摘要
目的观察ABT737作用于人卵巢癌顺铂耐药SKOV3/DDP细胞后,细胞凋亡和自噬的发生,探讨抑制自噬后对凋亡的影响。方法体外培养SKOV3/DDP细胞,按ABT737药物终浓度0、2.5、5.0和10.0μmol/L分为4组,分别作用12和24 h。流式细胞术检测各组细胞凋亡率,分别采用免疫荧光和免疫印迹法(Western blot)检测各组细胞自噬相关蛋白LC3和Beclin-1表达水平。将细胞分为对照组、ABT737组、3-Ma组和联合用药组,分别以细胞培养基、ABT737(5.0μmol/L)、3-Ma(1.0 mmol/L)和ABT737联合3-Ma作用于SKOV3/DDP细胞,Western blot法检测12和24 h后凋亡相关蛋白active Caspase-3和cyto C的表达水平。结果与对照组比较,12和24 h各剂量ABT737组细胞凋亡率均明显增高,差异有统计学意义(P<0.01)。间接免疫荧光法检测LC3和Beclin-1蛋白表达,结果显示随着ABT737药物终浓度增加和作用时间的延长,两蛋白阳性表达亮度均越来越强,提示蛋白表达增强。Western blot检测LC3和Beclin-1蛋白表达水平,结果显示随着ABT737药物终浓度增加和作用时间的延长,细胞中LC3(F=10.878,F=13.256,P<0.05)和Beclin-1(F=6.589,F=7.365,P<0.05)蛋白表达水平均逐渐升高。3-Ma抑制自噬后,与ABT737组相比,联合用药组active Caspase-3 12和24 h表达水平明显增强,12和24 h cyto C表达水平也明显增强,差异有统计学意义(P<0.05)。结论 ABT737诱导人卵巢癌顺铂耐药SKOV3/DDP细胞凋亡的同时伴随着自噬的发生,抑制自噬能够促进凋亡。
Objective To observe the effect of ABT737 on apoptosis and autophagy in human ovarian cancer SKOV3/DDP cells,and to explore the effect of inhibiting autophagy on apoptosis.Methods The SKOV3/DDP cells were cultured with various concentrations ABT737 of 0,2.5,5.0,10.0μmol/L for 12 and 24 h.The changes of apoptosis rates were detected by flow cytometry;the expressions of LC3 and Beclin-1 in various groups were detected by both indirect immunofluorescence method and Western blot.Then SKOV3/DDP cells were divided into control group,ABT737 group,3-Ma group and combined group,which were applied by cell culture medium,ABT737(5.0μmol/L),3-Ma(1.0 mmol/L)and ABT737 combined with 3-Ma.The expression levels of apoptosis related protein active Caspase-3 and cyto C were detected by Western blot.Result Compared with control group,the rates of apoptosis in 12 and 24 h groups were significantly increased.The immunofluorescence result showed that the expressions of LC3 and Beclin-1 were significantly increased in different doses of ABT737 groups with the increase of dose and lime.The expression levels of LC3(F=10.878,F=13.256,P<0.05)and Beclin-1(F=6.589,F=1.365,P<0.05)were significantly increased in different doses of ABT737 groups with the increase of dose and time.Following the inhibition of autophagy by 3-Ma,compared with ABT737 group,the expression levels of active Caspase-3 12 and 24 h were significantly increased in combined group,and the expression levels of cyto C 12 and 24 h were also increased.Conclusion ABT737 can induce both apoptosis and autophagy in SKOV3/DDP cells,and the inhibition of autophagy promotes apoptosis.
作者
于洋
徐路
刘师兵
李松岩
徐冶
YU Yang;XU Lu;LIU Shi-bing;LI Song-yan;XU Ye(Medical Research Laboratory,Jilin Medical University,Jilin Jilin 132013,China;Department of Histology andEmbryology,College of Basic Medical Sciences,Jilin Medical University,Jilin Jilin 132013,China)
出处
《毒理学杂志》
CAS
CSCD
2019年第1期7-11,共5页
Journal of Toxicology
基金
国家自然科学基金(81372793)
吉林省教育厅"十二五"科技研究重点项目资助课题[吉教科合字(2016)第237号]
吉林省卫计委青年科技骨干培养计划项目资助课题(2017Q049)
