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Am J Physiol Cell Physiol 295: C296-C301, 2008. First published June 4, 2008; doi:10.1152/ajpcell.00499.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

TRPM8 activation suppresses cellular viability in human melanoma

Hisao Yamamura,1,2 Shinya Ugawa,1 Takashi Ueda,1 Akimichi Morita,3 and Shoichi Shimada1

1Department of Molecular Morphology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan; 2Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan; and 3Department of Geriatric and Environmental Dermatology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan

Submitted 19 October 2007 ; accepted in final form 29 May 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The transient receptor potential melastatin subfamily (TRPM), which is a mammalian homologue of cell death-regulated genes in Caenorhabditis elegans and Drosophila, has potential roles in the process of the cell cycle and regulation of Ca2+ signaling. Among this subfamily, TRPM8 (also known as Trp-p8) is a Ca2+-permeable channel that was originally identified as a prostate-specific gene upregulated in tumors. Here we showed that the TRPM8 channel was expressed in human melanoma G-361 cells, and activation of the channel produced sustainable Ca2+ influx. The application of menthol, an agonist for TRPM8 channel, elevated cytosolic Ca2+ concentration in a concentration-dependent manner with an EC50 value of 286 µM in melanoma cells. Menthol-induced responses were significantly abolished by the removal of external Ca2+. Moreover, inward currents at a holding potential of –60 mV in melanoma cells were markedly potentiated by the addition of 300 µM menthol. The most striking finding was that the viability of melanoma cells was dose-dependently depressed in the presence of menthol. These results reveal that a functional TRPM8 protein is expressed in human melanoma cells to involve the mechanism underlying tumor progression via the Ca2+ handling pathway, providing us with a novel target of drug development for malignant melanoma.

Ca2+-permeable channel; cell viability; G-361; malignant melanoma; menthol; transient receptor potential melastatin

MALIGNANT MELANOMA is the most deadly form of skin cancer, and its incidence is steadily increasing worldwide. The American Cancer Society estimates that 62,480 new cases of cutaneous melanoma will be diagnosed in the United States in 2008 (4.3% of all cancer cases) and that 8,420 patients will die of the disease (1.5% of all cancer deaths). Skin melanoma is estimated to be the sixth and seventh most common cancer in men and women, respectively, among new cases of cancer in the United States in 2008 (12). In malignant melanoma, normal melanocytes transform into melanoma cells that are both rapidly proliferative and highly metastatic. Cutaneous melanoma is classified into clinical stages according to significant prognostic factors. Despite extensive research and clinical trials, the prognosis remains poor for patients with malignant melanoma. Localized melanoma is curable by surgery, whereas there is no therapy for metastatic melanoma or melanoma with metastatic potential. Because melanoma is typically resistant to traditional forms of chemotherapy and radiotherapy, several alternative methods of treatment, including immunotherapy, biochemotherapy, and gene therapy, have been investigated (5, 7, 21, 22, 24). In addition, drug screening has been conducted to find and develop efficient and specific compounds with inhibitory action on the progression of malignant melanoma. The plasma membrane in melanocytes and melanoma cells possesses a variety of ion channels, which are thought to be involved in the proliferation, differentiation, and malignant alteration of cells and thus may contribute to tumor development (2, 30). Therefore, modulators of these channels expressed in melanoma cells may be an attractive target for medical treatment for malignant melanoma.

The transient receptor potential (TRP) melastatin subfamily (TRPM) is relatively conserved through evolution from invertebrates to mammals. These members in mammals share 20% to 40% amino acid identity to homologues in Caenorhabditis elegans (C. elegans) and Drosophila, and all have six common transmembrane segments and a unique TRP domain (10, 28). These TRPM channels play pivotal roles in regulation of the cell cycle and Ca2+ handling. One such gene, C. elegans gon-2, is required for mitotic cell divisions of gonadal precursor cells (28, 29). Another C. elegans gene, ced-11, has a function in programmed cell death (28). In addition, these mammalian homologues include two major tumor-related proteins, TRPM1 and TRPM8. A putative tumor suppressor protein, TRPM1 (also described as melastatin), is correlated with the severity of melanoma metastasis (8). TRPM8 protein (also known as Trp-p8) is expressed primarily in the prostate, and, in contrast with TRPM1, its expression is elevated in tumors (25).

