Study On Influence Of Biological Behavior And Angiogenesis Of Prostate Cancer PC3Cells Via Transient Receptor Potential M8

Prostate cancer (PC) is a major health problem, totalling one fourth of all new cancer cases diagnosed in adult males in American each year, and accounting for about9%of all cancer-related death rates in the same population. 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, treatment options for advanced hormone-refractory prostate cancers (HRPC) are still relatively inefficient.The role of Ca2+is well established in the majority of the cell signalling pathways involved in carcinogenesis. Calcium-permeable channels are potential candidates for participating in Ca2+homeostasis into prostate cancer cells. One transient receptor potential (TRP) superfamily of cation channels is of particular interest. The human trpm8gene, initially known as trp-p8, has been shown to be mainly expressed in the prostate and overexpressed in prostate cancer. The precise physiological function of TRPM8channel in normal and cancerous prostate tissue is still unknown. TRPM8expression upregulates markedly in PC and in other tumors, suggesting an important role in carcinogenesis. TRPM8expression-silencing experiments using small interference RNA (siRNA) suggested that Ca2+influx through this channel plays an essential role in cellular Ca2+homoeostasis in prostate epithelial cells and is involved in cell survival.The aims of this study were to investigate the influence of biological behavior and angiogenesis of prostate cancer PC3cells via Transient Receptor Potential M8both in vitro and in vivo. Part I Study on Influence of Biological Behavior and Angiogenesis of Prostate Cancer PC3Cells via Transient Receptor Potential M8in vitroObjective To investigate the influence of biological behavior and angiogenesis of prostate cancer PC3cells via Transient Receptor Potential M8in vitro.Methods There were three groups, which are blank control group, experimental group and blank vector group, in our experiment. To culture PC3cells regularly and transfect the retrival vector pcDNA3/TRPM8and pcDNA3into prostate cancer PC3cells, and screen PC3cell line expressing gene TRPM8steadily by using G418at the concentration of400mg/L. The expression of TRPM8in PC3cells was investigated by RT-PCR and Western blot. Afterwards, the cell cycle distribution of three groups cells was analysised by flowcytometry for the effect of TRPM8on proliferation of PC3cell. Furthermore, the effects of TRPM8on apoptosis-resistant ability and migration of PC3cells were also analysised by flowcytometry for apoptosis rate,Western blot, and scratching assay. Finally, the effects of TRPM8on angiogenesis of PC3cell was investigated by Western blot.Results Retrovial vector was transfected into PC3cells successfully, and the PC3cells expressing retrovial vector steadily were screened successfully. The expression of TRPM8in experimental group cells was detected through PCR and Western blot using specific anti-TRPM8antibody while there were no expression of TRPM8in blank vector group cells. Cell cycle analysis indicated that percent of cell in G0/G1stage of blank control, blank vector group, and experimental group were54.54%,52.83%,and69.00%, respectively (p<0.05). Apoptosis rate of blank control, blank vector group, and experimental group were4.47%,4.88%, and11.40%, respectively (p<0.05). In the scratch motility assay,24h after scratching, the migration rate of blank control, blank vector group, and experimental group were100%,97%, and65.47%(p<0.05). Western blot analysis indicated that compared with blank vector group, protein expression of VEGF, Bcl-2were significantly decreased in experimental group cell. Conclusion In PC3cells, TRPM8showed negtive effects on cell proliferation, migration, angiogenesis, and the ability of anti-apoptosis. Part II Study on Influence of Biological Behavior and Angiogenesis of Prostate Cancer PC3Cells via Transient Receptor Potential M8in vivoObjective To investigate the influence of biological behavior and angiogenesis of prostate cancer PC3cells via Transient Receptor Potential M8in vivo.Methods There were three groups, which are blank control group, experimental group, and blank vector group, in our experiment. Three groups cells were respectively inoculated in the right flank to establish model of transplanted tumor. All mice were treated daily for four weeks and examined by histology and immunohistochemical staining for CD31and PCNA of each group. CD31marked microvascular density (MVD) test were performed. Western blot analysis were used to detect the expression of VEGF and Bcl-2in protein level.Results The nude models of prostate tumor were established successfully. Tumor growth rate of experoment group was slower than the other two groups, there are statistically significant differences between them. The weight of each group is as follows, experimental group was1.97±0.12g, blank vector group was2.62±0.10g, blank control group was2.78±0.05g, there are statistically significant differences between the experimental group and the others (p<0.05). The MVD value of each group is as follows, experimental group was29.66±6.04, blank vector group was37.50±9.97, blank control group was38.82±12.11, there are statistically significant differences between the experimental group and the others. The SPF value of each group is as follows, experimental group was67.92%, blank vector group was78.74%, blank control group was80.93%, there are statistically significant differences between the experimental group and the others. Compared to blank control group and blank vector group, there was decreased in Bcl-2of experimental group in protein level. The correlation between MVD and VEGF was positive (r=0.419, P=0.021).Conclusions Overexpression of TRPM8had negative consequences on the proliferation and angiogenesis progression of PC-3cells in vivo.