Increase in serum Ca2+/Mg2+ ratio promotes proliferation of prostate cancer cells by activating TRPM7 channels.

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg(2+) concentrations. Changes in Mg(2+) concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca(2+)/Mg(2+) ratio facilitated Ca(2+) influx in both control and prostate cancer cells, a significantly higher Ca(2+) influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca(2+)/Mg(2+) ratio per se. Additionally, an increase in the extracellular Ca(2+)/Mg(2+) ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca(2+)/Mg(2+) ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca(2+)/Mg(2+) ratio could be essential for the initiation/progression of prostate cancer.

Introduction

Ca2+ signaling is essential for regulating physiological functions such as cell proliferation and differentiation (1–3). Prostate cancer (PCa) is the second most lethal tumor among men, and Ca2+ has been shown to be essential for increased cell proliferation in prostate cells (4–6). However, the ion channel(s) involved in increased Ca2+ entry that can lead to an increase in cell proliferation is not fully understood. Additionally, the mechanism that leads to alterations in Ca2+ handling in PCa is still poorly defined. Understanding the factors that drive PCa toward increased cell proliferation is crucial for the development of new therapies that can prevent and/or inhibit initiation and/or progression of PCa. Importantly, early stage PCa depends on androgens that are needed for its growth, and because these androgens also regulate Ca2+ entry, it can be anticipated that abnormal Ca2+ signaling may be an essential step toward increased cell proliferation and in the development of PCa (7–9). In addition, besides Ca2+, other ions such as Mg2+ also play a critical role in cell proliferation (10, 11); however, the mechanism and the importance of the tight balance between these ions especially in PCa is still unclear.

Mg2+ is the second most common cation in intracellular fluids. Appropriate Mg2+ levels have been shown to be involved in physiological functions such as nucleic acid metabolism, protein synthesis, and energy production (12, 13). Interestingly, recently it has also been proposed that Mg2+ could also initiate cell proliferation upon mitogenic stimulus because cells are able to increase their intracellular Mg2+ content by activating Mg2+ influx, which is needed for initiation of protein synthesis (14, 15). Therefore, influx of both extracellular Ca2+ and Mg2+ needs to be tightly maintained for proper intracellular ion homeostasis. Furthermore, alterations in this homeostasis will likely increase cell proliferation and can lead to cancer. An increase in cytosolic Ca2+ has also been shown to regulate apoptosis (16, 17). Thus, a tight balance between Ca2+ and Mg2+ ions is needed. Additionally, loss of this balance can alter the normal functioning of the cell and promote cell proliferation, thereby inducing a more cancerous phenotype. Thus, identification of the ion channel that can regulate this tight balance is important to understand how cells adapt a more cancerous phenotype.

Melastatin-like transient receptor potential (TRPM) subfamilies are a diverse group of voltage-independent Ca2+-permeable cation channels that are expressed in mammalian cells (18–21). Two of these members, TRPM6 and TRPM7, are unique because they possess an enzyme domain in their C termini and have been shown to be regulated by intracellular levels of Mg2+-complexed nucleotides (22). Interestingly, TRPM6/7 have been shown to be spontaneously activated that will conduct Ca2+ and Mg2+ at negative membrane potentials, and the current is strongly activated when Mg-ATP falls below 1 mm (designated MagNuM for magnesium-nucleotide-regulated metal current) (20). TRPM6/7 channels are widely expressed ion channels and recently have been shown to be associated with cell proliferation and survival (22); however, their function and expression in prostate cancer cells is not known. Mutations in the TRPM6/7 gene have also been shown in patients suffering from a hereditary form of hypomagnesaemia caused by impaired Mg2+ reabsorption (23), suggesting an unequivocal role of these channels in Mg2+ homeostasis. Consistent with this, TRPM7-deficient DT40 cells showed decreased cell proliferation that was rescued by adding extracellular Ca2+ or Mg2+ (24). Nevertheless, despite considerable progress in the understanding of MagNuM currents, the molecular nature of the channels involved in proliferation of prostate cancer cells, which can contribute to the development of PCa, remains unknown.

The data presented here indicate that TRPM7 functions as an endogenous MagNuM channel in prostate cancer cells and that a decrease in extracellular Mg2+ concentration potentiates MagNuM currents specifically in prostate cancer cells. Interestingly, in prostate cancer cells, TRPM7 also potentiated Ca2+ currents, and an increase in extracellular Ca2+ or a decrease in extracellular Mg2+ further increased Ca2+ influx. Finally, this increase in Ca2+ influx due to a higher Ca2+/Mg2+ ratio was critical to promote cell proliferation without altering the expression levels of TRPM7. Consistent with these results, the serum Ca2+/Mg2+ ratio was also increased in prostate cancer patients, indicating that the Ca2+/Mg2+ ratio is perhaps critical for cell proliferation in prostate cancer cells.

