肽基脯氨酰顺反异构酶抑制剂胡桃醌对宫颈鳞癌SiHa细胞凋亡的作用

Pin1 inhibitor juglone induces apoptosis in human cervical cancer SiHa cells

目的:探讨肽基脯氨酰顺反异构酶抑制剂胡桃醌对宫颈鳞癌Si Ha细胞增殖和凋亡的影响及其促凋亡的机制。方法:选取处于对数生长期的Si Ha细胞,将其分为空白对照组和不同剂量(10、20、50、80及100μmol/L)胡桃醌给药组,共6组。采用MTT法观察胡桃醌对Si Ha细胞的抑制作用,并计算出半数抑制浓度(IC50=20.4μmol/L),依据IC50确定胡桃醌的有效浓度;选取与IC50接近的浓度(20μmol/L)通过Hoechst 33258及流式细胞术测定胡桃醌对Si Ha细胞凋亡的影响,Western blot检测凋亡相关蛋白caspase-3、8、9以及抑癌基因PTEN的蛋白水平。结果:MTT实验结果显示,与对照组比较,各给药组对细胞活力的抑制作用明显,差异有统计学意义(P<0.05);Hoechst 33258检测表明,20μmol/L胡桃醌处理细胞12 h后可出现明显的细胞核典型凋亡细胞形态,流式细胞术检测结果说明胡桃醌可诱导Si Ha细胞出现早期凋亡;Western blot检测显示与正常对照组相比,20μmol/L胡桃醌处理细胞12 h后,cleaved caspase-3、8、9及PTEN的蛋白水平明显增加。结论:胡桃醌可以抑制Si Ha细胞的活力,并诱导细胞凋亡,其机制主要是通过抑制caspase途径并增加抑癌基因的表达。

AIM：To explore the effect of peptidyl-prolyl cis/trans isomerase （Pin1） inhibitor juglone on apop-tosis of human cervical cancer SiHa cells.METHODS：Cultured SiHa cells were incubated with juglone at concentrations of 10, 20, 50, 80 and 100 μmol/L for 24 h.The SiHa cell activity was detected by methyl thiazolyl tetrazolium （ MTT） assay.The cell apoptosis was analyzed by flow cytometry with Hoechst 33258 staining.The protein levels of cleaved caspase-3,8,9 and PTEN was determined by Western blotting.RESULTS：In different doses of juglone groups, the SiHa cell growth was greatly inhibited （ P〈0.05） in a dose-dependent manner as compared with control group.The IC50 of ju-glone was 20.4 μmol/L.After treatment with juglone at concentration of 20 μmol/L for 12 h, the apoptosis of SiHa cells was induced, and the typical morphological changes of cell apoptosis such as karyopyknotic pyknic hyperfluorescence bolus, nuclear fragmentation and apoptotic body were observed by Hoechst 33258 staining.The early apoptotic rate was increased significantly as compared with the control.The protein levels of cleaved caspase-3, 8, 9 and PTEN were also increased sig-nificantly as compared with control group.CONCLUSION：Juglone significantly inhibits the cell activity and induces the apoptosis of SiHa cells in vitro by inhibiting the caspase pathway and increasing the expression of anti-oncogene.