Ion channels are involved in normal physiologic processes and in the pathology of various diseases. In this study, we investigated the presence and potential function of transient receptor potential melastatin 7 (TRPM7) channels in the growth and proliferation of FaDu and SCC25 cells, two common human head and neck squamous carcinoma cell lines, using a combination of patch-clamp recording, Western blotting, immunocytochemistry, small interfering RNA (siRNA), fluorescent Ca2+ imaging, and cell counting techniques. Although voltage-gated K+ currents were recorded in all cells, none of FaDu cells express voltage-gated Na+ or Ca2+ currents. Perfusion of cells with NMDA or acidic solution did not activate inward currents, indicating a lack of NMDA receptor and acid-sensing channels. Lowering extracellular Ca2+, however, induced a large nondesensitizing current reminiscent of Ca2+-sensing cation current or TRPM7 current previously described in other cells. This Ca2+-sensing current can be inhibited by Gd3+, 2-aminoethoxydiphenyl borate (2-APB), or intracellular Mg2+, consistent with the TRPM7 current being activated. Immunocytochemistry, Western blot, and reverse transcription-PCR detected the expression of TRPM7 protein and mRNA in these cells. Transfection of FaDu cells with TRPM7 siRNA significantly reduced the expression of TRPM7 mRNA and protein as well as the amplitude of the Ca2+-sensing current. Furthermore, we found that Ca2+ is critical for the growth and proliferation of FaDu cells. Blockade of TRPM7 channels by Gd3+ and 2-APB or suppression of TRPM7 expression by siRNA inhibited the growth and proliferation of these cells. Similar to FaDu cells, SCC25 cells also express TRPM7-like channels. Suppressing the function of these channels inhibited the proliferation of SCC25 cells. [Cancer Res 2007;67(22):10929–38]

For both excitable and nonexcitable cells, Ca2+ influx is critical for normal cell function. Unlike excitable cells where voltage-gated Ca2+ channels play an important role in Ca2+ entry, nonexcitable cells usually lack voltage-gated Ca2+ channels. The main Ca2+ entry pathway in these cells seems to be store-operated Ca2+ channels or Ca2+-permeable nonselective cation channels (13), of which the transient receptor potential (TRP) channels have recently been recognized as a major candidate. The TRP superfamily of ion channels are divided into six subfamilies according to their sequence homology (46). The melastatin-like transient receptor potential (TRPM) channel subfamily of TRP channels has eight members, TRPM1 to TRPM8. By mediating cations entry as well as membrane depolarization, activation of TRPM subfamily of ion channels has profound influence on various physiologic and pathologic processes (6, 7).

Because Ca2+ is an essential regulator for cell cycle and proliferation (8), it is of importance to identify Ca2+ entry pathways in nonexcitable cells including tumor cells. Although Ca2+ release–activated Ca2+ channel (CRAC), one of the store-operated channels, was initially considered to be the primary pathway for Ca2+ entry in nonexcitable cells (1, 9), more recent studies have recognized the importance of TRPM7 channels in Ca2+-dependent processes (10). In addition, activation of TRPM7 channels has been shown to be involved in cellular Mg2+ homeostasis, diseases caused by abnormal magnesium absorption, and anoxia-induced neuronal cell death (1113). However, the presence and potential function of TRPM7 channels in head and neck tumor cells are unknown.

In this study, we examined the presence and potential role of TRPM7 channels in the growth and proliferation of FaDu and SCC25 cells, two common human head and neck squamous carcinoma cell lines. Our data suggest that activation of TRPM7 channels is critical for the growth and proliferation of these tumor cells.

Cells. FaDu cells (ATCC HTB-43), a cell line of human hypopharyngeal squamous cell carcinoma, were cultured in Eagle's MEM supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin, and 50 μg/mL streptomycin at 37°C. Cells were plated in 35-mm poly-l-ornithine–coated dishes for electrophysiologic recording, Western blot, and fluorescence imaging 3 to 5 days after plating. SCC25 cells (ATCC CRL-1628), a cell line of human thyroid squamous cell carcinoma, were cultured in a 1:1 mixture of Dulbecco's MEM and F-12 medium supplemented with 10% FBS and 400 ng/mL hydrocortisone.

Electrophysiology. Whole-cell recordings were done as previously described (14). Patch electrodes were constructed from thin-walled borosilicate glass (WPI) and had resistances of 1 to 3 MΩ. Currents were recorded using Axopatch 200B amplifier with pCLAMP software (Axon Instruments). They were filtered at 2 kHz and digitized at 5 kHz using Digidata 1322A. Data were eliminated from statistical analysis when access resistance was >10 MΩ or leak current was >100 pA at −60 mV. A multibarrel perfusion system was used to achieve a rapid exchange of external solutions (14). All experiments were done at room temperature.

Immunocytochemistry. Immunocytochemistry was done as described (15). Cells were seeded on 25-mm coverslips. After 3 days, they were fixed with 10% formalin followed by permeabilization with 3% Triton X-100/PBS. They were then incubated in PBS containing 2% goat serum and 1% bovine serum albumin for 30 min, followed by overnight incubation with 1:100 dilution of TRPM7 primary antibody (Abgent) at 4°C. After extensive wash in PBS, cells were incubated with 1:750 dilution of Cy3-conjugated goat anti-rabbit antiserum (Jackson ImmunoResearch) for 2 h at room temperature. Coverslips were then mounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Immunostained cells were visualized under a fluorescent microscope. Excitation/emission wavelengths of 580/630 nm were used to visualize TRPM7-labeled cells. Images were collected with an Optronics DEI-750 camera and analyzed using an image analysis system (Bioquant).

Western blotting. Western blotting was done as described (15). Cells were harvested with 100 μL of lysis buffer. After centrifugation at 12,000 × g for 30 min (4°C), the supernatant was collected and mixed with Laemmli sample buffer and incubated at 37°C for 1 h. The samples were resolved by 8% SDS-PAGE, followed by electrotransfer onto polyvinylidene difluoride membranes (Bio-Rad Laboratories). For visualization, blots were probed with antibodies against TRPM7 (1:250), α-tubulin (Abcam), or actin (1:2,000; Abcam), and detected with horseradish peroxidase–conjugated secondary antibody (1:1,000; Cell Signaling) and an enhanced luminescence kit (Amersham).

