Cisplatin kills ovarium cancer cells through the TRPV1-mediated mitochondrial oxidative stress and apoptosis: TRPV1 inhibitor role of eicopentotaneoic acid

doi:10.21203/rs.3.rs-4850309/v1

Download PDF

Article

Cisplatin kills ovarium cancer cells through the TRPV1-mediated mitochondrial oxidative stress and apoptosis: TRPV1 inhibitor role of eicopentotaneoic acid

https://doi.org/10.21203/rs.3.rs-4850309/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The most commonly used treatment, cisplatin (Cisp), causes excessive Ca²⁺ influx mediated by TRPV1 and produces a high concentration of mitochondrial free reactive oxygen radicals (mROS). In clinical trials, it can be used with other adjuvant medicinal agents to increase safety and efficacy. Although there are contradictory findings, eicosapentaenoic acid (EPA) as an adjuvant has been demonstrated to suppress the proliferation of ovarian cancer cells. We assessed the effects of EPA and Cisp incubations on oxidant, lysosomal injury, and apoptotic values in the OVCAR-3 ovarian tumor cell line by activating TRPV1. Five groups were induced with Cisp (25 µM for 24h), EPA (100 µM for 24h), Cisp + EPA, and Cisp + TRPV1 antagonist (capsazepine, CPZ). We discovered that, in comparison to control cells, Cisp-mediated upregulation of TRPV1 protein is downregulated by EPA and CPZ, but Cisp mediates greater TRPV1-induced Ca²⁺ entry in cells. The major mROS in cells that cause Cisp-mediated TRPV1 activation include increases in mROS but decreases in glutathione, glutathione peroxidase, mitochondrial function, OVCAR-3 viability, and number. In response to capsaicin, Cisp-mediated TRPV1 stimulation causes mitochondrial Ca²⁺ and Zn²⁺ overload, which is followed by increases of caspase-3/-8/-9, lysosomal injury, and apoptosis, however, these effects were less pronounced in the Cisp + EPA and Cisp + CPZ groups. To sum up, we first showed that Cisp kills OVCAR-3 cells by stimulating TRPV1, even while blocking the channel reduced the anti-cancer effects of Cisp. Cisp and TRPV1 stimulators together may provide an alternative method of killing ovarian tumor cells.

Biological sciences/Cancer

Biological sciences/Cancer/Cancer prevention

Biological sciences/Cell biology/Cell death

Biological sciences/Cell biology/Cell death/Apoptosis

Health sciences/Molecular medicine

Apoptosis

Eicosapentaenoic acid

Ovarian cancer

Mitochondrial dysfunction

TRPV1 channel.

The incidence of ovarian cancer is rising globally, making it among the top five causes of death for women.¹ Ovarian cancer is distinct in that it can be difficult to diagnose and can spread quickly. The molecular processes behind ovarian cancer metastasis, however, remain unclear. Therefore, it would be important to understand the molecular mechanism of ovarian cancer metastasis in order to effectively manage ovarian cancer patients.

Cisplatin (Cisp)-based chemotherapy and cytoreductive surgery are the standard treatments for ovarian cancer.² Unfortunately, the initial response rate to this treatment is not sustained, and the disease recurs in 70% of women within months due to chemoresistance caused by increased Ca²⁺ influx and oxidative stress.^3,4 There have been suggestions that this high frequency of chemoresistance may contribute to the high death rate associated with ovarian cancer.⁵ Apoptotic and oxidant signaling pathways mediated by DNA damage are the sources of Cisp-induced anticancer action.^2,5 Cisp induces apoptosis, mitochondrial (mROS) and cytoplasmic (cROS) reactive free oxygen species, which destroy a variety of tumor cells, including ovarian cancer cells.^2,3 Thus, the results of cell culture investigations suggest that prooxidant therapies,^6,7 which limit the progression of the disease by augmenting excessive Ca²⁺ influx, cROS, and mROS in cancer cells, may represent a viable substitute for current systemic drugs in the fight against cancer.

Changes in the cytosolic free calcium ion concentration ([Ca²⁺]_c) decrease the signaling systems responsible for cell death and growth in both cancerous and normal cells.⁸ The transient receptor potential (TRP) vanilloid 1 (TRPV1) channel is a member of the Ca²⁺-permeable TRP superfamily. It can be activated by a variety of stimuli, such as oxidative stress and capsaicin (CAP).^9,10 TRPV1 is specifically inhibited by capsazepine (CPZ).¹¹ According to recent studies, TRPV1 is essential for both mitochondria membrane potential (mMP) loss-mediated apoptosis and mROS-mediated apoptosis in thyroid, glioblastoma, and breast cells.^12–14 Cisp activates caspases-3 (CSP/3), -8 (CSP/8), and − 9 (CSP/9) by raising [Ca²⁺]_c and cytosolic free zinc ion ([Zn²⁺]_c) concentrations through TRPV1 and TRPM2 channels in tumor cells.^12–15 As predicted, CPZ prevents tumor cells from receiving TRPV1-induced CSP/3, CSP/8, and CSP/9 activation.^12–14 The connection between mitochondria-mediated mROS and apoptotic interactions and Cisp-mediated TRPV1 stimulation-dependent Ca²⁺ influx in ovarian cancer cells is still unknown. Additionally, Ca²⁺ can be absorbed by the mitochondria via the inner mitochondrial membrane's mitochondrial Ca²⁺ channel uniporter (MCU), which depolarizes the mitochondria and helps in the open the mitochondrial permeability transition pore.¹⁵ By stimulating TRPV1, the PTP can also cause mitochondrial enlargement and outer membrane permeabilization, which can result in the production of mROS and caspase-activating factors as well as the triggering of cell death and apoptosis.¹⁶ Cisp has been shown to induce pro-apoptotic Ca²⁺ release from the cytosol and mitochondria of cells. This results in an excess of [Ca²⁺]_c and mitochondrial Ca²⁺, which in turn promotes TRPV1-mediated apoptosis and mitochondria-mediated apoptosis in a number of cancer cells, with the exception of cisplatin-sensitive OVCAR-3 ovarian cancer cells.^12–15 It is unknown, therefore, exactly how Cisp-induced TRPV1 activation and Ca²⁺ release access the mitochondria of ovarian cancer cells.

Eicosapentaenoic acid (EPA), a member of the omega-3 polyunsaturated fatty acids, has been shown to have a crucial role in the development of cancer, as evidenced by the fact that the plasma of cancer patients has been found to be significantly lower than that of healthy individuals.¹⁸ EPA has been linked in a number of studies to suppressing tumor metastasis and ovarian cancer cell proliferation.^19,20 Contrary to the reports, EPA blocked the migration and development of ovarian cancer cells mediated by the Nav1.5 sodium channel.²¹ In the various mouse brain regions, EPA reduced the expression of TRPV1.²² Through the suppression of TRPV1, EPA in mice reduced behavior associated with pain produced by capsaicin.²³ In order to affect the TRPV1 channel in ovarian cancer cells, EPA may either stimulate or inhibit it.

