocation to the mitochondrial membrane, consequently promoting the release of cytochrome c into the cytoplasm, followed by the activation of caspase-9 and -3, which caused cell apoptosis. In addition, naltrindole was able to regulate the expression levels of the cellular internal phosphorylated PKC proteins, which are closely associated with the inhibition of cell proliferation. In conclusion, the inhibition of δ-opioid receptors may inhibit brain glioma cell proliferation and lead to apoptosis, which is closely associated with the mitochondrial and PKC pathways. δ-opioid receptorgliomaapoptosismitochondriaprotein kinase C pathway ==== Body Introduction Brain glioma is a tumor that originates from the neuroepithelial tissues. Brain glioma is the most common malignant intracranial tumor and the most common tumor of the central nervous system, accounting for 70% of human primary malignant brain tumors (1,2). Glioma has become the focus of studies with regard to diseases of the central nervous system. However, the condition is difficult to study due to its high incidence and poor treatment results (3). At present, glioma is mainly treated with surgery, radiotherapy and chemotherapy, but the curative effect and prognosis are not optimistic. The results of such diseases have not improved significantly for the past 30 years. The median survival time of patients with glioblastoma is between 12 and 15 months (4,5). Therefore, glioma is of significant study value, and the mechanism of glioma cancer cell death has become a key area of research interest in order to search for drugs with breakthrough effects. Previous studies have shown that δ-opioid receptor activation may affect tumor cell proliferation and apoptosis (6), as well as the progression of human hepatocellular carcinoma and cholangiocarcinoma (7,8). It has been identified that activated δ-opioid receptors may promote the growth of certain malignant tumors (9–11), including neuroblastoma and lung or colon cancer. However, there is are no studies on whether the δ-opioid receptor inhibits the growth of human brain glioma cells. Furthermore, there is little knowledge with regard to the specific antitumor mechanism of the δ-opioid receptor. Certain studies have demonstrated that the apoptosis of brain glioma cells is closely associated with the mitochondrial and protein kinase C (PKC) pathways (12–14). Our previous study identified that the downregulation of the δ-opioid receptor may promote changes in Bax and Bcl-2 protein expression. The shifting of the Bax and Bcl-2 proteins results in the release of cytochrome c to activate the caspase family to cause apoptosis (15,16). PKC protein expression levels were also shown to decrease significantly. The present study aimed to investigate the impact of δ-opioid receptors on the proliferation of brain glioma cells and apoptosis and to explore the δ-opioid receptor-induced cell apoptosis signaling pathway. δ-opioid receptors were able to release cytochrome c and activate the caspase family to induce brain glioma cell apoptosis by regulating the Bax and Bcl-2 proteins. Materials and methods Cell culture Human brain glioma U87 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were inoculated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY, USA) containing 10% fetal calf serum (HyClone Laboratories, Inc., Logan, UT, USA), 100 U/ml penicillin and 100 U/ml streptomycin. The cells were then cultured in an incubator containing 5% CO2 and 95% oxygen at 37°C. Cell viability The U87 cells that were in a logarithmic growth phase were harvested and inoculated in 96-well culture plates at a density of 1×105 cells/ml. Once the cells had grown adherent, various doses of DADLE (Sigma, St. Louis, MO, USA) were administered to the groups, with 6 duplicate wells for each concentration. There was also a negative control group that did not contain any drug. All the cells were placed into a 5% CO2 incubator for a further culture of 24, 48 and 72 h prior to the color reaction. Each well was administered 20 μl MTT (5 mg/ml) and cultured in a CO2 incubator for 4 h prior to disposing of the culture solution. Dimethyl sulfoxide (DMSO; 150 μl) was added to each well for room temperature oscillation for 10 min, and the optical density (OD) values of each well were measured using a microplate reader (Asys Hitech GmbH, Eugendorf, Austria). Apoptosis test Trypsin (0.25%) was digested to collect the cells of all the experimental groups, and the cell density was adjusted to 1×106 cells/ml. Annexin V-fluorescein isothiocyanate (FITC; 5 μl) and 5 ml propidium iodide (PI) were added to dye the cells for 30 min at 4°C prior to the flow cytometry analysis. Mitochondrial membrane potential detection JC-1 staining and flow cytometry were used to detect the changes in the mitochondrial membrane potential, according to previously published instructions (17). The fluorescence signals of the JC-1 monomer and polymer were detected using FL1 and FL2 detectors, respectively. FL1-H and FL2-H represented the green and red fluorescence intensities, respectively. CellQuest software version 4.0.2 (Quest Software Inc., Aliso Viejo, CA, USA) was used for the quantification of the results. Hoechst 33342 nuclear staining The human brain glioma U87 cells were plated in a 6-well plate with polylysine-coated cover slips and cultured for 24 h. The cells were then treated with or without naltrindole for 48 h. The untreated and treated cells were washed twice with PBS and incubated with 8 μg/ml Hoechst 33342 (Sigma) at 37°C for 20 min. The fluorescence images were confirmed using a fluorescence microscope (EZ4D; Leica Microsystems, Mannheim, Germany). Western blot assay The cells of all the experimental groups were collected and allotted 2 ml lysis solution, which contained 50 mM Tris-HCl, 137 mM NaCl, 10% glycerin, 100 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml eupeptin, 1% NP-40 and 5 mM cocktail (pH 7.4), for cell lysis to obtain the proteins. The bicinchoninic acid (BCA) assay was used for quantitative measurement. The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then shifted to the PVDF membrane using the semi-dry method and sealed with 5% skimmed milk powder at 4°C overnight. The membranes were washed with TBST and the primary antibodies (cytochrome c rabbit polyclonal, Bax rabbit polyclonal, Bcl-2 rabbit monoclonal, Bcl-xL mouse monoclonal and PKC mouse monoclonal) were added at 37°C for hybrid for 1 h prior to washing with TBST. The secondary goat anti-rabbit β-actin and goat anti-mouse β-actin monoclonal antibodies were added at 37°C for hybridization for 1 h prior to washing with TBST. The color reaction was observed for 5 min using autoradiography. Quantity One software was used for the OD value analysis and measurement. The results were indicated using the OD value/β-actin OD value of the samples. Results Inhibition of δ-opioid receptor inhibits brain glioma cell growth Various concentrations of naltrindole (0, 0.01, 0.1, 1.0 and 10 μM) were administered to the U87 cells for 24, 48 and 72 h prior to using the MTT method to determine cell activity (Fig. 1). The A570 value of the U87 cells was shown to decrease when the concentration of naltrindole increased from 0.01 to 10 μM. The A570 value decreased most significantly when the concentration was 1.0 μM, indicating that naltrindole has an inhibitory effect on the proliferation of brain glioma cells in a concentration-dependent manner. Inhibition of δ-opioid receptor induces brain glioma cell apoptosis The U87 brain glioma cells were treated with various doses of naltrindole (0, 0.01, 0.1 and 1.0 μM) for 48 h, and Hoechst 33342 nuclear staining and flow cytometry were used to assess apoptosis (Fig. 2). As shown in the results, the condensed chromatin of the apoptotic cells in the 1.0 μM naltrindole-treated groups was significantly brighter than the chromatin of the normal cells in the control group (Fig. 2A). Furthermore, with a higher naltrindole dose, the quantity of the apoptotic U87 cells increased significantly in a dose-dependent manner (Fig. 2B and C). These results demonstrated that naltrindole induces the dose-dependent apoptosis of human brain glioma U87 cells. Inhibition of δ-opioid receptor induction of human brain glioma cell apoptosis through the mitochondrial pathway To further explore the signaling pathway of naltrindole-induced brain glioma apoptosis, JC-1 staining flow cytometry was used to analyze the changes in the mitochondrial membrane potential, and western blot analysis was used to analyze the changes in the expression levels of the relevant proteins, Bax, Bcl-2, Bcl-xL, Bak and cytochrome c (Fig. 3). The therapeutic dosage of naltrindole resulted in a decreased mitochondrial membrane potential (Fig. 3A). Naltrindole downregulated the expression levels of Bcl-2 and Bcl-xL in a dose-dependent manner. By contrast, the expression levels of Bax, Bak and cytochrome c proteins increased (Fig. 3B–E). The present data demonstrated that naltrindole is able to change the mitochondrial membrane potential to promote the shift of Bax and Bcl-2 and the release of cytochrome c into the cytoplasm, which results in the apoptosis of brain glioma cells. Inhibition of δ-opioid receptors on the expression levels of brain glioma cell apoptosis-related proteins In order to investigate the impact of the inhibition of δ-opioid receptors on the expression levels of brain glioma cell apoptosis-related proteins, various doses of naltrindole were administered to the U87 cells for 48 h and western blot analysis was used to analyze the expression levels of the procaspase-9 and -3 proteins (Fig. 4). Following the treatment with the various doses of naltrindole, the U87 cell procaspase-9 and -3 protein expression levels decreased significantly compared with the normal control group. The data demonstrated that naltrindole induces U87 apoptosis through the mitochondria-mediated caspase-9 and -3 pathways. Effect of the inhibition of δ-opioid receptors on the expression levels of brain glioma cell PKC proteins In order to investigate the impact that inhibiting the δ-opioid receptors had on the expression levels of the brain glioma cell PKC proteins, various doses of naltrindole were administered to the U87 cells for 48 h and western blot analysis was used to test the expression levels of the PKC and p-PKC proteins (Fig. 5). It was demonstrated that following the treatment with various doses of naltrindole, the expression levels of PKC and p-PKC in the U87 cells decreased significantly compared with the normal control group. The data showed that the inhibition of the proliferation of the U87 cells by naltrindole may be mediated by the PKC pathway. Inhibition of δ-opioid receptors induces brain glioma cell cycle blockade in the G0/G1 phase Flow cytometry was used to investigate whether naltrindole had an impact on the brain glioma cell cycle. The results revealed that 48 h after the administration of the various doses of naltrindole to the U87 cells, the cells were blockaded in the G0/G1 phase at higher levels than in the normal control group (Fig. 6) This indicated that naltrindole is able to inhibit the percentage of U87 cells in the G0/G1 phase in order to restrain cell proliferation. Discussion The concept of cell apoptosis was first proposed by Kerr et al(18) and is widely accepted. Cell apoptosis is widespread in all types of cells. Studies have demonstrated that apoptosis plays a significant role during the incidence and development of numerous kinds of tumors (19–21). Previous studies have shown that the common treatment among the vast majority of antitumoral regimens is the induction of tumor cell apoptosis to suppress the growth of the tumor (20,21). Therefore, tumor cell apoptosis induction for the treatment of tumors is a new target of action against the tumor that is already becoming a new developmental direction in tumor therapy. The present study aimed to discuss the functions and applied values of δ-opioid receptors during brain glioma treatment. Previous studies have confirmed that artificially excited or inhibited δ-opioid receptors may affect the proliferation and apoptosis of numerous types of tumor cells (22–24). Therefore, the antitumor effects of δ-opioid receptors are highly studied. However, it is not well acknowledged whether δ-opioid receptors play the same role in brain glioma or not. The present study observed that the specific inhibitor of δ-opioid receptors, naltrindole, inhibited glioma cell proliferation in a dose- and time-dependent manner. This indicates that δ-opioid receptors are closely associated with the occurrence and developmental processes of brain glioma, which is a new target for the potential treatment of this disease. A study by Kerros et al(25) revealed that opioid receptors and somatostatin may be used as a heterodimer assembly for separately regulating the proliferation of malignant cells, which contributes to U266 cells apoptosis of human multiple myeloma. A study by Marzioni et al(8) identified that the active state of the δ-opioid receptors had a close association with the occurrence and development of human cholangiocarcinoma, whose mechanism of action may be associated with signaling conduction pathways through phosphoinositide 3-kinase (PI3K) and ERK1/2. The results from the present study are consistent with these findings. Following the treatment with various doses of naltrindole in the brain glioma cells, the positive rate of annexin V staining increased according to the dose dependence. This illustrated that the inhibition of the δ-opioid receptors may induce brain glioma cell apoptosis, but not cell death. Naltrindole also significantly inhibited the periodical changes of the brain glioma cells and arrested the cells in the G0/G1 phase in order to change the cell cycling process and sequentially induce cell apoptosis. A study by Tang et al(26) demonstrated that DADLE was able to inhibit the proliferation of HepG2 of human liver cancer cells by specifically activating the δ-opioid receptors and improving the sensitivity of the tumor cells to the chemotherapy drug, cisplatin. The double effect of the δ-opioid receptors on the tumors may be associated with the subtypes of receptors and the inhomogeneity of the tumors. Triggering cell apoptosis involves the pathways of endogenous mitochondria and exogenous dead receptors, and this conclusion has been well recognized (27). In the present study, following the administration of the various doses of naltrindole for the treatment of brain glioma, Bax shifted from the cytoplasm to the mitochondrial membrane. Firstly, the mitochondrial membrane potential was reduced, then immediately after, cytochrome c was released into the cytoplasm. The aforementioned results indicated that brain glioma cell apoptosis induced by the inhibition of the δ-opioid receptor was likely to be mediated by the endogenous mitochondrial pathway. The Bcl-2/Bax families are the key regulation factors of the endogenous mitochondrial apoptosis pathway (28,29). Under apoptosis promoting effect factors, Bax shifted from the cytoplasm to the mitochondrial membrane, which altered the permeability of the mitochondrial membrane, facilitating the release of cytochrome c from the mitochondria into the cytoplasm (30) and consequentially activating the apoptosis cascade and finally, cell apoptosis. The activation of the caspase family was a significant prerequisite for cell apoptosis, as it activated the proteases that are associated with apoptosis when apoptosis occurred within the cells (31). Following the administration of naltrindole, the changes in the protein levels of procaspase-9 and -3 were analyzed. The expression levels of procaspase-9 and -3 decreased sharply when cell apoptosis occurred in the brain glioma cells. Cytochrome c was released from the mitochondria into the cytoplasm and produced biological effects to activate procaspase-9 and -3, which had a crucial role to play during the apoptosis pathway (32). The previous results suggested that the inhibition of the δ-opioid receptors resulted in brain glioma cell apoptosis and was closely associated with the mitochondrial pathways. Historical research demonstrated that PKC is a type of serine/threonine protein kinase, which has wide biological activities and plays a significant part in the regulation of the differentiation and proliferation of cells (33). Numerous other studies indicated that PKC activation facilitated tumor cell proliferation (34) and also took part in the brain glioma proliferation and differentiation processes (35). The present study demonstrated that naltrindole reduced the expression levels of PKC and p-PKC in brain glioma cells by concentration dependence and inhibited tumor cell proliferation. This illustrated that the PKC pathway participated in the process of naltrindole inhibition of brain glioma cell proliferation at the very least. However, it is worth further research to confirm which specific subtype of PKC was functioning. In conclusion, the present study revealed that the inhibition of δ-opioid receptors induced brain glioma cell apoptosis by regulating the effects of the Bcl-2/Bax families on the mitochondrial pathway, thus releasing cytochrome c and activating the caspase families, and by regulating the PKC signaling conduction pathway. The inhibition of δ-opioid receptors may be used in the future as a new means for the prevention and treatment of cerebral glioma, making an important contribution towards the therapy for this condition. Acknowledgements This study was supported by Natural Science Foundation of China funding (no. 81271278). The authors would like to thank Dr G Tang (Anhui Medical University, China) for advice on the manuscript. Figure 1 Naltrindole inhibits the growth of brain glioma cells. Cell reproductivity was tested using the MTT method following the administration of various concentrations of naltrindole (0, 0.01, 0.1, 1.0 and 10 μM), which acted on the U87 cells for 24, 48 and 72 h. The results are representative of three independent experiments. OD, optical density. Figure 2 Naltrindole induces brain glioma cell apoptosis. U87 cell apoptosis induced by various concentrations of naltrindole for 48 h was identified using (A) Hoechst 33342 nuclear staining and analyzed by (B) annexin V-FITC/PI double-stained flow cytometry. (C) The histogram shows the U87 apoptosis rate. P<0.05 vs. 0 μM. The data are representative of three independent experiments. FITC, fluorescein isothiocyanate; PI, propidium iodide. Figure 3 Naltrindole induction of brain glioma cell apoptosis through the mitochondrial pathway. The U87 cells were treated with various doses of naltrindole (0, 0.1 and 1.0 μM) for 48 h. (A) Flow cytometry was used to analyze the change in the mitochondrial membrane potential. (B) A western blot analysis was used to analyze the protein expression levels of Bax and cytochrome c. (C) A western blot analysis was used to analyze the protein expression levels of Bcl-2, Bcl-xL and Bak. (D and E) The histogram shows the results from B and C (%). P<0.05. Results are representative of three independent experiments. Figure 4 Impact of naltrindole on the expression levels of brain glioma cell apoptosis-related proteins procaspase-9 and -3. (A) A western blot analysis was used to analyze the protein expression levels of procaspase-9 and -3. (B) Quantitative analysis of western blotting results. P<0.05. The results are representative of three independent experiments. Figure 5 Impact of naltrindole on the expression levels of brain glioma cell PKC and p-PKC. (A) A western blot analysis was used to analyze the protein expression levels of PKC and p-PKC. (B) Quantitative analysis of western blotting results. P<0.05. The results are representative of three independent experiments. PKC, protein kinase C; p-PKC, phophorylated PKC. Figure 6 Impact of naltrindole on the brain glioma cell cycle. Following the administration of various doses of naltrindole (0, 0.1 and 1.0 μM) to the U87 cells for 48 h, flow cytometry was used to analyze the cell cycle. *P<0.05 vs. 0 μM. The data are representative of three independent experiments. ==== Refs References 1 Ricard D Idbaih A Ducray F Lahutte M Hoang-Xuan K Delattre JY Primary brain tumours in adults Lancet 379 1984 1996 2012 22510398 2 Johannesen TB Langmark F Lote K Cause of death and long-term survival in patients with neuro-epithelial brain tumours: a population-based study Eur J Cancer 39 2355 2363 2003 14556928 3 Zhang Y Chao T Li R Liu W Chen Y Yan X Gong Y Yin B Liu W Qiang B Zhao J Yuan J Peng X MicroRNA-128 inhibits glioma cells proliferation by targeting transcription factor E2F3a J Mol Med (Berl) 87 43 51 2009 18810376 4 Komotar RJ Otten ML Moise G Connolly ES Jr Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma-a critical review Clin Med Oncol 2 421 422 2008 21892310 5 Stupp R Mason WP van den Bent MJ Weller M Fisher B Taphoorn MJ Belanger K Brandes AA Marosi C Bogdahn U Curschmann J Janzer RC Ludwin SK Gorlia T Allgeier A Lacombe D Cairncross JG Eisenhauer E Mirimanoff RO European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma N Engl J Med 352 987 996 2005 15758009 6 Notas G Kampa M Nifli AP Xidakis K Papasava D Thermos K Kouroumalis E Castanas E The inhibitory effect of opioids on HepG2 cells is mediated via interaction with somatostatin receptors Eur J Pharmacol 555 1 7 2007 17113072 7 Tang B Li Y Yuan S Tomlinson S He S Upregulation of the δ opioid receptor in liver cancer promotes liver cancer progression both in vitro and in vivo Int J Oncol 7 31 2013 (Epub ahead of print) 8 Marzioni M Invernizzi P Candelaresi C Maggioni M Saccomanno S Selmi C Rychlicki C Agostinelli L Cassani B Miozzo M Pasini S Fava G Alpini G Benedetti A Human cholangiocarcinoma development is associated with dysregulation of opioidergic modulation of cholangiocyte growth Dig Liver Dis 41 523 533 2009 18948067 9 Heiss A Ammer H Eisinger DA delta-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation Exp Cell Res 315 2115 2125 2009 19362548 10 Madar I Bencherif B Lever J Heitmiller RF Yang SC Brock M Brahmer J Ravert H Dannals R Frost JJ Imaging delta- and mu-opioid receptors by PET in lung carcinoma patients J Nucl Med 48 207 213 2007 17268016 11 Debruyne D Leroy A DE Wever O Vakaet L Mareel M Bracke M Direct effects of delta opioid receptor agonists on invasion-associated activities of HCT-8/E11 colon cancer cells Anticancer Res 30 9 17 2010 20150612 12 Zhong J Kong X Zhang H Yu C Xu Y Kang J Yu H Yi H Yang X Sun L Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation PLoS One 7 e39378 2012 22761775 13 Ordys BB Launay S Deighton RF McCulloch J Whittle IR The role of mitochondria in glioma pathophysiology Mol Neurobiol 42 64 75 2010 20414816 14 Zhou J Cheng G Cheng G Tang HF Zhang X Novaeguinoside II inhibits cell proliferation and induces apoptosis of human brain glioblastoma U87MG cells through the mitochondrial pathway Brain Res 1372 22 28 2011 21147078 15 Zhang ZF Guo Y Zhang JB Wei XH Induction of apoptosis by chelerythrine chloride through mitochondrial pathway and Bcl-2 family proteins in human hepatoma SMMC-7721 cell Arch Pharm Res 34 791 800 2011 21656365 16 Du J Tang B Wang J Sui H Jin X Wang L Wang Z Antiproliferative effect of alpinetin in BxPC-3 pancreatic cancer cells Int J Mol Med 29 607 612 2012 22246103 17 Tang B Zhang Y Liang R Yuan P Du J Wang H Wang L Activation of the δ-opioid receptor inhibits serum deprivation-induced apoptosis of human liver cells via the activation of PKC and the mitochondrial pathway Int J Mol Med 28 1077 1085 2011 21874227 18 Kerr JF Wyllie AH Currie AR Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics Br J Cancer 26 239 257 1972 4561027 19 Chiarugi P Giannoni E Anoikis: a necessary death program for anchorage-dependent cells Biochem Pharmacol 76 1352 1364 2008 18708031 20 Ng CF Ng PK Lui VW Li J Chan JY Fung KP Ng YK Lai PB Tsui SK FHL2 exhibits anti-proliferative and anti-apoptotic activities in liver cancer cells Cancer Lett 304 97 106 2011 21377781 21 Yang TY Chang GC Chen KC Hung HW Hsu KH Sheu GT Hsu SL Sustained activation of ERK and Cdk2/cyclin-A signaling pathway by pemetrexed leading to S-phase arrest and apoptosis in human non-small cell lung cancer A549 cells Eur J Pharmacol 663 17 26 2011 21575631 22 Hatzoglou A Kampa M Castanas E Opioid-somatostatin interactions in regulating cancer cell growth Front Biosci 10 244 256 2005 15574365 23 Kampa M Bakogeorgou E Hatzoglou A Damianaki A Martin PM Castanas E Opioid alkaloids and casomorphin peptides decrease the proliferation of prostatic cancer cell lines (LNCaP, PC3 and DU145) through a partial interaction with opioid receptors Eur J Pharmacol 335 255 265 1997 9369381 24 Baldelli B Vecchio L Biggiogera M Vittoria E Muzzonigro G Gazzanelli G Malatesta M Ultrastructural and immunocytochemical analyses of opioid treatment effects on PC3 prostatic cancer cells Microsc Res Tech 64 243 249 2004 15452891 25 Kerros C Cavey T Sola B Jauzac P Allouche S Somatostatin and opioid receptors do not regulate proliferation or apoptosis of the human multiple myeloma U266 cells J Exp Clin Cancer Res 28 77 2009 19500423 26 Tang B Du J Gao ZM Liang R Sun DG Jin XL Wang LM DADLE suppresses the proliferation of human liver cancer HepG2 cells by activation of PKC pathway and elevates the sensitivity to cis-diammine dichloridoplatium Zhonghua Zhong Liu Za Zhi 34 425 429 2012 (In Chinese) 22967443 27 von Haefen C Wendt J Semini G Sifringer M Belka C Radetzki S Reutter W Daniel PT Danker K Synthetic glycosidated phospholipids induce apoptosis through activation of FADD, caspase-8 and the mitochondrial death pathway Apoptosis 16 636 651 2011 21437721 28 Burlacu A Regulation of apoptosis by Bcl-2 family proteins J Cell Mol Med 7 249 257 2003 14594549 29 Mattson MP Kroemer G Mitochondria in cell death: novel targets for neuroprotection and cardioprotection Trends Mol Med 9 196 205 2003 12763524 30 Saito M Korsmeyer SJ Schlesinger PH BAX-dependent transport of cytochrome c reconstituted in pure liposomes Nat Cell Biol 2 553 555 2000 10934477 31 Nicholson DW Ali A Thornberry NA Vaillancourt JP Ding CK Gallant M Gareau Y Griffin PR Labelle M Lazebnik YA Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis Nature 376 37 43 1995 7596430 32 Riedl SJ Shi Y Molecular mechanisms of caspase regulation during apoptosis Nat Rev Mol Cell Biol 5 897 907 2004 15520809 33 Capiati DA Vazquez G Tellez Iñón MT Boland RL Antisense oligonucleotides targeted against protein kinase c alpha inhibit proliferation of cultured avian myoblasts Cell Prolif 33 307 315 2000 11063133 34 Ali AS Ali S El-Rayes BF Philip PA Sarkar FH Exploitation of protein kinase C: a useful target for cancer therapy Cancer Treat Rev 35 1 8 2009 18778896 35 Colombo D Tringali C Franchini L Cirillo F Venerando B Glycoglycerolipid analogues inhibit PKC translocation to the plasma membrane and downstream signaling pathways in PMA-treated fibroblasts and human glioblastoma cells, U87MG Eur J Med Chem 46 1827 1834 2011 21388717	24179523	PMC3813693	CC BY	2021-01-04 23:20:31	yes	Oncol Lett. 2013 Nov 26; 6(5):1351-1357
