The epithelial calcium channel TRPV6 is upregulated in breast carcinoma compared with normal mammary gland tissue. The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy. Previously, we showed that tamoxifen inhibits calcium uptake in TRPV6-transfected Xenopus oocytes. In this study, we examined the effect of tamoxifen on TRPV6 function and intracellular calcium homeostasis in MCF-7 breast cancer cells transiently transfected with EYFP-C1-TRPV6. TRPV6 activity was measured with fluorescence microscopy using Fura-2. The basal calcium level was higher in transfected cells compared with nontransfected cells in calcium-containing solution but not in nominally calcium-free buffer. Basal influxes of calcium and barium were also increased. In transfected cells, 10 μmol/L tamoxifen reduced the basal intracellular calcium concentration to the basal calcium level of nontransfected cells. Tamoxifen decreased the transport rates of calcium and barium in transfected cells by 50%. This inhibitory effect was not blocked by the estrogen receptor antagonist, ICI 182,720. Similarly, a tamoxifen-induced inhibitory effect was also observed in MDA-MB-231 estrogen receptor–negative cells. The effect of tamoxifen was completely blocked by activation of protein kinase C. Inhibiting protein kinase C with calphostin C decreased TRPV6 activity but did not alter the effect of tamoxifen. These findings illustrate how tamoxifen might be effective in estrogen receptor–negative breast carcinomas and suggest that the therapeutic effect of tamoxifen and protein kinase C inhibitors used in breast cancer therapy might involve TRPV6-mediated calcium entry. This study highlights a possible role of TRPV6 as therapeutic target in breast cancer therapy. (Mol Cancer Res 2009;7(12):2000–10)
Breast cancer is still ranked third among all cancer deaths and had the highest incidence rate of all cancers of both sexes in Europe in 2006 (1). The survival chances of breast cancer patients depend to a great extent on the expression level and responsiveness of steroid hormone receptors in cancer tissues. Tumors that are estrogen receptor (ER) and progesterone receptor positive have a higher treatment success than those that are nonresponsive to estrogen and progesterone (2). The most widely used antiestrogen therapy is the application of tamoxifen, which belongs to the selective estrogen receptor modulators. Tamoxifen competitively inhibits estradiol binding to the ER and evokes a series of events such as conformational changes of the ER and dimerization and dissociation of heat shock proteins. This triggers binding of the ER to estrogen-responsive elements and transcriptional regulation. Additionally, many coregulator proteins can interact with the ER, but the exact mechanism of how tamoxifen effects the tumors and what role calcium plays in this process is not yet completely understood (3). There is also evidence that tamoxifen is cytotoxic for ER-negative breast cancer cells and that it can be effective in one third of ER-negative tumors (4, 5). A study by Bollig et al. (6) showed that tamoxifen upregulates protein phoshatase 1 α through an ER-independent pathway. Altered changes in intracellular Ca2+ were shown to be induced by tamoxifen at micromolar concentrations and to trigger death of breast cancer cells (7-10).
Changes in intracellular calcium homeostasis are a crucial step in tumor formation in every type of cancer because they influence several cellular functions such as cellular motility, differentiation, proliferation, and apoptosis. Furthermore, calcium is a key regulator of the cell cycle (11). Interestingly, there is an upregulation or downregulation of specific calcium channels or pumps associated with certain types of cancer. For instance, the plasma membrane ATPase 2 (PMCA2) is overexpressed in certain breast cancer cell lines such as ZR-75-1 compared with nontumorigenic 184B5 cells (12). In prostate cancer tumors, the expression level of TRPM8 mRNA is significantly increased in malignant tissue compared with healthy tissue (13). One other example is the altered expression of TRPV6 in prostate, pancreatic, thyroid, colon, ovary, and breast cancer (14).
