自噬抑制药物氯喹联合索拉菲尼抑制肝癌细胞的实验研究.docVIP

  自噬抑制药物氯喹联合索拉菲尼抑制肝癌 细胞的实验研究# 丁振斌，史颖弘，彭远飞，宋康* 5 10 15 20 25 30 35 40 45 （复旦大学附属中山医院肝癌研究所，上海，200032） 摘要：目的：研究小剂量氯喹（CQ）联合索拉菲尼（Sorafenib）的抑制肝癌细胞的效果， 并探索其机制。方法：首先应用 sorafenib 干预三种肝癌细胞 MHCC97-L、PLC/PRF/5 和 HepG2，应用 annexinV/PI 染色流式检测细胞凋亡，GFP-LC3 转染及 LC3 Western blot 技术 检测细胞自噬活性。应用自噬抑制剂 3-MA、小剂量 CQ 或 RNAi 沉默 Atg5 的表达抑制自噬 活性后，检测 sorafenib 诱导的 MHCC97-L 细胞凋亡。结果：Sorafenib 能够诱导肝癌细胞凋 亡，annexin V 阳性细胞显著增加，且具有浓度及时间依赖趋势。在 sorafenib 诱导细胞凋亡 的同时，我们发现细胞内的自噬相关蛋白 Atg5、Beclin 1 均随着时间的延长表达逐渐增加。 自噬标记性蛋白 LC3-II 在药物作用 6h 后即显著高表达。Sorafenib 干预 GFP-LC3 转染后的 细胞，6h 后即出现大量点状荧光聚集，提示自噬小泡的形成增加。而后我们发现通过 3-MA、 小剂量 CQ 或 RNAi 沉默 Atg5 的表达抑制自噬可以增强 sorafenib 诱导的细胞凋亡。结论： CQ 通过抑制自噬的降解机制可以促进 sorafenib 诱导的肿瘤细胞凋亡，增强 sorafenib 的抑瘤 作用。自噬作为细胞的一种保护性机制，可以保护肿瘤细胞逃避药物诱导的凋亡。 关键词：关键词 1；自噬 2；肝细胞癌 3；凋亡 4；氯喹 5；索拉菲尼 中图分类号：R73-36+11 Effect of sorafenib in combination with chloroquine on preventing HCC in vivo DING Zhenbin, SHI Yinghong, PENG Yuanfei, SONG Kang (Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032) Abstract: Purpose: To investigate the effect of sorafenib in combination with low-dose chloroquine (CQ) on HCC, and explore the underlying mechnism to provide a new route to improve the effect of HCC chemotherapy. Methods: MHCC97-L, PLC/PRF/5 and HepG2 cells were treated with sorafenib at different concentrations and differnt time courses. The extent of cell apoptosis was routinely assessed by annexin V/PI staining. The autophagic activity was determined by GFP-LC3 redistribution and LC3 Western blot. Then the autophagic activity was inhibited by silencing atg5, 3-MA or low-dose CQ, and the apoptosis induced by sorafenib was assessed again. Resuls: Sorafenib can induce apoptosis of HCC cell lines. And the annexin V-positive cells increased significantly with the concentrations and time courses of sorafenib treatment. Meanwhile, the expression of autophagy-related protein atg5 and beclin 1 also increased in the sorafenib treatment. LC3-II was found in a high level after 6 hours treatment