奥拉帕利联合GANT61对卵巢癌细胞增殖和凋亡的影响

Effects of PARP inhibitor olaparib combined with GLI1 inhibitor GANT61 on proliferation and apoptosis of ovarian cancer cells

目的探讨胶质瘤相关癌基因同源物(glioma-associated oncogene homolog 1,GLI1)抑制剂GANT61,聚腺苷二磷酸核糖聚合酶(poly-ADP Ribose polymerase,PARP)抑制剂奥拉帕利(olaparib)以及olaparib联合GANT61对卵巢癌细胞增殖和凋亡的影响。方法通过Western印迹法检测正常卵巢上皮组织和上皮性卵巢癌组织中GLI1的表达情况以及正常卵巢上皮细胞(IOSE-80)和卵巢癌细胞系(SKOV3、ES2、HEY、A2780)中GLI1的表达水平。选取高表达GLI1的卵巢癌细胞系进行实验,设置实验分组:对照组、GANT61组、olaparib组和olaparib联合GANT61组。通过CCK8法、平板克隆实验、EDU实验检测各处理组细胞增殖情况。通过流式细胞术检测各处理组细胞中Annexin V+/7AAD+细胞比例。通过Western印迹法以及免疫荧光分别检测DNA损伤标志物γ-H2AX的表达水平和灶点形成情况。结果与正常上皮性卵巢组织相比,GLI1在上皮性卵巢癌组织中显著高表达(P<0.001);与IOSE-80正常卵巢上皮细胞相比,卵巢癌细胞中GLI1蛋白表达水平显著升高(P<0.05)。CCK8、平板克隆实验、EdU实验以及流式细胞术结果表明,相较于对照组,GANT61组和olaparib组细胞抑制率和凋亡率均增高(P<0.05),且联合组较olaparib组细胞抑制率和凋亡率增高(P<0.05)。免疫荧光和Western印迹法的结果表明,相较于对照组,GANT61组和olaparib组细胞γ-H2AX的灶点数量与蛋白表达水平显著升高(P<0.05),且联合组较olaparib组细胞γ-H2AX的灶点数量与蛋白表达水平均显著升高(P<0.05)。结论olaparib联合GANT61能够显著诱导卵巢癌细胞DNA损伤、抑制细胞增殖以及诱导细胞凋亡。

Objective To investigate the effects of GLI1 inhibitor GANT61,PARP inhibitor olaparib and olaparib combined with GANT61 on the proliferation and apoptosis of ovarian cancer cells.Methods Western blotting was used to detect the expression of GLI1 in normal ovarian epithelial tissues and epithelial ovarian cancer tissues,as well as in normal ovarian epithelial IOSE-80 cells and ovarian cancer cell lines.The highly GLI1-expressing ovarian cancer ES2 cells were divided into control group,GANT61 group,olaparib group and olaparib combined with GANT61 group.The cell proliferation was detected by CCK8 method,the clone test and EdU assay.The proportion of Annexin V+/7AAD+cells was detected by flow cytometry.The expression level of DNA damage markerγ-H2AX and the formation of foci were detected by Western blotting and immunofluorescence,respectively.Results Compared with normal epithelial ovarian tissues,GLI1 was highly expressed in ovarian cancer tissues(P<0.001).Compared with IOSE-80 cells,GLI1 protein was highly expressed in ovarian cancer cells(P<0.05).The results of CCK8,the clone formation,EDU assay and flow cytometry showed that compared with the control group,the cell inhibition rate and apoptosis rate of the GANT61 group and the olaparib group were increased(P<0.05),while the combination group was higher than those of olaparib group and GANT61 group(P<0.05).The results of immunofluorescence and Western blotting showed that compared with the control group,the number of foci and the protein expression ofγ-H2AX in the GANT61 group and the olaparib group were significantly increased(P<0.05),and the combination group was increased more than the olaparib group and GANT61 group(P<0.05).Conclusion PARP inhibitor olaparib combined with GLI1 inhibitor GANT61 can significantly induce DNA damage,inhibit cell proliferation and induce cell apoptosis in ovarian cancer cells.