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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 effect of arsenic trioxide (As_2O_3) combined with 3’-azido-3’-deoxythymidine (AZT) on autophagy in human hepatoma HepG2 cells and its effects Possible mechanism of action, provide new ideas for the treatment of liver cancer. Methods HepG2 cells were treated with As2O3 (2μmol / L), AZT (20μmol / L) and two drugs respectively. Meanwhile, HepG2 cells without any drug treatment were used as blank control group. After 24 h treatment, the accumulation of acidic vesicles and autophagy lysosomes in HepG2 cells were observed by acridine orange staining. The expressions of Beclin1, lc3 and p62 m in HepG2 cells were detected by fluorescence quantitative PCR The expression of Beclin1, lc3Ⅱ, lc3Ⅱ / Ⅰ, p62 protein in HepG2 cells was detected by Western blotting. Results Acridine orange staining showed that the accumulation of acidic vesicles and autophagy lysosomes in combination group were more than those in control group and single drug group (P <0.01). Fluorescent quantitative PCR showed that Beclin1 , The expression of lc3 m RNA was significantly higher than that of the control group and the single drug group (P <0.01), and the expression of p62 m RNA was lower than that of the control group and the single drug group (P <0.01). Western blotting showed that HepG2 The expression of Beclin1, lc3Ⅱ and lc3Ⅱ / Ⅰ was higher than that of control group and each single drug group (P <0.01). The expression of p62 protein was lower than that of control group and single drug group (P <0.01). Conclusion The effect of As_2O_3 combined with AZT on autophagy in HepG2 cells is obviously stronger than that in single drug group, which may be related to the up-regulation of Beclin1.