Cyclopamine对人前列腺癌PC-3细胞增殖凋亡及Bcl-2,Bax,caspase-9蛋白表达的影响

目的研究cyclopamine对人前列腺癌PC-3细胞增殖和凋亡的作用及对Bcl-2,Bax,caspase-9蛋白表达的影响,并探讨其机制。方法体外培养的PC-3细胞应用cyclopamine处理后,通过MTT测定细胞增殖抑制情况;电镜下观察肿瘤组织形态学变化;流式细胞仪检测凋亡率;Western印迹技术检测Bcl-2,Bax,caspase-9蛋白的表达变化。结果MTT法结果显示4μmol/L,8μmol/L及12μmol/L浓度的cyclopamine对PC-3细胞增殖有显著抑制作用,与对照组相比差异有显著性(P0.05或P0.01),抑制效果都随着浓度的增大及时间的增加而增强。电镜下可观察到典型的凋亡细胞;流式细胞仪检测结果表明cyclopamine诱导PC-3细胞发生凋亡;Western印迹技术的结果:经培养72h后,随着药物浓度增大,Bcl-2蛋白表达呈逐渐减少,而Bax,有活性的caspase-9蛋白表达逐渐增多。结论Cyclopamine抑制PC-3细胞增殖,在一定剂量和时间范围内呈时间、浓度依赖关系,并可诱导其凋亡。Cyclopamine诱导PC-3细胞凋亡可能通过Bcl-2,Bax,caspase-9介导。

Effects of cyclopamine on the proliferation and apoptosis of PC-3 cellsand the expressions of Bcl-2, Bax and caspase-9 proteins in human prostate cancer

Objectives To study the function of poliferation and apoptosis and the expressions of Bcl-2 ,Bax and caspase-9 proteins on human prostate cancer cell line PC-3 induced by cyclopamine, and to further investigate this mechanism induced by them.Methods After PC-3 cells cultured in vitro were treated by cyclopamine, Methylthiazolyl tetrazolium (MTT) assay was used to measure the situation of the poliferation inhibition; pathomorphism change of PC-3 cells was observed by electron microscopy; flow cytometry (FCM) was employed to examine the apoptosis rate; the expressions of Bcl-2, Bax and caspase-9 proteins were determined by Western blotting.Results The result of MTT assay revealed that cyclopamine in 4, 8, 12 μmol/L significantly inhibited the poliferation of PC-3 cells,which was markedly different from that of controls (P<0.05, or P<0.01). Cyclopamine could inhibit the poliferation of PC-3 cells in a concentration-dependent and time-dependent manner. Transmission electron microscopy examination demonstrated the scattered apoptotic cells could be observed in PC-3 cells. The outcome of FCM indicated cyclopamine could induce apoptosis of PC-3 cells. The result of the Western blotting showed that after being cultured for 72 hours, the levels of Bcl-2 proteins expression were down-regulated in a concentration-dependent manner, and Bax and cleaved caspase-9 was up-regulated.Conclusions Cyclopamine could inhibit the poliferation of PC-3 cells and to a certain extent revealed time and concentration relationship, and induce their apoptosis. The apoptosis of PC-3 cells induced by cyclopamine was mediated by Bcl-2, Bax and caspase-9.