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. 2019 Feb 7:2019:7362875.
doi: 10.1155/2019/7362875. eCollection 2019.

Mechanism for Regulation of Melanoma Cell Death via Activation of Thermo-TRPV4 and TRPV2

Jiaojiao Zheng  1 , Fangyuan Liu  1   2 , Sha Du  1 , Mei Li  1 , Tian Wu  1 , Xuejing Tan  2 , Wei Cheng  1
Affiliations

Mechanism for Regulation of Melanoma Cell Death via Activation of Thermo-TRPV4 and TRPV2

Jiaojiao Zheng et al. J Oncol. .

Abstract

Background: Thermo-TRPs (temperature-sensitive transient receptor potential channels) belong to the TRP (transient receptor potential) channel superfamily. Emerging evidence implied that thermo-TRPs have been involved in regulation of cell fate in certain tumors. However, their distribution profiles and roles in melanoma remain incompletely understood.

Methods: Western blot and digital PCR approaches were performed to identify the distribution profiles of six thermo-TRPs. MTT assessment was employed to detect cell viability. Flow cytometry was applied to test cell cycle and apoptosis. Calcium imaging was used to determine the function of channels. Five cell lines, including one normal human primary epidermal melanocytes and two human malignant melanoma (A375, G361) and two human metastatic melanoma (A2058, SK-MEL-3) cell lines, were chosen for this research.

Results: In the present study, six thermo-TRPs including TRPV1/2/3/4, TRPA1, and TRPM8 were examined in human primary melanocytes and melanoma cells. We found that TRPV2/4, TRPA1, and TRPM8 exhibited ectopic distribution both in melanocytes and melanoma cells. Moreover, activation of TRPV2 and TRPV4 could lead to the decline of cell viability for melanoma A2058 and A375 cells. Subsequently, activation of TRPV2 by 2-APB (IC50 = 150 μM) induced cell necrosis in A2058 cells, while activation of TRPV4 by GSK1016790A (IC50 = 10 nM) enhanced apoptosis of A375 cells. Furthermore, TRPV4 mediated cell apoptosis of melanoma via phosphorylation of AKT and was involved in calcium regulation.

Conclusion: Overall, our studies revealed that TRPV4 and TRPV2 mediated melanoma cell death via channel activation and characterized the mechanism of functional TRPV4 ion channel in regulating AKT pathway driven antitumor process. Thus, they may serve as potential biomarkers for the prognosis and are targeted for the therapeutic use in human melanoma.

