高三尖杉酯碱对人急性髓系白血病干细胞的杀伤作用

Cytotoxicity of homoharringtonine on leukemic stem-like cells in AML cell line KG-1

目的:观察高三尖杉酯碱(HHT)对人急性髓系白血病(AML)干细胞的杀伤作用,探讨其分子学机制。方法:用流式细胞仪(FACS)分析AML细胞株白细胞干细胞(LSC)的表型特征,用MTT法检测HHT对具有LSC特征的KG-1细胞的生长抑制作用,用FACS观察HHT对CD34+CD38-CD96+的KG-1细胞数量的影响,Western blot法检测药物处理后Caspase途径和凋亡相关蛋白的改变。结果:KG-1和Kasumi-1细胞CD34+CD38-CD96+分别为69%和26.7%,U937细胞不表达CD96。HHT能显著抑制KG-1细胞的生长,并呈剂量依赖性(r2=0.9971,P<0.05)。48 h的半数抑制浓度为16.9 ng/ml。HHT作用KG-1细胞后,CD34+CD38-CD96+细胞的比例从63.6%下降到17.1%。HHT能显著激活Caspase-3、Caspase-9和PARP,抑制磷酸化Akt和Bcl-2的表达。结论:HHT能显著抑制人AML细胞KG-1的增殖,减少CD34+CD38-CD96+白血病干细胞数量;对Akt活性和Bcl-2的调控可能是其重要的作用机制。

Objective： To investigate the effect of homoharringtonine（HHT） on leukemic stem-like cells（LSC） in human acute myeloid leukemia（AML） cell lines.Methods： The phenotypes of AML cell lines U937,Kasumi-1,and KG-1 cells were analyzed by flow cytometry（FACS）.The effect of HHT on leukemia stem-like cells with immunophenotype of CD34＋CD38-CD96＋ was detected with FACS.Cell growth was measured by MTT assay.Activation of Caspase pathway and expression of apoptotsis-related regulator proteins were examined by Western blotting.Results： FACS demonstrated that the 69% of KG-1 cells expressed LSC phenotype CD34＋CD38-CD96＋,while 26.7% on Kasumi-1 cells expressed this marker.In contrast,U937 cells showed CD96 negative.HHT significantly inhibited cell growth of KG-1 cells with an IC50 of 16.9 ng/ml at 48 h.The ratio of CD34＋CD38-CD96＋ cells decreased from 63.6% to 17.1% after HHT treatment.Enhanced apoptosis was demonstrated in HHT group evidenced by strong activation of Caspase-9,Caspase-3 and PARP.HHT treatment resulted in down-regulation of expression of anti-apoptotic protein BCL-2 and phosphated-Akt.Conclusion： HHT can effectively kill the leukemic stem-like cells in human AML cell line KG1 by inhibiting cell growth and inducing apoptosis which is associated with activation of Caspase pathway and down-regulation of anti-apoptotic proteins and phosphated-Akt.