Apoptosis resistance and Cr(VI) carcinogenesis

细胞凋亡抵抗和 Cr(VI) 致癌作用

• 批准号: 9058060
• 负责人: Xianglin Shi
• 金额: $ 33.86万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9058060
• 关键词: Animal Model; Antioxidants; Apoptosis; Apoptotic; BCL2 gene; Binding; CASP9 gene; Carcinogens; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Chromates; Cleaved cell; Comparative Study; DNA Damage; Down-Regulation; Epithelial; Equilibrium; Event; Exhibits; Generations; Health; Heme; Human; Induction of Apoptosis; Industry; Lung; Malignant - descriptor; Mediating; Modification; Mutate; NQO1 gene; Neoplasm Metastasis; Normal Cell; Nuclear; Oxidases; Oxidoreductase; Oxygenases; Pathway interactions; Play; Poison; Poly(ADP-ribose) Polymerases; Process; Production; Proliferating; Promoter Regions; Proteins; Quinine; Reaction; Reactive Oxygen Species; Regulation; Resistance; Response Elements; Role; Staging; Superoxide Dismutase; System; Testing; Up-Regulation; antioxidant enzyme; carcinogenesis; catalase; cell transformation; chromium hexavalent ion; exposed human population; genetic regulatory protein; migration; overexpression; small hairpin RNA; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Chromate (Cr (VI)) compounds are toxic and carcinogenic on humans. Our preliminary studies show that exposure of human lung bronchial epithelial (BEAS-2B) cells to Cr (VI) generated reactive oxygen species (ROS). Through ROS reactions, Cr(VI) caused cell transformation, leading to tumorigenesis. However, once cells were transformed, the capability of those cells to generate ROS was decreased. Expressions of antioxidant regulative nuclear factor Nrf2, its positive regulator, p62, and several major antioxidant enzymes were increased. Apoptosis eliminates DNA-damaged or mutated cells. When cells acquire apoptosis resistance, they continue to proliferate, leading to carcinogenesis. Cr(VI)-transformed cells developed apoptosis resistance as indicated by reductions of cleaved poly(ADP-ribose) polymerase (C-PARP) and cleaved caspase 9 (C-caspase 9) and by elevation of anti- apoptotic protein Bcl-2. Binding of Nrf2 to antioxidant response element (ARE) of Bcl-2 gene was increased, indicating the possibility of Nrf2 in up-regulation of Bcl-2. In Cr (VI)-transformed cells, inhibition of p62 by its shRNA reduced Nrf2 expression, leading to induction of apoptosis. These results provide a linkage among p62, Nrf2, Bcl- 2, and apoptosis resistance. The central hypothesis of this application is that due to up-regulations of p62 and Nrf2 and decreased generation of ROS, Cr (VI)-transformed cells develop apoptosis resistance and increase cell survival, invasion, and migration, contributing to overall mechanism of Cr (VI)-induced carcinogenesis. Aim 1 will investigate the mechanism of decreased ROS generation of Cr(VI)-transformed cells. We will carry out comparative studies using non-transformed and Cr(VI)- transformed cells to study each key step of major Cr(VI)- induced ROS generation pathway and identify the specific step responsible for decreased ROS generation in Cr(VI)-transformed cells. We will also investigate the contribution of elevated antioxidant level by focusing on Nrf2 and Nrf2 targeting antioxidants. Aim 2 will investigate apoptosis resistance and its role in enhanced proliferation, invasion, and migration of Cr(VI)-transformed cells. We will alter ROS production by up-regulation of key proteins involved in ROS generation pathway and by down-regulation of key antioxidant enzymes to study the role of ROS in apoptosis resistance. We will investigate whether Nrf2-regulated Bcl-2 induction is a key event. We will alter apoptosis resistance by modifying Bcl-2, Bcl-xL, Mcl-1, or Bax expression to investigate the role of apoptosis resistance in Cr(VI)-enhanced cell proliferation, invasion, and migration. Aim 3 will investigate the role of apoptosis resistance in Cr(VI)-induced tumorigenesis and metastasis using animal models. We will investigate the role of ROS by altering ROS generation including modification of antioxidant enzymes and Nfr2. The role of p62 will be investigated by inhibiting its expression. The role of apoptosis resistance will be investigated by alternation of apoptosis regulative proteins, Bcl-2, Bcl-xL, Mcl-1, or Bax. The same approaches will be used to investigate the role of ROS, p62, and apoptosis resistance in metastasis of Cr(VI)-transformed cells using animal models.