TRPV6 belongs to the transient receptor potential (TRP) channels. The “TRPV” vanilloid subfamily comprises six channels of which TRPV5 and TRPV6 are relatively selective epithelial calcium channels expressed in renal tubular cells or in the apical membrane of enterocytes, respectively. TRPV6 was discovered in 1999 in rat duodenum using expression cloning (15). Structurely, TRPV6 comprises 730 amino acids and contains six transmembrane domains and a short hydrophobic region between TM5 and TM6, which functions as the pore-forming region. The COOH terminus contains a calmodulin binding site and the NH2 terminus has several ankyrin repeats on the intracellular side (16). TRPV6 is predominantly expressed in the duodenum where calcium is absorbed. In situ hybridization showed localization in the epithelial cells and on the villi tips (14). Additionally, TRPV6 expression was detected in pancreatic acinar cells, mammary gland duct cells, sweat glands, skin, and placenta (17). Examination of TRPV6 transport by electrophysiology revealed that the channel mediates passive Ca2+ transport with high calcium selectivity and an apparent Km value for Ca2+ of 0.44 mmol/L. The cation permeability decreases in the order Ca2+ > Ba2+ > Sr2+ > Mn2+ (18). At the present time, there is no selective blocker for TRPV6-mediated calcium influx available. Nonspecific blockage with ruthenium red, Gd3+, and La3+ was observed (19). TRPV6 mRNA expression is regulated by dietary calcium, 1,25-vitamin D3, dihydrotestosterone, and estrogen (20-22). The intestinal calcium absorption is significantly reduced in TRPV6 knockout mice (23). This shows the importance of TRPV6 as a major calcium uptake pathway of dietary calcium in the intestine.
The exact function of TRPV6 in exocrine tissues is still unknown and its role in cancer is not clear thus far. Numerous studies are published on the role of TRPV6 in prostate cancer. A correlation of TRPV6 expression to the prostate cancer Gleason grade was detected in different cancer stages, whereas there was only little expression in normal prostate tissue (24). Furthermore, certain prostate cancer cell lines such as LNCaP and PC-3 express high amounts of TRPV6 mRNA (20). A study in LNCaP cells showed that TRPV6 can influence the proliferation rate, the cell cycle, and the expression of the proliferating cell nuclear antigen. TRPV6 is the main channel responsible for calcium uptake in this cell line and it can activate the nuclear factor of activated T-cells. Lehen'Kyi et al. (25) showed in the same study that the androgen receptor is involved in TRPV6 regulation in a ligand-independent way. They found that androgen receptor knockdown by siRNA decreased TRPV6 mRNA and protein levels, but the ligands DHT, an androgen receptor–selective agonist, and Casodex, a selective antagonist, had no significant effect on TRPV6 mRNA expression.
This confirms earlier results of studies using HEK293 cells, which revealed an increased proliferation rate if the cells were stably expressed with TRPV6 (26). Of great importance is the fact that TRPV6 mRNA and protein expression is not only increased in prostate cancer, but also in human carcinomas of the colon, thyroid, ovary, and breast (14, 20, 27).
In the ductal epithelial cells of the mammary gland, TRPV6 was found to be expressed in the apical membrane (14).
Our former study confirmed that TRPV6 is expressed at higher levels in breast cancer samples compared with nontumorous samples (28). We also showed that TRPV6 expression in T47D breast cancer cells is increased by estrogen, progesterone, and 1,25-vitamin D3. In contrast, tamoxifen was found to downregulate TRPV6 expression in cancer cells and to inhibit radioactive calcium uptake into TRPV6-expressing Xenopus oocytes. Therefore, TRPV6 might be involved in the mechanism of tamoxifen in breast cancer cells. In T47D cells, TRPV6 is able to control proliferation as we showed using the siRNA expression knockdown targeting TRPV6. The studies indicated that it might be clinically useful to develop specific TRPV6 inhibitors as breast cancer drugs. Especially, in the case of ER-negative tumors, TRPV6 targeting could be promising (28).
As a continuation of our published article on the role of TRPV6 in breast cancer, in the present study, our goal was to show the inhibitory effect of tamoxifen on TRPV6-mediated calcium uptake in breast cancer cells and to investigate the mechanism and the subsequent effect on intracellular calcium homeostasis. Our findings help to clarify a potential mechanism of how tamoxifen may be useful in breast cancer therapy.
Expression of TRPV6 in MCF-7, T47D ER+, and MDA-MB-231 ER− Breast Cancer Cells
First, we tested the expression of TRPV6 mRNA in three widely used human breast cancer cell lines. TRPV6 mRNA was found to be expressed at a high level in T47D cells, whereas MCF-7 and MDA-MB-231 cells showed very low levels of TRPV6 (Fig. 1A). The amount of TRPV6 mRNA expressed in T47D was comparable with the mRNA level in LNCaP prostate cancer cells (data not shown). Immunoprecipitation of TRPV6 followed by Western blot technique revealed that T47D cells express higher levels of TRPV6 protein compared with the other two cell lines (Fig. 1B). When we examined the level of the expressed TRPV6 protein qualitatively in these three cell lines using immunofluorescence microscopy, our observation was similar as shown in Fig. 1C. Furthermore, in T47D cells, TRPV6 showed colocalization with PMCA, suggesting that it is expressed at the plasma membrane (Fig. 2).
