三氧化二砷诱导K562／ADM细胞凋亡过程中对bcl-2、survivin、ROS表达的影响

Impact of As_2O_3 on Bcl-2, Survivin and Reactive Oxygen Species (ROS) Expressions during the Process of Induced Apoptosis in Multidrug-Resistant Human Leukemia K562/ADM Cells

[目的]探讨As2O3诱导白血病K562/ADM耐药细胞凋亡的分子机制。[方法]采用MTT比色法检测K562/ADM增殖活性;细胞形态学和AnnexinⅤ/PI双染色检测细胞凋亡;RT-PCR检测bcl-2、survivin和caspase-3基因mRNA的表达水平;流式细胞法(FCM)测定bcl-2、caspase-3蛋白表达。激光共聚焦显微镜(LCSM)观察细胞内活性氧(ROS)产生情况。[结果]As2O3显著抑制K562/ADM细胞的增殖;经As2O3处理后细胞形态上出现典型的凋亡改变,AnnexinⅤ/PI双染显示凋亡细胞明显增加;凋亡抑制基因bcl-2、survivinmRNA及bcl-2蛋白表达下调,caspase-3 mRNA表达和caspase-3活性显著增强,ROS活性下降。[结论]As2O3诱导K562/ADM耐药细胞凋亡,与bcl-2和survivin表达下调,caspase-3活化有关,而与活性氧的氧化损伤无关。

[Purpose] To explore the apoptosis-indueing molecular mechanism of As203 on muhidrug-resistant leukemia K562/ADM cells. [Methods] The cell proliferating activity of K562/ADM cells was assessed with MTT assay. The cell apoptosis was determined by optical microscopic morphology and Annexin Ⅴ/PI double-staining. The expression of bel-2, survivin and caspase-3 mRNA were examined with reverse transcription polymerase chain reaction （RT-PCR）. Expression of bel-2 protein and caspase-3 activity were measured with flow cytomery （FCM）. Reactive oxygen species （ROS） was labeled by DCFH-DA and examined by LCSM. [Results] As2O3 significantly inhibited K562/ADM cells growth. After As2O3 treatment, the typical apoptotic morphological changes were obseawed in K562/ADM cells, and the apoptosis rate of the cells by AnnexinⅤ/Pl staining was obviously increased. The decreased expression of bel-2 and survivin gene mRNA and bcl-2 protein were shown in the As203 adminated K562/ADM cells. Further, As2O3 treatment could enhance easpase-3 gene mRNA expression and easpase-3 activity, and lower level of ROS. [Conclusionl Apoptosis induced by As2O3 in multidrug-resistant K562/ADM cells relates to downregulatlon of bel-2 and survivin expressions and activation of caspase-3, whereas ROS do not take part in the apoptosis mechanism.