Dietel M, Herzig I, Reymann A, Brandt I, Schaefer B, Bunge A, Heidebrecht H J, Seidel A
Institut of Pathology, Christian-Albrechts-Universität zu Kiel, Germany.
J Cancer Res Clin Oncol. 1994;120(5):263-71. doi: 10.1007/BF01236382.
Multidrug-resistant tumor cells can be resensitized by combined application of the selecting cytostatic drug and a chemosensitizer, such as cyclosporin A (CsA) or a calcium channel blocker. Since clinical trials on the circumvention of multidrug resistance (MDR) with chemosensitizers report disparate results, we investigated whether tumor cells of the MDR phenotype can develop additional resistance to the cytostatic chemosensitizer combination. Thus, the Adriamycin(ADR)-selected, P-glycoprotein-positive MDR Friend leukemia cell line F4-6RADR was exposed to stepwise increased concentrations of CsA at a constant level of 0.05 microgram/ml ADR. The initial CsA concentration (plus 0.05 microgram/ml ADR) to inhibit cell growth of F4-6RADR cells by 50% (IC50) was 0.04 microgram/ml. By continuous incubation for more than 6 months, the IC50 for CsA (at constant ADR) was elevated to 3.6 micrograms/ml (90-fold), thus generating the variant F4-6RADR-CsA. The F4-6RADR-CsA cells were cross-resistant for cyclosporin H (CsH), a non-immunosuppressive derivative of CsA. As shown by immunocytochemistry as well as by the polymerase chain reaction and by Western blotting including densitometry, P-glycoprotein was preserved in the F4-6RADR-CsA variant and was expressed at a 4-fold higher level than in F4-6RADR cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis could detect no new proteins in F4-6RADR-CsA as compared to F4-6RADR. Interestingly, resistance of F4-6RADR-CsA cells remained reversible for the calcium antagonists verapamil and dihydropyridine B859-35 (dexniguldipine-HCl), indicating that CsA and these compounds interfere with the P glycoprotein function by different pharmacodynamic mechanisms. Transport studies with [14C]ADR, performed in the presence and absence of chemosensitizers, confirmed the good correlation of P-glycoprotein function with the pattern of resistance found in proliferation assays. Cellular accumulation of [3H]cyclosporin was reduced to 71% of that of the F4-6 controls in F4-6RADR-CsA cells, but remained at the level of controls in F4-6RADR cells. Results indicate that increased amounts of the P-glycoprotein--besides other, perhaps more important mechanisms that are as yet unknown--partially mediate CsA resistance in F4-6RADR-CsA cells. We have designated this new form of resistance "secondary combined resistance" (SCR). The results suggest that at least some clinical cases of insensitivity to chemosensitizers or of relapse after reversing therapy could be explained by SCR, and that resensitizing treatment of tumor patients should be based on the consideration of several chemosensitizers of different pharmacodynamics.
多药耐药肿瘤细胞可通过联合应用选择的细胞抑制药物和化学增敏剂（如环孢素A（CsA）或钙通道阻滞剂）使其重新敏感。由于关于用化学增敏剂规避多药耐药（MDR）的临床试验报告结果不一，我们研究了具有MDR表型的肿瘤细胞是否会对细胞抑制性化学增敏剂组合产生额外耐药性。因此，将阿霉素（ADR）选择的、P-糖蛋白阳性的MDR弗氏白血病细胞系F4-6RADR暴露于在0.05微克/毫升ADR恒定水平下逐步增加浓度的CsA中。抑制F4-6RADR细胞生长50%（IC50）的初始CsA浓度（加0.05微克/毫升ADR）为0.04微克/毫升。通过连续培养6个月以上，CsA（在ADR恒定情况下）的IC50升高至3.6微克/毫升（90倍），从而产生变体F4-6RADR-CsA。F4-6RADR-CsA细胞对CsA的非免疫抑制衍生物环孢素H（CsH）具有交叉耐药性。免疫细胞化学、聚合酶链反应以及包括光密度测定的蛋白质印迹法显示，P-糖蛋白在F4-6RADR-CsA变体中得以保留，且表达水平比F4-6RADR细胞高4倍。与F4-6RADR相比，十二烷基硫酸钠/聚丙烯酰胺凝胶电泳分析在F4-6RADR-CsA中未检测到新蛋白质。有趣的是，F4-6RADR-CsA细胞对钙拮抗剂维拉帕米和二氢吡啶B859-35（盐酸右尼非地平）的耐药性仍然是可逆的，这表明CsA和这些化合物通过不同的药效学机制干扰P-糖蛋白功能。在有和没有化学增敏剂存在的情况下用[14C]ADR进行的转运研究证实，P-糖蛋白功能与增殖试验中发现的耐药模式具有良好相关性。F4-6RADR-CsA细胞中[3H]环孢素的细胞蓄积量降至F4-6对照的71%，但在F4-6RADR细胞中保持在对照水平。结果表明，除了其他可能更重要但尚未知晓的机制外，P-糖蛋白量增加部分介导了F4-6RADR-CsA细胞对CsA的耐药性。我们将这种新的耐药形式称为“继发性联合耐药”（SCR）。结果表明，至少某些对化学增敏剂不敏感或逆转治疗后复发的临床病例可能可用SCR来解释，并且肿瘤患者的重新敏感化治疗应基于对几种不同药效学的化学增敏剂的考虑。