Reverse transcription-PCR. Isolation of total RNA was done with RNeasy Mini kit (Qiagen). First-strand DNA was generated from 0.5 μg of total RNA using oligo(dT)15 and reverse transcriptase SuperScript II (Invitrogen) at a reaction volume of 20 μL. An oligonucleotide primer pair was synthesized over regions specific for human TRPM7 cDNA (GenBank accession no. NM017672). A primer pair for the detection of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GenBank accession no. M33197) was used as the internal control. Negative control in PCR reaction was done by replacing cDNA with ultrapure water. Reverse transcription-PCR (RT-PCR) amplification was done with Advantage cDNA polymerase mix (Clontech) using a thermal cycler (MJ Mini, Bio-Rad Laboratories). The protocol for PCR amplification consists of denaturation at 94°C for 3 min, 29 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 15 s, and extension at 72°C for 30 s. Products were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The primers used for RT-PCR were TRPM7 forward (5′-CCATACCATATTCTCCAAGGTTCC-3′), TRPM7 reverse (5′-CATTCCTCTTCAGATCTGGAAGTT-3′), GAPDH forward (5′-ATGCTGGTGCTGAGTATGTCGTG-3′), and GAPDH reverse (5′-TTACTCCTTGGAGGCCATGTAGG-3′).

Small interfering RNA silencing. To construct a plasmid for TRPM7 gene silencing, first two oligonucleotides were annealed and inserted into pSilencer 1.0-U6 small interfering RNA (siRNA) expression vector (Ambion). A fragment that was cut with BamHI was excised and inserted into BamHI site of pCAGGS-eGFP (kindly provided by Dr. J. Miyazaki; Division of Stem Cell Regulation Research, Osaka University Medical School, Osaka, Japan) to express both enhanced green fluorescence protein (eGFP) and short hairpin RNA (16). The oligonucleotide sequences were 5′-GTCTTGCCATGAAATACTCTTCAAGAGAGAGTATTTCATGGCAAGACTTTTTT-3′ for the sense strand and 5′-AATTAAAAAAGTCTTGCCATGAAATACTCTCTCTTGAAGAGTATTTCATGGCAAGACGGCC-3′ for the antisense strand (10). As a negative control, a fragment cut with BamHI from pSilencer 1.0-U6 siRNA expression vector was inserted into pCAGGS-eGFP. Transfection was done with Fugene 6 transfection reagent (Roche Applied Science). Cells were used 72 h after siRNA transfection.

For the study of cell proliferation, siPORT NeoFX transfection agent (Ambion) was used. Briefly, FaDu or SCC25 cells cultured in a 25-cm2 flask were trypsinized and diluted to ∼1 × 105/mL. siPORT NeoFX and TRPM7 siRNA (Ambion) were diluted separately (1:20) in Opti-MEM I reduced-serum medium (Invitrogen) for a final volume of 50 μL/well. After 10-min incubation, transfection complexes were formed by combining the diluted siPORT NeoFX and siRNA and were incubated for additional 10 min. The medium with transfection complexes was dispensed into empty wells of a 24-well plate and cell suspensions were added. Final siRNA concentration and cell number were 30 nmol/L and 1 × 105 per well, respectively. Cell counting and lactate dehydrogenase (LDH) assay were done 72 h after transfection. A negative control siRNA (Ambion) was used to verify that the effect seen with TRPM7 siRNA was not due to the transfection process.

Ca2+ imaging. Ca2+ imaging was done as described (14). Cells grown on glass coverslips were incubated with 10 μmol/L fura-2-acetoxymethyl ester for 45 min (22°C) and transferred to a perfusion chamber on an inverted microscope (Nikon). Images were acquired with a charge-coupled device camera (Sensys KAF 1401, Photometrics) and then digitized and analyzed in a computer controlled by Axon Imaging Workbench software (AIW2.1, Axon Instruments). The shutter and filter wheel were also controlled by AIW to allow timed illumination of cells at 340- and 380-nm excitation wavelengths. Fura-2 fluorescence was detected at 510 nm.

Cell count. Cell counting was done as described (10). The initial number of FaDu or SCC25 cells was adjusted to ∼1 × 105 per well. Test chemicals were added into culture medium at the time of plating. Seventy-two hours after plating, cells were resuspended and counted with a hemocytometer. For medium with different concentrations of free [Ca2+], appropriate amount of EGTA was added to yield the right concentrations using Cabuffer program. pH in each medium was readjusted to 7.2 after EGTA.

LDH assay. LDH assay was done as described (14). Seventy-two hours after plating, cells were washed with Neurobasal medium. Fifty microliters of medium were collected from each well and placed into 96-well plates for background LDH measurement. Triton X-100 (0.5%) was then used to permeabilize the cells for obtaining the maximal releasable LDH. After 30 min, 50-μL medium was collected from each well. Fifty microliters of assay mixture from Cytotoxicity Detection Kit (Roche Applied Science) were then added to each sample. After 30-min incubation at room temperature, the absorbance at 492 nm was read on a multiwell plate reader.

Solutions and chemicals. Standard extracellular solution contained (in millimolar) 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 20 HEPES, 10 glucose (pH 7.4 adjusted with NaOH; 320–335 mOsm). For divalent-free external solutions, CaCl2 and MgCl2 were removed and osmolarity was adjusted with glucose. Patch electrodes contained (in millimolar) 140 CsF, 35 CsOH, 10 HEPES, 2 MgCl2, 1 CaCl2, 11 EGTA, 2 TEA, 4 MgATP (pH 7.25 adjusted with CsOH, 290–300 mOsm). For recordings of voltage-gated K+ current, CsF was replaced with KF. For Mg2+-free pipette solution, Mg2+ and MgATP were omitted. 2-Aminoethoxydiphenyl borate (2-APB) was purchased from Calbiochem; gadolinium chloride and 1-heptamol were from Sigma.

Statistics. Data are expressed as mean ± SE. Student's t test and ANOVA were used as appropriate for the analysis of statistical significance.

