De Rosa María Fabiana, Ackerley Cameron, Wang Bernice, Ito Shinya, Clarke David M, Lingwood Clifford
Division of Molecular Structure and Function, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 2008 Feb 22;283(8):4501-11. doi: 10.1074/jbc.M705473200. Epub 2007 Nov 13.
Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.
通过ABC药物转运蛋白（ABCB1）、P-糖蛋白（P-gp/MDR1）过表达导致的多药耐药（MDR）是癌症化疗中的主要障碍。许多抑制剂可逆转MDR，但像环孢菌素A（CsA）一样，具有显著毒性。MDR1还是一种转位酶，可在高尔基体中将葡糖神经酰胺翻转至内侧，以增强中性糖鞘脂（GSL）合成。我们观察到部分MDR1/球三糖神经酰胺（Gb3）在细胞表面共定位，且去除GSL会使细胞表面MDR1减少。因此，MDR1可能与GSL相互作用。金刚烷基Gb3是一种水溶性Gb3模拟物，但其他GSL类似物则不然，它可逆转MDR1-MDCK细胞的耐药性。用CsA或adaGb3处理1小时后，细胞表面MDR1上调，但在72小时时，细胞表面表达消失。细胞内MDR1始终积累，提示质膜MDR1转运存在长期缺陷。AdaGb3或CsA可迅速降低罗丹明123的细胞外排。MDR1还介导胃肠道上皮细胞的药物外排，限制口服生物利用度。当在极化的人肠道C2BBe1细胞两侧或仅在顶端加入adaGb3时，长春碱的顶端至基底转运显著增加，与标准MDR1抑制剂维拉帕米相当。突变型MDR1的二硫键交联显示adaGb3不与跨膜片段（TM）6和TM7表面近端螺旋之间的MDR1维拉帕米/环孢菌素结合位点结合，而是与更靠近TM6中心和TM7细胞外表面的相邻位点结合，即靠近双层小叶界面。志贺毒素介导的Gb3内吞作用也会上调总MDR1并抑制药物外排。因此，膜Gb3与MDR1之间的功能相互作用为基于生理学的MDR1调节提供了一种方法，以提高化疗药物的生物利用度。
