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PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
In addition, a study revealing the cellular protective impact of α-LA on human bronchial epithelial cells exposed to paraquat through the reduction of ROS and MDA levels confirmed the antioxidant effects of α-LA (41).
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
There is a strong connection between redox regulation and programmed cell death, developmental senescence, and pathogen response.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
The p53 gene plays a crucial role not just in cancer but also in longevity and aging in humans and mammals (42).
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
Pro-apoptotic Bax and anti-apoptotic Bcl-2 genes are involved in mitochondria-controlled programmed cell death in mammals.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
The present findings revealed that the expression of p53 and Bax genes in SH-SY5Y cells in the D-gal group was significantly higher than that of the untreated group.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
However, the expression of the genes was significantly decreased in the α-LA + D-gal and Met + D-gal groups.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
In addition, Bcl-2 gene expression in the D-gal group decreased significantly compared to the untreated group, while it increased significantly in the α-LA + D-gal and Met + D-gal groups.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
Literature indicated that there is an upregulation of Bax and p53, as well as a downregulation of Bcl-2 mRNA levels, following the administration of D-gal (434445).
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
Furthermore, according to a study, α-LA further reduced cytochrome c loss in the body by decreasing p53 gene expression and preserving the integrity of the mitochondrial membrane, which reduced apoptosis in older rats (46).
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
However, this study only measured the effect of α-LA on the expression of Bax, Bcl-2, and p53 at the mRNA level, and it is necessary to confirm the effects at the protein level as well.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
The results showed that α-LA exerted its protective effects by increasing antioxidant capacity and reducing oxidative stress and modulating genes involved in apoptosis, which makes it a promising compound for preventing age-related diseases.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
H. Nadi Yazdi contributed to writing the original draft, investigation, methodology, and conceptualization; F. Mirzavi contributed to writing the original draft, reviewing, and editing; M. Kazemian Kakhki and A.R. Afshari contributed to methodology and formal analysis; S.H. Mousavi and M. Soukhtanloo contributed to the conceptualization, supervision, and funding acquisition.
PMC12614211
The protective effects of α-lipoic acid against D-galactose-induced cellular senescence in human SH-SY5Y neuroblastoma cell line
All authors read and approved the final version of the manuscript.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Neural tumors represent diverse malignancies with distinct molecular profiles and present particular challenges due to the blood-brain barrier, heterogeneous molecular etiology including epigenetic dysregulation, and the affected organ’s critical nature.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
KCC-07, a selective and blood-brain barrier penetrable MBD2 (methyl CpG binding domain protein 2) inhibitor, can suppress tumor development by inducing p53 signaling, proven only in medulloblastoma.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Here we demonstrate KCC-07 treatment’s application to other neural tumors.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
KCC-07 treatment reduced proliferation rates of U-87MG (glioma cell line) and SH-SY5Y (neuroblastoma cell line).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
p53 stabilization occurred in these cell lines without significantly affecting programmed cell death factors under KCC-07 exposure.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Furthermore, tumor cell growth inhibition was enhanced when combined with DNA damaging reagents.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Both phleomycin (radiomimetic agent inducing DNA double strand breaks) and etoposide (topoisomerase II inhibitor inducing DNA double strand breaks) treatment activated p53-dependent signaling for apoptosis and cell cycle arrest, consequently suppressing tumor cell growth.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Dual treatment with KCC-07 (epigenetic modifier) and DNA damaging reagents augmented tumor cell suppression, suggesting greater benefits of combinatorial therapy for neural tumors than previously demonstrated.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The hallmarks of cancer have been updated a couple of times since first proposed by Hanahan and Weinberg in 2000 .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The second generation of cancer hallmarks supplemented 4 additional factors, including ‘genomic instability and mutation’, which can result partly from defective DNA damage repair and DNA damage response (DDR) .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The expanded cancer hallmarks now contain 4 more entities: ‘unlocking phenotype plasticity’, ‘senescent cells’, ‘polymorphic microbiomes’, and ‘nonmutational epigenetic reprogramming’ .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
