Redox Mechanisms of Chemoprevention in Lung by Selenium Supplementation

描述（由申请人提供）：硒（Se）的癌症预防作用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（GPx 2））的表达来启动抗氧化防御系统。为了进一步研究新的和传统的硒化合物在预防肺癌的机制，我们建议测试的假设，不同的硒化合物改变氧化还原状态，与ARE/Nrf2激活和TrxRI和GPx2发挥作用的氧化还原调节与以下目的：目的1：确定硒化合物的HOP92人肺细胞系的氧化还原状态的调制。本研究的重点是大细胞肺癌细胞系HOP92，以研究细胞对硒的反应，以及这些变化与A549腺癌和BEAS-2B支气管上皮细胞系的初步数据相比如何。在硒化合物处理后48小时，使用细胞计数试剂盒-8测定法测定细胞活力。将使用游离巯基和ROS的细胞计数测定法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。目标二：研究Nrf2转录因子和硒蛋白TrxRI和GPx2在HOP92、A549和BEAS-2B细胞系中调节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的作用是否受Nrf2转录因子调节。将使用突变体ARE报告基因构建体和靶向Nrf2的siRNA进一步验证Nrf2活化和需求。基因敲除也将在三种细胞系中产生，以减少TrxRI和GPx2的转录，我们的实验室已经发现，在硒化合物处理后，TrxRI和GPx2在肺组织中差异表达。然后使用细胞计数法测定游离巯基、ROS水平和线粒体膜电位，评估与氧化还原状态相关的细胞应答。 公共卫生相关性：硒是饮食中的一种必需营养素，已被证明可以降低几种癌症的发病率和死亡率，包括肺癌。硒如何预防癌症目前还不清楚，但一种方法可能是通过打开细胞的防御系统对抗氧化应激。这项工作旨在促进对硒补充剂如何预防癌症的理解，并提供有关如何最好地预防和治疗肺癌的进一步信息。

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.