Lowering extracellular Ca2+ activates an inward current in FaDu cells. To determine the presence and potential function of ion channels in the growth and proliferation of human head and neck tumor cells, we first used patch-clamp technique to record the membrane current in FaDu cells, a cell line of human hypopharyngeal carcinoma (17). We examined whether these cells have voltage-gated Na+, Ca2+, or K+ current. From a holding potential of −60 mV, membrane was gradually depolarized from −80 to +80 mV by a series of voltage pulses at an increment of +20 mV. As shown in Fig. 1A,-a, no inward current was activated by the depolarizing pulses (n > 10), indicating a lack of voltage-gated Na+ and Ca2+ channels in these cells. However, in all cells recorded, membrane depolarization activated outward K+ current as in the majority of excitable and nonexcitable cells. Perfusion of FaDu cells with 100 μmol/L NMDA plus 3 μmol/L glycine or acidic solutions (pH 5.0) did not activate substantial current (Fig. 1A , b and c; n = 10), indicating the lack of NMDA channels and acid-sensing ion channels (14).

Figure 1.

Activation of Ca2+-sensitive currents in FaDu cells. A, a, representative traces showing the presence of outward K+ currents in FaDu cells. Currents were activated by a series of depolarizing voltage pulses from −80 to +80 mV at an increment of +20 mV. Holding potential was −60 mV. No obvious inward current was detected at any potential, indicating the lack of voltage-gated Na+ and Ca2+ channels. b, representative trace showing the lack of NMDA currents in FaDu cells. Following a stable baseline recording, cells were perfused with NMDA (100 μmol/L) and its co-agonist glycine (3 μmol/L). c, representative trace showing the lack of acid-activated current in FaDu cells. d, a rapid drop of [Ca2+]o from 2 to 0 mmol/L (nominally free) evoked a slow nondesensitizing inward current that rapidly recovered on restoration of [Ca2+]o. Holding potential was −60 mV. B, representative current traces and summary data showing concentration-dependent inhibition of the inward current in FaDu cells by external Ca2+. Dose-response curve was fit by Hill equation with an average IC50 of 0.07 ± 0.02 mmol/L (n = 9). C, a, representative traces showing the Ca2+-sensing current activated at different membrane potentials. Currents were activated by step reductions of [Ca2+]o from 2 to 0 mmol/L over a range of holding potentials from −80 to +20 mV. Pipette solution contains 0 mmol/L Mg2+. b, summary data showing the current-voltage relationship (I-V curve) of the Ca2+-sensing current (n = 7).

Figure 1.

Activation of Ca2+-sensitive currents in FaDu cells. A, a, representative traces showing the presence of outward K+ currents in FaDu cells. Currents were activated by a series of depolarizing voltage pulses from −80 to +80 mV at an increment of +20 mV. Holding potential was −60 mV. No obvious inward current was detected at any potential, indicating the lack of voltage-gated Na+ and Ca2+ channels. b, representative trace showing the lack of NMDA currents in FaDu cells. Following a stable baseline recording, cells were perfused with NMDA (100 μmol/L) and its co-agonist glycine (3 μmol/L). c, representative trace showing the lack of acid-activated current in FaDu cells. d, a rapid drop of [Ca2+]o from 2 to 0 mmol/L (nominally free) evoked a slow nondesensitizing inward current that rapidly recovered on restoration of [Ca2+]o. Holding potential was −60 mV. B, representative current traces and summary data showing concentration-dependent inhibition of the inward current in FaDu cells by external Ca2+. Dose-response curve was fit by Hill equation with an average IC50 of 0.07 ± 0.02 mmol/L (n = 9). C, a, representative traces showing the Ca2+-sensing current activated at different membrane potentials. Currents were activated by step reductions of [Ca2+]o from 2 to 0 mmol/L over a range of holding potentials from −80 to +20 mV. Pipette solution contains 0 mmol/L Mg2+. b, summary data showing the current-voltage relationship (I-V curve) of the Ca2+-sensing current (n = 7).

Close modal

Recent studies have suggested that TRPM7 is one of the ubiquitously expressed ion channels (12, 1820). Because of their Ca2+ permeability and the lack of voltage-dependent gating, they are well designed to contribute to numerous Ca2+-dependent cell processes in nonexcitable cells. For this reason, we explored the possibility that FaDu cells express TRPM7 channels. In the presence of physiologic concentrations of Ca2+ and Mg2+, a rapid drop of extracellular Ca2+ concentration ([Ca2+]o) from 2 to 0 mmol/L evoked a nondesensitizing inward current (Fig. 1A -d). The amplitude of this current measured right after the formation of whole-cell configuration ranged from 138.69 to 1,545.08 pA (mean, 642.12 ± 99.42 pA; n = 14). When Mg2+ was omitted from the pipette solution, the amplitude was 256.37 to 4,491.31 pA (mean, 898.19 ± 103.60 pA; n = 49). This Ca2+-sensitive current is reminiscent of the Ca2+-sensing nonselective cation current in central nervous system neurons and HEK293 cells (21), which was later identified as largely carried by TRPM7 channels (13).

We next recorded the currents activated at different [Ca2+]o to construct a dose-response relationship. As shown in Fig. 1B, the maximal inward current was recorded in the absence of [Ca2+]o and the current gradually decreased with increasing [Ca2+]o. A concentration-response analysis revealed an IC50 of 0.07 ± 0.02 mmol/L for Ca2+ blockade (Fig. 1B; n = 9).

To determine whether this Ca2+-sensing current shows any voltage dependency, currents activated by lowering [Ca2+]o from 2.0 to 0 mmol/L were recorded over a range of holding potentials from −80 to +20 mV. As shown in Fig. 1C,-a, the currents reversed near 0 mV (n = 7) and the current-voltage relationship seemed to be linear, illustrating a lack of voltage dependence (Fig. 1C -b).

