Redox Mechanisms of Chemoprevention in Lung by Selenium Supplementation

补硒预防肺部化学预防的氧化还原机制

• 批准号: 7751422
• 负责人: ROBYN POERSCHKE
• 金额: $ 3.17万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7751422
• 关键词: A549; Adenocarcinoma; Antioxidants; Binding; Biological Assay; Cancer cell line; Cell Count; Cell Line; Cell Wall; Cells; Chemoprevention; Competence; Complex; DNA; DNA delivery; DNA-Binding Proteins; Data; Diet; Dimensions; Electron Microscopy; Epithelial Cells; Escherichia coli; Fluorescence Microscopy; Genes; Genetic Transcription; Gold Colloid; Hour; Human; Immunoblotting; Incidence; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Membrane Potentials; Minor; Modeling; Molecular Structure; Nutrient; Oxidation-Reduction; Oxidative Stress; Pattern; Play; Polyacrylamide Gel Electrophoresis; Proteins; Reporter; Response Elements; Role; Selenium; Small Interfering RNA; Structure; Structure of parenchyma of lung; Sulfhydryl Compounds; Supplementation; System; Testing; Time; Toxic effect; Western Blotting; Work; cancer prevention; cancer type; chaperonin; disulfide bond; glutathione peroxidase; in vivo; mitochondrial membrane; monomer; mortality; mutant; novel; prevent; protein protein interaction; public health relevance; response; selenoprotein; thioredoxin reductase 1; transcription factor; uptake

DESCRIPTION (provided by applicant): Cancer prevention by selenium (Se) has been shown to be compound and model dependent, but the mechanisms are not well understood. One hypothesis points to priming of the antioxidant defense system by selenium via modulation of cellular redox parameters and increased expression of antioxidant response element (ARE) genes, including the selenoproteins thioredoxin reductase 1 (TrxRI) and glutathione peroxidase 2 (GPx2). To further investigate the mechanisms of novel and conventional selenocompounds in preventing lung cancer, we propose to test the hypothesis that distinct selenocompounds alter redox status, with ARE/Nrf2 activation and TrxRI and GPx2 playing a role in redox modulation with the following aims: Aim 1: Determine the modulation of redox status of the HOP92 human lung cell line by selenocompounds. This aim focuses on a large cell lung cancer cell line, HOP92, in order to investigate what cellular responses to selenium occur and how these changes compare to the A549 adenocarcinoma and BEAS-2B bronchial epithelial cell lines for which preliminary data exists. Cellular viability will be determined 48 hours after selenocompound treatment using a Cell Counting Kit-8 assay. Cellular redox status will be assessed using cytometric assays for free thiols and ROS. Mechanisms of toxicity will be investigated using a JC-1 cytometric assay for mitochondrial membrane potential and Western blotting for the chaperonin BiP/GRP78. Aim 2: Investigate the role of the Nrf2 transcription factor and selenoproteins TrxRI and GPx2 in modulating oxidative stress induced by selenocompounds in the HOP92, A549 and BEAS-2B cell lines. An ARE reporter construct will be utilized to determine if selenocompound effects are modulated by the Nrf2 transcription factor. Nrf2 activation and requirement will further be validated using mutant ARE reporter constructs and siRNA targeted to Nrf2. Gene knockdowns will also be produced in the three cell lines to reduce transcription of TrxRI and GPx2, which have been found by our lab to be differentially expressed in lung tissue after selenocompound treatment. Cellular responses related to redox status will then be assessed using cytometric assays for free thiols, ROS levels and mitochondrial membrane potential. PUBLIC HEALTH RELEVANCE: Selenium is an essential nutrient present in the diet that has been shown to decrease the incidence and mortality of several types of cancer, including lung cancer. How selenium prevents cancer is currently not well understood, but one way may be by turning on the cell's defense system against oxidative stress. This work seeks to advance the understanding of how selenium supplementation prevents cancer and provide further information on how lung cancer might best be prevented and treated.

描述（由申请人提供）：硒（Se）预防癌症已被证明是化合物和模型依赖性的，但其机制尚不清楚。一种假设指出，硒通过调节细胞氧化还原参数和增加抗氧化反应元件 (ARE) 基因（包括硒蛋白硫氧还蛋白还原酶 1 (TrxRI) 和谷胱甘肽过氧化物酶 2 (GPx2)）的表达来启动抗氧化防御系统。为了进一步研究新型和传统的含硒化合物预防肺癌的机制，我们建议测试以下假设：不同的含硒化合物改变氧化还原状态，ARE/Nrf2激活，TrxRI和GPx2在氧化还原调节中发挥作用，目的如下：目标1：确定含硒化合物对HOP92人肺细胞系氧化还原状态的调节。该目标重点关注大细胞肺癌细胞系 HOP92，以研究细胞对硒的反应，以及这些变化与已有初步数据的 A549 腺癌和 BEAS-2B 支气管上皮细胞系相比如何。硒化合物处理后 48 小时，使用 Cell Counting Kit-8 测定法测定细胞活力。将使用游离硫醇和 ROS 的细胞计数测定来评估细胞氧化还原状态。将使用 JC-1 细胞计数测定线粒体膜电位和蛋白质印迹法研究伴侣蛋白 BiP/GRP78 的毒性机制。目标 2：研究 Nrf2 转录因子以及硒蛋白 TrxRI 和 GPx2 在调节 HOP92、A549 和 BEAS-2B 细胞系中硒化合物诱导的氧化应激中的作用。 ARE 报告构建体将用于确定硒化合物效应是否受 Nrf2 转录因子调节。 Nrf2 的激活和需求将使用突变 ARE 报告基因构建体和针对 Nrf2 的 siRNA 进一步验证。三种细胞系中也会产生基因敲低，以减少 TrxRI 和 GPx2 的转录，我们实验室发现经过硒化合物处理后，TrxRI 和 GPx2 在肺组织中差异表达。然后，将使用细胞计数法测定游离硫醇、ROS 水平和线粒体膜电位来评估与氧化还原状态相关的细胞反应。 公共健康相关性：硒是饮食中存在的一种必需营养素，已被证明可以降低包括肺癌在内的多种癌症的发病率和死亡率。目前尚不清楚硒如何预防癌症，但一种方法可能是打开细胞针对氧化应激的防御系统。这项工作旨在增进对补硒如何预防癌症的了解，并提供有关如何最好地预防和治疗肺癌的进一步信息。