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PMC12842104
To check for the inhibition of the PI3K/AKT signaling pathway as one of the antiproliferative mechanisms of action of E. bicolor xylene extract, expression of PI3K, phosphorylated AKT, and non-phosphorylated proteins was evaluated.
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PMC12842104
Reduced or no expression of PI3K, AKT, and pAKT proteins was observed in both E. bicolor extract-treated T47D and MDA-MB-231 cell lines (Figure 8), suggesting inhibition of the PI3K/AKT signaling pathway by E. bicolor xylene extract.
[ { "end": 109, "label": "CellLine", "start": 105, "text": "T47D" }, { "end": 124, "label": "CellLine", "start": 114, "text": "MDA-MB-231" } ]
PMC12842104
Disruption of cytosolic calcium homeostasis and generation of excessive ROS induce ER stress .
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PMC12842104
To assess the effect of E. bicolor xylene extract on ER stress-dependent apoptotic protein kinase RNA-like ER kinase (PERK)—activating transcription factor 4 (ATF4)—C/EBP homologous protein (CHOP) (PERK/ATF4/CHOP) and activating transcription factor 6 (ATF6)—spliced X-box binding protein 1—C/EBP homologous protein (CHO...
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PMC12842104
E. bicolor extract treatment led to the expression of PERK and CHOP (endoplasmic apoptotic protein) (Figure 9A).
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PMC12842104
However, expression of phosphorylated PERK and ATF4 proteins was absent in capsaicin and E. bicolor xylene extract-treated T47D and MDA-MB-231 cells (Figure 9A), indicating the absence of PERK/ATF4/CHOP pathway.
[ { "end": 127, "label": "CellLine", "start": 123, "text": "T47D" }, { "end": 142, "label": "CellLine", "start": 132, "text": "MDA-MB-231" } ]
PMC12842104
We further checked the expression of XBP1s protein, which activates CHOP and can be induced by ER stress leading to the ER-stress mediated apoptotic ATF6/XBP1s/CHOP pathway.
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PMC12842104
Expression of XBP1s was found in both capsaicin and E. bicolor extract-treated cells, suggesting the presence of the ATF6/XBP1s/CHOP pathway.
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PMC12842104
Worldwide, breast cancer is the most prevalent cancer diagnosed in women .
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PMC12842104
Expanding advances in diagnosis is essential to reduce the global mortality from breast cancer.
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PMC12842104
In this context, numerous plant species contain natural compounds with demonstrated anticancer potential .
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Conducting focused research on bioactive plant chemicals, scientists could develop novel therapeutic options for breast cancer treatment.
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PMC12842104
The present study tested the antiproliferative activities of E. bicolor xylene extract on ER-positive T47D and triple-negative MDA-MB-231 breast cancer cell lines and found that the extract significantly reduced the proliferation of both breast cancer cell lines (Figure 1, Figure 2 and Figure 3).
[ { "end": 106, "label": "CellLine", "start": 102, "text": "T47D" }, { "end": 137, "label": "CellLine", "start": 127, "text": "MDA-MB-231" } ]
PMC12842104
Previously, it was reported that E. bicolor latex ethanol extract possesses antiproliferative activity on T47D and MDA-MB-231 breast cancer cell lines , but no mechanisms of action were provided.
[ { "end": 110, "label": "CellLine", "start": 106, "text": "T47D" }, { "end": 125, "label": "CellLine", "start": 115, "text": "MDA-MB-231" } ]
PMC12842104
This study proposes the mechanisms of action of E. bicolor diterpene extract in the two cell lines under study presented in Figure 10 and Figure 11.
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PMC12842104
Plants of Euphorbia genus are well-known for their antiproliferative effects.
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PMC12842104
E. hirta L. methanolic extract suppressed MCF-7 cell growth at 24 h with a GI50 value of 25.26 µg/mL .
[ { "end": 47, "label": "CellLine", "start": 42, "text": "MCF-7" } ]
PMC12842104
A study on the effect of E. macroclada acetone extract made from leaves and flowers reported significant cytotoxicity in MCF-7 breast cancer cells .
[ { "end": 126, "label": "CellLine", "start": 121, "text": "MCF-7" } ]
PMC12842104
E. macroclada tissues extracted with dichloromethane and ethyl acetate showed cytotoxic effects on MDA-MB-468 cells.
[ { "end": 109, "label": "CellLine", "start": 99, "text": "MDA-MB-468" } ]
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In contrast, the methanol and latex extracts in DMSO did not exhibit cytotoxic effects .
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PMC12842104
Another study on the biological activity of E. tirucalli L. stem extracts in methanol, butanol, and hexane resulted in inhibition of MCF-7 and MDA-MB-231 cell proliferation in a concentration-dependent manner .
