ANTITUMOR THERAPIES BASED ON INHIBITION OF SUPEROXIDE DISMUTASE

基于抑制超氧化物歧化酶的抗肿瘤疗法

• 批准号: 6203295
• 负责人: LARRY OBERLEY
• 金额: $ 11.34万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-31 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6203295
• 关键词: MCF7 cell; antineoplastics; antisense nucleic acid; bleomycin; catalase; complementary DNA; doxorubicin; drug resistance; electron spin resonance spectroscopy; enzyme activity; fatty acids; free radical oxygen; glutathione; glutathione peroxidase; glutathione reductase; ionizing radiation; lipid peroxides; neoplasm /cancer pharmacology; northern blottings; oxidoreductase inhibitor; southern blotting; superoxide dismutase; transfection; tumor necrosis factor alpha

The purpose of this proposal is to study the role of active oxygen species in cancer cell cytotoxicity and to determine if lipids are a major target of these species. In order to study the role of active oxygen, superoxide dismutase (SOD) levels of parental and doxorubicin- resistant human breast carcinoma MCF-7 cells will be modulated and the effect on cell killing by selected antitumor agents (doxorubicin, bleomycin, ionizing radiation, tumor necrosis factor) determined. Modulation of superoxide dismutase will be accomplished by transfection of sense and anti-sense SOD cDNA. After transfection, clones with lower and higher than control levels of SOD activity will be isolated. These clones will then be tested for sensitivity to the selected antitumor agents. Both CuZnSOD and MnSOD will be transfected. Verification that superoxide dismutase modulation has occurred will be accomplished using enzyme activity, immunoreactive protein, Northern and Southern blot assays. Catalase, glutathione peroxidase, glutathione reductase, and glutathione levels will also be measured in the stable clones to determine if any of the other common antioxidants have been altered. Similar studies will be performed after glutathione peroxidase (GPX) transfection. GPX will be transferred into wild type MCF-7 cells as well as an SOD-overexpressing clone. The role of oxygen and lipid radicals will also be tested directly by measuring qualitatively and quantitatively the radicals present after treatment of control and SOD-and/or GPX-modified cells with the selected antitumor agents. Measurement of radicals will be accompanied by using the electron paramagnetic resonance technique of spin trapping. Last, the role of lipids in the antitumor effect will be studied. This will be done in two different ways: 1) measuring the amount of lipid peroxidation that occurs during tumor cell killing by the above agents; and 2) modifying the lipids of the cell and determining the effect on tumor cell killing by selected antitumor combinations in control and SOD- and/or GPX-transfected cell lines. This research should provide valuable new information on the mechanisms and targets of antitumor agents. It is hoped this knowledge will eventually lead to more powerful antitumor protocols.

本提案的目的是研究活性氧的作用 癌细胞细胞毒性中的物种并确定脂质是否是一种 这些物种的主要目标。 为了研究主动的作用 氧、亲本和阿霉素的超氧化物歧化酶 (SOD) 水平 耐药人乳腺癌MCF-7细胞将被调节，并且 选定的抗肿瘤药物（阿霉素、阿霉素、 博莱霉素、电离辐射、肿瘤坏死因子）测定。 超氧化物歧化酶的调节将通过转染来完成 有义和反义SOD cDNA。 转染后，克隆具有较低的 高于对照水平的 SOD 活性将被分离出来。 这些 然后将测试克隆对所选抗肿瘤药物的敏感性 代理。 CuZnSOD 和 MnSOD 都将被转染。 验证超氧化物歧化酶调节已经发生将是 使用酶活性、免疫反应蛋白、Northern 和 Southern印迹分析。 过氧化氢酶、谷胱甘肽过氧化物酶、谷胱甘肽 还原酶和谷胱甘肽水平也将在稳定的环境中进行测量 克隆以确定其他常见抗氧化剂是否已被 改变了。 类似的研究将在谷胱甘肽过氧化物酶（GPX）之后进行 转染。 GPX 也将被转移到野生型 MCF-7 细胞中 作为 SOD 过表达克隆。 氧和脂质自由基的作用也将通过以下方法直接测试 定性和定量测量之后存在的自由基 用选定的处理对照和 SOD 和/或 GPX 修饰的细胞 抗肿瘤剂。 自由基的测量将伴随使用 自旋捕获的电子顺磁共振技术。 最后，将研究脂质在抗肿瘤作用中的作用。 这 将通过两种不同的方式完成：1）测量脂质的量 上述药物杀死肿瘤细胞过程中发生的过氧化作用； 2) 改变细胞的脂质并确定对 通过对照和 SOD- 中选定的抗肿瘤组合杀死肿瘤细胞 和/或GPX转染的细胞系。 这项研究应该提供有关机制的有价值的新信息 和抗肿瘤药物的靶点。 希望这些知识能够 最终导致更强大的抗肿瘤方案。