Cell survival and arsenic carcinogenesis

描述(申请人提供)：尽管砷致癌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ROS可引起DNA损伤、脂质过氧化和蛋白质修饰，导致多种致癌反应。我们的初步研究表明，与未转化的细胞相比，砷转化的人肺支气管上皮细胞(BEAS-2B)产生ROS的能力显著降低。这种ROS生成的减少使细胞具有癌前特征，包括快速生长。在砷暴露下，砷转化的细胞表现出减少的凋亡(抗凋亡)。这一假设认为，由于砷转化细胞产生ROS的能力较低，细胞产生了凋亡抵抗并增加了细胞存活率，这是砷诱导致癌的总体机制之一。为了检验这一假设，本文提出了三个目标。目的1探讨砷转移细胞中ROS生成减少的机制。我们将研究在砷转化的BEAS-2B细胞中，ROS产生途径的障碍是否与砷诱导的低水平ROS产生有关。我们还将研究转化细胞中抗氧化酶水平的增加是否与ROS生成水平的下降有关。这一目标的完成将建立减少砷转化细胞中ROS产生的机制。目的2探讨ROS生成减少在砷转化细胞抗凋亡中的作用。我们研究了：(A)砷转化细胞中ROS生成减少是否与砷转化细胞的凋亡抵抗有关；(B)砷转化细胞是否比传代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)抗氧化酶的下调或NADPH氧化酶在转化细胞中的过表达是否增加了ROS的生成并促进了砷刺激下的细胞凋亡；(D)下调Bcl-2是否增加了砷转化细胞的凋亡；以及(E)砷转化细胞是否会由于ROS生成和抗凋亡能力的降低而表现出快速生长和增强侵袭和迁移的能力。总体而言，这一目标将证明ROS在砷转化细胞抗凋亡中的作用。目的3研究砷转化细胞诱导的肿瘤发生及细胞凋亡的作用。我们将探讨凋亡抵抗在砷转化细胞肿瘤发生中的作用。我们还将使用皮肤肿瘤形成模型和原位肺癌模型来研究ROS和凋亡调节蛋白Bcl2在砷转化细胞诱导的肿瘤发生中的作用。预期通过下调Bcl2基因使砷转化细胞的凋亡率增加，从而抑制肿瘤生长。类似地，细胞产生ROS能力的改变，即通过异位过表达和敲除ROS清除和产生酶，将对砷转化细胞的肿瘤发生产生影响。

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.