三氧化二砷诱导K562/ADM耐药细胞凋亡中活性氧的作用

Reactive oxygen species play an important role in arsenic trioxide-induced apoptosis of multi-drug resistant K562/ADM cells

目的:探讨三氧化二砷(As2O3)诱导K562/ADM细胞凋亡中活性氧(ROS)的作用和其与耐药基因mdr1及其编码的P-糖蛋白(P-gp)表达的关系。方法:应用噻唑蓝(MTT)比色法检测K562/ADM细胞增殖活性;AnnexinⅤ/PI染色法检测细胞凋亡;RT-PCR检测mdr1基因mRNA的表达;流式细胞术测定P-gp表达、ROS活性和细胞内阿霉素(ADM)含量。结果:As2O3显著抑制K562/ADM细胞的增殖,AnnexinⅤ/PI双染检测显示凋亡细胞明显增加;ROS活性明显下降;mdr1mRNA和P-gp表达明显降低,P-gp功能受抑,细胞内ADM含量显著增高。结论:As2O3抑制K562/ADM耐药细胞的增殖活性和促进其凋亡,其机制可能为通过降低细胞ROS水平、抑制mdr1/P-gp的表达和功能,进而逆转mdr1/P-gp介导的多药耐药和凋亡抑制。

AIM： To discuss the role of the reactive oxygen species （ROS） and relationship between ROS and expression of mdr1/P-gp during apoptosis induced by arsenic trioxide （As2O3） in multidmg-resistant human leukemia K562/ADM cells. METHODS： The cell proliferaling activity was assessed with MTT assay. The cell apoptosis was determined by annexin V/PI staining. ROS was labelled by DCFH-DA and examined by flow cytometry. Expression of mdr1 mRNA and P-gp were detected by RT-PCR and flow cytometry, respectively. The contents of adriamycin （ADM） were detected by flow cytometry. PdESULTS： As2O3 inhibited K562/ADM cells growth effectively, and the apoptosis rate of the cells by Annexin V/PI staining was obviously increased. During apoptosis induced by 2 to 5 μmol·L^-1 As2 O3, the level of ROS was markedly decreased; the expression of mdr1 mRNA and P-gp were significantly down-regulated, and the function of P-gp was restrained so that the content of ADM increased in the cells. CONCLUSION： As2 O3 inhibits the proliferation activity and reverses phenomena of P-gp-mediated multidrug-resistance and apoptosis resistance in drug-resistant K562/ADM cells. The possible mechanism is down-regulation of mdr1/P-gp expression via declines of ROS activity.