| Prostate cancer (PC) is a significant malignancy of genitourinary system, and it is a big threat to man health. The morbidity of PC, which is up to28%, takes the first place among the malignancy happened in man. Also prostate cancer takes the second place in cancer-related man’s death. In America, The morbidity of PC has exceeded the morbidity of lung cancer, and among all the cancers, prostate cancer has been the first treat to man’s health. In China, with the improvement in living standards, aging of population and diet structure change, the incidence of prostate cancer is on the rising trend year by year. In the early stages, prostate cancer cells depend on androgens for growth and survival, so androgen-ablation therapy at this time may be effective in causing a tumor to regress. However, prostate cancer will evolve to be recurrent, incurable and androgen-independent if there is sufficient time to elapse. However, treatment options for advanced hormone-refractory prostate cancers (HRPC) are still relatively inefficient.Being the second intracellular messenger, Ca2+is involved in cell proliferation and apoptosis. The broken of Ca2+homoeostasis plays an important role in carcinogenesis, and Ca2+channels may be the new targets for cancer treatment. The transient receptor potential (TRP) family attracted wide attention for its calcium-permeability, especially TRPM8. TRPM8channel is Ca2+-permeable, and can be activated by cold temperature and menthol. TRPM8plays an important role in Ca2+homeostasis. In normal tissues, the human trpm8gene, initially known as trp-p8, has been shown to be mainly expressed in sensory nerves and prostate. Compared with normal prostate tissue, TRPM8is over-expressed in prostate cancer tissue, and the expression level is closely related with the grading of prostate cancer. So TRPM8may be a potential diagnostic and therapeutic marker in prostate cancer. The precise physiological function of TRPM8channel in normal and cancerous prostate tissue is still unclear. Henshall et al found that anti-androgen therapy can greatly reduce the expression of TRPM8, suggesting that TRPM8is regulated by androgens. TRPM8expression-silencing experiments using small interference RNA (siRNA) suggested that Ca+influx through this channel plays an essential role in cellular Ca2+homoeostasis in prostate epithelial cells and is involved in cell survival. The antagonist of TRPM8has the same result with siRNA targeted on TRPM8.Menthol is a naturally occurring compound, which has been widely used in cosmetics and pharmaceutical products, and also as flavoring in food. Menthol is considered to be Generally-Recognized-As-Safe and has been approved for over-the-counter external use in the concentration up to16%. The mechanisms by which menthol induces a cool sensation were unknown until TRPM8was recognized as a molecular target of menthol. In addition, it was reported that menthol could cause a sustained increase of [Ca2+]cyt in LNCaP cells and such effect can cause cell apoptosis. In recent years, it has been reported that TRPA1can be activated by menthol in low concentration and suppressed in high concentration. Being a member of TRP family, TRPA1is anther cold sensory receptor, different from TRPM8. The studies on TRPA1mainly focus on cold sensation not on tumor.The aim of this study was to investigate the possible effect of activation of TRPM8channel by menthol on the cell proliferation, motility and apoptosis of DU145cells.Part I Study on the Expression and Function of TRPM8and TRPA1Channels in DU145CellsObjective To investigate the expression of TRPM8and TRPA1in DU145cells and whether menthol can activate TRPM8channel.Methods RT-PCR, Western blot and immunohistochemical assay were performed to investigate the expression of TRPM8and TRPA1. Ca+imaging was performed to investigate whether menthol can activate TRPM8channel.Results The results of RT-PCR, Western blot and immunohistochemical assay suggested that TRPM8expressed in DU145cells while there was no TRPA1expression. Ca2+imaging showed that menthol could dramatically increase the intracellular Ca+concentration.Conclusion In DU145cells, the expression of TRPM8is abundant and active, however, there is no TRPA1expression. Menthol can activate TRPM8channel and induce Ca2+influx. Part Ⅱ Study on Influence of Biological Behavior of Prostate Cancer DU145Cells via activation of TRPM8by MentholObjective To investigate the possible effect of activation of TRPM8by menthol on cell proliferation, motility and apoptosis in DU145cells.Methods MTT assay was performed to investigate whether menthol can suppress the proliferation of DU145cells. Flow cytometry was applied to examine whether menthol can cause cell cycle arrest. Scratch assay and Transwell assay were performed to investigate whether menthol can suppress cell motility. Hochest33258staining assay and flow cytometry assay were performed to examine menthol-induced cell apoptosis. And Western blot was also applied to investigate the changes of cell cycle-related and cell motility-related proteins.Results The results of MTT assay showed that, compared to the nonmenthol-treated cells, the population of cells treated with menthol of different concentrations (25,50,75,100μM) decreased (the results are presented as percentages with the latter as100%)(100%±2.68%VS90.66%±6.60%,82.78%±7.24%,70.12%±9.96%,53.41%±6.45%, respectively). It’s obvious that the population decrease of menthol-treated cells was corrected by20min BCTC (20μM) treatment before the menthol (p<0.01). The results of flow cytometry assay showed that, compared with control, there is a significant increase in Go/G1phase cell fraction after24h (p<0.05),48h and72h (p＜0.01)100μM menthol treatment (49.12%±1.92%VS61.71%±2.70%,77.65%±1.63%,71.81%±2.46%, respectively). Hochest33258staining assay and flow cytometry assay showed that menthol did not induce cell apoptosis. The scratch assay and transwell assay revealed that menthol inhibited cell motility. The migration rate is expressed as a percentage with the value for nonmenthol-treated cells being100%(58.62%±11.55%for24h,p<0.05and48.21%±11.11%for48h, p＜0.01). Compared with the cells only treated with100μM menthol, the migration of the cells treated with BCTC (20μM) for20min first was partly corrected (p<0.05). And the result of transwell assay was similar with the result of scratch assay. Also menthol decreased the expression of cell cycle-related and cell motility-related proteins.Conclusions Menthol can inhibit cell proliferation and motility through the activation of TRPM8in DU145cells, but it cannot induce cell apoptosis. |
PDF Full Text Request