2-甲氧基雌二醇诱导K562细胞凋亡及机制研究

本研究探讨2-甲氧基雌二醇（2-ME2）对K562细胞的诱导凋亡作用及其机制。采用不同浓度2-ME2处理K562细胞，MTT法检测K562细胞的增殖活性，DNA琼脂糖凝胶电泳和Annexinv／PI双染色法检测K562细胞的凋亡效应，流式细胞术检测细胞线粒体膜电位的变化，RT-PCR和Western blot方法分别检测相关基因mRNA和／或蛋白的表达变化。结果表明：2-ME2抑制K562细胞增殖呈时间和剂量依赖性；48小时的半数抑制浓度（IC。）为2μmol／L；2μmol／L2-ME2作用于K562细胞24、48、72小时，DNA凝胶电泳分析可见典型DNA梯形条带；Annexinv／PI双染色法检测显示，细胞凋亡率分别为13．78％、22．32％和29．43％，而对照组凋亡率仅为1．78％（P〈0．05）；1、2和4μmol／L2-ME2分别处理K562细胞24小时，流式细胞术检测显示细胞线粒体膜电位下降；2μmol／L2-ME2处理K562细胞24、48和72小时，K562细胞中bcr／abl、bcl-2mRNA表达下调，而baxmRNA表达上调，Bcl-2、procaspase-3、procaspase-9、PARP（116 kD）和p-Akt蛋白表达下调，胞浆Cyto-C和PARP活性片段（85kD）表达上调，而对总Akt蛋白表达无影响。结论：2-ME2通过升高bax／bcl-2比值降低线粒体膜电位、促使细胞色素C（Cyto-c）释放入胞浆、触发K562细胞的线粒体凋亡途径，活化caspase-3，导致K562细胞凋亡并抑制其增殖；通过下调bcr／ab1 mRNA表达以阻断P13K／Akt信号通路也参与了该过程。

Mechanism Underlying 2-methoxyestradiol Inducing Apoptosis of K562 cells

The aim of this study was to investigate apoptotic effect of 2-methoxyestradiol （2-ME2） on K562 cells and its mechanism. K562 cells were treated with different concentrations of 2-ME2. MTT assay was used to examine the effect inducing growth inhibition. DNA fragmentation assay and Annexin V-FITC/PI staining were used to detect the effect of apoptosis. The change of mitochondrial transmembrane potential was analyzed by flow cytometry. The expres- sions of related gene mRNA and/or proteins were detected by RT-PCR and Western blot respectively. The results indica- ted that the 2-ME2 inhibited proliferation of K562 cells in a time- and dose-dependent manners and the concentration of 50% growth inhibition （ IC50 ） was 2μmol/L at 48 hours. 2-ME2 induced DNA ladder and significantly increased apoptosis in K562 cells when exposed to 2 μmol/L of 2-ME2 for 24,48 and 72 hours, the result of Annexin-V/PI staining showed that rates of the apoptotic cells were 13.78% ,22.32% and 29.43% respectively, which was remarkably higher than that of control （ 1.78% ） （p 〈 0.05 ）. The FCM analysis showed that the mitochondrial transmembrane potential in K562 cells lowered after exposed to 1,2 and 4 tzmol/L of 2-ME2 for 24 hours. 2-ME2 down-regulated the expression of bcr/abl and bcl-2, up-regulated the expression of bax mRNA, and down-regulated protein expressions of bcl-2, pro- caspase-3, procaspase-9, PARP（ 116 kD） and p-Akt, and up-regulated expression of cytoplasmic Cyto-C and PARP 85 kD apoptosis-related cleavage fragment protein, but had no effect on total Akt protein in K562 cells after treated with 2μmol/L of 2-ME2 for 24,48 and 72 hours. It is concluded that the 2-ME2 can induce the apoptosis and inhibit the prolif- eration of K562 cells by increasing the ratio of bax/bcl-2, reducing the mitochondrial transmembrane potential, releasing cytochrome C to cytoplasm, initiating the mitochondrial apoptosis pathway and leading in turn to caspase-3 activation. These findings suggest that interfere PI3K/Akt signal pathway via down-regulating the expression of bcr/abl mRNA is implicated in the effect of 2-ME2 on K562 cells.