氯化钴对人卵巢癌SKOV3细胞顺铂敏感性的影响

Influence of CoCl_2 in cisplatin sensitivity of human ovarian cancer SKOV3 cells

目的:观察氯化钴(CoCl_2)作用于人卵巢癌SKOV3细胞株后对SKOV3细胞顺铂敏感性的影响,并阐述其可能机制。方法:体外培养SKOV3细胞株,随机分为对照组、CoCl_2组、顺铂(DDP)组和CoCl_2联用DDP (联合)组,CoCl_2组细胞以200μmol·L^(-1) CoCl_2作用4h后换普通细胞培养基培养20h,DDP组细胞以含10 mg·L^(-1) DDP的细胞培养基培养24h,联合组细胞以200μmol·L^(-1) CoCl_2作用4h后更换含10mg·L^(-1) DDP的细胞培养基继续培养20h。MTT法检测各组细胞存活率,免疫荧光法检测各组细胞中低氧诱导因子1α(HIF-1α)和诱导型一氧化氮合酶(iNOS)阳性表达强度,Rhod 2-AM荧光探针检测各组细胞线粒体钙离子(Ca^(2+))水平,免疫蛋白印迹(Western blotting)法观察凋亡相关蛋白细胞色素C (cyto C)、半胱氨酸天冬氨酸蛋白酶3 (caspase 3)和活化半胱氨酸天冬氨酸蛋白酶3 (cleaved caspase 3)蛋白表达水平,Muse~凋亡试剂盒检测各组细胞凋亡率。结果:与对照组比较,CoCl_2组细胞存活率无明显变化(P>0.05),其余2组细胞存活率明显下降(P<0.05);且联合组高于DDP组(P<0.05)。与对照组比较,CoCl_2组和联合组细胞中HIF-1α阳性表达强度明显增强(P<0.05);DDP组和联合组细胞中iNOS阳性表达强度明显增强(P<0.05)。与对照组比较,DDP组和联合组细胞Ca^(2+)水平升高(P<0.05);且DDP组高于联合组(P<0.05)。与对照组比较,CoCl_2组细胞中cyto C、caspase 3和cleaved caspase 3蛋白表达水平无明显变化(P>0.05),DDP组细胞中cyto C、caspase 3和cleaved caspase 3蛋白表达水平明显升高(P<0.05);且联合组低于DDP组(P<0.05)。与对照组比较,DDP组细胞凋亡率明显升高(P<0.05);联合组细胞凋亡率较DDP组降低(P<0.05)。结论:CoCl_2可以通过抑制DDP诱导的人卵巢癌SKOV3细胞株iNOS表达增强来减少线粒体途径凋亡的发生,降低其顺铂敏感性。

Objective:To observe the effect of CoCl2 on the cisplatin sensitivity of human ovarian cancer SKOV3 cells,and to clarify the possible mechanism.Methods:The SKOV3 cells were cultured in vitro and randomly divided into control group,CoCl2 group,cisplatin(DDP)group and CoCl2 combined with DDP(combination)group.The cells in CoCl2 group were cultured in normal cell medium for 20 h after cultured in 200μmol·L^-1 CoCl2 for 4 h,the cells in DDP group were cultured in normal cell medium containing 10 mg·L^-1 DDP for 24 h,and the cells in combination group were cultured in 10 mg·L^-1 DDP for 20 h after cultured in 200μmol·L^-1 CoCl2 for 4 h.The survival rates of SKOV3 cells in various groups were detected by MTT method,and the positive expression intensities of hypoxia-inducible factor-1α(HIF-1α)and inducible nitric oxide synthase(iNOS)in the cells in various groups were detected by immunofluorescence method.Rhod 2-AM fluorescence probe was used to observe the levels of Ca^2+in mitochondria in the cells in various groups.Western blotting method was used to observe the expression levels of cytochrome C(cyto C),cysteinyl aspartase 3(caspase 3)and cleaved cysteinyl aspartase 3(cleaved caspase 3).Muse■apoptosis assay kit was used to detect the apoptotic rates of cells in various groups.Results:Compared with control group,the survival rate of the cells in CoCl2 group had no significant change(P>0.05),and the survival rates of the cells in DDP and combination groups were decreased(P<0.05);the survival rate in combination group was higher than that in DDP group(P<0.05).Compared with control group,the positive expression intensities of HIF-1αin CoCl2 and combination groups were increased(P<0.05).Compared with control group,the positive expressions of iNOS in DDP and combination groups were increased(P<0.05).The Ca^2+levels in the cells in DDP group and combination groups were higher than that in control group(P<0.05)and the Ca^2+level in DDP group was higher than that in combination group(P<0.05).Compared with control group,the expression levels of cyto C,caspase 3 and cleaved caspase 3 proteins in the SKOV3 cells in CoCl2 group had no significant changes(P>0.05),and the expression levels of cyto C,caspase 3 and cleaved caspase 3 in DDP group were increased significantly(P<0.05);compared with DDP group,they were lower than those in combination group(P<0.05).Compared with control group,the apoptotic rate of SKOV3 cells in DDP group was increased significantly(P<0.05);the apoptotic rate of SKOV3 cells in combination group was lower than that in DDP group(P<0.05).Conclusion:CoCl2 can redece the mitochondrial apoptosis of human ovarian cancer SKOV3 cells by inhibiting the DDP-induced enhancement of iNOS expression and decrease the sensitivity of SKOV3 cells to cisplatin.