Liu Liying, Gao Fei, Ye Yanqiong, Chen Zhiheng, Dai Yunpeng, Zhao Ping, Guan Guotao, Zhao Mingyi
Department of Pediatrics, Shandong Provincial Hospital Affiliated to Shangdong University, Jinan 250021, China.
Department of Cardiac Surgery, Guangdong General Hospital, Guangzhou 510010, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2016 Oct 28;41(10):1016-1023. doi: 10.11817/j.issn.1672-7347.2016.10.002.
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.
观察高迁移率族蛋白B1（HMGB1）对人白血病细胞系（K562）细胞自噬及化疗耐药性的影响，并探讨其潜在机制。方法：体外培养K562细胞，分为6组：化疗组、化疗对照组、HMGB1预处理组、HMGB1预处理对照组、HMGB1 siRNA组和siRNA对照组。化疗组进一步分为长春新碱（VCR）组、依托泊苷（VP-16）组、阿糖胞苷（Ara-C）组、多柔比星（ADM）组和三氧化二砷（As2O3）组。采用细胞计数试剂盒-8评估细胞活性。通过蛋白质免疫印迹法检测HMGB1、微管相关蛋白1轻链3（LC3）、AMP活化蛋白激酶（AMPK）和雷帕霉素靶蛋白（m-TOR）的蛋白水平。采用酶联免疫吸附测定（ELISA）评估血清HMGB1水平。通过单丹磺酰尸胺染色检测自噬，并在透射电子显微镜下观察。结果：与对照组相比，化疗（VCR、VP-16、Ara-C、ADM和As2O3）组细胞活性显著降低，血清HMGB1水平显著升高（均P<0.05）。与对照组相比，HMGB1预处理组细胞活性和血清HMGB1水平显著升高（均P<0.05）。与siRNA对照组相比，HMGB1 siRNA组细胞活性和血清HMGB1水平显著降低（均P<0.05）。与对照组相比，HMGB-1预处理组LC3-II表达和自噬体形成增加（均P<0.05），p-AMPK表达增加，p-mTOR表达降低（均P<0.05）。结论：HMGB1可通过AMPK/m-TOR途径增加K562细胞的自噬并促进化疗耐药性。
