吴茱萸碱联合RO3306对人肝癌细胞HepG2的协同抑制

Synergistic Inhibition of Evodiamine Combined with RO3306 on Human Hepatocarcinoma Cell Line HepG2

【目的】探讨吴茱萸碱(EVO)联合细胞周期蛋白依赖性激酶1(CDK1)的特异抑制剂RO3306对人肝癌细胞HepG2的生长抑制、诱导凋亡是否具有协同增效作用。【方法】采用四甲基偶氮唑盐(MTT)法、碘化丙啶(PI)单染流式细胞术检测EVO诱导不可逆凋亡的时间转折点;采用MTT法、集落形成法、PI单染流式细胞术检测EVO和RO3306联合用药与单独用药比较是否具有协同增效作用(q>1.15为协同作用)。【结果】MTT法结果显示:作用16 h起,再撤药培养12 h与撤药前比较抑制率明显上升。流式细胞术检测表明:细胞同步化处理后吴茱萸碱作用12 h大部分细胞发生M期阻滞(M-arrest);作用20 h细胞发生M期滑移(M-slippage)。MTT法检测2μmol/L吴茱萸碱、2μmol/L RO3306及两者联合作用的抑制率分别是33.64%、6.10%和52.74%,q值为1.32。集落形成法显示:1μmol/L吴茱萸碱、2μmol/LRO3306和两者联合作用的抑制率分别是48.21%、11.13%和78.13%,q值为1.83;2μmol/L吴茱萸碱、2μmol/LRO3306和两者联合作用的抑制率分别是9.75%、66.8%和91.2%,q值为1.3。流式细胞术显示:2μmol/L吴茱萸碱、2μmol/L RO3306和两者联合作用的抑制率分别是23.7%、6.6%和44.8%,q值为1.31。【结论】吴茱萸碱诱导M-arrest发生于12 h左右,诱导不可逆凋亡的时间转折点为20 h左右,吴茱萸碱联合RO3306对HepG2具有明显的协同抑制作用。

Objective To investigate the proliferation inhibition and the apoptosis-induction effect of evodiamine（EVO） combined with RO3306,a specific CDK1 inhibitor,on human hepatocarcinoma cell line HepG2,and to study whether the effects are synergistic.Methods Methyl thiazolyl tetrazolium（MTT） assay,and propidium iodide（PI） staining associated with flow cytometry（FCM） assay were used to observe the occurrence of an irreversible apoptosis induced by EVO.MTT assay,colony-forming assay and FCM assay were used to investigate the synergistic action of EVO combined with RO3306.The evaluation criteria of synergistic action were q〉1.15.Results The results of MTT assay showed that a higher inhibition rate for HepG2 was presented when HepG2 cells were treated with EVO for 16 h and then cultured for another 12 h with EVO removed.The results of FCM assay showed that M-arrest occurred when HepG2 cells were treated with EVO for about 12 h after cell synchronization,and M-slippage occurred when HepG2 cells were treated with EVO for about 20 h.Results of MTT assay showed that the inhibitive rates in 2 μmol/L EVO group,2 μmol/L RO3306 group and the combination group were 33.64%,6.09% and 52.74%,respectively,and q=1.32.Results of colony-forming assay showed the inhibitive rates in 1 μmol/L EVO group,2 μmol/L RO3306 group and the combination group were 48.21%,11.13% and 78.13%,respectively,and q=1.83;the inhibitive rates in 2 μmol/L EVO group,2 μmol/L RO3306 group and the combination group were 9.75%,66.80% and 91.20%,respectively,and q=1.3.Results of FCM assay showed that the inhibitive rates in 2 μmol/L EVO group,2 μmol/L RO3306 group,and the combination group were 23.7%,6.6% and 44.8%,respectively,and q=1.31.Conclusion M-arrest of HepG2 occurs after treatment with EVO for about 12 h,and the inconvertible apoptosis occurs after treatment with EVO for about 20 hours.It is indicated that EVO combined with RO3306 shows an obvious synergistic inhibition on HepG2.