新藤黄酸抑制人肝癌细胞BEL-7402自噬作用研究

Inhibitory Effect of Gambogenic Acid on the Autophagy of Human Hepatoma BEL-7402 Cells

目的观察新藤黄酸(gambogenic acid,GNA)对人肝癌细胞BEL-7402自噬的影响,为GNA抗肿瘤研究提供理论依据。方法体外培养人肝癌BEL-7402细胞株,采用MTT法检测GNA对人肝癌细胞BEL-7402存活率的影响;倒置显微镜观察给药前后细胞形态变化;单丹磺酰尸胺(monodansylcadaverine,MDC)染色,荧光显微镜观察细胞自噬小体;透射电子显微镜进一步观察自噬小体;Western blot法检测自噬相关蛋白微管相关蛋白1轻链3(microtubule associated protein 1light chain 3,LC3)和p62、Beclin1的表达水平及加入自噬诱导剂雷帕霉素(rapamycin,Rap)和自噬抑制剂氯喹(chloroquine,CQ)后自噬相关蛋白的表达情况。结果 GNA能显著抑制人肝癌细胞BEL-7402的增殖并呈时效及量效关系;倒置显微镜观察结果显示GNA组细胞随药物浓度增大,细胞悬浮增多,高浓度给药组细胞出现碎片化;荧光显微镜下观察结果显示,随药物浓度的增加,荧光点状聚集增多;透射电子显微镜观察发现,随GNA浓度的增加,细胞内自噬空泡数量增多,与对照组比较,加入自噬诱导剂Rap后,自噬空泡数量显著增多;Western blot法检测结果显示,随GNA浓度的增加,Beclin1、p62、LC3-Ⅱ蛋白表达水平均增加(P<0.05),LC3-Ⅰ无明显变化,表明BEL-7402细胞自噬前期被激活,后期进程被抑制。结论 GNA能抑制人肝癌细胞BEL-7402的增殖,其机制可能部分是通过抑制自噬后期自噬体与溶酶体的融合,抑制人肝癌BEL-7402细胞的保护性自噬。

Objective To investigate the influence of gambogenic acid(GNA)on the autophagy of human hepatoma BEL-7402 cells,and to provide a theoretical basis for anti-tumor studies of GNA.Methods Human hepatoma cell line BEL-7402 was cultured in vitro.MTT assay was used to observe the effect of GNA on the viability of BEL-7402 cells;inverted microscopy was used to observe cell morphology before and after administration;monodansylcadaverine(MDC)staining was performed and then a fluorescence microscope was used to observe autophagosomes;a transmission electron microscope was used for further observation of autophagosomes;Western blot was used to measure the expression of the autophagy-related proteins microtubule-associated protein 1 light chain 3(LC3),p62,and Beclin1,as well as their expression after the addition of the autophagy inducer rapamycin(Rap)and the autophagy inhibitor chloroquine(CQ).Results GNA significantly inhibited the proliferation of BEL-7402 cells in a time-and dose-dependent manner.Inverted microscopy showed that in the GNA group,cell suspension increased with the increase in drug concentration,and fragmented cells were observed in the high-concentration group.The fluorescence microscope showed that fluorescence point aggregation increased with the increase in drug concentration.The transmission electron microscope showed that the number of autophagic vacuoles increased with the increase in the concentration of GNA,and compared with the control group,there was a significant increase in the number of autophagic vacuoles after the addition of the autophagy inducer Rap.Western blot showed that with the increase in GNA concentration,there were significant increases in the protein expression of Beclin1,p62,and LC3-Ⅱ(P<0.05),while there was no significant change in LC3-Ⅰ,suggesting that the early stage of autophagy of BEL-7402 cells was activated and the late stage was inhibited.Conclusion GNA can inhibit the proliferation of human hepatoma BEL-7402 cells,possibly by inhibiting the fusion of lysosomes and autophagosomes in the late stage of autophagy and the protective autophagy of BEL-7402 cells.