吴茱萸碱通过TRIB2/AKT信号通路诱导人白血病细胞K562凋亡

目的探讨吴茱萸碱(evodiamine,EVO)对人白血病细胞K562增殖与凋亡的影响,并初步探究其潜在的分子机制。方法以CCK-8法测定不同浓度的EVO处理K652细胞不同时间后的增殖抑制作用,流式细胞术检测细胞凋亡,qRT-PCR检测TRIB2 mRNA表达,Western blot检测TRIB2/AKT信号通路;以EVO作用于AKT激活剂SC79预处理后的K562细胞,流式细胞术检测细胞凋亡,Western blot检测TRIB2/AKT信号通路。以EVO作用于Wnt激活剂SKL2001预处理后的K562细胞,Western blot检测TRIB2/AKT信号通路。结果EVO对K562细胞的增殖抑制作用呈时间及浓度依赖性;流式结果显示EVO体外可促细胞凋亡且呈浓度依赖性;EVO处理后,TRIB2mRNA的表达降低,TRIB2/AKT信号通路被抑制;SC79削弱了由EVO诱导的K562细胞凋亡,且SC79和SKL2001均能逆转EVO对AKT信号通路的抑制作用。结论EVO可诱导人白血病细胞K562凋亡,从而抑制其增殖,其机制可能是通过抑制TRIB2的表达,进而抑制AKT的磷酸化,最终抑制下游基因NF-κB p65的磷酸化,从而诱导细胞凋亡并抑制其生长。

Evodiamine induces apoptosis of leukemia cell line K562 via modulation of TRIB2/AKT pathway

Aim To investigate the effects of Evodiamine(EVO)on proliferation and apoptosis of human leukemia cell line K562 and its potential mechanisms.Methods K562 cells were treated with EVO at different concentrations(0,1,2,4,8,16,32,64μmol·L^-1)for different time periods(24,48h),respectively,and then the cell inhibition was detected by CCK-8 assay.Flow cytometry,qRT-PCR and Western blott were performed for analyzing the cell apoptosis,the mRNA level of TRIB2 and the expression of TRIB2,AKT,p-AKT,IκBα,p-IκBα,NF-κB p65,p-NF-κB p65,caspase-3,c-caspase-3,Bcl-2 respectively after exposure to different concentrations of EVO.K562 cells were pretreated with SC79 prior to treatment with EVO,and afterwards flow cytometry and Western blot were applied to detect the apoptosis in K562 cells and the expression of TRIB2,AKT,p-AKT,IκBα,p-IκBα,NF-κB p65,p-NF-κB p65,caspase-3,c-caspase-3,Bcl-2 respectively.K562 cells were pretreated with SKL2001 prior to treatment with EVO,and Western blot was applied to detect TRIB2,AKT,p-AKT.Results EVO inhibited the proliferation of K562 cells in a dose-and time-dependent manner and induced apoptosis in K562 cells in a dose-dependent manner.The qRT-PCR and Western blot revealed that the mRNA level of TRIB2(P<0.01),the expression of TRIB2,p-AKT,p-IκBα,p-NF-κB p65,Bcl-2(P<0.05)in K562 cells significantly decreased and the expression of caspase-3 and c-caspase-3(P<0.01)in K562 cells significantly increased compared to control group after treatment of EVO.In addition,SC79 inhibited the ability of EVO-induced apoptosis of K562 cells and SKL2001 attenuated down-regulation of p-AKT in K562 cells(P<0.01).Conclusions Evodiamine-induced apoptosis and proliferation inhibition of K562 cells might be mediated through its modulation activity of AKT pathway by inhibiting TRIB2 levels,which then regulates downstream gene NF-κB p65 and ultimately affects the apoptosis and proliferation process of leukemia cells.