EXPERIMENTAL PROCEDURES

Cell Culture Reagents and Silencing of TRPM7

Control prostate cell line RWPE (CRL 11609) and prostate cancer cell lines DU145 (HTB-81) and PC3 (CRL1435) were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in their respective medium along with various supplements as suggested by ATCC. Cells were maintained at 37 °C with 95% humidified air and 5% CO2 and passaged as needed. Culture medium was changed twice weekly, and cells were maintained in complete medium until reaching 90% confluence, then trypsinized, centrifuged, and resuspended in the same medium as described before (25). For various ion contents, cells were resuspended in DMEM devoid of calcium and magnesium ions, and both calcium and magnesium ions were supplemented as needed. For RNAi experiments, shRNA that targets the coding sequence of human TRPM7 was obtained from Origene Technologies, and a FITC-conjugated non-targeting shRNA was used as a control. Cells were transfected with individual shRNA (50 nm) using Lipofectamine 2000 in Opti-MEM according to the supplier's instructions (Invitrogen) and assayed after 48 h. For overexpression, HA-TRPM7 was used, and 5 μg of plasmid DNA was transformed using Lipofectamine 2000 in Opti-MEM according to the supplier's instructions and assayed after 24 h. Antibodies that were used in this study are described in the figures. All other reagents used were of molecular biology grade obtained from Sigma unless mentioned otherwise.

Cell Viability Assays

Cells were seeded on 96-well plates at a density of 0.5 × 105 cells/well. The cultures were grown for 24 h followed by addition of fresh medium prior to the experiment. Cell viability was measured by using the Vibrant MTT cell proliferation assay kit (Molecular Probes, Eugene, OR). 30 μl of MTT reagent (0.5 mg/ml MTT in PBS containing 10 μm Hepes) was added to each well and incubated in a CO2 incubator for 2 h. The medium was aspirated from each well, and the culture plate was dried at 37 °C for 1 h. The resulting formazan dye was extracted with 100 μl of 0.04 n HCl in isopropanol, and the absorbance was measured in a microplate reader (Molecular Devices, Sunnyvale, CA) at 570 and 630 nm. Cell viability was expressed as a percentage of the control culture. Trypan blue staining was also used to study cell proliferation and to differentiate between live and dead cells. Cells (5 × 106 cells/well) were grown in 24-well plates under different conditions for 48 h, trypsinized, stained using an equal volume of trypan blue, and counted using a light microscope.

Electrophysiology

For patch clamp experiments, coverslips with cells were transferred to the recording chamber and perfused with an external Ringer's solution of the following composition: 145 mm NaCl, 5 mm CsCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm Hepes, 10 mm glucose, pH 7.3 (NaOH). Hepes was replaced by Mes for the solutions at pH ≤6.0. Whole cell currents were recorded using an Axopatch 200B (Axon Instruments, Inc.). The patch pipette had resistances between 3 and 5 megaohms after filling with the standard intracellular solution of the following composition: 150 mm cesium methane sulfonate, 8 mm NaCl, 10 mm Hepes, 10 mm EGTA, pH 7.2 (CsOH). With a holding potential of 0 mV, voltage ramps ranging from −100 to +100 mV and of 100-ms duration were delivered at 2-s intervals after whole cell configuration was formed. Currents were recorded at 2 kHz and digitized at 5–8 kHz. pClamp 10.1 software was used for data acquisition and analysis. Basal leak was subtracted from the final currents, and average currents are shown. All experiments were carried out at room temperature.

Membrane Preparations and Western Blot Analyses

Cells were harvested and stored at −80 °C. Crude lysates were prepared from RWPE and DU145 cells as described previously (25). Protein concentrations were determined using the Bradford reagent (Bio-Rad), and 25–50 μg of proteins was resolved on 3–8% SDS-Tris acetate gels, transferred to PVDF membranes, and probed with respective antibodies. A 1:500 dilution for TRPM7 (Epitomics) and a 1:1000 dilution for actin (Santa Cruz Biotechnology) antibodies were used to probe respective proteins. Peroxidase-conjugated respective secondary antibodies were used to label the proteins. Proteins on the membrane were detected using ECL reagent (Pierce) and analyzed using a Lumi-Imager (Roche Applied Science) as described (25–28).

