Mcl-1在二烯丙基二硫诱导人白血病HL-60细胞G_2/M阻滞中的作用

Mcl-1 involves in G_2/M arrest of HL-60 cells induced by diallyl disulfide

目的:探讨Mcl-1在二烯丙基二硫(DADS)诱导人白血病HL-60细胞周期阻滞过程中的作用。方法:CCK-8检测DADS抑制HL-60细胞的作用;流式细胞术观察DADS对HL-60细胞的周期阻滞效应;West-ern blotting分析DADS处理HL-60细胞后Mcl-1、PCNA(细胞核增殖抗原)、CDK1(周期依赖激酶1)表达情况;RNAi干扰Mcl-1观察Mcl-1对白血病细胞周期的影响;免疫共沉淀分析Mcl-1与PCNA、CDK1结合作用。结果:CCK-8比色结果显示,15、30、60、120、240μmol/L DADS处理HL-60细胞,细胞增殖抑制率分别为31.15%、55.88%、66.14%、75.29%、80.35%,呈浓度依赖性增加(P<0.05)。流式细胞术检测显示,60μmol/L和120μmol/L DADS作用HL-60细胞24 h和48 h后,呈时间和浓度依赖性诱导HL-60细胞G2/M期阻滞(P<0.05)。Western blotting分析发现60μmol/LDADS处理HL-60细胞后PCNA、Mcl-1、CDK1表达下调(P<0.05)。Mcl-1基因沉默24 h后G2/M期百分率增加到19.4%,与对照组比较有显著差异(P<0.05)。而Mcl-1基因沉默后加入60μmol/L DADS作用时,G2/M期百分率比60μmol/L DADS处理组增加6.0%(P<0.05)。免疫共沉淀发现,HL-60细胞中存在Mcl-1与PCNA、CDK1的相互结合,DADS处理HL-60细胞后Mcl-1与PCNA、CDK1的结合作用减弱。结论:DADS能诱导HL-60细胞增殖抑制和G2/M阻滞,其抑制增殖作用与PCNA表达下调有关。Mcl-1基因沉默可增强DADS对HL-60细胞G2/M期阻滞作用。

AIM： To investigate the role of Mcl-1 in the G2/M arrest induced by diallyl disulfide（DADS） in leukemic HL-60 cells.METHODS： The inhibitory effect of DADS on human leukemic HL-60 cells was detected by CCK-8 method in vitro.Flow cytometry analysis was employed to observe the cycle arrest in HL-60 cells and the effect of DADS-induced G2/M arrest on HL-60 cells with Mcl-1 gene knockdown by RNAi silencing.The expression of Mcl-1,PCNA and CDK1 in HL-60 cells treated with DADS was determined by Western blotting.The binding of Mcl-1 with PCNA and CDK1 was detected by coimmuno-precipitation.RESULTS： HL-60 cells were treated with DADS at concentration of 15,30,60,120 or 240 μmol/L for 48 h.The inhibition rates of HL-60 cell proliferation were 31.15%,55.88%,66.14%,75.29% and 80.35%,respectively,and gradually enhanced with the increase in the concentration of DADS（P0.05）.Flow cytometry analysis revealed that the proliferation of HL-60 cells was blocked by DADS in the G2/M phase.Treatment with DADS for 24 h and 48 h at concentrations of 60 μmol/L and 120 μmol/L,the percentage of G2/M phase cells increased as compared to the untreated cells（P0.05）.DADS induced arrest of HL-60 cells in G2/M phase in a time-and dose-dependent manner（P0.05）.The results of Western blot analysis indicated that Mcl-1,PCNA and CDK1 in HL-60 cells were significantly reduced after treated with DADS（P0.05）.HL-60 cell cycle progression delayed by silencing Mcl-1 gene with siRNA technique,suggesting that silence of Mcl-1 gene led to G2/M arrest.Compared to the cells treated with DADS only,the percentage of G2/M cells raised in the cells with Mcl-1 gene silencing and treated with DADS（P0.05）,indicating that Mcl-1 gene silencing enhanced the effect of DADS-induced G2/M arrest in HL-60 cells.The binding of Mcl-1 with PCNA and CDK1 was detected by coimmuno-precipitation and the formation of heterodimers was observed,which was decreased after treated with DADS for 4 h.CONCLUSION： DADS inhibits the proliferation of HL-60 cells and induces its G2/M phase arrest.The decreased expression of PCNA is related to inhibiting the proliferation of leukemic cells.Knockdown of Mcl-1 gene enhances the effect of DADS-induced G2/M arrest.