描述（由申请人提供）：铬酸盐（Cr（VI））化合物对人类有毒且致癌。我们的初步研究表明，人肺支气管上皮 (BEAS-2B) 细胞暴露于 Cr (VI) 会产生活性氧 (ROS)。通过ROS反应，Cr(VI)引起细胞转化，导致肿瘤发生。然而，一旦细胞被转化，这些细胞产生ROS的能力就会下降。抗氧化调节核因子Nrf2、其正调节因子、p62和几种主要抗氧化酶的表达增加。细胞凋亡消除 DNA 损伤或突变的细胞。当细胞获得凋亡抵抗时，它们会继续增殖，导致癌变。 Cr(VI) 转化的细胞产生了细胞凋亡抗性，如裂解聚 (ADP-核糖) 聚合酶 (C-PARP) 和裂解 caspase 9 (C-caspase 9) 的减少以及抗凋亡蛋白 Bcl-2 的升高所示。 Nrf2与Bcl-2基因抗氧化反应元件(ARE)的结合增加，表明Nrf2上调Bcl-2的可能性。在 Cr (VI) 转化的细胞中，p62 的 shRNA 抑制会降低 Nrf2 的表达，从而诱导细胞凋亡。这些结果提供了 p62、Nrf2、Bcl-2 和细胞凋亡抗性之间的联系。该申请的中心假设是，由于 p62 和 Nrf2 的上调以及 ROS 生成的减少，Cr (VI) 转化的细胞产生凋亡抵抗并增加细胞存活、侵袭和迁移，从而促进 Cr (VI) 诱导癌变的总体机制。目标 1 将研究 Cr(VI) 转化细胞中 ROS 生成减少的机制。我们将使用非转化细胞和 Cr(VI) 转化细胞进行比较研究，以研究主要 Cr(VI) 诱导的 ROS 生成途径的每个关键步骤，并确定导致 Cr(VI) 转化细胞中 ROS 生成减少的具体步骤。我们还将通过关注 Nrf2 和 Nrf2 靶向抗氧化剂来研究抗氧化剂水平升高的贡献。目标 2 将研究细胞凋亡抵抗及其在 Cr(VI) 转化细胞增殖、侵袭和迁移增强中的作用。我们将通过上调参与ROS生成途径的关键蛋白和下调关键抗氧化酶来改变ROS的产生，以研究ROS在细胞凋亡抵抗中的作用。我们将研究 Nrf2 调节的 Bcl-2 诱导是否是一个关键事件。我们将通过修改 Bcl-2、Bcl-xL、Mcl-1 或 Bax 表达来改变细胞凋亡抵抗，以研究细胞凋亡抵抗在 Cr(VI) 增强的细胞增殖、侵袭和迁移中的作用。目标 3 将利用动物模型研究细胞凋亡抵抗在 Cr(VI) 诱导的肿瘤发生和转移中的作用。我们将通过改变 ROS 的生成（包括修饰抗氧化酶和 Nfr2）来研究 ROS 的作用。 p62 的作用将通过抑制其表达来研究。通过细胞凋亡调节蛋白 Bcl-2、Bcl-xL、Mcl-1 或 Bax 的交替来研究细胞凋亡抵抗的作用。相同的方法将用于使用动物模型研究 ROS、p62 和细胞凋亡抗性在 Cr(VI) 转化细胞转移中的作用。