Confocal Microscopy

For immunofluorescence, cells were grown overnight on coverslips, washed twice with PBS, and fixed for 30 min using 3% paraformaldehyde. Cells were then permeabilized using cold methanol and blocked for 20 min using 5% donkey serum. For TRPM7 staining, cells were treated with anti-TRPM7 antibody at a 1:100 dilution for 1 h. Cells were washed (three times with PBS, 0.5% BSA) and labeled with rhodamine-linked anti-rabbit secondary antibody (1:100 dilution) (24, 30). Confocal images were collected using an MRC 1024-krypton/argon laser-scanning confocal microscope equipped with a Zeiss LSM 510 Meta photomicroscope.

Quantitative RT-PCR

TRPM7 mRNA expression was assessed with real time RT-PCR using commercially available primers (Origene Technologies). cDNA was transcribed from 1 μg of total RNA with iScript cDNA (Bio-Rad). An equal amount of cDNA template was added to iQ SYBR Green Supermix together with appropriate primers at 0.2 μm each. Quantitative PCR was performed using an iCycler iQ real time detection system following the specifications of the manufacturer. The relative level of mRNA was interpolated from each sample. GAPDH was used for normalization of the transcripts. Specificity of PCR product formation was confirmed by monitoring melting peaks.

Study Design and Statistical Analyses

A retrospective analysis of medical charts of patients that were diagnosed with prostate cancer between 2000 and 2011 was performed. Cases were identified from the cancer registry at Altru hospital, North Dakota. Controls from the same hospital that were negative for prostate-specific antigen or prostate biopsies were also used. The study was approved by the Institutional Review Boards of the hospital and the University of North Dakota. Data on age, histology, prostate-specific antigen were abstracted using electronic records. The inclusion criterion for cases was men with histologically confirmed prostate cancer as a primary site with cancer diagnosed between 2000 and 2011. The exclusion criteria included diagnosis of any cancer other than primary prostate cancer. The inclusion criterion for controls was men who had an annual physical exam between 2000 and 2010 at the same hospital as cases without cancer. χ2 or Fisher's exact tests were used to analyze the differences between groups for categorical variables, and t tests were used for continuous variables. All p values are two-sided, and statistical significance was defined as p values <0.05. Analyses were performed using SAS software V9.1.3 (SAS Institute, Cary, NC).

RESULTS

Functional Characterization of MagNuM Channel in Prostate Cells

In prostate cells, Mg2+ ion is not only crucial for DNA regulation but is also essential for regulating other cellular functions such as cell proliferation. However, the identity of the channel that regulates intracellular Mg2+ levels in prostate cells is not known. We show here that both control prostate cells (RWPE) and prostate cancer cells (DU145 and PC3) showed inward and outward rectifying currents that were prompted using a voltage ramp protocol only in conditions in which intracellular Mg2+ was decreased. Using NaCl-based extracellular solutions containing physiological concentrations of CaCl2 and MgCl2, both control and prostate cancer cells showed a large outward rectifying current that reversed close to 0 mV. The current properties were consistent with previous recordings observed in different cells that have been shown to be linked with TRPM7 channels (20, 29–31). In control prostate cells, the outward rectifying currents appeared gradually and reached a plateau within 400 s after initiation (by eliminating intracellular Mg2+) of the whole cell recording. Interestingly, under these conditions, the amplitude of the current observed was not significantly different in control or prostate cancer cells (at −100 mV, 1.42 ± 0.55 pA/pF was observed in control cells, whereas 0.93 ± 0.16 pA/pF was observed in prostate cancer cells) (Fig. 1, A and C; IV curves are shown in Fig. 1, B and D). The average current density recorded at both positive and negative potentials is presented in Fig. 1G. Similarly, the outward current was also not significantly different in control and cancer cells (at +100 mV, the current density was 17.0 pA/pF for control, whereas 23.9 pA/pF was observed in DU145 and 16.3 pA/pF was observed in PC3 prostate cancer cells) (Fig. 1, E–G).

FIGURE 1.

