Although the transient receptor potential melastatin 8 (TRPM8) cold receptor is highly expressed in prostate cancer (PCa) and constitutes a promising diagnostic and prognostic indicator, the natural agonists of this channel in the prostate, as well as its physiological and pathological functions, remain unknown. In this study, we identified the well-known PCa marker, prostate-specific antigen (PSA), as a physiological TRPM8 agonist. Electrophysiological and Ca(2+) imaging studies demonstrated that PSA activated TRPM8-mediated current by the bradykinin 2 receptor signaling pathway. Further investigation of this mechanism by cell-surface biotinylation revealed that the increase in TRPM8 current induced by PSA was due to an increase in the number of functional TRPM8 channels on the plasma membrane. Importantly, wound-healing and migration assays revealed that TRPM8 activation by PSA reduced motility of the PC3 PCa cell line, suggesting that plasma membrane TRPM8 has a protective role in PCa progression. Consequently, PSA was identified as a natural TRPM8 agonist in the prostate and we propose a putative physiological role for both of these proteins in carcinogenesis, making this pathway a potentially important target for anticancer agent development.

PSA reduces prostate cancer cell motility by stimulating TRPM8 activity and plasma membrane expression.

// Swapna Asuthkar 1 , Kiran Kumar Velpula 1 , Pia A. Elustondo 2 , Lusine Demirkhanyan 1 and Eleonora Zakharian 1 1 University of Illinois College of Medicine, Department of Cancer Biology and Pharmacology, Peoria, IL, USA 2 Dalhousie University, Halifax, NS, Canada Correspondence to: Eleonora Zakharian, email: // Swapna Asuthkar, email: // Keywords : prostate cancer (PC), transient receptor potential melastatin 8(TRPM8) ion channel, testosterone, DHT (5α-dihydrotestosterone), androgen receptor (AR) Received : March 18, 2015 Accepted : April 07, 2015 Published : April 29, 2015 Abstract The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8 . Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PM TRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PM TRPM8 activity elicits Ca 2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PM TRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation.

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TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

Functional and physiopathological implications of TRP channels.

We investigated the expression of transient receptor potential canonical 6 (TRPC6) protein in benign and malignant human prostate tissues and in prostate cancer cell lines and the association with the stage, grade and androgen responsiveness of the tumors. Immunohistochemical techniques, Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to investigate TRPC6 expression. TRPC6 protein was detected in 9 of 20 (45.0%) of benign prostatic hyperplasia (BPH) cases, and there was a significant difference compared with prostate cancer (129 of 149 [86.6%]) (P < 0.01). TRPC6 expression was associated with the histological grade and extraprostatic extension (P < 0.01). Tumors of higher stage tended to have a higher frequency of TRPC6 protein staining, but the difference was not significant among T2, T3 and T4. TRPC6 expression difference between androgen-independent (AI) tumors and androgen-dependent (AD) tumors was not statistically significant. TRPC6 was also observed in prostate cancer cell lines. In summary, TRPC6 is detected in benign and malignant human prostate tissues and prostate cancer cell lines and is associated with the histological grade, Gleason score and extraprostatic extension of prostate cancer.

/pdf/expression-of-trpc6-in-benign-and-malignant-human-prostate-kc09ap36qy.pdf

Expression of TRPC6 in benign and malignant human prostate tissues.

Prostate cancer is a significant health concern. In the early stages, prostate cancer cells depend on androgens for growth and survival, hence androgen-ablation therapy at this time may be effective in causing tumor regression. However, treatment options for advanced hormone-refractory prostate cancers are still relatively inefficient. This study aimed to investigate the possible effects of TRPM8 on the proliferation and angiogenesis of androgen-independent cancer PC-3 cells in vivo. Thirty male nude mice were divided into three groups: the PC-3, PC-3-vector and PC-3-TRPM8 groups. PC-3, PC-3-vector and PC-3-TRPM8 cells were respectively inoculated in the right flank to establish a transplanted tumor model. The mice were treated daily for four weeks and each group was examined by histology and immunohistochemical staining for CD34, FAK and PCNA. A CD34 marked microvascular density (MVD) test was performed. Western blot analysis was used to detect the VEGF protein expression level. Compared to the PC-3 and PC-3-vector groups, the PC-3-TRPM8 group revealed a decrease in tumor volume (P=0.000 and P=0.000, respectively), MVD (P=0.045 and P=0.041, respectively), VEGF (P=0.000 and P=0.000, respectively), FAK and PCNA. The correlation between MVD and VEGF was positive (r=0.419; P=0.021). These data show that the overexpression of TRPM8 had a negative effect on the proliferation and angiogenesis progression of PC-3 cells in vivo.

https://www.spandidos-publications.com/10.3892/ol.2011.410/download

Effects of TRPM8 on the proliferation and angiogenesis of prostate cancer PC-3 cells in vivo

Objective To investigate the effects of transient receptor potential M8 (TRPM8) channel on the proliferation and motility of androgen-independent prostate cancer PC-3 cells.Methods After being permanently transfected with an empty vector and cDNA encoding the TRPM8 protein,cells were analysed for cell cycle distribution and motility using flow cytometry and scratch assay.Results Immunocytochemistry and Ca2+ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells.Cell cycle distribution and scratch assay analysis revealed that TRPM8 induced cell cycle arrest at the G0/G1 stage ( 71.89% 3.43% vs 54.67%±4.59%、51.48%±3.54%,P0.05 ) and facilitated the cell apoptosis induced by starvation ( 12.56%±1.78% vs 4.67%±1.49%、5.28±1.35%,P0.05 ).Furthermore,TRPM8 inhibited the migration of PC-3-TRPM8 cells (P0.01) through the inactivation of focal-adhesion kinase.Conclusions It appears that TRPM8 was not essential for the survival of PC-3 cells;however,the overexpression of TRPM8 had negative effects on the proliferation and migration of PC-3 cells.Thus,TRPM8 and its agonists may serve as important targets for the treatment of prostate cancer.

Liu Tong-z

Papers

Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells