Qian X D, Beck W T
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Hospital, Memphis, Tennessee 38101.
Cancer Res. 1990 Feb 15;50(4):1132-7.
Verapamil enhances anticancer drug cytotoxicity in multidrug resistant (MDR) cells, apparently by competing with these agents for binding to P-glycoprotein (Pgp). In this study, we provide direct evidence for this competition. We studied the binding of an optically pure photoaffinity analogue of verapamil, (S)-5-[(3-azidophenylethyl)-[N-methyl-3H]- methylamino]-2-(3,4,5-trimethoxyphenyl)-2-isopropylvaleronitrile (LU-49888), to Pgp from MDR cell lines. LU-49888 specifically labeled a single Mr 170,0000 protein that was identified as Pgp on Western blots and also by specific immunoprecipitation with monoclonal antibody C-219. A 200-fold molar excess of vinblastine or vincristine specifically inhibited this binding by greater than 98%. LU-49888 labeling of Pgp was also inhibited by actinomycin D (45%), podophyllotoxin (47%), and amsacrine (82%), marginally by doxorubicin (25%), colchicine (22%), daunorubicin (18%), and etoposide (14%), but not by teniposide. Modulators of Pgp-MDR also compete with LU-49888 for binding to Pgp: verapamil (82%), diltiazem (73%), quinidine (91%), reserpine (91%), rescinnamine (88%), and trimethoxybenzoylyohimbine (89%). Chloroquine was moderately inhibitory (25%), whereas chlorpromazine and yohimbine, which are not modulators in our MDR cell lines, did not inhibit the binding of LU-49888 to Pgp. LU-49888 labeling of Pgp was also completely inhibited by (R)-, (S)-, and racemic desmethoxyverapamil, all with the same efficiency. Our results demonstrate that the verapamil analogue LU-49888 specifically binds to Pgp and suggest that verapamil and some MDR modulators exert their effects by interacting with Pgp.
维拉帕米可增强多药耐药（MDR）细胞中抗癌药物的细胞毒性，显然是通过与这些药物竞争结合P - 糖蛋白（Pgp）来实现的。在本研究中，我们为这种竞争提供了直接证据。我们研究了维拉帕米的一种光学纯光亲和类似物，即（S）-5 - [（3 - 叠氮苯乙基）-[N - 甲基 - ³H]-甲氨基]-2 - （3,4,5 - 三甲氧基苯基）-2 - 异丙基戊腈（LU - 49888）与MDR细胞系中的Pgp的结合。LU - 49888特异性标记了一种分子量为170,000的单一蛋白质，该蛋白质在蛋白质免疫印迹上以及通过用单克隆抗体C - 219进行特异性免疫沉淀被鉴定为Pgp。200倍摩尔过量的长春碱或长春新碱可特异性抑制这种结合，抑制率大于98%。放线菌素D（45%）、鬼臼毒素（47%）和安吖啶（82%）也能抑制Pgp的LU - 49888标记，阿霉素（25%）、秋水仙碱（22%）、柔红霉素（18%）和依托泊苷（14%）有轻微抑制作用，而替尼泊苷则无抑制作用。Pgp - MDR调节剂也与LU - 49888竞争结合Pgp：维拉帕米（82%）、地尔硫卓（73%）、奎尼丁（91%）、利血平（91%）、利舍那明（88%）和三甲氧基苯甲酰育亨宾（89%）。氯喹有中度抑制作用（25%），而在我们的MDR细胞系中不是调节剂的氯丙嗪和育亨宾则不抑制LU - 49888与Pgp的结合。（R）-、（S）-和消旋去甲氧基维拉帕米对Pgp的LU - 49888标记也有完全抑制作用，且抑制效率相同。我们的结果表明，维拉帕米类似物LU - 49888能特异性结合Pgp，并提示维拉帕米和一些MDR调节剂通过与Pgp相互作用发挥作用。
