Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple negative breast cancer cells

Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells Received for publication, August 25, 2020, and in revised form, December 24, 2020 Published, Papers in Press, December 30, 2020, https://doi.org/10.1074/jbc.RA120.015769 Isaac Jardin 1, * ,‡ , Raquel Diez-Bello 1,‡ , Debora Falcon 2,‡ , Sandra Alvarado 1 , Sergio Regodon 3 , Gines M. Salido 1 , Tarik Smani 4 , and Juan A. Rosado 1 From the 1 Department of Physiology (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers (IMPB), University of Extremadura, Caceres, Spain; 2 Cardiovascular Physiopathology Group, Institute of Biomedicine of Sevilla, Sevilla, Spain; 3 Department of Animal Medicine, University of Extremadura, Caceres, Spain; and 4 Department of Medical Physiology and Biophysic, Institute of Biomedicine of Sevilla, Sevilla, Spain Edited by Roger Colbran Melatonin has been reported to induce effective reduction in growth and development in a variety of tumors, including breast cancer. In triple-negative breast cancer (TNBC) cells, melatonin attenuates a variety of cancer features, such as tumor growth and apoptosis resistance, through a number of still poorly characterized mechanisms. One biological process that is important for TNBC cells is store-operated Ca 2+ entry (SOCE), which is modulated by TRPC6 expression and function. We wondered whether melatonin might intersect with this pathway as part of its anticancer activity. We show that melatonin, in the nanomolar range, significantly attenuates TNBC MDA-MB-231 cell viability, proliferation, and migration in a time- and concentration-dependent manner, without having any effect on nontumoral breast epithelial MCF10A cells. Pretreatment with different concentrations of melatonin significantly reduced SOCE in MDA-MB-231 cells without altering Ca 2+ release from the intracellular stores. By contrast, SOCE in MCF10A cells was unaffected by melatonin. In the TNBC MDA-MB-468 cell line, melatonin not only attenuated viability, migration, and SOCE, but also reduced TRPC6 expression in a time- and concentration-dependent manner, without altering expression or function of the Ca 2+ channel Orai1. The expression of exogenous TRPC6 overcame the effect of melatonin on SOCE and cell proliferation, and silencing or inhibition of TRPC6 impaired the inhibitory effect of melatonin on SOCE. These findings indicate that TRPC6 downregulation might be involved in melatonin’s inhibitory effects on Ca 2+ influx and the maintenance of cancer hallmarks and point toward a novel antitumoral mechanism of melatonin in TNBC cells. Breast cancer is one of the most common tumors and the major cause of female cancer deaths in the Western world (1). Among the breast cancer subtypes, triple-negative breast cancer (TNBC) is characterized by the absence of estrogen and progesterone receptors and lack of excess of human epidermal growth factor receptor 2 (HER2) expression. TNBC accounts for about 15% of all breast cancer cases (2), and TNBC cells exhibit low levels of differentiation as well as rapid and aggressive proliferation (3). A number of cancer features in TNBC cells have been reported to depend on Ca 2+ influx across the plasma membrane. A major pathway for Ca 2+ influx in TNBC cells is store-operated Ca 2+ entry (SOCE), a mechanism regulated by the filling state of the agonist-sensitive intracellular Ca 2+ stores. SOCE in TNBC cells has been demonstrated to occur with the participation of the endoplasmic reticulum (ER) Ca 2+ sensor STIM1 and the Ca 2+ permeable channel Orai1 (4). The latter is a well-known regulator of proliferation and migration of TNBC cells (5,6). We have recently reported that the cation permeable channel TRPC6 interacts with Orai1 in the