Wang Wei, Ke Shunan, Chen Gang, Gao Qinglei, Wu Sufang, Wang Shixuan, Zhou Jianfeng, Yang Xiaokui, Lu Yunping, Ma Ding
Department of Obstetrics & Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China.
Oncol Rep. 2004 Dec;12(6):1365-70.
The mechanisms of drug-resistance in human ovarian cancer cells have not been entirely clarified. The purpose of this study was to investigate whether LRP is involved in the resistance of ovarian cancer cell lines to cisplatin and its molecular mechanism. Human ovarian cisplatin-resistant cancer cell lines (A2780/DDP and COC1/DDP) and their parental cisplatin-sensitive cell lines (A2780 and COC1), alone or transfected with antisense LRP-specific oligonucleotides (ODN) or sense ODN, were treated with cisplatin to induce differentiation. Expression of LRP was examined by RT-PCR and Western blot analysis. The sensitivities of cells to cisplatin were assessed using sulforhodamine B (SRB) assay and flow cytometry, and the accumulation and efflux of cisplatin in the cells and isolated nuclei were examined by high performance liquid chromatographic (HPLC) assay. The expressions of LRP in A2780/DDP and COC1/DDP cells were higher than those in A2780 and COC1 cells and conferred resistance to cisplatin. Transfection of LRP AsODN into A2780/DDP and COC1/DDP cells down-regulated LRP expression and reversed the resistance phenotype. Levels of cisplatin accumulating in cells were increased by LRP-specific AsODN and anti-LRP monoclonal antibody. Isolated nuclei from A2780 and COC1 cells or A2780/DDP and COC1/DDP cells incubated with anti-LRP antibody contained more cisplatin than the nuclei of A2780/DDP and COC1/DDP cells not treated with anti-LRP antibody. Efflux of cisplatin was greater from the nuclei of A2780/DDP and COC1/DDP cells than those of A2780 and COC1 cells, and was inhibited by anti-LRP monoclonal antibody. Thus, LRP was involved in the resistance of ovarian cancer cells to cisplatin and has an important role in the transport of cisplatin both in exocytotic vesicles and between the nucleus and cytoplasm.
人类卵巢癌细胞中的耐药机制尚未完全阐明。本研究的目的是探讨肺癌耐药相关蛋白（LRP）是否参与卵巢癌细胞系对顺铂的耐药及其分子机制。将人卵巢癌顺铂耐药细胞系（A2780/DDP和COC1/DDP）及其亲代顺铂敏感细胞系（A2780和COC1）单独培养，或转染反义LRP特异性寡核苷酸（ODN）或正义ODN后，用顺铂处理以诱导分化。通过逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析检测LRP的表达。使用磺酰罗丹明B（SRB）法和流式细胞术评估细胞对顺铂的敏感性，并通过高效液相色谱（HPLC）法检测顺铂在细胞和分离细胞核中的蓄积及流出情况。A2780/DDP和COC1/DDP细胞中LRP的表达高于A2780和COC1细胞，且赋予了对顺铂的耐药性。将LRP反义寡脱氧核苷酸（AsODN）转染到A2780/DDP和COC1/DDP细胞中可下调LRP表达并逆转耐药表型。LRP特异性AsODN和抗LRP单克隆抗体可增加细胞中顺铂的蓄积水平。与未用抗LRP抗体处理的A2780/DDP和COC1/DDP细胞核相比，用抗LRP抗体孵育的A2780和COC1细胞或A2780/DDP和COC1/DDP细胞分离出的细胞核含有更多顺铂。A2780/DDP和COC1/DDP细胞核中顺铂的流出量大于A2780和COC1细胞，且被抗LRP单克隆抗体抑制。因此，LRP参与了卵巢癌细胞对顺铂的耐药过程，并且在顺铂的胞吐小泡运输以及细胞核与细胞质之间的运输中起重要作用。
