Hendricks C B, Rowinsky E K, Grochow L B, Donehower R C, Kaufmann S H
Johns Hopkins Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Cancer Res. 1992 Apr 15;52(8):2268-78.
Topotecan (TPT, 9-dimethylaminomethyl-10-hydroxycamptothecin) is the first topoisomerase I-directed cytotoxic agent to enter clinical trials in the United States in two decades. The effect of P-glycoprotein (Pgp) overexpression on TPT cytotoxicity was examined in CHRC5 (colchicine-resistant) and AuxB1 (parental) Chinese hamster ovary cells. Examination of the IC50 values observed in colony-forming assays revealed that the CHRC5 cells were 15-fold (SD, +/- 3; n = 3) resistant to TPT after a 1-h exposure and 3.2-fold (SD, +/- 1.4; n = 4) resistant in continuous exposure experiments. Band depletion immunoblotting revealed that 4-fold higher concentrations of extracellular TPT were required to induce the formation of topo I-DNA complexes in CHRC5 cells as compared to AuxB1 cells. To assess the role of Pgp in this resistance, drug accumulation and cytotoxicity assays were repeated in the absence and presence of quinidine. Addition of quinidine enhanced TPT accumulation (measured by high-performance liquid chromatography) and diminished the IC50 for TPT to a greater extent in CHRC5 cells than in AuxB1 cells. To examine whether similar effects could be detected in Pgp-expressing human cells, MCF-7/Adriar breast cancer cells and KG1a human acute myelogenous leukemia cells were examined. Quinidine or verapamil enhanced TPT accumulation in both of these cell lines but had no effect in parental MCF-7 cells or a variety of human leukemia cell lines that do not overexpress Pgp. Cytotoxicity measurements performed by counting the number of surviving cells (MCF-7/Adriar) or employing a modified, highly stable tetrazolium dye reduction assay (leukemia cell lines) revealed that quinidine diminished the IC50 for TPT in the Pgp-overexpressing cell lines but not in the control lines. These results suggest that Pgp overexpression diminishes TPT accumulation and TPT cytotoxicity in hamster and human cells. It should be stressed, however, that these effects were substantially smaller than the effects of Pgp overexpression on the accumulation and cytotoxicity of the anthracycline daunorubicin and the epipodophyllotoxin etoposide in the same cell lines.
拓扑替康（TPT，9 - 二甲基氨基甲基 - 10 - 羟基喜树碱）是二十年来首个进入美国临床试验的拓扑异构酶I导向的细胞毒性药物。在CHRC5（秋水仙碱耐药）和AuxB1（亲代）中国仓鼠卵巢细胞中检测了P - 糖蛋白（Pgp）过表达对TPT细胞毒性的影响。对集落形成试验中观察到的IC50值进行检测发现，CHRC5细胞在1小时暴露后对TPT的耐药性为15倍（标准差，±3；n = 3），在连续暴露实验中为3.2倍（标准差，±1.4；n = 4）。条带缺失免疫印迹显示，与AuxB1细胞相比，CHRC5细胞中诱导拓扑异构酶I - DNA复合物形成所需的细胞外TPT浓度要高4倍。为了评估Pgp在这种耐药性中的作用，在不存在和存在奎尼丁的情况下重复进行药物积累和细胞毒性试验。添加奎尼丁可增强TPT的积累（通过高效液相色谱法测量），并且在CHRC5细胞中比在AuxB1细胞中更大程度地降低了TPT的IC50。为了检测在表达Pgp的人类细胞中是否能检测到类似的效应，对MCF - 7/Adriar乳腺癌细胞和KG1a人类急性髓性白血病细胞进行了检测。奎尼丁或维拉帕米可增强这两种细胞系中TPT的积累，但对亲代MCF - 7细胞或多种未过表达Pgp的人类白血病细胞系没有影响。通过计数存活细胞数量（MCF - 7/Adriar）或采用改良的、高度稳定的四氮唑染料还原试验（白血病细胞系）进行的细胞毒性测量显示，奎尼丁降低了过表达Pgp的细胞系中TPT的IC50，但在对照细胞系中没有。这些结果表明，Pgp过表达会降低仓鼠和人类细胞中TPT的积累及TPT细胞毒性。然而，应该强调的是，这些效应远小于Pgp过表达对相同细胞系中蒽环类药物柔红霉素和表鬼臼毒素依托泊苷的积累及细胞毒性的影响。
