Cell survival and arsenic carcinogenesis

描述（由申请人提供）：尽管砷诱导的致癌作用的分子机制仍有待研究，但砷产生的活性氧（ROS）被认为是重要的。砷产生的活性氧可引起DNA损伤、脂质过氧化和蛋白质修饰等多种致癌反应。我们的初步研究表明，砷诱导的ROS产生的能力大大降低砷转化的人肺支气管上皮细胞（BEAS-2B）相对于非转化细胞。ROS生成的这种减少赋予细胞癌前特征，包括快速生长。砷转化的细胞在砷暴露后表现出减少的凋亡（凋亡抗性）。该提议假设，由于ROS生成的低效力，砷转化的细胞产生凋亡抗性和增加的细胞存活，有助于砷诱导的致癌的整体机制。提出了三个目标来检验这一假设。目的1探讨砷对细胞内活性氧产生的抑制机制。我们将研究是否损伤的ROS产生途径是负责砷转化BEAS-2B细胞中的砷诱导的ROS产生的低水平。我们还将研究转化细胞中抗氧化酶水平的增加是否有助于ROS生成水平的降低。这一目标的完成将建立砷转化细胞中ROS生成减少的机制。目的2探讨活性氧生成减少在砷转化细胞抗凋亡中的作用。我们研究了（a）砷转化细胞的ROS产生减少是否是砷转化细胞抗凋亡的原因;（B）砷转化细胞是否比其与砷匹配的对照细胞具有更高的SOD和过氧化氢酶的抗氧化活性;（c）是否─在转化细胞中抗氧化酶的降低或NADPH氧化酶的过表达增加了ROS的产生，并增强了砷对细胞凋亡的反应刺激;（d）Bcl-2的敲低是否增加砷转化的细胞的凋亡;和（e）砷转化的细胞是否由于ROS产生减少和凋亡抗性而显示快速生长和增强的侵袭和迁移。总之，这一目标将证明活性氧在砷转化细胞的凋亡抗性中的作用。目的3探讨砷转化细胞诱导肿瘤发生及凋亡的作用。我们将研究砷转化细胞的肿瘤发生中凋亡抵抗的作用。我们还将使用皮肤肿瘤发生模型和原位肺癌模型来研究ROS和凋亡调节蛋白Bcl-2在砷转化细胞诱导的肿瘤发生中的作用。预期通过Bcl-2敲低增加砷转化细胞的凋亡将降低肿瘤生长。类似地，细胞的ROS产生能力的改变，即，通过ROS清除和产生酶的异位过表达和敲低，将对砷转化细胞的肿瘤发生产生影响。 公共卫生相关性：长期接触砷对健康的影响是主要的健康问题之一。本研究将探讨砷诱导的致癌机制，重点是减少活性氧的产生和砷转化细胞的凋亡抵抗。本研究结果将有助于了解砷的致癌机制和制定预防策略。

DESCRIPTION (provided by applicant): Although the molecular mechanism of arsenic-induced carcinogenesis remains to be investigated, reactive oxygen species (ROS) generated by arsenic are considered to be important. Arsenic-generated ROS could cause DNA damage, lipid peroxidation and protein modification, leading to various carcinogenic responses. Our preliminary studies have shown that the capacity of ROS generation induced by arsenic is substantially reduced in arsenic-transformed human lung bronchial epithelial (BEAS-2B) cells relative to the non-transformed cells. Such a reduction in ROS generation endows cells with premalignant features, including rapid growth. Arsenic- transformed cells exhibit a decreased apoptosis (apoptosis resistance) upon arsenic exposure. This proposal hypothesizes that due to a low potency of ROS generation, arsenic-transformed cells develop apoptosis resistance and increased cell survival, contributing to the overall mechanism of arsenic-induced carcinogenesis. Three aims are proposed to test this hypothesis. Aim 1 will investigate the mechanism of decreased ROS generation in the arsenic-transferred cells. We will investigate whether impairment of the ROS generating pathway is responsible for a low level of arsenic-induced ROS generation in arsenic transformed BEAS-2B cells. We will also investigate whether the increased level of antioxidant enzymes in transformed cells contributes to the decreased level of ROS generation. The completion of this aim will establish the mechanism of decreased ROS generation in arsenic-transformed cells. Aim 2 will investigate the role of reduced ROS generation in apoptosis resistance of arsenic-transformed cells. We investigate (a) whether reduced ROS generation of arsenic- transformed cells is responsible for apoptosis resistance in arsenic-transformed cells; (b) whether arsenic- transformed cells have higher antioxidant activities of SOD and catalase than their passage-matched control cells; (c) whether knock-down of antioxidant enzymes or overexpressing NADPH oxidase in the transformed cells increases ROS generation and enhances apoptosis in response to arsenic stimulation; (d) whether knocking down of Bcl-2 increases apoptosis of arsenic-transformed cells; and (e) whether arsenic- transformed cell will show fast growth and enhanced invasion and migration due to the decreased ROS generation and apoptosis resistance. Overall, this aim will demonstrate the role of ROS in apoptosis resistance of arsenic-transformed cells. Aim 3 will investigate the arsenic-transformed cells-induced tumorigenesis and the role of apoptosis. We will investigate the role of apoptosis resistance in tumorigenesis of arsenic-transformed cells. We will also investigate the roles of ROS and apoptosis regulatory proteins, Bcl-2, in arsenic-transformed cells-induced tumorigenesis using both skin tumorigenesis model and orthotopic lung cancer model. It is expected that the increase of apoptosis of arsenic-transformed cells by Bcl-2 knockdown will decrease tumor growth. Similarly, alterations in the ROS generating capacity of the cells, i.e., by ectopic overexpression and knockdown of the ROS-scavenging and producing enzymes, will have an effect on tumorigenesis of arsenic-transformed cells. PUBLIC HEALTH RELEVANCE: Health effects of chronic exposure to arsenic are one of the major health concerns. The present study will investigate the mechanism of arsenic-induced carcinogenesis with a focus on decreased generation of reactive oxygen species and apoptosis resistance in arsenic transformed cells. The results will help understand the mechanism of arsenic carcinogenesis and to develop preventive strategy.