Pharmacologic characterization of the Ca2+-sensing current. Previous studies have shown that Gd3+ is a potent nonspecific blocker for TRPM7 channels (10, 13, 22, 23). In the presence of 1, 3, or 10 μmol/L Gd3+ in the bath solution, the amplitude of the Ca2+-sensing current in FaDu cells was decreased by 24.53 ± 9.08% (n = 6, P < 0.05), 36.23 ± 7.12% (n = 9, P < 0.01), and 46.66 ± 10.89% (n = 9, P < 0.01), respectively (Fig. 2A). This potency of Gd3+ inhibition of the Ca2+-sensing inward current in FaDu cells is consistent with the effect of Gd3+ on Mg2+-inhibitable cation channels (MIC) or TRPM7 channels (10, 13, 23).

Figure 2.

Pharmacologic characterization of the Ca2+-sensing current in FaDu cells. A, dose-dependent inhibition of the Ca2+-sensing current by Gd3+. Representative traces of the Ca2+-sensing currents in the absence and presence of different concentrations of Gd3+. Holding potential, −60 mV. Pipette solution contained no Mg2+. *, P < 0.05; **, P < 0.01. B, effect of 2-APB on the Ca2+-sensing current. Representative traces and summary bar graph showing the Ca2+-sensing current in the absence and presence of 100 μmol/L 2-APB. **, P < 0.01. C, representative current traces showing the lack of inhibition on the Ca2+-sensing current by 1 mmol/L heptanol. D, a, time-dependent change in the amplitude of the Ca2+-sensing current with pipette solution containing either 8 or 0 mmol/L Mg2+. Currents were induced by a drop of [Ca2+]o from 2 to 0 mmol/L at −60 mV. The decrease of the current amplitude with time was significantly greater with pipette solution containing 8 mmol/L Mg2+ (P < 0.05, two-way ANOVA). b, current-voltage relationship of the Ca2+-sensing current in the presence and absence of physiologic concentrations of Ca2+ and Mg2+. Currents were activated by 400-ms voltage-ramp pulses ranging from −80 to +100 mV in the presence (a) and absence (b) of 2 mmol/L Ca2+ and 1 mmol/L Mg2+ in the extracellular solutions. Note that even in the presence of normal Ca2+ and Mg2+, substantial current can still be activated at negative membrane potentials.

Figure 2.

Pharmacologic characterization of the Ca2+-sensing current in FaDu cells. A, dose-dependent inhibition of the Ca2+-sensing current by Gd3+. Representative traces of the Ca2+-sensing currents in the absence and presence of different concentrations of Gd3+. Holding potential, −60 mV. Pipette solution contained no Mg2+. *, P < 0.05; **, P < 0.01. B, effect of 2-APB on the Ca2+-sensing current. Representative traces and summary bar graph showing the Ca2+-sensing current in the absence and presence of 100 μmol/L 2-APB. **, P < 0.01. C, representative current traces showing the lack of inhibition on the Ca2+-sensing current by 1 mmol/L heptanol. D, a, time-dependent change in the amplitude of the Ca2+-sensing current with pipette solution containing either 8 or 0 mmol/L Mg2+. Currents were induced by a drop of [Ca2+]o from 2 to 0 mmol/L at −60 mV. The decrease of the current amplitude with time was significantly greater with pipette solution containing 8 mmol/L Mg2+ (P < 0.05, two-way ANOVA). b, current-voltage relationship of the Ca2+-sensing current in the presence and absence of physiologic concentrations of Ca2+ and Mg2+. Currents were activated by 400-ms voltage-ramp pulses ranging from −80 to +100 mV in the presence (a) and absence (b) of 2 mmol/L Ca2+ and 1 mmol/L Mg2+ in the extracellular solutions. Note that even in the presence of normal Ca2+ and Mg2+, substantial current can still be activated at negative membrane potentials.

Close modal

Next, we test the effect of 2-APB. Over a concentration range of 10 to 100 μmol/L, 2-APB has broad inhibitory effects on TRP superfamily including TRPM7 channels (10, 2427). In the presence of 100 μmol/L 2-APB, the current in FaDu cells was inhibited by 31.80 ± 6.18% (Fig. 2B; n = 15, P < 0.01). The inhibition by 2-APB further suggests that the Ca2+-sensing current in FaDu cells is carried by TRP channels.

Lowering [Ca2+]o is known to activate hemi-gap junction channels (28). To rule out the possible involvement of the hemi-gap junction channel, the effect of 1-heptanol, a commonly used inhibitor for gap junction channels (29, 30), was tested. As shown in Fig. 2C, bath perfusion of 1 mmol/L 1-heptanol was not effective in modulating the currents (n = 5), indicating the lack of an involvement of the hemi-gap junction channels.

Nadler et al. (12) found that TRPM7 channel activity in HEK293 cells was strongly suppressed by internal Mg2+. Similarly, Kozak and Cahalan (31) found that MIC (or TRPM7) channels in rat basophilic leukemia cells were inhibited by internal divalent cations. The TRPM7-like current in rat brain microglia was also inhibited by the intracellular Mg2+ (18). In Jurkat E6-1 human leukemic T cells, internal Mg2+ has been used as a tool to isolate the CRAC current from that of TRPM7 channels (32). To provide further evidence that TRPM7 is involved in the generation of the Ca2+-sensing current, 8 mmol/L MgCl2 was included in the pipette solution and the amplitude of the Ca2+-sensing current that was recorded with or without internal Mg2+ was compared. As shown in Fig. 2D -a, the presence of internal Mg2+ significantly inhibited the current (n = 12 with [Mg2+]i, n = 6 without [Mg2+]i; P < 0.05), providing additional evidence that the Ca2+-sensing currents in FaDu cells are carried, at least partially, by TRPM7 channels.

Current-voltage relationship was then generated using a voltage ramp to see whether the Ca2+-sensing currents show outward rectification in the presence of normal Ca2+, an important feature of the TRPM7 current. As shown in Fig. 2D -b, in the presence of normal Ca2+ and Mg2+ in the external solution, small inward currents were activated at negative potentials whereas larger outward currents were recorded at positive potentials, showing a prominent outward rectification (n = 13). When the external solution was made divalent-free, the inward currents were dramatically increased, resulting in a near linear I-V relationship (n = 10).

Taken together, both electrophysiologic and pharmacologic data strongly indicate an involvement of TRPM7 channels in mediating the Ca2+-sensing current.

