小剂量As_2O_3联合AZT对人肝癌HepG2细胞自噬的影响及其作用机制

Effects of low-dose As_2O_3 combined with AZT on autophagy of human hepatocellular carcinoma HepG2 cells and its mechanism

目的探讨小剂量三氧化二砷(arsenic trioxide,As_2O_3)联合3’-叠氮-3’-脱氧胸腺嘧啶核苷(3’-azido-3’-deoxythymidine,AZT)对人肝癌HepG2细胞自噬的影响及其可能存在的作用机制,为肝癌的治疗提供新思路。方法分别用As_2O_3(2μmol/L)、AZT(20μmol/L)及两药联合处理人肝癌HepG2细胞,同时以未经任何药物处理的HepG2细胞作为空白对照组。药物作用24 h后,吖定橙染色法观察各组人肝癌HepG2细胞中酸性囊泡和自噬溶酶体的堆积情况;荧光定量PCR法检测各组人肝癌HepG2细胞的Beclin1、lc3和p62 m RNA的表达;蛋白质印迹法检测各组人肝癌HepG2细胞Beclin1、lc3Ⅱ、lc3Ⅱ/Ⅰ、p62蛋白的表达。结果吖啶橙染色法显示:联合用药组酸性囊泡和自噬溶酶体的堆积多于对照组及各单药组(P<0.01);荧光定量PCR法显示:联合用药组HepG2细胞的Beclin1、lc3 m RNA表达明显高于对照组及各单药组(P<0.01),p62 m RNA表达低于对照组及各单药组(P<0.01);蛋白质印迹法检测结果显示联合用药组HepG2细胞的Beclin1、lc3Ⅱ、lc3Ⅱ/Ⅰ蛋白表达量高于对照组及各单药组(P<0.01),p62蛋白表达低于对照组及各单药组(P<0.01)。结论 As_2O_3联合AZT促进人肝癌HepG2细胞自噬的作用明显强于各单药组,可能与两药联合导致Beclin1的表达上调有关。

Objective To investigate the effects of arsenic trioxide(As_2O_3) and 3'-azido-3'-deox ythymidine(AZT) on the autophagy of huamn liver cancer HepG2 cells, and to explore its possible mechanism. Methods Human liver cancer HepG2 cells were treated with As_2O_3(2 μmol/L), AZT(20 μmol/L), and As_2O_3 combined with AZT, respectively; the blank control group was not given any medicine. Acridine orange staining was used to observe the accumulation of acidic vesicular and autolysosomes in human liver cancer HepG2 cells in different groups. The expression levels of Beclin1,lc3, p62 m RNA in different groups were detected by real-time fluorescent quantitative-PCR. The protein expression levels of Beclin1, lc3Ⅱ, p62, lc3Ⅱ/Ⅰin different groups were detected by western blotting. Results As_2O_3 combined with AZT could significantly increase the accumulation of acidic vesicular and autolysosomes, up-regulate the expressions of Beclin1, lc3 m RNAs and down-regulate the expression of p62 m RNAs, as well as up-regulate the expression levels of Beclin1, lc3Ⅱ, lc3Ⅱ/Ⅰprotein and down-regulate the expression of P62 protein compared with the blank control group, As_2O_3 group and AZT group. Conclusion The level of autophagy in human hepatocellular carcinoma HepG2 cells treated with As_2O_3 combined with AZT was significantly higher than that in each single drug group.This effect may be related to the up-regulation of Beclin1.