Apoptosis resistance and Cr(VI) carcinogenesis

细胞凋亡抵抗和 Cr(VI) 致癌作用

• 批准号: 8765910
• 负责人: Xianglin Shi
• 金额: $ 33.75万
• 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/8765910
• 关键词: Animal Model; Antioxidants; Apoptosis; Apoptotic; BCL2 gene; Binding; Carcinogens; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Chromates; Cleaved cell; Comparative Study; DNA Damage; Down-Regulation; Enzymes; Epithelial; Equilibrium; Event; Exhibits; Generations; 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; carcinogenesis; caspase-9; catalase; cell transformation; chromium hexavalent ion; exposed human population; genetic regulatory protein; migration; overexpression; public health relevance; 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））化合物对人类有毒和致癌。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）产生的活性氧（ROS）。通过ROS反应，CR（VI）导致细胞转化，导致肿瘤发生。但是，一旦细胞转化，这些细胞产生ROS的能力就会降低。抗氧化剂调节性核因子NRF2的表达，其阳性调节剂P62和几种主要的抗氧化剂酶的表达增加了。凋亡消除了DNA受损或突变的细胞。当细胞获得抗凋亡耐药性时，它们会继续增殖，从而导致致癌。 CR（VI）转化的细胞在裂解的聚（ADP-核糖）聚合酶（C-PARP）和切割的caspase 9（C-Caspase 9）以及抗凋亡蛋白BCl-2的升高表明，形成了凋亡抗性。 NRF2与Bcl-2基因的抗氧化反应元件（IS）的结合增加了，表明NRF2在Bcl-2上调中的可能性。在CR（VI）转化的细胞中，通过其shRNA抑制p62降低了NRF2的表达，从而导致凋亡诱导。这些结果提供了P62，NRF2，BCL-2和抗凋亡耐药性之间的联系。该应用的核心假设是，由于p62和NRF2的上调以及ROS（VI）转化细胞的产生降低引起的凋亡耐药性并增加了细胞的存活，侵袭和迁移，这有助于CR（VI）诱导的癌变的整体机制。 AIM 1将研究降低Cr（VI）转化细胞的ROS生成降低的机制。我们将使用非转化和CR（VI）转化的细胞进行比较研究，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）转换细胞中ROS生成降低的特定步骤。我们还将通过关注靶向抗氧化剂的NRF2和NRF2来研究升高的抗氧化剂水平的贡献。 AIM 2将研究抗凋亡抗性及其在增强CR（VI）转化细胞增殖，侵袭和迁移中的作用。我们将通过上调参与ROS生成途径的关键蛋白以及下调关键抗氧化剂酶来改变ROS的产生，以研究ROS在凋亡耐药性中的作用。我们将研究NRF2调节的BCL-2诱导是否是关键事件。我们将通过修改Bcl-2，Bcl-XL，MCL-1或BAX表达来改变凋亡的抗性，以研究凋亡耐药性在CR（VI）增强细胞增殖，侵袭和迁移中的作用。 AIM 3将研究使用动物模型在CR（VI）诱导的肿瘤发生和转移中的凋亡耐药性的作用。我们将通过改变ROS的产生（包括抗氧化剂酶和NFR2的修饰）来研究ROS的作用。 p62的作用将通过抑制其表达来研究。凋亡耐药性的作用将通过凋亡调节蛋白（BCL-2，BCL-XL，MCL-1或BAX）的交替进行研究。使用动物模型，将使用相同的方法来研究ROS，p62和抗凋亡耐药性在CR（VI）转化细胞转移细胞中的作用。