triple-negative MDA-MB-231 cell line, where it is required for the expression of Orai1 in the plasma membrane and, thus, SOCE (7). TRPC6 has been found to be highly expressed in human breast ductal adenocarcinoma compared with the adjacent nontumor tissue (8,9), as well as in a number of breast cancer cell lines (9,10), including the TNBC cell line MDAMB-231 (7,9). The high TRPC6 expression in TNBC cells suggests that it might be relevant for cell function, and thus, TRPC6 might be a target for antitumoral therapeutic strategies. Concerning the functional role of TRPC6 in TNBC cells, it has been reported that functional TRPC6 is required for cell proliferation and both 2D and 3D migration (7,9). Consistent with this, treatment of MDA-MB-231 cells with the olive-oil-derived phenolic compound oleocanthal results in attenuation of TRPC6 expression and, consequently, reduced cell proliferation, migration, and viability, an effect that was absent in the nontumoral breast epithelial MCF10A cell line (11). Melatonin (N-acetyl-5-methoxy tryptamine) has been reported to exhibit effective reduction in growth and development in a variety of tumors (12–14), including breast cancer (15,16). The antitumoral actions of melatonin on breast cancer cells have been widely studied in cells of the luminal (estrogen receptor expressing) subtype (17,18). In TNBC, melatonin attenuates tumor growth (19) as well as the expression of NF-κB(20) in MDA-MB-231 cells in a xenograft tumor model. Furthermore, in vitro studies have reported that treatment with melatonin, in the millimolar range, decreases ‡ These authors contributed equally to this work. *For correspondence: Isaac Jardin, [email protected] RESEARCH ARTICLE J. Biol. Chem. (2021) 296 100254 1 © 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Figure 1. Melatonin attenuates MDA-MB-231 cell viability. MCF10A (A,Cand E) and MDA-MB-231 cells (B,D, and F) were treated with melatonin (10–1000 nM) or the vehicle, and 48, 72, or 168 h later, cells were loaded with calcein and propidium iodide (PI). Cell staining was visualized using an inverted microscope as described in Experimental procedures.G–L, scatter plots represent calcein (Cal) and propidium iodide (PI) staining under the different conditions expressed as the ratio between stained versus visible cells and are expressed as mean ± SD. Images shown are representative of 6–8 independent experiments. Analysis of statistical significance was performed using two-way ANOVA (F values for calcein and PI data were 1.34, 2.01, and 0.61 and pvalues were 0.26, 0.14, and 0.71 for concentration, time, and the interaction, respectively, for G,H, and I. F values for calcein and PI data were 106.5, 179.8, and 38.53, and pvalues were <0.0001 for concentration, time, and the interaction respectively for J,K, and L) with post-hoc Tukey’s multiple comparison test (p<0.0001 for J–L). *p<0.05 as compared with their respective controls (Dunnett’s test). Melatonin attenuates TRPC6 expression in breast cancer cells 2J. Biol. Chem. (2021) 296 100254 viability in MDA-MB-231 cells by inducing apoptosis under acute acidosis conditions (21). Melatonin has been reported to attenuate MDA-MB-231 cell invasion and migration in the nanomolar range, a mechanism that has been shown to be mediated by inhibition of the DJ-1/KLF17/ID-1 signaling pathway (22), as well as by the expression of kisspeptin (KiSS1), a well-known suppressor of metastasis (23). A role for melatonin in the modulation of Ca 2+ influx has been reported. Melatonin reduces dopamine release in rat hypothalamus by attenuation of Ca 2+ entry (24) and exerts neuroprotective effects on rat hippocampus by attenuating Ca 2+ entry via TRPM2 (25). Furthermore, administration of melatonin restores SOCE in aged mice pancreatic acinar