TRAIL及其受体在咖啡因抑制肝癌细胞系HepG2增殖中的作用

The role of TRAIL and its receptors in the inhibitory effect of caffeine on the proliferation of hepatocellular carcinoma cell line HepG2

目的探讨TRAIL及其受体在咖啡因(caffeine)抑制肝癌细胞系HepG2增殖中的作用。方法HepG2细胞分别经caffeine、TRAIL及caffeine+TRAIL作用24h,采用MTT法检测HepG2细胞增殖抑制情况,根据中效原理进行联合用药效应评价;流式细胞仪检测细胞凋亡和细胞周期分布;Westernblot法检测caffeine作用不同时间HepG2细胞中TRAIL受体相关蛋白的表达。结果在1.25～20mmol.L-1浓度范围内,caffeine明显抑制HepG2细胞增殖;在0.01275～0.2040μmol.L-1浓度范围内,TRAIL可明显抑制HepG2细胞增殖。Caffeine联合TRAIL在多数效应范围内的合用指数小于1,具有协同作用。Caffeine5mmol.L-1和TRAIL0.0510μmol.L-1联合用药组HepG2细胞凋亡率明显高于各单独用药组,且两者联合用药对HepG2细胞周期具有明显的影响,使G0/G1期细胞比例明显增加,S期及G2/M期细胞比例明显减少;caffeine5mmol.L-1作用HepG2细胞24h时,其DR4及DR5的表达量明显增加,而DcR1和DcR2的表达无改变。结论TRAIL在caffeine抑制HepG2细胞增殖过程中具有一定的协同作用,其机制可能与caffeine上调HepG2细胞表面DR4、DR5的表达,联合TRAIL后能够进一步诱导凋亡及调节细胞周期有关。

Aim To explore the roles of TRAIL and its receptors in the inhibitory effect of caffeine on the proliferation of human hepatocellular carcinoma cell line HepG2.Methods The HepG2 cells were incubated with different concentrations of caffeine,TRAIL and caffeine＋TRAIL for 24 hours.The proliferation inhibition of HepG2 was detected with MTT assay,and the efficacy of drug combination was evaluated according to the median-response principle.The apoptosis rate and the cell cycle distribution were analyzed with flow cytometry（FCM）.The expressions of TRAIL-receptor protein induced by caffeine at different time points were measured by Western blot.Results Caffeine and TRAIL could significantly inhibit proliferation of HepG2 cell within a certain range of treating concentration.In the range of majority responses,the combination indexes for caffeine plus TRAIL were less than 1 indicating synergic effects.The apoptosis rate of the combined group（caffeine 5 mmol·L^-1 plus TRAIL 0.051 0 μmol·L^-1）was higher than those of the group treated with caffeine 5 mmol·L^-1 or TRAIL 0.051 0 μmol·L^-1 alone.Furthermore,the cell cycle analysis by FCM revealed that the combined group could significantly induced G0/G1 arrest and decreased the cell ratio in S and G2/M phases compared with the control group.The expressions of DR4 and DR5 were increased significantly by caffeine at 5 mmol·L^-1 for 24 h.However,the expressions of DcR1 and DcR2 did not varied obviously.Conclusion TRAIL might play synergic role in the inhibitory effect induced by caffeine on the proliferation of HepG2 cells,the mechanism of which is maybe that caffeine up-regulates the expressions of DR4 and DR5,and coincubated with caffeine and TRAIL augmented apoptosis induction and cell cycle regulation in HepG2 cells.