Apoptosis resistance and Cr(VI) carcinogenesis

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，人肺支气管上皮细胞（BEAS-2B）暴露于Cr （VI）会产生活性氧（ROS）。Cr（VI）通过ROS反应引起细胞转化，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及几种主要抗氧化酶的表达均升高。细胞凋亡消除dna受损或突变的细胞。当细胞获得抗凋亡能力时，它们继续增殖，导致癌变。Cr（VI）转化的细胞产生了凋亡抗性，其表现为cleaved poly（adp -核糖）聚合酶（C-PARP）和cleaved caspase 9 （C-caspase 9）的减少以及抗凋亡蛋白Bcl-2的升高。Nrf2与Bcl-2基因抗氧化反应元件（anti - oxide response element， ARE）的结合增加，提示Nrf2可能参与上调Bcl-2的表达。在Cr （VI）转化的细胞中，通过其shRNA抑制p62可降低Nrf2的表达，从而诱导细胞凋亡。这些结果提供了p62、Nrf2、Bcl- 2和细胞凋亡抵抗之间的联系。本研究的中心假设是，由于p62和Nrf2的上调和ROS的减少，Cr （VI）转化的细胞产生凋亡抵抗，增加细胞存活、侵袭和迁移，从而参与了Cr （VI）诱导癌变的整体机制。目的1将探讨Cr（VI）转化细胞ROS生成减少的机制。我们将采用未转化和Cr(VI)-转化细胞进行对比研究，研究Cr(VI)-诱导的主要ROS生成途径的每个关键步骤，并确定Cr(VI)-转化细胞中导致ROS生成减少的具体步骤。我们还将通过Nrf2和Nrf2靶向抗氧化剂来研究抗氧化剂水平升高的贡献。目的2将研究细胞凋亡抵抗及其在增强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和下调关键抗氧化酶来改变ROS的产生，研究ROS在细胞凋亡抵抗中的作用。我们将研究nrf2调控的Bcl-2诱导是否是一个关键事件。我们将通过改变Bcl-2、Bcl-xL、Mcl-1或Bax的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。目的3将通过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，包括抗氧化酶和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。凋亡抵抗的作用将通过改变凋亡调节蛋白Bcl-2、Bcl-xL、Mcl-1或Bax来研究。同样的方法将用于研究ROS、p62和细胞凋亡抵抗在动物模型中Cr（VI）转化细胞转移中的作用。

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.