Detection of TRPM7 protein and mRNA in FaDu cells. Biochemical and molecular biological techniques were then used to further show the existence of TRPM7 channels. As shown in Fig. 3A, clear immunoreactivity against TRPM7 was detected in almost all FaDu cells tested. Western blotting showed a clear band of ∼220 kDa (Fig. 3B,-a). To verify the specificity of the antibody used, HEK293 cells with inducible expression of TRPM7 (13) were used as a positive control. Following induction of TRPM7 expression, a clear protein band of ∼220 kDa was detected in HEK293 cells (Fig. 3B,-a). RT-PCR also detected the presence of TRPM7 mRNA in FaDu cells (Fig. 3B -b).

Figure 3.

Detection of TRPM7 protein and mRNA in FaDu cells before and after siRNA-TRPM7 transfection. A, immunofluorescent image showing positive staining of FaDu cells with anti-TRPM7 antibody. Right, overlaid image with anti-TRPM7 in red and DAPI-stained nuclei in blue. B, detection of TRPM7 protein in FaDu cells by Western blot (a) and RT-PCR (b). For Western blot, HEK293 cells stably transfected with TRPM7 served as a positive control, whereas actin was used as an internal control. For RT-PCR, ultrapure water served as a negative control, whereas GAPDH was used as an internal control. C, a, summary data showing the inhibition of the Ca2+-sensing currents in FaDu cells by TRPM7 siRNA. Currents were induced by a drop of [Ca2+]o from 2 to 0 mmol/L at −60 mV with Mg2+-free pipette solution. Electrophysiologic recordings were done 72 h after TRPM7 siRNA transfection. **, P < 0.01. b, right, the specific loss of TRPM7 immunoreactivity was observed in cells transfected with TRPM7 siRNA as indicated by white arrows (top), compared with no immunoreactivity changes when cells were transfected with empty vector (bottom). Left, cells with eGFP fluorescence as an indication of successful transfection. D, a, Western blot images show the reduction of TRPM7 protein in FaDu cells transfected with TRPM7 siRNA compared with nontransfected or control siRNA–transfected cells. Actin served as an internal control. b, RT-PCR images show reduction of TRPM7 mRNA in cells transfected with TRPM7 siRNA. Replacement of TRPM7 cDNA with ultrapure water served as a negative control. GAPDH was used as an internal control.

Figure 3.

Detection of TRPM7 protein and mRNA in FaDu cells before and after siRNA-TRPM7 transfection. A, immunofluorescent image showing positive staining of FaDu cells with anti-TRPM7 antibody. Right, overlaid image with anti-TRPM7 in red and DAPI-stained nuclei in blue. B, detection of TRPM7 protein in FaDu cells by Western blot (a) and RT-PCR (b). For Western blot, HEK293 cells stably transfected with TRPM7 served as a positive control, whereas actin was used as an internal control. For RT-PCR, ultrapure water served as a negative control, whereas GAPDH was used as an internal control. C, a, summary data showing the inhibition of the Ca2+-sensing currents in FaDu cells by TRPM7 siRNA. Currents were induced by a drop of [Ca2+]o from 2 to 0 mmol/L at −60 mV with Mg2+-free pipette solution. Electrophysiologic recordings were done 72 h after TRPM7 siRNA transfection. **, P < 0.01. b, right, the specific loss of TRPM7 immunoreactivity was observed in cells transfected with TRPM7 siRNA as indicated by white arrows (top), compared with no immunoreactivity changes when cells were transfected with empty vector (bottom). Left, cells with eGFP fluorescence as an indication of successful transfection. D, a, Western blot images show the reduction of TRPM7 protein in FaDu cells transfected with TRPM7 siRNA compared with nontransfected or control siRNA–transfected cells. Actin served as an internal control. b, RT-PCR images show reduction of TRPM7 mRNA in cells transfected with TRPM7 siRNA. Replacement of TRPM7 cDNA with ultrapure water served as a negative control. GAPDH was used as an internal control.

Close modal

Effects of TRPM7 siRNA silencing on the Ca2+-sensing currents. To obtain more evidence that TRPM7 channels are involved in mediating the Ca2+-sensing currents in FaDu cells, we used RNA interference, a process of posttranscriptional gene silencing that inhibits the expression of native genes with high specificity (33, 34). A 21-nucleotide siRNA duplex specifically targeting human TRPM7 was selected (see Materials and Methods). In our experiments, ∼30% of FaDu cells were successfully transfected with siRNA, as indicated by eGFP fluorescence.

To test the effect of TRPM7 siRNA on the Ca2+-sensing currents, patch-clamp recording was done on GFP-positive cells 72 h after TRPM7 siRNA transfection. As shown in Fig. 3C -a, the amplitude of the Ca2+-sensing current was significantly reduced in cells transfected with TRPM7 siRNA. In six cells tested, the current density was decreased by 65.45 ± 26.72% in cells transfected with TRPM-siRNA, as compared with the control cells transfected with empty vector.

Immunocytochemistry and Western blot were used to confirm the reduction of TRPM7 protein. As shown in Fig. 3C,-b, in siRNA-transfected cells (as indicated by eGFP), the specific loss of TRPM7 immunoreactivity was observed, whereas in cells transfected with vector alone, strong immunoreactivity against TRPM7 exists. Western blotting also showed a significant reduction in the density of 220-kDa band in TRPM7 siRNA–transfected cells (Fig. 3D -a).

RT-PCR was further used to see whether transfection with TRPM7 siRNA suppresses the expression of TRPM7 mRNA in FaDu cells. As shown in Fig. 3D -b, transfection with TRPM7 siRNA induced an apparent suppression of TRPM7 mRNA, whereas transfection with vector alone had no effect.

Ca2+ is critical to FaDu cell growth. Ca2+ signaling is fundamental for cell cycling and proliferation. Given the fact that TRPM7 channel is Ca2+-permeable and activation of these channels is expected to affect the concentration of intracellular [Ca2+]i, we carried out Ca2+ imaging experiments to monitor changes in [Ca2+]i as a response to changes in [Ca2+]o. In a total of 73 cells tested, 34 (∼47%) cells responded to changes in [Ca2+]o with detectable changes in [Ca2+]i. As shown in Fig. 4A, over a range of 0 to 2 mmol/L [Ca2+]o, [Ca2+]i changes in parallel to [Ca2+]o. Because changing [Ca2+]o affects the driving force for Ca2+ entering the cells, these data suggest that there might be a basal activation of TRPM7 channels.