Understanding how mROS production, apoptosis induction through TRPV1 activation, and Ca²⁺ influx produced by Cisp and EPA enhance or inhibit oxidant-dependent OVCAR-3 killing is crucial for the development of alternative therapeutics. We speculate that enhancing the commonly used conventional Cisp treatment would be a viable alternative for synergistic TRPV1-mediated cytotoxicity of ovarian carcinoma cells.

TRPV1 protein expression amounts in the OVCAR-3 cells

Till today, the cells of the OVCAR-3 have not been reported to contain the TRPV1 channel. Using the Western blot method, we assessed the protein expression amounts of TRPV1 for each of the four groups (Fig. 1A; Supplementary Fig. 1A). As shown in Fig. 1B, the Cisp group exhibited a greater amount of TRPV1 protein compared to the control (Ctrl) and EPA groups (p < 0.05). Furthermore, in comparison to the Cisp group, its expression level was significantly (p < 0.05) diminished in the Cisp plus EPA groups.

Cell viability, number, and debris quantity in the OVCAR-3 cells

In comparison to the Ctrl and EPA groups, the Cisp group exhibited enhanced cell viability (Fig. 1C) and OVCAR-3 number (Fig. 1D) (p < 0.05). However, the incubation of EPA and CPZ resulted in a decrease in these parameters in the Cisp + EPA and Cisp + CPZ groups (p < 0.05). The number of debris (waste of dead cells) was higher (p < 0.05) in the Cisp group but decreased in the Cisp + EPA and Cisp + CPZ groups as a result of EPA and CPZ incubation (Fig. 1E).

The stimulation of TRPV1 enhanced the increases in [Ca²⁺]_c levels caused by Cisp

Utilizing the LSM-800, green pictures of Fluo-3/AM from each of the four groups were captured (Fig. 2A). Following CAP stimuli, the Cisp group had higher [Ca²⁺]_c than the Ctrl group (p < 0.05), while the Cisp + EPA group had considerably lower levels of [Ca²⁺]_c (Fig. 2B). Following CPZ treatments, the [Ca²⁺]_c concentration was reduced in the Ctrl, EPA, Cisp, and Cisp plus EPA groups. The contribution of TRPV1 to the Cisp-induced increase of [Ca²⁺]_c has been confirmed by the Fluo-3/AM outcomes.

The Cisp-induced increase in TRPV1 current density was diminished by EPA

We intended to further test the alterations using patch-clamp analysis in the SH-SY5Y cells in addition to the [Ca²⁺]_c (Fluo-3/AM) investigations. In the absence of CAP stimulation of OVCAR-3 (Fig. 3A), the TRPV1 current of cells remained unchanged. However, CAP (10 µM) stimulation activated the TRPV1 of cells [Figs. 3B and 3B (I-V)]. In the OVCAR-3, the Ctrl + CAP group had higher TRPV1 current densities (94.27 pA/pF) than the Ctrl group (3.80 pA/pF) (Fig. 3F) (p < 0.05). By stimulating CAP [Figs. 3C and 3C (I-V)], the current density of TRPV1 in the cells was further (p < 0.05) increased in the Cisp + CAP group (135.40 pA/pF). In comparison to the groups of Ctrl + CAP and Cisp + CAP, the current density of TRPV1 in the cells was considerably (p < 0.05) lower in the Ctrl + CAP + CPZ (4.39 pA/pF) and Cisp + CAP + CPZ (5.41 pA/pF) groups (Fig. 3F). There was no increase in the TRPV1 currents following the CAP stimulation in EPA (Fig. 3D) and Cisp + EPA (Fig. 3E). In comparison to the Cisp + CAP group (Fig. 2F), the mean densities of TRPV1 currents in the cells were significantly (p < 0.05) lower in the EPA + CAP (3.41 pA/pF) and Cisp + EPA + CAP (4.81 pA/pF) groups. The TRPV1 stimulator action of Cisp and the TRPV1 modulator activity of EPA on the excessive Ca²⁺ influx in the OVCAR-3 cells were further confirmed by the patch-clamp data.

Cisp treatments promote apoptosis and caspase activity, which subsequently stimulate Cisp-induced OVCAR-3 death

The stimulation of TRPV1 in malignant cells except OVCAR-3 by a rise in apoptosis and caspases was the process by which chemotherapeutic agents-induced alterations to cell viability, number, and debris were arranged.^6,12,13,14 The ovarian tumor cell killer effects of EPA were examined by stimulating CSP/3, CSP/9, and apoptosis.¹⁹ Contrary to the reports, EPA blocked the migration and development of ovarian cancer cells mediated by the Nav1.5 sodium channel.²¹ EPA, Cisp, and CPZ-induced alterations of apoptosis (Fig. 4A), CSP/3 (Fig. 4B), CSP/8 (Fig. 4C), and CSP/9 (Fig. 4D). In PI, Hoechst, BF, overlay (Fig. 5A), and 2.5D (Fig. 5B), we determined the cell death (PI positive cell) percentage in the current study (Fig. 5C). Apoptosis, CSP/3, CSP/8, CSP/9, and PI positive OVCAR-3 percentages were substantially higher in the Cisp than in the Ctrl and EPA (p < 0.05), despite being markedly decreased in the Cisp + EPA and Cisp + CPZ (p < 0.05).

Decrease in mitochondrial function

Loss of mMP is known to be related to early apoptosis because mitochondrial depolarization, an irreversible alteration during Cisp-induced apoptosis, occurs.¹⁵ We chose to use the JC-1 and LSM-800 confocal microscope to investigate the effects of EPA and Cisp on mMP in the OVCAR-3 cells after detecting an increase in apoptosis in the Cisp groups. JC-1 aggregates and releases red fluorescence in the mitochondria of non-apoptotic cells.¹⁵ JC-1 is present in the cytoplasm of apoptotic cells as a monomer and fluoresces green.¹⁶ Thus, a decrease in the red/green fluorescence intensity ratio could be used to evaluate mitochondrial depolarization.

According to Supplementary Figs. 2A and 2B, the current study's findings indicate that Cisp intervention in the cells led to a decrease in the ratios of JC-1 red/green, indicating a decrease in the reduction of mMP but an increase in mitochondrial damage and depolarization (p < 0.05). On the other hand, Cisp-induced cells treated with EPA and CPZ showed larger ratios of JC-1 red/green (Supplement Fig. 2C) (p < 0.05). These findings demonstrated that Cisp had both an apoptotic effect and a stimulating reduction in mitochondrial activity.

