环孢菌素D衍生物PSC833逆转K562/DOX细胞的凋亡抗性及其机制

 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。

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

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 ΔΨm decreased. 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.