伯舒替尼诱导急性髓系白血病细胞分化和细胞凋亡

Bosutinib induced cell differentiation and apoptosis in acute myeloid leukemia cell lines

目的探讨伯舒替尼对人急性髓系白血病（AML）细胞株HL-60、THP-1和U937细胞分化和凋亡的影响及其作用机制。方法伯舒替尼0~20μmol/L处理HL-60、THP-1和U937细胞48 h,MTT法检测细胞增殖;伯舒替尼0~5μmol/L处理HL-60、THP-1和U937细胞72 h,流式细胞仪检测细胞CD11b表达;伯舒替尼0~10μmol/L处理HL-60、THP-1和U937细胞72 h,胱天蛋白酶（caspase）广谱抑制剂benzyloxy-carbonyl-Val-Ala-Asp-fluoromethyl-ketone（Z-VAD-FMK）预处理1 h后加入伯舒替尼处理U937细胞48 h,Annexin V-FITC/PI双染法检测细胞凋亡;不同浓度伯舒替尼（0~5μmol/L）分别处理HL-60和U937细胞24 h后,Western印迹法检测细胞分化相关转录因子C/EBPβ、P21和c-Myc以及凋亡相关分子Mcl-1、Bax和Caspase 3蛋白表达的改变。结果伯舒替尼剂量依赖性抑制AML细胞增殖。与空白对照组相比,不同浓度伯舒替尼处理AML细胞72 h后,细胞CD11b表达和细胞凋亡率均明显增加（P〈0.05或P〈0.01）。伯舒替尼处理HL-60细胞有效上调C/EBPβ和P21的蛋白表达水平,引起U937细胞Mcl-1表达降低和Bax表达增强,并激活caspase 3,而Z-VAD-FMK预处理U937细胞显著抑制伯舒替尼诱导的细胞凋亡。结论伯舒替尼可有效抑制AML细胞增殖,诱导细胞分化和促进其凋亡,可作为有效治疗AML的潜在药物。

Objective To explore the effect of bosutinib on cell differentiation and apoptosis of human acute myeloid leukemia（AML）cell lines including HL-60,THP-1,and U937,and to clarify the related mechanisms. Methods MTT Assay was used to assess the proliferation of HL-60,THP-1,and U937 cells treated with various concentrations of bosutinib（0-20 μmol/L）for 48 h.Cell surface marker CD11 b expression was detected by flow cytometry in HL-60,THP-1,and U937 cells treated with bosutinib（0-5μmol/L）for 72 h. Apoptosis was evaluated by Annexin V-FITC/PI double stainning in the following two sets of experiments：1 HL-60,THP-1 and U937 cells were treated with various concentrations of bosutinib（0-10 μmol/L）. 2 U937 cells were pretreated with caspases inhibitor benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone（Z-VAD-FMK）for 1 h and then exposed to bosutinib for 48 h.Western blot was used to examine the protein expression of differentiation related transcription factors C/EBPβ,P21,and c-Myc,and apoptosis related protein Mcl-1,Bax,and Caspase 3 in HL-60 and U937 cells treated with bosutinib for 24 h. Results Bosutinib dose- dependently inhibited AML cell growth. Treatment with various concentrations of bosutinib for 72 h significantly increased CD11 b expression and apoptotic rate in AML cells as compared to blank control（P〈0.05 or P〈0.01）. Bosutinib effectively upregulated protein expression levels of C/EBPβ and P21 in HL-60 cells,and induced down-regulation of Mcl-1 with up-regulation of Bax protein expression,and activated Caspase 3 in U937 ells. Pretreatment of U937 cells with Z-VAD-FMK significantly inhibited apoptosis induced by bosutinib. Conclusion Bosutinib effectively inhibits cell proliferation and induces cell differentiation with apoptosis in AML cells. It could be a potent therapeutic agent against AML.