Characterization of intracellular Mg A, representative traces showing changes of whole cell currents in conditions of 1.2 mm Mg2+ and 1.5 mm Ca2+ external solution from normal prostate cells (RWPE) that were activated by the depletion of intracellular Mg2+. Outward currents (top curve) were measured at +100 mV; inward currents (bottom curve; red) were measured at −100 mV. Average IV curves (developed from maximum currents) under this condition are shown in B. C and E, changes of whole cell currents under similar conditions from prostate cancer cells (DU145 and PC3) are shown. Outward currents were again measured at +100 mV; inward currents were measured at −100 mV (bottom line; red). IV curves (developed from maximum currents) of these cells under this condition are shown in D and F. G, average (8–10 recordings) current intensity at +100 and −100 mV under these conditions is shown. H, representative IV curves of prostate cancer cells (DU145) under conditions of pH 7.3, pH 5.0, and pH 4.0. Time courses of membrane current recorded on prostate cancer cells (DU145) are shown in I. Removal of bath Ca2+ and Mg2+ (divalent ion-free (DVF)) induced a remarkable increase of inward and outward currents. IV curves of corresponding time points are shown in J. K, representative IV curves of prostate cancer cells (DU145) under conditions of standard external solution (SES) (1.2 mm Mg2+, 1.5 mm Ca2+, and 145 mm Na+) and NMDG-Cl (Na+ replaced with NMDG in the external solution). L, representative IV curves of prostate cancer cells (DU145) under conditions of 3 mm Ca2+ and 150 mm NMDG-Cl external solution or 3 mm Mg2+ and 150 mm NMDG-Cl external solution. The inset shows a magnified view of the reverse potential under different conditions. Error bars represent ±S.E.

To confirm that the channels are indeed mediated by TRPM7, channel properties were evaluated at various pH values, which again showed that lowering the pH increased the maximum current intensity (Fig. 1H). Furthermore, large inward currents were observed upon removal of all extracellular divalent cations (Fig. 1, I and J), consistent with previous studies (30). In addition, outward currents, but not inward currents, were reduced when NMDG was added in the extracellular solution (Fig. 1K), suggesting that the outward currents are only partially dependent on Na+ efflux. Finally, current-voltage relations were also evaluated in the presence of varying extracellular Ca2+ and Mg2+ solutions, which showed a preference toward Mg2+ entry over Ca2+. However, in the absence of Mg2+, the channels were able to conduct Ca2+ currents (Fig. 1L).

To investigate whether we can separate the MagNuM currents on the basis of their pharmacological properties, we studied the effects of 2APB on these cells. It has been shown previously that TRPM6 currents are potentiated by the addition of 2APB, whereas TRPM7 currents are significantly inhibited by addition of similar concentrations of 2APB (32, 33). Extracellular addition of 500 μm 2APB dramatically decreased MagNuM current amplitude in control cells (Fig. 2, A and C), indicating that the channel is mainly mediated by TRPM7. Consistent with these results, prostate cancer cells also showed a significant decrease in the channel conductance, and addition of 2APB also decreased MagNuM currents (Fig. 2, B and C). Importantly, removal of 2APB from the extracellular solutions further potentiated the currents (Fig. 2D), suggesting that TRPM7 rather than TRPM6 underlies this current. Surprisingly, unlike control cells, 2APB did not completely inhibit the MagNuM currents observed in cancer cells, suggesting that other channels can also play a role in these cells. Finally, to confirm that the channel properties are indeed dependent on TRPM7, we silenced TRPM7 expression using siRNA. As indicated in Fig. 2E, control cells transfected with TRPM7siRNA, but not control siRNA, showed a significant decrease in TRPM7 protein levels (Fig. 2E). Furthermore, both outward and inward currents were significantly decreased in both control and prostate cancer cells that expressed TRPM7 siRNA (Fig. 2, F and G). Importantly, the channel properties were not changed, suggesting that TRPM7 is the major MagNuM channel in these cells. In addition, we also overexpressed TRPM7 in DU145 cells, which showed an increase in the outward and inward currents (Fig. 2G).

FIGURE 2.

Decrease in intracellular Mg Bath application of 500 μm 2APB inhibited MagNuM current in RWPE and cancer cells, and average IV curves in control (A) and cancer cells (B) are shown. A dose-dependent inhibition of MagNuM currents by 2APB in RWPE and DU145 cells is shown in C. D represents outward currents in DU145 cells. Once the currents reached their peak, 500 μm 2APB was applied to the bath followed by recovery of the current by washing out 2APB. E, representative blots indicating DU145 cells expressing shRNA targeting TRPM7 or control non-targeting shRNA. Mock represents similar conditions without any shRNA plasmid. Cell lysates from DU145 cells were resolved on NuPAGE 3–8% Tris acetate gels and analyzed by Western blotting using TRPM7 antibodies (Epitomics). β-Actin was used as a loading control. Respective IV curves of cells transfected with shRNA targeting TRPM7 in RWPE and DU145 cells are show in F. Average (6–10 recordings) current intensity under these conditions in RWPE and DU145 cells are shown in G. ** indicates values (mean ± S.E.) that are significantly different from control (p < 0.01). H, IV curves from control or TRPM7-overexpressing prostate cancer cells (DU145). I, full blots of DU145 cells (control) or cells either overexpressing TRPM7 (HA-TRPM7) or TRPM7 shRNA (Sh-TRPM7). Western blots were performed either using TRPM7 or HA antibodies. IB, immunoblot. Error bars represent ±S.E.