cells (26) and enhances thrombin-evoked Ca 2+ entry and aggregation in sheep platelets (27). In the present study, we have investigated the possible modulation of TRPC6 function by melatonin and the role of SOCE in the antitumoral effects of melatonin in TNBC cells. Results Effect of melatonin in MDA-MB-231 and MCF10A cell viability and migration Melatonin exerts antitumoral effects in a number of cancer cells and tumoral tissues, including breast cancer (20,22). Hence, we have explored the effect on cell viability of the treatment of TNBC MDA-MB-231 cells and nontumoral breast epithelial MCF10A cells with melatonin by using the cell-permeant dye calcein and propidium iodide. MDA-MB- 231 and MCF10A cells were treated with increasing concentrations of melatonin (10–1000 nM) or the vehicle, as control, and 48, 72, and 168 h (1 week) later, calcein and propidium iodide fluorescences were assessed. As shown in Figure 1,A,C, and E, our results indicate that almost 100% of the MCF10A cells show that calcein fluorescence and propidium iodide staining were unaffected by treatment with melatonin at any of the concentrations tested, at least for 1 week (n = 7). Figure 2. Melatonin attenuates MDA-MB-231 cell migration. MCF10A and MDA-MB-231 cells were treated for 48, 72, or 168 h with melatonin (10–1000 nM) or the vehicle (control) and were subjected to wound healing assay as described in Experimental procedures.A, representative images were acquired at 0, 24, and 48 h from the beginning of the assay. Bars represent 200 μm. The dotted lines define the areas lacking cells. Band C, quantification of the wound size, in micrometers, at the different conditions, expressed as the mean ± SD of four independent experiments. Analysis of statistical significance was performed using two-way ANOVA (for B: 0 h, F values were 0.21, 0.02, and 0.58 and pvalues were 0.88, 0.97, and 0.73 for concentration, time, and the interaction respectively; for B: 24 h, F values were 1.11, 0.60, and 1.14 and pvalues were 0.34, 0.54, and 0.33; for B: 48 h, F values were 1.72, 0.86, and 0.62 and pvalues were 0.16, 0.42, and 0.71; for C: 0 h, F values were 0.35, 0.31, and 0.17 and pvalues were 0.78, 0.72, and 0.98; for C: 24 h, F values were 1570, 616.6, and 447.6 and pvalues were <0.0001 in all cases; for C: 48 h, F values were 6019, 958.4, and 608.1 and pvalues were <0.0001 in all cases) with posthoc Tukey test (*p<0.0001 compared with the scratch time = 0 h. $ p<0.01 compared with the corresponding time in vehicle-treated cells). Melatonin attenuates TRPC6 expression in breast cancer cells J. Biol. Chem. (2021) 296 100254 3 Figure 3. Melatonin attenuates store-operated Ca 2+ entry but not Ca 2+ release in MDA-MB-231 breast cancer cells. A–F, MCF10A (A,C, and E) and MDA-MB-231 (B,D, and F) cells were treated for 48, 72, or 168 h with melatonin (10–1000 nM) or the vehicle (control). Fura-2-loaded cells were perfused with a Ca 2+ -free medium (100 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca 2+ (final concentration 1 mM) to initiate Ca 2+ entry. Gand H, quantification of TG-evoked Ca 2+ release (G) or entry (H) determined as described in Experimental procedures. Scatter plots are represented as mean ± SD. Dots represent single experiments including 20–30 cells. Analysis of statistical significance was performed using two-way Melatonin attenuates TRPC6 expression in breast cancer cells 4J. Biol. Chem. (2021) 296 100254 Treatment of MDA-MB-231 cells with melatonin only significantly attenuates calcein staining after pretreatment with 1000 nM for 72 h (p= 0.0008; one-way ANOVA combined with Dunnett’s test) and 168 h (p= 0.0002; Dunnett’s test) or after treatment with 100 nM for 168 h (p= 0.0004; Dunnett’s test; see Fig. 1). Consistently, pretreatment with 100 nM melatonin