[ { "end": 138, "label": "CellLine", "start": 133, "text": "MCF-7" }, { "end": 153, "label": "CellLine", "start": 143, "text": "MDA-MB-231" } ]
PMC12842104
Our study found that E. bicolor ethanol extract possesses antiproliferative effects only at higher concentrations in ER-positive T47D cells.
[ { "end": 133, "label": "CellLine", "start": 129, "text": "T47D" } ]
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However, no antiproliferative effect was observed on the triple-negative MDA-MB-231 cells (Figure 1).
[ { "end": 83, "label": "CellLine", "start": 73, "text": "MDA-MB-231" } ]
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Different solvent extracts of Euphorbia species, as well as extract concentration used, show a wide range of antiproliferative effects in breast cancer cell lines.
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In our study, we do not claim that diterpenes are the exclusive constituents of the xylene extract, nor do we assign the observed biological effects to a single compound.
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The results are interpreted as arising from an extract whose activity reflects the combined effects of its components.
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Compared to E. bicolor ethanol extract, the xylene extract was toxic to HMEC at high concentrations.
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PMC12842104
Previous research showed that the diterpene RTX was not toxic to normal cells .
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However, E. bicolor xylene extract contains other chemicals besides the diterpene RTX.
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Biochemical identification results from our lab, previously published, showed that E. bicolor latex extract contains the diterpenes RTX and abietic acid .
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PMC12842104
The HMEC cytotoxicity may be a combined effect of RTX, abietic acid, and other chemicals in the extract.
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Therefore, future research should focus on chemical identification and isolation of other diterpenes in E. bicolor extracts, to individually be used to study antiproliferative effects, thus selecting the nontoxic ones for possible drug development.
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PMC12842104
In our attempt to understand the antiproliferative mechanisms of action of E. bicolor xylene extract in T47D and MDA-MB-231 cells, we took into consideration the activation of TRPV1, calcium influx, and apoptotic markers.
[ { "end": 108, "label": "CellLine", "start": 104, "text": "T47D" }, { "end": 123, "label": "CellLine", "start": 113, "text": "MDA-MB-231" } ]
PMC12842104
Calcium ions as secondary messengers have important regulatory functions, also being involved in cellular processes of cancer development .
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PMC12842104
TRPV superfamily of plasma membrane ion channels is one of the most active calcium-permeable superfamilies in regulating Ca influx .
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PMC12842104
It is known that capsaicin activates TRPV1 in cancer cells and has potent anticancer activity against certain cancer types through both Ca-dependent and -independent mechanisms .
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PMC12842104
In our study, capsaicin showed antiproliferative activity in T47D and MDA-MB-231 cells at much higher concentrations than E. bicolor xylene extract, which may be because the extract contains various diterpenes, and they may synergistically work to induce the observed antiproliferative effects.
[ { "end": 65, "label": "CellLine", "start": 61, "text": "T47D" }, { "end": 80, "label": "CellLine", "start": 70, "text": "MDA-MB-231" } ]
PMC12842104
Blocking TRPV1 and chelating calcium increased the T47D and MDA-MB-231 cell viability but did not completely counteract the effect of E. bicolor xylene extract.
[ { "end": 55, "label": "CellLine", "start": 51, "text": "T47D" }, { "end": 70, "label": "CellLine", "start": 60, "text": "MDA-MB-231" } ]
PMC12842104
On the other hand, inhibiting ROS formation with NAC completely blocked the effect of E. bicolor extract, resulting in increased cell viability of only T47D cells, suggesting that increased ROS level is the underlying mechanism of cell death in T47D cells.
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Other antiproliferative studies employing Euphorbia species, such as E. lathyrism L., E. antiquorum L., and E. fischeriana Steud.,
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have also found that increased ROS levels induced apoptosis .
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Our results also revealed an immediate (2 s after treatment) release of calcium from the ER and mitochondrial calcium overload (27 s after treatment) in T47D cells, indicating that increased ROS levels trigger the accumulation of calcium in mitochondria.
[ { "end": 157, "label": "CellLine", "start": 153, "text": "T47D" } ]
PMC12842104
Other studies also revealed crosstalk between ROS levels and calcium signaling in different diseases, including cardiac physiology, pathologies, and neurodegeneration .
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PMC12842104
Blocking TRPV1 using capsazepine and chelating calcium with BAPTA-AM led to increased cell viability of MDA-MB-231 cells, suggesting that the activation of TRPV1 by E. bicolor xylene extract is the primary mechanism responsible for cell death in that cell line.
[ { "end": 114, "label": "CellLine", "start": 104, "text": "MDA-MB-231" } ]
PMC12842104
Consequently, activation of TRPV1 causes an excessive influx of calcium in the cells, ultimately leading to apoptosis in MDA-MB-231 cells.
