二氯乙酸盐激活ROS-JNK通路增强索拉非尼对肝癌细胞增殖的抑制作用  

Dichloroacetate enhances sorafenib’s inhibitory effect on proliferation of hepatocellular carcinoma cells by activating ROS-JNK pathway

作　　者：孙梁博 姚洁 李涛[1,3] 陈岺曦 闫小晶[1] 何凤田[1] 连继勤[1] SUN Liangbo;YAO Jie;LI Tao;CHEN Linxi;YAN Xiaojing;HE Fengtian;LIAN Jiqin(Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences,Army Medical University ( Third Military Medical University), Chongqing, 400038;Department of Digital Medicine, Faculty of Biomedical Engineering and Imaging Medicine, Army Medical University ( Third Military Medical University), Chongqing, 400038;Cancer Institute, Second Affiliated Hospital, Army Medical University ( Third Military Medical University), Chongqing, 400037, China)

机构地区：[1]陆军军医大学(第三军医大学)基础医学院生物化学与分子生物学教研室,重庆400038 [2]陆军军医大学(第三军医大学)生物医学工程与影像医学系数字医学教研室,重庆400038 [3]陆军军医大学(第三军医大学)第二附属医院全军肿瘤诊治研究所,重庆400037

出　　处：《第三军医大学学报》2019年第17期1627-1634,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81572375);重庆市自然科学基金面上项目(CSTC2018jcyjA2018)~~

摘　　要：目的探讨二氯乙酸盐(dichloroacetate,DCA)与索拉非尼(sorafenib)联合使用对肝癌细胞Hep3B增殖抑制的效果及可能机制。方法将Hep3B细胞分为对照组(DMSO)、DCA处理组(5 mmol/L)、索拉非尼处理组(10μmol/L)和联合组(5 mmol/L DCA联合10μmol/L索拉非尼)4个组,处理24 h后,在显微镜下观察各组细胞形态;采用CCK-8检测细胞增殖,流式细胞仪检测细胞凋亡;通过Western blot检测凋亡相关蛋白PARP的表达和p-JNK的水平;采用活性氧(ROS)检测试剂盒检测细胞ROS的变化;用流式细胞仪检测加入抗氧化剂和阻断JNK通路后的细胞凋亡的变化。结果 DCA和索拉非尼联合处理24 h能够显著改变细胞形态,杀伤细胞。与对照组和单独用药组比较,联合组显著增强对肝癌Hep3B细胞的增殖抑制效果(P<0.05),明显增加Hep3B细胞中PARP的剪切与JNK的磷酸化水平,胞内ROS水平也明显升高;联合使用抗氧化剂NAC可显著抑制DCA和索拉非尼处理导致的JNK磷酸化水平升高和对Hep3B细胞的增殖抑制效果。结论 DCA和索拉非尼联合使用可显著抑制肝癌细胞Hep3B的增殖,其机制可能与激活ROS-JNK通路有关。Objective To investigate the synergistic effect of dichloroacetate(DCA) and sorafenib on the inhibition of proliferation in hepatocellular carcinoma(HCC) cells. Methods Hep3 B cells were treated respectively, with DMSO(control), DCA(5 mmol/L), sorafenib(10 μmol/L), and DCA(5 mmol/L) combined with sorafenib(10 μmol/L) for 24 h, then the cells were stained with crystal violet and observed under a microscope for cell morphology. CCK-8 assay was used to detect the changes of cell proliferation, and flow cytometry was employed to measure cell apoptosis. Then, Western blotting was utilized to detect the protein expression of apoptosis-related protein PARP and p-JNK, and the production of reactive oxygen species(ROS) was measured by ROS detection kits, Finally, flow cytometry was performed to detect the changes of apoptosis after addition of anti-oxidant, NAC and JNK inhibitor, SP600125. Results DCA combined with sorafenib significantly changed the cell morphology and killed the cells. Compared with DCA alone or sorafenib alone, DCA combined sorafenib significantly enhanced the inhibitory effect on the proliferation in Hep3 B cells(P<0.05), increased the cleavage of PARP and the phosphorylation of JNK. Moreover, the combination induced the production of intracellular ROS(P<0.05). Co-treatment of antioxidant NAC resulted in significant inhibition of elevated JNK phosphorylation and inhibitory effect on proliferation induced by DCA combined sorafenib in Hep3 B cells. Conclusion Combination of DCA and sorafenib significantly inhibits proliferation in Hep3 B cells, which may be related with the activation of ROS/JNK signal pathway.

关 键 词：二氯乙酸盐 索拉非尼 肝癌 细胞增殖 ROS-JNK通路 

分 类 号：R735.7[医药卫生—肿瘤] R969.2[医药卫生—临床医学]