自噬调节在结肠癌细胞化疗耐药性的作用机制研究

Mechanism of autophagy regulating chemoresistance in colon cancer cells

目的:探究自噬调节在结肠癌细胞化疗过程中耐药性的作用机制。方法:以顺铂诱导人结肠癌细胞系EC9706细胞自噬,观察自噬抑制剂3-MA对EC9706细胞的影响。CCK8法测定细胞生长情况。流式细胞术检测细胞凋亡和细胞周期。MDC检测自噬。Western blot法检测Beclin-1、PI3KⅢ、LC3-Ⅰ、LC3-Ⅱ蛋白表达情况。结果:联合组EC9706细胞抑制率为(57.34±0.55)%,明显高于顺铂组的(32.00±0.32)%;而联合组与顺铂组EC9706细胞抑制率均明显高于对照组的(5.22±0.06)%。顺铂组MDC染色自噬空泡数为(1.72±0.12),明显高于对照组的(1.00±0.06)和联合组的(0.99±0.35)。联合组EC9706细胞凋亡率为(29.12±0.34)%,明显高于顺铂组的(19.76±0.15)%;且G0/G1、G2/M期细胞凋亡率分别为(54.68±0.35)%、(2.33±0.05)%,均明显低于顺铂组的(58.45±0.12)%、(7.56±0.16)%;而S期细胞凋亡率为(44.78±0.45)%,明显高于顺铂组的(38.25±0.65)%(P<0.05)。联合组及顺铂组Beclin-1、PI3KⅢ、LC3-Ⅰ、LC3-Ⅱ蛋白表达倍数均明显高于对照组,且联合组Beclin-1、PI3KⅢ、LC3-Ⅰ、LC3-Ⅱ蛋白表达倍数均明显低于顺铂组(P<0.05)。结论:自噬可能是顺铂诱导的结肠癌细胞的一种自我保护机制,抑制其自噬可能是结肠癌辅助化疗的新策略。

Objective:To explore the mechanism of autophagy regulation in drug resistance of colon cancer cells during chemotherapy.Methods:Autophagy of human colon cancer cell line EC9706 was induced by cisplatin,and the effect of autophagy inhibitor 3-MA on EC9706 cells was observed.The cell growth was determined by CCK8 method.Cell apoptosis and cell cycle were detected by flow cytometry.MDC detects autophagy.Western blot method to detect Beclin 1,PI3KⅢ,LC3-Ⅰ,LC3-Ⅱ protein expression.Results:The inhibition rate of EC9706 cells in combination group was(57.34±0.55)%,which was significantly higher than that in cisplatin group(32.00±0.32)%.The inhibitory rate of EC9706 cells in combination group and cisplatin group was significantly higher than that in control group(5.22±0.06)%.The number of autophagy vacuoles in MDC staining of cisplatin group was(1.72±0.12),which was significantly higher than that in control group(1.00±0.06)and combined group(0.99±0.35).The apoptosis rate of EC9706 cells in the combined group was(29.12±0.34)%,significantly higher than that in the cisplatin group(19.76±0.15)%,and the apoptosis rate of cells in the G0/G1 and G2/M stages were(54.68±0.35)% and(2.33±0.05)%,respectively,which were significantly lower than that in the cisplatin group(58.45±0.12)% and(7.56±0.16)%,respectively.The apoptosis rate of cells in the S stage was(44.78±0.45)%,significantly higher than that in the cisplatin group(38.25±0.65)%(P<0.05).Joint and cisplatin group Beclin 1,PI3KⅢ,LC3-Ⅰ,multiples LC3-Ⅱ protein expression were significantly higher than that of control group,and the joint group of Beclin 1,PI3KⅢ,LC3-Ⅰ,multiples LC3-Ⅱ protein expression were significantly lower than that of cisplatin group(P<0.05).Conclusion:Autophagy may be a self-protection mechanism of cisplatin induced colon cancer cells,and inhibition of autophagy may be a new strategy of adjuvant chemotherapy for colon cancer cells.