双氢青蒿素和达沙替尼联合用药对肝癌HepG2细胞的抑制作用  

Inhibition effect of combined treatment of dihydroartemisinin and dasatinib on HepG2 liver cancer cells

作　　者：安姿旖 周兆[1] 刘革修[1] AN Ziyi;ZHOU Zhao;LIU Gexiu(Institute of Hematology, Jinan University, Guangzhou 510010, China)

机构地区：[1]暨南大学血液研究所,广东广州510010

出　　处：《暨南大学学报（自然科学与医学版）》2020年第3期214-223,共10页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家自然科学基金资助项目(81270568)。

摘　　要：目的:探讨双氢青蒿素(DHA)和达沙替尼(Das)联合用药对人肝癌细胞的抑制作用及其分子机制.方法:采用CCK8法、克隆形成实验、吖啶橙/溴化乙锭(AO/EB)染色法、流式细胞术分别进行细胞活性、细胞凋亡、细胞周期、线粒体膜电位、细胞内活性氧(ROS)含量进行检测评价双青蒿素和达沙替尼协同用药的药效,采用Western blot研究其作用机制.结果:(1)DHA和Das单独使用均抑制HepG2细胞的生长,两者在一定的浓度下联合发挥协同作用;联合用药结果显示细胞克隆数量少且集落小;(2)联合用药后,周期调控蛋白c-Myc、Cyclin E1和E2F1表达明显降低,细胞周期在G0/G1阻滞;(3)联合用药后细胞内ΔΨm明显下降且凋亡更加显著,抗凋亡蛋白Bcl-2和PARP表达明显减少,促凋亡蛋白Bim、Cleaved-Caspase 9和Cleaved-PARP的表达显著增加;(4)DHA应用可显著促进胞内ROS的产生,并且显著下调抗氧化蛋白Nrf2表达.结论:DHA和Das联合用药显著抑制HepG2的增殖活性,二者在一定浓度下发挥协同作用,促使周期阻滞,诱导细胞凋亡,分子机制与G1期周期阻滞及线粒体依赖的凋亡信号通路有关.Objective:To explore the depressant effect of combined treatment of dihydroartemisinin(DHA)and dasatinib(Das)on human hepatocellular carcinoma cells(HCC)and its molecular mechanism.Methods:The cytotoxicity of drug(DHA or/and Das)was determined by CCK8 and plate clone formation assay.Cell cycle,cell apoptosis,the reactive oxygen(ROS)and the transformation of mitochondrial membrane potential(ΔΨm)was observed by flow cytometry,morphological change and AO/EB staining.The expression levels of some related protein was examined by Western blotting method.Results:DHA and Das were toxic to HepG2 cells,and the majority of HepG2 cells treated with both DHA and Das were inhibited.The colony formation capacity of HepG2 cells was also significantly inhibited by the co-treatment group.The co-treatment of DHA and Das reduced the cycle proteins and the cell cycle was arrest in G0/G1 phase.The co-treatment of DHA and Das induced decreasedΔΨm as well as more apoptosis,which could down-regulated the expression of Bcl-2 and Poly ADP-ribose polymerase(PARP)and unregulated the expression of Bim,Cleaved-Caspase 9 and Cleaved-PARP.Morphological change and AO/EB staining were accordance with the results of flow cytometry.The ROS levels in the cells was elevated significantly by DHA.The co-treatment down-regulated the expression of antioxidant proteins Nrf2.Conclusion:The combination of DHA and Das could significantly inhibit the proliferation of HepG2 cells,and exerted a synergistic effect at a certain concentration to promote cell cycle arrest and induce cell apoptosis.The mechanism may be related to G1 cycle arrest and mitochondrial dependent apoptotic signaling pathway.

关 键 词：双氢青蒿素 达沙替尼 联合用药 肝癌HEPG2细胞 

分 类 号：R730.23[医药卫生—肿瘤]