Oxidative stress, Cr(VI) carcinogenesis, and prevention

 说明(申请人提供)：铬(VI)是众所周知的环境致癌物质。其作用机制或预防机制尚待研究。我们的初步研究表明，人支气管上皮(BEAS-2B)细胞暴露于铬(VI)会产生活性氧(ROS)，这是导致铬(VI)诱导细胞转化的原因。转化后，这些细胞表现出Nrf2及其目标抗氧化蛋白的结构性表达，导致ROS生成减少。Nrf2的结构性表达通过Nrf2与Bcl2基因启动子的抗氧化反应元件(ARE)结合来激活Bcl2，从而提高转化细胞的存活率和致瘤性。这些初步研究表明，在未转化的细胞中，Nrf2的激活可能会减少ROS的产生，从而抑制铬(VI)诱导的细胞转化。相反，在铬(VI)转化的细胞中，Nrf2的结构性表达失活可能会降低Bcl2，增加ROS，从而抑制转化细胞的存活和致瘤性。我们筛选了各种天然化合物，试图选择具有两种特性的化合物：(A)激活未转化细胞中的Nrf2并降低ROS，以防止铬(VI)诱导的细胞转化；(B)抑制Nrf2的结构性表达，并增强转化细胞中的ROS和细胞凋亡，以防止肿瘤的发生。我们发现，木犀草素这一天然化合物具有这样的双重性质。中心假说是，木犀草素通过诱导未转化细胞中Nrf2的瞬时激活，从而抑制铬(VI)诱导的细胞转化，以及通过抑制铬(VI)转化细胞中Nrf2的结构性表达，从而抑制铬(VI)转化细胞的存活、肿瘤发生和血管生成，从而保护铬(VI)诱导的癌变。目的1研究木犀草素对六价铬诱导的细胞转化的预防作用。我们将研究木犀草素诱导Nrf2及其目标抗氧化剂基因的激活，以及减少铬(VI)产生的ROS。这些分子事件的整合很可能是木犀草素对铬(VI)诱导的细胞转化的保护作用。目的2在铬(VI)转化细胞中，木犀草素能够抑制Nrf2的结构性表达，提高ROS水平，降低铬(VI)转化细胞的存活率和致瘤性。此外，还将研究木犀草素对饮水中铬(VI)诱导的动物血管生成的保护作用。目的3利用最近建立的“终生”慢性金属暴露动物模型，研究木犀草素对铬(VI)致CD1小鼠致癌的保护作用。这些动物从繁殖到成年，在存在和不存在木犀草素的情况下，都会通过饮用水暴露于铬(VI)。将研究Nrf2的作用及其关键靶点。肿瘤将在成年后2岁的后代中进行评估。本研究不仅对铬(VI)的致癌机理有了新的认识，而且对于使用针对Nrf2信号通路的化学预防化合物对抗铬(VI)诱导的致癌作用也具有潜在的翻译作用。

 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.