Effect of Tamoxifen on Calcium Entry in T47D Cells
The basal calcium influx was determined by measuring the rate of the increase of the fura-2 fluorescence ratio following administration of 1 mmol/L calcium. In T47D cells, this calcium influx was very small (Fig. 3A and C). When we investigated the effect of 10 μmol/L tamoxifen on basal calcium influx, we found a remarkable increase in calcium entry (Fig. 3B and C). Additionally, tamoxifen induced a large transient increase in intracellular calcium in nominally calcium-free buffer (Fig. 3B).
These effects can be explained by the fact that tamoxifen induces calcium depletion of the endoplasmic reticulum calcium stores, which in turn activates store-operated calcium channels (SOCC) expressed in the plasma membrane. This phenomenon was observed in several other cell types. Unfortunately, the activation of SOCCs masks any possible effect of tamoxifen on TRPV6. Therefore, we had to turn to another approach namely overexpressing EYFP-tagged TRPV6 in MCF-7 cells.
Effect of Tamoxifen on TRPV6-Mediated Calcium Entry in MCF-7 Cells
MCF-7 cells were chosen for transfection of TRPV6 because they express only very low levels of endogenous TRPV6 (Fig. 1A). Confocal, fluorescence images of transfected MFC-7 cells suggested that exogenous TRPV6 is expressed at the plasma membrane (Fig. 4, arrows). The transfected cells were identified based on EYFP fluorescence (Fig. 5A and B).
As expected, the basal intracellular calcium concentration was highly increased in transfected cells, compared with nontransfected cells (ratio of 2.027 ± 0.023 versus 1.23 ± 0.004; P < 0.001; Figs. 5C, 6A and B). When extracellular calcium was omitted, this difference was no longer detectable (1.16 ± 0.004 versus 1.116 ± 0.002). Furthermore, basal calcium and barium influx was 100- and 9- fold larger in transfected cells compared with nontransfected cells, respectively (Fig. 6C and D).
Basal intracellular calcium concentrations and the basal calcium influx rate correlated closely, as shown in Fig. 6E and F. Both of the influxes were almost completely inhibited by 100 μmol/L Gd3+, a nonspecific TRPV6 inhibitor (data not shown). Our functional data and confocal images provided solid evidence that the expressed EYFP-TRPV6 is fully functional at the plasma membrane.
Incubation with 10 μmol/L tamoxifen for 10 minutes induced a transient calcium increase followed by a sustained decrease in intracellular calcium. The intracellular calcium in transfected cells finally reached the calcium level of nontransfected cells (Fig. 7A). In nominally calcium-free medium, the tamoxifen-induced calcium increase was reduced to insignificant levels (Fig. 7B).
The application of 10 μmol/L tamoxifen for 10 minutes induced a prominent decrease in initial calcium and barium influx rate down to 37.6% (ratio of 440.84 ± 45.84 versus 165.77 ± 26.39, respectively) and 39.7%, respectively (ratio of 90.75 ± 9.00 versus 36.04 ± 3.20; Fig. 6C and D). Tamoxifen at 1 μmol/L for 1 hour also decreased TRPV6 activity to 24.4% (ratio of 440.84 ± 45.84 versus 107.63 ± 16.04) but not when applied for 10 minutes (Fig. 8). Furthermore, the tamoxifen metabolite, 4-hydroxy-tamoxifen (10 μmol/L for 10 minutes), decreased TRPV6 activity to 17.77% (ratio of 440.84 ± 45.84 versus 78.32 ± 13.74; Fig. 8).
Determination Whether the Estrogen Receptor Is Involved in the Observed Effect of Tamoxifen
When the ER antagonist ICI 182,780 (1 μmol/L) was applied together with tamoxifen to MCF-7 cells, we observed no significant change of the tamoxifen-induced inhibition in the presence of ICI 182,780 (Fig. 9A). To confirm these findings, we examined the effect of 10 μmol/L tamoxifen in the ER-negative, human breast cancer cell line, MDA-MB-231, transiently transfected with EYFP-C1-TRPV6. Tamoxifen exerted a similar effect such as in transfected MCF-7 cells. The initial calcium influx was decreased to 30.5% by tamoxifen in transfected cells compared with the control group (ratio of 542.28 ± 157.58 versus 165.73 ± 32.29; Fig. 9B).
