Oxidative stress, Cr(VI) carcinogenesis, and prevention

 描述（由申请人提供）：Cr（VI）是众所周知的环境致癌物。其作用或预防机制仍有待研究。我们的初步研究表明，暴露于铬（VI）的人支气管上皮细胞（BEAS-2B）产生活性氧（ROS），这是负责铬（VI）诱导的细胞转化。转化后，这些细胞表现出Nrf 2及其靶抗氧化蛋白的组成型表达，导致ROS产生减少。Nrf 2的组成型表达通过Nrf 2与Bcl-2基因启动子的抗氧化反应元件（ARE）结合来激活Bcl-2，导致这些转化细胞的存活和致瘤性增加。这些初步研究表明，在非转化细胞中，Nrf 2的活化可以减少ROS的产生，从而抑制Cr（VI）诱导的细胞转化。相反，在Cr（VI）转化细胞中，Nrf 2组成型表达的失活可能会降低Bcl-2和增加ROS，导致转化细胞的存活和致瘤性抑制。我们筛选了各种天然化合物，试图选择具有两种性质的化合物：（a）在非转化细胞中激活Nrf 2并降低ROS以防止Cr（VI）诱导的细胞转化，和（B）在Cr（VI）转化细胞中抑制Nrf 2的组成型表达并增强ROS和细胞凋亡以防止肿瘤发生。我们发现，毛地黄黄酮，一种天然化合物，具有这样的双重性质。中心假设是毛地黄黄酮通过诱导非转化细胞中Nrf 2的瞬时激活，导致抑制Cr（VI）诱导的细胞转化，以及通过使Cr（VI）转化细胞中Nrf 2的组成型表达失活，导致抑制Cr（VI）转化细胞的存活和肿瘤发生以及血管生成，来保护Cr（VI）诱导的致癌作用。目的1研究毛地黄黄酮对Cr（VI）诱导的细胞转化的抑制作用。我们将研究木犀草素诱导的Nrf 2及其靶抗氧化基因的激活，以及Cr（VI）产生的ROS的减少。这些分子事件的整合可能是负责保护毛地黄黄酮对铬（VI）诱导的细胞转化。目的2证明在Cr（VI）转化细胞中，毛地黄黄酮能够抑制Nrf 2的组成性表达，增加ROS水平，并降低Cr（VI）转化细胞的存活率和致瘤性。还将研究毛地黄黄酮对通过饮用水暴露于Cr（VI）的动物中Cr（VI）诱导的血管生成的保护作用。目的3将探讨毛地黄黄酮对铬（VI）诱导的致癌性在CD 1小鼠使用最近建立的动物模型的“终身”慢性金属暴露的保护。在存在和不存在毛地黄黄酮的情况下，动物将在从繁殖到成年的整个生命周期内通过饮用水暴露于Cr（VI）。将研究Nrf 2及其关键靶点的作用。将在长达2岁的成年后代中评估肿瘤。该研究不仅将获得Cr（VI）致癌作用的新机制，而且还将对使用靶向Nrf 2信号通路的化学预防化合物对抗Cr（VI）诱导的致癌性产生潜在的转化影响。

 DESCRIPTION (provided by applicant): Cr(VI) is well known environmental carcinogen. Its mechanism of action or prevention remains to be investigated. Our preliminary studies have shown that exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) generates reactive oxygen species (ROS) which are responsible for Cr(VI)-induced cell transformation. After transformation, these cells exhibit constitutive expressions of Nrf2 and its target antioxidant proteins, resulting in decreased ROS generation. The constitutive expression of Nrf2 activates Bcl-2 through binding of Nrf2 to antioxidant responsive element (ARE) of Bcl-2 gene promoter, leading to increased survival and tumorigenicity of these transformed cells. These preliminary studies indicate that in non-transformed cells activation of Nrf2 may decrease ROS generation, thus inhibit Cr(VI)-induced cell transformation. In contrast, in Cr(VI)-transformed cells inactivation of constitutive expression of Nrf2 may decrease Bcl-2 and increase ROS, leading to inhibitions of survival and tumorigenicity of transformed cells. We have screened various natural compounds attempting to select those with two properties: (a) to activate Nrf2 and decrease ROS in non- transformed cells to prevent Cr(VI)-induced cell transformation and (b) to inhibit constitutive expression of Nrf2 and enhance ROS and apoptosis in Cr(VI)-transformed cells to prevent tumorigenesis. We have found that luteolin, a natural compound, has such dual properties. The central hypothesis is that luteolin protects Cr(VI)- induced carcinogenesis both by inducing transient activation of Nrf2 in non-transformed cells, resulting in inhibition of Cr(VI-induced cell transformation and by inactivating constitutive expression of Nrf2 in Cr(VI)- transformed cells, resulting in inhibitions of survival and tumorigenesi of Cr(VI)-transformed cell and angiogenesis. Aim 1 will study prevention of luteolin against Cr(VI)-induced cell transformation. We will study luteolin-induced activations of Nrf2 and its target antioxidant genes, and decreases of Cr(VI)-generated ROS. The integration of these molecular events is likely to be responsible for protection of luteolin against Cr(VI)- induced cells transformation. Aim 2 will demonstrate that in Cr(VI)-transformed cells luteolin is able to inhibit constitutive expressions of Nrf2, increase ROS level, and decrease survival and tumorigenicity of Cr(VI)- transformed cells. The protection of luteolin against Cr(VI)-induced angiogenesis in animal exposed to Cr(VI) via drinking water will also be investigated. Aim 3 will investigate protection o luteolin against Cr(VI)-induced carcinogenicity in CD1 mice using a recently established animal model of "whole-life" chronic metal exposure. The animals will be exposed to Cr(VI) in the presence and absence of luteolin via drinking water in the whole life time from breeding to adulthood. The roles of Nrf2 and its key target will be studied. Tumors will be assessed in the offspring up to 2 years of adulthood. The proposed study will not only gain novel mechanistic insight in Cr(VI) carcinogenesis, but also have potential translational impact of using chemopreventive compounds targeting Nrf2 signaling pathway to combat Cr(VI)-induced carcinogenicity.