组蛋白去乙酰化酶抑制剂SAHA联合伊马替尼对K562细胞的协同杀伤作用

Synergistic effect of histone deacetylase inhibitor suberoylanilide hydroxamic acid with imatinib on K562 cells

目的:观察组蛋白去乙酰化酶(HDAC)抑制剂SAHA联合伊马替尼(imatinib,IM)对人慢性髓系白血病(CML)细胞株的协同杀伤作用,并探讨其分子学机制。方法:将不同浓度的SAHA和IM分别或联合作用于对数生长期的K562细胞,MTT比色法检测药物对细胞的生长抑制作用;应用Hoechst荧光染色法和流式细胞仪(FACS)检测细胞凋亡,Western blot法检测药物处理后Bcr-Abl及其下游信号途径蛋白的表达、Caspase途径活化和凋亡相关蛋白表达的改变。结果:SAHA和IM单独作用K562细胞呈剂量依赖性抑制细胞生长(SAHA:F=433.8,P<0.001;IM:F=54.14,P<0.001);两药联合对细胞的生长有协同杀伤作用,联合指数CI<1。FACS分析和Hoechst荧光染色证实,SAHA和IM两药联合能加速K562细胞的凋亡,激活Caspase-3、Caspase-8和PARP,下调抗凋亡蛋白Mcl-1表达。此外,SAHA和IM联用能抑制Bcr-Abl及其磷酸化水平,下调JAK2和磷酸化STAT5表达。结论:HDAC抑制剂SAHA联合IM能协同杀伤CML细胞、加速细胞凋亡。其作用机制可能是两药联用协同抑制了Bcr-Abl及其磷酸化水平,抑制JAK2/STAT5信号途径和下调下游靶基因Mcl-1。

Objective： To investigate synergistically killing effect of histone deacetylase（HDAC） inhibitor suberoylanilide hydroxamic acid（SAHA） combined with imatinib on human chronic myeloid leukemia（CML） cell line.Methods： K562 cells were co-treated with SAHA and imatinib.Cell growth was measured by MTT assay.Apoptosis was determined using Hoechst staining apoptosis detection kit and flow cytometric analysis.Activation of Caspase pathway,expression of Bcr-Abl and its downstream target genes,and expression of anti-apoptotic proteins were detected by Western blot.Results： SAHA synergized the cytotoxicity of imatinib against leukemia K562 cells,concomitantly with increased apoptosis and enhanced activation of Caspase-3,-8 and PRAP.The combination therapy resulted in significantly lower levels of Bcr-Abl,phosphorylated Bcr-Abl compared to treatment with either SAHA or imatinib alone.Furthermore,the co-treatment resulted in down-regulation of anti-apoptotic protein Mcl-1 expression.Also,marked down-regulated expression of JAK2,STAT5,and phosphorylated STAT5 was detected in the combination therapy.Conclusions： Combining HDAC inhibitor SAHA with imatinib can kill CML cells synergistically by inhibiting cell growth and inducing apoptosis,which is associated with activation of Caspase pathway and regulation of anti-apoptotic proteins.