Determination Whether Protein Kinase C Plays a Role in the Observed Effect of Tamoxifen
Because tamoxifen was shown to affect protein kinase C (PKC) activity in MCF-7 cells and several possible serin/threonine phosphorylation sites can be predicted on TRPV6, we tested whether PKC inhibition could be involved in the effect of tamoxifen on TRPV6 activity (29). We found that activation of PKC with 200 nmol/L phorbol 12-myristate 13-acetate (PMA) promptly elevated intracellular calcium in transfected cells, whereas the effect was much smaller in nontransfected cells. Furthermore, in some transfected cells, slow calcium oscillations could be observed (Fig. 10A). When extracellular calcium was removed, this robust calcium increase vanished. Also, basal calcium influx was significantly increased in the transfected cells (Fig. 10A). The inhibitory effect of tamoxifen was completely abolished in the presence of PMA (Fig. 10B). When we applied the PKC inhibitor calphostin C (100 nmol/L), we observed that calphostin C alone or in combination with tamoxifen decreased the initial calcium influx in transfected MCF-7 cells to 40.5% and 46.4%, respectively (from 440.84 ± 45.84 to 178.57 ± 21.99, to 204.34 ± 30.56; Fig. 10B). Therefore, tamoxifen exerted no additional inhibitory effect when PKC was inhibited.
In spite of intensive efforts toward development of novel therapeutic approaches for the prevention and treatment of breast cancer, the incidence rate of breast adenocarcinomas is still highest among all cancer types and the mortality rate was third among all cancers in Europe in 2006 (1). Because intracellular calcium plays a critical role in many fundamental cellular processes such as proliferation, apoptosis, and secretion, calcium levels are spatially and temporally tightly controlled in cells. Disturbances in intracellular calcium homeostasis are a crucial factor in the process of tumor progression in all cancer types. Many studies have been conducted to evaluate how the regulatory system of intracellular calcium homeostasis and the associated calcium signaling pathways are altered in breast cancer cells compared with normal mammary gland cells. A recent review summarizes the possible roles of T-type calcium channels in breast cancer progression (30). The essential roles of the SOCC Orai1 and the signaling molecule Stim1 in breast cancer metastasis formation was recently reported (31). Monteith and his group (12) observed that the expression of different isoforms of the PMCA is changed in breast cancer cells and that PMCA2 expression is significantly increased (32). Furthermore, inhibition of PMCA using siRNA decreased proliferation of MCF-7 human breast cancer cells (33). In recent years, there has been accumulating evidence that the TRPV6 epithelial calcium entry channel is involved in breast cancer progression. In human and mouse breast cancer samples, TRPV6 protein expression was increased (14).
In addition, our laboratory recently showed that a knockdown of TRPV6 expression using siRNA decreased basal calcium influx and proliferation of T47D human breast cancer cells (28). As a novel finding, we also found that tamoxifen, at micromolar levels, inhibits TRPV6-mediated calcium influx in human TRPV6-expressing Xenopus oocytes. In the present study, we addressed the mechanism of this inhibitory effect and its consequences on intracellular calcium homeostasis using transfected breast cancer cell lines.
We selected two widely used human, ER-positive breast cancer cell lines, T47D and MCF-7, and MDA-MB-231, which are ER-negative. First, we examined the expression of TRPV6 in these cell lines. The mRNA levels were low in MCF-7 and MDA-MB-231 and high in T47D cells. The expression of TRPV6 in T47D cells was comparable with LNCaP prostate cancer cells, which are known to have high TRPV6 levels (data not shown). Immunofluorescence staining and Western blotting of immunoprecipitated samples also showed much higher expression of TRPV6 in T47D cells compared with MCF-7 or MDA-MB-231 cells. Unfortunately, in T47D cells, a high dose of tamoxifen above 1 μmol/L induced depletion of the ER calcium stores followed by a robust increase in SOCC activity. Because, in nondepolarizing cells, the majority of the basal calcium influx occurs through SOCC, the possible effect of tamoxifen on native TRPV6 activity was masked. Tamoxifen also exerts the same effect on SOCC activity in several cell types such as human oral cancer cells, CHO-K1 cells, human osteosarcoma cells, and ZR-75-1 human breast cancer cells (34-37). This effect was shown not to be mediated by ERs.
