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.

这项建议的目的是研究活性氧的作用。 肿瘤细胞的细胞毒性和确定脂类是否是一种 这些物种的主要目标。为了研究主动的作用 氧，超氧化物歧化酶(SOD)水平的亲本和阿霉素- 耐药的人乳腺癌MCF-7细胞将被调制并 部分抗肿瘤药物(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控将通过转染法实现 正义和反义超氧化物歧化酶基因的表达。转染后，低表达的克隆 并且高于对照水平的超氧化物歧化酶活性将被分离出来。这些 然后将测试克隆对所选抗肿瘤药物的敏感性 探员们。铜锌超氧化物歧化酶和锰超氧化物歧化酶都将被转导。 已发生超氧化物歧化酶调节的核实将是 使用酶活性、免疫活性蛋白、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 还原酶和谷胱甘肽的水平也将在稳定中进行测定 克隆以确定是否有其他常见的抗氧化剂 被更改了。 类似的研究将在谷胱甘肽过氧化物酶(GPX)之后进行 转染法。GPX也将转入野生型MCF-7细胞 作为一个过量表达超氧化物歧化酶的克隆。 氧和脂自由基的作用也将直接通过 定性和定量测量后存在的自由基 用所选细胞处理对照细胞和SOD和/或GPX修饰的细胞 抗肿瘤药物。自由基的测量将伴随着使用 自旋捕获的电子顺磁共振技术。 最后，将研究脂类在抗肿瘤作用中的作用。这 将通过两种不同的方式来完成：1)测量脂类的数量 上述药物在杀死肿瘤细胞时发生的过氧化反应； 2)修饰细胞的脂类，测定其对 对照组和超氧化物歧化酶联合用药对肿瘤细胞的杀伤作用 和/或转GPX基因的细胞系。 这项研究将提供有关这一机制的有价值的新信息。 以及抗肿瘤药物的靶标。希望这一知识将会 最终导致更强大的抗肿瘤方案。