自噬抑制药物氯喹在诱导肝癌细胞死亡中的机制研究.docVIP

  自噬抑制药物氯喹在诱导肝癌细胞死亡中 的机制研究# 丁振斌，史颖弘，彭远飞，宋康* 5 10 15 20 25 30 35 40 （复旦大学附属中山医院肝癌研究所，上海，200032） 摘要：目的：研究自噬抑制药物氯喹（CQ）诱导肝癌细胞死亡中的作用及其相关机制，探 索自噬相关药物可能的临床应用价值。方法：选取三种公认的肝癌细胞系，给予不同浓度 CQ 干预 24h，应用 MTT 法检测增殖，应用 PI 染色法检测细胞死亡，应用 Hochest33342 染 色、TUNEL 染色、Caspase9 Western blot 法检测凋亡。应用 GFP-LC3 转染、LC3 Western blot 及细胞电镜技术检测细胞自噬功能。结果：MTT 检测显示 CQ 作用 24h 能够显著抑制肝癌 细胞增殖。CQ（≥20uM）干预细胞 24h 后，PI 染色阳性细胞明显增多，Hochest33342 核 染色发现核浓聚、核碎裂，TUNEL 染色阳性细胞明显增多，Caspase9 活化；而 CQ（5uM、 10uM）干预后 PI 染色阳性细胞无明显变化。CQ 处理细胞后 GFP-LC3 阳性细胞显著增 多,LC3-II 蛋白表达的显著增高。沉默 Atg5 的表达以及自噬抑制剂 3-MA 的预处理不能显著 抑制 40uM CQ 诱导的 MHCC97-L 细胞的死亡，而同时运用 Caspase 抑制剂 Z-VAD-FMK 和 3-MA 或沉默 Atg5 的表达，PI 染色发现细胞的死亡显著受到抑制。结论：小剂量 CQ（0~10uM） 可以抑制自噬降解，而大剂量 CQ（≥20uM）更显著地导致自噬小泡及自噬标志性蛋白在肿 瘤细胞内积聚，并直接诱导肝癌细胞死亡。其死亡形式既具有凋亡的特征又具有自噬样死亡 的特征。 关键词：自噬；肝细胞癌；凋亡；氯喹 中图分类号：R73-36+11 The death mechanism of HCC cells indued by autophagy-related agent chloroquine DING Zhenbin, SHI Yinghong, PENG Yuanfei, SONG Kang (Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032) Abstract: Purpose: To investigate the role of chloroquine (CQ) in inducing the death of HCC and its underlying mechanism, and to explore the clinical application of autophagy-related agents. Method: Three well-known HCC cell lines were treated with CQ at different concentrations for 24 hours. Then MTT assay was applied to determine the proliferation, and PI staining assay was used to dectect the cell death. The specific apoptosis was examined by Hochest 33342 staining, TUNEL staining and Caspase9 Western blot. The autophagic activity was explored, using GFP-LC3 redistribution, LC3 Western blot, and electron microscopy. Results: MTT assay revealed that the proliferation was significantly inhibited by CQ. The cell death and PI-positive cells increased significantly after HCC cells were treated with CQ (≥20uM) for 24 hours. However, low-dose CQ (5uM and10uM) can not induce cell death of HCC and the increase of PI-positive cells. After MHCC97-L was treated with CQ (40uM and 80uM