To evaluate the effect of tamoxifen on TRPV6, we increased the number of TRPV6 channels residing in the plasma membrane of the MCF-7 cells, which expresses a low level of endogenous TRPV6. To this end, we overexpressed EYFP-tagged human TRPV6 in MCF-7 cells to reach expression levels of TRPV6 comparable with those of SOCCs. In such TRPV6-overexpressing cells, we observed a significantly higher basal calcium level and increased calcium and barium influxes. Confocal immunofluorescence studies confirmed that TRPV6 is expressed at the plasma membrane. A short, 10-minute incubation of 10 μmol/L tamoxifen induced a much higher transient calcium response in transfected cells compared with nontransfected cells, the effect of which was diminished when extracellular calcium was omitted.
It should be noted that we selected this initial tamoxifen concentration because the IC50 value of tamoxifen was 7.5 μmol/L in Xenopus oocytes (28).
In transfected MCF-7 cells, after transient calcium elevation, basal intracellular calcium returned to the same level as in nontransfected cells. As described above, this temporary calcium elevation is probably due to ER calcium store depletion and subsequent activation of SOCCs. In transfected cells, both basal calcium and barium influxes were reduced by tamoxifen to 37.6% and 39.7%, respectively. In nontransfected cells, there was either no effect (barium influx) or a small increase (calcium influx) in response to tamoxifen. Hydroxy-tamoxifen (10 μmol/L) and prolonged incubation with lower (1 μmol/L) dose of tamoxifen also reduced TRPV6-mediated calcium influx, suggesting that in tamoxifen-treated breast cancer patients, inhibition of TRPV6 could be involved in the antitumor effect of tamoxifen. After treatment of patients with 5 or 20 mg tamoxifen citrate daily for 28 days, the concentration of tamoxifen can reach between 0.73 and 2 μmol/L in the breast cancer tissue (38). In our experiments, we used 10 μmol/L tamoxifen and, to further ensure the physiologic significance, also a lower concentration of tamoxifen (1 μmol/L).
As we expected, ERs were not involved because inhibition of ERs with ICI 182,780 in ER+ MCF-7 cells was ineffective. Also, tamoxifen reduced TRPV6 activity in the ER-negative cell line, MDA-MB-231, to the same extent as in the ER-positive cell line, MCF-7.
Previously, it was shown that tamoxifen is a potent PKC inhibitor in vitro (39). However, in MCF-7 cells, the reported findings on the effect of tamoxifen are controversial. Rowlands et al. tested the inhibitory potential of several tamoxifen analogues in MCF-7 cells (29). In 2003, one group observed a reduction of PKC activity by tamoxifen, whereas in another report, an increased PKC activity and induction of PKC translocation to the plasma membrane in response to tamoxifen was observed (40, 41). The phosphorylation of T702 on TRPV6 by PKC was also shown by Flockerzi and his group (42). This threonine residue is localized in the calmodulin binding site of TRPV6. Phosphorylation at this position blocks inactivation of the channel by calcium-calmodulin complex. In our experiments, activation of PKC by PMA completely abolished the inhibitory effect of tamoxifen on TRPV6. When the PKC inhibitor calphostin C (100 nmol/L) was applied, TRPV6 activity was diminished. Furthermore, tamoxifen had no inhibitory effect in the presence of calphostin C. It was shown previously that calphostin C can reduce MCF-7 cell viability very potently (43). However, because TRPV6-mediated barium entry was also inhibited by tamoxifen, and because barium does not bind to calmodulin (44), we conclude that tamoxifen does not inhibit TRPV6 function through augmentation of calmodulin-mediated inactivation. Nevertheless, there are several other possible sites on TRPV6 predicted to be phosphorylated by PKC. Phosphorylation of one or more of these could enhance TRPV6-mediated ion influx.
In summary, we show that tamoxifen inhibits TRPV6 activity via an ER-independent way. This finding may help to explain the mechanism behind the success in therapy of ER-negative breast tumors with tamoxifen. Although the detailed mechanism of how tamoxifen affects TRPV6 still needs to be elucidated, we conclude that PKC activity modulates the inhibitory effect of tamoxifen. We also found that PKC activation enhances, whereas PKC inhibition decreases, TRPV6 activity, and that these effects are not mediated by calmodulin. It is tempting to suggest that PKC inhibitors used for breast cancer treatment affect TRPV6 as well. Our findings suggest that TRPV6 might be a possible target for the development of new breast cancer medications.