Cisp increases generations of lipid peroxidation (LPx), mROS, and cROS and the accumulation of [Zn ²⁺ ] _c in OVCAR-3 cells, although EPA and CPZ decrease the changes in the cells

Mitochondrial Zn²⁺ and Ca²⁺ overload is a characteristic of apoptotic cell death that results in a decrease in mMP and the opening of the mitochondrial permeability transition pore, which is then followed by the formation of oxidants such as LPx, cROS, and mROS.^7,12,15 By increasing LPx, mROS, and cROS in tumor cells, cisp-mediated Zn²⁺ and Ca²⁺ influx ultimately caused tumor cell apoptosis and death.^7,24,25

Utilizing the Mito Tracker ret dye, the mitochondria in the [Zn²⁺]_c imaging (RhodZin3) analysis were identified. Using a laser confocal microscope, the red, green, overlay (Fig. 6A) and 2.5D images (Fig. 6B) of [Zn²⁺]_c were obtained. The Cisp group exhibited significantly greater levels of both cytosolic zinc (cZn²⁺) (Fig. 6C) and mitochondrial zinc (mZn²⁺) (Fig. 6D) in comparison to the Ctrl and EPA groups. Conversely, cZn²⁺ and mZn²⁺ levels were decreased in Cisp-induced cells treated with EPA and CPZ (p < 0.05).

MitoSOX (red), DCFH-DA (green), and their overlay (Fig. 7A) were obtained using the LSM-800 confocal microscope, along with 2.5D pictures (Fig. 7B). Our findings showed that the Cisp group had significantly (p < 0.05) higher levels of MitoSOX (Fig. 7C), DCFH-DA (Fig. 7D), and LPx (Fig. 7E) than the Ctrl and EPA groups. Treatment with EPA and CPZ reduced MitoSOX, DCFH-DA, and LPx levels in the Cisp + EPA and Cisp + CPZ groups (p < 0.05).

These results showed that Cisp-mediated accumulation of cZn²⁺ and mZn²⁺ in addition to the mitochondrial Ca²⁺ accumulation triggered excessive formation of cROS and mROS, but TRPV1 inhibition with CPZ therapy lowered the accumulations and generations.

The GSH concentrations and GSH-Px activity were decreased by increasing TRPV1 stimulation during Cisp incubation

Growing evidence suggests that TRPV1-mediated mitochondrial Ca²⁺ buildup increases mROS and LPx, thus lowering GSH and GSH-Px.^26,27 TRPV1 is activated by both malignant and normal cells in the presence of low or insufficient GSH.^6,28,29 The treatment of Cisp decreased GSH levels and GSH-Px activity in breast cancer cells by stimulating TRPV1, despite the fact that OVCAR-3 tumor cells were not examined.^6,12 We recorded pictures of ThiolTracker (GSH) and 2.5D of OVCAR-3 cells (Fig. 8A), and we assessed the differences in fluorescence intensity of GSH in the pictures (Fig. 8B). We manually carried out GSH (Fig. 8C) and GSH-Px (Fig. 8D) measurements in the cells in addition to the imaging analysis. When comparing the Cisp group to the Ctrl and EPA groups, the fluorescence intensity-concentration of GSH, and activity of GSH-Px were reduced (p < 0.05). However, the incubations of CPZ and EPA resulted in an increase (p < 0.05) in the Cisp + EPA and Cisp + CPZ groups.

Cisp induces lysosomal injury through the stimulation of TRPV1 in OVCAR-3 cells

TRPV1-stimulated Ca²⁺ influx induces mROS and lysosomal function injury in the skin of mice.³⁰ After observing the increase of mROS, the effect of Cisp on lysosomal function was investigated. LysoTracker Green DND-26 dye is an acidotropic probe that accumulates in acidic organelles, such as lysosomes, and exhibits a pH-dependent increase in fluorescence intensity upon acidification.³¹ Cisp increased the LysoTracker Green DND-26 fluorescence intensity (Supplementary Fig. 3A), suggesting lysosomal pH alteration in OVCAR-3. In addition to the iROS and mROS results, these results suggested that Cisp induced lysosomal activity in OVCAR-3. However, CPZ and EPA pretreatment abolished Cisp-induced lysosomal activity (Supplementary Fig. 3B). Together, these results, combined with the above data, collaboratively demonstrate that TRPV1 is a potential target for the treatment of Cisp-caused ovarium tumor toxicity. Cisp-induced lysosomal autophagy via activating the TRPV1 signaling pathway in the OVCAR-3 cells.

In this study, we firstly reported that Cisp activated the redox-sensitive TRPV1 channel and caused mZn²⁺ and Ca²⁺ influx, which in turn stimulated mitochondrial and lysosomal failure as well as the activation of CASP-3/-8/-9 in ovarium tumor (OVCAR-3) cells. Nonetheless, the tumor cell death effect of Cisp was lessened due to the suppression of TRPV1 caused by the CPZ and EPA incubations. Cisp via TRPV1 activation may therefore be an additional therapeutic approach to increase the susceptibility of ovarium tumor cells to mROS-dependent cell death.

Emerging as a redox-sensitive TRP channel, TRPV1 is found in both normal and malignant microenvironments.^32–34 The TRPV1 protein is expressed more frequently in a number of malignant cancers, such as cervical squamous cell carcinoma,³⁵ thyroid carcinoma cells,¹³ breast cancer cells, ^6,12 and colorectal tumor cells.²⁶ Furthermore, TRPV1, cROS, and mROS stimulation were enhanced by Cisp therapy, leading to the death of breast cancer cells.^6,12 The impact of TRPV1 stimulation on oxidative damage can differ depending on the type of tumor. In retinoblastoma, for example, TRPV1-mediated Ca²⁺ influx increases the antioxidant defense program,³⁶ whereas in breast and colorectal cancer cells, it stimulates mitochondrial mROS activity.^6,12,26 Cisp promotes oxidative stress and apoptosis in a range of cancer types via stimulating TRPV1.^2,6,7,12,15 Here, we found that OVCAR-3 cell lines, which provide a suitable model to investigate the impact of intracellular Ca²⁺ signals on a therapeutically relevant ovarian cancer type, have a notably elevated expression of the TRPV1 protein.¹ Furthermore, a persistent increase in [Ca²⁺]_c was induced by the TRPV1 agonist CAP and was receptive to pharmacological TRPV1 inhibition (EPA and CPZ). It's interesting to note that whereas increases in [Ca²⁺]^c in cancer cells have long been known to promote cell survival and proliferation, sustained elevations in [Ca²⁺]_c result in apoptotic cell death.^37,38,39 Consequently, OVCAR-3 cell survival was reduced by TRPV1-induced cytosolic Ca²⁺ overload, even while the number of cell debris increased. Similar to the current findings, a study found that EPA inhibited the Nav1.5 sodium channel, which is responsible for the migration and growth of ovarian cancer cells.²¹ In contrast to the present findings, EPA has been associated in several studies with the inhibition of tumor spread and proliferation of ovarian cancer cells.^19,20 It appears that the effects of EPA on TRPV1 and cell proliferation in tumor cells are therefore cell-specific.

