氯化钴诱导人卵巢癌SKOV3细胞顺铂敏感性下降及机制研究

Mechanism of CoCl2 on reducing cisplatin sensitivity in human ovarian cancer SKOV3 cells

目的将氯化钴作用于人卵巢癌SKOV3细胞,观察其顺铂(DDP)敏感性变化,并阐述可能机制。方法体外培养SKOV3细胞,随机分为对照组、氯化钴组、DDP组和联合给药组,氯化钴组以200μmol/L氯化钴作用4 h后更换普通细胞培养液继续培养20 h,DDP组以含10 mg/L DDP的培养基作用24 h,联合给药组以200μmol/L氯化钴作用4 h后更换含10 mg/L DDP的细胞培养液继续培养20 h。MTT法检测各组细胞存活率;Muse^■凋亡检测试剂盒检测各组细胞凋亡率;Hoechst33342染色观察各组细胞核形态;蛋白免疫印迹法(Western blot)观察细胞色素C(cyto C)、半胱氨酸天冬氨酸蛋白酶9(Caspase-9)、缺氧诱导因子(HIF-1α)、p-糖蛋白(p-GP)、磷酸化的信号转导子和转录激活子3(p-STAT3)表达水平。结果与对照组比较,DDP组细胞存活率明显下降;与DDP组比较,联合给药组细胞存活率明显升高。与对照组比较,DDP组细胞凋亡率升高;与DDP组比较,联合用药组细胞凋亡率明显降低,上述差异均有统计学意义(P<0.05)。与对照组比较,DDP组细胞核异常比例明显升高;与DDP组比较,联合用药组细胞核异常比例降低,差异均有统计学意义(P<0.05)。与对照组比较,DDP组细胞中cyto C和Caspase-9表达水平明显升高,氯化钴组和联合用药组HIF-1α、p-STAT3和p-GP表达水平明显升高;与DDP组比较,联合用药组cyto C和Caspase-9表达水平降低,氯化钴组和联合用药组HIF-1α、p-STAT3和p-GP表达水平明显升高,差异均有统计学意义(P<0.05)。结论氯化钴诱导缺氧可导致HIF-1α表达水平升高,同时伴随p-STAT3和p-GP表达水平的升高,人卵巢癌SKOV3细胞DDP敏感性下降。

Objective To observe the effect of CoCl2 on reducing cisplatin sensitivity in human ovarian cancer SKOV3 cells,and to explore the possible mechanism.Methods The SKOV3 cells were cultured in vitro and were randomly divided into control group,CoCl2 group,DDP group and combined group.The survival rates of cells in various groups were detected by MTT assay.The apoptoic rates of cells in various groups were detected by Muse^■ apoptosis detection kit.The cell nucleus morphology was observed by Hoechst33342 staining.The expression levels of cyto C,Caspase-9,HIF-1α,p-GP and p-STAT3 were observed by Western blot.Results Compared with control group,the survival rate of cells in DDP group was decreased significantly(P<0.05);compared with DDP group,the survival rate of cells in combined group was increased(P<0.05).Compared with control group,the apoptotic rate of cells in DDP group was increased(P<0.05);compared with DDP group,the apoptotic rate of cells in combined group was decreased(P<0.05).Compared with control group,the rate of abnormal nuclears in DDP group was increased significantly(P<0.05);compared with DDP group,the rate of abnormal nuclears in combined group was decreased(P<0.05).Compared with control group,the expression levels of cyto C and Caspase-9 in cells of DDP group were increased(P<0.05);compared with DDP group,the expression levels of cyto C and Caspase-9 in cells of combined group were decreased(P<0.05).Compared with control group and DDP group,the expression levels of HIF-1α,p-STAT3 and p-GP in cells of CoCl2 group and combined group were increased(P<0.05).Conclusion Hypoxia induced by CoCl2 can increase the expression levels of HIF-1α,p-STAT3 and p-GP,which lead to a decrease in cisplatin sensitivity in human ovarian cancer SKOV3 cells.