Redox Mechanisms of Chemoprevention in Lung by Selenium Supplementation

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的，但其机制尚不清楚。一种假说指出，硒通过调节细胞氧化还原参数和增加抗氧化反应元件（ARE）基因的表达，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)，启动抗氧化防御系统。为了进一步研究新型硒化合物和传统硒化合物预防肺癌的机制，我们提出验证不同硒化合物改变氧化还原状态的假设，ARE/Nrf2激活，TrxRI和GPx2在氧化还原调节中发挥作用，目的如下：目的1：确定硒化合物对HOP92人肺细胞系氧化还原状态的调节。本研究的重点是大细胞肺癌细胞系HOP92，目的是研究细胞对硒的反应，以及这些变化如何与A549腺癌和BEAS-2B支气管上皮细胞系进行比较，这些细胞系已有初步数据。使用细胞计数试剂盒-8测定硒化合物处理后48小时的细胞活力。细胞氧化还原状态将评估使用细胞测定游离硫醇和活性氧。研究人员将使用JC-1细胞测定法检测线粒体膜电位和Western blotting检测伴侣蛋白BiP/GRP78的毒性机制。目的2：探讨Nrf2转录因子和硒蛋白TrxRI和GPx2在硒化合物诱导的HOP92、A549和BEAS-2B细胞系氧化应激中的作用。一个ARE报告结构将被用来确定硒化合物效应是否被Nrf2转录因子调节。Nrf2的激活和需求将使用突变的ARE报告结构和靶向Nrf2的siRNA进一步验证。在这三种细胞系中也会产生基因敲低，以减少TrxRI和GPx2的转录，我们实验室已经发现，硒化合物处理后，TrxRI和GPx2在肺组织中存在差异表达。与氧化还原状态相关的细胞反应将使用细胞测定法评估游离硫醇、活性氧水平和线粒体膜电位。

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.