In this investigation, we show that the TRPM8 channel was expressed in human melanoma G-361 cells and that channel activation by menthol, a naturally occurring ligand for TRPM8 (14, 18), caused sustainable increments in both intracellular Ca2+ concentration ([Ca2+]i) and current amplitude in melanoma cells. The most interesting finding was that exposure to menthol prominently depressed the survival of melanoma cells. Thus these results provide a novel profile for the TRPM8 channel that channel activity with Ca2+ permeability may be involved in tumor progression in melanoma.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. All experiments were approved by the Ethics Committee of the Nagoya City University Graduate School of Medical Sciences. A human malignant melanoma cell line (G-361) was obtained from Health Science Research Resources Bank (Osaka, Japan) and cultured in McCoy's 5A modified medium supplemented with 10% heat-inactivated fetal bovine serum, 20 U/ml penicillin G, and 20 µg/ml streptomycin.

Molecular biology. Total RNA was extracted from homogenates of melanoma cells to perform reverse transcription-polymerase chain reaction (RT-PCR), electrophoresis, cloning, and subsequent sequencing, as described previously (32, 33). PCR amplification was carried out for 35 cycles. To detect a human TRPM8 transcript (GenBank accession number NM_024080), specific oligonucleotide primers were designed as follows: (+) 5'-TCT ACA TCG CGC AGT CCA AAG GT-3' and (–) 5'-ATA GGA ATT CTT GGC GAT CTG CA-3' (base 506 to 1548).

In situ hybridization. Melanoma cells were fixed with 4% formaldehyde in 0.1 M phosphate buffer. Cell-based in situ hybridization with specific [35S]-labeled riboprobes was performed as described previously (34). The riboprobes of human TRPM8 (base 506 to 1548) were synthesized by in vitro transcription with T7 or SP6 RNA polymerase (Promega, Madison, WI).

[Ca2+]i measurement. [Ca2+]i was recorded using a Ca2+ imaging system (Argus/HiSCA; Hamamatsu Photonics, Hamamatsu, Japan) equipped with a fluorescent microscope (IX-70; Olympus, Tokyo, Japan), an objective lens (Fluor 40 x0.75 NA; Nikon, Tokyo, Japan), and an Argus/HiSCA software (version 1.70; Hamamatsu Photonics). Experiments were performed 48 to 72 h after subculture. Melanoma cells were loaded with 10 µM fura-2 acetoxymethyl ester (fura-2/AM; Molecular Probes, Eugene, OR) for 30 min and excessive fura-2/AM was washed thoroughly for 10 min. The recording solution had an ionic composition of 137 mM NaCl, 5.9 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 14 mM glucose, 10 mM HEPES, and pH 7.4. The external Ca2+-free solution was prepared by removal of 2.2 mM CaCl2. The filters for excitation wavelengths were 340 and 380 nm, and the filter for emission wavelength was 510 ± 25 nm. The [Ca2+]i was presented as the ratio of fluorescence intensities (F340/F380). The Ca2+ images were scanned every 1 to 2 s. The recording chamber was continuously perfused with solution at a flow rate of 2 ml/min. All experiments were carried out at room temperature (24 ± 1°C).

Electrophysiological recording. Electrophysiological studies were carried out using a whole cell voltage-clamp technique with an Axopatch 200B amplifier and pCLAMP software (version 8; Axon Instruments, Foster City, CA) in melanoma cells, as described previously (31). The extracellular solution had an ionic composition of 137 mM NaCl, 5.9 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 14 mM glucose, 10 mM HEPES, and pH 7.4. The pipette solution contained 140 mM KCl, 1 mM MgCl2, 10 mM HEPES, 2 mM Na2ATP, 5 mM EGTA, and pH 7.2. The current recordings were performed at a holding potential of –60 mV.

Cellular viability assay. The viability of melanoma cells was evaluated using a Cell Counting Kit-8 (Dojin, Kumamoto, Japan) based on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Melanoma cells were subcultured in 96-well plates and incubated at 37°C for 72 h (to approximately 1 x 104 cells/well). The cellular viabilities in the absence and presence of menthol were quantitated colorimetrically as the absorbance at 450 nm (A450) and are expressed as follows: viability (%) = (A450 of menthol-treated cells/A450 of control cells) x 100. These media always contained 0.3% dimethyl sulfoxide as a solvent throughout the experiments, regardless of the absence or presence of menthol.

Drugs. All reagents were obtained from Sigma-Aldrich (St. Louis, MO). (L)-menthol was dissolved in dimethyl sulfoxide at the concentration of 1 M as a stock solution. It was confirmed that up to 0.3% of dimethyl sulfoxide did not affect the responses.