DNA modification, particularly methylation, is one form of nonmutational epigenetic event, and CpG methylation causes gene silencing .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Several enzymes and proteins are involved in DNA methylation, including DNA methyltransferases (DNMTs), 5’-methylcytosine hydroxylases (TETs), and methyl-CpG binding domain (MBD) proteins .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The MBD protein family consists of eleven proteins including MBD1 to MBD6, which function as DNA methylation readers in gene repression .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Alterations of MBD proteins, particularly MBD1 to MBD4, have been found in several tumors including prostate, endometrial, and brain tumors .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
In particular, MBD2 has functional impacts on hematopoietic and solid tumors.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Reduction or inhibition of MBD2 is advantageous in tumor treatment .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The approach using an MBD2 inhibitor has been studied for the first time, and only so far, in medulloblastoma, the most frequent pediatric brain tumor.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
In medulloblastoma, inhibition of MBD2 binding to methylated DNA by KCC-07, a specific MBD2 inhibitor that is blood-brain barrier penetrable, leads to MDM2 sequestration and p53 stabilization, and eventually suppression of tumor growth in a p53 dependent manner, suggesting that epigenetic modification of DNA could be one of the therapeutic targets for tumor treatment .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Other MBD2 inhibitors including 8, 8-ethylenebistheophylline, CID3100583, APC ([R]-[3-(2-Amino-acetylamino)-pyrrolidine-1-carboxylic acid tert-butyl ester]), and ABA (2-amino-N--acetamide) were suggested as novel candidates, yet the biological effects of these potential MBD2 inhibitors were not verified , which places KCC-07 in a unique position for this purpose.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Another approach to tumor treatment via p53 signaling is by inducing DNA damage, particularly DNA double strand breaks (DSBs), the most deleterious type of DNA damage .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
DSBs are immediately recognized by early responding proteins including ATM (Ataxia telangiectasia mutated), which phosphorylates numerous downstream proteins such as H2AX, KAP1, CHK2, and p53 to trigger appropriate responses to DNA damage.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Chemotherapeutic agents inducing DNA damage activate p53 signaling, leading to cell cycle arrest and programmed cell death to prevent tumor progression [11-14].
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
While targeting epigenetic modification or DNA structure itself is beneficial for tumor treatment, a combinatory effect of KCC-07 and DNA damaging reagents has not been explored.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Here we tested whether the effect of DNA damaging reagents on neural tumor cells can be augmented with KCC-07 exposure.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The results showed additive effects of genotoxic drugs and KCC-07 on tumor cell growth inhibition and programmed cell death, demonstrating the remarkable therapeutic efficacy achieved by combining DNA damage induction and MBD2 inhibition for neural tumor treatment.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Two neural tumor cell lines, U-87MG (originated from malignant glioma, wildtype p53) and SH-SY5Y (a subline of neuroblastoma, wildtype p53), were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
These cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM, high glucose with pyruvate, Gibco, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and Penicillin-Streptomycin (Gibco) at 37°C with 5% CO2.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
KCC-07 (3-[4-(2-Pyridinyl)-2-thiazolyl]amino]phenol (CAS 315702-75-1)) was obtained from Selleckchem (TX, USA).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Phleomycin (Phleo, CAS 11006-33-0) and etoposide (ETP, CAS 33419-42-0) were purchased from Sigma (MO, USA).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
When necessary, reagents were dissolved in DMSO (dimethyl sulfoxide).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Each experimental condition of drug treatments is indicated in the figures.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Total RNA from cell lines was isolated using TRIzol reagent (Invitrogen/Ambion, TX, USA) according to the manufacturer’s protocol, and cDNA was generated from the isolated total RNA using ReverTra Ace qPCR RT master mix with gDNA remover (Toyobo, Osaka, Japan).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The primers to measure gene expression levels were adapted from previous reports .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Gene expression was measured by real-time PCR using a Rotor-Gene Q PCR machine and SYBR green (Qiagen, Hilden, Germany).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
