P-糖蛋白线粒体转位与线粒体DNA缺失肿瘤细胞多药耐药产生的关系

P-glycoprotein Mitochondrial Translocation on Multidrug Resistance in the Mitochondrial DNA-depleted Cells

线粒体DNA缺失细胞(ρ~0细胞)拮抗化疗药物诱导的凋亡,但其确切机制尚不明确。本研究探讨P-gp线粒体转位与人肝癌细胞(SK-Hepl)mtDNA缺失细胞(ρ~0SK-Hep1)多药耐药产生的关系。以SK-Hep1、ρ~0SK-Hep1和转线粒体细胞SK-Hep1Cyb为研究对象,CCK-8方法检测细胞对药物敏感性;AnnexinV/PI双染法及DAPI染色法检测细胞凋亡;Westernblot检测P-gp表达;激光共聚焦显微镜结合免疫荧光检测P-gP细胞内分布。结果显示,SK-Hep1、ρ~0SK-Hep1和SK-Hep1Cyb细胞对多柔比星(DOX)的IC_(50)分别为0.62±0.02μg/ml、4.93±0.17μg/ml和0.57±0.02μg/ml。SK-Hep1、ρ~0SK-Hep1和SK-Hep1Cyb细胞凋亡率分别为1 1.25%±1.36%、4.75%±0.98%和14.50%±1.57%,ρ~0SK-Hep1对细胞凋亡有明显抗性。Western blot检测发现ρ~0细胞内P-gP、Bax、Bcl-2表达增加,Bcl-2/Bax比值增加。免疫荧光共定位显示,ρ~0细胞线粒体内P-gP表达增加。结果表明,ρ~0细胞对化疗药物诱导的凋亡有明显抵抗,这种现象可能与ρ~0细胞P-gP、Bax、Bcl-2表达增加有关。ρ~0细胞P-gP线粒体转位可发挥外排泵作用将药物排出线粒体,改变化疗药物的亚细胞分布,减少细胞内药物浓度,使细胞产生多药耐药。

Cells that express the multidrug resistance（MDR） phenotype are resistant to the mitochondrial related apoptosis induced by several anticancer drugs.Why the mitochondrial DNA depleted cells showed MDR phenotype is still unknown.This study focused on P-glycoprotein（P-gp） mitochondrial translocation on multidrug resistance in the mitochondrial DNA-depleted cells.SK-Hepl,p^0SK-Hep1 and the transmitochondria cells SK-Hep1Cyb were used.Sensitivity of the cells to chemotherapeutic drugs was assessed by CCK-8 assays.Apoptosis ratio of the cells were measured by Annexin V/PI double staining.Expression of P-gp was detected by Western blot,and distribution of P-gp within the cells was assayed by inununofuorescence combined with laser scanning confocal microscopy.Results showed,the IC_（50） of SK-Hepl,p^0SK-Hep1 and SK-Hep1Cyb to Doxorubicin were 0.62±0.02μg/ml,4.93±0.17μg/ml and 0.57±0.02μg/ml,respectively,and the apoptosis ratio of SK- Hep1, p^0SK-Hepl and SK-Hep1Cyb were 11.25%±1.36%、4.75%±0.98%and 14.50%±1.57%,respectively.The p^0SK-Hep1 exhibited resistance to apoptosis induced by the chemotherapeutic drugs.P-gp、Bax and Bcl-2 were overexpressed and Bcl-2/Bax increased.p^0 cells showed resistance to apoptosis induced by chemotherapeutic drugs,and this might be related to increased Bax/Bcl-2,and increased P-gp translocation to mitochondria in the p^0 cells.Therefore,P-gp could be involved in the protection of apoptosis due to antiproliferative drugs.