One of the most promising therapeutic methods employed by the current drug development strategy is to produce oxidative damage through TRP channel stimulation. Numerous medications have been used as agents for aberrant Ca²⁺ influx-induced increases of LPx, cROS, and mROS by selectively causing lipid, protein, and DNA damage to tumor cells and inhibiting antioxidant systems, including GSH and GSH-Px.^8,10,12,15 Cisp inhibited the antioxidant defense mechanisms of OVCAR-3 cells by promoting oxidative damage through LPx, cROS, and mROS, which decreased GSH and GSH-Px. Additionally, there is mounting evidence that TRPV1-mediated mitochondrial Ca²⁺ accumulation reduces GSH and GSH-Px via elevating mROS and LPx.^26,27 Both cancerous and normal cells can activate TRPV1 when there is a small quantity of GSH present.^6,28,29 Even though OVCAR-3 tumor cells were not studied, the administration of Cisp reduced GSH levels and GSH-Px activity by upregulating LPx, cROS, and mROS in breast cancer cells by activating TRPV1.^6,12 We discovered that TRPV1 activation by Cisp resulted in oxidative damage, whereas TRPV1 suppression by the EPA and CPZ groups increased the survival of tumor cells while lowering apoptosis, LPx, mROS, and cROS in the OVCAR-3 cells. Furthermore, mMP parameters were lowered. These results support a prior study that investigated how EPA inhibits oxidative damage to block TRPV1 in specific mouse brain areas and reduces capsaicin-induced pain.^21,22

Ca²⁺ influx via TRPV1 stimulation is a crucial sensitizing signal in the pro-apoptotic transition of mitochondria and lysosome injury, which is important in the regulation of cell death.^26,40,41 The production of mROS primarily happens during the ATP synthesis process through stimulation of the mitochondrial electron transport chain, where electron leakage in complexes I and III leads to a partial reduction of oxygen to form superoxide.⁸ The mitochondria have the highest concentration of [Zn²⁺]_c when compared to other subcellular compartments.⁴² Overloading cellular or mitochondrial Zn²⁺ will decrease mMP, enhance mROS and apoptosis, but decrease cellular ATP levels.⁴² The elevation of [Ca²⁺]_c and [Zn²⁺]_c in cancer cells caused lysomal damage when TRPM7 was stimulated.⁴³ The antioxidant and TRPV1 antagonist treatments inhibited TRPV1 in glioblastoma cells, which regulated the alterations.⁴⁴ Pro-apoptotic mechanisms that cause mitochondria to swell include mitochondrial Ca²⁺ and Zn²⁺ overloads via TRPV1 stimulation. This can cause the outer membrane to rupture or be disturbed, which releases mitochondrial proapoptotic factors like CSP/3, CSP/9, and cROS into the cytosol.^{17, 26} An elevation in TRPV1 stimulation-mediated mMP and mROS is frequently linked to the onset and spread of cancer.²⁷ Thus, one of the main roles of mitochondria is not only their involvement in energy metabolism but also their control over cell death. One possible approach to treating ovarian cancer could be to target the increase in mROS levels in cancer cells that goes beyond a certain point.^1–3 Although TRPV1 channel blockers (EPA and CPZ) blocked these elevations in the cells, we observed that activating TRPV1 with Cisp significantly increased tumor cell death (PI positive cell percentage) and apoptosis in OVCAR-3 cells via the upregulation of cZn²⁺, mZn²⁺, apoptosis, and CSP-3/-8/-9, but downregulation of mMP. It was thus shown that by preventing TRPV1-mediated Ca²⁺ entry, the abrupt rise in apoptosis and caspase (CASP-3/-8/-9) activations caused by Cisp incubation could be reduced. Similarly, TRPV1 was found to increase mMP-dependent CSP-3/-8/-9 activations, cell death, and apoptosis in Cisp-treated breast cancer and neuroblastoma cells.^6,12,14 Targeting the increase in mROS levels in cancer cells beyond a threshold could be a potential strategy for ovarian cancer therapy.^1–3 We noticed that activating TRPV1 with Cisp substantially induced tumor cell death and apoptosis in OVCAR-3 cells via the upregulation of cZn²⁺, mZn²⁺, CSP-3/-8/-9 but downregulation of mMP, despite the fact that these rises were inhibited in the cells by TRPV1 channel blockers (EPA and CPZ). The sudden increase in CSP-3/-8/-9 activations resulting from Cisp incubation was therefore demonstrated to be decreased through blocking TRPV1-mediated Ca²⁺ entry. Likewise, in Cisp-treated breast cancer and neuroblastoma cells, TRPV1 was discovered to increase mMP-dependent CSP-3/-8/-9 activations, cell death, and apoptosis.^6,12,14 TRPV1 activation in OVCAR-3 cells promotes mMP decrease-dependent apoptosis and tumor cell death rather than cell survival, as has been previously shown in breast cancer and neuroblastoma cells.^6,12,14

In conclusion, TRPV1 activation has therefore increased the chemo-sensitizing effects of Cisp in ovarian carcinoma cells. The current experiment's findings indicate that TRPV1 stimulation increased the sensitivity of ovarian carcinoma cells to Cisp. Apoptosis induction has been connected to the fundamental processes of tumor cell death. After receiving Cisp treatment, cells showed increases in apoptotic signs such as CSP-3/-8/-9; however, these indicators were reduced by TRPV1 inhibition with EPA and CPZ. Lysosomal damage, LPx, cROS, mROS, and TRPV1-stimulated Ca²⁺ and Zn²⁺ influx while lowering mMP, GSH, and GSH-Px preventative antioxidant systems (Supplementary Fig. 4). This discovery offers a novel method of treating ovarian tumor cells by combining a TRPV1 agonist with Cisp; however, additional testing in lab animals is necessary to verify the possible chemo-sensitizing impact of Cisp.

Cell culture. The OVCAR-3 ovarian cancer epithelial cell line from the American Type Culture Collection (ATCC) was used in this study. OVCAR-3 cells were cultured in 5 ml RPMI cell culture media in the identical the amount. In addition, the medium also includes one percent penicillin-streptomycin combination and ten percent fetal bovine serum (Gibco Inc., Istanbul, Türkiye). The connected cells were investigated using a laser confocal microscope (LSM-800) (Carl Zeiss, Oberkochen, Germany) in glass bottom dishes (35 mm) from Mattek Corporation Inc., Ashland, MA, USA. For the remaining analyses, the 2x10⁶ cells were seeded in T25 cm² flasks with filter lids.