==== Front Diagn PatholDiagn PatholDiagnostic Pathology1746-1596BioMed Central 1746-1596-8-1792415294110.1186/1746-1596-8-179ResearchAstragalus saponins affect proliferation, invasion and apoptosis of gastric cancer BGC-823 cells Wang Tao 1wang371@126.comXuan Xiaoyan 2xuanxiaoyanzzu@sina.comLi Min 2limin75@163.comGao Ping 1wang37127070@tom.comZheng Yuling 3zhengyuling1@sina.comZang Wenqiao 2zangwenqiao@sina.comZhao Guoqiang 2zhaogq@zzu.edu.cn1 Department of Hemato-tumor, The First Affiliated Hospital of Henan University of TCM, Zhengzhou, People’s Republic of China2 Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, People’s Republic of China3 Henan University of TCM, Zhengzhou, People’s Republic of China2013 24 10 2013 8 179 179 5 10 2013 22 10 2013 Copyright © 2013 Wang et al.; licensee BioMed Central Ltd.2013Wang et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Background Astragalus memebranaceus is a traditional Chinese herbal medicine used in treatment of common cold, diarrhea, fatigue, anorexia and cardiac diseases. Recently, there are growing evidences that Astragalus extract may be a potential anti-tumorigenic agent. Some research showed that the total saponins obtained from Astragalus membranaceus possess significant antitumorigenic activity. Gastric cancer is one of the most frequent cancers in the world, almost two-thirds of gastric cancer cases and deaths occur in less developed regions. But the effect of Astragalus membranaceus on proliferation, invasion and apoptosis of gastric cancer BGC-823 cells remains unclear. Methods Astragalus saponins were extracted. Cells proliferation was determined by CCK-8 assay. Cell cycle and apoptosis were detected by the flow cytometry. Boyden chamber was used to evaluate the invasion and metastasis capabilities of BGC-823 cells. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. Results The results demonstrated that total Astragalus saponins could inhibit human gastric cancer cell growth both in vitro and in vivo, in additional, Astragalus saponins deceased the invasion ability and induced the apoptosis of gastric cancer BGC-823 cells. Conclusions Total Astragalus saponins inhibited human gastric cancer cell growth, decreased the invasion ability and induced the apoptosis. This suggested the possibility of further developing Astragalus as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent in gastric cancer therapy. Gastric cancerProliferationInvasionApoptosis ==== Body Introduction Astragalus memebranaceus (AST) is a traditional Chinese herbal medicine used in treatment of common cold, diarrhea, fatigue, anorexia and cardiac diseases [1-4]. In recent years, radix Astragalus membranaceus has also been used to ameliorate the side effects of cytotoxic antineoplastic drugs [5]. The active pharmacological constituents of radix Astragalus membranaceus include various polysaccharides, saponins and flavonoids [6]. Among these, Astragalus polysaccharides have been most widely studied, mainly on their immunopotentiating properties like stimulation of murine B-cell proliferation and cytokine production [7]. Apart from these, clinical studies also showed that Astragalus polysaccharides could counteract the side effects of chemotherapeutic drugs, such as a significant reduction in the degree of myelosuppression in cancer patients [8]. Recently, there are growing evidences that Astragalus extract may be a potential anti-tumorigenic agent. For instance, hepatocarcinogenesis could be prevented in rats fed with the aqueous extract of Astragalus, which is mainly composed of Astragalus polysaccharides [9]. There are also reports that describe the potentiating effect of Astragalus extract in recombinant interleukin-2-generated lymphokine-activated cells upon the anti-tumorigenic action of drugs against murine renal carcinoma [10]. Saponins isolated from radix Astragalus membranaceus consist of astragalosides (I–VIII) and some of their isomer isoastragalosides (I,II and IV) [11,12]. Similar to the polysaccharides obtained from the same herb, Astragalus saponins have been found to possess immunomodulating effects. The pure isolated saponin astragaloside IV could increase murine B and T cell proliferation [13] and possess cardioprotective properties [14,15]. Some research showed that the total saponins obtained from Astragalus membranaceus (AST) possessed significant antitumorigenic activity in HT-29 human colon cancer cells and tumor xenograft [16]. AST suppressed cancer cell growth by inhibiting proliferation through phase-specific cell cycle arrest and promotion of caspase-dependent apoptosis. In nude mice xenograft, the AST induced reduction in tumor volume was comparable with that produced by the conventional chemotherapeutic drug 5-fluorouracil (5-FU), of which the side effects (including mortality) associated with the drug combo 5-FU 1 oxaliplatin could be largely alleviated when AST was used along with 5-FU in replacement of oxaliplatin [17]. Gastric cancer is one of the most frequent cancers in the world, almost two-thirds of gastric cancer cases and deaths occur in less developed regions. Gastric cancer is a significant cancer burden currently and be one of the key issues in cancer prevention and control strategy in China. But the effect of Astragalus membranaceus on proliferation, invasion and apoptosis of gastric cancer BGC-823 cells remains unclear. In the present study, we investigated the effect of Astragalus saponins on proliferation, invasion and apoptosis of gastric cancer BGC-823 cells. Materials and methods Materials Radix Astragalus membranaceus had been obtained from the province of Henan, China. The authenticity and quality of the crude herb were then tested in the Quality Assurance Laboratory, The First Affiliated Hospital of Henan University of TCM. Antibodies were from Santa Cruz Biotechnology, USA. The human gastric cancer BGC-823 cell line was purchased from the Chinese Academy of Sciences Cell Bank. All cells were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and grown in a 37°C, 5% CO2 incubator. Preparation of AST extract Astragalus saponins were extracted according to the method of Ma et al. [18] with slight modifications. In brief, 500 g of crude herb was refluxed with 2% potassium hydroxide in methanol for 1 h. Butan-1-ol was added to the reconstituted residue from above for phase separation to obtain total saponins. The dried and lyophilized AST powder (0.6% w/w) was reconstituted in ultrapure water to form a 10 mg/ml stock and stored at −20°C. CCK-8 assay The cells in the logarithmic phase of growth were seeded in 96-well plates at a cell density of 5 × 104/well. Cells were cultured at 37°C in 5% CO2. After 12 hours, replace medium containing 0 μg/ml, 20 μg/ml, 40 μg/mland 80 μg/ml, 0 μg/ml as the control. after 0 h、24 h、48 h、72 h, 10 μL CCK-8 were mixed. Cells were cultured at 37°C in 5% for 3 hours, and then optical density was measured at 450 nm. All experiments were performed in triplicate. Cell-cycle analysis For cell cycle analysis by flow cytometry (FCM), cells in the logarithmic phase of growth were harvested by trypsinization, washed with PBS, fixed with 75% ethanol overnight at 4°C and incubated with RNase at 37°C for 30 min. Nuclei were stained with propidium iodide for 30 min. A total of 104 nuclei were examined in a FACS Calibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). All experiments were performed in triplicate. Transwell invasion assay Transwell filters (Costar, USA) were coated with matrigel (3.9 μg/μl, 60–80 μl) on the upper surface of the polycarbonic membrane (6.5 mm in diameter, 8 μm pore size). After 30 min of incubation at 37°C, the matrigel solidified and served as the extracellular matrix for tumor cell invasion analysis. Cells transfected were harvested in 100 μl of serum free medium and added to the upper compartment of the chamber. The cells that had migrated from the matrigel into the pores of the inserted filter were fixed with 100% methanol, stained with hematoxylin, mounted, and dried at 80°C for 30 min. The number of cells invading the matrigel was counted from three randomly selected visual fields, each from the central and peripheral portion of the filter, using an inverted microscope at 100× magnification. All experiments were performed in triplicate. Apoptosis assay The Annexin V-FITC/PI Apoptosis Detection Kit I (Abcam, USA) was used to detect and quantify apoptosis by flow cytometry. In brief, cells in the logarithmic phase of growth were harvested in cold PBS and collected by centrifugation for 5 min at 1000 × g. Cells were resuspended at a density of 1 × 106 cells/ml in 1 × binding buffer, stained with FITC-labeled annexin V for 5 min and immediately analyzed in a FACScan Flow Cytometer (Becton Dickinson). Data were analyzed by Cell Quest software. Tests were repeated in triplicate. Nude mouse tumor xenograft model Fifteen immunodeficient female BALB/C nude mice, 5–6 weeks old were purchased from the Experimental Animal Center of the Henan province, China, They were bred under aseptic conditions and maintained at constant humidity and temperature, according to standard guidelines under a protocol approved by Zhengzhou University. Mice in the different groups were subcutaneously injected in the dorsal scapular region with the BGC-823 cells. After transplantation, the skin was closed and the mice were divided randomly into three groups (five mice per group). In 2 days, AST (100 mg/kg body weight) (Group1) and AST (200 mg/kg body weight) (Group2) [15] were administered once a day. PBS was administered as the control group. Primary tumors were allowed to develop for 28 days. The tumors formed were measured with a caliper every 7 days, and tumor volume was calculated using the formula: volume = π(length × width2)/6. Tumors were harvested after 4 weeks. Statistical analysis SPSS17.0 was used for statistical analysis. One-way analysis of variance (ANOVA) and the χ2 test were used to analyze the significance between groups. Multiple comparisons between the parental and control vector groups were made using the Least Significant Difference test when the probability for ANOVA was statistically significant. All data represent mean ± SD. Statistical significance was set at p < 0.05. Results Astragalus saponins inhibited proliferation of gastric cancer BGC-823 cells CCK-8 assay was used to measure the cell growth and viability of BGC-823 cells. The effects of Astragalus saponins on proliferation of gastric cancer BGC-823 cells were determined. As shown in Figure 1A, Compared to the control, Astragalus saponins inhibited proliferation of BGC-823 cells at a dose- and time-dependent manner. These results suggested that Astragalus saponins might function as a tumor suppressor in BGC-823 in vitro. Figure 1 Effect of Astragalus saponins on proliferation and cell cycle of BGC-823 cells. A. Astragalus saponins inhibited proliferation of BGC-823 cells. Cells were treated with, 20 μg/ml, 40 μg/ml, and 80 μg/ml Astragalus saponins, 0 μg/ml as a control, and cell proliferation was assessed using the CCK8 assay. Data are presented as the mean of triplicate experiments. The growth inhibitory effect of the Astragalus saponins was time and dose dependent, with the maximum inhibition detected 72 h after treatment. Significant difference (p < 0.05). B. Astragalus saponins impaired cell cycle progression in BGC-823 cells. Cell cycle distribution was analyzed by flow cytometry. Data are presented as the mean of triplicate experiments. Administration of Astragalus saponins significantly increased the percentage of cells in the G0/G1 phase, and significantly decreased the S and G2/M phase fractions. Astragalus saponins induced G1 arrest in BGC-823 cells The cell cycle distribution was analyzed by flow cytometry. The fractions of BGC-823 cells in the G0/G1 phase of the cell cycle in the 0 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml Astragalus saponins groups were 55.99%, 65.44%, 70.89%, and 78.82% respectively (Figure 1B). These results indicated that Astragalus saponins induced cell cycle arrest in the G0/G1 phase, delayed the progression of the cell cycle, and inhibited cell proliferation. Astragalus saponins decreased the invasive and migration ability of BGC-823 cells Transwell invasion assay was used to evaluate the impact of Astragalus saponins on cell invasion and migration. BGC-823 cells were treated with 0 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml Astragalus saponins, and then placed in a Transwell chamber. The number of Astragalus saponins treated BGC-823 cells migrating through the matrigel was significantly lower (p < 0.05) than those of the 0 μg/ml Astragalus saponins group (the control group), (Figure 2). The effect was more obvious for 80 μg/ml Astragalus saponins. These results demonstrated that Astragalus saponins inhibited the invasive ability of BGC-823 cells in vitro. Figure 2 Astragalus saponins decreased the invasion and migration ability of BGC-823 cells. A. BGC-823 cell invasion was determined using the transwell invasion assay, which measures the number of cells that migrate through the matrigel into the lower surface of the polycarbonic membrane (100×). Data are presented as the mean of triplicate experiments. B. Cell invasion decreased significantly (p < 0.05) in a dose-dependent manner in Astragalus saponins -treated cells compared with the control group. Astragalus saponins induced apoptosis of BGC-823 cells BGC-823 cells apoptosis was measured by flow cytometry. Statistically significant (p < 0.05)increases in annexin V + apoptotic cells were observed in 20 μg/ml(6.53 ± 0.62%), 40 μg/ml(12.14 ± 0.69%) and 80 μg/ml (18.2 ± 0.79%)Astragalus saponins–treated BGC-823 cell lines compared to control group (3.56 ± 0.45%) (Figure 3A). These results demonstrated that Astragalus saponins induced apoptosis of BGC-823 cells in vitro. Figure 3 BGC-823 cell apoptosis and xenograft tumor experiment. A. Astragalus saponins induced apoptosis in BGC-823 cells. Cell apoptosis was analyzed by flow cytometry. Statistically significant (p < 0.05)increases in annexin V + apoptotic cells were observed in Astragalus saponins–treated BGC-823 cells compared to controls. Data are presented as the mean of triplicate experiments. B. BGC-823 cell xenograft tumor experiment. Injection of Astragalus saponins inhibited tumor growth in a BGC-823 xenograft model compared with the blank control group. Significant difference (p < 0.05). Astragalus saponins inhibited gastric cancer xenograft growth Because Astragalus saponins play an important role in cell survival, we performed a proof-of-principle experiment using a BGC-823 cell xenograft model. As shown in Figure 3B, a significant decrease in tumor volume was observed in the Astragalus saponins -treated group (P < 0.05). These findings further suggest the therapeutic potential of Astragalus saponins for the gastric cancer. Discussion Despite recent advancement in understanding the carcinogenic processes of gastric cancer, the increasing incidence and relatively low remission rate of chemotherapy have urged the scientific community to establish more effective treatment regimens by adopting novel and innovative approaches. The discovery and use of active medicinal compounds from herbal/natural sources have provided alternative treatment choices for patients [19,20]. Tumor metastasis starts with breakdown of epithelial integrity, followed by malignant cells invading into the surrounding stroma and lymphovascular space, by which tumor cells travel to distant target organs. Some researchers showed that Ezrin, HER2 and c-MET abnormal expression were related to the poor prognosis of gastric adenocarcinoma [21-23]. Astragalus membranaceus (Radix Astragali) has a long history of medicinal use in Chinese herbal medicine. It has been formulated as an ingredient of herbal mixtures to treat patients with deficiency in vitality, which symptomatically presents with fatigue, diarrhea and lack of appetite. Radix Astragali is also commonly used as immunomodulating agent to stimulate the immune system of immunodeficient patients. Moreover, it has been reported that herbal formulations containing Radix Astragali and some of its constituents could produce hepatoprotective [24], antiviral [25] and antioxidative effects [26]. Recently, evidence from various animal and clinical studies has demonstrated that Radix Astragali may possess anticarcinogenic property [27], which could attenuate the systemic side effects of conventional antineoplatic drugs [28]. In the present study, we have shown that the total saponins obtained from radix Astragalus membranaceus could be established as effective chemotherapeutic agent to suppress gastric cancer cell growth through promotion of apoptosis and inhibition of cell proliferation. This is the first report that clearly characterizes the anti-tumor properties of Astragalus saponins in gastric cancer cells and tumor xenograft. Astragalus saponins affect proliferation, invasion and apoptosis of gastric cancer BGC-823 cells. The mechanisms remain unclear. Some researchers considered the anticancer properties of Astragalus species could be explained by immunological mechanisms on the basis of the results obtained in a study with urological neoplasm cells and bladder murine carcinomas, Rittenhouse et al. [29] reported that A. membranaceus may exert its antitumor activity by abolishing tumor-associated macrophage suppression. The potentiation of the natural killer cytotoxicity of peripheral blood mononuclear cells in patients with systemic lupus erythematosus was demonstrated by Zhao [30] using an enzyme-release assay. The activity was increased in the samples of healthy donors and patients with the pathology. The release of a natural killer cytotoxic factor by peripheral blood mononuclear cells was higher in the control group, and the levels of that factor correlated well with natural killer activity, and correlated negatively with the clinical effect. Others thought the anticancer properties of Astragalus was associated with RSK2. The Ras-ERKs-RSK2 pathway regulates cell proliferation, survival, growth, motility and tumorigenesis. RSK2 is a direct substrate kinase of ERKs and, functionally speaking, is located between ERKs and its target transcription factors [31]. Studies have demonstrated that the total cellular RSK2 protein level is significantly higher in cancer cells compared with normal tissues and premalignant cell lines [32-37]. In summary, we have demonstrated in the present study that total Astragalus saponins could inhibit human gastric cancer cell growth both in vitro and in vivo. This suggested the possibility of further developing Astragalus as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent in gastric cancer therapy. Conclusions Total Astragalus saponins could inhibit human gastric cancer cell growth both in vitro and in vivo. This suggested the possibility of further developing Astragalus as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent in gastric cancer therapy. Competing interests The authors declare that they have no competing interests. Authors’ contribution XYX, ML and PG: conceived of the study, and participated in its design and coordination and helped to draft the manuscript. WQZ and GQZ: carried out part of experiments and wrote the manuscript. YLZ performed the statistical analysis. All authors read and approved the final manuscript. Acknowledgments This study was supported by Henan province science and technology research projects (122102310550). ==== Refs Di N Liu F-N Miao Z-F Du Z-M Xu H-M Astragalus extract inhibits destruction of gastric cancer cells to mesothelial cells by anti-apoptosis World J Gastroenterol 2009 15 5 570 577 10.3748/wjg.15.570 19195058 Sun Y Jin L Wang T Xue J Liu G Li X Polysaccharides from astragalus membranaceus promote phagocytosis and superoxide anion (O2-) production by coelomocytes from sea cucumber apostichopus japonicus in vitro Comp Biochem Physiol C Toxicol Pharmacol 2008 147 293 298 10.1016/j.cbpc.2007.11.003 18221918 Wang PC Zhang ZY Zhang J Tong TJ Two isomers of HDTIC isolated from astragali radix decrease the expression of p16 in 2BS cells Chin Med J (Engl) 2008 121 231 235 18298915 Yuan C Pan X Gong Y Xia A Wu G Tang J Han X Effects of astragalus polysaccharides (APS) on the expression of immune response genes in head kidney, gill and spleen of the common carp, cyprinus carpio L Int Immunopharmacol 2008 8 51 58 10.1016/j.intimp.2007.10.009 18068100 Zee-Cheng RK Shi-quan-da-bu-tang (ten significant tonic decoction), SQT. Apotent Chinese biological response modifier in cancer immunotherapy, potentiation and detoxification of anticancer drugs Methods Find Exp Clin Pharmacol 1992 14 725 736 1294861 Liu J Zhang JF Lu JZ Zhang DL Li K Su K Wang J Zhang YM Wang N Yang ST Bu L Ou-Yang JP Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation Acta Pharmacol Sin 2013 34 1 137 145 10.1038/aps.2012.133 23103623 Shao BM Xu W Dai H Tu P Li Z Gao XM A study on the immune receptors for polysaccharides from the roots of astragalus membranaceus, a Chinese medicinal herb Biochem Biophys Res Commun 2004 320 1103 1111 10.1016/j.bbrc.2004.06.065 15249203 Duan P Wang ZM Clinical study on effect of astragalus in efficacy enhancing and toxicity reducing of chemotherapy in patients of malignant tumor Zhongguo Zhong Xi Yi Jie He Za Zhi 2002 22 515 517 12592686 Liu J Chen HB Guo BL Zhao ZZ Liang ZT Yi T Study of the relationship between genetics and geography in determining the quality of astragali radix Biol Pharm Bull 2011 34 9 1404 1412 10.1248/bpb.34.1404 21881225 Wang Y Qian XJ Hadley HR Lau BH Phytochemicals potentiate interleukin-2 generated lymphokine-activated killer cell cytotoxicity against murine renal cell carcinoma Mol Biother 1992 4 143 146 1445669 Liu J Hu X Yang Q Yu Z Zhao Z Yi T Chen H Comparison of the immunoregulatory function of different constituents in radix astragali and radix hedysari J Biomed Biotechnol 2010 2010 479426 20224658 Zheng Z Liu D Song C Cheng C Hu Z Studies on chemical constituents and immunological function activity of hairy root of astragalus membranaceus Chin J Biotechnol 1998 14 2 93 97 10196633 Wang YP Li XY Song CQ Hu ZB Effect of astragaloside IV on T, B lymphocyte proliferation and peritoneal macrophage function in mice Acta Pharmacol Sin 2002 23 263 266 11918853 Zhang WD Chen H Zhang C Liu RH Li HL Chen HZ Astragaloside IV from Astragalus membranaceus shows cardioprotection during myocardial ischemia in vivo and in vitro Planta Med 2006 72 4 8 10.1055/s-2005-873126 16450288 Zhang BQ Hu SJ Qiu LH Shan QX Sun J Xia Q Bian K Diphasic effects of Astragalus membranaceus BUNGE (Leguminosae) on vascular tone in rat thoracic aorta Biol Pharm Bull 2005 28 8 1450 1454 10.1248/bpb.28.1450 16079491 Tin MMY Cho CH Chan K James AE Ko JKS Astragalus saponins induce growth inhibition and apoptosis in human colon cancer cells and tumor xenograft Carcinogenesis 2007 28 1347 1355 10.1093/carcin/bgl238 17148504 Kathy KW Auyeung1, Chi-Hin Cho2 and Joshua K.S. Ko A novel anticancer effect of astragalus saponins: transcriptional activation of NSAID-activated gene Int J Cancer 2009 125 1082 1091 10.1002/ijc.24397 19384947 Ma XQ Shi Q Duan JA Dong TT Tsim KW Chemical analysis of radix astragali (Huangqi) in China: a comparison with its adulterants and seasonal variations J Agric Food Chem 2002 50 17 4861 4866 10.1021/jf0202279 12166972 Yang B Xiao B Sun T Antitumor and immunomodulatory activity of astragalus membranaceus polysaccharides in H22 tumor-bearing mice Int J Biol Macromol 2013 62C 287 290 24060282 Wu JJ Sun WY Hu SS Zhang S Wei W A standardized extract from paeonia lactiflora and astragalus membranaceus induces apoptosis and inhibits the proliferation, migration and invasion of human hepatoma cell lines Int J Oncol 2013 43 5 1643 1651 24002667 Shan L Ying J Lu N HER2 expression and relevant clinicopathological features in gastric and gastroesophageal junction adenocarcinoma in a Chinese population Diagn Pathol 2013 8 76 83 10.1186/1746-1596-8-76 23656792 Jin J Jin T Quan M Piao Y Lin Z Ezrin overexpression predicts the poor prognosis of gastric adenocarcinoma Diagn Pathol 2012 7 135 148 10.1186/1746-1596-7-135 23039327 Sotoudeh K Hashemi F Madjd Z Sadeghipour A Molanaei S Kalantary E The clinicopathologic association of c-MET overexpression in Iranian gastric carcinomas; an immunohistochemical study of tissue microarrays Diagn Pathol 2012 7 57 62 10.1186/1746-1596-7-57 22640970 Jia R Cao L Xu P Jeney G Yin G In vitro and in vivo hepatoprotective and antioxidant effects of astragalus polysaccharides against carbon tetrachloride-induced hepatocyte damage in common carp (cyprinus carpio) Fish Physiol Biochem 2012 38 3 871 881 10.1007/s10695-011-9575-z 22089693 Tang L Liu Y Wang Y Long C Phytochemical analysis of an antiviral fraction of Radix astragali using HPLC-DAD-ESI-MS/MS J Nat Med 2010 64 2 182 186 10.1007/s11418-009-0381-1 20037801 Karimi P Khavari-Nejad RA Niknam V Ghahremaninejad F Najafi F The effects of excess copper on antioxidative enzymes, lipid peroxidation, proline, chlorophyll, and concentration of Mn, Fe, and Cu in Astragalus neo-mobayenii Sci World J 2012 2012 615670 Cui R He J Wang B Zhang F Chen G Yin S Shen H Suppressive effect of astragalus membranaceus Bunge on chemical hepatocarcinogenesis in rats Cancer Chemother Pharmacol 2003 51 75 80 10.1007/s00280-002-0532-5 12497209 Kim W Kim SH Park SK Chang MS Astragalus membranaceus ameliorates reproductive toxicity induced by cyclophosphamide in male mice Phytother Res 2012 26 9 1418 1421 10.1002/ptr.4756 22674751 Rittenhouse JR Lui PD Lau BH Chinese medicinal herbs reverse macrophage suppression induced by urological tumors J Urol 1991 146 2 486 490 1856958 Zhao XZ Effects of astragalus membranaceus and tripterygium hypoglancum on natural killer cell activity of peripheral blood mononuclear in systemic lupus erythematosus Zhongguo Zhong Xi Yi Jie He Za Zhi 1992 12 11 669 671 645 1301849 Shiner RJ Schofield E Bates PA Waelkens E Dallman M Lamb J Zicha D Downward J Seckl MJ Pardo OE A siRNA screen identifies RSK1 as a key modulator of lung cancer metastasis Oncogene 2011 30 32 3513 3521 10.1038/onc.2011.61 21423205 Gawecka JE Young-Robbins SS Sulzmaier FJ Caliva MJ Heikkilä MM Matter ML Ramos JW RSK2 protein suppresses integrin activation and fibronectin matrix assembly and promotes cell migration J Biol Chem 2012 287 52 43424 43437 10.1074/jbc.M112.423046 23118220 van Jaarsveld MT Blijdorp IC Boersma AW Pothof J Mathijssen RH Verweij J Wiemer EA The kinase RSK2 modulates the sensitivity of ovarian cancer cells to cisplatin Eur J Cancer 2013 49 2 345 351 10.1016/j.ejca.2012.08.024 23041051 Eisinger-Mathason TS Andrade J Lannigan DA RSK in tumorigenesis: connections to steroid signaling Steroids 2010 75 3 191 202 10.1016/j.steroids.2009.12.010 20045011 Hilinski MK Mrozowski RM Clark DE Lannigan DA Analogs of the RSK inhibitor SL0101: optimization of in vitro biological stability Bioorg Med Chem Lett 2012 22 9 3244 3247 10.1016/j.bmcl.2012.03.033 22464132 Cho YY Lee MH Lee CJ Yao K Lee HS Bode AM Dong Z RSK2 as a key regulator in human skin cancer Carcinogenesis 2012 33 12 2529 2537 10.1093/carcin/bgs271 22918890 Peng C Zhu F Wen W Yao K Li S Zykova T Liu K Li X Ma WY Bode AM Dong Z Tumor necrosis factor receptor-associated factor family protein 2 is a key mediator of the epidermal growth factor-induced ribosomal S6 kinase 2/cAMP-responsive element-binding protein/Fos protein signaling pathway J Biol Chem 2012 287 31 25881 25892 10.1074/jbc.M112.359521 22685297	24152941	PMC3818446	CC BY	2021-01-05 01:30:42	yes	Diagn Pathol. 2013 Oct 24; 8:179
