甲异靛对K562细胞bcr-abl信号通路的影响以及诱导细胞凋亡机制研究

目的 探讨甲异靛对K562细胞bcr-abl信号通路的影响以及甲异靛诱导K562细胞凋亡的机制.方法 应用流式细胞术检测甲异靛诱导K562细胞凋亡的情况以及线粒体膜电位稳定性.应用Western blot技术检测bcr-abl信号通路相关蛋白、caspase以及Bcl-2家族成员在甲异靛作用前后的表达情况.RT-PCR技术检测甲异靛作用前后bcr-abl mRNA表达水平,用电泳移动迁移速度分析检测STAT3、STATS的DNA结合能力.结果 20 μmol/L甲异靛町降低ber-abl融合蛋白的表达及其磷酸化水平,但并不改变其mRNA表达水平,甲异靛可降低STATS以及CRKL的磷酸化水平,抑制STAT3、STATS的DNA结合能力.5-20μmoL/L甲异靛可诱导K562细胞凋亡并且具有浓度依赖性.甲异靛诱导K562细胞凋亡中伴随caspase 3、8、9活化以及细胞线粒体膜电位降低,caspase 3、8、9特异抑制剂z-DEVD-fmk、z-IETD-cho和z-LETD-fmk可阻断甲异靛诱导的K562细胞凋亡.甲异靛诱导K562细胞凋亡前后不伴有Bcl-2、Bax、Bid蛋白表达水平的改变.结论 甲异靛通过降低bcr-abl融合蛋白的表达并抑制bcr-abl信号通路中相关蛋白活性从而抑制K562细胞的增殖.甲异靛诱导K562细胞凋亡依赖于caspase活化以及线粒体途径,但Bcl-2家族成员不参与凋亡的调控.

Effect on bcr-abl signaling pathway and the mechanisms of apoptosis induction by meisoindigo in K562 cells

Objective To investigate the effect of meisoindigo on bcr-abl signaling pathway and to explore the mechanism of meisoindigo inducing apoptosis in K562 cells.Methods Apoptosis and mitochon dria membrane potential (MMP) were evaluated by flow cytometry.In K562 cells,the expression level of Bcl-2 family members,cleaved caspase members,bcr-abt,STATS and CRKL were determined by Western blot and bcr-abl mRNA expression level was measured by RT-PCR before and after meisoindigo treatment.The DNA binding potential of STAT3 and STAT5 was checked by electronic mobility shift assay (EMSA).Resuits Down-regulation of total and phosphorylated bcr-abl protein level in K562 cells was observed when treated with 20μmol/L meisoindigo,but its mRNA level was not changed.The expression level of phosphorylated STAT5 and CRKL was decreased and the DNA binding potential of STAT3 and STAT5 were inhibited in K562 cell after exposure to meisoindigo.Exposure to 5-20μmol/L meisoindigo induced apoptosis accompanied with activating of caspase 3,8,9 and decreasing of MMP in K562 cells in a dose-dependent manner.The apoptosis was blocked by 50 μmol/L z-DEVD-fmk,z-IETD-cho,z-LETD-fmk,the specific inhibitors of caspase3,8,9,respectively.No change in Bcl-2,Bax and Bid protein expression levels were observed before and after meisoindigo inducing apoptosis.Conclusion Meisoindigo can inhibit the proliferation of K562 cells by affecting the bcr-abl signaling transduction pathway.Meisoindigo induces K562 cell apoptosis through a novel caspase dependent pathway in addition to the contribution of mitochondria.The Bcl-2 family members are not involved in the apoptosis induction by meisoindigo in K562 cells.