Statistical analysis. Pooled data are shown as means ± SE. Statistical significance between the two groups and among groups was determined by Student's t-test and Scheffé's test after one-way analysis of variance, respectively. Significant difference is expressed in the figures (P < 0.05 or P < 0.01). The data of the relationship between menthol concentrations and responses were fitted using the following equations: A = Amax/{1 + [Kd/(menthol)]n} (Fig. 3B) and relative value (%) = 100 – (100 – C)/{1 + [Kd/(menthol)]n} (Fig. 6B), where Amax is the maximum value of the response, Kd is the apparent dissociation constant of menthol, [menthol] is the concentration of menthol, n is the Hill coefficient, and C is the component resistant to menthol.


Figure 3
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  		Fig. 3. Ca2+ influx by menthol in human melanoma cells. The effects of menthol on cytosolic Ca2+ mobilization were examined using a Ca2+ imaging system with a Ca2+ fluorescent probe, 10 µM fura-2/AM, in human melanoma cells. A: the application of 100 µM menthol caused a slight increase in intracellular Ca2+ concentration ([Ca2+]i) from the resting level in a melanoma cell, and the increase was maintained during exposure to menthol. The accumulative addition of 300 µM menthol markedly increased [Ca2+]i in the same cell. The [Ca2+]i elevation evoked by menthol recovered to the resting level by the removal of menthol. B: the dependency on menthol concentration of the [Ca2+]i response is plotted. The [Ca2+]i response was significantly increased by the application of menthol at a concentration of 300 µM and more (P < 0.01), and the [Ca2+]i rise was in a concentration-dependent manner in melanoma cells. The EC50 value for menthol on [Ca2+]i in melanoma cells was 286 µM, and the Hill coefficient was 1.46. Experimental data were obtained from 42 cells.

 

Figure 6
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  		Fig. 6. Cellular viability of human melanoma in the presence of menthol. The effects of TRPM8 channel activation by the application of menthol on cellular viability were investigated in human melanoma cells. The viability of melanoma cells is quantitated colorimetrically by absorbance at 450 nm in the absence or presence of menthol. The density of melanoma cells before beginning the viability assay was uniform, about 1 x 104 cells/well in 96-well plates. Relative viability (%) = (A450 of menthol-treated cells/A450 of control cells) x 100. A: the relationship between cell viability and culture time after the treatment with menthol is summarized. In the presence of 300 µM menthol, cell survival was dramatically suppressed after 12 h culture (P < 0.05). B: the concentration dependency of growth inhibition by exposure to menthol was analyzed after 24 h culture in melanoma cells. Changing the concentration of menthol in the range of 10 to 3,000 µM showed that cellular vitality was significantly reduced by menthol at a concentration of 300 µM and more (P < 0.05), and reduction was in a concentration-dependent manner. The IC50 value of menthol on the viability in melanoma cells was 682 µM, and the Hill coefficient was 1.04. Experimental data were obtained from 16 to 32 wells. The statistical significance of the difference is expressed as *P < 0.05 or **P < 0.01 vs. in the absence of menthol.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of TRPM8 transcript in human melanoma cells. At first, we examined whether the TRPM8 channel was expressed in human melanoma cells by RT-PCR method. RT-PCR analysis using total RNA extracted from human melanoma cells and specific primers for human TRPM8 channel showed a clear expression of TRPM8 transcript (1043 bp) with an RT procedure, but not without RT (Fig. 1). We also confirmed that the RT-PCR fragment was in fact derived from TRPM8 mRNA by sequencing the PCR products. Similar results were obtained from five independent experiments.


Figure 1
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  		Fig. 1. Expression of transient receptor potential melastatin subfamily member 8 (TRPM8) transcript in human melanoma cells. TRPM8 transcript was observed by RT-PCR with gene-specific primers in human melanoma G-361 cells. A typical gel image of PCR product originated from melanoma cells before (right lane) and after (middle lane) RT procedure is presented. The clear expression of TRPM8 transcript was detected in melanoma cells (1043 bp) but not with a negative control RT (–). Similar results were obtained from five independent experiments. Numbers shown at left indicate the base size obtained from Ready-Load {phi}X174 RF DNA/Hae III fragments (Invitrogen, Carlsbad, CA) as a DNA size marker (M; left lane).

