Apoptosis resistance and Cr(VI) carcinogenesis

描述（由申请人提供）：铬酸盐（Cr（VI））化合物对人体有毒且致癌。我们的初步研究表明，暴露于Cr（VI）的人肺支气管上皮细胞（BEAS-2B）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的能力就会降低。抗氧化调节核因子Nrf 2及其正调节因子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。当细胞获得凋亡抗性时，它们继续增殖，导致癌变。Cr（VI）转化的细胞产生凋亡抗性，如通过裂解的聚（ADP-核糖）聚合酶（C-PARP）和裂解的半胱天冬酶9（C-caspase 9）的减少以及通过抗凋亡蛋白Bcl-2的升高所指示的。Nrf 2与Bcl-2基因抗氧化反应元件（ARE）的结合增强，提示Nrf 2可能参与了Bcl-2基因的上调。在Cr（VI）转化的细胞中，通过其shRNA抑制p62降低了Nrf 2的表达，导致诱导凋亡。这些结果提供了p62、Nrf 2、Bcl- 2和凋亡抗性之间的联系。本申请的中心假设是，由于p62和Nrf 2的上调和ROS的产生减少，Cr（VI）转化的细胞产生凋亡抗性并增加细胞存活、侵袭和迁移，从而促进Cr（VI）诱导的致癌的总体机制。目的1探讨Cr（VI）转化细胞减少ROS产生的机制。我们将使用未转化和Cr（VI）转化的细胞进行比较研究，以研究主要Cr（VI）诱导的ROS产生途径的每个关键步骤，并确定负责减少Cr（VI）转化细胞中ROS产生的特定步骤。我们还将通过关注Nrf 2和Nrf 2靶向抗氧化剂来研究抗氧化剂水平升高的贡献。目的2将研究凋亡抵抗及其在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，以研究ROS在抗凋亡中的作用。我们将研究Nrf 2调节的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的表达来改变凋亡抵抗，以研究凋亡抵抗在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的产生来研究ROS的作用，包括抗氧化酶和Nfr 2的修饰。p62的作用将通过抑制其表达来研究。凋亡抗性的作用将通过凋亡调节蛋白Bcl-2、Bcl-xL、Mcl-1或Bax的交替来研究。同样的方法将被用来研究的作用，ROS，p62，和细胞凋亡的阻力，转移的Cr（VI）转化细胞使用动物模型。

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.