Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF)

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells, and cancer‐associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N‐terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co‐cultured with CAF, the RES‐induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial‐stromal crosstalk in the TME leading to resistance to the RES‐induced apoptosis.


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However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME.
Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells.
Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis. treatment over the recent decades. Despite early success in suppressing prostate tumor growth, in most cases, the emergence of hormonerefractory cancer cells leads to tumor growth in a hormone-refractory manner associated with an increased risk of metastasis. In these cases of advanced PCa, hormonal manipulations or chemotherapies are essentially palliative with no effective cure for advanced PCa. There is thus an urgent need to investigate the mechanisms of resistance in PCa in order to design new therapeutic drugs.
In recent years, the importance of dietary components such as anti-oxidants in disease prevention has become an important area of research. Resveratrol (RES), a natural polyphenol found in red wine and foods such as grapes and peanuts, has been suggested to have potent chemo-preventive properties and has been reported to inhibit distinct phases of carcinogenesis in vitro. 1 Previous studies have demonstrated that resveratrol could not only induce apoptosis directly in PC-3 and LNCaP PCa cells, 2,3 but could also sensitize LNCaP and other cell types to apoptosis induced by TRAIL and chemotherapeutic agents. [4][5][6] The mechanism of sensitization has been reported to involve down-regulation of the expression of survivin, a member of the IAP family 4,5 and altered the expression of Bcl-2 family proteins. [4][5][6] Thus, apart from a role in chemoprevention, RES has been suggested to have a potential to be used therapeutically to enhance the effects of chemotherapeutic agents by regulating sensitivity to apoptosis.
However, all these studies were only performed on isolated prostate cancer cells without taking into consideration the TME where in the PCa chemotherapies and PCa treatments in general. Ion channels are integral membrane proteins that form a pore to allow the passage of specific ions by passive diffusion. A rise in free cytosolic Ca 2+ concentration ([Ca 2+ ] c ) near the plasma membrane due to Ca 2+ influx through the membrane Ca 2+ channels is the main mechanism inducing exocytosis. [10][11][12] In the context of cancer, alterations in Ca 2+ homeostasis with modulation of ion channel expression and/or activity could be essential during carcinogenesis or its progression by modulating processes including cell growth, differentiation, migration, or secretion.
Previously published data show that environmental factors could modulate ion channel activity or expression. 13  we showed the expression of TRPA1 channel PCa CAF and its involvement in growth factors secretion by these cells. 15 The activation of the channel by RES induced an increase in intracellular calcium concentrations and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells.
Interestingly, we also show here that when the prostate cancer cells were co-cultured with CAF, the RES-induced apoptosis in epithelial cancer cells was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. These data show the importance of the CAF cells on the effects of RES on prostate cancer cells in the TME. Moreover, the present work highlights CAF TRPA1 channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk leading to the resistance to RES-induced apoptosis. Our data suggest the targeting of the CAF TRPA1 channel along with the use of RES as an anti-cancer agent to induce the apoptosis of the prostate cancer cells.   2.5 | RT-PCR analysis of mRNA expression PS30 cells and primary cultured CAF cells were treated by RES and or TRPA1 inhibitors at the indicated concentrations for 48 h and then total RNA were isolated and RT-PCR experiments were performed as described earlier. 17 The PCR primers and the target sequences for siRNA in this study were designed on the basis of established GenBank sequences and synthesized by Eurogentec (Angers, France) and are detailed in Table 1.

| Immunofluorescence studies
The protein expression studies in PCa cells were carried out using

| Western blot assay
Primary cultured CAF cells and PS30 cells were cultured to 80% confluence and total proteins were extracted. Protein samples (20 μg) were subjected to SDS-PAGE on either 6% or 10% acrylamide gels and transferred to a PVDF membrane by semi-dry Western blotting (Bio-Rad).

| Apoptosis assays
Two techniques were used to study the apoptosis in epithelial cells: Student's t-test was used for statistical comparison of the differences and P < 0.05 was considered significant. hepatocyte growth factor (HGF) (Fig. 1E) and vascular endothelial growth factor (VEGF) (Fig. 1F) compared to control. Moreover, intracellular Ca 2+ chelation with the incubation of the cells with BAPTA/AM decreased the basal secretion of HGF and VEGF by more than twofold (Fig. 1E,F) suggesting that the basal secretion is a Ca 2+ -dependent process in CAF. It is thus possible that RES is able to induce a modulation of the ion channels activity leading to a cytosolic calcium rise and subsequent secretion.

| Resveratrol induces Ca 2+ entry in prostate CAFs
In order to study whether the effects of RES on CAF gene expression and growth factor secretions are mediated by the activation of calcium signaling pathway in these cells, we performed Ca 2+ imaging experiments. As shown in Fig. 2   | 1857 a dose-dependent manner (EC 50 = 6.49 μM) (Fig. 2B). The RESinduced calcium response consisted in a rapid rise followed by a sustained plateau obtained for the concentrations of RES > 1 μM.
For these experiments, we used Fura-2/AM, a ratiometric fluores- and not a mobilization from intracellular Ca 2+ stores (Fig. 2F). This implies that RES activates plasma membrane ion channels to induce a Ca 2+ entry in prostate CAFs.