 
In situ hybridization evidence of TRPM8 channel in human melanoma cells. The confirmed antibody for human TRPM8 channel was not available, unfortunately, so we performed a [35S]-radioactive cell-based in situ hybridization approach for detection of the channel expression instead of an immunocytochemical method. With a cell-based in situ hybridization technique using a specific antisense riboprobe for human TRPM8 channel, the abundant expression of TRPM8 mRNA was observed in melanoma cells, but not with a control sense probe (Fig. 2). The positive signals of TRPM8 mRNA were distributed in the cytosolic region of melanoma cells. Most cells expressed TRPM8 channels despite the cell density, cultured days, and passages. These factors were not influenced by the expression degree of TRPM8 channel. Similar results were obtained from 10 independent paired preparations.


Figure 2
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  		Fig. 2. In situ hybridization evidence of TRPM8 channel in human melanoma cells. Expression of TRPM8 channel was detected using a [35S]-radioactive cell-based in situ hybridization with specific probes in human melanoma G-361 cells. A typical paired image from cell-based in situ hybridization targeting TRPM8 channel in melanoma cells is shown. Ten to fifteen cells (~20 µm in diameter) are fixed per image (170 x 170 µm). Apparently, most melanoma cells express abundant TRPM8 mRNA (antisense; accumulation of particles), but not with a control sense probe (sense). Similar results were obtained from 10 independent paired preparations.

 
Ca2+ influx by menthol in human melanoma cells. To identify the functional expression of TRPM8 channel protein, the effects of menthol, an agonist for TRPM8, on cytosolic Ca2+ regulation were examined using a Ca2+ imaging system with a Ca2+ fluorescent probe, 10 µM fura-2/AM, in melanoma cells. The application of 100 µM menthol caused a slight increase in [Ca2+]i from the resting level of 0.38 ± 0.01 to 0.47 ± 0.02 (n = 72), and the [Ca2+]i increase was kept during the exposure to menthol (Fig. 3A). The sequential addition of 300 µM menthol markedly increased [Ca2+]i to 0.67 ± 0.02 (n = 72, P < 0.01). The [Ca2+]i rise evoked by 300 µM menthol was recovered to the resting level by the removal of menthol in most cells tested (65 of 72 cells). After washout for a few minutes, the readministration of 300 µM menthol caused [Ca2+]i increase with similar responses to first challenge (n = 17). Next, the concentration dependence of menthol-induced [Ca2+]i elevation was examined in melanoma cells. Changing the concentration of menthol at the range from 10 to 3,000 µM showed that [Ca2+]i was significantly increased by menthol at a concentration of 300 µM (by 0.29 ± 0.02, n = 42, P < 0.01 vs. control) and greater, and the [Ca2+]i increment was in a concentration-dependent manner (by 0.55 ± 0.04 at 3 mM, n = 42, P < 0.01; Fig. 3B). The EC50 value of menthol on [Ca2+]i in melanoma cells was 286 µM, and the Hill coefficient was 1.46.

Effects of external Ca2+ removal on menthol-evoked response in human melanoma cells. To address whether menthol-induced [Ca2+]i elevation mediated Ca2+ influx through a Ca2+-permeable TRPM8 pathway, the effects of Ca2+ removal in extracellular solution (2.2 mM Ca2+) on [Ca2+]i changes in the presence of menthol were examined in melanoma cells. The [Ca2+]i rise induced by 300 µM menthol (by 0.33 ± 0.01 in the presence of Ca2+, n = 14) was dramatically blocked in the absence of external Ca2+ (84 ± 2% decrease, n = 14, P < 0.01; Fig. 4). After thorough washout for over 3 min in the presence of external 2.2 mM Ca2+, the menthol-induced [Ca2+]i increase was recovered (P > 0.05 vs. first application).


Figure 4
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  		Fig. 4. Effects of external Ca2+ removal on menthol-evoked response in human melanoma cells. The effects of removing Ca2+ from extracellular solution on [Ca2+]i changes in the presence of menthol were examined in human melanoma cells. A: a typical [Ca2+]i trace in a melanoma cell in response to menthol in the presence and absence of external Ca2+ is represented. The [Ca2+]i increase induced by 300 µM menthol was dramatically influenced by the removal of external 2.2 mM Ca2+. After thorough washout for over 3 min in the presence of external Ca2+, the third application of menthol in the presence of external Ca2+ caused a [Ca2+]i increase similar to the first application. B: the effects of 300 µM menthol on melanoma cells in the absence (open column) and presence (solid column) of extracellular Ca2+ are summarized. The statistical significance of the difference is expressed as **P < 0.01 vs. normal. Experimental data were obtained from 14 cells.

