环孢菌素D衍生物PSC833逆转K562/DOX细胞的凋亡抗性及其机制

Reversal of apoptosis resistance of doxorubicin-resistant human myelogenous leukemia cell line K562 /DOX by a cyclosporin D analogue PSC833

目的:研究环孢菌素D衍生物PSC833对K562/DOX细胞多药耐药的逆转作用。方法:MTT法进行阿霉素(doxorubicin,DOX)和长春新碱(vincristine,VCR)的细胞毒测定;流式细胞术测定细胞周期;Annexin V/PI双染流式细胞术检测细胞凋亡;活性氧测定以DCFH-DA标记,线粒体跨膜电位测定用JC-1标记,细胞内钙测定用Fluo-3/AM标记,均以流式细胞术检测;Western blotting法测定细胞色素C(Cyt C)、Bcl-2、Bax和cleaved caspase-3蛋白表达。结果:环孢菌素D衍生物PSC833能显著增强DOX/VCR的细胞毒作用,使细胞阻滞在G2/M期;通过降低线粒体膜电位,升高细胞内活性氧和Ca2+水平,释放Cyt C、下调Bcl-2、上调Bax、激活cleaved caspase-3,增加DOX诱导的耐药细胞凋亡。结论:PSC833与DOX合用后,可阻滞细胞周期于G2/M期,通过线粒体通路诱导K562/DOX细胞凋亡。

AIM： To study the reversal effect of a cyclosporin D analogue PSC833 on multidrug resistance of doxorubicin-resistant human myelogenous leukemia（ K562 /DOX） cells. METHODS： The reversal effects of PSC833 on resistance to doxorubicin（ DOX） /vincristine（ VCR） in K562 /DOX cells were observed by MTT assay. The cell cycle analysis was performed by flow cytometry. Annexin V /PI staining was used to identify PSC833-induced apoptosis in K562 /DOX cells. These cells underwent incubation with DCFH-DA,JC-1 and Fluo-3 /AM followed by flow cytometry for the measurement of reactive oxygen species（ ROS）,mitochondrial membrane potential（ ΔΨm）,and intracellular calcium,respectively. The protein levels of cytochrome C（ Cyt C）,Bcl-2,Bax,and cleaved caspase-3 were detected by Western blotting. RESULTS： The DOX /VCR-induced cytotoxicity was significantly potentiated by PSC833. PSC833 arrested the cells in G2/M phase and increased the apoptosis induced by DOX in K562 /DOX cells. During the apoptosis,the level of ROS and intracellular calcium increased,while the level of ΔΨmdecreased. Furthermore,the release of Cyt C,activation of caspase-3,up-regulation of Bax and down-regulation of Bcl-2 were observed in K562 /DOX cells treated with PSC833 and DOX. CONCLUSION： The reversal effect of PSC833 on multidrug resistance in K562 /DOX cells is associated with the induction of apoptosis through a mitochondria-dependent pathway.