Akt特异性抑制剂MK2206诱导U937及RS4;11细胞凋亡及其机制

Inducing Effect of Akt Kinase Inhibitor MK2206 on Apoptosis in U937 Cells and RS4;11 Cells,and Its Mechanism

目的:本研究探讨Akt激酶抑制剂MK2206对U937及RS4;11细胞增殖、凋亡的影响,并分析其可能的作用机制。方法:以不同浓度MK2206处理U937及RS4;11细胞24、48 h,用CCK-8法绘制细胞增殖曲线;Annexin V/7-氨基放线菌素D(7-AAD)双标记法分析细胞凋亡情况;流式细胞术检测细胞周期的变化;实时定量PCR检测Bax、Bcl-2、XIAP、CDK1、caspase-3基因mRNA表达的变化。结果:MK2206对U937及RS4;11细胞增殖具有抑制作用,且抑制效应呈一定的时间和剂量依赖性,U937细胞24、48 h的半数抑制浓度(IC50)分别为(0.48±0.15)和(0.09±0.01)μmol/L,RS4;11细胞24、48 h的半数抑制浓度(IC50)分别为(0.91±0.02)和(0.68±0.11)μmol/L。0.5μmol/L MK2206作用于U937细胞及1.0μmol/L MK2206作用于RS4;11细胞24 h、48 h,Annexin V/7-AAD标记的阳性细胞升高,U937细胞组24 h细胞凋亡率为(4.18±0.70)%,48 h细胞凋亡率为(22.53±4.67)%,均高于对照组的(1.35±0.34)%(P<0.05),且48 h细胞凋亡率较24 h更为明显(P<0.05);RS4;11细胞组24 h和48 h细胞凋亡率分别为(5.74±0.58)%和(10.07±1.24)%,均高于对照组的(1.32±0.31)%(P<0.05),且48 h细胞凋亡率较24 h更为明显(P<0.05)。流式细胞术检测细胞周期结果显示,U937细胞组G2/M期细胞比例为(96.78±9.11)%,高于对照组的(9.64±0.91)%(P<0.05);RS4;11细胞组G2/M期细胞比例为(14.19±3.82)%,高于对照组的(5.75±1.28)%(P<0.05)。荧光定量PCR检测结果示,两种细胞中Bax、caspase-3 mRNA相对表达水平升高,而Bcl-2、XIAP表达水平降低,同时伴CDK1 mRNA水平的减少,与各自对照组相比差异有统计学意义(P<0.05)。结论:MK2206能有效抑制U937及RS4;11细胞增殖及促进细胞凋亡,使细胞周期阻滞于G2/M期,其促凋亡机制与上调Bax与caspase-3基因表达、下调Bcl-2与XIAP基因表达有关,细胞周期G2/M期阻滞与CDK1基因表达水平下降有关。

Objective： This study was purposed to investigate the effect of Akt kinase inhibitor MK2206 on proliferation and apoptosis of U937 cells and RS4 ; 11 cells, and to explore its possible mechanism. Methods ： U937 and RS4 ; 11 cells were cultured with different concentrations of MK2206 for 24 h and 48 h, and cell growth curve was analyzed by CCK-8; cell apoptosis was analyzed by Annexin V/7-AAD double labeling; cell cycle changes were analyzed by flow cytometry. The BAX,BCL-2 ,XIAP, CDK1, caspase-3 mRNA expressions were determined by real time PCR. Results： MK2206 significantly inhibited the growth of U937 and RS4 ; 11 cells in a time-and dose-dependent manner, and the ICs0 values of U937 cells for 24 h and 48 h were （0.48 ± 0.15 ）μ mol/L and （0.09 ± 0.01 ） panol/L respectively, while IC50 values of RS4 ; 11 cells for 24 h and 48 h were（ 0.91 ± 0.02 ） p, mol/L and （ 0.68±0.11 ） p, mol/L respectively. U937 were cultured with 0.5 μmol/L MK2206 and RS4 ; 11 cells were cultured with 1.0 p, mol/L MK2206 for 24 h and 48 h, and the both apoptosis rates were higher for 24 h or 48 h than that in control group（ P 〈 0.05 ）, meanwhile the apoptosis rates for 48 h were higher than 24 h. The results of cell cycle detection showed that the both cells were arrested in G2/M phase compared with control group. The real time PCR assay revealed that the expressions of BAX, caspase-3 mRNA in cells treated with MK2206 were increased, while BCL-2 ,XIAP, CDK1 were reduced compared with control group. Condusion ： MK2206 can inhibit proliferation and induce apoptosis of U937 and RS4 ; 11 ceils, and the both cells are arrested in G2/M phase. The mechanism of promoting apoptosis may be related with up-regulating BAX, caspase-3 and down-regulating BCL-2 ,XIAP, meanwhile the cell cycle arrested in G2/M phase may be associated with down-regulating CDK1.