柔红霉素在耐药细胞株K562/ADR内的异常分布

摘要

Objective　To examine subcellular distribution of daunorubicin (DNR) in P-glycoprotein-mediated multidrug-resistant cell line K562/ADR and its relation to multidrug resistance.Methods　The subcellular distribution of DNR in K562/ADR was studied by confocal scanning laser microscopy (CSLM), fluorometry, RT-PCR. Rhodamine 123, NBD-ceramide and neutral red as fluorescent probes to stain the mitochondria, Golgi apparatus and lysosomes respectively were used to identify the subcellular compartments wherein DNR was sequestered. Effect of verapamil, chloroquine and brefeldin A on DNR distribution and accumulation was examined. Results　Compared with the drug-sensitive cell K562/S in which DNR fluorescence diffusely appeared in the nucleus and cytoplasm, DNR in K562/ADR cells was distributed to the perinuclear region and peripheral cytoplasm, It was scenty in the nucleus and other cytoplasmic regions, as suggested by the distribution of Rhodamine123. Only verapamil, but not chloroquine and brefeldin A, could markedly restore diffuse cytoplasmic and nuclear fluorescence distribution in the resistant cell line. Conclusion　Altered subcellular distribution of DNR in durg resistant cell line may participate in the generation of multidrug resistance in which P-glycoprotein plays an important role.%目的了解化疗药物在糖蛋白(Pgp)耐药细胞株K562/ADR亚细胞结构的异常分布及其与耐药的关系。方法采用共聚焦激光扫描显微镜、荧光法和RT-PCR等方法, 研究柔红霉素(DNR)在K562/ADR细胞亚细胞结构的分布及其与耐药的关系，以及维拉帕米尔(verapamil)、布雷菲尔得菌素(brefeldin A)、氯喹对细胞内DNR异常分布的影响。将3种特异染色线粒体、高尔基体、溶酶体的荧光物质Rh123、NBD-ceramide、中性红作为探针，鉴定分隔储留DNR的细胞器。结果在K562/ADR细胞中，DNR荧光主要集中在核周和周边胞浆，核内及胞浆其他部位荧光很少。这种分布方式与Rh123荧光在该细胞的分布极其相似，与DNR在敏感细胞K562/S细胞核、浆均匀分布不同。verapamil可逆转DNR在K562/ADR的异常分布，而氯喹、brefeldin A无此作用。结论 DNR在耐药细胞的异常分布参与了肿瘤细胞耐药的形成，Pgp在此过程中发挥了重要作用。