柚皮素增强抗人DR5单抗对人肝癌细胞系HepG2的凋亡作用

Synergistic effect of anti-human DR5 monoclonal antibody and naringenin on human hepatocellular carcinoma HepG2 cells

探讨柚皮素增强抗人DR5单克隆抗体对人肝癌细胞系HepG2的凋亡作用及可能机制。MTT法检测柚皮素及抗人DR5单抗对人肝癌细胞系HepG2的生长抑制作用;Annexin V/PI双染分析细胞凋亡;蛋白免疫印记检测Caspase 8的表达;流式细胞术检测HepG2细胞表面DR5的表达。结果显示,10 mg/L的mDRA-6作用于HepG2细胞12 h后细胞增殖抑制率为39%;10 mg/L的mDRA-6分别与200μmol/L、400μmol/L、800μmol/L柚皮素共同作用于HepG2细胞后细胞增殖抑制率分别增加至58%、79%、85%;Annexin V/PI双染及流式细胞术检测结果表明,2 mg/L的mDRA-6作用细胞12 h后细胞凋亡率为16%,2 mg/L的mDRA-6和400μmol/L柚皮素共同作用于HepG2细胞12 h后细胞凋亡率增加为63%;mDRA-6和柚皮素共同作用于HepG2细胞后,蛋白免疫印记结果显示Caspase 8的激活片段明显显现,流式细胞术检测HepG2细胞表面DR5的表达,其基础表达率为49%,400μmol/L柚皮素作用于HepG2细胞12后,HepG2细胞表面DR5的表达率增加为81%。实验显示柚皮素可增强抗人DR5单克隆抗体对人肝癌细胞系HepG2的凋亡作用,其可能机制为柚皮素诱导HepG2细胞表面DR5受体表达上调所致。

To study the synergistic effect of mDRA6 and naringenin on hepatocellular carcinoma HepG2 cells and its possible mechanism, HepG2 cells were cultured with RPMI1640 medium in regular condition. Cytotoxicity was examined by MTT as say. The rate of apoptosis were detected by flow cytometry with Annexin VFITC/PI staining. DR5 expression on the surface of HepG2 cells was determined by flow cytometry. Casepase 8 expression on HepG2 cells was determined by WB. MTT analy sis showed the death rates of HepG2 cells was 390/oo in the presence of 10mg/L mDRA6 for 12 hours. When mDRA 6 with na ringenin at 200, 400, 800 μmol/ L were used to treat HepG2 cells, the death rates of HepG2 cells increased to 58%, 79%, 85 % respectively. Flow cytometry detection indicated that the apoptosis rate of the cells was 16 % in the presence of 2 mg/L mDRA6 for 12 hours with Annexin VFITC/PI, but the apoptosis rate of the cells increase to 63% after 2 mg/L mDRA6 with 400 ttmol/ L naringenin treated for 12 hours, WB detection indicated that Caspase8 were actived after mDRA6 with nar ingenin treated together. The natural expression of DR5 on the surface of the HepG2 cells is 49%, whereas it increased up to 81% after naringenin treated. Therefore, naringenin can synergistically enhance the apoptotic effect of DR5 monoclonal anti body on HepG2 cells, The possible mechanism may be that naringenin can increase DR5 expression on HepG2 cell surfaces.