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Figures

Figure 1
Figure 1
The distribution profiles of six thermo-TRPs in human melanoma cells and melanocytes. (a) Western blot analysis of TRPV1 (i), TRPV2 (ii), TRPV3 (iii), TRPV4 (iv), TRPA1 (v), and TRPM8 (vi) ion channels expression level in protein samples collected from primary epidermal melanocytes, and melanoma cells of A375, G361, A2058, and SK-MEL-3. (b) Droplet digital PCR detection of six thermo-TRPs for TRPV1 (i), TRPV2 (ii), TRPV3 (iii), TRPV4 (iv), TRPA1 (v), and TRPM8 (vi) in primary epidermal melanocytes, and melanoma cells of A375, G361, A2058, and SK-MEL-3. Total mRNA from human primary epidermal melanocytes and melanoma cells of A375, G361, A2058, and SK-MEL-3 were isolated, and digital PCR screening analysis for the indicated genes was performed. Determination of copy numbers per genome of six samples. Concentration values for indicated genes (□). Error bars represented 95% confidence intervals, NTC represented nontemplate control. β-actin was used as a positive control, and all tests were performed in at least three independent experiments.
Figure 2
Figure 2
Functional TRPVs mediated proliferation of melanoma cells. (a) Cells morphologically changed after GSK1016790A (20 nM and 50 nM) application to A375 melanoma cells and melanocytes via microscopy imaging (i) & (ii). (b) Cell viability test by MTT experiment exhibited inhibition of cell proliferation for melanoma A375 cells but not for primary epidermal melanocytes by treatment of either DMSO (control) or TRPV4 specific activator of GSK1016790A (1 nM, 10 nM, 20 nM, 50 nM) (i) & (ii). (c) Cell viability test by MTT experiment exhibited inhibition of cell proliferation for melanoma A2058 cells but not for primary epidermal melanocytes by treatment of either DMSO (control) or 2-APB (50 μM, 100 μM, 150 μM, 200 μM, 400 μM) (i) & (ii). (d) Measurement of calcium imaging in GSK1016790A application with 2 nM concentration in A375 melanoma cells was carried out, and intracellular calcium that dramatically enhanced was observed and calcium signal receded after a blocker of ruthenium red was applied (i). A representative single cell (ii) recording by time course showed calcium influx fluctuation before and after GSK1016790A application and channel blocker was added (iii), (n = 12, p < 0.01). (e) Measurement of calcium imaging showed 2-APB could induce calcium influx which could be blocked by TRP channel blocker of ruthenium red in A2058 cells (i), (n = 12, p < 0.01). When TRPV1 agonist of capsaicin was applied, there was no apparent calcium signal observed in A2058 melanoma cells (ii), (n = 19).
Figure 3
Figure 3
TRPV2 activation dominated A2058 melanoma cells undergoing necrosis. (a) Flow cytometry analysis via FITC-Annexin V and PI staining showed A2058 melanoma cells with 2-APB (100 μM, 200 μM, and 400 μM) treatment undergoing necrosis and enhanced apoptosis (i) & (ii). (b) Morphology measurement by microscopic imaging for A2058 melanoma cells with 2-APB (1 μM, 10 μM, 100 μM, 200 μM, and 400 μM) treatment exhibited prominent necrotic and apoptotic cells.
Figure 4
Figure 4
Silencing TRPV4 ion channel suppressed GSK1016790A mediating signals. (a) A representative western blot indicated the successful knockdown of TRPV4 protein by shRNA (i) & (ii). (b) Loss of TRPV4 fully suppressed the inhibition of proliferation for the treatment of GSK1016790A (1 nM, 10 nM, 20 nM, and 50 nM) to A375 melanoma cells. (c) Suppression of TRPV4 attenuated the inhibition of proliferation for the treatment of 4α-PDD (100 nM, 200 nM, 400 nM, and 1 μM) in A375 melanoma cells. (d) Inhibition of TRPV4 by GSK2193874 (10 nM, 50 nM, 100 nM and 200 nM) did not affect melanoma cell proliferation. (e) Treatment with GSK1016790A (1 nM, 10 nM, 20 nM and 50 nM) and GSK2193874 (100 nM) did not affect cell viability of melanoma. (f) Pretreatment of ruthenium red fully suppressed the inhibition of proliferation for the application of GSK1016790A with 20 nM and 50 nM to A375 melanoma cells. All tests were performed in at least three independent experiments.
Figure 5
Figure 5
GSK1016790A application induced apoptosis of human A375 melanoma cells. (a) Flow cytometry test via FITC-Annexin V and PI staining showed increasing apoptotic signals by treatment with GSK1016790A to A375 melanoma cells (i) & (ii). (b) Flow cytometry analysis via FITC-Annexin V and PI staining for 4α-PDD treatment exhibited enhancing apoptotic cells in melanoma A375 cells (i) & (ii). (c) Pretreatment of ruthenium red prominently attenuated apoptotic cells for the application of GSK1016790A with 20 nM and 50 nM to A375 melanoma cells; cells were stained with Hochest33342 and PI. All tests were performed in at least three independent experiments.
Figure 6
Figure 6
TRPV4 functionally mediated melanoma cells apoptosis via AKT pathway and was influenced by calcium. (a) Western blot analysis of MAPK as well as AKT signals was collected for activation of TRPV4 in A375 melanoma cells. Cells were treated by GSK1016790A with 20 nM and 50 nM for 24 h and lysates were then probed with the indicated antibodies, β-actin was used as a loading control, and pAKT was observed upregulated. (b) Assessments of pAKT changes triggered by GSK1016790A with 20 nM and 50 nM within 10 min as well as 24 h in melanoma A375 cells. (c) Influences of calcium ion on GSK1016790A-induced, TRPV4-mediated signal transduction. A375 cells were untreated or pretreated with EGTA or BAPTA-AM for 1 h before being stimulated with GSK1016790A. All tests were performed in at least three independent experiments.
Figure 7
Figure 7
Functional TRPVs influenced melanoma cell fate in different profiles. (a) Flow cytometry assessment of 4α-PDD triggered apoptosis (i) of melanoma A375 cells via staining with FITC - Annexin V and PI. Activation of TRPV4 by 4α-PDD induced human melanoma A375 cells undergoing early apoptosis (ii). A slight degree of necrotic cells was observed as well (iii). (b) Flow cytometry assessment of 2-APB triggered cell death of melanoma A2058 cells via staining with Hochest33342 and PI. Activation of TRPV2 by 2-APB in melanoma A2058 cells underwent late apoptosis as well as necrosis (i). 2-APB application induced a slight degree of early apoptotic cells (ii) and dominated cells undergoing necrosis and late apoptosis (iii).
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References

    1. Zhou Z. H., Song J. W., Li W., et al. The acid-sensing ion channel, ASIC2, promotes invasion and metastasis of colorectal cancer under acidosis by activating the calcineurin/NFAT1 axis. Journal of Experimental & Clinical Cancer Research. 2017;36(130):1–12. doi: 10.1186/s13046-016-0484-y. - DOI - PMC - PubMed
    1. Peters A. A., Jamaludin S. Y. N., Yapa K. T. D. S., et al. Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells. Oncogene. 2017;36(46):6490–6500. doi: 10.1038/onc.2017.234. - DOI - PubMed
    1. Adapala R. K., Thoppil R. J., Ghosh K., et al. Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy. Oncogene. 2016;35(3):314–322. doi: 10.1038/onc.2015.83. - DOI - PMC - PubMed
    1. Ko J.-H., Ko E. A., Gu W., Lim I., Bang H., Zhou T. Expression profiling of ion channel genes predicts clinical outcome in breast cancer. Molecular Cancer. 2013;12(1, article 106) doi: 10.1186/1476-4598-12-106. - DOI - PMC - PubMed
    1. Fraser S. P., Grimes J. A., Djamgoz M. B. A., et al. Effects of voltage-gated ion channel modulators on rat prostatic cancer cell proliferation: Comparison of strongly and weakly metastatic cell lines. The Prostate. 2000;44(1):61–76. doi: 10.1002/1097-0045(20000615)44:1<61::AID-PROS9>3.0.CO;2-3. - DOI - PubMed

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