GLUTATHIONE & RELATED ENZYMES IN CELULAR DRUG RESISTANCE

谷胱甘肽

• 批准号: 3459578
• 负责人: Shivendra Singh
• 金额: $ 7.88万
• 依托单位: MERCY HOSPITAL OF PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3459578
• 关键词: alkylating agents; antineoplastics; athymic mouse; bladder neoplasm; crosslink; cytogenetics; detoxification; drug resistance; electron spin resonance spectroscopy; enzyme induction /repression; free radical oxygen; free radical scavengers; glutathione; human tissue; isozymes; mitomycin C

Mitomycin C (MMC) is commonly used to treat bladder cancer, the third most prevalent malignant disease among males in USA. The clinical effectiveness of MMC is often limited by the emergence of drug resistant tumor cells through mechanism(s) not completely understood. The overall purpose of this research proposal is to gain insight into the mechanism(s) of resistance to MMC with particular emphasis on understanding the role of glutathione (GSH) and related enzymes in the process. GSH levels will be compared in sensitive and resistant bladder cancer cells and correlated with MMC cytotoxicity. In order to further substantiate that the GSH levels affect MMC response, effect of BSO induced GSH depletion on MMC cytotoxicity will be studied in vitro in colony forming assay and in vivo in nude mouse xenografts. In addition, sorting of subsets of cells on the basis of GSH content will also be attempted to seek correlation between GSH content and MMC cytotoxicity. The levels of GSH related enzymes, particularly GSH peroxidases including the novel monomeric form will be determined in these cells and correlated with MMC cytotoxicity. Since interrelationship of monomeric peroxidase with other peroxidases is not known, this enzyme will be purified and characterized. Expression and characteristics of GSH S-transferase (GST) isoenzymes of normal human bladder will be investigated to identify tissue specific GSTs. The levels of GST isoenzymes will be compared in sensitive and resistant cells in order to see alterations in specific class-toxicity. In order to gain insight into the mechanism by which GSTs may contribute to MMC resistance, GST mediated catalytic and non-catalytic detoxification of MMC will be studied in vitro using purified GST and in situ in sensitive and resistant cells. Studies will be performed to see if MMC resistance can be reversed by irreversible inhibitors of GST. MMC induced DNA cross-links will be quantitated in sensitive and resistant cells by alkaline elution assay. The extent and levels of enzymes involved in removal of DNA cross-link will also be compared in these cells. In addition, the contribution of MMC induced free radical scavengers on free radical generation by ESR spectroscopy and MMC cytotoxicity in colony forming assay. In long term, the proposed studies may be helpful in devising strategies to circumvent cellular resistance to MMC.

丝裂霉素C（MMC）通常用于治疗膀胱癌，是第三大治疗方法。 在美国男性中流行的恶性疾病。 临床有效性 丝裂霉素C的应用常常受到耐药肿瘤细胞出现的限制 通过机制（S）不完全理解。 的总体目的 本研究计划旨在深入了解 对MMC的抵抗力，特别强调了解 谷胱甘肽（GSH）和相关酶在这个过程中。 GSH水平将 在敏感和耐药膀胱癌细胞中进行比较， MMC细胞毒性。 为了进一步证实GSH 水平影响MMC反应，BSO诱导的GSH耗竭对MMC的影响 将在体外集落形成试验和体内研究细胞毒性 在裸鼠异种移植物中。 此外，在细胞上分选细胞亚群也是一个重要的步骤。 GSH含量的基础上，也将试图寻求GSH之间的相关性 含量和MMC细胞毒性。 GSH相关酶的水平， 特别是包括新单体形式的GSH过氧化物酶， 在这些细胞中测定并与MMC细胞毒性相关。 以来 单体过氧化物酶与其它过氧化物酶相互关系不 已知，这种酶将被纯化和表征。 表达及 正常人谷胱甘肽S-转移酶（GST）同工酶特性 将研究膀胱以鉴定组织特异性GST。 水平 GST同工酶将在敏感和耐药细胞中进行比较， 以观察特定类别毒性的变化。 为了获得 深入了解GST可能有助于MMC抵抗的机制， GST介导的MMC的催化和非催化解毒将是一个新的研究方向。 在体外使用纯化的GST和在敏感和耐药细胞中原位研究 细胞 将进行研究，以了解MMC耐药性是否可以逆转 不可逆的GST抑制剂。 MMC诱导的DNA交联将是 在敏感和抗性细胞中通过碱性洗脱测定进行定量。 参与去除DNA交联的酶的程度和水平将 也可以在这些细胞中进行比较。 此外，MMC的贡献 诱导型自由基清除剂对自由基生成的影响 光谱学和MMC细胞毒性。 从长远来看， 拟议的研究可能有助于制定规避战略， 细胞对MMC的抵抗力。