for 168 h (p= 0.0004; Dunnett’s test) or with 1000 nM melatonin for 72 h (p= 0.0008) and 168 h (p= 0.0002) significantly enhanced propidium iodide staining (Fig. 1;n=6–8). These findings indicate that melatonin reduces MDA-MB-231 cell viability in a time- and concentration-dependent manner without affecting the viability of MCF10A cells. We have further explored the effect of melatonin on MCF10A and MDA-MB-231 cells migration using the wellestablished wound healing assay (28). Cells were seeded, manually scratched, and cultured in culture medium, supplemented with 1% serum to prevent further cell growth, and cell migration was determined as described in Experimental procedures. MCF10A and MDA-MB-231 cells were treated with increasing concentrations of melatonin (10–1000 nM), or the vehicle (Control), for 48, 72, and 168 h, and then scratched. The wound size was determined 0, 24, and 48 h after scratching, as indicated (Fig. 2A). In the absence of melatonin, all the cells significantly reduced the wound size during the first 48 h (Fig. 2;p<0.05; n = 4). Addition of melatonin to MCF10A cells did not significantly attenuate cell migration at the times and concentrations investigated (Fig. 2B; n = 4). By contrast, treatment of MDA-MB-231 cells with melatonin significantly attenuated cell migration in a concentrationdependent manner (Fig. 2,Aand C;p<0.05; n = 4). The effect observed after 168 h of treatment at any concentration of melatonin or after 72 h with 1000 nM melatonin might be attributed to a reduction in cell viability (as shown in Fig. 1), but the inhibitory effects of melatonin at 10 or 100 nM for 48 or 72 h pretreatment (Fig. 2C, left and middle panel) cannot be attributed to changes in cell viability, thus suggesting that treatment with melatonin impairs TNBC cell migration without affecting the ability of MCF10A cells to migrate. Melatonin attenuates store-operated Ca 2+ entry in MDA-MB- 231 cells Since Ca 2+ entry has been reported to play a relevant role supporting different cancer hallmarks in TNBC cells (4,7), we have assessed the ability of melatonin to impair SOCE in these cells. MCF10A and MDA-MB-231 cells were pretreated for 48, 72, and 168 h with increasing concentrations of melatonin (10–1000 nM), and SOCE was evoked by Ca 2+ store depletion using the SERCA inhibitor TG. As depicted in Figure 3, cell treatment with TG (1 μM) resulted in a transient increase in cytosolic free-Ca 2+ concentration due to Ca 2+ release from the intracellular Ca 2+ stores. Subsequent addition of CaCl 2 (1 mM) to the extracellular medium resulted in a further increase in cytosolic free-Ca 2+ concentration, indicative of SOCE. In MCF10A cells, treatment with melatonin did not significantly alter Ca 2+ release or entry induced by TG (Fig. 3,A,C,E, and G; n = 50 cells/day/5–7 days). Interestingly, pretreatment of MDA-MB-231 cells with melatonin, exclusively for 72 or 168 h, significantly attenuated TG-induced Ca 2+ influx in a concentration-dependent manner, without having any significant effect on Ca 2+ release from the intracellular stores (Fig. 3, B,D,F, and H; n = 50 cells/day/5–7 days). Altogether, these findings indicate that melatonin specifically reduces the activation of SOCE in MDA-MB-231 breast cancer cells while it does not have detectable effect in nontumoral MCF10A cells. Melatonin attenuates TRPC6 expression in MDA-MB-231 TRPC6 expression has been found to be enhanced in breast tumoral samples from patients as well as in both luminal breast cancer cell lines and TNBC cell lines (7,9). In MDA-MB-231 cells, SOCE is highly dependent on TRPC6 expression (7). Hence, in order to ascertain the molecular basis of the effect of melatonin on SOCE in MDA-MB-231 cells, we have assessed its effect on TRPC6 channel expression. MCF10A and