[ { "end": 131, "label": "CellLine", "start": 121, "text": "MDA-MB-231" } ]
PMC12842104
Therefore, a potential approach for developing new drug therapies for triple-negative breast cancer could involve channel activators to induce calcium influx and cell death .
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PMC12842104
In T47D cells, blocking TRPV1 with CAPZ inhibited the effect of E. bicolor xylene extracts but only at small concentrations of extract (2–16 µg/mL), suggesting that TRPV1 may still be involved in reducing cell viability.
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PMC12842104
Experimental evidence indicates that when calcium homeostasis is disrupted, it leads to the breakdown of the mitochondrial membrane potential.
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PMC12842104
This disruption also increases ROS levels and the release of proapoptotic proteins, resulting in apoptosis .
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PMC12842104
Mitochondrial Ca overload was observed after E. bicolor xylene extract treatments of both T47D and MDA-MB-231 cells in our study (Figure 6), and therefore, we pursued the examination of apoptotic protein markers expression for both the mitochondrial intrinsic and extrinsic apoptotic pathways.
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PMC12842104
It is known that mitochondrial depolarization releases cytochrome c into the cytoplasm, activating caspase-3 via caspase-9 .
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PMC12842104
Activation of caspase-3 is a well-recognized hallmark of apoptosis induced by other Euphorbia plant extracts such as E. tirucalli and E. esula L. .
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PMC12842104
E. bicolor xylene extract activated downstream caspases-3/9 (Figure 7), indicating the activation of the apoptotic pathways, as presented in the proposed models of apoptotic mechanisms in Figure 10 and Figure 11.
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PMC12842104
Anti-apoptotic BCL-2 protein protects cancer cells by preventing apoptosis .
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We observed reduced expression of BCL-2 protein in E. bicolor extract-treated T47D and MDA-MB-231 cells, suggesting that E. bicolor constituents of the xylene extract could be excellent candidates for targeting BCL-2 proteins in different cancers.
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Our results indicate that the BAX protein is expressed at a high molecular weight on Western blot.
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Studies indicated that BAX forms oligomers, necessary for its proapoptotic activity.
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The high molecular weight BAX oligomers bind to the mitochondrial membrane, leading to the mitochondrial intrinsic apoptotic pathway .
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PMC12842104
The xylene extract of E. bicolor activated the extrinsic apoptotic pathway, as evidenced by the expression of FAS and caspase-8 proteins (Figure 7, Figure 10 and Figure 11).
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PMC12842104
These findings are supported by previous studies, which further suggest that the TRPV1 N-terminus can interact with proapoptotic FAS-associated proteins to trigger extrinsic apoptotic pathways .
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PMC12842104
It has been shown that TRPV1 is part of the ER membrane structure .
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PMC12842104
Its activation can lead to calcium disruption and cause ER stress , which can be an active source of calcium release in the cytoplasm.
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PMC12842104
ER stress and compromised mitochondria can subsequently release apoptotic signals .
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PMC12842104
Our results show that E. bicolor xylene extract induced ER stress and ultimately triggered apoptosis in MDA-MB-231 breast cancer cells.
[ { "end": 114, "label": "CellLine", "start": 104, "text": "MDA-MB-231" } ]
PMC12842104
In T47D cells, increased ROS levels may trigger ER stress and activate mitochondrial apoptosis as a result of E. bicolor xylene extract treatment.
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PMC12842104
Expression of XBP1s, an activated (by splicing) transcription factor that facilitates ER stress , was observed in both E. bicolor extract treated T47D and MDA-MB-231 cells as an indication of ER-mediated stress, which stimulated the expression of CHOP, a transcription factor that increases the expression of other apopt...
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PMC12842104
Our results also suggest that E. bicolor xylene extract downregulates the PI3K/AKT signal transduction pathway (Figure 8, Figure 10 and Figure 11).
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PMC12842104
The serine/threonine kinase AKT, also referred to as protein kinase B (PKB), is a key regulator of numerous cellular processes, including survival, growth, proliferation, cell cycle progression, and metabolism.
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PMC12842104
Proper regulation of AKT activity is essential, since the balance between loss or gain of AKT activation contributes significantly to the pathophysiology of cancer .
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PMC12842104
We observed significantly low total and phosphorylated AKT and PI3K protein expressions in both E. bicolor xylene extract-treated T47D and MDA-MB-231 cells (Figure 8).
[ { "end": 134, "label": "CellLine", "start": 130, "text": "T47D" }, { "end": 149, "label": "CellLine", "start": 139, "text": "MDA-MB-231" } ]
PMC12842104
It has been shown that PI3K/AKT pathway activation contributes to tumorigenesis, and inhibition of PI3K and AKT can decrease cellular proliferation and increase cell death .
