p27Kip1在As2O3诱导白血病细胞凋亡中的作用机制

目的:研究三氧化二砷(arsenic trioxide,As2O3)及转录生长因子(transforming growth factor-β1,TGF-β1)对人HL-60细胞增殖的影响,并探讨相关信号通路.方法:流式细胞术检测细胞周期及凋亡,RT-PCR检测p27Kip1、TGF-β1、Cyclin E和Bcl-2表达,免疫组织化学方法和Western blot方法检测p27Kip1、TGF-β1、Cyclin E和Bcl-2蛋白水平.结果:As2O3、TGF-β1单药或联合处理均可诱导细胞凋亡,以联合处理组凋亡最明显,并且As2O3单药组及联合处理组细胞周期阻滞于G1期.As2O3单药及联合处理组可见p27Kip1、内源性TGF-β1表达上调,Cyclin E和Bcl-2表达下调,外源性TGF-β1处理组可见p27Kip1 mRNA及蛋白表达上调,内源性TGF-β1 mRNA表达上调,内源性TGF-β1蛋白水平与对照组比较无明显变化,Cyclin E和Bcl-2表达下调,联合处理组p27Kip1及内源性TGF-β1表达上调及Cyclin E和Bcl-2表达下调程度强于单药处理组.结论:As2O3诱导细胞凋亡的机制可能是通过上调TGF-β1,从而上调p27Kip1,拮抗Cyclin E和Bcl-2的作用,抑制细胞增殖,使细胞周期阻滞于G1期,从而诱导细胞发生凋亡,外源性TGF-β1通过上调内源性TGF-β1,从而上调p27Kip1,增强As2O3诱导细胞凋亡的作用.

The mechanism of p27Kip1 in leukemia cell apoptosis induced by As2O3

Objective:To study the proliferation and apoptosis of arsenic trioxide (arsenic trioxide,As2O3) and (transforming growth factor beta 1,TGF-β1) on HL-60 cell,as well as discuss the related mechanism.Methods:HL-60 cells treared with different concentrations As2O3,trypan blue staining detect cell proliferation inhibition rate,inhibition rate of HL-60 treated with As2O3 and (or) TGF-β1 was deteated with CCK-8,and cell cycle was detected with flow cytometry,p27Kip1,endogenous TGF-β1,Cyclin E and Bcl-2 were detected by SYBR GreenRT-PCR,immunohistochemical method and Western blotting.Results:Apoptosis of HL-60 cells induced by different concentrations of As2O3 produced a time-dose dependent relationship.As2O3 and (or) TGF-β1 can induce apoptosis on HL-60 cells,and the combination group had the most obvious apoptosis.Cell cycle was arrested at G1 phase in As2O3 single treatment group and combined treatment group.p27Kip1 and endogenous TGF-β1 of HL-60 cells treated by As2O3 single and combined with exogenous TGF-β1 were increased,accompaning with decreased expression of Cyclin E and Bcl-2,p27Kip1 was upregulated after treated exogenous TGF-β1,and the mRNA expression of endogenous TGF-β1 was upregulated,but the protein expression of endogenous TGF-β1 has no significant change compared with the control group,Cyclin E and Bcl-2 were downregulated in the exogenous TGF-β1 group,the expression change was stronger in combined group than in the single drug treatment.Conclusion:Apoptosis of HL-60 cells was induced by As2O3 and (or) TGF-β1,and the cell cycle was arrested at G1 phase in As2O3 single treatment group and combined treatment group.Apoptosis of HL-60 cells induced by As2O3 may be caused by up-regulating TGF-β1,then p27 Kip1,which was antagonistic to Cyclin E and Bcl-2,leading to the cell cycle arrested at G1 phase.Exogenous TGF-β1 can enhance the role of As2O3 by upregulate the endogenous TGF-β1.