硫利达嗪增强伊马替尼对慢性粒细胞白血病细胞 K562的抑制作用及机制

摘要

Objective To study the effects of thioridazine (THZ)and imatinib (IM)on chronic myeloid leukemia (CML)cells (K562 cells).Methods The K562 cells were treated by different concentrations of IM(0,0.1,1, 10 and 100 μmol /L)and THZ (0,2,4,8 and 16 μmol /L)for 24 and 48 h.The effects of each drug on the inhi-bition of cells were examined by the MTT assay.Then K562 cells were treated by different concentrations of IM(2, 4,8 and 16 μmol /L)and THZ (0.1,1 and 10 μmol /L)for 24 h alone or in combination.The inhibition of cells was examined by the MTT assay.The combinational index (CI)was calculated by the CompuSyn software.Next K562 cells were treated by IMof 1 μmol /L and THZ of 2 μmol /L for 24 h alone or in combination.Apoptosis was detected by the Annexin V /PI staining and flow cytometry.Apoptosis related proteins,pPI3K and pAKT were de-tected by the Western blotting.Results Results of the MTT assay indicated that being treated by THZ of low con-centrations alone had no significant influence on the inhibition of K562 cells,while being treated by THZ of 16μmol /L showed significant effect on inducing death of K562 cells.IMalone had effect on inducing death of K562 cells of a low concentration.K562 cells being treated by IMcombined with THZ had a good synergistical effect on inducing death.Then K562 cells were treated with selected drug concentration according to CI values for 24 h,it revealed that thioridazine group had no significant apoptosis effect by flow cytometry,while in imatinib group,two-drug combination group there had different degree of apoptosis effect,and the control group was statistically signifi-cant (P <0.01).Meanwhile,results of the Western blot showed that the increased expression of Cleaved Bid in each experimental group cells,while decreased expression of Procaspase 3.Up-regulation of anti apoptosis protein (Bcl-2,pPI3K and pAKT)expression and downregulation of apoptosis protein Bax expression demonstrated that the difference was statistically significant (P <0.01)comparing each experimental groups with the control group. Conclusion IM plus THZ can synergistically induce caspase-independent apoptosis of K562 cells.The killing mechanism may be associated with the mitochondrial pathway and inhibition of the PI3K-AKT pathway.%目的：观察硫利达嗪（THZ）、伊马替尼（IM）单药及联合对人慢性粒细胞白血病（CML）细胞 K562的诱导作用，并探讨其机制。方法采用四甲基偶氮唑蓝（MTT）法测定THZ、IM单药及联合对细胞的抑制作用，计算两药协同指数（CI）。检测 THZ、IM单药及联合对细胞凋亡的影响。West-ern blot 法检测凋亡蛋白 Cleaved-Bid、Procaspase 3，凋亡调节蛋白 Bcl-2、Bax、pPI3K、pAKT 表达的变化。结果 THZ 16μmol／L 作用24 h 后 K562细胞增殖明显抑制，低剂量细胞毒性作用不明显。IM低剂量即有较强的细胞毒性作用，两药联合后经计算有较好的协同作用。根据 CI 值选定药物浓度作用 K562细胞24 h，流式细胞术结果显示，THZ 单药组无明显细胞凋亡，IM单药组、IM联合 THZ 组均存在不同程度的细胞凋亡，与对照组比较差异有统计学意义（P ＜0．01）。Western blot 法结果显示各实验组细胞胞内 Cleaved-Bid 表达量增加，Procaspase 3表达量下降。抗凋亡蛋白 Bcl-2、pPI3K、pAKT 下调、促凋亡蛋白 Bax 表达明显上调，各实验组与对照组比较，差异有统计学意义（P ＜0．01）。结论低剂量 THZ 联合 IM对 CML 细胞 K562具有显著的增殖抑制作用，其作用较 IM单用作用强，THZ 有明确增敏 IM的作用。与 IM、THZ 单药组比较，IM联合 THZ 组 Cleaved Bid 表达量上调、Procaspase 3表达量下降，其杀伤机制可能与线粒体通路及抑制 PI3K-AKT 通路均有关。