Figure 4.

Ca2+ and TRPM7 are critical to FaDu cell growth and proliferation. A, fura-2 fluorescence imaging showing changes in the intracellular Ca2+ concentration as indicated by 340/380 nm ratio in response to changes in [Ca2+]o (left), and summary data showing changes in [Ca2+]i as indicated by 340/380 nm ratio in response to changes in [Ca2+]o (right; n = 5–9). **, P < 0.01, compared with 2.0 mmol/L [Ca2+]o. B, effect of [Ca2+]o on the growth and proliferation of FaDu cells as measured by direct cell counting (left) or total LDH release (right). Over a range of [Ca2+]o from 1 to 0.1 mmol/L, the rate of proliferation of the FaDu cells decreased in parallel with decreasing [Ca2+]o. For each data point, 16 to 32 wells of cells from four to eight independent experiments were analyzed. *, P < 0.05; **, P < 0.01, compared with 2.0 mmol/L [Ca2+]o. C, concentration-dependent inhibition of the proliferation of FaDu cells by 2-APB, measured by cell count (left) or total LDH release (right). D, inhibition of the proliferation of FaDu cells by TRPM7 siRNA, evaluated by cell count (left) and LDH assay (right). Con, cells grew in normal medium; siRNA(+), cells were transfected with TRPM7 siRNA; siRNA(−), cells were transfected with negative siRNA. For each data point, 16 to 32 wells of cells from four to eight independent experiments were analyzed. *, P < 0.05; **, P < 0.01.

Figure 4.

Ca2+ and TRPM7 are critical to FaDu cell growth and proliferation. A, fura-2 fluorescence imaging showing changes in the intracellular Ca2+ concentration as indicated by 340/380 nm ratio in response to changes in [Ca2+]o (left), and summary data showing changes in [Ca2+]i as indicated by 340/380 nm ratio in response to changes in [Ca2+]o (right; n = 5–9). **, P < 0.01, compared with 2.0 mmol/L [Ca2+]o. B, effect of [Ca2+]o on the growth and proliferation of FaDu cells as measured by direct cell counting (left) or total LDH release (right). Over a range of [Ca2+]o from 1 to 0.1 mmol/L, the rate of proliferation of the FaDu cells decreased in parallel with decreasing [Ca2+]o. For each data point, 16 to 32 wells of cells from four to eight independent experiments were analyzed. *, P < 0.05; **, P < 0.01, compared with 2.0 mmol/L [Ca2+]o. C, concentration-dependent inhibition of the proliferation of FaDu cells by 2-APB, measured by cell count (left) or total LDH release (right). D, inhibition of the proliferation of FaDu cells by TRPM7 siRNA, evaluated by cell count (left) and LDH assay (right). Con, cells grew in normal medium; siRNA(+), cells were transfected with TRPM7 siRNA; siRNA(−), cells were transfected with negative siRNA. For each data point, 16 to 32 wells of cells from four to eight independent experiments were analyzed. *, P < 0.05; **, P < 0.01.

Close modal

We next examined whether a change in [Ca2+]o affects the growth and proliferation of FaDu cells. As shown in Fig. 4B, the degree of proliferation of FaDu cells, as analyzed by cell counts, is closely regulated by the concentration of [Ca2+]o (Fig. 4B,, left). In a range between 1 and 0.1 mmol/L [Ca2+]o, the rate of proliferation of FaDu cells decreased gradually with a parallel reduction in [Ca2+]o. In addition to cell counting, we used the lactate dehydrogenase (LDH) assay to acquire more objective data on cell proliferation. LDH is a ubiquitous enzyme in all living cells, and the total amount of LDH releasable is proportional to the number of cells tested (35). As shown in Fig. 4B  (right), LDH measurement also showed a clear dependence of the proliferation of FaDu cells on [Ca2+]o.

Inhibition of cell proliferation by TRPM7 blockade and TRPM7 siRNA. We next determined whether the activities of TRPM7 channels influence the growth and proliferation of FaDu cells. First, we tested the effect of Gd3+, a nonspecific TRPM7 channel inhibitor, on the proliferation of FaDu cells. Addition of 50 and 100 μmol/L Gd3+ in the culture medium inhibited the growth of FaDu cells by 18.29 ± 2.59% (P < 0.01) and 39.44 ± 7.20% (P < 0.01) with cell counting (n = 16 wells) and by 36.99 ± 7.28% (P < 0.01) and 50.37 ± 7.80% (P < 0.01) with LDH assay (n = 16 wells; data not shown). Similar to Gd3+, 2-APB also inhibited the proliferation of FaDu cells (Fig. 4C).

TRPM7 siRNA was then used to selectively suppress the expression of TRPM7 channels. As shown in Fig. 4D, transfection of cells with 30 nmol/L TRPM7 siRNA significantly inhibited cell proliferation. Cells transfected with negative control siRNA showed no difference in cell proliferation compared with nontransfected cells. Taken together, our data suggest that activation of TRPM7 channels plays an important role in the growth and proliferation of FaDu cells.

