摘要
目的探讨大麻素受体激动剂WIN55,212-2(WIN)联合金诺芬(AF)对人肝癌细胞Hep G2增殖和凋亡的影响,并对其机制进行初步研究。方法分别用5μmol/L WIN、0.25μmol/L AF、5μmol/L WIN联合0.25μmol/L AF处理Hep G2细胞24 h和48 h后,MTS法检测细胞活力;采用Annexin V-FITC和PI双染色法并通过流式细胞仪分析细胞凋亡;Western blot检测凋亡相关蛋白(Bcl-2、Bip、ATF4、PARP、Caspase-3、Caspase-8、Caspase-9)表达的变化。分别用5μmol/L WIN联合0.25μmol/L AF、Z-VAD-FMK以及Z-VAD-FMK预处理1 h后再用5μmol/L WIN联合0.25μmol/L AF处理Hep G2细胞24 h,Western blot检测蛋白PARP、Caspase-3、Caspase-8、Caspase-9的变化。结果与两药单独使用相比,WIN联合AF明显抑制细胞增殖,诱导细胞凋亡,可以引起Bcl-2蛋白表达水平下调,内质网应激反应相关蛋白Bip、ATF4蛋白表达水平上调,以及活化Caspase-3、Caspase-8、Caspase-9,引起PARP蛋白的切割。加入Caspase广谱抑制剂Z-VAD-FMK处理细胞后,能部分逆转联合用药的细胞凋亡比例。结论大麻素受体激动剂WIN联合AF能有效诱导肝癌细胞凋亡,其机制可能与Bcl-2表达下调、内质网应激反应和Caspase系统活化相关。
Objective To investigatethe effects of the combination of WIN55,212- 2（ WIN） and Auranofin（ AF） on the proliferation and apoptosis of Hep G2 human hepatocellular carcinoma（ HCC） cell line and its mechanism.Methods Hep G2 cells cultured in vitro were treated with 5 μmol / L WIN55,212- 2（ WIN）,0. 25 μmol / L Auranofin（ AF） or the combination of 5 μmol/L WIN and 0. 25 μmol/L AF for 24 h and 48 h,respectively. Survival rate of Hep G2 cells was detected by MTS assay. Double staining of Annexin V- FITC and PI,and flow cytometry was used to detect the change of cell apoptosis. Western blot was used to analyze the expression of apoptosis- related proteins（ Bcl- 2,Bip,ATF4,PARP,Caspase- 3,Caspase- 8,Caspase- 9） of Hep G2 cell line. Hep G2 cells cultured in vitro were treated with the combination of 5 μmol / L WIN and 0. 25 μmol / L AF,Z- VAD- FMK or pretreated with Z- VAD- FMK for 1 h and then exposed to WIN and AF for 24 h,Western blot was used to analyzed the expression of PARP,Caspase- 3,Caspase- 8,Caspase- 9. Results Compared with single drug treatment,WIN combined with AF could significantly inhibit proliferation and induce apoptosis in Hep G2 cells,down- regulate the expression of Bcl- 2 and increase the expression of Bip and ATF4,and further activate Caspase- 3,Caspase- 8,Caspase- 9 to initiate PARP cleavage. Pretreatment of Hep G2 cells with caspase inhibitor Z- VAD- FMK for 1 h partially reversed the apoptosis caused by the co- treatment. Conclusion The combination of WIN55,212- 2 and Auranofin effectively induces apoptosis of Hep G2 cell line,which was associated with Bcl- 2 down- regulation,endoplasmic reticulum（ ER） stress and caspase activation.
出处
《广东医学》
CAS
北大核心
2016年第8期1109-1112,共4页
Guangdong Medical Journal
基金
国家自然科学基金资助项目(编号:81372446)
广东省研究生培养创新计划项目(编号:2014JGXM-MS20)
广东省高等教育教学改革项目(编号:GDJG20141136)
广州市教育科学规划课题(编号:11A033)