==== Front Case Rep EndocrinolCase Rep EndocrinolCRIM.ENDOCRINOLOGYCase Reports in Endocrinology2090-65012090-651XHindawi Publishing Corporation 10.1155/2013/747898Case ReportA Newborn with Genital Ambiguity, 45,X/46,XY Mosaicism, a Jumping Chromosome Y, and Congenital Adrenal Hyperplasia http://orcid.org/0000-0002-2013-102XZhang Lei 1 Cooley Linda D. 1 Chandratre Sonal R. 2 Ahmed Atif 3 Jacobson Jill D. 2 1Cytogenetics Laboratory, Department of Pathology and Laboratory Medicine, Children's Mercy Hospitals and Clinics, University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USA2Division of Endocrinology and Diabetes, Department of Pediatrics, Children's Mercy Hospitals and Clinics, University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USA3Department of Pathology and Laboratory Medicine, Children's Mercy Hospitals and Clinics, University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USAJill D. Jacobson: jjacobson@cmh.eduAcademic Editors: E. Hershkovitz and R. Swaminathan 2013 22 10 2013 2013 74789823 8 2013 12 9 2013 Copyright © 2013 Lei Zhang et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Disorders of sex development (DSD), formerly termed “intersex” conditions, arise from numerous causes. CAH secondary to 21-hydroxylase deficiency is the most common cause of DSD. Sex chromosome disorders, including sex chromosome mosaicism, are the second most common cause of DSD. We discuss a medically complex neonate with DSD presenting with ambiguous genitalia. Hormone levels suggested 21-hydroxylase deficiency. Molecular analysis revealed compound heterozygous mutations in the 21-hydroxylase gene (CYP21A2), confirming the diagnosis of CAH. Chromosome analysis revealed sex chromosome mosaicism with three cell lines: 45,X[8]/45,X,tas(Y;16)(p11.32;p13.3)[8]/45,X,t(Y;8)(p11.32;p23.3)[4] with the Y chromosome in telomere association with chromosomes 8p and 16p in different cell lines, a “jumping translocation.” Histologically, the right gonad had irregular, distended seminiferous tubules with hyperplastic germ cells contiguous with ovarian stroma and primordial follicles. The left gonad had scant ovarian stroma and embryonic remnants. Chromosome analyses showed mosaicism in both gonads: 45,X[17]/45,X,tas(Y;8)(p11.32;p23.3)[3]. This is the first case of coexisting CAH and 45,X/46,XY mosaicism reported in the English literature and the third case of a constitutional chromosome Y “jumping translocation.” Our report documents the medical and genetic complexity of children such as this one with ambiguous genitalia and discusses the need for a multidisciplinary team approach. ==== Body 1. Introduction Ambiguous genitalia in a newborn represent a medical and family psychological emergency. Ambiguous genitalia, a common presenting feature in most disorders of sex development (DSD), suggest a complex differential diagnosis that must be promptly and thoroughly investigated to determine the etiology. Guidelines for a quick, accurate diagnosis are critical for optimal patient management and gender assignment. The recognition of the complexity in diagnosis, terminology, and management of these conditions prompted the Lawson Wilkins Pediatric Endocrine Society (LWPES) and the European Society for Pediatric Endocrinology (ESPE) to issue a consensus statement on the classification of these disorders. The 2006 document proposed a new terminology nomenclature and classified DSD in three broad categories: (1) sex chromosome DSD (45,X Turner and variants, 47,XXY Klinefelter and variants, 45,X/46,XY mixed gonadal dysgenesis (MGD) and ovotesticular DSD (OT-DSD), 46,XX/46,XY chimeric type, or mosaic type OT-DSD), (2) 46,XY DSD (disorders of testicular development or disorders in androgen synthesis/action), and (3) 46,XX DSD (disorders of ovarian development or fetal androgen excess) [1, 2]. Congenital adrenal hyperplasia (CAH) falls into the 46,XX DSD consensus statement DSD category. CAH comprises a group of autosomal recessive disorders that result from either partial or complete deficiency of specific enzymes in the steroidogenesis pathway. The most common form, 21-hydroxylase deficiency, accounts for 90% to 95% of cases and is caused by mutations within the CYP21A2 gene [3]. CAH leads to virilization and genital ambiguity in females, whereas males exhibit no genital ambiguity. 45,X/46,XY mosaicism is classified within the sex chromosome DSD category. The clinical phenotype of patients with 45,X/46,XY mosaicism is broad, ranging from women, with or without Turner syndrome stigmata, to apparently normal males, with intervening variable ambiguous phenotypes [4]. Gonad histology associated with 45,X/46,XY mosaicism is also variable with partial, complete, mixed, or asymmetric gonadal dysgenesis [5]. “Jumping translocations” (JTs) are rare chromosomal phenomena where the same portion of one donor chromosome translocates to two or more different recipient chromosomes. JTs are mainly described in hematological malignancies and rarely observed in the constitutional karyotype. So far approximately 50 cases of constitutional JTs have been reported with the majority involving at least one acrocentric chromosome [6, 7]. Here we describe a full term neonate with genital ambiguity, CAH resulting from compound heterozygous mutations in the CYP21A2 gene, a mosaic 45,X/46,XY karyotype, a jumping chromosome Y translocation to nonacrocentric chromosomes, and a bilateral mosaic 45,X/46,XY gonadal karyotype exhibiting the same chromosome Y translocation. 2. Case Report The patient was born at 39-week gestation to a 28-year-old G1P1 mother. Father was 27 years old. Pregnancy was complicated by chorioamnionitis. Mother had a history of polycystic ovarian syndrome with no other androgen exposure. The family history was negative for consanguinity or infantile deaths. The birth weight, length, and head circumference were 2830 g (50% ile), 49.5 cm (8% ile), and 33.5 cm (14.5% ile), respectively. BP was normal. Physical examination was normal with the exception of the genitourinary system. The external genitalia were ambiguous. The phallus was 2 cm in length with a blind dimple on the glans and a urethra that opened ventrally at the base, consistent with Prader 4 hypospadias. The clitoral index was 0.8. The anogenital ratio was 0.83 (Figure 1). No gonads were palpable in the labial/scrotal tissue or in either inguinal canal. Initial blood work showed normal electrolytes and thyroid function and undetectable levels of LH and FSH. Peripheral blood (PB) fluorescence in situ hybridization (FISH) analysis using probes for chromosomes X and Y centromeres and the SRY gene found a single X chromosome in 64–74% of nuclei and an XY SRY+ genotype in 26–36% of nuclei. FISH results became available on day of life 3 (DOL 3) (Figure 2(a)). Total testosterone on DOL 5 was 808 ng/dL (normal newborn female 20–64 ng/dL). A care conference with the family to discuss the hormonal and cytogenetic results, which appeared to fully explain the genital ambiguity, was scheduled. However, the state newborn screen returned suspicious for congenital adrenal hyperplasia (CAH). An unstimulated 17-hydroxyprogesterone was 2,800 ng/dL (normal < 90 ng/dL), plasma renin activity was 29.8 ng/mL/hr (normal 2–35 ng/mL/hr), and 11-deoxycorticosterone was slightly elevated at 72 ng/dL (7–49 ng/dL) (as may paradoxically be seen in 21-hydroxylase deficiency). The infant was provisionally diagnosed with coexisting CAH and mixed gonadal dysgenesis (MGD). Hydrocortisone and fludrocortisone were initiated. The total testosterone fell to 89 ng/dL on DOL 10, 4 days after the initiation of glucocorticoids. Conventional G-banded chromosome analysis showed a mosaic karyotype with three cell lines: 8 cells with a 45,X karyotype, 8 cells with a 46,X,tas(Y;16)(pter;pter) karyotype, and 4 cells with a 46,X,tas(Y;8)(pter;pter) karyotype: mos 45,X[8]/45,X,tas(Y;16)(p11.32;p13.3)[8]/45,X,tas(Y;8)(p11.32;p23.3)[4] (Figures 2(b), 2(c) and 2(d)). The Y chromosome was in telomeric association (tas) with chromosome 16 in 8 cells and with chromosome 8 in 4 cells. This unusual chromosome anomaly is the result of a chromosome Y “jumping translocation.” A 46,XY cell line was not found with analysis of additional 44 metaphase cells. Metaphase FISH analysis using a Yqh probe, Xp/Yp subtelomeric probes, and 8p and 16p subtelomeric probes showed all subtelomeric regions present with no loss of chromosome material secondary to the telomeric associations, tas(Y;16) and tas(Y;8) (Figures 3(a), 3(b), 3(c), and 3(d)). Paternal chromosome analysis was normal. Microarray CGH analysis using Baylor 4 × 180 Exon Array v8.1 chip showed sex chromosome mosaicism (arr Xpterqter(pter-qter)x1,Ypterqter(pter-qter)x0-1) with no other clinically relevant copy number variation. CAH gene-targeted mutation analysis for 21-hydroxylase-related CAH revealed the patient to be a compound heterozygote for the following mutations in the CYP21A2 gene: IVS2-13C/A>G and p.R356W, a gene pattern expected to result in CAH with classical symptoms. Echocardiogram and renal ultrasound studies to assess cardiac, aortic, and kidney structure were negative for anomalies associated with the 45,X karyotype. Cystoscopy, vaginoscopy, and laparoscopy on DOL 9 revealed bilateral fallopian tubes, streak gonads, a normal-appearing uterus, vagina, and cervix. The gonads were removed and sent for histological analysis. The right gonad measured 1.5 × 0.3 × 0.2 cm, and the left gonad measured 1.4 × 0.7 × 0.3 cm. The histological examination of the right gonad revealed slightly irregular and distended seminiferous tubules with hyperplastic germ cells contiguous with ovarian stroma exhibiting several primordial follicles. The pathologist interpreted these findings as consistent with ovotestis (Figure 4). The left gonad showed scant ovarian stroma and embryonic remnants focally present with no seminiferous tubules or peritesticular adnexal structures consistent with gonadal dysgenesis. Chromosome analysis of tissues from both gonads showed a mosaic karyotype: mos 45,X[17]/45,X,tas(Y; 8)(p11.32; p23.3)[3]. The tas(Y;16) was not found in gonadal tissues. FISH analysis of the gonads using FFPE tissue showed a single X chromosome in 80% of nuclei and an XY genotype in 20% of nuclei. This mosaic genotype was seen in both the areas with the seminiferous tubules and the areas with the ovarian stroma. A gender assignment care conference was held with the family and our multidisciplinary team. Parents had bonded with the infant as a female, as the second trimester ultrasound had identified the fetus as a girl. The team concurred with the family's wish for female gender assignment. The possibility of gender dysphoria was discussed, and the team recommended strongly against early feminizing or tissue reduction surgery. The possibility of future gender reassignment was also discussed. The patient is followed in both the pediatric endocrinology clinic and the multidisciplinary clinic for disorders of sexual development. She continues on hydrocortisone and fludrocortisone. Her height is at the 3rd percentile at 6 months, which may reflect the 45,X cell line. Growth hormone therapy is anticipated in the near future [8]. The patient and family are reassessed with the help of a psychologist and a social worker at each multidisciplinary clinic visit. 3. Discussion No reports of a patient with coexisting 45,X/46,XY mosaicism and CAH were found in the English language literature. One report in the French literature [9] described a newborn with 45,X/46,XY mosaicism and ambiguous genitalia with a moderate elevation of blood 17-OH progesterone, consistent with CAH secondary to 21-hydroxylase deficiency. One gonad was palpable, and histological examination revealed the presence of a testis and a streak gonad. The authors considered mixed gonadal dysgenesis as the cause of sexual ambiguity in their patient. Both the congenital adrenal hyperplasia and the sex chromosome mosaicism could have contributed to the genital ambiguity in our patient. The initial total testosterone was in the range for a newborn male at 808 ng/dL at 5 days of age. It fell to 89 ng/dL by DOL 10, the day of surgery (which was also 4 days after the initiation of glucocorticoids). A repeat testosterone level two days after surgery was 110 ng/dL. Normally the nadir of testicular testosterone production occurs between 4 and 7 days of life. Thus, it is difficult to determine whether the fall in testosterone was a result of suppression of adrenal androgen in response to hydrocortisone treatment or the normal physiological nadir. The existence of two separate diagnoses, either of which could have contributed to genital ambiguity, complicated gender assignment. Psychosexual follow-up studies of individuals with disorders of sexual development have yielded varied results [10–13]. CAH secondary to 21-hydroxylase deficiency is the best-studied form of DSD with respect to long-term psychological follow-up. Studies have shown a small, but clinically substantial risk for gender dysphoria in patients with CAH. Dessens et al. performed a meta-analysis of the literature on gender identity, gender identity problems, gender dysphoria, and gender change in chromosomal females with congenital adrenal hyperplasia from years 1950 to 2005. The authors showed that gender dysphoria varied depending on the sex of rearing. Of the 250 CAH 46,XX individuals raised as female, 13 (5.2%) reported gender dysphoria, whereas, of 33 raised as male, 4 (12.1%) exhibited gender dysphoria. The authors recommended female gender assignment in 46,XX newborns with CAH even in the presence of significant virilization [14]. Less is known about gender dysphoria in patients with sex chromosome mosaicism, as few studies exist. In a psychosexual follow-up study with sex chromosome mosaicism, 19 young adults were studied. Nine were raised as females and 10 as males. All patients raised as male exhibited male gender identity. Two out of nine women raised as female (22%) did not identify with the female gender [11]. Sex chromosome mosaicism is among the more difficult of all DSDs with respect to gender assignment. By definition, there are variations in the numbers of X and Y chromosomes in different tissues leading to the entire spectrum of male and female phenotypes, hormonal levels, and gender identity in patients with this single diagnosis [4, 5]. Histopathological examination of the gonads from our patient showed ovarian stroma with primordial follicles and seminiferous tubules with germ cells on the right and ovarian stroma without testicular tubules or peritesticular adnexa on the left. The pathognomonic histologic feature of OT-DSD is the presence of seminiferous tubules and ovarian follicles or oocytes, representing testicular and ovarian tissue in the same individual. MGD may feature streak gonads, dysgenetic testes, or asymmetric gonads with streak on one side and dysgenetic testis on the other. The interpretation of a right ovotestis stimulated a query regarding the classification of ovotestis given the pathological findings in this very young child. A study that found a greater density of primordial follicles in the youngest of girls with TS and 45,X karyotypes [15] raises the question of whether the gonad would continue to show follicles and still be consistent with an ovotestis if examined at an older age. In addition to having a mosaic sex chromosome complement, our patient's Y chromosome showed telomeric association with two different chromosomes, such that the child possessed three constitutional cell lines. The attachment of one chromosome to two or more other chromosomes in different cell populations is referred to as a jumping translocation (JT). This type of anomaly may be seen in various types of malignancies, but it is a very unusual constitutional chromosome anomaly. A literature search found two reports of a jumping translocation (JT) involving the Y chromosome. Sawyer et al. described the first chromosome Y JT in an Ullrich-Turner syndrome patient with a mosaic 45,X/46,X,tas(Y;21)(q12;p13) karyotype in tissue samples from skin, peritoneum, fascia, and left and right gonads. Two additional cell lines were seen in the left gonad: 46,X,tas(Y; 21)(q12; p13),−22 and 46,X,tas(Y; 21)(q12; p13),+tas(Y; 14)(q12; p13),−22. The Yqter was in telomere association with chromosome 21 short arm in the skin, peritoneum, fascia, and right gonad and in tas with chromosomes 14 and 21 short arms in the left gonad [16]. Huang et al. described a unique telomeric association involving the Y chromosome that “jumped” during meiosis from chromosome 19 in the father, tas(Y;19)(pter;pter), to chromosome 15 in his son, tas(Y;15)(pter;pter). Father and son were normal phenotypic males [17]. In our patient, the Ypter was in telomeric association with the nonacrocentric chromosomes 8 and 16 in different cell lines. Both gonads showed the tas(Y;8). Paternal chromosome analysis was normal indicating that the telomeric associations were de novo. The genetic content of the cells with 46,X,tas(Y;8) and 45,X,tas(Y;16) in our patient is equivalent to normal 46,XY cells as microarray CGH and subtelomeric FISH analyses showed no evidence for gain or loss of genetic material at the associated telomeres. The exact mechanism that leads to chromosome telomeric association is unknown. It is known, however, that telomeres are very important for maintenance of chromosome stability. Telomeres cap the ends of chromosomes and prevent chromosome fusion [7, 18, 19]. We can only speculate on how the Yp telomere had become attached to the chromosome 8 or 16 telomere. Possibly the association was mediated through telomere recombination to gain stability during meiosis or when the zygote was formed. Chromosomes in telomeric association have two centromeres (dicentric). During mitosis, the configuration of the telomerically associated dicentric chromosome would pose problems to segregation. The chromosomes in association may be “pulled apart” with detachment of the Y chromosome. The detached Y chromosome may be lost with the formation of a 45,X cell line or may reattach to the telomere of a second chromosome, resulting in the formation of a 45,X cell line and either a 46,X,tas(Y;16) or 46,X,tas(Y;8) cell line. In summary, disorders of gonadal development exhibit a wide clinical, cytogenetic, and histopathological spectrum making gender assignment difficult. An experienced multidisciplinary team approach is necessary early in the course of DSDs, as there are many factors that must be taken into account prior to assigning gender. These include gonadal function, phenotype, internal genitalia (i.e., presence of uterus), potential of fertility and sexuality, gonadal histopathology and risk of future gonadal malignancy, and prenatal brain virilization. Our patient with ambiguous genitalia exemplifies the complexity that may be found during the complete endocrine and genetic evaluation and highlights the necessity for a multidisciplinary team in making gender assignment and management decisions. Consent Full written consent has been obtained from the parents of the patient for the publication of this paper. A copy of the written consent is available for review. Conflict of Interests The authors have no conflict of interests and have not received funding or grants for this paper. Acknowledgments The authors would like to thank Wayne V. Moore, M.D., Ph.D., John M. Gatti, M.D., Emily M. McNellis, M.D., Jennifer L. Kussmann, M.S.G.C., and Anna Egan, Ph.D., Clinical Psychologist, for their involvement in the multidisciplinary team care of the patient. Figure 1 Photo of ambiguous genitalia in this patient. Figure 2 Illustration of mosaicism for different cell lines. (a) FISH analysis of interphase nuclei with probes for chromosome X centromere (green) and the SRY gene (red). Partial karyotypes (b, c, d) show (b) chromosomes 8, t(Y;8), 16, and X in 46,X,tas(Y;8) cell line. (c) Partial karyotype shows chromosomes 8, 16, t(Y;16) and X in 46, X,tas(Y;16) cell line. (d) Partial karyotype shows chromosomes 8, 16, and X in 45,X cell line. Figure 3 FISH analysis of tas(Y;8) and tas(Y;16) with subtelomeric probes. (a) FISH with 8pter (green) and 8qter (red) probes shows signal on the junction of tas(Y;8) (arrow) and normal chromosome 8. (b) FISH with XYpter (green) and XYqter (red) probes shows signal on the junction of tas(Y;8) (arrow) and normal chromosome X. (c) FISH with 16pter (green) and 16qter (red) probes shows signal on the junction of tas(Y;16) (arrow) and normal chromosome 16. (d) FISH with XYpter (green) and XYqter (red) probes shows signal on the junction of tas(Y; 16) (arrow) and normal chromosome X. Figure 4 Gonadal histology. (a) Ovarian tissue identified in the right gonad revealed characteristic ovarian stroma and the presence of several scattered primordial follicles (H&E ×100). (b) In addition to the ovary, the right gonad also revealed testicular tissue with numerous seminiferous tubules that appeared dysmorphic (H&E ×200). (c) Immunohistochemistry with inhibin stain highlights the seminiferous tubules that display irregular branching and anastomosis (Inhibin ×200). ==== Refs 1 Hughes IA Houk C Ahmed SF Lee PA Consensus statement on management of intersex disorders Archives of Disease in Childhood 2006 91 7 554 563 2-s2.0-33745773870 16624884 2 Lee PA Houk CP Ahmed SF Hughes IA Consensus statement on management of intersex disorders. International consensus conference on intersex Pediatrics 2006 118 2 488 500 3 Speiser PW White PC Congenital adrenal hyperplasia The New England Journal of Medicine 2003 349 8 776 788 2-s2.0-0042466547 12930931 4 El Moussaif N Haddad NE Iraqi N Gaouzi A 45,X/46,XY mosaicisme: report of five cases and clinical review Annales d’Endocrinologie 2011 72 3 239 243 2-s2.0-79959352699 5 Ocal G Berberoglu M Siklar Z The clinical and genetic heterogeneity of mixed gonadal dysgenesis: does “disorders of sexual development (DSD)” classification based on new Chicago consensus cover all sex chromosome DSD? European Journal of Pediatrics 2012 171 10 1497 1502 22644991 6 Iwarsson E Sahlén S Nordgren A Jumping translocation in a phenotypically normal male: a study of mosaicism in spermatozoa, lymphocytes, and fibroblasts The American Journal of Medical Genetics A 2009 149 8 1706 1711 2-s2.0-68049093005 7 Reddy KS The conundrum of a jumping translocation (JT) in CVS from twins and review of JTs The American Journal of Medical Genetics A 2010 152 11 2924 2936 2-s2.0-78049257955 8 Davenport ML Punyasavatsut N Gunther D Savendahl L Stewart PW Turner syndrome: a pattern of early growth failure Acta Paediatrica 1999 88 433, supplement 118 121 2-s2.0-0033431710 9 Del Pino O Carel JC Barbet JP Morel Y Chaussain JL Association of mixed gonadal dysgenesis and non-classical 21-hydroxylase deficiency Archives de Pediatrie 1996 3 12 1258 1261 2-s2.0-0030332591 9033792 10 Wallien MSC Cohen-Kettenis PT Psychosexual outcome of gender-dysphoric children Journal of the American Academy of Child and Adolescent Psychiatry 2008 47 12 1413 1423 2-s2.0-57749201356 18981931 11 Szarras-Czapnik M Lew-Starowicz Z Zucker KJ A psychosexual follow-up study of patients with mixed or partial gonadal dysgenesis Journal of Pediatric and Adolescent Gynecology 2007 20 6 333 338 2-s2.0-37049013546 18082854 12 Reiner WG Gender identity and sex-of-rearing in children with disorders of sexual differentiation Journal of Pediatric Endocrinology and Metabolism 2005 18 6 549 553 2-s2.0-21444442552 16042322 13 Kojima Y Mizuno K Nakane A Kato T Kohri K Hayashi Y Long-term physical, hormonal, and sexual outcome of males with disorders of sex development Journal of Pediatric Surgery 2009 44 8 1491 1496 2-s2.0-67650941189 19635293 14 Dessens AB Slijper FME Drop SLS Gender dysphoria and gender change in chromosomal females with congenital adrenal hyperplasia Archives of Sexual Behavior 2005 34 4 389 397 2-s2.0-22144443443 16010462 15 Hreinsson JG Otala M Fridström M Follicles are found in the ovaries of adolescent girls with Turner’s syndrome Journal of Clinical Endocrinology and Metabolism 2002 87 8 3618 3623 2-s2.0-18544386684 12161485 16 Sawyer J North P Swanson C Telomeric fusion and chromosome instability in multiple tissues of a patient with mosaic Ullrich-Turner syndrome The American Journal of Medical Genetics 1997 69 4 383 387 9098487 17 Huang B Martin CL Sandlin CJ Wang S Ledbetter DH Mitotic and meiotic instability of a telomere association involving the Y chromosome The American Journal of Medical Genetics 2004 129 2 120 123 2-s2.0-4344636821 18 Hatakeyama S Fujita K Mori H Omine M Ishikawa F Shortened telomeres involved in a case with a jumping translocation at 1q21 Blood 1998 91 5 1514 1519 2-s2.0-0032032299 9473214 19 Saltman D Morgan R Cleary ML de Lange T Telomeric structure in cells with chromosome end associations Chromosoma 1993 102 2 121 128 2-s2.0-0027476057 8432193	24251047	PMC3819822	CC BY	2021-01-05 10:32:45	yes	Case Rep Endocrinol. 2013 Oct 22; 2013:747898
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 24244539PONE-D-13-3207110.1371/journal.pone.0079655Research ArticlemiR-122 Regulates Tumorigenesis in Hepatocellular Carcinoma by Targeting AKT3 miR-122 Targets AKT3 in HCCNassirpour Rounak Mehta Pramod P. Yin Min-Jean * Oncology Research, Pfizer Worldwide Research and Development, San Diego, California, United States of America Cheng Jin Q. Editor H.Lee Moffitt Cancer Center & Research Institute, United States of America * E-mail: min-jean.yin@pfizer.comCompeting Interests: All authors are current full time employees of Pfizer Inc., whose company funded this study. There are no patents, products in development or marked products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: RN MJY. Performed the experiments: RN PPM. Analyzed the data: RN PPM MJY. Contributed reagents/materials/analysis tools: RN PPM. Wrote the paper: RN MJY. 2013 7 11 2013 9 1 2018 8 11 e796555 8 2013 3 10 2013 © 2013 Nassirpour et al2013Nassirpour et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.MicroRNAs (miRNAs) have been implicated in the orchestration of diverse cellular processes including differentiation, proliferation, and apoptosis and are believed to play pivotal roles as oncogenes and tumor suppressors. miR-122, a liver specific miRNA, is significantly down-regulated in most hepatocellular carcinomas (HCCs) but its role in tumorigenesis remains poorly understood. Here we identify AKT3 as a novel and direct target of miR-122. Restoration of miR-122 expression in HCC cell lines decreases AKT3 levels, inhibits cell migration and proliferation, and induces apoptosis. These anti-tumor phenotypes can be rescued by reconstitution of AKT3 expression indicating the essential role of AKT3 in miR-122 mediated HCC transformation. In vivo, restoration of miR-122 completely inhibited xenograft growth of HCC tumor in mice. Our data strongly suggest that miR-122 is a tumor suppressor that targets AKT3 to regulate tumorigenesis in HCCs and a potential therapeutic candidate for liver cancer. The authors have no support or funding to report. ==== Body Introduction MiRNAs are small non-coding RNAs that act as agents of the RNA interference pathway to regulate protein expression by destabilization and/or translational inhibition of target messenger RNAs (mRNAs) [1]. Because miRNAs usually bind to their targets with incomplete complementarity, one single miRNA may have multiple gene targets and numerous effectors within the same functional pathway, producing a coherent physiological response via multiple parallel perturbations [2], [3]. In fact, miRNAs have been implicated in the regulation of a variety of complex biological functions including cellular proliferation, differentiation and apoptosis and are therefore attractive cancer therapeutic targets [4], [5]. Additionally, miRNAs are frequently located at fragile sites and genomic regions susceptible to amplification, deletion, or translocation during tumor development [6] and growing evidence suggests that miRNAs can function as tumor suppressors or oncogenes [4]. Interestingly miRNA expression profiling analyses have revealed characteristic miRNA signatures for various human cancers [7], [8]. Hepatocellular carcinoma (HCC) is the fifth most common human cancer, affecting over 500,000 people each year worldwide with major risk factors such as hepatitis B and C infections, alcohol abuse, xenobiotics, primary billiary cirrhosis, diabetes, non-alcoholic fatty liver disease, and genetic disorders such as hemochromatosis and α1-antitrypsin deficiency [9]. Although recent advances in functional genomics provide an increasingly comprehensive understanding of hepatocarcinoma development [10], [11], the molecular pathogenesis of HCC remains poorly understood and the clinical heterogeneity of HCC combined with lack of sensitive and early diagnostic biomarkers and treatment strategies have led to a high mortality rate for HCC patients. Therefore, research and development for effective targeted therapies are in strong need to combat this aggressive cancer. miR-122 is the most abundant miRNA in the liver, constituting 70% of the total hepatic miRNAs [12], [13]. Not only is miR-122 crucial to normal liver development and function, including fatty acid and cholesterol metabolism, but it also seems to play pivotal roles in various liver diseases such as the Hepatitis C Virus (HCV) replication [14], [15]. Additionally, this liver specific miRNA has been reported to be dramatically down regulated in most HCCs, where it is often inversely associated with poor prognosis and metastasis [16]–[18]. In fact, mice with germline knockout or liver specific knockout of miR-122 develop steatohepatitis, fibrosis and spontaneous tumors resembling HCC [19], [20]. Although Cyclin G1, MDR, ADAM17, and CUTL1 have been proposed as targets of miR-122, the mechanism behind miR-122 regulation of tumorigenesis in HCCs remains poorly understood [18], [21]–[23]. In this study, we identify AKT3 as a novel and direct target of miR-122 in human HCCs. In summary, our data demonstrate that AKT3 expression is inversely correlated to miR-122 levels in HCC cell lines, and that over-expression of miR-122 in a subset of HCC cell lines decreases AKT3 mRNA and protein levels by directly binding to the 3’UTR of AKT3, which subsequently leads to inhibition of cell proliferation and migration. Consequently, we were able to rescue these miR-122 induced anti-tumor activities by reconstituting AKT3 expression. In vivo, over-expression of miR-122 in a HCC cell line, SNU-182, also inhibited xenograft tumor growth in nude mice. Therefore, our data strongly suggest that miR-122 is a tumor suppressor by targeting AKT3 expression to modulate HCC cell transformation, and that over-expression of miR-122 or down-regulation of AKT3 may prove beneficial as therapeutic potentials for HCC patients. Results miR-122 directly binds to the 3’UTR of hs-AKT3 to regulate its expression We first confirmed that miR-122 is exclusively expressed in normal human liver tissue by comparing its expression in other normal tissues (Figure 1A). miR-122 expression in tumor cell lines from other organs was very low (Figure 1B), further confirming that miR-122 is a liver-specific miRNA as reported. Using real-time RT-PCR, we also confirmed that miR-122 expression was significantly down-regulated or completely abolished in a variety of human HCC cell lines including Hep-3B2, SNU-182, SNU-475, as well as a hepatoblastoma cell line Hep-G2. Although the HCV-transformed HCC cell line Huh-7 also showed reduced expression of miR-122, it still maintained a significant level of miR-122 expression, as shown in Figure 1B. Therefore, miR-122 expression is specific to liver and is highly suppressed in the human HCC cell lines tested. 