YM155增敏Lexatumumab诱导肝癌细胞株LH86凋亡的实验研究

The study on YM155-sensitized lexatumumab-induced apoptosis in hepatocellular carcinoma cells

目的探讨小分子化合物YM155增敏死亡受体单克隆抗体Lexatumumab激活诱导肝癌细胞株LH86凋亡的效果及分子机制。方法体外培养肝癌LH86细胞株,分为对照组(DMSO)、YM155处理组(1μmol/L)、Lexatumumab处理组(1μg/ml)和YM155(1μmol/L)联合Lexatumumab(1μg/ml)组。荧光显微镜观察上述各组细胞核凋亡形态变化,计数凋亡细胞,计算凋亡率。Western blotting检测各组中细胞凋亡标志性蛋白caspase-3和Bax的表达。结果荧光显微镜下,1μmol/L YM155或1μg/ml Lexatumumab单独处理均未能诱导LH86细胞出现胞核固缩凝聚或染色质断裂,而1μmol/L YM155预处理细胞30 min能够逆转Lexatumumab诱导的肝癌细胞核固缩凝聚和染色质断裂。YM155和Lexatumumab联合处理12 h,细胞凋亡率达60%,高于其余3组(P<0.05)。Western blotting检测显示,仅YM155联合Lexatumumab组出现明显的caspase-3蛋白切割条带;YM155和Lexatumumab单独处理均未能诱导Bax构象变化;经1μmol/L YM155预处理后,Lexatumumab能够有效诱导Bax构象变化激活。结论 YM155能够增敏单克隆抗体Lexatumumab诱导肝癌细胞株LH86细胞凋亡,YM155和Lexatumumab联合处理诱导凋亡可能与Bax构象变化激活相关。

Objective To investigate the roles of small molecule compound YM155 in lexatumumab-induced apoptosis in hepatocellular carcinoma（HCC） LH86 cells and the possible mechanism. Methods HCC LH86 cells were cultured in vitro. Control group（DSMO）, YM155-treated group（1 μmol/ L）, lexatumumab-treated group（1 μg/ ml） and YM155 plus lexatumumab-treated group（1 μmol/ L YM155＋1 μg/ ml lexatumumab） were set up. Cells untreated or treated with various conditions were observed under microscope to show necleus fragmentation and calculate cell apoptotic rate. Western blotting was performed to detect apoptotic marker molecule caspase-3 cleavage activation and Bax conformational activation. Results YM155（1 μmol/ L） and lexatumumab（1 μg/ ml） did not induce nucleus fragmentation in LH86 cells. Interestingly, 1 μmol/ L YM155 significantly sensitized lexatumumab-induced ap-optosis in HL86 cells. The apoptotic rate of YM155 plus lexatumumab-treated group was 60%,higher than the other 3 groups（ P〈0. 05）. The combination treatment effectively increased cleaved caspase-3 protein levels. Mechanism analysis revealed that YM155 and lexatumumab could promote Bax activation through its conformational change. Conclusion YM155 is able to sensitize monoclonal anti-body lexatumumab-induced apoptosis in HCC cells,which may be mediated by Bax conformational activation.