抑制自噬对贝伐单抗诱导的结肠癌HCT116细胞凋亡的影响

摘要

目的:探讨抑制自噬对贝伐单抗诱导的结肠癌HCT116细胞凋亡的影响.方法:用贝伐单抗、自噬抑制剂3-甲基腺嘌呤(3-MA)处理人结肠癌HCT116细胞48 h,Annexin V-FITC染色、流式细胞术检测细胞凋亡,吖啶橙染色、荧光显微镜观察细胞酸性自噬泡的变化,Western blot法检测细胞内自噬相关蛋白Beclin-1以及凋亡相关蛋白Caspase-9的表达.结果:贝伐单抗作用48 h对HCT116细胞的半数抑制剂量(IC50)为16 mg/L,3-MA的IC50为5 mmol/L.在此浓度下,3-MA单独作用可降低细胞中Beclin-1的表达,贝伐单抗单独作用可增强细胞中Beclin-1的表达,二者联用有拮抗作用(P<0.05).3-MA和贝伐单抗单独作用均可提高细胞凋亡率(P<0.05),增强细胞中Caspase-9蛋白的表达(P<0.05),二者联用有协同作用(P<0.05).结论:抑制自噬可增强贝伐单抗对结肠癌HCT116细胞的杀伤作用.%Aim: To study the effect of autophagy inhibition on bevacizumab-induced apoptosis of HCT116 cells.Methods: Human colon cancer HCT116 cells were treated with bevacizumab and autophagy inhibitor 3-methyl adenine(3-MA) alone or combined for 48 h, cell apoptosis rate was detected by Annexin V-FITC staining and flow cytometry, the changes of acidic autophagic vacuoles observed by acridine orange staining, the expressions of autophagy-related protein Beclin-1 and apoptosis-related protein Caspase-9 were detected by Western blot.Results: The median inhibition dose of bevacizumab for 48 h was 16 mg/L, and that of 3-MA was 5 mmol/L.At these doses,3-MA alone could decrease the expression of Beclin-1 in the cells(P<0.05), while bevacizumab alone could enhance Beclin-1 expression(P<0.05), and the effects of bevacizumab and 3-MA were antagonistic(P<0.05).3-MA and bevacizumab alone could increase the apoptosis rate(P<0.05) and enhance the expression of Caspase-9 protein in the cells(P<0.05), and they had synergistic effect(P<0.05).Conclusion: Autophagy inhibition may enhance the killing effect of bevacizumab on the human colon cancer HCT116 cells.