10.1371/journal.pone.0079655.g001Figure 1 miR-122 is down regulated in human hepatocellular carcinoma cell lines. (A) miR-122 level, normalized to RNU-48, was measured in RNA samples collected from normal stomach, lung, colon, and breast tissues. (B) Normalized miR-122 expression in a variety of human cancer cell lines. Next we searched bioinformatic prediction algorithms such as miRanda, TargetMiner, DIANA-MicroT, UPennrna22, and miRDB for predicted targets of this miRNA. AKT3 was identified as one of the candidate targets for hsa-miR-122-5p. Using a different bioinformatic algorithm, Tsai and colleagues also had previously listed AKT3 as a potential target of miR-122, although they did not explore this interaction [18]. Since AKT is a key regulator in many cancers, we decided to investigate the sequence alignments between AKT3 3’UTR further, and found that in three species, the human miR-122 in fact shows partial complementarity (Figure 2A). We then amplified the human AKT3 3’UTR by PCR and sub-cloned it into a luciferase reporter vector as illustrated in Figure 2B. This construct was used for co-transfection with miR-122 construct in SNU182 (cells lacking endogenous miR-122 expression) and Huh7 (cells harboring some endogenous miR122 expression) cell lines. A luciferase assay was then used in determining whether miR-122 can bind to the 3’UTR of AKT3. Results demonstrate that miR-122 expression remarkably decreased the firefly luciferase activity in SNU-182 cells indicating miR-122 binding to 3’UTR (Figure 2C). As expected, we observed a lower basal firefly luciferase activity in Huh-7 relative to SNU-182, due to the endogenous expression of miR-122 in Huh-7 cells, as indicated in Figure 1B. Transfection of miR-122 into Huh-7 cells did however decrease the luciferase activity, albeit to a lower degree (Figure 2C). Since the 3’UTR of the other 2 members of the mammalian AKT family, AKT1 and AKT2, lack any predicted binding sites for miR-122, we focused our binding luciferase assays on the AKT3-isoform. Therefore, the luciferase reporter assay confirms direct binding of miR-122 to hsa-AKT3 3’UTR. 10.1371/journal.pone.0079655.g002Figure 2 miR-122 directly binds to the 3’UTR of hsa-AKT3. (A) Sequence alignments of miR-122 with 3’UTR of AKT3 from 3 mammalian species shows partial complementarity. (B) Schematic representation describing the 3’UTR luciferase reporter assay. The assay was carried out simultaneously in SNU-182 and Huh-7 cells, over-expressing miR-122 GFP or the GFP vector alone, as well as parental cells co-transfected with the pGL3-3’UTR construct containing AKT3 3’UTR. Luciferase assays were performed 48 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity to account for variations in transfection efficiency. Firefly luciferase activity will be reduced if there is a direct binding between miR-122 and the 3’UTR of AKT3 sequence inserted in the vector. (C) Luciferase activity was measured in SNU-182 and Huh-7 parental, miR-122-GFP and GFP over-expressing cells transfected with the luciferase reporter 3’UTR construct or vector alone. Results represent at least three different independent experiments, and statistical significance between indicated groups is depicted as P<0.01, * P<0.005. AKT3 expression is inversely correlated to miR-122 levels in HBV transformed HCC cell lines We next examined the expression levels of miR-122 and AKT3 in the normal human liver and human HCC cell lines. As shown in Figure 1 and Figure 3A, miR-122 expression is greatly reduced in the HCC cell lines compared to that in normal liver. Concurrently, AKT3 expression level is up-regulated in all three HCC cell lines (Hep3B2, SNU-182 and SNU-475) with little or no expression of miR-122 (Figure 3A). Interestingly, in the hepatoblastoma HepG2 cells and the HCV-transformed Huh-7 lines, AKT3 is not over-expressed in comparison to normal liver tissue (Figure 3A). The Huh-7 AKT3 levels are not surprising considering the endogenous expression of miR-122 in these cells. As expected due to a lack of miR-122 binding site, although highly homologous, AKT1 and AKT2 mRNA levels only showed slight increases in the HCC cell lines in comparison to normal liver (Figure 3B). Similar to the observations made for AKT3 transcript levels, AKT3 protein levels were also significantly higher in SNU-182 and SNU-475 HCC cell lines (Figure 3C). These results indicate that miR-122 level is inversely correlated to the AKT3 mRNA and protein levels in the HCC cell lines. 10.1371/journal.pone.0079655.g003Figure 3 AKT3 expression is inversely correlated to miR-122 levels in HCC cell lines. (A) The AKT3 transcript level normalized to its expression in normal liver (right Y axis) and normalized miR-122 expression (left Y axis) were measured in various HCC cell lines. (B) The relative expression level of closely homologous isoforms AKT1 and AKT2 were measured in HCC cell lines. (C) Western blot analysis of total AKT and AKT3 protein levels in various HCC cell lines. Actin was used as the loading control in these studies. Over-expression of miR-122 in HCC down-regulates AKT3 We next examined the effects of miR-122 over expression in human HCC cell lines, SNU-182, SNU-475, Hep3B2, and Huh-7. miR-122 was sub-cloned in a lentiviral expression vector and was successfully over expressed in these cell lines (Figure 4A). As expected, over-expression of miR-122 decreased both the mRNA and protein levels of AKT3 in SNU-182 cells as shown in Figure 4A. Similar data was collected from the SNU-475, and Hep3B2 (data not shown). In Huh-7 cells, which express some endogenous miR-122, over-expression of miR122 also decreased AKT3 protein levels but this change was only visible on the immunoblot with long exposure time due to the low endogenous AKT3 levels in this cell line (Figure 4A). To confirm specificity, we also examined alterations in the other 2 AKT family members in these miR122 transduced cells. Over-expression of miR-122 in SNU-182 and Huh-7 did not significantly alter the AKT1 or AKT2 expression, as shown in Figure 4B, again suggesting that miR-122 specifically targets AKT3. Therefore, these results support the hypothesis that miR-122 negatively regulates AKT3 translation in HCC cell lines. 10.1371/journal.pone.0079655.g004Figure 4 Restoring miR-122 expression decreased AKT3 translation. (A) AKT3 mRNA and protein levels were measured in SNU-182 and Huh-7 cells stably over-expressing miR-122-GFP or GFP alone. The membrane blot of the Huh-7 cells required unusually long exposures before the AKT3 bands could be visualized. (B) AKT1 and AKT2 transcript levels were measured in SNU-182 and Huh-7 cells stably over-expressing miR-122-GFP or GFP alone. Over-expression of miR-122 inhibits cell migration and induces apoptosis AKT kinases regulate diverse cellular processes including cell proliferation and survival, cell size and response to nutrient availability, as well as tissue invasion and angiogenesis in both normal and tumor cells [24]. Since AKT3 is highly expressed in SNU-182 and SNU-475 cells (Figure 3), it is likely that AKT3 plays an essential role in the tumorigenesis of these cell lines. Therefore, we next investigated whether inhibition of AKT3 by restoring miR-122 expression would have anti-tumor effects in SNU-182 and SNU-475 in comparison to a HCC cell line (Huh-7) with endogenous miR122 expression. SNU-182, SNU-475, and Huh-7 cells are able to migrate across the polycarbonate membrane upon HGF-1 stimulation, a well established characteristic of highly transformed HCC cells. Over-expression of miR-122 decreased the HGF-induced cell migration in the HBV-transformed SNU-182 and SNU-475 but not in the HCV-transformed Huh-7 cells (Figure 5A) indicating the critical role of AKT3 in regulating migration in SUN-182 and SNU-475 cells. Since Huh7 already expresses miR-122, over-expression of this miRNA did not alter cell migration in these cells. To confirm that miR-122 induced inhibition in cell migration is due to the decreased level of AKT3 in SNU-182 and SNU-475 cells, we performed a rescue experiment by transiently transfecting a vector encoding the human AKT3 cDNA in the SNU-182 cells, which stably expressed GFP or miR-122-GFP. Results shown in Figure 5B clearly indicate that transient over expression of AKT3 in miR-122-GFP expressing SNU-182 cells rescues the migratory inhibition described above by approximately 70%. Taken together, the migration assays suggest that miR-122 over-expression in SNU-182 cells down regulates AKT3, which in turn inhibits the HGF-induced cell migration in these cells. Furthermore, these miR-122 inhibited migratory responses were rescued by partial restoration of AKT3 expression. Therefore, miR-122 regulation of AKT3 expression is necessary and sufficient in modulating HCC cellular migration in HBV-transformed cells. 10.1371/journal.pone.0079655.g005Figure 5 Restoring miR-122 expression in HBV-transformed HCC cell lines inhibited cell migration and induced apoptosis. These miR122 induced anti-tumor activities were rescued by ectopic expression of AKT3. (A) Cell migration assays were performed on SNU-182 or Huh-7 cells over-expressing miR-122 GFP or GFP alone. Migratory responses to the bottom chamber with and without addition of stimulator (10% HGF) are shown. (B) Cell migration assays were performed using miR-122-GFP or GFP alone over-expressing SNU-182 cells with or without AKT3 reconstitution. (C) Phosphorylation of BAD, total BAD level and cleaved caspase 3 were measured in SNU-182 and Huh-7 cells over-expressing miR-122-GFP or GFP alone. (D) Transient reconstitution effects of AKT3 in SNU-182 cells over-expressing miR-122-GFP or GFP alone were also measured. Statistical significance between the indicated groups is depicted as * P<0.005. AKT family members have also been shown to regulate the apoptotic pathways mainly by a phosphorylation dependent inhibition of the pro-apoptotic Bcl-2 family member, BAD, to promote cell survival [25]. We had noticed that the HCC cells transduced with miR-122 showed slower growth rates in culture relative to their parental cell lines. Therefore, we next studied the effects of miR-122 over-expression on apoptosis/proliferation. SNU-182 cells over-expressing miR-122 exhibited decreased phosphorylation of BAD, in addition to an increase in total BAD levels in comparison to the parental cells and Huh-7 cells over-expressing miR-122 (Figure 5C). Furthermore, HBV-transformed cell lines SNU-182 and Hep-3B2 (data not shown) cells over-expressing miR-122 showed elevations of cleaved caspase 3 levels, another pro-apoptotic protein marker (Figure 5C). These data indicate that restoration of miR-122 in HCC cell lines mediates phosphorylation and up-regulation of BAD to promote apoptosis in SNU-182 cells but not in Huh-7 cells, which endogenously express miR-122. To further confirm that the decreased pBAD and increased cleaved caspase 3 in miR-122 over-expressing SNU-182 cells is due to AKT3 down regulation, AKT3 rescue experiments were performed and data showed that ectopic transient expression of AKT3 is able to partially rescue the effects of miR-122 over-expression in SNU-182 cells (Figure 5D). These data taken together strongly suggest that miR-122 over-expression in SNU-182 cells decreases cell migration and increases apoptosis through its direct regulation of AKT3 translation. Over-expression of miR-122 induces in-vitro as well as in-vivo anti-tumor activities in aggressive HCC cell line, SNU-182 cells After establishing the modulation of the apoptotic pathways by miR-122 in HCC cell lines, we next explored the effects of miR-122 on cell proliferation. As expected and in agreement with our apoptotic assays, over expression of miR-122 dramatically slowed down cell proliferation in SNU-182 but not in Huh-7 cells (Figure 6A and 6C). The inhibition of cell proliferation was rescued by ectopic expression of AKT3 in miR-122 harboring SNU-182 cells (Figure 6B). The lack of regulation observed in Huh-7 cells with miR-122 over-expression could again be contributed to the maintained endogenous miR-122 expression in these cells indicating that increasing miR-122 expression in these cells is not sufficient to alter their tumorigenic abilities. We finally investigated the effects of miR-122 over-expression on in-vivo tumor growth. SNU-182 cells stably over-expressing miR-122-GFP were established and subcutaneously implanted in nude mice, and tumor growth was monitored over time (SNU182 cells stably expressing GFP alone as well parental cell lines were used as control). Figure 6D shows a dramatic reduction in tumor growth in miR-122 over-expressing SNU-182 xenograft models. Therefore, over-expression of miR-122 in the highly transformed SNU-182 HCC cell line induced in-vitro and in-vivo anti-tumor activity classifying miR-122 as a HCC tumor suppressor. 10.1371/journal.pone.0079655.g006Figure 6 miR-122 over-expression inhibited in-vitro cell proliferation and in-vivo tumor growth in a highly transformed HCC SNU-182 xenograft mouse model. Cell proliferation was measured in (A) SNU-182 and (C) Huh-7 parental cells and cells stably over-expressing miR-122-GFP or GFP alone. (B) Cell proliferation was measured in SNU-182 cells over-expressing miR-122-GFP or GFP with or without the reconstitution of AKT3 expression. (D) Nude mice were implanted with SNU-182 parental lines as well as cells over-expressing miR-122-GFP or GFP vector alone, and tumor growth was monitored and plotted as tumor volume (mm3) over time. Statistical significance between the indicated groups are depicted as P<0.01, and * P<0.005. Discussion miR-122 has previously been shown to be dramatically down regulated in most HCCs and is generally indicative of poor prognosis and higher risk of metastasis [16]–[18]. This study investigates the role of miR-122 in the tumorigenesis of HCCs. Here we show that miR-122 functions as a tumor suppressor in the HBV-transformed HCC human cell lines and report AKT3 as a novel and direct target of miR-122. Importantly, restoring miR-122 expression suppresses HCC cell migration and in vivo tumor growth and induces apoptosis by its direct and specific regulation of AKT3. Although several targets have been reported for miR-122 to date [18], [21]–[23], none can fully account for the wide range of cellular transformation and tumorigenic characteristics observed in the miR-122 down regulated HCCs. AKT, also known as Protein Kinase B (PKB), is a serine/threonine kinase that plays a key role in multiple cellular transformation processes such as apoptosis, cell proliferation, and cell migration. In this study, we demonstrate that miR-122 directly targets AKT3 to regulate the cellular transformations and tumorigenesis in non-HCV transformed human HCC cell lines (Figure 2). Furthermore, restoring miR-122 expression in these cell lines not only induced apoptosis and inhibited migration, but also dramatically suppressed tumorigenesis. Since the phenotypes induced by miR-122 over expression were rescued by a transient expression of ectopic AKT3, we propose that miR-122 regulation of AKT3 expression is necessary and sufficient for modulating tumorigenesis and cellular transformation in human HCC cell lines. The AKT family is comprised of three closely related isoforms: AKT1, AKT2, and AKT3, which have a highly conserved domain structure and presumably play similar roles in cell proliferation, survival, metabolism, and many other cellular functions [26]. However, there are functional distinctions among the AKT isoforms in mediating tumor development and progression which seems to be orchestrated in a tissue specific manner [27], [28]. Gene knockout and siRNA studies on AKT1 and AKT2 have revealed isoform-specific functions of AKT family members in their regulation of cell migration, which generally correlate with tumor invasiveness and metastasis. Interestingly, very little is known regarding the role of AKT3 in cell migration [27]. Additionally, although all three AKT isoforms are able to transform cells in-vitro [29], amplification or gene mutation of AKT3 have not been reported in human cancers [26]. A growing number of recent publications suggest that HBV enhances the expression of the mTOR and PI3K/Akt pathways (e.g. Wang et al. 2013). In this study, we show that the HBV transformed cells show both significantly decreased miR-122 expression as well as an enhanced expression of AKT3 which seems to regulate tumorigenesis in this subclass of highly aggressive and transformed HCCs. Our data clearly demonstrate that targeting and specific down-regulation of AKT3 by miR-122 over expression (as shown in Figure 4B) was able to block migration and this inhibition was rescued by reconstitution of AKT3 expression. Interestingly, sustained AKT1 and AKT2 expression in SNU-182 cells was not sufficient in maintaining cell migration in miR-122 over-expressing cells, suggesting that AKT3, but not AKT1 or AKT2, is necessary and sufficient in regulating migration and metastasis in some HCCs. AKT family members are also established regulators of apoptosis. They can promote growth factor-mediated cell survival both directly and indirectly by phosphorylating a variety of apoptotic substrates, such as Bad, a pro-apoptotic Bcl-2 family member [25]. AKTs have been shown to phosphorylate Bad, which in turn inhibits its pro-apoptotic functions to promote cellular proliferation and decreases caspase activity. Not surprisingly, down regulation of AKT3 by miR-122 over-expression decreased Bad phosphorylation, increased total Bad accumulation, elevated cleaved caspase 3 levels, and induced programmed cell death. This miR-122 induced apoptosis was rescued by partially restoring AKT3 expression, indicating that AKT3 is not only essential in regulating cellular migration, but also plays pivotal roles in apoptosis and proliferation. Therefore, restoration of miR-122 can induce anti-tumor activities through specific targeting of AKT3, suggesting that miR-122 can function as a tumor suppressor in HCCs which harbor diminished miR-122 expression. The tumor suppressor functions of miR-122 restoration were observed in all three HBV transformed cell lines tested (Hep3B2, SNU-182, and SNU-475). Interestingly, in the hepatoblastoma cells tested (HepG2), AKT3 is not expressed even though miR-122 is highly down regulated. Interestingly, this cell line is not as aggressive or tumorigenic as the HCC transformed cell lines. This suggests that this cell lines likely lacks the mechanism necessary for tumorigenesis and down regulation of miR-122 is not sufficient to induce that change. Even though miR-122 and AKT3 expression are inversely correlated in the HBV-transformed cell lines tested, whether this correlation is specifically related to the HBV-transformation needs to be investigated in more detail. In conclusion, we have shown that miR-122 directly and specifically binds to the 3’UTR of human AKT3, and over-expression of miR-122 in HBV-transformed HCC cell lines is able to decrease AKT3, at both the transcript and protein level, to block cell migration, induce apoptosis, and inhibit cell proliferation and tumor growth in mice. Ectopically expressed AKT3 is able to rescue these anti-tumor characteristics induced by miR-122 over-expression indicating that the regulation of tumorigenesis by miR-122 is mediated through targeting AKT3 in these HCCs. Materials and Methods Cell Culture All cell lines (except for Huh-7 which was acquired from Japan’s Health Science Research Resource Bank) were procured from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with L-glutamine and grown in a humidified incubator with 5% CO2 at 37°C. Stable cell line generation We used the System Biosystem’s LentimiR™ vectors, which consist of the native stem loop structure, for stable expression of our miRNAs. The lentiviral expression vector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the specific mature miRNAs. For production of a high titer of viral particles, we used the ViraPower™ Lentiviral Support Kits (Invitrogen) together with Lipofectamine™ 2000 (Invitrogen) for tranfecting the vectors into HEK293T cells. Because infected cells stably express copGFP, we used FACS sorting to select for the infected cells harboring the miRNA of interest. RT-PCR TaqMan miRNA assays (Life Technologies, CA) were used to quantify the expression levels of mature miR-122 as well mRNAs for AKT1, 2, 3. Total RNA extracted by miRvana (life technologies) was reverse transcribed in reaction mixture containing miR-specific stem-loop RT primers. Quantitative real time polymerase chain reaction (qPCR) was performed in triplicate reactions containing the prepared cDNA and TaqMan specific primers in Universal Master Mix without AmpErase UNG (Applied Biosystems). The qPCR was conducted at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds in a 7900 Real Time PCR machine (Applied Biosystem) and threshold cycles (C T) were calculated using Sequence Detection Software (SDS v2.2.1, Applied Biosystem). All mRNA quantification data were normalized to 18S RNA. All miRNA data are expressed relative to a RNU48 small nuclear (sn) RNA TaqMan PCR performed on the same samples, unless otherwise specified. Fold expression was calculated from the mean C T values using the 2−ΔΔCt method. Immunoblotting Cells were lysed in buffer containing 50 mM NaCl, 1.5 mM MgCl2, 50 mM HEPES, 10% glycerol, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40 supplemented with 1 mM Na3VO4, 1 mM PMSF, 1 mM NaF, 1 mM β-glycerophosphate. Protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche) was added prior to use. Protein concentration was determined using the BCA Protein Assay (Pierce/Thermo Fisher Scientific) following the manufacturer’s instructions. Protein (30–50 µg) was resolved by SDS-PAGE and transferred onto nitrocellulose membrane. Blots were probed with primary antibodies to detect proteins of interest. After incubation with secondary antibodies, membranes were visualized by chemiluminescence (Pierce/Thermo Fisher Scientific). All antibodies were from Cell Signaling Technology, Inc with the exception of Actin and AKT2 (Santa Cruz Biotechnology). Cell proliferation, apoptosis, and migration assays Resazurin Fluorescent Assay was used for the proliferation assays. Briefly, Cells are seeded at 3000–5,000 cells/100 ul/well in DMEM +10% FBS in a 96 well plate, and were incubated overnight at 37°C in 5% CO2. Resazurin (Sigma) fluorescent dye was added (1∶100) to each well. The cells were incubated at 37°C in 5% CO2 for 4 hours at which point the plate was read for fluorescence at 530/590 nm on the HTS 7000 plate reader. Cell Signaling Technologies PathScan® Apoptosis Multi-Target Sandwich ELISA Kits were used in the apoptosis assays. Briefly, antibodies for cleaved caspase 3 and phosphorylated BAD had been coated onto microwells by the manufacturer. After incubation with the cell lysates, the target protein was captured by the coated antibodies. Following extensive washing, a detection antibody was added to detect the captured target protein. An HRP-linked secondary antibody was then used to recognize the bound detection antibody. HRP substrate, TMB, was finally added for color development which is proportional to the quantity of bound target protein. Cell Biolab’s CytoSelect™ Cell Migration Assay Kit containing polycarbonate membrane inserts (8 µm pore size) in a 24-well plate was used in our migration assays. Migratory cells are able to extend protrusions towards HGF (Hepatic Growth Factor), and pass through the pores of the polycarbonate membrane. Non-migratory cells are removed from the top of the membrane and the migratory cells are stained and quantified. Transfection of DNA constructs and miR-122 mimics The entire 3′UTR of the hsa-AKT3 gene was amplified from a human cDNA clone obtained from Origene using the following primers incorporating the NheI and SalI restriction sites: AKT3 3′UTR forward, CGGCTAGCCGCGTCTCTTTCATTCTGCTACTTCACTGTC; AKT3 3′UTR reverse, CCGGTCGACCGCTTCACTCAGGTAGAAATATGAAAAAGAAGG . The AKT3 3′UTR amplicon was ligated into the pmirGLO Dual-Luciferase miRNA target expression vector (Promega) between the NheI and SalI restriction sites. Transfection of DNA constructs into cell lines was performed using Lipofectamin 2000 reagent (Life Technologies) according to the manufacturer's protocols. The double-stranded RNA that mimics endogenous human miR-122a, and a scrambled miRNA used as a non-targeting control, were obtained from Dharmacon. The introduction of miRNA mimics was accomplished by lipofection using Lipofectamine (Life Technologies), with a 30 nM miRNA mimic concentration per condition. Luciferase assay Three days after transfection with appropriate constructs, the cells were lysed and processed for luciferase luminescence measurements. For detection of luciferase activity the Dual-Glo luciferase assay system (Promega) was used as described by the manufacturer. Briefly, an appropriate amount of Dual-Glo reagent was added to the cell medium enabling cell lysis and subsequent detection of firefly luminescence in a luminometer. Normalization of the samples were performed by addition of the Dual-Glo Stop & Glo reagent enabling the detection of renilla luminescence (measured to normalize data for transfection efficiency variability), and the luciferase activity in relative light units (RLU) was subsequently calculated. Statistics Quantitative data are presented as the mean ± SD. Student's t test was used to determine significant differences between two groups. One-way ANOVA with Bonferroni's multiple comparison test was used to analyze significant differences among multiple groups; p≤0.05 was considered significant unless otherwise stated. Animal studies Six to eight-week-old nu/nu athymic female mice were obtained from Jackson Labs; the mice were maintained in pressurized ventilated caging at the Pfizer La Jolla animal facility. All studies were done under the approval of Pfizer Institutional Animal Care and Use Committee’s guidelines. Tumors were established by injecting 5×106 cells suspended 1:1 (v/v) with reconstituted basement membrane (Matrigel, BD Biosciences). Tumor dimensions were measured with vernier calipers, and tumor volumes were calculated using this formula: π/6 x (larger diameter) x (smaller diameter)2. Tumor growth inhibition percentage (TGI %) was calculated as 100 x (1-▵T/▵C). One way ANOVA Statistical analysis were performed and noted as * for p value is less than 0.001. The authors would like to thank Tod Smeal, Timothy S. Fisher, and Lars Engstrom for their help and support. ==== Refs References 1 Bartel DP (2004 ) MicroRNAs: genomics, biogenesis, mechanism, and function . Cell 116 : 281 –297 .14744438 2 Lim LP , Lau NC , Garrett-Engele P , Grimson A , Schelter JM , et al (2005 ) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs . Nature 433 : 769 –773 .15685193 3 Lewis BP , Shih IH , Jones-Rhoades MW , Bartel DP , Burge CB (2003 ) Prediction of mammalian microRNA targets . Cell 115 : 787 –798 .14697198 4 Esquela-Kerscher A , Slack FJ (2006 ) Oncomirs - microRNAs with a role in cancer . Nat Rev Cancer 6 : 259 –269 .16557279 5 Ambros V (2004 ) The functions of animal microRNAs . Nature 431 : 350 –355 .15372042 6 Calin GA , Sevignani C , Dumitru CD , Hyslop T , Noch E , et al (2004 ) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers . Proc Natl Acad Sci U S A 101 : 2999 –3004 .14973191 7 Calin GA , Croce CM (2006 ) MicroRNA signatures in human cancers . Nat Rev Cancer 6 : 857 –866 .17060945 8 Lu J , Getz G , Miska EA , Alvarez-Saavedra E , Lamb J , et al (2005 ) MicroRNA expression profiles classify human cancers . Nature 435 : 834 –838 .15944708 9 El-Serag HB , Rudolph KL (2007 ) Hepatocellular carcinoma: epidemiology and molecular carcinogenesis . Gastroenterology 132 : 2557 –2576 .17570226 10 Thorgeirsson SS , Lee JS , Grisham JW (2006 ) Functional genomics of hepatocellular carcinoma . Hepatology 43 : S145 –150 .16447291 11 Varnholt H , Drebber U , Schulze F , Wedemeyer I , Schirmacher P , et al (2008 ) MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma . Hepatology 47 : 1223 –1232 .18307259 12 Chang J , Guo JT , Jiang D , Guo H , Taylor JM , et al (2008 ) Liver-specific microRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells . J Virol 82 : 8215 –8223 .18550664 13 Lagos-Quintana M , Rauhut R , Yalcin A , Meyer J , Lendeckel W , et al (2002 ) Identification of tissue-specific microRNAs from mouse . Curr Biol 12 : 735 –739 .12007417 14 Esau C , Davis S , Murray SF , Yu XX , Pandey SK , et al (2006 ) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting . Cell Metab 3 : 87 –98 .16459310 15 Jopling CL , Yi M , Lancaster AM , Lemon SM , Sarnow P (2005 ) Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA . Science 309 : 1577 –1581 .16141076 16 Bai S , Nasser MW , Wang B , Hsu SH , Datta J , et al (2009 ) MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib . J Biol Chem 284 : 32015 –32027 .19726678 17 Coulouarn C , Factor VM , Andersen JB , Durkin ME , Thorgeirsson SS (2009 ) Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties . Oncogene 28 : 3526 –3536 .19617899 18 Tsai WC , Hsu PW , Lai TC , Chau GY , Lin CW , et al (2009 ) MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma . Hepatology 49 : 1571 –1582 .19296470 19 Hsu SH , Wang B , Kota J , Yu J , Costinean S , et al (2012 ) Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver . J Clin Invest 122 : 2871 –2883 .22820288 20 Tsai WC , Hsu SD , Hsu CS , Lai TC , Chen SJ , et al (2012 ) MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis . J Clin Invest 122 : 2884 –2897 .22820290 21 Gramantieri L , Ferracin M , Fornari F , Veronese A , Sabbioni S , et al (2007 ) Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma . Cancer Res 67 : 6092 –6099 .17616664 22 Xu Y , Xia F , Ma L , Shan J , Shen J , et al (2011 ) MicroRNA-122 sensitizes HCC cancer cells to adriamycin and vincristine through modulating expression of MDR and inducing cell cycle arrest . Cancer Lett 310 : 160 –169 .21802841 23 Xu H , He JH , Xiao ZD , Zhang QQ , Chen YQ , et al (2010 ) Liver-enriched transcription factors regulate microRNA-122 that targets CUTL1 during liver development . Hepatology 52 : 1431 –1442 .20842632 24 Altomare DA , Testa JR (2005 ) Perturbations of the AKT signaling pathway in human cancer . Oncogene 24 : 7455 –7464 .16288292 25 Datta SR , Dudek H , Tao X , Masters S , Fu H , et al (1997 ) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery . Cell 91 : 231 –241 .9346240 26 Hers I , Vincent EE , Tavare JM (2011 ) Akt signalling in health and disease . Cell Signal 23 : 1515 –1527 .21620960 27 Stambolic V , Woodgett JR (2006 ) Functional distinctions of protein kinase B/Akt isoforms defined by their influence on cell migration . Trends Cell Biol 16 : 461 –466 .16870447 28 Dillon RL , Muller WJ (2010 ) Distinct biological roles for the akt family in mammary tumor progression . Cancer Res 70 : 4260 –4264 .20424120 29 Bellacosa A , Testa JR , Staal SP , Tsichlis PN (1991 ) A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region . Science 254 : 274 –277 .1833819	24244539	PMC3820664	CC BY	2021-01-05 17:47:15	yes	PLoS One. 2013 Nov 7; 8(11):e79655