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PMC12842104
Therefore, our results suggest that E. bicolor diterpenes could become potential candidates for targeting the PI3K/AKT signaling pathways in breast cancers.
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PMC12842104
The current study revealed two different antiproliferative mechanisms for the two breast cancer types, ER-positive and triple-negative, which most likely are because of their distinct molecular profiles.
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T47D cancer cells express estrogen and progesterone hormone receptors, the primary targets for current hormone therapies.
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MDA-MB-231 cancer cells lack hormone receptors, and therefore, they are not responsive to hormone therapies .
[ { "end": 10, "label": "CellLine", "start": 0, "text": "MDA-MB-231" } ]
PMC12842104
It is known that natural products have multiple molecular targets .
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E. bicolor xylene extract may affect different targets depending on the cancer cells’ molecular characteristics, resulting in distinct antiproliferative molecular mechanisms.
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PMC12842104
In conclusion, our study presents for the first time the ROS-mediated antiproliferative mechanisms of action of E. bicolor xylene extract in T47D cells (Figure 10) and TRPV1-dependent mechanisms of action in MDA-MB-231 cells (Figure 11).
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PMC12842104
E. bicolor xylene extract generates high ROS levels in T47D cells and triggers several apoptotic pathways.
[ { "end": 59, "label": "CellLine", "start": 55, "text": "T47D" } ]
PMC12842104
In MDA-MB-231 cells, E. bicolor xylene extract activates TRPV1 and induces mitochondrial and ER stress-mediated apoptotic pathways.
[ { "end": 13, "label": "CellLine", "start": 3, "text": "MDA-MB-231" } ]
PMC12842104
In addition, the E. bicolor extract downregulates the PI3K/AKT signaling pathway in both T47D and MDA-MB-231 cells.
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PMC12842104
Our findings suggest that E. bicolor xylene extract could be used to design specific therapeutics for cancer cell types.
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PMC12842104
For ER-positive cell types, therapeutic agents could target the ROS-inducing apoptotic mechanism.
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PMC12842104
For MDA-MB-231 cell types, the therapeutic mechanism could target the TRPV1-dependent apoptotic pathways.
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E. bicolor plants were collected from fields around Denton, TX, USA, during September and October 2023 and a voucher specimen has been deposited in the TWU Herbarium.
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PMC12842104
The ethanol extraction of aerial plant tissues (50 g) followed the procedure we employed before in our laboratory .
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Xylene, a nonpolar solvent, was used to extract hydrophobic plant chemicals, especially diterpenes, following the protocol from Tidgewell (2007) with slight modifications .
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PMC12842104
Plant aerial tissues (50 g) were extracted in 200 proof xylene (Fisher Scientific, Newington, NH, USA) (1:4 w/v) at room temperature for one day.
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PMC12842104
Filtered supernatants were placed in pre-weighed vials and dried under nitrogen .
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Dried extract was dissolved in dimethyl sulfoxide (DMSO) (Fisher Scientific, Newington, NH, USA) in a 1:1 ratio and stored at −20 °C until use.
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PMC12842104
The extract collected using ethanol solvent is named ethanol extract, and the extract collected using xylene solvent is named xylene extract.
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PMC12842104
The ethanol extract represents a crude extract containing multiple classes of phytochemicals identified in our previous studies , whereas the xylene extract contains hydrophobic constituents.
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PMC12842104
The presence of diterpenes in this fraction was qualitatively confirmed by TLC using resiniferatoxin and abietic acid as reference standards (Supplementary Materials ).
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PMC12842104
The breast cancer cell lines under study, and adult human mammary epithelial cells (HMECs) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).
[ { "end": 82, "label": "CellLine", "start": 52, "text": "human mammary epithelial cells" } ]
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The cell culture media and conditions were as previously reported .
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PMC12842104
After 24 h, cells seeded into 96-well cell culture plates at 10,000 cells/well in a phenol-red-free medium were treated with increasing concentrations of E. bicolor ethanol, xylene extracts in DMSO (2, 4, 8, 16, 62.5, 125, 250, 500 µg/mL) or capsaicin at 0.5, 1, 5, 10, 50, 100, 250, and 500 µM concentrations.
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PMC12842104
The final concentration of DMSO was <0.1%.
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PMC12842104
Due to the limited availability and high cost of RTX, we chose to use capsaicin as a positive control for TRPV1 activation, a well-characterized TRPV1 activator and agonist of resiniferatoxin.
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This approach allowed us to reliably validate pathway activation while maintaining experimental consistency.
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PMC12842104
Antiproliferative activity of cells treated with increasing concentrations of E. bicolor ethanol, xylene extracts, or capsaicin was evaluated by performing MTS assays (Abcam, Waltham, MA, USA) as described before .
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