| Resveratrol (RES) activates TRPA1 in prostate CAFs
In order to identify the ion channels involved in the effects of the RES on PS30 cells, different voltage-dependent (nifedipin, flunarizin, Nickel) (Fig. S2) (Fig. 3). Indeed, the application of HC-030031 (50 μM) on RES-induced Ca 2+ entry suppressed the calcium response (Fig. 3A). Similarly, pretreatment of PS30 cells or primary cultured prostate CAF with the TRPA1 inhibitor for 5 min completely inhibited the RES-induced Ca 2+ entry (Fig. 3B,C), the response being restored when the inhibitor was removed from the medium (Fig. 3B). Similar results were obtained by using A-967079 (1 μM), another specific inhibitor of TRPA1 (data not shown). In the same manner, knockdown of TRPA1 expression by short interference RNA (siRNA) prevented the Ca 2+ entry induced by RES (Fig. 3E). These data show that TRPA1 channel is the main component of RES-induced calcium entry in PCa CAF.
These observations are surprising because RES is known to inhibit mouse and rat TRPA1 channel. 21,22 Using calcium imaging experiments, we, therefore, studied the effects of the TRPA1 activator

| Resveratrol affects epithelia-stromal paracrine interactions in prostate cancer through TRPA1 channel functionality
As mentioned, RES induced a [Ca 2+ ] c increase by activating TRPA1 channel (Fig. 3) and the secretion of growth factors (HGF and VEGF) in human prostate CAF cells (Fig. 1E,F). We, therefore, investigated whether the channel was involved in the secretion of these factors. As shown in Fig. 5, TRPA1 inhibition by HC-030031 (50 μM) prevented the RES-induced secretion of HGF (Fig. 5A) and VEGF (Fig. 5B).
Interestingly, the TRPA1 inhibitor blocked the secretion of the growth factors by at least 50% suggesting the involvement of the channel in the basal secretion of growth factors. As cytoplasmic calcium increase   Table 1  is also known to induce gene expression, we studied whether RES induced growth factors gene expression, and whether TRPA1 channel is implicated.

RT-PCR experiments showed that RES (1 and 10 μM, 48 h) is able
to increase HGF and VEGF expression, an effect prevented by TRPA1 inhibition (HC-030031, 50 μM) (Fig. 5C). Taken together, these data suggest that the modulation of TRPA1 channel activity by RES could impact the epithelium-stroma paracrine interactions.
RES is known to induce apoptosis in PCa epithelial cells LNCaP, PC-3, and DU145. We, therefore, studied whether the impact of RES on epithelial cancer cells is modified when co-cultured with CAF. We first studied the impact of RES on the apoptotic rate of LNCaP cells

| Resveratrol activates mutated TRPA1 in CAF cells
As mouse, rat, 20,21 and human (present study, Fig. 3F with wt-hTRPA1 (Fig. 7B). In the light of our results, these mutations/ SNP could be involved in the sensitivity of the TRPA1 channel to RES.
If this hypothesis is correct, since wt-TRPA1 is inhibited by RES, then   Our data show that the basal and RES-induced secretions were Ca 2+ -dependent as shown by the use of BAPTA/AM, an intracellular calcium chelator (Fig. 1E,F) and the TRPA1 calcium-permeable channel inhibitor HC-030031 (Fig. 5A,B). These data show that CAFs secretion involves ion channel and/or transporters activation. Ion channels are integral membrane proteins that form a pore to allow the passage of specific ions by passive diffusion. A rise in free cytosolic Ca 2+ concentration ([Ca 2+ ] c ) near the plasma membrane due to Ca 2+ influx through the membrane Ca 2+ channels is the main mechanism inducing exocytosis. [10][11][12] In cancers, the alterations of Ca 2+ homeostasis due to the modulation of ion channels expression and activity can be essential during carcinogenesis and/or its progression by modulating processes including cell growth, differentiation, migration or secretion. In this context, we have shown that TRPA1 was activated by RES (Figs. 2 and 3), leading to growth factor expression and secretion.
Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1) is a Ca 2+ -permeable ion channel that acts as a nociceptor, sensing acute and inflammatory pain signals. 34 Concerning the modulation of TRPA1 activity by RES, it has been shown that the mouse and rat ortholog of TRPA1 channels are inhibited by the polyphenol. 21,22 In the present work, we show that RES also inhibits the wild type (WT) human TRPA1 (Fig. 3F)  In the present study, in order to better understand the discrepancy between wt-hTRPA1 (inhibited by RES) and the PCa CAF-expressed TRPA1 (activated by RES), we cloned and sequenced the entire cDNA sequence of the TRPA1 channel expressed in human PCa CAF cells.
Compared to wt-hTRPA1, we observed three mutations in the N-terminal cytoplasmic domain of the channel (Fig. 7). These mutations seem to be involved in the effects of the RES on CAF

CONFLICTS OF INTEREST
The authors declare no competing financial interests