==== Front PLoS GenetPLoS GenetplosplosgenPLoS Genetics1553-73901553-7404Public Library of Science San Francisco, USA 24244175PGENETICS-D-13-0150010.1371/journal.pgen.1003791Research ArticleRNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells Regulation of LINE1 Mobility in mESCsCiaudo Constance 1 2 ¤a Jay Florence 1 3 Okamoto Ikuhiro 2 ¤b Chen Chong-Jian 2 4 Sarazin Alexis 1 Servant Nicolas 4 5 6 Barillot Emmanuel 4 5 6 Heard Edith 2 Voinnet Olivier 1 3 * 1 Swiss Federal Institute of Technology Zurich, Department of Biology, Chair of RNA biology, Zurich, Switzerland2 Institut Curie, CNRS UMR3215, Paris, France3 Life Science Zurich Graduate School, Plant Sciences program, University of Zurich, Zurich, Switzerland4 Institut Curie, Paris, France5 INSERM U900, Paris, France6 Mines ParisTech, Fontainebleau, FranceReik Wolf EditorThe Babraham Institute, United Kingdom* E-mail: voinneto@ethz.chThe authors have declared that no competing interests exist. Conceived and designed the experiments: CC OV. Performed the experiments: CC FJ IO. Analyzed the data: CC FJ CJC AS NS OV. Contributed reagents/materials/analysis tools: EH EB. Wrote the paper: CC OV. ¤a Current address: Swiss Federal Institute of Technology Zurich, Department of Biology, Chair of RNAi and Genome Integrity, Zurich, Switzerland. ¤b Current address: Kyoto University, Kyoto, Japan. 11 2013 7 11 2013 9 11 e10037913 6 2013 29 7 2013 © 2013 Ciaudo et al2013Ciaudo et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Retrotransposon Silencing During Embryogenesis: Dicer Cuts in LINE In most mouse tissues, long-interspersed elements-1 (L1s) are silenced via methylation of their 5′-untranslated regions (5′-UTR). A gradual loss-of-methylation in pre-implantation embryos coincides with L1 retrotransposition in blastocysts, generating potentially harmful mutations. Here, we show that Dicer- and Ago2-dependent RNAi restricts L1 accumulation and retrotransposition in undifferentiated mouse embryonic stem cells (mESCs), derived from blastocysts. RNAi correlates with production of Dicer-dependent 22-nt small RNAs mapping to overlapping sense/antisense transcripts produced from the L1 5′-UTR. However, RNA-surveillance pathways simultaneously degrade these transcripts and, consequently, confound the anti-L1 RNAi response. In Dicer−/− mESC complementation experiments involving ectopic Dicer expression, L1 silencing was rescued in cells in which microRNAs remained strongly depleted. Furthermore, these cells proliferated and differentiated normally, unlike their non-complemented counterparts. These results shed new light on L1 biology, uncover defensive, in addition to regulatory roles for RNAi, and raise questions on the differentiation defects of Dicer−/− mESCs. Author Summary A basal network of gene regulation orchestrates the processes ensuring maintenance of genome integrity. Eukaryotic small RNAs generated by the RNAse-III Dicer have emerged as central players in this network, by mediating gene silencing at the transcriptional or post-transcriptional level via RNA interference (RNAi). To gain insight into their potential developmental functions in mammals, we have characterized small RNA expression profiles during mouse Embryonic Stem Cell (mESCs) differentiation, a model for early mammalian development. Long interspersed elements 1 (L1) are non-long-terminal-repeat retrotransposons that dominate the mouse genomic landscape, and are expressed in germ cells or during early development and mESCs. Based on clear precedents in plants and fission yeast, we investigated a role for RNAi and other RNA-based pathways in the regulation of L1 transcription and mobilization. Our work uncovered the existence of small (s)RNAs that map to active L1 elements. Some have characteristics of cognate siRNA produced by Dicer, while others display strand biases and length heterogeneity that evoke their biogenesis through RNA surveillance pathways, in a Dicer-independent manner. Furthermore, genetic ablation of DICER or of ARGONAUTE proteins has complex and profound consequences on L1 transcription and mobilization, indicating that endogenous RNAi do indeed maintain genomic integrity against L1 proliferation. This work was supported by the ANR grant “RNA ES”; to both EH and OV. OV's laboratory was further supported by an ERC Young Investigator Award (Grant ERC-210890 “Frontiers of RNAi”) and Prix Liliane Bettencourt for Life Sciences. EH is a member of the European Network of excellence “Epigenesis”. CC was supported by a post-doctoral fellowship from the Federation of European Biochemical Societies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Long-interspersed elements-1 (LINE-1 or L1) belong to the most abundant class of autonomous transposable elements (TEs) in mammalian genomes. While most L1s are truncated and unable to transcribe or retrotranspose, a fraction of young, full-length L1s are capable of mobilization [1]. Active and inactive L1s influence the evolution of mammalian genomes, yet L1 insertions are also linked to disease [1], raising the issue of how L1 expression and retrotransposition are controlled. In plants, fungi and metazoans, silencing small (s)RNAs suppress TEs at both transcriptional and post-transcriptional levels [2]. In mice, germline-specific, 26–31-nt PIWI-associated RNAs (piRNAs) derived from TE-enriched clusters are loaded into ARGONAUTE-like PIWI proteins directing de-novo cytosine methylation and RNA degradation of active TEs, including L1 [3]. In most healthy somatic tissues, L1s are silenced via 5′-UTR promoter methylation, established from 7.5 days of embryogenesis [4]. In pre-implantation embryos, by contrast, L1 methylation progressively decreases, to reach 13–23% in blastocysts [5], which accumulate full-length L1 transcripts and undergo mosaic retrotransposition, a potential source of heritable and non-heritable mutations [6], [7]. Pre-implantation embryogenesis thus defines a critical window during which L1s should be tightly controlled despite their hypo-methylated status and the lack of piRNAs. In plants, RNA interference (RNAi) at the post-transcriptional level can operate as a surrogate to cytosine methylation and heterochromatinization in TE-silencing [8], [9]. RNAi relies on populations of small interfering (si)RNAs, processed sequentially by the RNase-III Dicer (DCR) from long, perfectly double-stranded (ds)RNA precursors [10]; these are commonly produced by TEs due to their complex insertion patterns or intrinsic bi-directional transcription. Processed siRNAs load into ARGONAUTE (Ago)-family effector proteins and guide sequence-specific degradation of complementary target transcripts. The existence of an endogenous (endo)-siRNA pathway in mammals has been debated, notably because long dsRNA triggers the non-specific interferon (INF) response in most cells [11]. In mouse oocytes, which lack an INF response, heterogeneous sRNA populations map to L1 and LTR elements, among other loci, but their DCR-dependency is unknown; additionally, L1 accumulation is unchanged in oocytes of conditional Dcr −/− animals [12]. Mouse Embryonic Stem Cells (mESCs) also lack an INF response and their ability to produce DCR-dependent endo-siRNAs was clearly established genetically [13]. Being isolated from the blastocyst's inner mass, cultured mESCs are thus potentially suited to study the mechanism(s) that might restrict L1 retrotransposition during pre-implantation, including, possibly, RNAi. Supporting this view, several classes of young, full-length endogenous L1 are hypo-methylated and transcriptionally active in undifferentiated mESCs [14], [15] but become re-methylated and silenced upon differentiation [15], [16]. Moreover, substantially increased L1 transcript levels were reported in undifferentiated Dcr −/− mESCs [17], although this was not confirmed in separate analyses of a distinct KO cell line [18]. Shallow RNA sequencing (15–50-nt size-range) in undifferentiated mESCs revealed that L1 transcription correlates with accumulation of sense and antisense sRNAs of undetermined nature/function, mapping mostly to the L1_5′-UTR [15]. In Human L1s, this region displays overlapping sense-antisense transcription with the potential to form dsRNA and, as such, was proposed to generate anti-L1 endo-siRNAs [19], [20]. In a pioneering study, attempts to substantiate this idea in somatic human cells yielded, however, indecisive conclusions: discrete 21–23-nt L1-derived sRNAs could indeed be detected in some cell lines but not others, and their DCR-dependency was not established; moreover, knocking-down human Dcr-1 caused only marginal increases in endogenous L1 transcription and retrotransposition [20], [21]. Here, we have investigated the possible link between RNAi and endogenous L1 regulation in undifferentiated mESCs. Uniquely, these cells can withstand full genetic ablation of DCR or the AGO proteins, albeit at the cost of proliferation and differentiation defects tentatively ascribed, at least partly, to an inability of Dcr −/− mESCs to produce micro- (mi)RNAs [17], [22]. Unlike siRNA populations, DCR-dependent miRNAs accumulate as discrete, imperfect duplexes excised from stem-loop-containing precursor transcripts produced from numerous independent transcription units. Mature miRNAs are thought to regulate hundreds of cellular transcripts displaying partial miRNA-complementarity, which include mRNAs important for cell fate specification but also pluripotency [23]. Undifferentiated mESCs contain relatively few, albeit highly abundant miRNAs, that can be genetically discriminated from endo-siRNAs and other rare DCR-dependent sRNAs using mutations in the generic miRNA biogenesis factor DGCR8; Dgcr8_KO mESCs contain, nonetheless, few non-canonical miRNAs produced by diverse means [13]. Combining the use of deep-sequencing and cell lines carrying null mutations in Dcr, Agos and Dgcr8, we have investigated the distribution, biochemical origin(s) and ability of L1-derived sRNAs to silence L1 transcript accumulation and retro-transposition in undifferentiated mESCs. Our study reveals an unexpected level of complexity in L1 silencing in these cells, where siRNA-directed RNAi processes are confounded by the overlapping effects of general RNA-surveillance pathways. These findings reveal a novel level of mammalian L1 regulation and shed new light on the proliferation defects and inability of Dcr −/− mESCs to differentiate. Results LINE-1 mRNA and proteins overaccumulate in Dicer knockout mESCs To further explore the L1-derived sRNAs in undifferentiated mESCs, we combined ILLUMINA deep-sequencing and the use of the ncPRO pipeline [24] enabling genomic mapping of repeat-derived sRNAs. A population of abundant, sense and antisense sRNAs was detected, mapping as a majority to the L1-Tf_5′-UTR, consistent with our previous observations (Figure 1A and Figure S1A) [15]. As seen previously with human L1 [19], strand-specific RT-PCR revealed that the 5′-UTR of the L1-Tf subfamily [25] displays overlapping sense-antisense transcription (Figure S1B) with the potential, therefore, to generate dsRNA as a possible source of DCR-dependent siRNAs. Because constitutive DCR depletion is detrimental to cultured mESCs [22], we pursued the above idea by generating inducible Cre-ERT2 Dcr knockouts. Although Dcr deletion was already achieved 24 h post-tamoxifen treatment (Figure 1B), reduced accumulation of miR-295, one of the most abundant mESC miRNAs, was only visible 6 days post-tamoxifen treatment, presumably reflecting the high DCR protein stability [22]. By 12 d post-tamoxifen treatment, miR-295 was below detection levels of quantitative qRT-PCR, indicating full depletion of DCR activity, also confirmed by quantitation of previously validated mESC miRNA target transcripts (Figure 1C and Figure S1C). Strikingly, decreased DCR levels were inversely correlated with accumulation of mRNA and ORF1 protein derived from all L1 classes (Figure 1D and 1E) or from distinct L1-subtypes displaying 5′_UTR polymorphisms [25] (Figure S1D). Analysis of a specific, polymorphic L1 element on chromosome 17 [15] yielded similar results (Figure 1F). Dicer −/− mESCs have been reported to display hypomethylation due to decrease levels in DNA methy-transferases (DNMTs) [26]. However, DNMT1 and DNMT3b proteins were expressed to the same levels in wild type and Dicer −/− cells (Figure S2A). In addition the L1 mRNA was not up-regulated in a cell line carrying a triple-KO for DNMT1, 3a and 3b (Figure S2B) [27]. Investigating the methylation status of the L1_5′-UTR through bisulfite sequencing revealed nonetheless that Dicer −/− mESCs are hypomethylated, which could contribute to the observed up-regulation of the L1 mRNA (Figure S2C). 10.1371/journal.pgen.1003791.g001Figure 1 L1 elements are up-regulated in Dcr−/− mESCs. A. Sequencing reads, from WT mESCs, within the 19–32-nt range were aligned against the mouse genome (version mm9). The distinct sequences coverage (Reads per Million (RPM) normalized) is depicted for the full length L1Md_Tf L1 [24]. B. PCR-based genotyping of the Dcr deletion 24 h and 48 h post-tamoxifen (Tam) treatment. C. qRT-PCR analysis of miR-295 levels in the tamoxifen treated mESCs, as depicted in (B). D. L1_ORF2 mRNA accumulation detected by qRT-PCR before and after Dcr deletion. E. Western analysis of L1_ORF1 protein levels before and after Dcr deletion; CM: Coomassie staining of total protein. F. Semi-quantitative RT-PCR analysis of RNA levels from a single L1-Tf copy on chromosome 17 before and after Dcr deletion. Retrotransposition of LINE-1 in Dicer knockout mESCs Moreover, this increase in L1 transcript/protein in Dcr −/−, but not Dcr Flx/Flx mESCs, was paralleled by a marked gain in endogenous L1 copy-number, estimated by Q-PCR using PCR primers specific for the L1_Tf subfamily. The promoter activity of mouse L1 elements lies in tandemly repeated, 200 bp monomeric units within the 5′-UTR. These monomers are distinct between different LINE-1 families [28]. We used promoter-specific primers to discriminate, by Q-PCR, the three active families of murine L1 elements designated Tf-, Gf-, and A-type (Table S1). For copy number analysis, we focused exclusively on L1_Tf, which was the subfamily we found mostly associated with small RNAs accumulation (Figure S1A) [15]. Using RepeatMasker (AFA. Smit and R. Hubley. RepeatModeler Open-1.0. http://www.repeatmasker.org, 2008–2010), we identified 22,506 sequences annotated L1Md_T (L1_Tf), 15,286 annotated L1Md_A and 819 annotated L1Md_Gf. Among these “fragment” population of L1 elements, we identified 2,291 L1Md_T, 1,338 L1Md_A and 35 L1Md_Gf, which have a length matching at least 95% of their corresponding L1 reference sequence. For 1 512 L1Md_T, we were able to identify at least one amplicon using the L1_Tf specific PCR primers (See Materials & Methods and Table S1), with an average of 3.9 amplicons per elements. We thus calculated 2,770 full length L1_Tf elements in the mm9 genome, which is in line with the 2000–3000 L1_Tf elements previously estimated by Naas et al. [29], of which 60% are putatively active. To evaluate the gain in copy-number, Dcr Flx/Flx mESCs were analysed at passage 10, upon which the Dcr deletion was induced; the L1_Tf copy number was then re-assessed after 20 additional passages in Dcr Flx/Flx and Dcr −/− background (Figure 2A). About 2,452 active L1_Tf copies were found in Dcr Flx/Flx mESCs at P10 and 2,707 copies at P30. A gain of approximately 860 new copies was detected in the Dcr −/− cell line after 20 passages. Therefore, we estimate that between 1 and 20 new active copies of L1_Tf were generated per day (i.e. 2 cell divisions) in the Dcr− /− background. 10.1371/journal.pgen.1003791.g002Figure 2 L1 elements retrotranspose in Dcr−/− mESCs. A. qPCR-based copy-number analysis of L1_Tf elements in Dcr Flx/Flx P10 (5 replicates), P30 (2 replicates) and Dcr −/− P30 (5 replicates) mESCs. *: p-value<0.01. B. qRT-PCR-based analysis of eGFP mRNA levels in WT and Dcr −/− mESCs stably transformed with a WT human eGFP-tagged L1 transgene, 24 h post-transfection and 6 passages (P6) after puromycin selection. C. Integrated eGFP as a diagnostic of retrotransposition detected by PCR in the genomic DNA of Dcr −/− mESCs carrying the human eGFP-tagged L1 transgene after 6 passages post-puromycin treatment. To ascertain the above PCR-based results, we adapted the gain-of-GFP retrotransposition assay previously developed by Prak and colleagues [30] and validated in WT mESCs [31]. To score de novo L1 retrotransposition events in the Dcr Flx/Flx and Dcr −/− mESC lines, a human L1 modified to contain an intronic, split eGFP reporter was stably integrated into the genome of Dcr Flx/Flx and Dcr −/− mESCs. During early propagation (passages 4 to 6 after selection of puromycin-resistant cells), only in Dcr −/− cells was the eGFP mRNA significantly increased, although the same quantity of plasmid was transfected in each cell lines and was similarly expressed 24 h post-transfection (Figure 2B and S2D); moreover, this increase was not visible in Dcr −/− cells transformed with RT-deficient point-mutation alleles of the eGFP-tagged L1 (Figure S2D) or in WT cells (data not shown). Amplification of eGfp DNA was also only observed in Dcr −/− cells transformed with WT eGFP-tagged L1 (passage 6; Figure 2C), a method also previously employed to validate active retrotransposition [32]. Finally, similar results were also obtained using a gain-of-Luciferase retrotransposition assay [33] (data not shown). We conclude that DCR negatively controls L1 transcript accumulation and retrotransposition in undifferentiated mESCs. L1 sRNAs form overlapping populations of DCR-dependent and -independent species Given the DCR-dependent control of L1, we next investigated whether L1_5′-UTR sRNAs are DCR products. Total RNA from Dcr −/− mESCs was subjected to ILLUMINA sequencing. As expected, loss-of-DCR activity caused a dramatic decline in cellular 21–23-nt RNAs including, chiefly, miRNAs, representing most sRNAs in WT mESCs. Consequently, the relative proportion of repeat-derived sRNAs was seemingly increased in Dcr −/− compared to WT cells (Figure S3A–C). However, read-size analysis and genomic mapping revealed a specific depletion in sense and antisense L1-derived 22-nt sRNAs in Dcr −/− mESCs (Figure 3A); the remaining, abundant DCR-independent L1-derived sRNAs were heterogeneous in size, ranging from 19- to 32-nt, had both sense and antisense orientations and mapped mostly to the L1_5′-UTR, as in WT cells (Figure 3B and S3D). To test if some L1-derived sRNAs were effectively loaded into cognate RNA silencing effectors, we analysed the sRNA content of immunoprecipitates from endogenous AGO1 and AGO2, the only Agos we found significantly expressed at the protein level in undifferentiated mESCs, in agreement with available mESC RNA-seq data [34]. Using qRT-PCR, we found that the most abundant sense and antisense L1_5′-UTR sRNAs were specifically loaded into AGO2, as were several abundant miRNAs tested (Figure 3C). To obtain a comprehensive and unbiased view of AGO2-loaded L1-derived sRNAs, we used a mESC line overexpressing Flag-HA-tagged human Ago2 (FHA-hAgo2), and subjected RNA isolated from anti-Flag immunoprecipitates to ILLUMINA sequencing (Figure 3E–G). The fraction of sense and antisense L1-derived sRNAs loaded into FHA-hAgo2 had a genomic distribution resembling that of total RNA from WT or Dcr −/− cells (Figure 3B); it was, however, clearly enriched in 22-nt sRNAs, the size range of cognate DCR products (Figure 3A). Sequential processing, by DCR, of long dsRNA substrates produces perfect siRNA duplexes with diagnostic 2-nt 3′ overhangs. These features were indeed displayed by approx. 10% of all 22-nt sRNA sequences derived from L1-Tf in WT mESCs, as illustrated in Figure 3D with the 5′_UTR region of a single, near-consensus L1-Tf locus on the X chromosome. The abundance of these species was reduced by approx. 20% in Dcr −/− compared to WT mESCs, consistent with the read-size analysis in Figure 3A. Furthermore, they showed a 7-fold enrichment in IPs of AGO2, the cognate effector of siRNAs (Figure 3D). The most straightforward interpretation of these results is that L1-derived sRNAs form overlapping and complex populations of DCR-dependent and -independent molecules. These likely derive from longer sense- and antisense-RNA, or long dsRNA hybrids thereof, which are produced mostly from the L1_5′-UTR. While DCR-independent sRNAs dominate the overall population and display broader and heterogeneous size ranges, DCR-dependent sRNAs with cognate features of siRNAs load preferentially into AGO2, owing to the known 22-nt size preference of this silencing effector protein [35]. 10.1371/journal.pgen.1003791.g003Figure 3 L1 sRNAs are partially produced by DCR and 22-nt L1-sRNAs loaded into AGO2. A. Size distribution of sequencing reads mapping to L1_Tf elements in Dcr −/− compared to WT mESCs. Note the deficit in 21–23-nt sRNAs in the former, and their enrichment in immunoprecipitated FHA-hAgo2, used in (B). B. Sequence coverages of L1_Tf elements from WT, Dcr −/− and immunoprecipitated E14-FHA-hAgo2 mESCs, as in Figure 1A. this peak is an artefact found in all libraries sequenced. C. qRT-PCR analysis of the most abundant sense and antisense L1_5′-UTR-derived sRNAs and of two mESC miRNAs in immunoprecipitates of endogenous AGO1 and AGO2. Lower panel: control Western analysis of endogenous AGO1 and AGO2 levels after immunoprecipitation; CM: Coomassie staining of total protein. D. Snapshot representation of perfect, 22-nt-long sRNA duplexes with 2-nt 3′ overhangs (in red) mapped on the 5′-UTR of a single L1-Tf element located on chromosome X. E. Relative proportions of all L1-Tf-derived sequences forming perfect duplexes with 2-nt overhangs in WT versus Dcr−/− mESCs (left panel) and their enrichment in AGO2 immunoprecipitates (right panel). The numbers of 22-nt distinct sequences were normalized by the L1-Tf family coverage of RPM and expressed as proportion compare to WT. Neither the RNA surveillance nor the microRNA pathway regulate L1 mobilization in mESCs To gain further insights into the nature and origin of DCR-independent L1 sRNAs and their possible involvement in L1 control, we tested the impact of 5′-3′ and 3′-5′ RNA-surveillance pathways operated by the Xrn class of exoribonucleases and the exosome, respectively. In diverse organisms, these pathways degrade aberrant pre-mRNA, transposon-derived and non-coding RNA, including antisense RNA [33], [34]. We thus generated stable mESC lines displaying knockdown (KD) of nuclear Xrn2, cytoplasmic Xrn1 or exosome co-factor Rrp6, using new and previously established shRNA constructs [36] (Figure 4A and S4A). The results presented are for Xrn2; similar effects were observed in Rrp6_KD cells (Figure S4A and S4C), while they were much less pronounced in Xrn1_KD cells (data not shown). L1 transcripts and ORF1 protein were found significantly up-regulated in Xrn2_KD mESCs, (Figure 4A–B and S4D), correlating with reduced accumulation of the most abundant sense and antisense L1-derived sRNAs (Figure S4B). Copy-number analysis (as in Figure 2A) showed, however, that neither the Xrn2_KD nor the Rrp6_KD cell lines showed enhanced L1 mobilization (Figure 4C and S4C). Thus, RNA-surveillance pathways likely contributed to the heterogeneous mESC L1-derived sRNAs, which are presumably degradation intermediates of exonucleolysis of the longer sense and antisense transcripts derived from the L1_5′-UTR. Loss of these RNA-surveillance pathways did not, however, impact on L1 retrotransposition. These results thus support a role for siRNA-mediated RNAi in the control of L1 mobility, although we could not rule out an indirect contribution of DCR-dependent miRNAs, which regulate hundreds of cellular transcripts. To address this, we examined Dgcr8_KO mESCs, in which production of even the most abundant mESC miRNAs, including miR-295, is abrogated [37] (Figure 4D). Accumulation of endo-siRNAs and potential DGCR8-independent (i.e. non-canonical) miRNAs should remain in these cells [13], [35] (Figure S4E). As shown in Figure 4E and 4F, Dgcr8_KO cells unexpectedly displayed enhanced L1 mRNA accumulation, possibly explained by the hypomethylation status of L1_5′-UTR in these cells (Figure S2A–C). However, there was no increase in L1 copy number (Figure 4F), ruling out the contribution of canonical miRNAs to the observed DCR-dependent control of L1 retrotransposition. 10.1371/journal.pgen.1003791.g004Figure 4 L1 mRNA levels and genomic copy-number in various knock-out and knock-down mESC lines. A. Western analysis of XRN2 and L1_ORF1 accumulation in WT and Xrn2_KD mESCs; CM: Coomassie staining of total protein. B. qRT-PCR analysis of L1_ORF2 mRNA levels in WT and Xrn2_KD mESCs. C. qPCR analysis of L1_Tf copy-number in WT and Xrn2_KD mESCs. D–E. qRT-PCR analysis of miR-295 (D) and L1_ORF2 mRNA (E) levels in WT and Dgcr8_KO mESCs. F. qPCR analysis of L1_Tf copy-number in WT and Dgcr8_KO mESCs. G–H. qRT-PCR analysis of miR-295 (G) and L1_ORF2 mRNA (H) levels upon hAgo2 deletion in Tamoxifen-treated Ago1,2,3,4_KO mESCs. I. qPCR analysis of L1_Tf copy-number in Ago1,2,3,4_KO_hAgo2 mESCs before and after hAgo2 deletion. *: p-value<0.1. AGO2 is crucial for L1 silencing and strongly destabilized in Dcr−/− mESCs Although we could not formally exclude a role for some unknown DGCR8-independent miRNAs in L1 retrotransposition control, the above results pointed to the likely contribution of DCR-dependent, 22-nt siRNAs derived from the L1_5′-UTR region of overlapping sense-antisense transcription (Figure 3A and S3B); their strong loading-bias in AGO2 thus predicted a crucial role for this silencing effector in L1 regulation (Figure 3C and 3E). To test this idea and avoid functional redundancy with AGO1 as previously observed with miRNAs [38], [39], we used an established quadruple Ago1,2,3,4_KO mESC line, in which a stably expressed hAgo2 transgene can be deleted upon tamoxifen treatment [38] (Figure S4F). MiR-295 levels were strongly reduced at 2 d, and at 5 d post-tamoxifen treatment; a corresponding increase in microRNA target levels confirmed cellular depletion of hAgo2, as reported [38] (Figure 4G and S4G). As in Dcr −/− cells, tamoxifen-induced Ago1,2,3,4_KO mESCs displayed strong up-regulation of L1 transcripts and increased L1 copy-number, unlike their untreated counterparts (Figure 4H–I and S4H–I), supporting a key contribution of AGO2 in L1 silencing in undifferentiated mESCs. The L1 copy-number increase in Ago1,2,3,4_KO cells was noticeably less pronounced than in Dcr −/− cells (Figure 4I and 2A) however, possibly reflecting intrinsic differences in the relative initial L1 copy-number of non-treated Ago1,2,3,4_KO and Dcr Flx/Flx cell lines. Alternatively, cellular depletion of Dcr may have had additional, unanticipated effects that would lead to more potent L1 retrotransposition (Figure 2A). A survey of several key RNAi components indeed revealed a specific and dramatic reduction of AGO2 in Dcr −/− mESCs in multiple experiments, also reported recently in Dgcr8_KO mESCs [40] (Figure 5A). This effect was observed only at the protein level, as Ago2 mRNA remained expressed (Figure S5A); it was also specific, since AGO1 protein levels remained unchanged (Figure 5A). As the prevalent effector of DCR-dependent miRNAs (Figure 3C), representing alone up to 70% of all mESC miRNAs (Figure S3G), we reasoned that AGO2 might have been destabilized and degraded in Dcr −/− mESCs due to the loss of its main sRNA cargoes; such an effect was documented for the Arabidopsis miRNA-effector AGO1 [41]. Indeed, we found AGO2 levels to be significantly up-regulated upon treatment of Dcr −/− mESCs with the 26S-proteasome inhibitor MG132, an effect previously reported for miRNA-depleted hAgo2 [40], [42] (Figure S5B). Also consistent with a loss-of-miRNA-dependent effect, AGO2 levels, unlike those of AGO1, were also reduced in Dgcr8_KO mESCs, albeit consistently less than in Dcr −/− cells (Figure 5A). This, incidentally, possibly explained the results on L1 silencing obtained in Dgcr8_KO mESCs (Figure 4E–F): while a deficit in the main effector of 22-nt L1-derived siRNAs likely increased L1 mRNA accumulation, the remaining AGO2 levels were presumably still sufficient to restrict L1 retrotransposition. We further infer that, in the absence of canonical miRNAs, loading of DCR-dependent endo-siRNAs (including L1-derived siRNAs) and possibly non-canonical miRNAs, explains the residual levels of AGO2 in Dgcr8_KO mESCs (Figure 5A). These levels are considerably lower in Dcr −/− mESCs, because neither sRNA class is produced in this background. 