MDA-MB-231 cells were treated with increasing concentrations of melatonin (10–1000 nM) for 48, 72, or 168 h or the vehicle, as control, and then subjected to western blotting with specific anti-TRPC6 antibody. Interestingly, treatment of MDA-MB-231 cells with melatonin for 72 or 168 h significantly attenuated TRPC6 channel expression in a concentration-dependent manner as compared with vehicle-treated cells where the expression of TRPC6 was unaffected during the time investigated (Fig. 4,F and J;p<0.0001; n = 8–10). No significant effect on TRPC6 expression was detected upon treatment with melatonin for 48 h (Fig. 4B;n=8–10). By contrast, treatment of MCF10A cells with melatonin did not significantly alter TRPC6 expression at any concentration or pretreatment time investigated (Fig. 4,A,E,andI; n = 10). In order to assess whether melatonin has a nonspecificeffectonplasma membrane proteins, we tested its effect on the PMCA expression in MDA-MB-231 cells. As shown in Figure 4,C, G, and K, melatonin was unable to alter the PMCA expression at any of the experimental procedures tested (n = 5). Due to the relevance of Orai1 in SOCE in MDA-MB-231 cells (4), we have further explored whether melatonin alters its expression at the time and concentrations tested. As depicted in Figure 4,D,H, and L, pretreatment with melatonin (10–1000 nM) for 48, 72, and 168 h was without effect on the expression of Orai1 (n = 5). These findings indicate that melatonin downregulates TRPC6 expression in MDAMB-231 breast cancer cells, without having any effect on Orai1 expression, which might be responsible for the inhibition of cell viability and migration. ANOVA (F values were 0.97, 0.20, and 0.85 and pvalues were 0.40, 0.81, and 0.52 for concentration, time, and the interaction respectively for Ca 2+ release in MCF10A; F values were 2.73, 0.40, and 5.54 and pvalues were 0.64, 0.91, and 0.79 for Ca 2+ release in MDAMB231; F values were 1989, 1777, and 1145 and p values were 0.11, 0.17, and 0.33 for Ca 2+ entry in MCF10A; F values were 459.4, 891.8, and 125.8 pvalues were <0.0001 in all cases for Ca 2+ entry in MDAMB231) with post-hoc Tukey test (*p<0.0001 compared with the response observed in vehicle-treated cells). Melatonin attenuates TRPC6 expression in breast cancer cells J. Biol. Chem. (2021) 296 100254 5 Overexpression of TRPC6 attenuates the inhibitory effect of melatonin on SOCE in MDA-MB-231 cells We have recently reported that TRPC6 knockdown significantly attenuates Ca 2+ influx, as well as cell proliferation and migration in MDA-MB-231 cells (7). Hence, we have further investigated whether TRPC6 downregulation by melatonin underlies the effect of the hormone in SOCE in these cells by overexpressing TRPC6 or a pore-dead dominant-negative Figure 4. Melatonin downregulates TRPC6 expression in MDA-MB-231 cells. MCF10A (A,Eand I) and MDA-MB-231 cells (B–D,F–Gand J–L) were treated for 48, 72, and 168 h with melatonin (10–1000 nM) or the vehicle (control) and lysed. Whole-cell lysates were subjected to 10% SDS-PAGE and western blotting with the anti-TRPC6 (A,B,E,F,I, and J), anti-PMCA (C,G, and K), anti-Orai1 (D,Hand L), or anti-β-actin antibodies, as described in Experimental procedures. Blots are representative of five to ten separate experiments. Scatter plots represent TRPC6, Orai1, or PMCA expression. Analysis of statistical significance was performed using one-way ANOVA (0.001 <F<2.04 and 0.14 <p<0.99 for all panels, except (F) (F = 86.34; p<0.0001) and (J) (F = 182.2; p<0.0001)) with post-hoc Dunnett’s test for (Fand J)(*p<0.0001 compared with control). Melatonin attenuates TRPC6 expression in breast cancer cells 6J. Biol. Chem. (2021) 296 100254 Figure 5. Overexpression of TRPC6 attenuates melatonin-evoked inhibition of store-operated Ca 2+ entry in