阿的平联合硫利达嗪对慢性粒细胞白血病细胞的诱导凋亡作用

Effects of quinacrine combined with thioridazine on inducing apoptosis of chronic myelogenous leukemia cells

目的探讨阿的平(QC)联合硫利达嗪(THZ)对慢性粒细胞白血病(CML)细胞(K562)的影响。方法以不同浓度QC(0、1、2、3和4μmol/L)和THZ(0、2、4、8和16μmol/L)处理K562细胞不同时间(0、24和48 h)后,锥虫蓝排斥实验检测两药单加对K562细胞活性的影响。以不同浓度的QC(2、3和4μmol/L)和THZ(2、4和8μmol/L)分别单加及合加处理K562细胞24 h,锥虫蓝排斥实验检测K562细胞活力,利用CompuSyn软件计算两药合加的协同指数。3μmol/L的QC与4μmol/L的THZ分别单加及合加处理K562细胞,用或不用caspase不可逆抑制剂Z-VAD-FMK(20μmol/L)处理,RH-123/PI双染后流式细胞术检测线粒体跨膜电位的变化,Western blotting检测凋亡相关蛋白的表达。结果锥虫蓝排斥实验结果显示:QC与THZ单加,低浓度时对K562细胞的抑制效应并不明显;两药合加对K562细胞有良好的协同杀伤效应。Western blotting检测结果显示:K562经两药合加处理后出现PARP-1的剪切及凋亡相关蛋白Bcl-2、Mcl-1表达的减少,且这种效应不能被Z-VAD-FMK逆转。流式细胞术检测结果表明:相对于两药单加,3μmol/L的QC与4μmol/L的THZ合加处理24 h后K562细胞线粒体膜电位明显降低,并且这种效应不能被Z-VADFMK逆转。结论 QC与THZ两药联合能协同诱导K562细胞凋亡,这种效应为非caspase依赖,线粒体跨膜电位下降可能参与了这一过程。

Objective To study the effects of Q uinacrine （QC） and Thioridazine （THZ） on chronic myelogenous leukemia （CML） cells （K562 cells）. Methods K562 ceils were treated by different concentrations of QC （0, 1, 2, 3, and 4 μmol/L） and THZ （0, 2, 4, 8, and 16 μmol/L） for 0, 24, and 48 h. The effects of each drug on the viability of cells were examined by the trypan blue exclusion assay. Then K562 cells were treated by different concentrations of QG （2, 3, and 4 larnol/L） and THZ （2, 4, and 8 μmol/L） for 24 h alone or in combination. The viability of cells was examined by the trypan blue exclusion assay. The combinational index was calculated by the CompuSyn software. Next K562 cells were treated by O C of 3 μmol/L and THZ of 4 μmol/L for 24 h alone or in combination, with or without being treated by pan- caspase inhibitor Z-VAD-FMK of 20 μmol/L. Variations of the mitochondrial transmembrane potential were detected by the Rh123/PI staining and flow cytometry. Apoptosis related proteins were detected by the Western blotting. Results Results of the trypan blue exclusion assay indicated that being treated by Q,G or THZ of low concentrations alone had no significant influence on the viability of K562 cells, while being treated by QC combined with THZ had a good synergistical effect on inducing death of K562 cells. Results of the Western blotting showed that the cleavage of PARP-1 and the reduction of expressions of apoptosis-related proteins Mcl-1and Bcl-2 were induced after being treated by two drugs in combination and could not be reversed by Z-VAD- FMK. Results of the flow cytometry showed that compared to being treated by one drug, the mitochondrial transmembrane potential of K562 cells treated by QC of 3 tamol/L and THZ of 4 μrnol/L in combination for 24 h was significandy decreased and could not be reversed by Z-VAD-FMK. Conclusion QG plus THZ can synergistically induce caspase-independent apoptosis of K562 cells. The decrease of mitochondrial transmembrane potential may involve in this process.