Liu Z, Lheureux F, Pouliot J F, Heckel A, Bamberger U, Georges E
Institute of Parasitology, McGill University, Quebec, Canada.
Mol Pharmacol. 1996 Sep;50(3):482-92.
The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.
肿瘤细胞中P-糖蛋白（P-gp）的表达会导致多药耐药（MDR）表型。P-gp已被证明能以能量依赖的方式介导结构不同的药物跨细胞膜转运。在本报告中，我们表明苯并蝶啶类似物BIBW22 BS以剂量依赖的方式逆转CEM人淋巴瘤细胞的MDR表型。使用BIBW22 BS的光活性类似物{[3H]叠氮基-4-[N-(2-羟基-2-甲基丙基)-乙醇胺]-2,7-双（顺式-2,6-二甲基-吗啉基）-6-苯基蝶啶}，我们展示了在用P-gp特异性单克隆抗体免疫沉淀的耐药细胞中一种170 kDa蛋白的光亲和标记。[3H]叠氮基-BIBW22 BS对P-gp的光标记具有特异性且可饱和。此外，BIBW22 BS、长春碱和维拉帕米（而非秋水仙碱）抑制了[3H]叠氮基-BIBW22 BS对P-gp的光标记。药物结合研究表明，多药耐药细胞的膜比亲代药物敏感细胞结合更多的BIBW22 BS，且这种结合被长春碱抑制，在较小程度上被尿苷抑制。然而，药物转运研究表明BIBW22 BS不是P-gp外排泵的底物。有趣的是，BIBW22 BS在耐药细胞中积累更多。此外，BIBW22 BS在药物敏感和耐药细胞中的积累不依赖能量。这些结果与在相同多药耐药细胞中观察到的[3H]长春碱积累减少或外排增强形成对比。通过用金黄色葡萄球菌V蛋白酶进行克利夫兰图谱分析比较[3H]叠氮基-BIBW22 BS或[3H]叠氮平光标记的P-gp，显示出光标记肽段存在差异。综上所述，本研究结果表明BIBW22 BS是一种有效的多药耐药逆转剂，它直接与P-gp结合，但不会从耐药细胞中排出。