Materials and Methods
The breast cancer cell lines, MCF-7, MDA-MB-231 and T47D were obtained from American Type Culture Collection, Health Protection Agency, and NIH National Cell, respectively. Culture Center. Fugene 6 transfection reagent was used from Roche Applied Biosystems. The EYFP-C1-hTRPV6 vector was a generous gift from Prof. Christoph Romanin, Institute for Biophysics, Johannes Kepler Universität Linz, Austria.
Rabbit anti-human TRPV6 antibody was obtained from ProteinTech Group, Inc. Mouse anti-human PMCA antibody was purchased from Abcam. Hoechst 33342, goat anti-rabbit IgG conjugated to Alexa 594, goat anti-mouse IgG conjugated to Alexa 488, Fura-2 AM, cell culture medium RPMI 1640, and cell culture reagents were obtained from Invitrogen. The mounting medium Citifluor AF2 was from LucernaChem. Tamoxifen citrate was purchased from Acros Organics, and ICI 182,780 was from Tocris Bioscience. Phorbol-12-myristate-13-acetate and calphostin C were purchased from Calbiochem. All other chemicals were obtained from Sigma-Aldrich). Anti-rabbit IgG horseradish peroxidase conjugate (Promega), Amersham enhanced chemiluminescence Western Blotting Detection Reagents (GE Healthcare), Complete Mini (protease inhibitors; Roche Diagnostics GmbH), Immobilon-P Transfer Membrane (Milian), and 1% Nonidet P-40 (Roche Diagnostics GmbH) were used for the Western blotting and immunopreciptation.
The human breast cancer cells lines MCF-7 and T47D were cultured in RPMI medium supplemented with 10% fetal bovine serum, 1 mmol/L HEPES, and 1% penicillin/streptomycin. The cells were kept in a cell culture incubator at 5% CO2 at 37°C and were passaged twice a week.
cDNA was prepared for every sample by reverse transcription of total RNA isolated with Trizol using TaqMan Reverse Transcription Reagents (Applied Biosystems) according to the manufacturer's manual. For all experiments, mRNA expression was measured by real-time PCR using an Applied Biosystems 7500 Real-time PCR System. Reactions consisted of 1× Mastermix, 0.9 μmol/L forward and reverse primers, and 0.2 μmol/L dual-labeled fluorescent probes each for TRPV6 and β-actin. The sequences of the forward and reverse primers for TRPV6 were 5′-GGT TCC TGC GGG TGG AA-3′ and 5′-CCT GTG CGT AGC GTT GGA T-3′, respectively, with the resulting amplicon being 62 bp with a temperature of 60°C. The sequence of the probe for TRPV6 was 5′-ACA GGC AAG ATC TCA ACC GGC AGC-3′. The sequences of the forward and reverse primers for β-actin were 5′-CCT GGC ACC CAG CAC AAT-3′ and 5′-GCC GAT CCA CAC GGA ATA CT-3′, respectively, with the resulting amplicon being 69 bp with a temperature of 60°C. The sequence of the probe for β-actin was 5′-ATC AAG ATC ATT GCT CCT CCT GAG CGC-3′. The specificity of all primers was confirmed by BLAST search. Primer Express (Applied Biosystems) was used for designing primers for TRPV6 and β-actin. All primers were designed to cross exon-exon boundaries of the coding sequence. Primers were optimized and validated for the comparative Ct method, as described in the manufacturer's manual. ABI Prism SDS software version 1 was used for the analysis of the amplification plots. The fold change ± SD in TRPV6 expression was normalized to β-actin.