10.1371/journal.pgen.1003791.g005Figure 5 Rescue of L1 silencing in hDcr-complemented Dcr −/− mESCs. A. Western analysis of endogenous AGO1, AGO2 and L1_ORF1 protein levels in Dcr Flx/Flx, Dcr −/− and Dgcr8_KO ESCs; CM: Coomassie staining of total protein. B. Western analysis of endogenous AGO2 and L1_ORF1 protein levels in Dcr Flx/Flx, Dcr−/− mESCs and one representative stable line of hDcr-complemented Dcr −/− mESC; CM: Coomassie staining of total protein. C–E. qRT-PCR analysis of L1_ORF2 mRNA levels (C), miR-295 and miR-16 levels (D), and Hmga2 and Btg2 mRNA levels (established targets of mmu-miR-196a and mmu-let-7a and mmu-miR-132, respectively) in the various cell lines depicted in (E). Rescue of AGO2 levels and L1 silencing in miRNA-depleted Dcr −/− mESCs The above results prompted us to assess further the extent to which L1 silencing could be rescued in Dcr−/− mESCs in a miRNA-independent manner. We repeatedly failed to reintroduce hAgo2 transgenically into Dcr−/− mESCs, including a catalytic null (slicer deficient) allele of hAgo2, in our initial attempts to differentiate potential siRNA-mediated effects (slicer-dependent) and miRNA-mediated (slicer-independent) on L1 silencing. We ascribe this failure to an inability to sufficiently re-stabilize Ago2 in the absence of its main cargoes, the miRNAs [40]. We thus resorted to stably complement Dcr−/− mESCs with a human Dcr (hDcr) transgene. During their early propagation, several independent puromycin-selected clones displayed endogenous AGO2 levels consistently higher than in non-complemented Dcr−/− mESCs; moreover, L1 silencing, measured by ORF1 and mRNA accumulation, was restored in these cells to almost the levels seen in Dcr Flx/Flx cells (Figure 5B and 5C). Strikingly, however, in nearly all these clones, mature miRNA levels were only rescued to approximately 10% of WT levels, which is likely below physiological significance, because all validated miRNA targets tested accumulated ectopically in these cells (Figure 5D, 5E and Figure S5C). The levels of miRNAs and their targets were eventually restored to WT levels, as previously described [17], but only after extended periods of culture involving more than ten cell passages, during which the poor fitness of cultured Dcr−/− mESCs [22] presumably resulted in selection for cell variants with higher DCR levels. Nonetheless, the early passage data demonstrate that L1 silencing could be achieved in undifferentiated mESCs displaying as little as 10% total miRNAs, suggesting that RNAi via endo-siRNAs, including L1-derived siRNAs, is sufficient to silence L1s. Complemented, miRNA-defective Dcr −/− mESCs differentiate normally These data prompted us to re-evaluate the proliferation and differentiation defects of Dcr−/− cells [17], [22], [37]. Having now uncovered a potential role for DCR and AGO2 in L1 silencing in addition to their known regulatory functions via miRNAs, we explored to what extent the Dcr−/− cell defects were attributable to defective miRNA, as opposed to siRNA, biogenesis or action. Dcr Flx/Flx, Dcr−/− and hDcr-complemented Dcr−/− cells (early passages) could all undergo the formation of Embryoid Bodies (EBs), although this was achieved with a 1–2 d delay in Dcr−/− cells. 6 d after the onset of differentiation, EBs were plated onto adherent flasks and monitored until d10 of differentiation. Microscopy and quantitation of key pluripotency markers confirmed that Dcr−/− cell-derived EBs were unable to differentiate, even if they attached to the flasks' surface [17] (Figure 6A, 6B and Figure S6A). In contrast, the growth rate and morphology of cells differentiated around attached EBs were similar in Dcr Flx/Flx and hDcr-complemented Dcr−/− cells (Figure 6A). Furthermore, 10 d post-differentiation of hDcr-complemented Dcr−/− cells, AGO2 accumulation was partially rescued, and the levels of pluripotency and differentiation markers were similar to those of Dcr Flx/Flx cells (Figure 6B and Figure S6A). MiRNA accumulated to only 5–7% of WT levels in hDcr-complemented Dcr−/− cells, which displayed, accordingly, ectopic miRNA target accumulation (Figure S6A and S6B). However, L1 transcript accumulation in these cells was as low as in differentiated Dcr Flx/Flx cells (Figure S6A), demonstrating rescue of L1 silencing. These results strongly suggest that miRNAs alone are unlikely to underpin mESC differentiation in the above experimental setting. Additional processes likely entail the production of DCR-dependent endo-siRNAs, of which some might contribute to protecting the mESC genome integrity by silencing active retrotransposons, including L1. Consistent with this idea, Dgcr8_KO cells that lack all canonical miRNAs but, unlike Dcr−/− cells, suppress L1 mobilization, could partially differentiate in the same experimental setting, agreeing with previous findings [37]; Xrn2_KD cells, which also are L1-silencing proficient, differentiated similarly to Dcr Flx/Flx cells (Figure 6C). 10.1371/journal.pgen.1003791.g006Figure 6 hDcr-complemented Dcr −/− ESCs differentiate normally despite accumulating 5–7% total miRNAs compared to WT. A. Visualization of Embryoid bodies from Dcr Flx/Flx, Dcr −/− and hDcr-complemented Dcr −/− mESCs after 1, 6 and 10 days of differentiation. B. Western analysis of OCT4 and endogenous AGO2 protein levels in the cells depicted in (A) before (d0) and after 10 days of differentiation (d10); CM: Coomassie staining of total protein. C. Same as (B) but in WT, Xrn2_KD and Dgcr8_KO mESCs. Discussion Our results support a role for DCR-dependent L1-derived siRNAs in taming endogenous L1 retrotransposition in undifferentiated mESCs. They are thus consistent with RNAi safeguarding genome integrity during a time window of mouse development when DNA hypo-methylation coincides with L1 mobilization [7]. A role for RNAi in correcting DNA methylation defects of TEs is fully supported by previous work in Arabidopsis [8], [9], in which intricate and opposing interactions between the RNAi and RNA-surveillance pathways have also been documented [43]. A parallel can be further established between our results and those of recent work in S. pombe, showing that RNAi at several loci, including Tf2 retroelements, is confounded by the 3′-5′ exonuclease activity of the exosome; genetic ablation of Rrp6 was, accordingly, sufficient to uncover siRNAs accumulating at these loci, showing, in that case, selective competition between the two pathways [44]. Human L1 transcription has been associated with the production of abundant non-polyadenylated and possibly uncapped RNAs of both strands accumulating in the nucleus [45]. Similar RNAs produced from the mouse L1_5′-UTR region likely provide the bulk of templates for Xrn2 and the Rrp6-associated exosome, known to degrade aberrant transcripts in the nucleus [46]; the resulting degradation intermediates form an important source of sRNA, heterogeneous in size, that confound detection of bona fide siRNAs. Full-length L1 transcripts may also undergo degradation by both exonucleases since a gain in ORF1 protein accumulation was observed in Xrn2_KD and Rrp6_KD mESCs. Alternatively, it is possible that different subsets of LINE1 elements are affected differently by the various genetic backgrounds tested here Figure S4D). For instance, highly transcribed but transposition-deficient L1 variants may accumulate elevated levels of specific transcripts that are more sensitive to the action of Xrn2 and/or Rrp6. This would rationalize why elevated L1 transcription, as seen in the Xrn2_KD and Rrp6_KD backgrounds, does not necessarily correlate with retrotransposition. The overall complexity uncovered here with L1 probably explains, more generally, why the existence and distribution endo-siRNAs at the whole-genome scale has been difficult to establish in mammalian cells so far, despite the pervasive nature of aberrant and antisense transcription in mammalian genomes [47]. A role for DCR in silencing L1 and perhaps other TEs could also shed light on the previously reported failure of Dcr−/− mESCs to differentiate. The hDcr rescue experiment (Figure 6A) shows, notably, that miRNA biogenesis/activity is unlikely to contribute alone to mESC differentiation in this experimental setting and that endo-siRNAs are likely also important. Production of TE-derived siRNAs, in particular, might be crucial in preventing widespread double-strand break lesions and insertional mutagenesis, foreseeably detrimental to mESC proliferation and differentiation. This might also explain why Dgcr8_KO cells, unlike Dcr−/− cells, retain an ability to differentiate partially [17], [37] (Figure 6C). Indeed, Dgcr8_KO cells, unlike their Dcr−/− counterparts, exhibit detectable levels of AGO2 (Figure 5A), which must be stabilized by the loading of endo-siRNA including L1-derived siRNAs and/or non-canonical miRNAs. Transposon taming and/or endogenous regulation by these molecules might be sufficient to rescue, at least partly, the differentiation defects of Dgcr8_KO cells. Assessing the extent to which RNAi-dependent control of active TEs, including L1s, contributes to the integrity of mESCs proliferation and differentiation, and thus to early mammalian embryogenesis, is an attractive prospect for future investigation. Conclusions This study unravels how multiple RNA silencing pathways might cooperate to dampen the expression and mobilization of an active family of transposable element family in mammalian cells. It not only echoes previously findings made in plants, fungi and invertebrates [44], [48], [49], but also rationalizes the complex patterns of small RNAs uncovered in studies originally conducted in mouse oocytes [12], [50] and, more recently, in Human stem cells [51]. On a final note, although the mechanisms uncovered here in mESCs were tied in within the frame of early embryonic development, they may well also apply to other stem cell niches formed post-embryonically and present in many tissues of adult mammals. The states of pluri- and multi-potency seem generally associated with a deficit or absence of protein-based immunity, which includes the IFN response to exogenous and endogenous dsRNA. This might explain, at least partly, why these cells, unlike many other somatic cells, appear to accommodate RNAi triggered by long dsRNA [52], [53]. In this context, we contend that siRNA-based RNAi has persisted in vertebrates as a primordial mechanism that protects progenitor cells of developing and adult organisms against the harmful effects of transposons and exogenous viruses [54]. This proposed RNAi-based defence is anticipated to be important, because genomic instability or viral infections in progenitor cells would have long-lasting detrimental consequences throughout the entire lineages derived from them. Defensive, as opposed to regulatory roles for mammalian RNAi have been somewhat overlooked thus far, but we are optimistic that the work reported here and elsewhere [54] will shed new light on this specific and fascinating aspect of RNA silencing. Materials and Methods Culture and in vitro differentiation of ESC mESCs were cultured in Dulbecco's Modified Eagle Media (DMEM) (Invitrogen), containing 15% of a special selected batch of fetal bovine serum (FBS; Life Technologies) tested for optimal growth of mESCs, 1000 U/ml LIF (Millipore), 0.1 mM 2-β mercaptoethanol (Life Technologies), 0.05 mg/mL of streptomycin, and 50 U/mL of penicillin (Sigma) on a gelatin coated support in the absence of feeder cells. Embryoid body cultures were established by aggregation of mESCs in a low-adherent tissue culture dish into LIF-free DMEM, 10% FBS medium, from day 1 until day 6 and reattached on adherent flasks at day 10 of differentiation. The culture medium was changed daily. All cells were grown at 37°C in 8% CO2. The male E14 mESC line (129/Ola background) [55] was used for Illumina deep sequencing of WT mESC. E14_FHA-hAgo2 cells was created by stable transfection of plasmid pIRESneo-FLAG/HA Ago2 corrected (Addgene plasmid 10822) [56] and selection on G418-containing medium. TKO mESCs were described in ref. [27]. Dgcr8_KO mESCs were purchased from Novus Biologicals (NBA1-19349). New CreERT2-Dcr Flx/Flx mESCs were isolated from the cross of floxed Dcr Flx/Flx mice [22], [57] and ROSA-CreERT2 mice [58]. The Dcr −/− mESC line was established from Dcr Flx/Flx mESC after induction with Tamoxifen (more than 15 days) and routinely control for their loss of microRNA accumulation. Genotyping primers used for the characterization of these cell lines are presented in Table S1. The inducible mESC line deficient for the four mouse Argonautes and carrying a floxed human Ago2 transgene (Ago1,2,3,4_KO) was described previously [38] and validated in the laboratory according to the author instructions. Dcr and hAgo2 deletions were induced with 4-OHT (Tam) stock solution (1 mM, dissolved in 100% ethanol) diluted 1∶1000 in cell culture medium to a final concentration of 1 µM for 6 and 12 days (Dcr) and 2 and 5 days (hAgo2). Transgenesis of WT and mutated version of the human L1-eGFP (RP = TgWT, JM111 = TgΔORF1 and 2980 = TgΔ5′UTR) [30] was carried out using 5 µg of each plasmids and Lipofectamine 2000 (Life Technologies) in E14 and Dcr −/− mESCs. Stable clones were selected on puromycin-containing medium (1 µg/mL; Sigma) 24 h post-transfection and analysed after 4 and 6 passages of the cells. Xrn2_KD mESC lines were generated using the pSUPER-puro vector (OligoEngine, http://www.oligoengine.com) engineered to produce the active shRNA 5′- CTCCAGAAGAGAACAGGAGAAAT-3′. Upon transfection of PGK mESCs [59], cells were selected on puromycin-containing medium. Control cell lines were generated by integration of an empty pSUPER-puro vector into PGK mESCs. Dgcr8_KO/Xrn2_KD mESC lines were generated using the pSUPER-puro vector engineered to produce the active shRNA 5′-CTCCAGAAGAGAACAGGAGAAAT-3′ and 5′- CTCGGGAAGATACAGTTGGAATT -3′. Upon transfection of the Dgcr8_KO lines, cells were selected on puromycin-containing medium. The hDcr-complemented Dcr −/− cell line was created by the transfection of plasmid pDESTmycDicer (Addgene plasmid 19873) [60] into Dcr −/− mESCs. Stable Dcr −/−hDcr mESCs were selected on G418-containing medium and analysed at early passages (<P5) and late passages (>P10). MG132 (Z-Leu-Leu-Leu-al; Sigma, C2211) was dissolved DMSO and added to the cells for 7 h to a final concentration of 0.5 µM. Deep-sequencing Total cellular RNA (5 µg), extracted using Isol-RNA Lysis Reagent (5PRIME) was processed into sequencing libraries using adapted Illumina protocols and sequenced at Fasteris (http://www.fasteris.com, Switzerland) using the HiSeQ 2000 sequencer. All next-generation sequencing data have been deposited to the NCBI Gene Expression Omnibus (GEO) and are accessible with the accession n° GSE43110 (WT and Dcr −/−) and GSE43153 (IP_FHA-hAgo2). sRNA analysis The sRNA-seq analyses were performed using the ncPRO pipeline [24]. Briefly, the reads were aligned on the mm9 genome using the Bowtie software and allowing multiple matches. Profiling of repeats was estimated from the intersection of the mapped reads with the RepeatMasker annotation. As annotated L1Md_Tf L1 repeats are often truncated or have different full length, the median size of full length was considered, and all LdMd_Tf L1 repeats were scaled to this median size when computing positional read coverage. The positional read coverage was computed by summing up the normalized counts (RPM) of reads covering each position, which was further normalized to the number of L1Md_Tf L1 repeats in the genome containing the position. PCR Strand-specific RT-PCR was performed using the Transcriptor Reverse Transcriptase kit (Roche) using 1 µg total RNA and following the manufacturer's instructions. PCR using primer for the specific Tf LINE from chromosome 17 were conducted at 95°C for 10 min, followed by 35 cycles at 95°C for 15 s, 60°C for 30 s, 72°C for 30 s and 10 min at 72°C and revealed on 1% agarose gel. Real-time PCR reagents for miRNAs, 5′_UTR sRNAs and control U6 snRNA were from Qiagen. 5′_UTR sRNAs sense and antisense discrete sequences have been extracted from deep-sequencing data and chose because their higher level of expression. For RT reactions, 1 µg total RNA was reverse-transcribed using the miScript Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. Following the RT reactions, cDNA products were diluted five times in distilled water, and 2 µL of the diluted cDNAs was used for PCR using QuantiTect SYBR Green PCR Master Mix and miScript Universal Primer (Qiagen). PCR reactions were conducted at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s on a LightCycler 480 real-time PCR machine (Roche). Real-time PCR for mRNAs was performed as described in [36] using the Rrm2 as a reference gene. L1 copy-number analysis was conducted on 50 ng of DNA and normalized with Rrm2 gene (a single-copy gene). Differences between samples and controls were calculated based on the 2−ΔCT method. Each Real-time PCR reaction was carried out in triplicates using samples from three or five independent differentiation events or cell lines for all mESC experiments, unless otherwise stated. Student's T-Test was used to evaluate the statistical significance of Q-PCR analysis of L1 copy Number. Primers used in this study are all listed in Table S1. L1 copy number analysis Using the ePCR package from NCBI (http://www.ncbi.nlm.nih.gov/sutils/e-pcr/) we identified 10 806 hits on the mouse mm9 reference genome using the L1_Tf specific primers as designed on the L1spa N°. AF016099 [29]. These primers generate 67 bp amplicons, present in the L1_Tf 5′_UTR repeated regions. Real-time quantitative PCR analysis of the L1_Tf copy number in Dicer Flx/Flx mESCs provided a figure of 9,478 at P10 and 10,465 at P30 PCR hits, remarkably close to the ePCR estimation (10,806). The small difference could be explained by the hybrid background of the Dicer mESC line used compared to the genome reference in mm9. The copy number assay shown in Figure 2A, involved a comparison of Dcr Flx/Flx mESCs sampled at passage 10 (9,478 amplicons detected) and at passage 30 (10,465 amplicons detected) with Dcr −/− mESCs sampled at passage 30 (13,821 amplicons detected). Therefore, we estimate that (13,821-10,465) = 3,356 new PCR amplicons were generated, corresponding to 3,356/3.9 = 860 new full length insertion after 20 passages in the Dcr −/− background. Since each passage represents 2 days of culture (40 days in total), 860/40 = 21.51 full length L1_Tf insertions were generated on average every day in Dcr −/− mESCs, although the fraction of active copies among these insertions is unknown. We conclude, therefore, that Dcr −/− mESCs undergo between 1 and 20 L1_Tf retrotransposition events per day. Cell lysates and immunoprecipitations E14_FHA-hAgo2 mESCs were scraped in cell lysis buffer (25 mM Tris, pH 7.9, 250 mM KCl, 0.2 mM EDTA, 20% glycerol and Roche Complete Protease Inhibitor without EDTA). Cells were lysed 10 min on ice, sonicated and centrifuged (10 000 rpm, 10 min at 4°C) before Western analysis or immunoprecipitation. Lysates were incubated at 4°C with 20 µL of FLAG-beads (Invitrogen) for 12 h. Beads were collected by centrifugation (2,000 rpm, 1 min). After at least three washes in 1 mL lysis buffer, beads were incubated with 100 µL 0.1 M glycine pH 2.5 for 10 min RT on a shaker. Ten µL 1 M Tris–HCl pH 8 was added to neutralize the elution buffer. Immunoprecipitated RNAs was then extracted from eluted proteins with Isol-RNA Lysis Reagent (5PRIME). Bisulfite sequencing-based DNA methylation analysis Genomic DNA was extracted using Isol-RNA Lysis Reagent (5PRIME). Bisulfite treatment was performed using the EpiTect Bisulfite Kit (Qiagen). Bisulfite-treated DNA was then amplified using the DreamTaq DNA Polymerase and primers listed in see accompanying primer list. PCR cycling conditions and primers design were made following the recommendations in [61]. PCR fragments were purified and cloned into pGEM-T Easy (Promega) and individual colonies were sequenced using M13 primers. Sequences were then analysed using Kismeth and BISMA softwares [62], [63] to obtain the percentage of methylated sites for each sequence context. Results shown were obtained in two independent experiments. Antibodies The following antibodies were used: anti-L1_ORF1 (gift of Dr Alex Bortvin, Carnegie Institution for Science, USA), anti-AGO1 (D84G10, Cell Signaling Technology, Beverly, MA, USA), anti-mouse AGO2 (clone 6F4, gift of Dr Gunter Meister, University of Regensburg, Germany), anti-XRN2 (A301-101A, Lubio Science, Switzerland), anti-EXOSC10 (Rrp6) (ab50558, Abcam, Cambridge, UK) and anti-OCT4 (ab19857, Abcam, Cambridge, UK). Supporting Information Figure S1 L1 elements are up-regulated in Dcr−/− mESCs. A. Number of sRNA reads and distinct sequences matching full length retrotransposon of LINE1 from L1Md_T, L1Md_A and L1Md_Gf families. B. Detection of overlapping sense and antisense L1 transcription at the L1_5′-UTR region using strand-specific RT-PCR in WT mESCs. The primer sets used are depicted. C. Accumulation of the Hmga2 and Btg2 mRNAs, respectively known targets for mmu-miR-196a and mmu-let-7a/mmu-miR-132, analyzed by qRT-PCR before and after Dcr deletion. D. L1_Tf, Gf and A sub-type mRNAs accumulation detected by qRT-PCR before and after Dcr deletion. Polymorphism in the repeated region indicated in the scheme was used to distinguish subtypes. (EPS) Click here for additional data file. Figure S2 Methylation and retrotransposition in Dcr −/− ESCs. A. Western analysis of DNMT1 & 3b proteins levels in Dcr Flx/Flx, Dcr −/− and Dgcr8_KO mESCs; CM: Coomassie staining of total protein. B. L1_ORF2 mRNA accumulation detected by qRT-PCR in Dcr Flx/Flx, Dcr −/−, Dgcr8_KO and TKO mESCs. C. Bisulfite sequencing-based methylation analysis at the L1 5′_UTR in Dcr Flx/Flx, Dcr −/−, Dgcr8_KO and TKO mESCs. Data were analysed with the Kismeth and BISMA online softwares [62], [63]. D. Expression of eGFP detected by qRT-PCR in WT and Dcr −/− mESCs carrying the human eGFP-tagged L1 transgene after 4 (P4) and 6 (P6) passages post-puromycin treatment for selection of stable transformants. L1 constructs lacking 5′UTR (TgΔ5′UTR) or ORF1 (TgΔORF1) were used as negative controls for retrotransposition. Note that TgWT = RP, TgΔORF1 = JM111 and TgΔ5′UTR = 2980 according to the previous nomenclature established in [30]. (EPS) Click here for additional data file. Figure S3 Deep-sequencing analysis of small RNA libraries. A. Compared size distribution of all deep sequencing reads mapping to the mm9 genome in WT and Dcr −/− sRNAs libraries. B. Pie chart distributions of non-coding RNAs, as annotated by the ncPRO pipeline, in WT and Dcr −/− sRNAs libraries. C. Relative proportions of reads mapping to pre-miRNAs in WT and Dcr −/− sRNAs libraries, as annotated by the ncPRO pipeline. D. 22-nt sequence coverages of L1_Tf elements from WT, Dcr −/− and immunoprecipitated E14-FHA-hAgo2 mESCs, normalized to the total amount of 22-nt reads from corresponding library. E. Size distribution of all reads of RNA isolated from hAgo2 immunoprecipitates mapping to the mm9 genome. F. Same as in (B) for hAgo2-bound sRNAs. G. Same as in (C) for hAgo2-bound sRNAs. (EPS) Click here for additional data file. Figure S4 L1 expression and genomic copy-number in various knock-out and knock-down mESC lines. A. Western analysis of RRP6 and L1_ORF1 accumulation in WT and Rrp6_KD mESCs; CM: Coomassie staining of total protein. B. Accumulation of Tf_5′-UTR (+) and (−) sRNAs detected by qRT-PCR in WT and Xrn2_KD mESCs. C. qPCR analysis of L1_Tf copy-number in WT and Rrp6_KD mESCs. D. L1_ORF2, Tf, Gf and A sub-type mRNAs accumulation detected by qRT-PCR in Xrn2_KD and Rrp6_KD mESCs. E. Accumulation of miR-320 detected by qRT-PCR in WT and Dgcr8_KO mESCs. F. Western analysis of AGO2 accumulation in WT and Ago1,2,3,4_KO_hAgo2 mESCs before and after hAgo2 deletion induced by tamoxifen; CM: Coomassie staining of total protein. G. Accumulation of the Hmga2 and Btg2 mRNAs, respectively targeted by mmu-miR-196a and mmu-let-7a/mmu-miR-132, analyzed by qRT-PCR before and after deletion of hAgo2. H. mRNA accumulation of L1_Tf, _Gf and _A sub-types detected by qRT-PCR before and after hAgo2 deletion. I. mRNA accumulation of a single Tf_L1 subtype located on chromosome 17, analyzed by semi-quantitative RT-PCR before and after hAgo2 deletion. (EPS) Click here for additional data file. Figure S5 Expression of AGO2 in Dcr −/− ESCs and microRNA expression in hDcr-complemented Dcr −/− ESCs. A. Accumulation of the Ago2 mRNA analyzed by qRT-PCR in WT, Dcr −/− and Dgcr8_KO mESCs. B. Endogenous AGO2 protein accumulation in DMSO- and MG132-treated in Dcr −/− mESCs. The data depicted are from two independent treatments. C. MiR-302d and miR-21 accumulation detected by qRT-PCR in Dcr Flx/Flx, Dcr −/− and hDcr-complemented Dcr −/− mESCs. (EPS) Click here for additional data file. Figure S6 mRNA and microRNA expression in hDcr-complemented Dcr −/− ESCs before and after differentiation. A. Accumulation of Fgf5 (ectoderm marker), Hmga2, Sox2 and L1_ORF2 mRNAs detected by qRT-PCR before (d0) and 10 days after differentiation (d10) of Dcr Flx/Flx, Dcr −/− and hDcr-complemented Dcr −/− mESCs. B. Accumulation of miR-295, miR-302d, miR-21 and miR-16 analyzed by qRT-PCR before and 10 days after differentiation of Dcr Flx/Flx, Dcr −/− and hDcr-complemented Dcr −/− mESCs. (EPS) Click here for additional data file. Table S1 Primers table. (DOCX) Click here for additional data file. We thank members of the Voinnet laboratory for critical reading of the manuscript and for stimulating discussions, Dr. G. Meister and Dr. A. Bortvin for the gift of the mouse AGO2 (6F4) antibody, and L1_ORF1 antibodies, respectively, Dr. X. Wang for the gift of the inducible Ago1,2,3,4_KO mESC line and Dr. G. Hannon for the gift of Dcr Flx/Flx mice. Isabelle Grandjean and Patricia Diabangouaya are deeply acknowledged for their key contribution to mice crosses and genotyping at the Curie Institute. ==== Refs References 1 Beck CR , Garcia-Perez JL , Badge RM , Moran J V (2011 ) LINE-1 elements in structural variation and disease . Annu Rev Genomics Hum Genet 12 : 187 –215 .21801021 2 Zaratiegui M , Irvine D V , Martienssen R (2007 ) Noncoding RNAs and gene silencing . Cell 128 : 763 –776 .17320512 3 Aravin AA , Sachidanandam R , Bourc'his D , Schaefer C , Pezic D , et al (2008 ) A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice . Mol Cell 31 : 785 –799 .18922463 4 Smith ZD , Chan MM , Mikkelsen TS , Gu H , Gnirke A , et al (2012 ) A unique regulatory phase of DNA methylation in the early mammalian embryo . Nature 484 : 339 –344 .22456710 5 Howlett SK , Reik W (1991 ) Methylation levels of maternal and paternal genomes during preimplantation development . Development 113 : 119 –127 .1764989 6 Packer AI , Manova K , Bachvarova RF (1993 ) A Discrete LINE-1 Transcript in Mouse Blastocysts . Dev Biol 157 : 281 –283 .7683285 7 Kano H , Godoy I , Courtney C , Vetter MR , Gerton GL , et al (2009 ) L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism . Genes Dev 23 : 1303 –1312 .19487571 8 Teixeira FK , Heredia F , Sarazin A , Roudier F , Boccara M , et al (2009 ) A role for RNAi in the selective correction of DNA methylation defects . Science 323 : 1600 –1604 .19179494 9 Slotkin RK , Vaughn M , Borges F , Tanurdzić M , Becker JD , et al (2009 ) Epigenetic reprogramming and small RNA silencing of transposable elements in pollen . Cell 136 : 461 –472 .19203581 10 Bernstein E , Caudy AA , Hammond SM , Hannon GJ (2001 ) Role for a bidentate ribonuclease in the initiation step of RNA interference . Nature 409 : 363 –366 .11201747 11 Stark GR , Kerr IM , Williams BR , Silverman RH , Schreiber RD (1998 ) How cells respond to interferons . Annu Rev Biochem 67 : 227 –264 .9759489 12 Watanabe T , Totoki Y , Toyoda A , Kaneda M , Kuramochi-Miyagawa S , et al (2008 ) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes . Nature 453 : 539 –543 .18404146 13 Babiarz JE , Ruby JG , Wang Y , Bartel DP , Blelloch R (2008 ) Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs . Genes Dev 22 : 2773 –2785 .18923076 14 Arand J , Spieler D , Karius T , Branco MR , Meilinger D , et al (2012 ) In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases . PLoS Genet 8 : e1002750 .22761581 15 Chow JC , Ciaudo C , Fazzari MJ , Mise N , Servant N , et al (2010 ) LINE-1 activity in facultative heterochromatin formation during X chromosome inactivation . Cell 141 : 956 –969 .20550932 16 Weng A , Magnuson T , Storb U (1995 ) Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells . Development 121 : 2853 –2859 .7555712 17 Kanellopoulou C , Muljo SA , Kung AL , Ganesan S , Drapkin R , et al (2005 ) Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing . Genes Dev 19 : 489 –501 .15713842 18 Calabrese JM , Seila AC , Yeo GW , Sharp P a (2007 ) RNA sequence analysis defines Dicer's role in mouse embryonic stem cells . PNAS 104 : 18097 –18102 .17989215 19 Mätlik K , Redik K , Speek M (2006 ) L1 antisense promoter drives tissue-specific transcription of human genes . J Biomed Biotech 2006 : 1 –16 . 