To estimate cell proliferation, the SRB assay was applied.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Two cell lines were exposed continuously to KCC-07 for 2 days prior to DNA damage induction by either etoposide (ETP) or phleomycin (Phleo).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
At the indicated time points, cells were fixed with 10% trichloroacetic acid (Sigma-Aldrich).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The 0 time point represents 1 hour after either ETP or Phleo treatments.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Fixed cells were stained with 0.4% SRB (in 1% acetic acid solution, Sigma-Aldrich) followed by dissolving in 10 mM Tris base (Millipore, MA, USA), and the color intensity was measured with a microplate spectrophotometer (BioTek plate reader, Agilent Technologies, CA, USA) at 540 nm.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Alternatively, cell viability was measured by MTT assay (AkrivisBio, CA, USA).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The cells were treated with KCC-07 and/or DNA damaging reagents as indicated in the figures.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The MTT procedure was followed as the manufacturer suggests, and the final metabolite was quantified at 590 nm.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Changes in cell cycle were analyzed after 72 hour exposure to KCC-07 and/or DNA damage reagent treatment for 24 hours.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Cells were fixed with ethanol and then treated with RNase A (100 μg/ml, Macherey-Nagel, Duren, Germany).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Propidium iodide (PI, Sigma-Aldrich) was used to stain cells followed by flow cytometry analysis (Fluorescence-Activated Cell Sorting, FACS) using FACSCanto II (BD Biosciences, NY, USA) with FACSDiva software (v9.2, BD Biosciences).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
For cell death analysis in vitro, two cell lines were seeded onto 12-well plates and exposed to the drug conditions described in the figure.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Cells were maintained at 37°C with 5% CO2 during live cell monitoring.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
PI stained cells, which are indicative of disintegrated cell membranes, were imaged and quantified by the IncuCyte live cell analysis system (Sartorius, Gottingen, Germany).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Cell lysates from KCC-07 and/or DNA damaging reagent treatments were obtained in a lysis buffer containing 60 mM Tris buffer pH 6.8, 2.4% sodium dodecyl sulfate (SDS), 6% β-mercaptoethanol, 0.12% bromophenol blue, and 12% glycerol.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Proteins were separated in SDS-PAGE gels and transferred onto nitrocellulose membranes (Cytiva, MA, USA).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Western blot bands were visualized by enhanced chemiluminescence (ECL, Bio-Rad Laboratories, CA, USA or Cytiva, MA, USA).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
When necessary, nuclear and cytoplasmic fractionation was applied.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Cells were lysed in 10 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100, 1 mM dithiothreitol (DTT).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
After centrifugation at 1,300 rcf, the soluble supernatants were further centrifuged at 13,000 rpm for the cytosolic fraction, and the insoluble pellet was lysed in 20 mM Tris, pH 8.0, 0.4 M NaCl, 15% glycerol, 1.5% Triton X-100, also containing MNase (micrococcal nuclease, Worthington Biochemical, NJ, USA) followed by centrifugation at 16,000 rcf.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The soluble fraction from this step constituted the nuclear fraction.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The antibodies used for Western blot analysis were ATM (Abcam, Cambridge, UK), ATM-pS1981 (Cell Signaling Technology, MA, USA), KAP1 (Novus Biologicals, CO, USA), KAP1-pS824 (Novus Biologicals), CHK2 (Millipore, MA, USA), CHK2-pT68 (Novus Biologicals), γ-H2AX (Cell Signaling Technology), p53 (Santa Cruz Biotechnology, TX, USA), p53-pS15 (Cell Signaling Technology), MDM2 (Santa Cruz Biotechnology), GAPDH (Genetex, CA, USA), and histone H3 (Abcam).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Quantified data were analyzed statistically using Prism Software (GraphPad, CA, USA).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
P values less than 0.05 were considered statistically significant (*<0.05, **<0.01, ***<0.001).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
First, we tested whether KCC-07 could affect cell growth of U-87MG and SH-SY5Y cell lines, which are neural origin tumors.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Although the two cell lines showed different sensitivity to KCC-07, both cell lines displayed dose dependent cell viability in response to KCC-07 using MTT assay (Fig. 1A).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