Study groups. Five groups of OVCAR-3 cells were generated: (1) the control group (Ctrl), which was maintained in cell culture conditions for 24h; (2) the EPA group, which was exposed to 100 µM of EPA (Cat #: NC9892897, Cayman Chemical) for 24h; ⁴⁵ (3) the Cisp group, which was exposed to 25 µM Cisp for 24h;^6,12 and (4) the Cisp plus EPA group, which was exposed to both of them. In the fifth group, TRPV1 antagonist (CPZ) was added and the cells were cultured for an additional hour.⁴⁴

Western blotting for the TRPV1 channel in OVCAR-3 cells. Currently, information regarding TRPV1 expression in OVCAR-3 cells is not available. Consequently, we initiated the utilization of standard methods for conducting Western blotting protein analysis of expression in the frozen OVCAR-3 cells.⁴¹ Protein samples from the cells were separated on SDS-PAGE gels and then placed onto PVDF membranes. The primary antibodies against TRPV1 (1:200; ab11168, Abcam, Istanbul, Türkiye) and α-actin (1:1 000; Abcam) were then incubated on the cell membranes for two nights, one at 4°C and the other overnight. After that, the membranes were inspected with an improved chemiluminescence apparatus (G:Box Gel Imagination System, Syngene, UK). The signal intensity was measured using the program ImageJ. The protein band intensities were shown as comparison densities in the row graphics.

Tests for the proliferation, debris, and viability of cells. The MTT test was used to analyze the cell viability, however the CASY TT counting cell equipment (Roche, Mannheim, Germany) was used to count the number of cells and debris (waste of dying cells).²⁷ After being suspended in 10 milliliters of the diluting solution, the OVCAR-3 cells were examined in accordance with the manufacturer's instructions. The CASY method combines resistance detection for particle detection with pulse region assessment based on electronic pulse process methodology.⁴⁶ Therefore, it is possible to distinguish between living and dead cells. Cell viability results were shown as percentage changes, and cell and debris counts were expressed as x10⁷/per milliliter and x10⁶/per milliliter, respectively.

Evaluation of the concentration of cytoplasmic free Ca ²⁺ concentration ([Ca²⁺]_c). Following the incubation of the cells in the Fluo-3/AM (Cell Signaling Technology) solution, the cells adhering in the dishes were washed and then given 1 ml of extracellular buffer. The green images of Fluo-3/AM were acquired for the LSM-800 confocal microscopy analysis using a 40x1.3 DICIII oil objective, and the diode laser stimulation was kept at 488 nm. Following the application of the TRPV1 stimulator (10 µM CAP), the OVCAR-3 cells were subjected to the TRPV1 antagonist (100 µM CPZ) in order to inhibit Ca²⁺ entrance. The results were expressed as a.u. for the mean Fluo-3/AM fluorescence intensity.

Electrophysiology. Conventional whole-cell patch-clamp electrophysiological measurements were conducted with a patch-clamp set at room temperature (23 ± 2°C) and an EPC 10 amplifier (HEKA, Lamprecht, Germany).¹⁰ The electrode cell resistances obtained from a puller (PC-10, Narishige, Tokyo, Japan) utilized for whole cell recording ranged from 4 to 6 MΩ. Previous research detailed the components of intracellular and extracellular buffers.^26,44 In order to produce an extracellular solution devoid of Na⁺, we introduced 150 mM N-methyl-D-glucamine (NMDG⁺) and used HCl to correct the pH. The voltage of the cells was capped at -60 millivolts. An external TRPV1 antagonist (100 µM CPZ) was used in the study to reduce the TRPV1 currents that extracellular 10 µM CAP generated in cells. The results of the TRPV1 current were displayed as pA/pF.

The CSP/3, CSP/8, CSP/9, and apoptosis tests. Cell apoptosis was measured in accordance with the manufacturer's instructions using an APOPercentage detection kit (Biocolor Ltd., Co Antrim, UK) in a plate reader (Infinite PRO 200; Tecan Austria GmbH, Groedig, Austria) analysis (at 550 nm). As previously reported, caspase substrates were used in the plate reader (Infinite PRO 200) to evaluate the activities of CSP/3, CSP/8, and CSP/9.^26,44 In the Infinite PRO 200, the caspase substrate cleavages were examined between 380 and 460.

Fluorescence units per milligram of protein were computed in the cells following the Biuret method's determination of protein levels. The outcomes of CSP/3, CSP/8, CSP/9, and apoptosis were shown as percentage increases over the level observed prior to treatment.

mROS and cROS detections. While the production of mROS (ThermoFischer Scientific, Istanbul, Türkiye) was carried out using the MitoSOX Red reagent (Cat #: M7513), the production of cROS was detected using 2,7-dichlorofluorescein diacetate (DCFH-DA, Cat #: C6827). Grown in 35-mm dishes, OVCAR-3 cells were loaded for 15–20 minutes at 37°C with 20 mM DCFH-DA and MitoSOX Red. Using an LSM-800 confocal microscope, cell fluorescence variations of MitoSOX and DCFH-DA as an arbitrary unit (a.u.) were examined immediately after two washes of ice-cold 1xPBS washing. The diode laser stimulation in the MitoSOX was maintained at 561 nm, although DCFH-DA was adjusted to 488 nm to stimulate the OVCAR-3 cells.²⁶

Measurements of mitochondrial membrane potential (mMP). Detections of mitochondrial membrane potential (mMP). The Mito Probe JC-1 (Cat #: T3168, ThermoFischer Scientific) dye was used to detect the changes in mMP. The OVCAR-3 cells in the dishes from treatment groups and controls were incubated in cell culture medium with 10 uM JC-1 for 15–20 min in the dark. Then, the cells were washed twice by staining extracellular buffer and analyzed by LSM-800 confocal microscope for 1 h. The red aggregation showed normal mMP, while the green monomer indicated a reduction in MMP due to mitochondrial dysfunction. Results were presented as the rate of red aggregation/green monomer stained by JC-1.

Cell death percentage. Ten µM Hoechst 33342 and 20 µM propidium iodide were added to the cell medium in the dishes. Propidium iodide (PI), which only stains chromatin DNA in dead cells and fluoresces red, can be used to determine the percentage of dead cells (PI-positive cells). Hoechst is a blue fluorescent dye that stains chromatin DNA. Using a 40x oil objective, images were captured with the LSM confocal microscope and processed with Blue ZEN software. PI was turned on at 561 nm and Hoechst 33342 at 405 nm using a diode laser. In total, 25–50 cells were counted across distinct fields (n = 5–8). At least three separate inductions were used in each experiment. It was calculated to find the percentage of cell death (PI) relative to living cells.

A reduced glutathione (GSH) imaging analysis. To determine the GSH concentrations in OVCAR-3 cells, the ThiolTracker Violet GSH measuring assay (Cat #: T10095, ThermoFisher Scientific) was used. ThiolTracker Violet green images in the OVCAR-3 were captured using the ZEN software (blue edition 3.2) and an LSM confocal microscope equipped with a 40x1.3 DICIII oil objective (Ext: 405 nm, Emi: 526 nm). ⁴⁷ The green color images' fluorescence intensity variations were expressed as a.u.