20 Yang N , Kazazian HH (2006 ) L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells . Nat Struct Mol Biol 13 : 763 –771 .16936727 21 Kazazian H (2011) Mobile DNA: Finding Treasure in Junk. 1st ed. FT Press Science. 22 Murchison EP , Partridge JF , Tam OH , Cheloufi S , Hannon GJ (2005 ) Characterization of Dicer-deficient murine embryonic stem cells . PNAS 102 : 12135 –12140 .16099834 23 Tay Y , Zhang J , Thomson AM , Lim B , Rigoutsos I (2008 ) MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation . Nature 455 : 1124 –1128 .18806776 24 Chen C , Servant N , Toedling J , Sarazin A , Marchais A , et al (2012 ) ncPRO-seq: a tool for annotation and profiling analysis of ncRNAs from small RNA-seq . Bioinformatics 28 : 3 –5 . 25 Goodier JL , Ostertag EM , Du K , Kazazian HH (2001 ) A novel active L1 retrotransposon subfamily in the mouse . Genome Res 11 : 1677 –1685 .11591644 26 Ip J , Canham P , Choo KHA , Inaba Y , Jacobs Sa , et al (2012 ) Normal DNA Methylation Dynamics in DICER1-Deficient Mouse Embryonic Stem Cells . PLoS Genet 8 : e1002919 .22969435 27 Tsumura A , Hayakawa T , Kumaki Y , Takebayashi S , Sakaue M , et al (2006 ) Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b . Genes Cells 11 : 805 –814 .16824199 28 Ostertag EM , Kazazian HHJ (2001 ) BIOLOGY OF MAMMALIAN L1 RETROTRANSPOSONS . Annual review of genetics 35 : 501 –538 . 29 Naas TP , DeBerardinis RJ , Moran J V , Ostertag EM , Kingsmore SF , et al (1998 ) An actively retrotransposing, novel subfamily of mouse L1 elements . The EMBO journal 17 : 590 –597 .9430649 30 Prak ET , Dodson AW , Farkash EA , Kazazian HH Jr (2003 ) Tracking an embryonic L1 retrotransposition event . PNAS 100 : 1832 –1837 .12569170 31 Ostertag EM , Prak ET , DeBerardinis RJ , Moran JV , Kazazian HH (2000 ) Determination of L1 retrotransposition kinetics in cultured cells . NAR 28 : 1418 –1423 .10684937 32 Ostertag EM , DeBerardinis RJ , Goodier JL , Zhang Y , Yang N , et al (2002 ) A mouse model of human L1 retrotransposition . Nat Genet 32 : 655 –660 .12415270 33 Xie Y , Rosser JM , Thompson TL , Boeke JD , An W (2011 ) Characterization of L1 retrotransposition with high-throughput dual-luciferase assays . NAR 39 : e16 .21071410 34 Xiao S , Xie D , Cao X , Yu P , Xing X , et al (2012 ) Comparative epigenomic annotation of regulatory DNA . Cell 149 : 1381 –1392 .22682255 35 Dueck A , Ziegler C , Eichner A , Berezikov E , Meister G (2012 ) microRNAs associated with the different human Argonaute proteins . NAR 40 : 9850 –9862 .22844086 36 Ciaudo C , Bourdet A , Cohen-Tannoudji M , Dietz HC , Rougeulle C , et al (2006 ) Nuclear mRNA degradation pathway(s) are implicated in Xist regulation and X chromosome inactivation . PLoS Genet 2 : e94 .16789828 37 Wang Y , Medvid R , Melton C , Jaenisch R , Blelloch R (2007 ) DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal . Nat Genet 39 : 380 –385 .17259983 38 Su H , Trombly MI , Chen J , Wang X (2009 ) Essential and overlapping functions for mammalian Argonautes in microRNA silencing . Genes Dev 23 : 304 –317 .19174539 39 Wang D , Zhang Z , O'Loughlin E , Lee T , Houel S , et al (2012 ) Quantitative functions of Argonaute proteins in mammalian development . Genes Dev 26 : 693 –704 .22474261 40 Smibert P , Yang J-S , Azzam G , Liu J-L , Lai EC (2013 ) Homeostatic control of Argonaute stability by microRNA availability . NSMB 1 –9 . 41 Derrien B , Baumberger N , Schepetilnikov M , Viotti C , Cillia J De (2012 ) Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway . PNAS 109 : 15942 –15946 .23019378 42 Johnston M , Geoffroy M , Sobala A , Hay R , Hutvagner G (2010 ) HSP90 Protein Stabilizes Unloaded Argonaute Complexes and Microscopic P-bodies in Human Cells . Mol Biol Cell 21 : 1462 –1469 .20237157 43 Gy I , Gasciolli V , Lauressergues D , Morel J-B , Gombert J , et al (2007 ) Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors . The Plant cell 19 : 3451 –3461 .17993620 44 Yamanaka S , Mehta S , Reyes-Turcu FE , Zhuang F , Fuchs RT , et al (2013 ) RNAi triggered by specialized machinery silences developmental genes and retrotransposons . Nature 493 : 557 –560 .23151475 45 Swergold GD (1990 ) Identification, Characterization, and Cell Specificity of a Human LINE-1 Promoter . Mol Cell Biol 10 : 6718 –6729 .1701022 46 Houseley J , Tollervey D (2009 ) The many pathways of RNA degradation . Cell 136 : 763 –776 .19239894 47 Rosenbloom KR , Sloan C a , Malladi VS , Dreszer TR , Learned K , et al (2012 ) ENCODE Data in the UCSC Genome Browser: year 5 update . NAR 41 : 56 –63 . 48 Fagegaltier D , Bouge AL , Berry B , Poisot E , Sismeiro O , et al (2009 ) The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila . PNAS 106 : 21258 –21263 .19948966 49 Moreno AB , Martínez de Alba AE , Bardou F , Crespi MD , Vaucheret H , et al (2013 ) Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants . NAR 41 : 4699 –4708 .23482394 50 Tam OH , Aravin AA , Stein P , Girard A , Murchison EP , et al (2008 ) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes . Nature 453 : 534 –538 .18404147 51 Hu Q , Tanasa B , Trabucchi M , Li W , Zhang J , et al (2012 ) DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation . NSMB 19 : 1168 –1175 . 52 Paddison PJ , Caudy A a , Hannon GJ (2002 ) Stable suppression of gene expression by RNAi in mammalian cells . PNAS 99 : 1443 –1448 .11818553 53 Billy E , Brondani V , Zhang H , Müller U , Filipowicz W (2001 ) Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines . PNAS 98 : 11443 –14428 . 54 Maillart P, Ciaudo C, Marchais A, Li Y, Jay F, et al.. (2013) Antiviral RNA interference in mammalian cells. In press. 55 Hooper M , Hardy K , Handyside A , Hunter S , Monk M (1987 ) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells . Nature 326 : 292 –295 .3821905 56 Meister G , Landthaler M , Patkaniowska A , Dorsett Y , Teng G , et al (2004 ) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs . Mol Cell 15 : 185 –197 .15260970 57 Bernstein E , Kim SY , Carmell M a , Murchison EP , Alcorn H , et al (2003 ) Dicer is essential for mouse development . Nat Genet 35 : 215 –217 .14528307 58 Vooijs M , Jonkers J , Berns a (2001 ) A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent . EMBO reports 2 : 292 –297 .11306549 59 Penny GD , Kay GF , Sheardown SA , Rastan S , Brockdorff N , et al (1996 ) Requirement for Xist in X chromosome inactivation . Nature 379 : 131 –137 .8538762 60 Landthaler M , Gaidatzis D , Rothballer A , Chen PY , Soll SJ , et al (2008 ) Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs . RNA 14 : 2580 –2596 .18978028 61 Henderson IR , Chan SR , Cao X , Johnson L , Jacobsen SE (2010 ) Accurate sodium bisulfite sequencing in plants . Epigenetics 5 : 47 –49 .20081358 62 Gruntman E , Qi Y , Slotkin RK , Roeder T , Martienssen Ra , et al (2008 ) Kismeth: analyzer of plant methylation states through bisulfite sequencing . BMC bioinformatics 9 : 371 .18786255 63 Rohde C , Zhang Y , Reinhardt R , Jeltsch A (2010 ) BISMA–fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences . BMC bioinformatics 11 : 230 .20459626	24244175	PMC3820764	CC BY	2021-01-05 07:39:56	yes	PLoS Genet. 2013 Nov 7; 9(11):e1003791
==== Front Int J OncolInt. J. OncolIJOInternational Journal of Oncology1019-64391791-2423D.A. Spandidos 2404244110.3892/ijo.2013.2103ijo-43-05-1420ArticlesSIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2 LI HONGQI 12FENG ZHIQIANG 1WU WEIZHANG 1LI JING 1ZHANG JINQIAN 3XIA TINGYI 141 Department of Radiation Oncology, Air Force General Hospital, Beijing 100142;2 Department of Radiation Oncology, Daping Hospital, The Third Military Medical University, Chongqing 400030;3 Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015;4 Department of Radiation Oncology, The General Hospital of Chinese People’s Liberation Army, Beijing 100853, P.R. ChinaCorrespondence to: Dr Tingyi Xia, Department of Radiation Oncology, Air Force General Hospital, Beijing 100142, P.R. China, E-mail: xiatingyi1959@21cn.comDr Jinqian Zhang, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, P.R. China, E-mail: jingwanghou@yahoo.com.cn11 2013 16 9 2013 16 9 2013 43 5 1420 1430 18 7 2013 04 9 2013 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.Lung cancer is the leading cause of death worldwide and associated with dismal prognoses. As a major mitochondrial deacetylase, SIRT3 regulates the activity of enzymes to coordinate global shifts in cellular metabolism and has important implications for tumor growth. Its role as a tumor suppressor or an oncogene in lung cancer is unclear, especially in non-small cell lung carcinoma (NSCLC). To identify the mechanism of SIRT3-interacting proteins, we performed a yeast two-hybrid screen using a human lung cDNA library. One of the positive clones encoded the full-length cDNA of the nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) gene and the interaction between SIRT3 and NMNAT2 was identified. The interaction on growth, proliferation, apoptosis of NSCLC cell lines, and energy metabolism related to SIRT3 were investigated. Screening from the library resulted in NMNAT2 gene. We found that NMNAT2 interacts with SIRT3 both in vitro and in vivo; SIRT3 binds to NMNAT2 deacetylating it. Downregulation of SIRT3 inhibited acetylation of NMNAT2 and NAD+ synthesis activity of the enzyme. Low expression of SIRT3 significantly inhibited mitotic entry, growth and proliferation of NSCLC cell lines and promoted apoptosis, which was related to energy metabolism involving in the interaction between SIRT3 and NMNAT2. Taken together, our results strongly suggest that the binding of SIRT3 with NMNAT2 is a novel regulator of cell proliferation and apoptosis in NSCLC cell lines, implicating the interaction between SIRT3 and NMNAT2, energy metabolism associated with SIRT3. SIRT3 proteinenergy metabolismlung neoplasmsacetylationNMNAT2 protein ==== Body Introduction Lung cancer is the most common cause of cancer deaths worldwide, ∼1.2 million deaths people die from it per year (1). It is the leading type of new cancer cases leading to death (2). Non-small cell lung carcinoma (NSCLC) is the most frequent subtype, ∼85% of all cases. Most of NSCLC patients have locally advanced or distant metastatic disease (stage III/IV) from onset of symptoms. It is strongly associated with poor prognosis and has a 5-year survival rate of <10 and 5% in male and female patients, respectively (3). Platinum-doublet regimen remains the standard with modest survival benefits, although also improved response rates (RRs) have been reported. However, the RRs is ranging from 17 to 32% and progression-free survival (PFS) of 3.1–5.5 months, overall survival (OS) of 7.4–11.3 months (4–7). To improve clinical outcome for patients with lung cancer, targeted therapies are increasingly being used, particularly in patients with specific molecular features. Altered metabolism is a hallmark of tumor cells supporting rapid cell proliferation (8). Many metabolic intermediates that support cell growth are provided by mitochondria; consequently, there is great interest in elucidating how mitochondrial metabolic pathways are regulated. The role of the conserved sirtuin family of deacetylases in nutrient-sensitive regulation of metabolic pathways is important, especially on the mitochondrial sirtuin, SIRT3. By deacetylating proteins involved in multiple mitochondrial processes, SIRT3 can co-ordinate global shifts in mitochondrial activity, with important implications for tumor growth (9). The vision of Warburg on cancer cell metabolism, that sidelined mitochondria as dysfunctional bystanders, must be revised. It includes the critical contribution of mitochondria supporting tumor growth. The main function of the mitochondrion is the production of energy, in the form of adenosine triphosphate (ATP). ATP from non-proliferating cells are rewired to serve as biosynthetic factories in rapidly proliferating cells and SIRT3 could serve as an important regulator of the balance between glycolytic and anabolic pathways and mitochondrial oxidative metabolism to regulate tumor cell growth (9,10). In nutrient-deprived conditions, SIRT3 deacetylates numerous mitochondrial proteins to promote nutrient oxidation and ATP production. Conversely, low SIRT3 activity increases ROS production, which signals through HIF1α to increase glycolytic metabolism and cellular proliferation. Increasing evidence indicates that SIRT3 plays a unique regulatory role, integrating mitochondrial regulation with intracellular signaling cascades. By targeting more than a half a dozen key metabolic enzymes, SIRT3 is perfectly positioned to orchestrate coordinated shifts in mitochondrial metabolism, with potential implications for diseases that rely on mitochondrial metabolic activities. It will be important for future studies to increase our understanding of how mammalian sirtuins integrate metabolism with signaling cascades by post-translational modification of diverse substrates and how modulation of sirtuin activity can influence health and disease (9,10). However, the role of SIRT3 as a tumor suppressor or an oncogene in lung cancer is unclear, especially in NSCLC. Herein, to address this question, the interaction between SIRT3 and NMNAT2 and its involvement in growth, proliferation and apoptosis of NSCLC cell lines was studied. Energy metabolism associated with SIRT3 was implicated and will be further studied on the interactive mechanisms in NSCLC cell lines with the interaction and possible applications in clinical treatment for lung cancer, especially NSCLC. Materials and methods Culture cells Human NSCLC cell lines A549, H1299 and PC-9 from ATCC were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycinneomycin (PSN) Antibiotic Mixture (Invitrogen, USA) at 37°C in a humidified 5% CO2/95% air environment. Plasmids For the yeast two-hybrid assay, the bait plasmid pGBKT7-SIRT3 was generated by inserting a PCR-amplified cDNA fragment containing SIRT3 into pGBKT7 (Clontech). SIRT3 was cloned into pBIND (Promega), pEGFP-C1 (Clontech), pGEX-2T vector (GE). Human full-length NMNAT2 cDNA and the cDNAs for its truncated mutants (NMNAT2 N and NMNAT2 C1–2) were PCR-amplified and subcloned in-frame into pCMV-5a (Sigma). NMNAT2 was cloned into pACT (Promega), pDsRed1-N1 (Clontech), pCDNA-3.1(−) myc/his (A) vector (Invitrogen). All plasmids were verified by restriction enzyme analysis and DNA sequencing. The hSIRT3 adenovirus was from Vector BioLabs (Philadelphia, PA, USA). Cell transfection and adenovirus infection At 24–30 h after seeding, NSCLC cells were used for adenovirus infection or immunostaining as described below. The adenoviruses were used at a multiplicity of infection (MOI) of 10. Cos7 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with penicillin-streptomycin and 10% fetal bovine serum (complete growth medium). Cells were transfected with appropriate plasmids using Superfect transfection reagent (Qiagen) according to the manufacturer’s protocol. For the RNA interference experiments, NSCLC cells were transfected with 200 nM On-Target-plus small interfering RNA (siRNA) specific for hSIRT3 using Dharma-FECT transfection reagents according to the manufacturer’s protocol (11). Yeast two-hybrid assay The bait plasmid pGBKT7-SIRT3 was used to screen a human lung cDNA library in pGADT7 according to the manufacturer’s instructions (Clontech). Transformants were placed on synthetic medium lacking tryptophan, leucine, adenine and histidine but containing 1 mM 3-aminotriazole. Approximately 4 million transformants were screened. The screened positive clones were also verified by one-on-one transformations and selection on agar plates lacking tryptophan and leucine, or adenine, histidine, tryptophan and leucine, respectively and were also processed by β-galactosidase assay. Co-immunoprecipitation For transfection-based co-immunoprecipitation assays, cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen), lysed in 0.5 ml lysis buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 0.25% NP-40, 1 mM DTT and protease inhibitor tablets from Roche) and immunoprecipitated with Protein G Plus/Protein A Agarose Suspension beads (Calbiochem) for 3 h at 4°C. The beads were washed 4 times with the lysis buffer and eluted in SDS sample buffer. The eluted proteins were separated by SDS-PAGE, followed by western blotting with antibody. Mammalian two-hybrid analysis To test the hypothesis of NMNAT2 interacting with SIRT3 in vivo using the CheckMate™ Mammalian Two-Hybrid system (Promega), plasmids pACT-NMNAT2 and plasmids pBIND-SIRT3 were constructed that were used for cotransfections of cells cultured in 6-well plates. Two micrograms of pACT-NMNAT2 plasmid and 2 μg of pBIND-SIRT3 plasmid were used for cotransfections by Lipofectamine 2000. The plasmids pACT-NMNAT2+pBIND, pACT+pBIND-SIRT3, pACT, pBIND and blank were used for transfections, respectively, as a negative control. The plasmids pBIND-Id and the pACT-MyoD Control Vector were cotransfectioned as a positive control. At 48 h after transfection, cells were washed 3 times with PBS and then lysed with passive buffer. Firely luciferase assays were performed using the Dual-Glo Luciferase assay system (Promega) following the manufacturer’s instructions. The firefly results were corrected for transfection efficiency using the renilla luciferase. Significance was determined using the paired Student’s t-test on the mean of three different experiments. GST pull-down assay The GST- and His-tagged fusion proteins were expressed and purified by glutathione-Sepharose 4B beads (GE) and Ni-NTA agarose (GE), respectively. The expression plasmid for NMNAT2 was used for in vitro transcription and translation in the TNT system (Promega). The NMNAT2 or the purified His-tagged fusion protein was incubated with GST fusion protein bound to glutathione-Sepharose beads in 0.5 ml of the binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3 mM DTT, 0.1% NP-40) at 4°C. The beads were precipitated, washed 4 times with the binding buffer, eluted by boiling in SDS sample buffer and analyzed by SDS-PAGE. Western blotting was performed with anti-His (Santa Cruz). A quantitative measurement of the band intensity was performed using the GE Typhoon Trio (GE, USA). Colocalization Cells were grown on glass coverslips in culture plates. Cells were co-transfected with plasmids, 2 μg of pEGFP-C1-SIRT3 (green fluorescent protein, GFP) and 2 μg of pDS-RED1-N1- NMNAT2 by Lipofectamine™ 2000 for 48 h and then cells were treated with 4% paraformaldehyde (PFA) for 10 min, washed 3 times with PBS, stained with PBS including 0.1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 30 min at 30°C. Confocal images were acquired using Zeiss 510 META confocal microscope. Western blot analyses Western blot analyses were prepared as described (12). NSCLC cell cultures were lysed and placed in RIPA buffer (Cell signaling) with 1 mM PMSF on ice for 30 min. Cell lysates were centrifuged at 14,000 g for 10 min and cell extracts were mixed with a 1:4 volume of SDS-PAGE loading buffer (10% β-mercaptoethanol, 10% glycerol, 4% SDS, 0.01% bromophenol blue and 62.5 mM Tris-HCl, pH 6.8) and heated to 65°C for 15 min. Samples were loaded on a 10% resolving SDS-polyacrylamide gel and transferred to polyvinyldifluoridine membranes. Membranes were incubated overnight at 4°C with rabbit polyclonal anti-SIRT3 (1:500; Abcam), NMNAT2 antibody (Santa Cruz, 1:1,000 dilution), β-actin (Santa Curz, 1:1,000), GST (Santa Cruz, 1:1,000), Flag (Santa Cruz, 1:1,000), His (Santa Cruz, 1:1,000), SIRT3 (Cell Signaling, 1:1,000), Myc (Santa Cruz, 1:1,000). All affinity-purified and species-specific fluorophore-conjugated secondary antibodies were obtained from Santa Cruz and used at dilutions between 1:500 and 1:800. Immunoreactivity was detected with luminol reagent (GE). In vitro acetylation-deacetylation assay Unlabeled Flag-NMNAT2 was in vitro translated using the TNT coupled transcription-translation rabbit reticulocyte lysate kit (Promega) and immunoprecipitated using anti-Flag M2 affinity beads. Beads with bound protein were washed 4–5 times with radioimmunoprecipitation assay buffer followed by a phosphate-buffered saline (PBS) wash. The final wash was performed in 1X HAT buffer (50 mM Tris, pH 8.0, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol). A typical acetylation reaction mixture contained 1 μg active p300/CBP-associated factor (PCAF) enzyme (Upstate Biotechnology), 0.3 mM acetyl coenzyme A (Sigma) and 10 mM sodium butyrate in 1X HAT buffer. Reaction mixtures were incubated at 30°C for 2 h on a rotator. For the deacetylation assay, acetylated Flag-NMNAT2 that bound to the beads was washed as described above and resuspended in 1X HDAC buffer (50 mM Tris, pH 8.0, 4 mM MgCl2, 0.2 mM dithiothreitol). The acetylated Flag-NMNAT2 substrate was either incubated only in 1X HDAC buffer (control) or with equal amounts of Myc-SIRT3 with or without NAD (1 mM) in 1X HDAC buffer. Myc-SIRT3 catalytic was produced in a prokaryotic expression system as described (15). Reaction mixtures were incubated for 2–3 h at 37°C on a rotator. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by western blotting (11). Cellular oxygen consumption assay Steady state cell respiration in cells was measured in non-buffered DMEM containing 5.5 mM glucose for cells with XFe24 analyzer (Seahorse Bioscience) according to the manual. ATP production assay Steady state cellular ATP levels were measured by using ATP bioluminescence assay kit CLS II in accordance with the protocol (Roche). The optimal detection range is between 10−7 and 10−10 M. The pH of the sample should be in the range of 7.6–8.0. ATP standard was diluted with redist water by serial dilution in the range of 10−5 and 10−10 M. Luciferase reagent was added to the samples/standards by automated injection and the measurement was started after a 1-sec delay and integrated for 1–10 sec. The blank (no ATP) was subtracted from the raw data and ATP concentrations calculated from a log-log plot of the standard curve data. NAD assay NAD assay was performed as previously described (16–18). Cells were extracted in 0.5 N HClO4, neutralized with 3 M KOH/125 mM Gly-Gly buffer (pH 7.4) and centrifuged at 10,000 × g for 5 min. Supernatants were mixed with a reaction medium containing 0.1 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 0.9 mM phenazine methosulfate, 13 U/ml alcohol dehydrogenase, 100 mM nicotinamide and 5.7% ethanol in 61 mM Gly-Gly buffer (pH 7.4). The A560 nm was determined immediately and after 10 min and results were calibrated with NAD standards. Cell synchronization, BrdU labeling and mitotic index To avoid potential carry-over effects of plasmids transfection-induced cell cycle defects in the previous cycle on the following mitotic entry during the next cycle, we transfected plasmids into cells during the interval between two thymidine blocks, so that we were able to evaluate direct impact of interaction of NMNAT2 with SIRT3 on mitotic entry (13). Cells were synchronized by double thymidine block. Briefly, cells were plated at 40% confluency and arrested with 2 mM thymidine. After 19-h incubation, cells were washed 4 times with fresh medium and treated with siRNA (NMNAT2 and control). After incubation with DNA-lipid mixture for 3 h, cells were washed twice and incubated in fresh medium for an additional 5 h. Subsequently, cells were cultured in medium containing 2 mM thymidine and 2 μg/ml puromycin for the second arrest and drug selection. After 16-h incubation, cells were released into the cell cycle by incubation in fresh medium. Cells were collected or fixed at indicated time-points and subjected to specific analyses. BrdU labeling for evaluation of DNA synthesis Following release from the second thymidine arrest at indicated time-points, cells grown in 12-well plate were pulse labeled with BrdU (50 μM) for 30 min. After three washes of PBS, cells were fixed with 1 ml of Carnoy’s fixative (3 parts methano 1:1 part glacial acetic acid) at −20°C for 20 min and followed by three washes of PBS. Subsequently, DNA was denatured by incubation of 2M HCl at 37°C for 60 min, followed by three washes in borate buffer (0.1 M borate buffer, pH 8.5). After incubation with the blocking buffer, cells were stained with anti-BrdU antibody (BD Biosciences, 1:100) overnight at 4°C. After washes in PBS, cells were incubated with Texas Red-conjugated anti-mouse goat IgG for 30 min at RT. After washes, cells were mounted and BrdU positive cells were manually scored under immunofluorescence microscope. Mitotic events were scored by time-lapse videomicroscopy and DNA staining. Cells were synchronized as described above. Real-time images were captured every 10 min with Openlab software. Mitotic events of cells were scored by their morphological change (from flat to round-up). For each experiment, ≥800 cells were videotaped, tracked and analyzed. Alternatively, nocodazole (100 ng/ml) was added into the medium after release, cells were collected, fixed and stained with DNA dye (Hoechst 33258). Mitotic cells were scored by nuclear morphology and DNA condensation. Cell growth and proliferation assay Cell growth for 48 h was determined by the colorimetric tetrazolium derived XTT (sodium 3′-(1-(phenylaminocarbonyl)-3, 4-tetrazolium)-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay (Roche Applied Science, Mannheim, Germany) and DNA synthesis of cells was assessed by the BrdU (bromodeoxyuridine) incorporation assay (Roche Applied Science). For the cell growth and proliferation assay, at 48 h after culture, the cells of each group were re-seeded in SWNHs-coated 96-well plates at a density of 0.3–1×104 cells per well. After 48 h, XTT and incorporated BrdU were measured colorimetrically using a microtiter plate reader (Bio-Rad) at a wavelength of 450 nm (14). Cell viability assay Cell viability was determined using a CCK-8 cell viability assay kit (Dojindo Laboratories, Japan). The cells (5×103 cells/well) were pretreated with various methods as indicated and then incubated for 16 h in a 96-well plate. Cell viability assay kit solution (10 μl) was added to each well of the plate. After incubation for 1 h at 37°C in the dark, absorbances were measured at 450 nm using a multi-well plate reader (15). Determimation of apoptosis Apoptotic cells were identified by fluorescence-activated cell sorting (FACS) using Annexin V-Fluos (Biolegend) following the protocol of the manufacturer. Statistical analyses Continuous normally distributed variables are presented as the mean ± standard deviation (SD). For statistical comparison of quantitative data between groups, analysis of variance (ANOVA) or t-test was performed. To determine differences between groups not normally distributed, medians were compared using Kruskal-Wallis ANOVA. χ2 test was used when necessary for qualitative data. The degree of association between variables was assessed using Spearman’s non-parametric correlation. All statistical analyses were carried out using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Probability values of ≤0.05 or less were considered to be statistically significant. Results Identification of NMNAT2 as a SIRT3-interacting protein by yeast two-hybrid system To identify novel SIRT3-interacting proteins, we performed a yeast two-hybrid screening of a human lung cDNA library using full-length SIRT3 as bait. Screening of 4 million transformants resulted in the isolation of several positive clones, which were identified to encode the full-length cDNA of NMNAT2 gene. To further identify the interaction of NMNAT2 with SIRT3 in yeast, we next examined the ability of SIRT3 protein to bind to NMNAT2 in yeast cells (Fig. 1A). These data demonstrated that SIRT3 interacts with NMNAT2 in yeast cells. Interaction of SIRT3 with NMNAT2 in vivo and in vitro To identify the interaction between SIRT3 and NMNAT2, we next examined the ability of SIRT3 protein to bind to NMNAT2 in mammalian cells. Cells were co-transfected with Flag-tagged SIRT3, Myc -tagged NMNAT2, or Myc control vector. Immunoprecipitation (IP) of cell lysates with an anti-Flag monoclonal antibody was followed by immunoblotting (IB) with anti-Myc. Results showed specific interaction between the Flag-SIRT3 and Myc-NMNAT2 (Fig. 1B). To test the hypothesis of SIRT3 interacting with NMNAT2 in vivo, the CheckMate™ Mammalian Two-Hybrid system (Promega) was used. The plasmids pACT-SIRT3 and pBIND-NMNAT2 were used for cotransfections by Lipofectamine 2000. The plasmids