脱氧佛波醇乙酸酯联合阿糖胞苷对急性髓系白血病细胞分化的影响

Effect of 12-deoxyphorbol-13-acetate in combination with cytarabine on cell differentiation in acute myeloid leukemia cell lines

目的研究脱氧佛波醇乙酸酯(12-deoxyphorbol 13-acetate,DPA)联合化疗药阿糖胞苷(cytarabine,Ara-C)对人急性髓系白血病(AML)细胞株HL-60、NB4和U937细胞分化的影响,并初步探讨其相关作用机制。方法用DPA 250 nmol/l和Ara-C 100 nmol/L单独及联合处理HL-60细胞72 h,姬姆萨染色观察细胞形态学改变;不同剂量DPA和Ara-C单独及联合处理HL-60、NB4和U937细胞,将蛋白激酶C抑制剂bisindolymaleimide I(GFX)和丝裂原活化蛋白激酶激酶抑制剂U0126分别预先加入DPA和Ara-C联合处理的HL-60细胞,流式细胞仪检测细胞CD11b表达;DPA和Ara-C单独及联合处理HL-60细胞24 h后,Western印迹法检测C/EBPα、C/EBPβ和c-Myc蛋白表达的改变。结果 DPA 250 nmol/L与Ara-C 100 nmol/L联合处理HL-60细胞,可引起细胞体积增大、细胞浆增多及细胞核/浆比例减小等细胞分化的典型形态变化。流式检测结果表明,与Ara-C单独给药组相比,DPA和Ara-C联合处理HL-60细胞72 h后,细胞CD11b表达明显增加(P<0.01);DPA 250nmol/L联合Ara-C 50 nmol/L处理NB4和U937细胞48 h后,细胞CD11b表达明显增强(P<0.01)。GFX或U0126分别预处理HL-60细胞能够完全阻断DPA联合Ara-C诱导的细胞分化。与空白对照组相比,DPA与Ara-C共处理明显降低HL-60细胞的c-Myc蛋白表达水平(P<0.01)。结论 DPA可增强Ara-C诱导的AML细胞分化,其作用机制可能与PKC/ERK通路激活和c-Myc表达降低相关。

Objective To study the effects of the combination treatment of 12-deoxyphorbol 13-acetate（DPA） and chemotherapy agent cytarabine（Ara-C） on differentiation in human acute myeloid leukemia（AML） cell lines HL-60,NB4 and U937,and to explore the related mechanisms.Methods HL-60 cells were treated for 72 h with DPA（250 nmol / L） or Ara-C（100 nmol / L） alone or in combination,the morphological changes were then determined by Giemsa staining.Cell surface marker CD11b expression was detected by flow cytometry in the following two sets of experiments： 1.HL-60,NB4 or U937 cells were treated with various concentrations of DPA or Ara-C alone or in combination.2.HL-60 cells were pretreated with protein kinase C inhibitor bisindolymaleimide Ⅰ（GFX） or mitogen-activated protein kinase kinase inhibitor U0126 and then exposed to DPA combined with Ara-C.Western blot analysis was used to examine the protein expression of C / EBPα,C / EBPβ,and c-Myc in HL60 cells which were treated for 24 h with DPA or Ara-C alone or in combination.Results Combined treatment using 250 nmol / L DPA and 100 nmol / L Ara-C resulted in the typical appearance of differentiated cells with morphologic features such as larger cell size,more cytoplasm and decreased nucleus-cytoplasm ratio.As shown by flow cytometry analysis,combination treatment with DPA and Ara-C for 72 h significantly increased CD11b expression（P 0.01） in HL-60 cells as compared to treatment with Ara-C alone.Coadministration of 250 nmol/L DPA and 50 nmol/L Ara-C for 48 h resulted in a significant increase in CD11b expression（P 0.01） in NB4 and U937 cells.Pretreatment of HL-60 cells with GFX or U0126 completely blocked HL-60 cell differentiation induced by DPA in combination with Ara-C.The protein expression of c-Myc in HL-60 cells was significantly downregulated by the combined treatment of DDA and Ara-C as compared with blank control（P 0.01）.Conclusion DPA could enhance Ara-C-induced differentiation of AML cells,and its mechanisms may be related to the activation of PKC / ERK pathway and the downregulated expression of c-Myc.