Mg-ATP Levels Inhibit MagNuM Currents, Which Are Mediated by TRPM7, in Prostate Cells

To further characterize these currents, we also used Mg-ATP because these channels have been shown to be activated by reduction in free intracellular Mg2+ or Mg-ATP concentrations per se. Under similar extracellular conditions, both inward and outward currents were decreased with intracellular application of 2 mm Mg-ATP (Fig. 3, A–E). Addition of intracellular Mg-ATP completely blocked MagNuM currents in normal prostate cells (Fig. 3, A and B; average density is shown in Fig. 3E). Surprisingly, similar concentrations of Mg-ATP did not completely inhibit the MagNuM currents in prostate cancer cells; however, the overall current density was still decreased (Fig. 3, C–E) when compared with cells without Mg-ATP (Fig. 1, C and D), suggesting that perhaps a higher concentration of Mg-ATP is needed to completely block MagNuM channels in cancer cells. Finally, TRPM7 localization was also observed using confocal microscopy, which showed expression of TRPM7 at the plasma membrane in both cell types (Fig. 3F). Interestingly, a higher TRPM7 staining in the plasma membrane was observed in DU145 cells, further indicating that the differences in the properties could just be due to their variable expression patterns. Overall, the data presented in Figs. 1–3 strongly indicate that the currents observed here are similar to those shown with TRPM7 (30, 31) for which under physiological concentrations a small inward flux of divalent cations and a large outward flux of monovalent cations were observed.

FIGURE 3.

Effect of intracellular Mg-ATP on prostate cells. Individual representative traces showing inhibition of cation currents by intracellular application of 2 mm Mg-ATP on normal prostate cells (A) and prostate cancer cells (C) are shown. Respective IV curves (developed from maximum currents) under these conditions on normal prostate cells (B) and prostate cancer cells (D) are shown. E, average (6–10 recordings) current intensity at +100 and −100 mV under these conditions is shown. F, representative confocal images showing TRPM7 staining in control RWPE and DU145 cancer cells. Controls shown here are respective cells that did not receive the primary antibodies but were treated with the secondary antibodies. Error bars represent ±S.E.

Increase in Ca2+/Mg2+ Ratio Facilitates Ca2+ Entry via the MagNuM Currents in Prostate Cancer Cells

We further investigated the importance of the Ca2+/Mg2+ ratio in control and prostate cancer cells. An increase in the Ca2+/Mg2+ ratio to 3.0 did not significantly increase the MagNuM currents in normal prostate cells (−1.47 ± 0.29 pA/pF at −100 mV and +21.20 ± 1.82 pA/pF at +100 mV) (Fig. 4, A and B; the average data are shown in Fig. 4E). In contrast, a significant increase in the MagNuM currents was observed in prostate cancer cells when the Ca2+ to Mg2+ ratio was increased to 3.0 (−1.51 ± 0.42 pA/pF at −100 mV and +35.10 ± 2.68 pA/pF at +100 mV) (Fig. 4, C–E). Importantly, no change in the IV properties of the channel was observed, suggesting that an increase in the Ca2+/Mg2+ ratio affects Ca2+ permeability more in cancer cells as compared with control prostate cells. Interestingly, with a further increase in the Ca2+/Mg2+ ratio to 5.0, the MagNuM currents in prostate cancer cells showed a greater increase in MagNuM channel activity (−3.87 ± 0.61 pA/pF at −100 mV and +42.67 ± 2.86 pA/pF at +100 mV) (Fig. 4, H–J, and supplemental Fig. S1, A and B), whereas the currents observed in normal prostate cells were not affected, and no further increase in MagNuM currents was observed (−2.11 ± 0.26 pA/pF at −100 mV and +28.03 ± 2.47 pA/pF at +100 mV) (Fig. 4, F, G, and J). These results suggest that the extracellular Ca2+/Mg2+ ratio has a profound effect on MagNuM currents in prostate cancer cells but not in normal prostate cells, and in the absence of external Mg2+, these cells will facilitate Ca2+ entry, which can promote cell proliferation.

