Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, Japan.
Biol Pharm Bull. 2010;33(3):504-11. doi: 10.1248/bpb.33.504.
The aim of this study was to reveal the expression and function of P-glycoprotein and multidrug resistance-associated proteins (MRP), members of the ATP-binding cassette (ABC) superfamily of drug transporters, in cultured human Y79 retinoblastoma cells. ABC transporter mRNA expression was evaluated by conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses. Cellular accumulation of rhodamine 123 (P-glycoprotein substrate), calcein (MRP substrate), and doxorubicin (P-glycoprotein/MRP substrate) was analyzed by fluorometry. Conventional RT-PCR analysis showed the expression of multidrug resistance 1 (MDR1), MRP1, MRP2 and lung resistance-related protein (LRP) mRNAs. Real-time RT-PCR analysis revealed that the expression levels of the MDR1 and MRP2 genes in Y79 cells were much lower than those in human intestinal cell line Caco-2, while the expression level of MRP1 was higher than that in Caco-2 cells. The accumulation of rhodamine 123 was not enhanced by verapamil or reversin 205, inhibitors of P-glycoprotein, indicating no function of P-glycoprotein in Y79 cells. The accumulation of calcein was significantly increased by various MRP inhibitors including probenecid, indicating that MRP functions in Y79 cells. The accumulation of doxorubicin was increased in the presence of metabolic inhibitors (10 mM 2-deoxyglucose and 5 mM sodium azide). However, most MRP inhibitors such as probenecid and indomethacin did not affect doxorubicin accumulation, while cyclosporin A and taclorimus significantly increased doxorubicin accumulation. These results suggest that MRP, but not P-glycoprotein, functions in Y79 cells, and that the efflux of doxorubicin from Y79 cells may be due to an ATP-dependent transporter, which has not been identified yet.
本研究旨在揭示 ABC 超家族药物转运体成员 P-糖蛋白和多药耐药相关蛋白（MRP）在培养的人 Y79 视网膜母细胞瘤细胞中的表达和功能。采用常规逆转录-聚合酶链反应（RT-PCR）和实时 PCR 分析评估 ABC 转运体 mRNA 的表达。通过荧光法分析罗丹明 123（P-糖蛋白底物）、钙黄绿素（MRP 底物）和阿霉素（P-糖蛋白/MRP 底物）在细胞内的积累。常规 RT-PCR 分析显示多药耐药 1（MDR1）、MRP1、MRP2 和肺耐药相关蛋白（LRP）mRNA 的表达。实时 RT-PCR 分析显示，Y79 细胞中 MDR1 和 MRP2 基因的表达水平明显低于人肠细胞系 Caco-2，而 MRP1 基因的表达水平高于 Caco-2 细胞。维拉帕米或逆转素 205（P-糖蛋白抑制剂）不能增强罗丹明 123 的积累，表明 Y79 细胞中 P-糖蛋白无功能。钙黄绿素的积累被各种 MRP 抑制剂显著增加，包括丙磺舒、吲哚美辛，表明 MRP 在 Y79 细胞中起作用。在代谢抑制剂（10mM 2-脱氧葡萄糖和 5mM 叠氮化钠）存在下，阿霉素的积累增加。然而，大多数 MRP 抑制剂，如丙磺舒和吲哚美辛，不影响阿霉素的积累，而环孢素 A 和他克莫司显著增加阿霉素的积累。这些结果表明，MRP 而非 P-糖蛋白在 Y79 细胞中起作用，阿霉素从 Y79 细胞中的外排可能是由于一种尚未确定的 ATP 依赖性转运体。