MDA-MB-231 cells. A,left panel, MDAMB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids. Forty-eight hours later, TRPC6 expression was determined by fluorescence microscopy. A,right panel, MDA-MB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids or empty vector (control). Forty-eight hours later cells were loaded with fura-2. Fura-2-loaded cells were perfused with a Ca 2+ -free medium (100 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca 2+ (final concentration 1 mM) to initiate Ca 2+ entry. Scatter plots represent the quantification of TG- Melatonin attenuates TRPC6 expression in breast cancer cells J. Biol. Chem. (2021) 296 100254 7 TRPC6 mutant (TRPC6dn) (7). As shown in Figure 5A, overexpression of TRPC6 has no additional effects on SOCE in MDA-MB-231 cells; by contrast, expression of TRPC6dn significantly inhibited SOCE. Interestingly, as depicted in Figure 5,Band C, in cells overexpressing TRPC6, the ability of melatonin to inhibit SOCE was significantly attenuated as compared with control cells (Fig. 3; for comparison see scatter plots in Fig. 5,Dand E). These findings suggest that TRPC6 downregulation is at least partially involved in the inhibitory effect of melatonin on SOCE in MDA-MB-231 cells. Overexpression of TRPC6dn and treatment with melatonin resulted in a significant decrease in cell viability, which prevented SOCE determination. The percentage of cells stained with propidium iodide after overexpression of TRPC6dn, in the absence and presence of 10, 100, and 1000 nM melatonin, was 40 ± 3, 52 ± 4, 57 ± 2, and 61 ± 4% after treatment for 72 h with melatonin and 45 ± 4, 42 ± 1, 53 ± 3, and 65 ± 1% after treatment for 168 h with melatonin, respectively (Fig. 5,Fand G; n = 3; analysis of statistical significance was performed using one-way ANOVA [F = 169.1 and 263.6 for Fig. 5Fand Fig. 5G, respectively, p<0.0001] combined with post-hoc Tukey test [p<0.0001 as compared with control]). Overexpression of TRPC6 attenuates the inhibitory effect of melatonin on MDA-MB-231 cell proliferation MDA-MB-231 cell proliferation has been reported to be strongly dependent on SOCE (7,29). Hence, we have investigated the effect of melatonin on MDA-MB-231 cell proliferation. Cells were treated with increasing concentrations of melatonin (10–1000 nM) for 48, 72, and 168 h, and subsequently, cell proliferation was assessed at time = 0h,aswellas24,48,and72hlater.Asshownin Figure 6,Dand G, treatment with melatonin for 72 and 168 h significantly attenuated MDA-MB-231 cell proliferation at almost all the concentrations investigated, as compared with cells treated with vehicle (p<0.0001; n = 6). No significant effect was observed after treatment with melatonin for 48 h (Fig. 6A). Interestingly, expression of exogenous TRPC6 completely reversed the effect of melatoninoncellproliferation(Fig. 6,B,Eand H) while expression of TRPC6dn was without effect on the inhibitory effect of melatonin on MDA-MB-231 cell proliferation (Fig. 6,C,F, and I). These findings indicate that the antiproliferative effect of melatonin in MDA-MB-231 cells is overcome by the expression of functional TRPC6. Pharmacological inhibition or silencing of TRPC6 impairs the effect of melatonin on SOCE in MDA-MB-231 cells To further support the involvement of TRPC6 in the inhibitory effect of melatonin on SOCE, we have impaired TRPC6 function either by pharmacological inhibition or by silencing the expression of the channel. As shown in Figure 7A, pretreatment of MDA-MB-231 cells for 10 min with the TRPC6 inhibitor SAR7334 (1 μM) significantly attenuates Ca 2+ influx induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG). In agreement with our previous studies (7), treatment of MDA-MB-231 cells with SAR7334 significantly attenuated SOCE without having any effect on