FREE RADICALS AND CHOLINE DEFICIENT LIVER CARCINOGENESIS

活性氧（ROS）多年来一直与癌症的发展有关。与ROS密切相关的一个主要例子是模型系统，在该系统中，给大鼠喂食胆碱缺乏（CD）饮食导致肝细胞癌（HCC）的发展，即完全没有暴露于任何外源性已知致癌物。利用这一模型，我们进行了新的观察，使活性氧与关键信号转导途径之间的联系成为可能，这些信号转导途径已被证明是癌症发生和发展的基础。我们首次证明了cd -肝脏的线粒体发生了变化，从而介导了H202的显著提高。此外，我们首次证明了PBN （a-苯基-丁基硝基），一种基于硝基的自由基诱捕剂，在极低水平的化合物下显着减少瘤前结节的发展并抑制肝细胞癌（HCC）的形成。PBN是该模型中研究过的最有效的抗癌物质。为了解释这些观察结果，我们假设cd方案介导线粒体膜的变化，使它们产生更高水平的H2O2，而PBN通过作用于复合物1显著抑制过量的H202产生。我们进一步假设过量的H202会导致PTEN肿瘤抑制蛋白的失活增强，从而导致其磷酸酶活性的丧失，从而介导akt激酶途径的激活，导致细胞凋亡药物过程减少，但致癌事件增加。我们进一步提出，由于过量的H202和某些生长因子（最有可能是TGFbeta1）的作用，在cd肝脏中形成的瘤前结节中的细胞更容易发生肿瘤（而不是凋亡），而PBN通过抑制过量的H202产生和抑制增强的信号转导过程来改变这些过程。我们提出PBN的作用是使癌前结节细胞倾向于凋亡过程，从而抑制肿瘤的发展。为了验证这一假设，我们提出了3个具体目标。简而言之，它们是：A)我们将确定导致它们产生过量H202的线粒体膜变化的性质。B)我们将重点关注H2O2作为信号分子的机制以及PBN在细胞模型中改变信号转导过程的作用。C)我们将确定PBN（或其代谢物4-OH-PBN）是否抑制cd介导的HCC发展，并确定这些化合物是否增加了该模型中肿瘤前细胞的凋亡。

Reactive oxygen species (ROS) have been implicated in cancer development for many years. A prime example where ROS are strongly implicated is the model system where feeding a choline deficiency (CD) diet to rats leads to hepatocellular carcinoma (HCC) development, i.e., in the complete absence of exposure to any exogenous known carcinogen. Utilizing this model, we have made novel observations that make it possible to link ROS with key signal transduction pathways that have been shown to be fundamental in cancer initiation and development. For the first time we have shown that mitochondria from CD-livers are changed such that they mediate a significantly higher yield of H202 production. Additionally, for the first time we have shown that PBN (a- phenyl-tent-butyl nitrone), a nitrone-based free radical trap, significantly reduces preneoplastic nodule development as well as inhibits hepatocellular carcinoma (HCC) formation at very low levels of the compound. PBN is the most potent anti-carcinogen ever studied in this model. To explain these observations we postulate that the CD-regimen mediates changes in mitochondrial membranes such that they produce enhanced levels of H2O2 and that PBN significantly inhibits the excess H202 production by acting at Complex 1. We further postulate that excess H202 causes an enhanced inactivation of the PTEN tumor suppressor protein, which causes a loss of its phosphatase activity and thereby mediates a shift toward the activation of the AKT-kinase pathway resulting in a decrease in apoptosis medicated processes but an increase in oncogenic events. We further propose that the cells in preneoplastic nodules that develop in CD-livers are predisposed toward oncogenesis (as opposed to apoptosis) because of the action of excess H202 and certain growth factors (most likely TGFbeta1) and that PBN alters these processes through both its inhibition of excess H202 production and also by suppression of enhanced signal transduction processes. We propose that PBN acts to cause the prenoplastic nodule cells to become predisposed toward apoptic processes thus leading to inhibition of tumor development. To test this hypothesis we have proposed 3 specific aims. Briefly they are: A) We will determine the nature of the alterations in mitochondrial membranes that cause them to produce excess H202. B) We will focus on the mechanisms of how H2O2 acts as a signaling molecule and the action of PBN in altering signal transduction processes in cell models. C) We will determine if PBN (or its metabolite 4-OH-PBN) inhibits CD-mediated HCC development and ascertain if these compounds increase apoptosis in preneoplastic cells in this model.