Analysis of cytosolic (cZn ²⁺ ) and mitochondrial (mZn ²⁺ ) Zn ²⁺ concentrations. After 15–20 min of incubation with 1 µM Mito Tracker red (MitoTr), analysis of cZn²⁺ and mZn²⁺ concentration in OVCAR-3 cells was stained using RhodZin3/AM (1 M per milliliter) (Cat #: T24195, ThermoFisher Scientific), a fluorescent probe for Zn²⁺ labeling. Using the ZEN program and an LSM-800 confocal microscope with a 40x oil objective (Ext: 404 nm, Emi: 526 nm), green pictures of RhodZin3/AM in the cells were taken. The fluorescence intensity changes in the green images were calculated using the ZEN program (blue edition 3.2), and the results were expressed as a.u.

Lysosome staining. Using LysoTracker Green DND-26 (Cat. No: #8783, Cell Signaling), the vesicles seen in the cytoplasm were stained during a 24-hour incubation period with Cisp and EPA. Following a 15–20-minute incubation period with 1 µM Mito Tracker red (MitoTr), 50 nM of LysoTracker Green DND-26 (LysoTr) was added to the culture medium of the dishes. Following an additional 15–20-minute incubation period, live cell observations were made using an LSM-800 confocal microscope. Diode laser stimulations of MitoTr and LysoTr were kept at 561 nm and 488 nm, respectively. After digitally recording and consecutively seeing the red-emitting MitoTr and the green-emitting LysoTr, the fluorescence intensity variations as a.u. were examined using blue ZEN software.

Analysis of LPx, glutathione peroxidase (GSH-Px), and GSH. Using a spectrophotometer (Shimadzu-UV1800, Kyoto, Japan), the total amount of protein, LPx, GSH-Px, and GSH optic density (absorbance) values in the OVCAR-3 samples were calculated.26,44 GSH, LPx, and protein concentrations in OVCAR-3 cells have been determined using the measurement of M/g. The GSH-Px activity of OVCAR-3 cells has been expressed as a protein with an IU/g value.

Analyses with statistics Every result was expressed as the means ± standard deviation (SD) for the specified number of experiments. The statistical significance was ascertained by using a one-way analysis of variance (ANOVA) in the SPSS program (version 24.0). The significance threshold for each experiment set, when compared to the control, EPA, and Cisp groups, is p < 0.05.

Acknowledgements. The author acknowledges appreciation to the technicians at BSN Health, Analysis, and Innovation Ltd. Inc. Göller Bölgesi Teknokenti, Isparta, Türkiye, Muhammet ŞAHIN and Fatih ŞAHIN, for their assistance with the patch-clamp and plate reader analyses.

Authors’ contribution. The present study's cell culture, cell viability, and caspase examinations were carried out by Dr. Mevlüt BUCAK. In the current work, Dr. Mustafa NAZIROĞLU produced the text and carried out the studies using a laser confocal microscope and western blot. Critical edits to the text were made by Dr. Mevlüt BUCAK, who also backed funding for the research.

Funding. The project was funded by BSN Health, Analysis, and Innovation Ltd., Isparta, Türkiye (Project no: 2023-5). Dr. Mevlüt BUCAK is the project's owner.

Availability of data and materials. The data employed for supporting the present investigation's conclusions are accessible upon request from Prof. Dr. Mustafa NAZIROĞLU.

Ethics approval. This article does not contain human participants or animal samples.

Competing interest The authors report no statement of disclosure.

Disclosure statement The author reports no statement of disclosure.

Declaration of Conflicting Interests The author states that she doesn’t have any conflicts of interest. The current study contains no human or animal samples.