TG-evoked Ca 2+ release from the intracellular stores (Fig. 7B). As shown in Figure 3, treatment with 100 and 1000 nM melatonin for 72 h significantly reduced SOCE, but, interestingly, in cells pretreated with SAR7334 SOCE was not significantly different in control and upon treatment with 100 or 1000 nM melatonin (one-way ANOVA; F = 1.97 and p= 0.14), thus indicating that inhibition of TRPC6 channels impairs the inhibitory effect of melatonin on SOCE (Fig. 7,B–D). Similarly, we have found that cell transfection with shTRPC6, which significantly attenuates SOCE (see Fig. 7B versus Fig. 7E) as previously reported (7), abolished any further inhibitory effect of melatonin, as observed by treatment with 100 and 1000 nM melatonin for 72 h. Altogether, these findings support a relevant role for TRPC6 in the inhibitory effect of melatonin on SOCE in MDA-Mb-231 cells. As previously shown in Figure 4, treatment with melatonin did not significantly alter Orai1 expression at the time and concentrations used in this study. We have further explored whether melatonin is able to alter Orai1 function. To address this issue, we have cotransfected cells with Orai1 and the Orai1-activating small fragment (OASF; amino acids 233–474) of STIM1, which is able to activate Orai1 independently on store depletion. Coexpression of Orai1 and OASF in MDAMB-231 cells resulted in a robust activation of Ca 2+ influx, which was unaffected by pretreatment for 72 h with melatonin 100 or 1000 nM (Fig. 7F). These findings indicate that melatonin does not significantly affect Orai1 function in these cells. Melatonin enhances the degradation of TRPC6 in MDA-MB- 231 cells We have further explored the mechanism involved in TRPC6 downregulation by melatonin. Protein expression is subjected to protein synthesis and degradation. First, we have evoked Ca 2+ release and entry determined as described in Experimental procedures. Data are presented as mean ± SD. Dots represent a single experiment including 20–30 cells. Analysis of statistical significance was performed using one-way ANOVA (F = 1.82; p= 0.19 for Ca 2+ release and F = 546.5 and p< 0.0001 for Ca 2+ entry, with post-hoc Dunnett’s test; *p<0.0001 compared with control). B–E, MDA-MB-231 cells were treated with melatonin (10–1000 nM) or the vehicle for 72 and 168 h. Forty-eight hours before the end of the treatment period, cells were transfected with TRPC6-YFP expression plasmid. Fura-2- loaded cells were perfused with a Ca 2+ -free medium (100 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca 2+ (final concentration 1 mM) to initiate Ca 2+ entry. Dand E, quantification of TG-evoked Ca 2+ release and entry. Scatter plots are represented as mean ± SD. Dots represent single experiments including 20–30 cells. Data obtained from cells transfected with TRPC6-YFP are compared with those obtained in control cells (gray bars; presented in Fig. 3) performed in parallel. Analysis of statistical significance was performed using two-way ANOVA (F values were 0.28, 0.59, and 0.32 and pvalues were 0.83, 0.61, and 0.96 for concentration, time, and the interaction respectively for Ca 2+ release; F values were 769.6, 2479, and 80.87 and pvalues were <0.0001 in all cases for Ca 2+ entry) with post-hoc Tukey test (*p<0.0001 compared with the response observed in vehicle-treated cells). F–G, MDA-MB-231 cells were treated with melatonin (10–1000 nM) or the vehicle for 72 and 168 h. Forty-eight hours before the end of the treatment period, cells were transfected with TRPC6dn-YFP expression plasmid or empty vector. Cells were loaded with propidium iodide, and cell staining was visualized using an inverted microscope as described in Experimental procedures. Images shown are representative of three independent experiments. Melatonin attenuates