Muñoz-Martínez Francisco, Lu Peihua, Cortés-Selva Fernando, Pérez-Victoria José María, Jiménez Ignacio A, Ravelo Angel G, Sharom Frances J, Gamarro Francisco, Castanys Santiago
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Cancer Res. 2004 Oct 1;64(19):7130-8. doi: 10.1158/0008-5472.CAN-04-1005.
Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.
多药外排泵ABCB1（MDR1）P-糖蛋白的过表达是肿瘤细胞产生多药耐药性（MDR）的一种机制，这会妨碍癌症化疗的成功进行。卫矛科的倍半萜是先前已证明能在几种人类癌细胞系和利什曼原虫菌株中逆转多药耐药性的天然化合物。然而，它们逆转耐药性的分子机制尚未明确。在本研究中，我们研究了28种二氢-β-agarofuran倍半萜逆转P-糖蛋白依赖性多药耐药表型的能力，并阐明了它们的分子作用机制。使用转染了人MDR1的NIH-3T3细胞进行的细胞毒性试验使我们能够筛选出最有效的倍半萜，这些倍半萜可逆转对柔红霉素和长春碱的体外耐药性。流式细胞术实验表明，上述活性化合物以饱和的、浓度依赖性方式特异性抑制P-糖蛋白的药物转运活性（抑制常数低至0.24±0.01 μmol/L），但不抑制ABCC1（多药耐药相关蛋白1；MRP1）、ABCC2（MRP2）和ABCG2（乳腺癌耐药蛋白；BCRP）转运蛋白的活性。此外，倍半萜在亚微摩尔浓度下抑制P-糖蛋白介导的[(3)H]秋水仙碱和四甲基罗丹明在CH(R)B30细胞膜以及富含P-糖蛋白的蛋白脂质体中的转运，这支持了P-糖蛋白是它们的分子靶点。质膜中的光亲和标记以及对纯化蛋白进行的荧光光谱实验表明，倍半萜与P-糖蛋白的跨膜结构域相互作用。最后，倍半萜以双相的、浓度依赖性方式调节P-糖蛋白的ATP酶活性：它们在极低浓度下刺激ATP酶活性，但在较高浓度下作为非竞争性抑制剂抑制ATP酶活性。由于卫矛科倍半萜具有逆转多药耐药性的效力以及对P-糖蛋白的特异性，它们是有前景的P-糖蛋白调节剂，在癌症化疗中具有潜在应用价值。