Ding DN, Xie LZ, Shen Y, Li J, Guo Y, Fu Y, Liu FY, Han FJ. Insights into the Role of Oxidative Stress in Ovarian Cancer. Oxid Med Cell Longev. 2021, 8388258, (2021). https://doi.org/10.1155/2021/8388258.
Kleih M, Böpple K, Dong M, Gaißler A, Heine S, Olayioye MA, Aulitzky WE, Essmann F. Direct impact of cisplatin on mitochondria induces ROS production that dictates cell fate of ovarian cancer cells. Cell Death Dis. 10(11), 851, (2019). https://doi.org/10.1038/s41419-019-2081-4.
Ma L, Wang H, Wang C, Su J, Xie Q, Xu L, Yu Y, Liu S, Li S, Xu Y, Li Z. Failure of Elevating Calcium Induces Oxidative Stress Tolerance and Imparts Cisplatin Resistance in Ovarian Cancer Cells. Aging Dis. 7(3), 254-266, (2016). https://doi.org/10.14336/AD.2016.0118.
Belotte J, Fletcher NM, Awonuga AO, Alexis M, Abu-Soud HM, Saed MG, Diamond MP, Saed GM. The role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer. Reprod Sci. 21(4), 503-508, (2014). https://doi.org/10.1177/1933719113503403.
Shen L, Xia M, Zhang Y, Luo H, Dong D, Sun L. Mitochondrial integration and ovarian cancer chemotherapy resistance. Exp Cell Res. 401(2), 112549, (2021). https://doi.org/10.1016/j.yexcr.2021.112549.
Nur G, Nazıroğlu M, Deveci HA. (2017) Synergic prooxidant, apoptotic and TRPV1 channel activator effects of alpha-lipoic acid and cisplatin in MCF-7 breast cancer cells. J Recept Signal Transduct Res. 37(6), 569-577, (2017). https://doi.org/10.1080/10799893.2017.
Abdalbari FH, Martinez-Jaramillo E, Forgie BN, Tran E, Zorychta E, Goyeneche AA, Sabri S, Telleria CM. Auranofin Induces Lethality Driven by Reactive Oxygen Species in High-Grade Serous Ovarian Cancer Cells. Cancers (Basel). 15(21), 5136, (2023). https://doi.org/10.3390/cancers15215136.
Nazıroğlu M. New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-ribose. Neurochem Res 32, 1990-2001, (2007). https://doi.org/10.1007/s11064-007-9386-x.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824, (1997). https://doi.org/10.1038/39807.
Nazıroğlu M. Activation of TRPM2 and TRPV1 Channels in Dorsal Root Ganglion by NADPH Oxidase and Protein Kinase C Molecular Pathways: a Patch Clamp Study. J Mol Neurosci. 61(3), 425-435, (2017). https://doi.org/10.1007/s12031-017-0882-411.
Szallasi A, Blumberg PM, Annicelli LL, Krause JE, Cortright DN. The cloned rat vanilloid receptor VR1 mediates both R-type binding and C-type calcium response in dorsal root ganglion neurons. Mol Pharmacol. 56(3), 581-587, (1999). https://doi.org/10.1124/mol.56.3.581.
Sakallı Çetin E, Nazıroğlu M, Çiğ B, Övey İS, Aslan Koşar P. Selenium potentiates the anticancer effect of cisplatin against oxidative stress and calcium ion signaling-induced intracellular toxicity in MCF-7 breast cancer cells: involvement of the TRPV1 channel. J Recept Signal Transduct Res. 37(1), 84-93, (2017). https://doi.org/10.3109/10799893.2016.1160931.
Xu S, Cheng X, Wu L, Zheng J, Wang X, Wu J, Yu H, Bao J, Zhang L. Capsaicin induces mitochondrial dysfunction and apoptosis in anaplastic thyroid carcinoma cells via TRPV1-mediated mitochondrial calcium overload. Cell Signal. 75, 109733, (2020). https://doi.org/10.1016/j.cellsig.2020.109733.
Ertilav K. Levetiracetam modulates hypoxia-induced inflammation and oxidative stress via inhibition of TRPV1 channel in the DBTRG glioblastoma cell line. J Cell Neurosci Oxid Stress 11(3), 885-894, (2019). https://doi.org/10.37212/jcnos.715227
Ertilav K, Nazıroğlu M. Honey bee venom melittin increases the oxidant activity of cisplatin and kills human glioblastoma cells by stimulating the TRPM2 channel. Toxicon. 222, 106993, (2023). https://doi.org/10.1016/j.toxicon.2022.106993.
Alevriadou BR, Patel A, Noble M, Ghosh S, Gohil VM, Stathopulos PB, Madesh M. Molecular nature and physiological role of the mitochondrial calcium uniporter channel. Am J Physiol Cell Physiol. 320(4), C465-C482, (2021). https://doi.org/10.1152/ajpcell.00502.2020.
Sukumaran P, Nascimento Da Conceicao V, Sun Y, Ahamad N, Saraiva LR, Selvaraj S, Singh BB. Calcium Signaling Regulates Autophagy and Apoptosis. Cells. 10(8), 2125, (2021). https://doi.org/10.3390/cells10082125.
Goupille C, Frank PG, Arbion F, Jourdan ML, Guimaraes C, Pinault M, et al. Low Levels of Omega-3 Long-Chain Polyunsaturated Fatty Acids Are Associated with Bone Metastasis Formation in Premenopausal Women with Breast Cancer: A Retrospective Study. Nutrients 15, 12(12):3832, (2020). https://doi.org/10.3390/nu12123832.
Han L, Zhang Y, Meng M, Cheng D, Wang C. Eicosapentaenoic acid induced SKOV-3 cell apoptosis through ERK1/2-mTOR-NF-κB pathways. Anticancer Drugs. 27(7), 635-642, (2016). https://doi.org/10.1097/CAD.0000000000000373.
Udumula MP, Poisson LM, Dutta I, Tiwari N, Kim S, Chinna-Shankar J, Allo G, Sakr S, Hijaz M, Munkarah AR, Giri S, Rattan R. Divergent Metabolic Effects of Metformin Merge to Enhance Eicosapentaenoic Acid Metabolism and Inhibit Ovarian Cancer In Vivo. Cancers (Basel). 14(6), 1504, (2022). https://doi.org/10.3390/cancers14061504.
Liu J, Liu D, Liu JJ, Zhao C, Yao S, Hong L. Blocking the Nav1.5 channel using eicosapentaenoic acid reduces migration and proliferation of ovarian cancer cells. Int J Oncol. 53(2), 855-865, (2018). https://doi.org/10.3892/ijo.2018.4437.
Liao HY, Yen CM, Hsiao IH, Hsu HC, Lin YW. Eicosapentaenoic Acid Modulates Transient Receptor Potential V1 Expression in Specific Brain Areas in a Mouse Fibromyalgia Pain Model. Int J Mol Sci. 25(5), 2901, (2024). https://doi.org/10.3390/ijms25052901.
Matta JA, Miyares RL, Ahern GP. TRPV1 is a novel target for omega-3 polyunsaturated fatty acids. J Physiol. 578(Pt 2):397-411, (2007). https://doi.org/10.1113/jphysiol.2006.121988..
Das A, Sankaralingam M. Are Zn(II) pincer complexes efficient apoptosis inducers? a deep insight into their activity against A549 lung cancer cells. Dalton Trans. 52(40), 14465-14476, (2023). https://doi.org/10.1039/d3dt02419a.
Wang ZF, Zhou XF, Wei QC, Qin QP, Li JX, Tan MX, Zhang SH. Novel bifluorescent Zn(II)-cryptolepine-cyclen complexes trigger apoptosis induced by nuclear and mitochondrial DNA damage in cisplatin-resistant lung tumor cells. Eur J Med Chem. 238, 114418, (2022). https://doi.org/10.1016/j.ejmech.2022.
Kaya MM. Silver nanoparticles stimulate 5-Fluorouracil-induced colorectal cancer cells to kill through the upregulation TRPV1-mediated calcium signaling pathways. Cell Biol Int. 48(5), 712-725, (2024). https://doi.org/10.1002/cbin.12141.
Güzel KGU, Nazıroğlu M, Ceyhan D. Bisphenol A-Induced Cell Proliferation and Mitochondrial Oxidative Stress Are Diminished via Modulation of TRPV1 Channel in Estrogen Positive Breast Cancer Cell by Selenium Treatment. Biol Trace Elem Res. 198(1), 118-130, (2020). https://doi.org/10.1007/s12011-020-02057-3.
Güzel M, Akpınar O. Hydroxychloroquine Attenuates Acute Inflammation (LPS)-Induced Apoptosis via Inhibiting TRPV1 Channel/ROS Signaling Pathways in Human Monocytes. Biology (Basel). 10(10), 967, (2021). https://doi.org/10.3390/biology10100967.
Badr H, Kozai D, Sakaguchi R, Numata T, Mori Y. Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line. Front Pharmacol. 7, 19, (2016). https://doi.org/10.3389/fphar.2016.00019.
Chen M, Dong X, Deng H, Ye F, Zhao Y, Cheng J, Dan G, Zhao J, Sai Y, Bian X, Zou Z. Targeting TRPV1-mediated autophagy attenuates nitrogen mustard-induced dermal toxicity. Signal Transduct Target Ther. 6(1), 29, (2021). doi: https://doi.org/10.1038/s41392-020-00389-z.
Zhang L, Guo J, You Q, Xu Y. A water-soluble fluorescent pH probe and its application for monitoring lysosomal pH changes in living cells. Anal Methods. 15(25), 3057-3063, (2023). https://doi.org/10.1039/d3ay00343d.
Piciu F, Balas M, Badea MA, Cucu D. TRP Channels in Tumoral Processes Mediated by Oxidative Stress and Inflammation. Antioxidants (Basel). 12(7), 1327, (2023). https://doi.org/10.3390/antiox12071327.
Köse SA, Nazıroğlu M. N-acetyl cysteine reduces oxidative toxicity, apoptosis, and calcium entry through TRPV1 channels in the neutrophils of patients with polycystic ovary syndrome. Free Radic Res. 49(3), 338-346, (2015). https://doi.org/10.3109/10715762.2015.1006214.
Sakaguchi R, Mori Y. Transient receptor potential (TRP) channels: Biosensors for redox environmental stimuli and cellular status. Free Radic Biol Med. 146, 36-44, (2020). https://doi.org/10.1016/j.freeradbiomed.2019.10.415.
Wang Z, Dong J, Tian W, Qiao S, Wang H. Role of TRPV1 ion channel in cervical squamous cell carcinoma genesis. Front Mol Biosci. 9, 980262, (2022). https://doi.org/10.3389/fmolb.2022.980262.
Oronowicz J, Reinhard J, Reinach PS, Ludwiczak S, Luo H, Omar Ba Salem MH, Kraemer MM, Biebermann H, Kakkassery V, Mergler S. Ascorbate-induced oxidative stress mediates TRP channel activation and cytotoxicity in human etoposide-sensitive and -resistant retinoblastoma cells. Lab Invest. 101(1), 70-88, (2021). https://doi.org/10.1038/s41374-020-00485-2
Astesana V, Faris P, Ferrari B, Siciliani S, Lim D, Biggiogera M, De Pascali SA, Fanizzi FP, Roda E, Moccia F, Bottone MG. (2021) Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells. Cell Mol Neurobiol. 41(3), 563-587, (2021). https://doi.org/10.1007/s10571-020-00873-8.
Faris P, Rumolo A, Pellavio G, et al. (2023) Transient receptor potential ankyrin 1 (TRPA1) mediates reactive oxygen species-induced Ca²⁺ entry, mitochondrial dysfunction, and caspase-3/7 activation in primary cultures of metastatic colorectal carcinoma cells. Cell Death Discov. 9(1), 213, (2023). https://doi.org/10.1038/s41420-023-01530-x.
Hausmann D, Hoffmann DC, Venkataramani V, Jung E, Horschitz S, Tetzlaff SK, et al. Autonomous rhythmic activity in glioma networks drives brain tumour growth. Nature. 613(7942), 179-186, (2023). https://doi.org/10.1038/s41586-022-05520-4.
Liu L, Sun X, Guo Y, Ge K. Evodiamine induces ROS-Dependent cytotoxicity in human gastric cancer cells via TRPV1/Ca²⁺ pathway. Chem Biol Interact. 2022 Jan 5;351:109756. doi: 10.1016/j.cbi.2021.109756.
Kaya MM, Kaya İ, Nazıroğlu M. (2023) Transient receptor potential channel stimulation induced oxidative stress and apoptosis in the colon of mice with colitis-associated colon cancer: modulator role of Sambucus ebulus L. Mol Biol Rep. 50(3):2207-2220. https://doi.org/10.1007/s11033-022-08200-8.
Wang X, An P, Gu Z, Luo Y, Luo J. Mitochondrial Metal Ion Transport in Cell Metabolism and Disease. Int J Mol Sci. 22(14), 7525, (2021). https://doi.org/10.3390/ijms22147525.
Xing Y, Wei X, Wang MM, Liu Y, Sui Z, Wang X, Zhang Y, Fei YH, Jiang Y, Lu C, Zhang P, Chen R, Liu N, Wu M, Ding L, Wang Y, Guo F, Cao JL, Qi J, Wang W. Stimulating TRPM7 suppresses cancer cell proliferation and metastasis by inhibiting autophagy. Cancer Lett. 525, 179-197, (2022). https://doi.org/10.1016/j.canlet.2021.10.043.
Ertilav K, Nazıroğlu M, Ataizi ZS, Yıldızhan K. Melatonin and Selenium Suppress Docetaxel-Induced TRPV1 Activation, Neuropathic Pain and Oxidative Neurotoxicity in Mice. Biol Trace Elem Res. 199(4), 1469-1487, (2021). https://doi.org/10.1007/s12011-020-02250-4.
Öcal Ö, Nazıroğlu M. Eicosapentaenoic acid enhanced apoptotic and oxidant effects of cisplatin via activation of TRPM2 channel in brain tumor cells. Chem Biol Interact. 359, 109914, (2022). https://doi.org/10.1016/j.cbi.2022.109914.
Roche Innovatis AG. CASY Cell Counter + Analyser System Model TT Operator Manual. Reutlingen, Roche Innovatis, 2014. www.ibgm.med.uva.es/files/upload/manual//casyoperatormanual.pdf.
Mandavilli BS, Janes MS. Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy. Curr Protoc Cytom. Chapter 9:Unit 9.35, (2010). https://doi.org/10.1002/0471142956.cy0935s53.