 
Menthol-induced currents in human melanoma cells. Further evidence for the menthol-evoked activation of TRPM8 channels was provided by whole cell voltage-clamp studies in melanoma cells. Cell capacitance was 39 ± 4 pF (n = 12). At a holding potential of –60 mV, the application of 300 µM menthol induced larger inward currents by 50 ± 4 pA (n = 6; Fig. 5A). The potentiation of inward current evoked by menthol was returned to the resting level by the removal of menthol from all cells examined (n = 6). The responses to menthol on inward currents were concentration dependent in melanoma cells: 0.18 ± 0.08 pA/pF at 100 µM menthol (n = 6) and 1.38 ± 0.32 pA/pF at 300 µM (n = 6, P < 0.05; Fig. 5B).


Figure 5
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  		Fig. 5. Menthol-induced currents in human melanoma cells. Whole cell currents were recorded at a holding potential of –60 mV in human melanoma cells. A: a typical trace of menthol sensitivity on inward current in a melanoma cell is plotted. The application of 300 µM menthol induced larger inward currents, and menthol-elicited inward currents were reversed to the resting level by the removal of menthol. B: the stimulatory effects elicited by 100 or 300 µM menthol in melanoma cells are summarized. The effects of menthol on inward currents are in a concentration-dependent manner. Experimental data were obtained from 12 cells. The statistical significance of the difference is expressed as *P < 0.05 vs. before application.

 
Cellular viability of human melanoma in the presence of menthol. It has been thought that Ca2+ influx via a Ca2+-permeable channel on the plasma membrane causes apoptosis and necrosis followed by morphological damage involving cell death. Since TRPM8 protein is a type of Ca2+-permeable channel, we examined the influence of Ca2+ influx through a TRPM8 pathway on cell survival using a quantitative colorimetric assay kit based on MTT test for viability in melanoma cells. In the presence of 300 µM menthol, melanoma cells were dramatically reduced after 12 h culture (67 ± 7%, n = 16, P < 0.05 vs. 0 h; Fig. 6A). Related to the decrease in a value of cell viability, these cells apparently altered morphological features such as cell damage and death.

In the next set of experiments, the concentration dependency of the growth inhibition by menthol was analyzed in melanoma cells. As the inhibitory effect by menthol at 24 h culture was most prominent, as shown in Fig. 6A (maximum inhibition to 51 ± 2%, n = 16, P < 0.01), we evaluated the viability of melanoma cells at 24 h culture in the absence or presence of menthol. These cells were incubated with medium containing various concentrations of menthol at the range from 10 to 3,000 µM for 24 h, when cellular viability was significantly reduced by exposure to menthol at a concentration of 300 µM (75 ± 5%, n = 32, P < 0.05 vs. in the absence of menthol) and greater (Fig. 6B). The menthol-induced growth inhibition was in a concentration-dependent manner (33 ± 4% at 3 mM, n = 32, P < 0.01). The IC50 value of menthol for the viability in melanoma cells was 682 µM, and the Hill coefficient was 1.04.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The incidence of malignant melanoma is one of the highest among all new cancer cases worldwide, although the medical treatment for malignant melanoma is largely unestablished despite extensive research and clinical trials (12). Localized melanoma is surgically resectable, whereas there is no therapy for metastatic melanoma or melanoma with metastatic potential. Because melanoma is typically resistant to traditional forms of chemotherapy and radiotherapy, alternative methods by novel therapies including immunotherapy, biochemotherapy, and gene therapy as well as by drug treatment with specific inhibitors of tumor progression have been investigated (5, 7, 21, 22, 24). In this investigation, we focused on a Ca2+-permeable channel expressed abundantly in human melanoma cells, and we examined, using Ca2+ imaging analysis, electrophysiological recording, and a biochemical approach, whether the regulation of channel activity was able to lead to an inhibitory effect on cellular viability of malignant melanoma.

