Study On The Mechanism Of Cyclopamine On Human Prostate Cancer Cell Line Du145

SectionⅠ:The Effects of Cyclopamine on Cell Proliferation and Cell Cycle of Prostate Cancer Cell Line DU145Abstract:Objective:To investigate the inhibitory effect of Hedgehog signal pathway blocker (cyclopamine) on Du145 cells. Methods:We interfered Du145 cells with cyclopamine at the concentration of 1,10,50 and 100μmol/L and detected its inhibitory effect by MTT colorimetry assay at 24,48 and 72 hours,as well as its effect on the cell cycle by flow cytometry, we also determined the difference in the mRNA expression of cyclin E between experiment and control group by RT-PCR at 48 hours after 50μmol/L cyclopamine intervention. Results:Cyclopamine inhibited the Du145 cells in a time- and dose-dependent manner, with inhibition rates of 7.42%,12.70% and 59.15% in the 10,50 and 100μmol/L groups respectively at 24 hours, significantly from that of the blank control group (P<0.05), It markedly suppressed the proliferation of the DU145 cells when cyclopamine over 10μmol/L at 24 hours. Flow cytometry showed an obviously increased proportion of G1 phase cells when the concentration over 10μmol/L after 48-hour intervention, with statistically significant differences from the proportion of G1 phase cells in the control, 10μmol/L and 50μmol/L groups,which were (52.17±2.21)%, (60.13±2.75)% and (74.30±3.52)% respectively (P< 0.01), and apoptotic peak was elevated with the increased concentration of cyclopamine; The cyclin E mRNA expression of Du145 cells was decreased by 61.90% at 48 hous after 50μmol/L cyclopamine intervention as compared with the blank control group(P< 0.01). Conclusion:Cyclopamine can inhibit the proliferation of Du145 cells, and the mechanism may be related with its effect of down-regulating the cyclin.E mRNA expression of Du145 cells and blocking them in stage G1. Cyclopamine can also induce the apoptosis of Du145 cells. Objective:To investigate the effect of cyclopamine induced apoptosis on human prostate cancer cell line Du145, and its impact on GLi-1 and Bcl-2 mRNA expression, make a preliminary explore to the mechanism of cyclopamine induced apoptosis on Du145; Methods:The morphological ultrastructural changes in Du145 cells after treatment with cyclopamine were studied using transmission electron microscopy. determined the difference in the mRNA expression of GLi-1 and Bcl-2 between experiment and control group by RT-PCR; Results:After treatment with cyclopamine for 72h. The characteristic of untreated Du145 cells surface showed a typical morphological feature of a cancer cell such as numerous microvilli and cyclopamine-treated Du145 cells showed distinct morphological changes corresponding to typical apoptosis, including karyopycnosis, chromatin margination, microvilli disappearance or reduction. The expression levels of Gli-1 and Bcl-2 mRNA in 5μmol/L cyclopamine group were decreased but no statistical significance(P>0.05), While 10μmol/L cyclopamine group was down- regulated significantly compare with control and DMSO group(P<0.01). Conclusion:Cyclopamine can induce the apoptosis of Du145 cells, and the mechanism may be related with its effect of down-regulating the Gli-1 and Bcl-2 mRNA expression of Du145 cells, then active the mitochondrial pathways of apoptosis.