TRPC6 expression in breast cancer cells 8J. Biol. Chem. (2021) 296 100254 investigated the effect of melatonin on TRPC6 synthesis by testing its possible effect on the expression of TRPC6 at the transcript level. As depicted in Figure 8A, qRT-PCR analysis indicates that melatonin (100 nM) was unable to alter TRPC6 mRNA expression after treatment for 48 or 72 h (n = 4–7). Hence, we have further explored the ability of melatonin to alter the expression of two miRNAs that have been reported to suppress TRPC6 expression at the protein level, miR26a-5p and miR181b (30,31). The latter was found to be scarcely expressed in MDA-MB-231 cells, and the expression of miR26a was found to be unaffected after treatment with melatonin for 48 or 72 h (Fig. 8B). These findings suggest that melatonin does not alter the synthesis of TRPC6 in MDA-MB- 231 cells. Therefore, we explored the effect of melatonin on TRPC6 protein degradation by analyzing TRPC6 ubiquitination. Ubiquitination was analyzed by immunoprecipitation of cell lysates with anti-TRPC6 antibody followed by western blotting with antiubiquitin antibody. Cells were treated in the absence and presence of MG132, a proteasome inhibitor that allows accumulation of the ubiquitinated form of the protein. As shown in Figure 8C(right panel), a small amount of ubiquitin was found to be associated to TRPC6 in untreated cells (control). Treatment with 100 nM melatonin for 72 h significantly increases the amount of ubiquitin associated to TRPC6. As expected, MG132 enhances the amount of ubiquitinated TRPC6 in cells treated with melatonin or the vehicle. Figure 6. Expression of exogenous TRPC6 reverses the antiproliferative effect of melatonin in MDA-MB-231 cells. MDA-MB-231 cells were treated for 48, 72, and 168 h with melatonin (10–1000 nM) or the vehicle (control). Forty-eight hours before the end of the treatment period, cells were transfected with TRPC6-YFP (B,E, and H) or TRPC6dn-YFP (C,F, and I) expression plasmid or empty vector (A,Dand G). Cell proliferation was assessed at time = 0, 24, 48, and 72 h using the BrdU cell proliferation assay kit, as described in Experimental procedures. Scatter plots represent cell proliferation 0, 24, 48, and 72 h after the onset of the experiment, presented as BrdU uptake rate (n = 6). Analysis of statistical significance was performed using two-way ANOVA (for A,D,G:0h,F values were 0.14, 0.14, and 1.35 and pvalues were 0.93, 0.86, and 0.24 for concentration, time, and the interaction respectively; for A,D,G: 24 h, F values were 593.8, 1570, and 199.6 and pvalues were <0.0001 in all cases; for A,D,G: 48 h, F values were 512.2, 1309, and 201.4 and pvalues were <0.0001 in all cases; for A,D,G: 72 h, F values were 1332, 5690, and 619.2 and pvalues were <0.0001 in all cases; for B,E,H: 0 h, F values were 0.03, 0.0.05, and 0.03 and p values were 0.99, 0.94, and 0.99; for B,E,H: 24 h, F values were 0.09, 0.09, and 0.05 and pvalues were 0.90, 0.98, and 0.99; for B,E,H: 48 h, F values were 0.09, 0.10, and 0.11 and pvalues were 0.89, 0.98, and 0.99; for B,E,H: 72 h, F values were 0.15, 0.05, and 0.07 and pvalues were 0.96, 0.94, and 0.99; for C,F,I:0h,F values were 0.05, 0.22, and 0.02 and pvalues were 0.99, 0.79, and 0.99; for C,F,I: 24 h, F values were 78.10, 2894, and 66.80 and pvalues were <0.0001 in all cases; for C,F,I: 48 h, F values were 704,6, 2442, and 169 and pvalues were <0.0001 in all cases; for C,F,I: 72 h, F values were 893.70, 3707, and 240.1 and p values were <0.0001 in all cases) with post-hoc Tukey’s test for comparison between the data corresponding to the same time of evaluation of proliferation (0, 24, 48, or 72 h) (*p<0.0001 compared with the corresponding control [cells not treated with melatonin]). 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