FIGURE 4.

MagNuM currents were increased when Ca A, representative traces showing changes of whole cell currents from normal prostate cells under conditions of a Ca2+/Mg2+ ratio of 3.0 (or 3 mm Ca2+ and 0 mm Mg2+) in external solution. Top, outward current measured at +100 mV; bottom, inward current measured at −100 mV. The IV curve under this condition is shown in B. C, representative traces showing changes of whole cell currents under similar conditions from prostate cancer cells. Top, outward current measured at +100 mV; bottom, inward current measured at −100 mV. The IV curve under this condition is shown in D. E, average (8–10 recordings) current intensity at +100 and −100 mV under these conditions. ** indicates values (mean ± S.E.) that are significantly different from control (p < 0.01). F, representative traces showing changes of whole cell currents from normal prostate cells under conditions of a Ca2+/Mg2+ ratio of 5.0 (or 5 mm Ca2+ and 0 mm Mg2+) in the external solution. Top, outward current measured at +100 mV; bottom, inward current measured at −100 mV. The IV curve under this condition is shown in G. H, changes of whole cell currents under similar conditions from prostate cancer cells. Top, outward current measured at +100 mV; bottom, inward current measured at −100 mV. The IV curve under this condition is shown in I. J, average (8–10 recordings) current intensity under these conditions. ** indicates values (mean ± S.E.) that are significantly different from control (p < 0.01). Error bars represent ±S.E.

Decreasing Extracellular Mg2+ and Increasing Ca2+/Mg2+ Ratio Increase Cell Proliferation in Prostate Cancer Cells

The results shown above indicate that the MagNuM currents observed in prostate cells are mediated via TRPM7. In addition, our results also show that a decrease in intracellular Mg2+/Mg-ATP levels activates these channels and facilitates Ca2+ entry when the Ca2+/Mg2+ ratio is increased. Thus, we initially examined the expression of TRPM7 to determine whether its expression is also altered in different Ca2+/Mg2+ conditions. As indicated in Fig. 5, A and C, TRPM7 was significantly increased in cancer cells when compared with control cells; however, no change in expression of TRPM7 was observed under various Ca2+/Mg2+ conditions. To establish the relevance of the Ca2+/Mg2+ ratio, we further assayed cell proliferation using varying Ca2+/Mg2+ conditions. As shown in supplemental Fig. S1C, a significant increase in the rate of cell proliferation was observed in cancer cells that were incubated with increasing Ca2+ concentrations. We next investigated the consequences of the increased Ca2+ to Mg2+ ratio in cell growth and proliferation in these two different cell types. Importantly, although an increase in the Ca2+ to Mg2+ ratio significantly increased cell proliferation in both normal and cancer cells, a significantly higher cell proliferation was observed in prostate cancer cells that were supplemented with higher Ca2+/Mg2+ concentrations (increasing Ca2+/Mg2+ ratio; Fig. 5B). One possibility could be that control cells could have more dying/dead cells when compared with cancer cells. Thus, we quantified live versus dead cells under these conditions. As shown in Fig. 5B, no significant change in the number of live versus dead cells was observed in either condition, further suggesting that increased MagNuM activity (as shown in Fig. 4) could be the reason for increased cell proliferation.

FIGURE 5.

Increased calcium entry via the MagNuM channels increased cell proliferation. A, representative blots indicating the expression of TRPM7 under various Ca2+/Mg2+ conditions. Cell lysates from control (RWPE) and DU145 cells were resolved on NuPAGE 3–8% Tris acetate gels and analyzed by Western blotting. Antibodies used are labeled in the figure; β-actin was used as a loading control. B, cell proliferation (MTT assays) under different Ca2+/Mg2+ ratios in control RWPE and DU145 cells. Values are normalized and expressed as percentages. * indicates significance (p < 0.05) versus control. The number of live cells in each condition was quantified using trypan blue and is shown as a percentage at the bottom of the bar graph. C, representative Western blots indicating the expression of TRPM7 in RWPE and DU145 cells expressing either control or TRPM7 shRNA. The top panel was probed with TRPM7 antibodies; the bottom panel was probed with control actin antibodies. D, cell proliferation (MTT assay) under different conditions of varying Ca2+/Mg2+ ratios in RWPE and DU145 cells. Values are expressed as percent change (mean ± S.E.). * indicates significance (p < 0.05) versus control cells. The number of live cells in each condition was quantified using trypan blue and is shown as a percentage. IB, immunoblot. Error bars represent ±S.E.

