MRP/GST SYNERGY AND MDR

MRP/GST 协同和 MDR

• 批准号: 2414451
• 负责人: CHARLES S MORROW
• 金额: $ 13.17万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-10 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2414451
• 关键词: HeLa cells; MCF7 cell; P glycoprotein; antineoplastics; antitoxins; chemical conjugate; complementary DNA; cytotoxicity; drug interactions; drug metabolism; drug screening /evaluation; enzyme activity; enzyme substrate; gene expression; glutathione; glutathione transferase; isozymes; messenger RNA; multidrug resistance; pharmacokinetics; synthetic peptide; toxin metabolism; transfection /expression vector

We propose that the multidrug resistance-associated protein (MRP) and glutathione S-transferases (GSTs) cooperate synergistically to confer high level multidrug resistance (MDR) to many anticancer drugs. By synergy we mean that the total effect on drug resistance of increases in both MRP and GST is greater than the sum of the effects on drug resistance of MRP and GST taken independently. We suggest that the mechanism of this synergy involves the conjugation of drugs, or their toxic metabolites, to glutathione (GSH) by GSTs. It is believed that these GSH-toxin conjugates are better substrates for the MRP-associated drug efflux pump than are the unconjugated drugs or metabolites. This hypothesis is appealing because, while GSTs can conjugate a number of anticancer drugs with GSH, the effect of increased GST on drug resistance has been inconsistent. Consequently, it has been suggested that a second gene product may be necessary for GSTs to fully confer drug resistance. Recent reports have strongly indicated that MRP is an ATP-dependent, GSH- conjugate exporter. Therefore, MRP is the prime candidate for the second gene product involved in GST-mediated drug resistance. To test this hypothesis, we will use model parental cell lines that express low basal levels of MRP and GST. By stable transfection of these cells, derivative cell lines will be established that express increased levels of MRP with or without concomitantly increased levels of the isozymes of GST. These cell lines will be examined for relative resistance to the cytotoxic effects of a panel of drugs and xenobiotic toxins. Comparison of cytotoxicity dose-response data for cells that express only increased MRP versus cells that express increased levels of MRP-GST isozyme combinations will allow the identification and quantitation of pharmacodynamic interactions between MRP and GSTs. Additionally, the relationship between resistance and maintenance of intracellular GSH pools will be explored. With these derivative cell lines, the relative contributions to resistance of drug conjugating enzymes, such as GSTs, and MRP can be evaluated. These cells will be used to test our hypothesis that GST and MRP can cooperate synergistically to confer high level resistance to select antineoplastic drugs. In order to examine the mechanism of any observed synergy between MRP and the isozymes of GST, the kinetics of drug accumulation and efflux will be determined in the derivative cell lines. These pharmacokinetic studies will establish whether or not increased GST levels facilitate drug export, by the formation GSH-drug conjugates, in cells having increased MRP.

我们认为多药耐药相关蛋白（MRP）和 谷胱甘肽S-转移酶（GST）协同合作，赋予高 对多种抗癌药物产生多药耐药（MDR）。通过协同作用，我们 这意味着MRP和MRP的增加对耐药性的总体影响， GST对耐药性的影响大于MRP和 GST独立进行。我们认为这种协同作用的机制 涉及药物或其有毒代谢物的结合， 谷胱甘肽（GSH）。据信这些GSH-毒素缀合物 是MRP相关药物外排泵的更好底物， 未结合的药物或代谢物。 这一假设是有吸引力的，因为GST可以结合许多 抗肿瘤药物与GSH、GST升高对耐药的影响 一直不一致因此，有人建议， 基因产物可能是GST完全赋予耐药性所必需的。 最近的报道强烈表明MRP是一种ATP依赖性的，GSH- 共轭输出器。因此，MRP是第二个的主要候选人 参与GST介导的耐药性的基因产物。 为了检验这一假设，我们将使用模型亲本细胞系， 表达低水平的MRP和GST。通过稳定转染这些 细胞，将建立表达增加的衍生细胞系， MRP水平，伴随或不伴随增加的 GST同工酶。将检查这些细胞系的相对耐药性 一组药物和异生物毒素的细胞毒性作用。 仅表达的细胞的细胞毒性剂量-反应数据比较 相对于表达增加水平的MRP-GST的细胞， 同工酶组合将允许鉴定和定量 MRP和GST之间的药效学相互作用。另夕h 抗性与细胞内GSH池维持的关系 将被探索。有了这些衍生细胞系， 对药物缀合酶如GST的抗性的贡献，以及 MRP是可以评估的。这些细胞将被用来验证我们的假设， GST和MRP可以协同作用，赋予高水平的抗性 来选择禁药为了研究任何 观察到MRP和GST同工酶之间的协同作用，药物动力学 将在衍生细胞系中测定累积和外排。 这些药代动力学研究将确定是否增加GST 水平促进药物出口，通过形成GSH-药物缀合物， MRP增加的细胞。