环巴胺对DU145细胞增殖和细胞周期的影响

Effects of cyclopamine on the proliferation and cycle of prostate cancer cell line DU145

目的:研究Hedgehog信号通路阻断剂(环巴胺)对DU145细胞增殖的抑制作用。方法:不同浓度(1、10、50、100μmol/L)环巴胺干预DU145细胞,分别在24、48、72h后采用噻唑蓝比色法检测其对细胞增殖的抑制作用;流式细胞术检测环巴胺对细胞周期的影响;RT-PCR检测50μmol/L环巴胺作用48h后实验组和对照组细胞周期蛋白E(cyclinE)mRNA表达水平的差异。结果:环巴胺对DU145细胞的抑制作用呈时效和量效依赖关系,当浓度>10μmol/L作用24h后会显著抑制细胞增殖,10、50、100μmol/L浓度组对细胞的抑制率分别为7.42%、12.70%和59.15%,与空白对照组相比,差异有统计学意义(P<0.05)。流式细胞术检测发现,当环巴胺浓度达10μmol/L以上,干预48h后G1期细胞比例明显升高。对照组、10、50μmol/L浓度组的G1期细胞百分比分别为:(52.17±2.21)%、(60.13±2.75)%和(74.30±3.52)%,差异有统计学意义(P<0.01);凋亡峰也随环巴胺浓度增加而逐渐增高。50μmol/L环巴胺作用48h后DU145细胞cyclinEmRNA表达显著降低,与空白对照组相比降低61.90%(P<0.01)。结论:环巴胺可以抑制DU145细胞的增殖能力,其机制可能与下调DU145细胞cyc-linEmRNA表达水平,从而将DU145细胞阻滞于G1期有关。环巴胺亦可以诱导DU145细胞凋亡。

Objective：To investigate the inhibitory effect of the Hedgehog signal pathway blocker（cyclopamine）on DU145 cells.Methods：We interfered DU145 cells with cyclopamine at the concentrations of 1,10,50 and 100 μmol/L and detected its inhibitory effect on the cells 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 the experimental and control groups by RT-PCR at 48 hours after 50 μmol/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 100 μmol/L groups respectively at 24 hours,significantly different from that of the blank control group（P 0.05）.It markedly suppressed the proliferation of the DU145 cells at 10 μmol/L at 24 hours.Flow cytometry showed an obviously increased proportion of stage G1 cells at the concentration of 10 μmol/L after 48-hour intervention,with statistically significant differences from the G1 cell proportions in the control,10 μmol/L and 50 μmol/L groups,which were（52.17 ± 2.21）%,（60.13 ± 2.75）% and（74.30 ± 3.52）% respectively（P0.01）.The apoptotic peak was elevated with the increased concentration of cyclopamine.The cyclin E mRNA expression of the DU145 cells was decreased by 61.90% at 48 hours after 50 μmol/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.