TRPM7-like current in SCC25 cell lines. To provide additional evidence that TRPM7 channels play an important role in the growth and proliferation of human head and neck tumor cells, we carried out similar experiments in SCC25 cells, a common cell line of human thyroid squamous cell carcinoma. Similar to FaDu cells, lowering [Ca2+]o activated inward current (Fig. 5A,, inset). The amplitude of the Ca2+-sensing current decreases with membrane depolarization, resulting in a linear current-voltage relationship and a reversal potential of ∼0 mV (Fig. 5A; n = 5). Addition of 10 μmol/L Gd3+ inhibited the current from 535.7 ± 99.3 to 113.8 ± 87.7 pA (n = 4, P < 0.05; Fig. 5B,-a), whereas addition of 100 μmol/L 2-APB inhibited the current from 521.3 ± 90.4 to 378.9 ± 90.9 pA (n = 5, P < 0.05; Fig. 5B,-b). Inclusion of Mg2+ in the intracellular solution suppressed the current (Fig. 5C,-a). When normalized to the current recorded at 5 min, the relative amplitude of the Ca2+-sensing current at 20 min was 0.89 ± 0.11 for 0 mmol/L Mg2+ and 0.54 ± 0.12 for 8 mmol/L Mg2+ (n = 5–6; P < 0.01). Similar to FaDu cells, current-voltage relationship in SCC25 cells showed outward rectification in the presence of Ca2+ but became linear in the absence of Ca2+ (Fig. 5C,-b; n = 4). Western blot detected a protein band of ∼220 kDa in SCC25 cells, and the expression of this protein is reduced following the transfection with siRNA-TRPM7 (Fig. 6A). Consistent with the reduction of protein level, the amplitude of the Ca2+-sensing current was significantly decreased in SCC25 cells transfected with TRPM7 siRNA (37.2 ± 2.3 pA/pF in control cells transfected with vector alone versus 8.7 ± 2.8 pA/pF in cells transfected with TRPM7 siRNA; n = 5 for each group, P < 0.05). Finally, addition of Gd3+, 2-APB, or siRNA all inhibited the growth and proliferation of the SCC25 cells (Fig. 6C and D).

Figure 5.

Electrophysiologic and pharmacologic characterization of the Ca2+-sensitive currents in SCC25 cells. A, left, representative traces (inset) and summary I-V relationship showing the Ca2+-sensitive current recorded at different holding potentials. A rapid drop of [Ca2+]o from 2 to 0 mmol/L (nominally free) evoked slow nondesensitizing currents that rapidly recovered on restoration of [Ca2+]o. B, representative traces showing inhibition of the Ca2+-sensitive current in SCC25 cells by Gd3+ and 2-APB. Addition of 10 μmol/L Gd3+ reduced the amplitude of the inward current from 535.7 ± 99.3 to 113.8 ± 87.7 pA (n = 4, P < 0.05), whereas addition of 100 μmol/L 2-APB inhibited the current from 521.3 ± 90.4 to 378.9 ± 90.9 pA (n = 5, P < 0.05). C, a, summary data showing the time-dependent inhibition of the Ca2+-sensitive current by intracellular Mg2+. The amplitude of the peak inward current activated at −60 mV was normalized to that recorded 5 min after the formation of whole-cell configuration (n = 5 and 6). The relative amplitude of the peak inward current at 20 min was 0.89 ± 0.11 and 0.54 ± 0.12 for current recorded in the absence of intracellular Mg2+ and in the presence of 8 mmol/L Mg2+, respectively (P < 0.01). b, representative I-V curve generated by depolarizing ramp pulses in the presence (a) or absence (b) of 2 mmol/L extracellular Ca2+. An outward rectification is apparent in the presence of 2 mmol/L Ca2+ (trace a).

Figure 5.

Electrophysiologic and pharmacologic characterization of the Ca2+-sensitive currents in SCC25 cells. A, left, representative traces (inset) and summary I-V relationship showing the Ca2+-sensitive current recorded at different holding potentials. A rapid drop of [Ca2+]o from 2 to 0 mmol/L (nominally free) evoked slow nondesensitizing currents that rapidly recovered on restoration of [Ca2+]o. B, representative traces showing inhibition of the Ca2+-sensitive current in SCC25 cells by Gd3+ and 2-APB. Addition of 10 μmol/L Gd3+ reduced the amplitude of the inward current from 535.7 ± 99.3 to 113.8 ± 87.7 pA (n = 4, P < 0.05), whereas addition of 100 μmol/L 2-APB inhibited the current from 521.3 ± 90.4 to 378.9 ± 90.9 pA (n = 5, P < 0.05). C, a, summary data showing the time-dependent inhibition of the Ca2+-sensitive current by intracellular Mg2+. The amplitude of the peak inward current activated at −60 mV was normalized to that recorded 5 min after the formation of whole-cell configuration (n = 5 and 6). The relative amplitude of the peak inward current at 20 min was 0.89 ± 0.11 and 0.54 ± 0.12 for current recorded in the absence of intracellular Mg2+ and in the presence of 8 mmol/L Mg2+, respectively (P < 0.01). b, representative I-V curve generated by depolarizing ramp pulses in the presence (a) or absence (b) of 2 mmol/L extracellular Ca2+. An outward rectification is apparent in the presence of 2 mmol/L Ca2+ (trace a).

Close modal
Figure 6.

Inhibition of the growth and proliferation of SCC25 cells by pharmacologic blockade of TRPM7-like current or suppression of TRPM7 expression. A, Western blot images showing the detection of TRPM7 protein in SCC25 before and after TRPM7 siRNA transfection. HEK293 cells with induced expression of TRPM7 channels were used as a positive control and actin served as an internal control. B, summary data showing the reduction of TRPM7-like current in SCC25 cells 72 h after transfection with TRPM7 siRNA. C, a, concentration-dependent inhibition of the proliferation of SCC25 cells by Gd3+, measured by cell count. b, concentration-dependent inhibition of the proliferation of SCC25 cells by 2-APB, measured by cell count. D, inhibition of the proliferation of SCC25 cells by TRPM7 siRNA, evaluated by cell count (a) and LDH assay (b). Con, cells grew in normal medium; siRNA(+), cells were transfected with TRPM7 siRNA; siRNA(−), cells were transfected with negative control siRNA. *, P < 0.05; **, P < 0.01.

Figure 6.

Inhibition of the growth and proliferation of SCC25 cells by pharmacologic blockade of TRPM7-like current or suppression of TRPM7 expression. A, Western blot images showing the detection of TRPM7 protein in SCC25 before and after TRPM7 siRNA transfection. HEK293 cells with induced expression of TRPM7 channels were used as a positive control and actin served as an internal control. B, summary data showing the reduction of TRPM7-like current in SCC25 cells 72 h after transfection with TRPM7 siRNA. C, a, concentration-dependent inhibition of the proliferation of SCC25 cells by Gd3+, measured by cell count. b, concentration-dependent inhibition of the proliferation of SCC25 cells by 2-APB, measured by cell count. D, inhibition of the proliferation of SCC25 cells by TRPM7 siRNA, evaluated by cell count (a) and LDH assay (b). Con, cells grew in normal medium; siRNA(+), cells were transfected with TRPM7 siRNA; siRNA(−), cells were transfected with negative control siRNA. *, P < 0.05; **, P < 0.01.