In addition, we examined the response of cell viability upon DNA damage induction.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Both cell lines were susceptible to Phleo or ETP, and these susceptibilities were not affected by KCC-07 treatment (Fig. 1B).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
These data suggest the possibility of expanding KCC-07 usage to other types of brain tumor cells.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
We also confirmed that the SH-SY5Y cell line is more susceptible to DNA damage than U-87MG .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Since KCC-07 could trigger p53 signaling , p53 status after treatment with KCC-07 and/or DNA damaging reagents was examined by Western blot.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
As a transcriptional activator and tumor suppressor, p53 must accumulate in the nucleus upon exposure to stress , which we observed in U-87MG and SH-SY5Y cell lines after KCC-07 treatment as well as DNA damage induction (Fig. 1C).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The location of MDM2, a specific E3 ubiquitin ligase for p53, was not changed after stress exposure.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
These data demonstrated that KCC-07’s therapeutic expansion to other brain tumor types, including glioblastoma and neuroblastoma, likely via p53 stabilization in both cell lines, which consequently reduced tumor cell proliferation rates.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Next, we examined what changes were induced in this experimental condition regarding p53 signaling.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
KCC-07 treatment induced CDKN1A (also known as p21, which is involved in cell cycle arrest in a p53 dependent manner) expression in both cell lines.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The increased CDKN1A expression was also detected after DNA damage induction, with a greater increment observed in SH-SY5Y cells, suggestive of higher sensitivity to DNA damage.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
However, no additive effect on CDKN1A expression was detected in combined treatment with KCC-07 and DNA damage induction (Fig. 2A).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
On the other hand, BBC3 (also known as PUMA, which is involved in cell death in a p53 dependent manner) expression was not increased by KCC-07 treatment, but was significantly increased following treatments with DNA damaging reagents.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Combined exposure to KCC-07 and DNA damaging reagents did not increase BBC3 expression further, except in U-87MG with combined treatment, which rather reduced its expression (Fig. 2B).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
These data indicate that the main consequence of KCC-07 treatment on U-87MG and SH-SY5Y cell lines is likely related to cell cycle progression rather than activation of cell death, similar to the previous report .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Furthermore, the immediate event upon DNA damage, which is called DDR, was investigated.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Upon DNA damage, particularly DNA DSBs which can be induced by either Phleo or ETP treatment, ATM is immediately auto-phosphorylated at serine (S) 1981, then phosphorylates several substrates including KAP1 at S824, CHK2 at threonine (T) 68, p53 at S15, and H2AX at S139 (called γH2AX) .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Western blot analyses indicate that proper phosphorylation modifications of ATM kinase substrates occurred after DNA damage induction, which was not affected by co-treatment with KCC-07 (Fig. 2C).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Of note, γH2AX signals were higher or maintained over time in cell line samples exposed to both KCC-07 and DNA damaging reagents.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
γH2AX signals are indicative of DNA strand breaks; therefore, these data suggest that the DNA repair process was likely compromised by combined treatment with KCC-07 (Fig. 2C), implying additive effects of the combined treatment.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The possible outcomes of DNA damage induction are either cell cycle arrest or cell death .
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
Therefore, we looked into both consequences.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
By applying FACS analysis, cell populations at each cell cycle phase were calculated.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
In general, DNA damage triggered G2/M arrest in both U-87MG and SH-SY5Y cell lines (Fig. 3A).
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
The DNA damaging conditions we applied to cell lines did not significantly increase cell populations arrested at G1 phase.
PMC12426421
KCC-07, MBD2 Inhibitor, Expands the Therapeutic Window of DNA Damage Inducing Reagents in Neural Tumor Cells
However, combined treatments with KCC-07 and DNA damaging reagents shifted the cell populations toward the G1 phase except for U-87MG with Phleo treatment, whose cell line was relatively resistant to this type of DNA damaging reagent (Fig. 3A), similar to previous reports .