To observe the effect of high-mobility group box 1 (HMGB1) on autophagy and chemotherapy resistance in human leukemiacell line (K562) cells, and to explore the underlying mechanisms. Methods: The K562 cells were cultured in vitro and divided into 6 groups: a chemotherapeutic group, a chemotherapeutic control group, a HMGB1 preconditioning group, a HMGB1 preconditioning control group, a HMGB1 siRNA group and a siRNA control group. The chemotherapeutic group was further divided into a vincristine (VCR) group, an etoposide (VP-16) group, a cytosine arabinoside (Ara-C) group, a adriamycin (ADM) group and a arsenic trioxide (As2O3) group. The cell activity was evaluated by cell counting kit-8. The protein levels of HMGB1, microtubule-associate protein1light chain3 (LC3), AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (m-TOR) were determined by Western blotting. The level of serum HMGB1 was evaluated by enzyme-linked immunosorbent assay (ELISA). The autophagy was examined by monodansylcadaverine staining and observed under transmission electron microscopy. Results: Compared with the control group, the cell activity was significantly decreased and the level of serum HMGB1 was significantly increased in the chemotherapeutic (VCR, VP-16, Ara-C, ADM and As2O3) groups (all P<0.05). Compared with the control group, the cell activity and the level of serum HMGB1 were significantly increased in the HMGB1 preconditioning group (both P<0.05). Compared with the siRNA control group, the cell activity and the level of serum HMGB1 were significantly decreased in the HMGB1 siRNA group (both P<0.05). Compared with the control group, the expression of LC3-II and the formation of autophagic bodies were increased in the HMGB1 preconditioning group (both P<0.05), the p-AMPK expression was increased and p-mTOR expression was decreased (both P<0.05). Conclusion: HMGB1 can increase the autophagy and promote chemotherapy resistance through the pathway of AMPK/m-TOR in K562 cells.
目的：观察高迁移率族蛋白1(high-mobility group box 1，HMGB1)对急性髓系白血病细胞株(human leukemiacell line，K562)自噬及化学治疗(化疗)耐药的影响，并探讨其相关的分子机制。方法：体外培养K562细胞，分为化疗药物处理组、化疗药物处理对照组、HMGB1纯化蛋白预处理组、HMGB1纯化蛋白预处理对照组、HMGB1 siRNA转染组和HMGB1 siRNA转染对照组，其中化疗药物处理组又分为长春新碱(vincristine，VCR)、足叶乙甙(etoposide，VP-16)、阿糖胞苷(cytosine arabinoside，Ara-C)、阿霉素(adriamycin，ADM)和三氧化二砷(arsenic trioxide，As2O3)处理组。细胞计数试剂盒-8检测细胞活性；Western印迹检测HMGB1，微管相关蛋白1轻链3(microtubule-associate protein1light chain3，LC3)，腺苷酸活化蛋白激酶(AMP-activated protein kinase，AMPK)和哺乳动物雷帕霉素靶点(mammalian target of rapamycin，m-TOR)磷酸化蛋白表达水平；ELISA法检测细胞上清液HMGB1蛋白含量；单丹磺酰尸胺染色和透射电镜观察细胞自噬状态。结果：与相应对照组比较，各化疗药物处理组(VCR，VP-16，Ara-C，ADM和As2O3)的细胞活性均显著降低，HMGB1蛋白表达均显著上调(均P<0.05)。与相应对照组比较，HMGB1纯化蛋白预处理组的细胞活性显著增加(P<0.05)，HMGB1蛋白表达显著上调(P<0.05)。与HMGB siRNA转染对照组比较，HMGB1 siRNA转染组的细胞活性显著降低(P<0.05)，HMGB1蛋白表达显著下调(P<0.05)；与相应对照组比较，HMGB1纯化蛋白处理组中LC3-II表达明显增强(P<0.05)，光镜下观察到自噬小体和自噬泡数量明显增加。同时，与相应对照组比较，HMGB1纯化蛋白处理组p-AMPKa的蛋白表达明显增强，而p-mTOR表达明显减弱(均P<0.05)。结论：HMGB1可能通过AMPK/m-TOR信号通路增强K562细胞自噬发挥化疗耐药性。.