Close modal

It has long been known that ion channels are of great importance for a large variety of physiologic functions including electrical signaling, gene expression, cell volume regulation, hormone secretion, etc. In contrast to these beneficial effects, certain channel proteins have been proposed to promote the growth and proliferation of tumor cells (3638). It is suggested that, during the change from a normal cell toward cancer, a series of genetic alterations take place, which may affect the expression of ion channels or induce changes in channel property/activity. The abnormal ion channel property/activity is then able to promote the growth and proliferation of the tumor (37). Melastatin, for example, functions as a tumor suppressor, which is down-regulated in highly metastatic cells. For this reason, assessment of melastatin mRNA has been used as a prognostic marker for metastasis in patients with localized malignant melanoma (39). Similarly, TRPM8 channel protein has been used as a prostate-specific marker and the loss of TRPM8 is considered as a sign of poor prognosis (40, 41). In addition, TRPV6 expression has been considered a prognostic marker and a promising target for new therapeutic strategies for the treatment of advanced prostate cancer (42). Therefore, the pathophysiologic function of ion channels is one of the new interesting research areas that may offer alternative prognostic and therapeutic strategies for cancer patients. In line with this argument, our studies showed that activation TRPM7 channels influence the growth and proliferation of human head and neck tumor cell lines.

We first showed the presence of a Ca2+-sensing current in FaDu and SCC25 cells, two common cell lines of human head and neck tumor. Several lines of evidence indicate that this Ca2+-sensing current is carried, at least partially, by TRPM7 channels. (a) The current is modulated by [Ca2+]o, with increased amplitude recorded in low Ca2+ external solutions. This feature is similar to TRPM7 currents recorded in other cells and in heterologous expression systems (12, 13, 18, 32). (b) The current is voltage independent, with a reversal potential ∼0 mV and a strong outward rectification in the presence of [Ca2+]o, consistent with the characteristics of TRPM7 channels (12, 13). (c) Inhibition of the Ca2+-sensing current by 2-APB, a nonspecific TRPM7 blocker, along with its sensitivity to Gd3+, also suggests the involvement of TRPM7 channels (13, 32, 43). (d) Inhibition of this current by intracellular Mg2+ is another important feature of TRPM7 channels (12, 31). (e) Reduction of the current by TRPM7 siRNA further supports the involvement of TRPM7 channels in mediating the Ca2+-sensing current. (f) Finally, Western blot and RT-PCR detected the presence of TRPM7 protein and mRNA in these cells.

CRAC channels share some electrophysiologic features with TRPM7 (32, 43, 44). For example, both channels are Ca2+-permeable and inhibited by Gd3+. However, at physiologic [Ca2+]o and [Na+]o, the reversal potential for TRPM7 current is ∼0 mV, whereas it is ∼40 mV for CRAC current (44). Moreover, TRPM7 channels have a much larger single-channel conductance, and the whole-cell currents show a strong outward rectification in contrast to inward rectification for CRAC current.

The exact mechanism for TRPM7 activation/modulation is still unclear. An early study (45) suggested that receptor-mediated breakdown of phosphatidylinositol-4,5-bisphosphate mediates negative feedback inhibition of TRPM7, whereas a different study suggested that TRPM7 is regulated by changes in the level of intracellular cyclic AMP (46). Our recent studies suggested that oxygen free radicals may directly activate the TRPM7 channels (13). Jiang et al. (47) also showed that in TRPM7-transfected HEK293 cells and in rat basophilic leukemia cells, TRPM7 current could be potentiated by acid. It is thus likely that under pathologic conditions such as various forms of injury, inflammation, and tumor progression, TRPM7 may become activated by combined oxidative stress and acidosis. Our findings that pharmacologic inhibition of TRPM7 channels or knockdown of the expression of TRPM7 mRNA suppresses the growth and proliferation of FaDu and SCC25 cells indicate an important role of these channels in the growth and proliferation of malignant head and neck tumor cells.

Tumor cells may exhibit a lower extracellular pH (6.5–6.9) than normal tissues (7.0–7.5; refs. 48, 49). Given the fact that overgrowth of many tumor cells is also associated with hypoxia in their microenvironment, one would expect that TRPM7 currents in tumor cells may be activated due to the oxidative stress (13) and potentiated by the pathologic acidic conditions. In the present study, we did not examine the possible consequence of acid potentiation of TRPM7-like current on the proliferation of FaDu cells. Future studies will mimic the in vivo microenvironment of tumor cells in which both acid accumulation and hypoxia will be implemented.

There is a lack of close correlation between doses used for Gd3+ and 2-APB to inhibit the Ca2+-sensing current and those used to prevent cell growth. For Gd3+, higher concentration is required for inhibiting the cell growth than the current, whereas the opposite is true for 2-APB. This lack of correlation is likely due the nonspecificity of these blockers. As mentioned above, 2-APB has broad inhibitory effects on TRP superfamily of ion channels. It may be possible that other TRP channels exist and partially contribute to the growth of FaDu and SCC25 cells. In addition to inhibiting TRPM7, Gd3+ is known to affect the activities of other ion channels or receptors. For example, it stimulates the Ca2+-sensing receptor (50). Activation of this receptor is known to induce intracellular Ca2+ release, which may stimulate the cell growth and counteract the inhibition of cell growth by TRPM7 inhibition. Future studies will determine whether FaDu and SCC25 cells do express other TRP channels and the Ca2+-sensing receptors. Nevertheless, our conclusion that TRPM7 is involved, at least partially, in the growth and proliferation of human head and neck tumor cells was based on the effect of Gd3+, 2-APB, and TRPM7 siRNA all together.

Note: J. Jiang, M-H. Li, and K. Inoue contributed equally to this work.

Grant support: NIH grants R01NS049470 and R01NS42926 (Z-G. Xiong).

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.

We thank Jingquan Lan, Theresa Lusardi, and Debbie Branigan for technical assistance, and Eric Kratzer for reading the manuscript.

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