Apoptosis resistance and Cr(VI) carcinogenesis

说明(申请人提供)：铬酸盐(六价铬)化合物对人体有毒致癌。我们的初步研究表明，人肺支气管上皮细胞(BEAS-2B)暴露于铬(VI)会产生活性氧(ROS)。通过ROS反应，铬(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。抗氧化剂调节核因子Nrf2及其正调控因子p62和几种主要抗氧化酶的表达增加。细胞凋亡消除了DNA受损或突变的细胞。当细胞获得抗凋亡能力时，它们会继续增殖，导致癌症的发生。通过裂解多聚ADP核糖聚合酶(C-PARP)和裂解caspase9(C-caspase9)的减少和抗凋亡蛋白Bcl2的升高，铬(VI)转化的细胞表现出对凋亡的抵抗。Nrf2与Bcl2基因的抗氧化反应元件(ARE)结合增加，提示Nrf2可能上调Bcl2的表达。在铬(VI)转化的细胞中，p62的shRNA抑制了Nrf2的表达，导致了细胞凋亡的诱导。这些结果提供了p62、Nrf2、Bcl2和细胞凋亡抵抗之间的联系。这一应用的中心假设是，由于p62和NRF2的上调以及ROS生成的减少，铬(VI)转化的细胞产生了凋亡抵抗，并增加了细胞的存活、侵袭和迁移，从而参与了铬(VI)诱导的致癌的总体机制。目的1探讨铬(VI)转化细胞ROS生成减少的机制。我们将利用未转化细胞和转化细胞进行对比研究，以研究铬(VI)诱导的主要ROS产生途径的每个关键步骤，并确定导致转化细胞中ROS产生减少的具体步骤。我们还将通过重点研究针对抗氧化剂的Nrf2和Nrf2来研究抗氧化剂水平升高的贡献。目的2研究细胞凋亡抵抗及其在促进铬(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在抗细胞凋亡中的作用。我们将研究Nrf2调节的Bcl2诱导是否是一个关键事件。我们将通过改变Bcl2、Bclxl、Mcl-1或Bax的表达来改变细胞的凋亡抵抗，以探讨凋亡抵抗在铬(VI)促进细胞增殖、侵袭和迁移中的作用。目的3利用动物模型研究细胞凋亡抵抗在六价铬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，包括抗氧化酶和NFR2的修饰来研究ROS的作用。我们将通过抑制p62的表达来研究其作用。通过细胞凋亡调节蛋白Bcl2、Bclxl、Mcl-1或Bax的变化来研究细胞凋亡抵抗的作用。同样的方法将被用来通过动物模型来研究ROS、p62和凋亡抵抗在铬(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.