To further establish that the increase in cell proliferation was dependent on TRPM7 expression, we silenced TRPM7 in these cells. As shown in Fig. 5C, expression of TRPM7 siRNA in RWPE or DU145 cells led to a significant decrease in TRPM7 protein levels. Consistent with these results, a significant decrease in cell proliferation was also observed in cells expressing TRPM7 siRNA. Importantly, inhibition of TRPM7 activity by either 2APB or a decrease in the extracellular concentration of divalent cation (by adding EGTA) also inhibited cell proliferation. These results further support the data presented in previous figures that a decreased Mg2+ concentration along with a subsequent increase in Ca2+ will increase cell proliferation and thus could lead to the cancerous phenotype in prostate cells.

Higher Serum Ca2+/Mg2+ Ratio and Increased TRPM7 Expression Were Observed in Prostate Cancer Patients

To further establish that indeed a decrease in the Ca2+ to Mg2+ ratio could lead to cancer, we next evaluated the serum Ca2+ and Mg2+ concentrations in age-matched control and prostate cancer patients. Records from medical charts of 84 patients newly diagnosed with prostate cancer and 65 patients without any cancer for whom serum Ca2+ and Mg2+ were measured were included in the analyses of prostate cancer risk. The median age (range) was 68 (52–91) years for men with prostate cancer and 72 (52–91) years for controls. Importantly, although no significant change in the corrected serum Ca2+ or Mg2+ levels was observed in prostate cancer patients, a significant increase in the Ca2+ to Mg2+ ratio was observed in prostate cancer patients (Fig. 6A). Overall, the results presented here suggest that an increase in the serum Ca2+ to Mg2+ ratio, which will increase Ca2+ entry by the activation of TRPM7 channels, can lead to an increase in cell proliferation and a cancer phenotype. We further assayed the expression of TRPM7 in age-matched control and prostate cancer samples. As indicated in Fig. 6B, a significant increase in TRPM7 expression was observed in prostate cancer samples when compared with controls. These results further suggest that increased TRPM7 expression in prostate cancer could lead to an altered Ca2+ to Mg2+ ratio, which can play a critical role in either prostate cancer initiation or its progression. More research is needed to evaluate its role in prostate cancer.

FIGURE 6.

Higher serum calcium to magnesium ratio and TRPM7 expression were observed in newly diagnosed prostate cancer patients. A, serum calcium and magnesium levels (in mg/dl) were obtained from age-matched control (cases that had measured magnesium; a total of three cases) and prostate cancer patients (positive biopsies that had measured magnesium at the date of their diagnosis; a total of 25 cases). Total serum calcium was adjusted using albumin that was estimated to be at 4 g/dl by a standard formula (corrected serum calcium = total calcium + 0.8 × (4 − patients albumin)). Values are expressed as mean ± S.D. * indicates significance (p < 0.05). B, RNA was extracted from four controls and age-matched prostate cancer samples, and quantitative RT-PCR was performed. Values represent mean ± S.E. of -fold change in TRPM7 expression when compared with GAPDH from at least two independent experiments. Values are expressed as mean ± S.E.

DISCUSSION

In the present study, we have characterized the functional characteristics of MagNuM currents in prostate cells. TRPM channels are widely expressed in cells including prostate tissues (34). Here we found that TRPM7 channels were expressed and functional in both normal prostate cells (RWPE) and prostate cancer cells (DU145 and PC3). Interestingly, the characteristics of the MagNuM currents observed in these cells were similar to those of TRPM7 (21, 32, 35). In addition, we also present novel evidence that currents of prostate cancer cells are more sensitive to alterations in the Ca2+/Mg2+ ratio. Under the physiological extracellular Mg2+ condition, prostate cells exhibited large TRPM7-like currents that were capable of Ca2+ influx. Moreover, we also found that TRPM7 channel activity was dependent on intracellular Mg-ATP levels, and a decrease in intracellular Mg-ATP facilitated divalent cation entry through TRPM7 channels. Although there are conflicting reports with regard to the effect of Mg-ATP on TRPM7 channels (36), our results indicate that an increase in Mg-ATP inhibits TRPM7 currents in prostate cells. One caveat in these results is that although these cell lines are used extensively they do not truly represent the prostate cancer phenotype, and thus more research is needed to establish the role of TRPM7 in prostate cancer.