These analyses with RT-PCR and cell-based in situ hybridization techniques clearly showed that melanoma cells possess TRPM8 channel at the levels of mRNA. Originally, TRPM8 (also known as Trp-p8) was identified as a prostate-specific gene, and its expression was elevated in tumors (25). TRPM8 channel is described as also being distributed in sensory neurons containing trigeminal and dorsal root ganglia (14, 18), urinary bladder (23), and taste papillae (1) as well as prostate cancer cell lines (25, 35). TRPM channels are conserved through evolution from invertebrates to mammals and have pivotal roles in cell cycle process and the regulation of Ca2+ signaling (10, 28). For example, gon-2 and ced-11 genes of this family in C. elegans are required for the determination of cell death such as gonadal cell divisions and programmed cell death (28, 29). In addition, this mammalian homologue includes a putative tumor suppressor protein, TRPM1 (also described as melastatin), which is downregulated in correlation with the potential for melanoma metastasis (8) and an upregulated protein in prostate tumors, TRPM8 (25). Despite the potential importance of these proteins, these physiological and pathological profiles have unfortunately been poorly adopted, except for functional expression in sensory neurons of TRPM8 as a cold and menthol receptor (14, 18).

In addition to morphological evidence of TRPM8 expression in melanoma cells, we showed that TRPM8 channel was functionally expressed using Ca2+ imaging analysis and electrophysiological recording in melanoma cells. The application of menthol induced a concentration-dependent Ca2+ influx and potentiation of inward currents at –60 mV in melanoma cells. Menthol is a naturally occurring compound of plant origin (4, 9) and is bound to TRPM8 channels as an agonist (14, 18). In a variety of bioassays in vitro and in vivo, it has been reported that TRPM8 activation by menthol at submillimolar concentrations evokes cooling responses in neurons of rat trigeminal and dorsal root ganglia (3, 14, 16), guinea pig urinary bladder (26), and human prostate cancer cells (35) as well as in TRPM8-recombinant cells (14, 18). In addition, menthol-induced TRPM8 activation evokes Ca2+ release from presynaptic Ca2+ stores of rat sensory neurons (27) and modulates cell survival in cultured cells of prostate cancer (35). Exposure to menthol causes cold sensation as a psychophysical effect via the TRPM8 pathway in humans (4, 15). The present study revealed that menthol-elicited Ca2+-influx and current potentiation were observed in melanoma cells, mediated through the activation of TRPM8 channels, suggesting that the functional expression of TRPM8 partly contributed to cytosolic Ca2+ handling for cell survival in melanoma cells.

Of most interest in the present study was that the viability of melanoma cells was dramatically suppressed in the presence of menthol. Cell survival was significantly reduced by exposure to menthol in a concentration-dependent manner. This menthol-induced growth inhibition in melanoma cells implied that Ca2+ permeability via TRPM8 channels partly contributed to the regulation of cellular viability. Similar inhibitory effects were induced via capsaicin-induced Ca2+ overload through a Ca2+-permeable channel, transient receptor potential vanilloid subfamily 1 in sensory neurons, bronchiolar epithelial cells, and recombinant cells (6, 19, 20). Therefore, Ca2+ signals via Ca2+-permeable channels on the plasma membrane are potential candidates for regulating proliferation and differentiation processes, which, in part, result in malignant alteration and tumor development. For tumor cells, it is known that an increase of cytosolic Ca2+ is required at two stages of the cell cycle, G0/G1 and G1/S transitions (30). The plasma membrane in melanoma cells expressed a variety of ion channels, which are thought to contribute to cell proliferation and differentiation and thus may be involved in tumor development (2, 30). So far, nonspecific K+ channel blockers, tetraethylammonium, quinidine, and imipramine, have been reported to inhibit the proliferation of melanoma cells (11, 13, 17). Our data suggest that the TRPM8 channel may be involved in the regulation of cell cycle processes, in addition to the sensation of cold temperature.

These data support the hypothesis that the influx of Ca2+ via activation of TRPM8 channels elicited by menthol is likely to trigger cell death in melanoma cells. These results provide a novel profile of the TRPM8 channel that elucidates how it may contribute to cell survival in melanoma cells. The investigation of the functional properties of melanoma cells is an important step in the development of novel strategies for anti-tumor therapeutics, including malignant melanoma.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This investigation was supported by Grants-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (to H. Yamamura) and for Scientific Research (B) and Exploratory Research from the Japan Society for the Promotion of Sciences (to S. Shimada).


   ACKNOWLEDGMENTS
 
We thank Katsuyuki Tanaka and Kenji Kajita for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Yamamura, Dept. of Molecular Morphology, Graduate School of Medical Sciences, Nagoya City Univ., 1 Kawasumi Mizuhocho Mizuhoku, Nagoya 467-8601, Japan (e-mail: yamamura{at}med.nagoya-cu.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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