Immunoprecipitation and Western Blotting
Three confluent 100-mm cell culture dishes of T47D, MDA-MB-231, and MCF-7 cells were washed thrice with ice-cold PBS. Cells were lysed in lysis buffer (composition: 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 10 mmol/L MgCl, 1% Nonidet P-40, and protease inhibitor cocktail) for 30 min at 4°C under constant agitation. Samples were centrifuged at 12,000 rpm for 20 min at 4°C, and the protein concentration of the saved supernatant was adjusted to 2 mg/mL. Thereafter, 1 mL of the supernatant was incubated with 4 μg of rabbit anti-TRPV6 antibody for 1 h under rotary agitation at 4°C. The samples were then mixed with 30 μL of agarose protein G beads, and the mixture was incubated overnight at 4°C. Beads were washed thrice with lysis buffer, and afterwards, 40 μL of 2× loading buffer were added. The samples were boiled at 95°C for 5 min and run on a 6% SDS-PAGE. The samples were transferred with a semidry method to a polyvinylidene difluoride membrane, and blocked with 5% milk, 0.5% bovine serum albumin, and 0.02% sodium azide in PBS overnight at 4°C. The blocked membrane was probed with the same anti-TRPV6 antibody at 1:500 dilution for 2 h at room temperature and washed thrice in PBS with 0.1% Tween 20. An horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (1:20,000) was applied for 1 h at room temperature. After three washes in PBS with 0.1% Tween 20 and one final wash in PBS, enhanced chemiluminescence was used to visualize the bands on the membrane.
Three days before staining, MCF-7 and T47D cells (150.000 cells per well) were seeded on poly-D-lysine–coated coverslips in six-well plates. All the following steps were done at RT. First, the cells were rinsed twice with TBS and then fixed with 4% PFA in TBS for 15 min. Subsequently, they were washed thrice for 10 min with TBS and blocked with TBS supplemented with 10% goat serum for 1 h. Then, incubation with rabbit anti-TRPV6 (1:100) in TBS with 0.5% bovine serum albumin and 0.02% NaN3 for 90 min followed by three washes with TBS for 10 min was done. The cells were stained with mouse anti-PMCA (1:500) for 1 h in the same buffer followed by the same washing procedure. The cells were incubated first with a goat anti-rabbit secondary antibody conjugated to Alexa 594 and then with a goat anti-mouse secondary antibody conjugated to Alexa 488 at 1:4,000 dilution in TBS with 0.5% bovine serum albumin and 0.02% NaN3 for 1 h separated by three 10-min washes with TBS. Then, the cells were washed thrice for 10 min and mounted with CityFluor AF2. For staining of EYFP-TRPV6–transfected cells, Fugene 6 was applied 48 h after seeding according to the manufacturer's manual. After transfection, cells were incubated for 24 to 48 h before fixing and mounting the cells as described above.
The slides were imaged with a confocal, laser scanning microscope setup using a Nikon Eclipse TE2000-E fully automatized inverted, epifluorescence microscope outfitted with Nikon D-Eclipse C1 laser confocal optics. The system equipped with a violet-diode (405 nm) and a multiline Argon (457-515 nm) from Melles Griot, and a Helium/Neon (594 nm) lasers from JDS Uniphase. Nikon EZ-C1 3.6 confocal imaging software installed on a HP xw4400 workstation was used for image acquisition. Brightness and contrast were adjusted with ImageJ.
For calcium imaging, breast cancer cells (300,000 cells per well) were seeded on poly-D-lysine–treated coverslips in 35-mm dishes. After 24 h, they were transfected with EYFP-TRPV6 plasmid using Fugene 6 according to the manufacturer's manual. After transfection, cells were incubated for 24 to 48 h before ionic imaging.
Cell were loaded with 2.5 μg/mL Fura-2 acetoxy-methyl ester dissolved in DMSO containing PluroniC F-1278 20% in serum-free RPMI in cell culture incubator for 1 h. Cells were incubated in calcium containing Krebs buffer for 20 min. For calcium and barium measurement, Fura-2 ratios (F340/F380) were monitored. All measurements were background corrected. The calcium imaging was done on a Nikon Eclipse Ti-U microscope equipped with a Polychrome V+ monochromator (TILL Photonics). A Nikon ×40 SFluor objective was used for visualization. Images were captured with a Hamamatsu Orca-ER monochrome CCD camera. Image acquisition and analysis was done using Simplepci version 6.2 from CImaging. Basal calcium or barium entry was measured as the rate of the increase of Fura-2 ratio in response to the administration of 1 mmol/L divalent cation into nominally calcium-free solution.
Data are presented as mean ± SEM. Nontransfected groups were compared with Mann-Whitney rank sum test. Spearmans rank correlation test and Fisher's z transformation were used in case of testing significance between transfected groups. Differences in the comparison tests lower than P < 0.05 or Z0.05 > 1.96 were considered significant.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest are disclosed.
We thank statistician Pasquale Cirillo PhD, Institute of Mathematical Statistics and Actuarial Sciences, University of Bern, for helpful discussion about data analysis.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.