TRPM7 has been shown to be expressed in prostate cells; however, its function in these cells has not been established. Importantly, unlike TRPM6, which is expressed only in epithelial cells, TRPM7 expression is widespread. Although both TRPM6 and TRPM7 have been shown to be permeable to both Ca2+ and Mg2+ cations, our data strongly suggest that TRPM7 is essential for prostate cells. Additionally, although there is general consensus that the channel is inhibited by free intracellular Mg2+, the functional roles of intracellular levels of Mg-ATP and the ability to bring in Ca2+ along with its physiological consequence have not yet been identified. Additionally, TRPM6 and TRPM7 channels have unique kinase domains, and although the function of the kinase domain is not fully understood (20, 21, 37), the permeability of these channels to divalent cations seems to be essential for cell viability (20, 21, 24, 31, 33, 37) and maintenance of the Mg2+ homeostasis in humans (23, 38, 39). Functional characteristics of TRPM7 channels have been studied in several cells, but their role in cancer cells and specifically in prostate cancer is still unknown.

Mg2+ plays essential physiological roles as a cofactor of numerous enzymes, as a modulator of ion channels and membrane transporters, and in the regulation of cell proliferation (12, 13, 15). Mg2+ deficiency is also a known risk factor for predisposition to leukemias, and 46% of tertiary cancer patients present with hypomagnesia. Additionally, Mg2+ deficiency seems to be carcinogenic, and in the case of solid tumors, a high level of supplemented Mg2+ inhibits carcinogenesis (13); however, the mechanism is not known (15). One of the most important findings that we have reported here is that a decrease in intracellular Mg2+ concentration led to an influx of Ca2+. Although both control and prostate cancer cells showed an increase in Ca2+ influx, the amount of Ca2+ influx in prostate cancer cells was higher as compared with normal cells. Consistent with these results, an increase in extracellular Ca2+ concentrations also led to a significant increase in cell proliferation in prostate cancer cells. In addition, a subsequent decrease in serum Mg2+ concentration along with an increase in Ca2+ concentrations to obtain a higher Ca2+/Mg2+ ratio not only increased MagNuM currents but also increased cell proliferation. This was specifically evident in prostate cancer cells, which showed a significant increase in cell proliferation as compared with control cells. Collectively, these results suggest that alterations in the Ca2+/Mg2+ ratio facilitate Ca2+ influx via the MagNuM channel and that this increase in Ca2+ promotes cell proliferation that can lead to cancer. These results are consistent with previous reports that have shown the relationship of MagNuM currents in regulating cell proliferation (40). Consistent with these results, an increase in the serum Ca2+ to Mg2+ ratio and TRPM7 expression was observed in prostate cancer patients, further indicating that the serum Ca2+ to Mg2+ ratio, rather than individual Ca2+ or Mg2+ concentrations in the serum, is the deciding factor leading to the increase in cell proliferation. These results are also consistent with a recent report that also showed a higher Ca2+/Mg2+ ratio in prostate cancer patients (10); however, the mechanism was not identified in this report. Importantly, in addition, these two reports suggest that the Ca2+/Mg2+ ratio can also be used for the diagnosis of prostate cancer. More research is needed to correlate its significance with regard to prostate-specific antigen levels and different grades of prostate cancer.

To functionally characterize the identity of the MagNuM channel, we used both pharmacological (2APB) and genetic approaches (silencing and overexpression). It has been shown previously that 2APB facilitates TRPM6-mediated magnesium currents, whereas TRPM7 inhibits the MagNuM currents. The results presented here showed an inhibition in the magnesium currents upon addition of 2APB, suggesting that TRPM7 rather than TRPM6 underlies this current. Consistent with this, silencing of TRPM7 not only decreased MagNuM currents but also inhibited cell proliferation. Conversely, increased expression of TRPM7 was also observed in prostate cancer cells when compared with control cells. This increase in TRPM7 could also account for the difference in the channel characteristics. Interestingly, no change in the expression levels of TRPM7 was observed in different Ca2+ to Mg2+ ratios, suggesting that the functional differences in MagNuM currents are not due to altered expression of TRPM7 but rather are dependent on the extracellular Ca2+ or Mg2+ levels. Overall, the results presented here suggest that in prostate cells MagNuM currents are mediated by TRPM7. Furthermore, the Ca2+ to Mg2+ ratio, which facilitates Ca2+ entry, was increased in cancer cells and led to an increase in cell proliferation. Thus, inhibiting TRPM7 activity (by using 2APB) can limit cell proliferation.