vibsanin A联用酪氨酸激酶抑制剂通过降低c-Myc表达诱导HL-60细胞分化

Vibsanin A combined with tyrosine kinase inhibitors induces HL-60 cell differentiation by down-regulating c-Myc expression

目的探究天然小分子化合物vibsanin A联用酪氨酸激酶抑制剂(TKI)诱导急性髓系白血病(AML)细胞HL-60分化的分子机制。方法 vibsanin A联用酪氨酸激酶抑制剂伊马替尼(imatinib)或塞卡替尼(saracatinib)处理HL-60细胞72 h后,流式细胞术检测细胞CD11b表达,Wright-Giemsa染色观察细胞形态变化;联合用药处理HL-60细胞不同时间(0~24 h)后,采用q RT-PCR和Western印迹法检测细胞分化相关转录因子C/EBPα、C/EBPβ和c-Myc表达变化;构建c-Myc表达重组慢病毒载体质粒,转染HL-60细胞,建立c-Myc异位过表达细胞株,并用于分析c-Myc表达对vibsanin A联用酪氨酸激酶抑制剂诱导HL-60细胞分化的影响。结果 vibsanin A分别联用伊马替尼或塞卡替尼均可明显增强HL-60细胞分化,并显著下调c-Myc在m RNA和蛋白水平的表达(P<0.01);在c-Myc异位过表达HL-60细胞实验中,c-Myc异位过表达可显著对抗联合用药对c-Myc表达的下调作用,并明显抑制联合用药诱导的HL-60细胞分化。结论 Vibsanin A联用伊马替尼或塞替尼可能通过下调c-Myc表达水平诱导HL-60细胞分化。

Objective To explore the molecular mechanisms underlying the acute myeloid leukemia(AML)HL-60 cell differentiation induced by natural compound vibsanin A combined with tyrosine kinase inhibitors(TKI).Methods Cell surface marker CD11b expression was detected by flow cytometry in HL-60 cells after treatment of the cells with vibsanin A in combination with imatinib or saracatinib for 72 h,and the cell morphology was examined by Wrigh-Giemsa staining.qRT-PCR and Western blot were used to examine the expression of differentiation-related C/EBPα,C/EBPβ,and c-Myc at both mRNA and protein levels in HL-60 cells after the cells were treated with the two drug combinations for various time(0-24 h).The recombinant lentiviral vector expressing c-Myc was constructed and used to transfect HL-60 cells in which the c-Myc cDNA was ectopically over expressed.The effect of c-Myc expression on the HL-60 cell differentiation induced by vibsanin A combined with TKI was investigated using the transfected HL-60 cells.Results Vibsanin A combined with imatinib or saracatinib significantly enhanced HL-60 cell differentiation.Both drug combinations downregulated the expression of c-Myc at both mRNA and protein levels(P<0.01)in the HL-60 cells.In the transfected HL-60 cells,the ectopic c-Myc overexpression could significantly counteract the down-regulated c-Myc expression and inhibit the cell differentiation induced by the vibsanin A/TKI combination.Conclusion The combination of vibsanin A with imatinib or saracatinib could induce the HL-60 cell differentiation,probably via the downregulation of c-Myc expression.