Hazlehurst L A, Foley N E, Gleason-Guzman M C, Hacker M P, Cress A E, Greenberger L W, De Jong M C, Dalton W S
Department of Biochemistry, Pharmacology, and Internal Medicine, H. Lee Moffitt Cancer Center, University of South Florida, Tampa, Florida 33612, USA.
Cancer Res. 1999 Mar 1;59(5):1021-8.
Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.
体外耐药性选择可导致一种复杂的表型，其中一种以上的耐药机制同时或相继出现。我们研究了在人骨髓瘤细胞系8226中用米托蒽醌进行选择时出现的耐药机制。一种新的转运机制在选择过程早期出现，这与8226/MR4细胞系对米托蒽醌产生10倍的耐药性有关。细胞内药物浓度的降低是ATP依赖的且对哇巴因不敏感。8226/MR4细胞系对荧光氮杂蒽吡唑BBR 3390有34倍的交叉耐药性。对BBR 3390的耐药性与细胞内药物浓度降低50%相一致。使用BBR 3390的共聚焦显微镜显示耐药细胞系中核质比降低了64%。一种新型化学增敏剂烟曲霉震颤素C可逆转米托蒽醌和BBR 3390细胞内药物浓度的降低。相比之下，烟曲霉震颤素C对P-糖蛋白阳性的8226/DOX6细胞系对米托蒽醌或BBR 3390的耐药性没有影响。将8226细胞系对米托蒽醌的耐药程度从10倍提高到37倍（8226/MR20）并没有进一步降低细胞内药物浓度。然而，与亲代药物敏感细胞系相比，8226/MR20细胞系中拓扑异构酶IIβ和α的表达分别降低了88%和70%。在低水平耐药的8226/MR4细胞系中未观察到拓扑异构酶表达和活性的这种降低。这些数据表明，低水平米托蒽醌耐药性是由于存在一种类似于P-糖蛋白和多药耐药相关蛋白的新型能量依赖型药物外排泵。用烟曲霉震颤素C阻断药物外排来逆转耐药性，应该能够对癌细胞系或临床肿瘤样本中的这种新型转运蛋白进行功能分析。对米托蒽醌耐药性的增加可能是由于细胞内药物积累减少、核质药物分布改变以及拓扑异构酶II活性改变所致。