pACT-SIRT3+pBIND, pACT+pBIND-NMNAT2, pACT, pBIND and blank were used for transfections, respectively, as a negative control. The plasmids pBIND-Id and the pACT-MyoD Control Vector were cotransfected as a positive control. After transfection for 48 h, cells were lysed. Luciferase assays were performed using the Dual-Glo Luciferase assay system (Promega) following the manufacturer’s instructions. The results were corrected for transfection efficiency using renilla luciferase. Significance was determined using the paired Student’s t-test on the mean of three different experiments. The luciferase levels of pACT-SIRT3 and pBIND-NMNAT2 were ≥2.64 times higher than negative control (P<0.05) (Fig. 1C). The expression of SIRT3 and NMNAT2 in lysate of those cells was detected by WB with anti-SIRT3 and anti-NMNAT2 monoclonal antibody (Fig. 1D). To demonstrate the interaction of SIRT3 and NMNAT2 in vitro, GST pull-down assays were performed in which in vitro translated His-SIRT3 was incubated with full-length GST-NMNAT2 or Flag. As shown in Fig. 1E, SIRT3 interacted with GST-NMNAT2 but not with Flag alone (Fig. 1E). To test the colocalization of SIRT3 and NMNAT2 in cells, cells were grown on glass coverslips in culture plates then co-transfected with plasmids pEGFP-C1-SIRT3 and pDS-RED1-N1-NMNAT2. After 48 h, cells were stained with PFA and DAPI, confocal images were acquired using Zeiss 510 META confocal microscope. NMNAT2 (red, Fig. 1F) and SIRT3 (green, Fig. 1G) protein, all localized to the cytoplasma. The nuclear of cells (blue, Fig. 1H) were stained by DAPI. The overlaid images indicated that SIRT3 overlapped partly with NMNAT2 (Fig. 1I) in the cytoplasma. These results indicate that SIRT3 interacted with NMNAT2 in vivo and in vitro. Mapping of the NMNAT2 and SIRT3 interaction regions To identify the region of SIRT3 required for its interaction with NMNAT2, Co-IP experiments were performed in which three deletion mutants. SIRT3 is cleaved by caspase-3 at two sites (D28 and D107, Fig. 2A). As shown in Fig. 2A, both full-length and the carboxy-terminal fragments of SIRT3 C1 protein interacted with Myc-NMNAT2, but the amino-terminal fragment of SIRT3 (SIRT3N, residues 1–28) did not interact with NMNAT2 (Fig. 2A). As shown in Fig. 2B, the Flag-SIRT3 C1 (29–307) bound specifically to NMNAT2, but the Flag-N (1–28) and FLAG-SIRT3 C2 (107–307) did not, suggesting that the 28–107 region is required for the interaction with NMNAT2. Among C-terminal cleavage fragments, SIRT3-C1 appeared to bear the highest affinity to Myc-NMNAT2, indicating that the region between residues 28–107 may be important for SIRT3-NMNAT2 interaction (Fig. 2B). We also tested the NMNAT2-SIRT3 interaction in vitro using purified SIRT3 proteins which were in agreement with our Co-IP results. SIRT3 deacetylates NMNAT2 under in vitro and in vivo assay conditions To test whether SIRT3 deacelylated NMNAT2, in an acetylation buffer Flag-NMNAT2 was incubated with PCAF. Acetylation of the protein was determined by western blotting with antiacetyllysine antibody (Fig. 3A). Flag-NMNAT2 was acetylated in vitro with PACF and it was precipitated with Flag M2 beads. Acetylated Flag-NMNAT2 was then incubated with beads containing SIRT3 in a deacetylation buffer with or without NAD. SIRT3 was immunoprecipitated from stable A549 cells. This indicated that SIRT3 deacetylated of NMNAT2 in vitro is dependent on the NAD level (Fig. 3B and C). Stable cells expressing SIRT3 were induced to overexpress with Flag-NMNAT2 and treated with NAM (10 mM for 24 h) and/or TSA (5 μM for 6 h) as indicated. Flag-NMNAT2 was immunoprecipitated and the level of acetylation was analyzed by probing with anti-acetyllysine antibody. It indicated that in vivo SIRT3 deacetylated NMNAT2 dependent on the TSA and NAM levels, especially related to TSA (Fig. 3D and E). Together, these data demonstrated that SIRT3 targets the enzyme NMNAT2, which catalyzes the formation of NAD (+) from nicotinamide mononucleotide (NMN) and ATP. Interaction of NMNAT2 with SIRT3 increases mitochondrial functions of A549 cells To study the role interaction between SIRT3 and NMNAT2 on mitochondrial function, intact cellular basal oxygen consumption rates (OCR) of A549 cells were measured by Seahorse XF24 analyzer. The OCR of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compared to control (P<0.01), whereas, the OCR of A549 cells decreased significantly with SIRT3 interference (P<0.01) (Fig. 4A). Steady-state cellular ATP levels of A549 cells were also measured. The steady-state cellular ATP level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compared to the A549 cells. The OCR of A549 cells decreased significantly (P<0.01) (Fig. 4B) with downregulation of SIRT3. There was significant expression of SIRT3 in NSCLC cell line A549. Adenovirus infection caused significant increase of SIRT3 in NSCLC cell lines. Transfection with SIRT1 siRNA caused reduction of protein after transfection (Fig. 4C). This suggested that SIRT1 siRNA selectively silences SIRT3 expression. NAD level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compared to A549 cells. Downregulation of SIRT3, the OCR of A549 cells decreased significantly (P<0.01) (Fig. 5). The result showed that the interaction between NMNAT2 and SIRT3 promotes mitochondrial function levels of A549 cells. Interaction of NMNAT2 with SIRT3 promotes mitotic entry, cell growth and proliferation of A549 cells To investigate the role of interaction between NMNAT2 and SIRT3 on mitotic entry, cell growth and proliferation, A549 cells were transfected with plasmids and synchronized at the G1/S transition as described in Materials and methods. Cells were pulse labeled with BrdU (50 μM) for 30 min at indicated time-points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of three independent experiments (Fig. 6A). Cell cycle progression of >1,000 cells was recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells (Fig. 6B). The results demonstrated that the interaction of NMNAT2 with SIRT3 promoted mitotic entry (Fig. 6). By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P<0.001). The growth of cells were significantly inhibited by interfering SIRT3 (P<0.001) (Fig. 7A). Cell viability was evaluated by CCK-8 assay. The result showed that the proliferation of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significantly than in A549 cells (P<0.001). The growth of cells were significantly inhibited by interfering SIRT3 (P<0.001) (Fig. 7B). Our results indicated that the interaction of NMNAT2 with SIRT3 did not affect DNA synthesis, but low expression of SIRT3 significantly inhibited mitotic entry, growth and proliferation of A549 cells. Interaction of NMNAT2 with SIRT3 inhibits apoptosis of A549 cells Our result showed that the apoptosis of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was less than A549 cells (P>0.05). The apoptosis of cells were significantly promoted by interfering SIRT3 (P<0.001). This indicated that the interaction of NMNAT2 with SIRTs inhibited apoptosis of A549 cells (Fig. 8). Low expression of SIRT3 significantly promoted A549 cell apoptosis (the results from H1299 and PC-9 cells were similar to those from A549 cells). Discussion NSCLC tumorigenesis follows a stepwise progression from hyperplasia to metaplasia, dysplasia, carcinoma in situ and, finally, to invasive tumors. To study the molecular pathogenesis of cancer during tumorigenesis, genes that inhibited lung carcinogenesis were identified. There are seven SIRT isoforms in mammals, with diverse biological functions including gene regulation, metabolism and apoptosis. SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways (10). However, its role in response to NSCLC remains unknown. In this study, we examined the role of SIRT3 in NSCLC. At first, we investigated the novel SIRT3-interacting protein; we performed a yeast two-hybrid screening of a human lung cDNA library using full-length SIRT3 as a bait. Screening of transformants resulted in the isolation of several positive clones, which were identified encoding the full-length cDNA of NMNAT2 gene. We next examined the ability of SIRT3 protein to bind to NMNAT2 in yeast cells (Fig. 1A). These data demonstrated that SIRT3 interacts with NMNAT2 in yeast cells. We examined the ability of SIRT3 protein to bind to NMNAT2 in mammalian cells. Our results showed a specific interaction between SIRT3 and NMNAT2 in NSCLC cells (Fig. 1B–D). To demonstrate the interaction of SIRT3 and NMNAT2 in vitro, GST pull-down assays were performed in which in vitro translated His-SIRT3 was incubated with full-length GST-NMNAT2 or Flag. As shown in Fig. 1E, SIRT3 interacted with GST-NMNAT2 but not with Flag alone (Fig. 1E). To test the colocalization, the cells were co-transfected with plasmids pEGFP-C1-SIRT3 and pDS-RED1-N1-NMNAT2. NMNAT2 (red, Fig. 1F) and SIRT3 (green, Fig. 1G) protein all localized to the cytoplasma. The overlaid images indicated that SIRT3 overlapped partly with NMNAT2 (Fig. 1I) in cytoplasma. These results indicate that SIRT3 interacted with NMNAT2 in vivo and in vitro. As shown in Fig. 2A, both full-length and the carboxy-terminal SIRT3 C1 fragments (residues 29–307) of SIRT3 protein interacted with Myc-NMNAT2. As shown in Fig. 2B, the results suggest that the 28–107 region is required for the interaction with NMNAT2. Among C-terminal cleavage fragments, SIRT3-C1 appeared to bear the highest affinity to Myc-NMNAT2, indicating that the region between residues 28–107 may be important for SIRT3-NMNAT2 interaction (Fig. 2B). NMNAT2 belongs to the nicotinamide mononucleotide adenylyltransferase (NMNAT) enzyme family, members of which catalyze an essential step in the NAD (NADP) biosynthetic pathway. Unlike the other human family member, which is localized to the nucleus and is ubiquitously expressed; this enzyme is cytoplasmic and is predominantly expressed in the brain. Two transcript variants encoding different isoforms have been found for this gene (16). NMNAT2 is expressed predominantly in neurons (17–21). Its protein product has been shown to localize to the trans-Golgi complex (17,18), where it is packaged and transported down axons to the synapse (22). In addition to differences in tissue expression and intracellular localization, there is an isofrom specific domain on each of the NMNAT genes (23). In NMNAT2 this region is palmitoylated at two cysteine residues and, when cleaved, the NAD+ synthesis activity of the enzyme increases significantly (18,24). This provides a mechanism to increase the cytosolic pool of NAD+ quickly in response to a stimulus such as cell stress. We tested our hypothesis that SIRT3 deacetylated NMNAT2. Our study showed an association of NMNAT2 with SIRT3 suggesting that NMNAT2 is the deacetylation target of SIRT3, which had the ability to deacetylate NMNAT2 in an NAD-dependent manner (Fig. 3). NMNAT2 was highly acetylated in cells treated with the HDAC inhibitors TSA and NAM compared to non-treated controls. When NMNAT2 was analyzed from SIRT3-expressing cells, we found that it was substantially deacetylated in these cells but not in cells expressing SIRT3. These data thus indicated that SIRT3 is one of the major regulators of NMNAT2 acetylation in vivo. SIRT3 was the only sirtuin implicated in extension of life span in human (25) and recent evidence has shown its involvement in mitochondrial energy metabolism and biogenesis (26) and preservation of ATP biosynthetic capacity in the heart (26). SIRT3 was shown to regulate the activity of acetyl-CoA synthetase 2 (AceCS2), an important mitochondrial enzyme involved in generating acetyl-CoA for the tricarboxylic acid (TCA) cycle. In these studies, SIRT3 knockout resulted in a marked decrease of basal ATP level in vivo (27). Recent studies in cardiomyocytes demonstrated the protective role of SIRT3 from oxidative stress and hypertrophy (28,29). Accordingly, the evidence suggests that SIRT3 could also have a pivotal role in protecting neurons from injury due to conditions that promote bioenergetic failure, such as excitotoxicity. Mitochondrial localization of SIRT3 plays a role in various mitochondrial functions, such as maintaining basal ATP level and regulating apoptosis. SIRT3 has been shown to regulate energy homeostasis (26). As a critical factor in energy metabolism for cell survival, nicotinamide adenine dinucleotide (NAD) has drawn considerable interest. NAD is an essential molecule playing a pivotal role in energy metabolism, cellular redox reaction and mitochondrial function. Recent studies have revealed that maintaining intracellular NAD is important in promoting cell survival in various types of diseases, including axonal degeneration, multiple sclerosis (MS), cerebral ischemia and cardiac hypertrophy (28,30–39). Loss of NAD decreases the ability of NAD-dependent cell survival factors to carry out energy-dependent processes, leading to cell death. In this study, we identified NMNAT2 as a new target of SIRT3 deacetylase. NMNAT2 is found in mitochondria and identified to target SIRT3. The function of the interaction between SIRT3 and NMNAT2 have been identified. The results showed that the interaction of NMNAT2 with SIRT3 increases mitochondrial functions of A549 cells (Figs. 4 and 5). the roles of SIRT3 and NMNAT2 on NSCLC cell lines related to energy metabolism were associated with the interaction between SIRT3 and NMNAT2. Moreover, the interaction of NMNAT2 with SIRT3 promoted mitotic entry, cell growth and proliferation of A549 cells and inhibited apoptosis of A549 cells (Figs. 6–8). Lung cancer is the leading cause of cancer-related mortality in the worldwide, which is the third most common malignant disease and ∼75% of all diagnosed lung cancers are non-small cell lung carcinoma (NSCLC) and is associated with dismal prognoses. Chemotherapy in addition to surgical resection and radiotherapy remains a basic strategy for treatment of malignant tumors. Unfortunately, it has been shown that NSCLC only has a limited sensitivity to chemotherapeutic drugs. A major obstacle in the treatment of patients is the inherent resistance to chemotherapeutic agents. Inhibition of cell apoptosis pathway was reported as one important cellular mechanism responsible for the resistance of NSCLC cells to treatment. Thus, our results strongly suggest that the binding of SIRT3 with NMNAT2 is a novel regulator of cell proliferation and apoptosis in NSCLC cell lines implicating the interaction between SIRT3 and NMNAT2 and energy metabolism associated with it. This study demonstrates that the interaction between SIRT3 and NMNAT2 may be a novel opportunity for future research for treatment of NSCLC. This study was supported by grants from the National Natural Science Foundation of China (nos. 30600524, 81071990 and 81201758), Science and Technology Planning Project of Guangdong Province (nos. 2012A030400055, 2010B080701088, 2011B080701096 and 2011B031800184), Science and Technology projects of Guangzhou (nos. 2011J410010 and 2011J4300066). Figure 1. NMNAT2 interacts with SIRT3 in yeast, in mammalian cells and in vitro. (A) Identification of NMNAT2 as a SIRT3-interacting protein by the yeast two-hybrid assay. Yeast AH109 cells were transformed with different plasmids and grown on SD/-Trp-Leu-His-Ade. +, grown within 96 h; −, no growth within 96 h. Positive colonies were tested for β-galactosidase activity. +, turned blue within 2 h; −, did not turn blue within 2 h. (B) Interaction of NMNAT2 with SIRT3 in mammalian cells. A549 cells, cultured in regular medium, were transfected with expression plasmids as indicated. Immunoprecipitation (IP) and immunoblotting (IB) were performed using anti-FLAG or anti-Myc monoclonal antibody respectively. (C) To test NMNAT2 interacting with SIRT3 in vivo by Mammalian Two-Hybrid system. A549 cells were transfected with expression plasmids as indicated and cultured in regular medium. At 48 h after transfection, cells were evaluated by firely luciferase assays. Significance was determined on the mean of three different experiments. The luciferase levels of pACT-NMNAT2 and pBIND-SIRT3 were ≥2.64 times higher than negative control (P<0.05). These results indicate that NMNAT2 interacted with SIRT3 in vivo and in vitro. Values are means of three experiments. All data are presented as the mean ± SEM. (D) The expressing of NMNAT2 and SIRT3 in lysate of the cells. The lysates of A549 cells transfected with those plasmids were detected by IB with anti-NMNAT2 and anti-SIRT3 monoclonal antibody. (E) Interaction of SIRT3 with NMNAT2 in vitro. Glutathione-Sepharose beads bound with FLAG-SIRT3 or with FLAG were incubated with His-NMNAT2. After washing the beads, the bound proteins were eluted and subjected to SDS-PAGE and autoradiography. To demonstrate the colocalization of NMNAT2 and SIRT3 in A549 cells, they were cultured, then co-transfected with plasmids. After 48 h, cells were stained with PFA and DAPI; confocal images were acquired using Zeiss 510 META confocal microscope. NMNAT2 protein (red) localized to the cytoplasma (F). SIRT3 (green) localized in the cytoplasma (G). The nuclear of cells (blue) were stained by DAPI (H). The overlaid images indicated that NMNAT2 overlapped partly with SIRT3 in the cytoplasma (I). Figure 2. Map of the NMNAT2 and SIRT3 interaction regions. (A) Mapping of SIRT3 interaction region in NMNAT2. (B) Co-immunoprecipitation of NMNAT2 and SIRT3. Map of the NMNAT2 SIRT3-interacting domains. Myc-SIRT3 and NMNAT2-Flag and its derivatives were overexpressed in A549 cells. NMNAT2-Flag protein was pulled down by Protein G Plus/Protein A Agarose Suspension beads. The presence of SIRT3 was detected by Myc immunoblotting. Figure 3. SIRT3 deacetylates NMNAT2 under in vitro and in vivo assay conditions. (A) In an acetylation buffer Flag-NMNAT2 was incubated with PCAF and acetylation of protein was determined by western blotting with antiacetyllysine antibody. (B) Deacetylation of NMNAT2 by SIRT3 in vitro. Flag-NMNAT2 was acetylated in vitro with PACF and it was precipitated with Flag M2 beads. Acetylated Flag-NMNAT2 was then incubated with beads containing SIRT3 in a deacetylation buffer with or without NAD. SIRT3 was immunoprecipitated from stable A549 cells. (C) Quantification of NMNAT2 deacetylation by SIRT3. (D) In vivo deacetylation of NMNAT2 by SIRT3. Stable cells expressing SIRT3 were induced to overexpress with Flag-NMNAT2 and treated with NAM (10 mM for 24 h) and/or TSA (5 μM for 6 h) as indicated. Flag-NMNAT2 was immunoprecipitated and the level of acetylation was analyzed by immunoblotting with antiacetyllysine antibody. (E) Quantification of NMNAT2 deacetylation by SIRT3 in vivo. Values are means of three experiments. All data are presented as the mean ± SEM. Figure 4. Interaction of NMNAT2 with SIRT3 boosted strongly mitochondrial functions of A549. Intact cellular basal oxygen consumption rates (OCR) of A549 cells were measured by Seahorse XF24 analyzer. The OCR of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compare to A549 cells (P<0.01). SIRT3 interference decreased significantly the OCR of A549 cells (A) (P<0.01). The steady-state cellular ATP level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compared to A549 cells, whereas, downregulation of SIRT3, decreased the OCR significantly in A549 cells (B) (P<0.01). Adenovirus infection caused significant increase of SIRT3 in NSCLC cell lines. Transfection with SIRT3 siRNA caused reduction of proteins after 48 h (C). Values are the means of three experiments. All data are presented as the mean ± SEM. Figure 5. Interaction of NMNAT2 with SIRT3 increases NAD levels of A549. NAD level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in the A549 cells alone. Downregulation of SIRT3, decreased significantly (P<0.01) the OCR of A549 cells. Values are the means of three experiments. All data are presented as the mean ± SEM. Figure 6. Interaction of NMNAT2 with SIRT3 promotes mitotic entry of A549. (A) Interaction of NMNAT2 with SIRT3 did not affect DNA synthesis. A549 cells were transfected with plasmids and synchronized at the G1/S transition as described in Materials and methods. Cells were pulse-labeled with BrdU (50 μM) for 30 min at indicated time-points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of the three experiments. (B) Interaction of NMNAT2 with SIRT3 promoted mitotic entry. Cell cycle progression of >1,000 cells was recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells. Values are means of three experiments. All data are presented as the mean ± SEM. Figure 7. Interaction of NMNAT2 with SIRT3 promoted mitotic growth and proliferation of A549 cells. (A) By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P<0.001). The growth of cells were significantly inhibited by interfering SIRT3 (P<0.001). (B) Cell viability was evaluated by CCK-8 assay. The result showed that the proliferation of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P<0.001). The growth of cells were significantly inhibited by interfering SIRT3 (P<0.001). Values are means of three experiments. All data are presented as the mean ± SEM. Figure 8. Interaction of NMNAT2 with SIRT3 inhibited apoptosis of A549 cells. The result showed that the apoptosis of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was less than in A549 cells (P>0.05). The apoptosis of cells were significantly promoted by interfering SIRT3 (P<0.001). Values are means of three experiments. All data are presented as the mean ± SEM. ==== Refs References 1. Parkin DM Bray F Ferlay J Pisani P Global cancer statistics, 2002 CA Cancer J Clin 55 74 108 2005 15761078 2. Jemal A Siegel R Xu J Ward E Cancer statistics CA Cancer J Clin 60 277 300 2010 20610543 3. Reungwetwattana T Weroha SJ Molina JR Oncogenic pathways, molecularly targeted therapies and highlighted clinical trials in non-small-cell lung cancer (nsclc) Clin Lung Cancer 13 252 266 2012 22154278 4. Fossella F Pereira JR von Pawel J Pluzanska A Gorbounova V Kaukel E Mattson KV Ramlau R Szczesna A Fidias P Millward M Belani CP Randomized, multinational, phase III study of docetaxel plus platinum combinations versus vinorelbine plus cisplatin for advanced non-small-cell lung cancer: the TAX 326 study group J Clin Oncol 21 3016 3024 2003 12837811 5. Kelly K Crowley J Bunn PA Jr Presant CA Grevstad PK Moinpour CM Ramsey SD Wozniak AJ Weiss GR Moore DF Israel VK Livingston RB Gandara DR Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced non-small-cell lung cancer: a Southwest Oncology Group trial J Clin Oncol 19 3210 3218 2001 11432888 6. Kelly K Crowley J Bunn PA Jr Presant CA Grevstad PK Moinpour CM Ramsey SD Wozniak AJ Weiss GR Moore DF Israel VK Livingston RB Gandara DR Phase III randomized trial comparing three platinum-based doublets in advanced non-small-cell lung cancer J Clin Oncol 20 4285 4291 2002 12409326 7. Schiller JH Harrington D Belani CP Langer C Sandler A Krook J Zhu J Johnson DH Eastern Cooperative Oncology Group Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer N Engl J Med 346 92 98 2002 11784875 8. Hanahan D Weinberg RA Hallmarks of cancer: the next generation Cell 144 646 674 2011 21376230 9. Lydia WSF Marcia CH Metabolic regulation by SIRT3: implications for tumorigenesis Trends Mol Med 18 516 523 2012 22749020 10. Finley LW Carracedo A Lee J Souza A Egia A Zhang J Teruya-Feldstein J Moreira PI Cardoso SM Clish CB Pandolfi PP Haigis MC SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization Cancer Cell 19 416 428 2011 21397863 11. Sundaresan NR Samant SA Pillai VB Rajamohan SB Gupta MP SIRT3 is a stress-responsive deacetylase in cardiomyocytes that protects cells from stress-mediated cell death by deacetylation of Ku70 Mol Cell Biol 28 6384 6401 2008 18710944 12. Li H Bergeron L Cryns V Pasternack MS Zhu H Shi L Greenberg A Yuan J Activation of caspase-2 in apoptosis J Biol Chem 272 21010 21017 1997 9261102 13. Hirota T Kunitoku N Sasayama T Marumoto T Zhang D Nitta M Hatakeyama K Saya H Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells Cell 114 585 598 2003 13678582 14. Jack F Ming J Jo M Cancer-specific functions of SIRT1 enable human epithelial cancer cell growth and survival Cancer Res 65 10457 10463 2005 16288037 15. Hamamoto R Furukawa Y Morita M Iimura Y Silva FP Li M Yagyu R Nakamura Y SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells Nat Cell Biol 6 731 740 2004 15235609 16. Sood R Bonner TI Makalowska I Stephan DA Robbins CM Connors TD Morgenbesser SD Su K Faruque MU Pinkett H Graham C Baxevanis AD Klinger KW Landes GM Trent JM Carpten JD Cloning and characterization of 13 novel transcripts and the human RGS8 gene from the 1q25 region encompassing the hereditary prostate cancer (HPC1) locus Genomics 73 211 222 2001 11318611 17. Kusumanchi P Zhang Y Jani MB Jayaram NH Khan RA Tang Y Antony AC Jayaram HN Nicotinamide mononucleotide adenylyltransferase 2 overexpression enhances colorectal cancer cell-kill by Tiazofurin Cancer Gene Ther 20 403 412 2013 23764899 18. Berger F Lau C Dahlmann M Ziegler M Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms J Biol Chem 280 36334 36341 2005 16118205 19. Mayer PR Huang N Dewey CM Dries DR Zhang H Yu G Expression, localization and biochemical characterization of nicotinamide mononucleotide adenylyltransferase 2 J Biol Chem 285 40387 40396 2012 20943658 20. Raffaelli N Lorenzi T Emanuelli M Amici A Ruggieri S Magni G Nicotinamide-mononucleotide adenylyltransferase from Sulfolobus solfataricus Methods Enzymol 331 281 292 2001 11265470 21. Raffaelli N Sorci L Amici A Emanuelli M Mazzola F Magni G Identification of a novel human nicotinamide mono-nucleotide adenylyltransferase Biochem Biophys Res Commun 297 835 840 2002 12359228 22. Yan T Feng Y Zheng J Ge X Zhang Y Wu D Zhao J Zhai Q Nmnat2 delays axon degeneration in superior cervical ganglia dependent on its NAD synthesis activity Neurochem Int 56 101 106 2010 19778564 23. Gilley J Coleman MP Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons PLoS Biol 8 e1000300 2010 20126265 24. Lau C Dölle C Gossmann TI Agledal L Niere M Ziegler M Isoform-specific targeting and interaction domains in human nicotinamide mononucleotide adenylyltransferases J Biol Chem 285 18868 18876 2010 20388704 25. Rose G Dato S Altomare K Bellizzi D Garasto S Greco V Passarino G Feraco E Mari V Barbi C BonaFe M Franceschi C Tan Q Boiko S Yashin AI De Benedictis G Variability of the SIRT3 gene, human silent information regulator Sir2 homologue and survivorship in the elderly Exp Gerontol 38 1065 1070 2003 14580859 26. Shi T Wang F Stieren E Tong Q SIRT3, a mitochondrial sirtuin deacetylase, regulates mitochondrial function and thermogenesis in brown adipocytes J Biol Chem 280 13560 13567 2005 15653680 27. Ahn BH Kim HS Song S Lee IH Liu J Vassilopoulos A Deng CX Finkel T A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis Proc Natl Acad Sci USA 105 14447 14452 2008 18794531 28. Hallows WC Lee S Denu JM Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases Proc Natl Acad Sci USA 103 10230 10235 2006 16790548 29. Pillai VB Sundaresan NR Kim G Gupta M Rajamohan SB Pillai JB Samant S Ravindra PV Isbatan A Gupta MP Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway J Biol Chem 285 3133 3144 2010 19940131 30. Sundaresan NR Gupta M Kim G Rajamohan SB Isbatan A Gupta MP Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice J Clin Invest 119 2758 2771 2009 19652361 31. Sokoloff L Relationships among local functional activity, energy metabolism and blood flow in the central nervous system Fed Proc 40 2311 2316 1981 7238911 32. Mattson MP Liu D Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders Neuromolecular Med 2 215 231 2002 12428812 33. Du L Zhang X Han YY Burke NA Kochanek PM Watkins SC Graham SH Carcillo JA Szabó C Clark RS Intramitochondrial Poly (ADP-ribosylation) contributes to NAD+ depletion and cell death induced by oxidative stress J Biol Chem 278 18426 18433 2003 12626504 34. Zeng J Yang GY Ying W Kelly M Hirai K James TL Swanson RA Litt L Pyruvate improves recovery after PARP-1-associated energy failure induced by oxidative stress in neonatal rat cerebrocortical slices J Cereb Blood Flow Metab 27 304 315 2007 16736046 35. Araki T Sasaki Y Milbrandt J Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration Science 305 1010 1013 2004 15310905 36. Wang J Zhai Q Chen Y Lin E Gu W McBurney MW He Z A local mechanism mediates NAD-dependent protection of axon degeneration J Cell Biol 170 349 355 2005 16043516 37. Kaundal RK Shah KK Sharma SS Neuroprotective effects of NU1025, a PARP inhibitor in cerebral ischemia are mediated through reduction in NAD depletion and DNA fragmentation Life Sci 79 2293 2302 2006 16935310 38. Ying W Wei G Wang D Wang Q Tang X Shi J Zhang P Lu H Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia Front Biosci 12 2728 2734 2007 17127275 39. Liu D Pitta M Mattson MP Preventing NAD (+) depletion protects neurons against excitotoxicity: bioenergetic effects of mild mitochondrial uncoupling and caloric restriction Ann NY Acad Sci 1147 275 282 2008 19076449	24042441	PMC3823398	CC BY	2021-01-04 23:23:52	yes	Int J Oncol. 2013 Sep 16; 43(5):1420-1430

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.