No competing interests reported.

Supplement.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Cisplatin kills ovarium cancer cells through the TRPV1-mediated mitochondrial oxidative stress and apoptosis: TRPV1 inhibitor role of eicopentotaneoic acid

Status:

Version 1

Abstract

Figures

Introduction

Results

TRPV1 protein expression amounts in the OVCAR-3 cells

Cell viability, number, and debris quantity in the OVCAR-3 cells

The stimulation of TRPV1 enhanced the increases in [Ca²⁺]_c levels caused by Cisp

The Cisp-induced increase in TRPV1 current density was diminished by EPA

Cisp treatments promote apoptosis and caspase activity, which subsequently stimulate Cisp-induced OVCAR-3 death

Decrease in mitochondrial function

The GSH concentrations and GSH-Px activity were decreased by increasing TRPV1 stimulation during Cisp incubation

Cisp induces lysosomal injury through the stimulation of TRPV1 in OVCAR-3 cells

Discussion

Material and Methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Cisplatin kills ovarium cancer cells through the TRPV1-mediated mitochondrial oxidative stress and apoptosis: TRPV1 inhibitor role of eicopentotaneoic acid

Status:

Version 1

Abstract

Figures

Introduction

Results

TRPV1 protein expression amounts in the OVCAR-3 cells

Cell viability, number, and debris quantity in the OVCAR-3 cells

The stimulation of TRPV1 enhanced the increases in [Ca2+]c levels caused by Cisp

The Cisp-induced increase in TRPV1 current density was diminished by EPA

Cisp treatments promote apoptosis and caspase activity, which subsequently stimulate Cisp-induced OVCAR-3 death

Decrease in mitochondrial function

The GSH concentrations and GSH-Px activity were decreased by increasing TRPV1 stimulation during Cisp incubation

Cisp induces lysosomal injury through the stimulation of TRPV1 in OVCAR-3 cells

Discussion

Material and Methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

The stimulation of TRPV1 enhanced the increases in [Ca²⁺]_c levels caused by Cisp