Mechanistic Studies on Lung Cancer Prevention by Tea Polyphenols

我们的长期目标是了解茶叶成分的癌症预防活性及其在人类癌症预防中的应用。茶多酚的防癌活性已在动物模型中得到证实，但其作用机制尚不清楚。本项目旨在阐明主要绿茶多酚-表没食子儿茶素没食子酸酯(EGCG)预防肺癌的机制。我们的目标是建立一个完整的体外和体内实验系统来验证我们的中心假设，即EGCG通过与特定的致癌靶分子结合来抑制肺癌的发生，这种结合触发了信号转导途径的改变，从而导致癌前和恶性细胞的生长抑制和凋亡。为了有效地检验我们的假设，我们计划使用体外和体内实验系统。具体目标如下： 1.建立并比较体内(异种移植和同种异体移植)和体外实验系统对肺癌细胞株的作用，探讨EGCG的作用机制。肺癌细胞系H1299(人)以及CL13和CL30(小鼠)将在培养和异种/同种移植中进行研究。我们将在体内和体外比较EGCG治疗对肿瘤生长、细胞凋亡、血管生成和相关分子变化(如ERK1/2、AKT、ASK1、JNK、VEGF和氧化应激参数)的影响。 2.通过亲和蛋白质组学和功能研究确定EGCG的直接分子靶点。为了确定EGCG的直接分子靶点，我们将与明尼苏达大学的董子刚博士合作，使用亲和层析和质谱仪。在工程细胞和目标1中建立的异种移植/同种移植模型中，将通过功能丧失或获得实验来研究假定的EGCG目标蛋白在介导EGCG行动中的功能作用。 3.在4-(甲基亚硝基)-1-(3-吡啶)-1-丁酮(NNK)诱导的A/J小鼠肺癌模型中，阐明EGCG的防癌作用机制。携带腺瘤的小鼠将接受EGCG的长期或短期治疗。与抑制肿瘤进展相关的细胞和分子变化将使用免疫组织化学、Western blotts和其他方法进行研究。我们将测试基于目标1和目标2的结果开发的关于假定的EGCG分子靶标和相关分子事件的特定假设。更好地了解EGCG预防癌症的机制和剂量-反应关系将有助于我们设计未来在人类身上的肺癌预防试验，例如，在前吸烟者身上。

Our long-term goal is to understand the cancer preventive activities of tea constituents and their applications to human cancer prevention. The cancer preventive activity of tea polyphenols has been demonstrated in animal models, but the mechanisms of action are not clearly understood. This project aims to elucidate the mechanisms of lung cancer prevention by (-)-epigallocatechin-3-gallate (EGCG), the major green tea polyphenol. Our goal is to establish an integrated in vitro and in vivo experimental system to test our central hypothesis that EGCG inhibits lung carcinogenesis by binding to specific target molecules which are vital to carcinogenesis and this binding triggers alterations in signal transduction pathways that lead to growth inhibition and apoptosis of premalignant and malignant cells. In order to effectively test our hypothesis, we plan to use both in vitro and in vivo experimental systems. The specific aims are as follows: 1. Establish and compare in vivo (xenograft & allograft) and in vitro experimental systems with lung cancer cell lines and study the mechanisms of action of EGCG. Lung cancer cell lines H1299 (human) and CL13 and CL30 (mouse) will be studied in culture and in xenografts/allografts. The effects of EGCG treatment on tumor growth, apoptosis, angiogenesis, and related molecular changes (e.g., ERK1/2, AKT, ASK1, JNK, VEGF, and oxidative stress parameters) will be compared in vivo vs. in vitro. 2. Identify direct molecular targets of EGCG by affinity-proteomics and functional studies. To identify direct molecular targets for EGCG, we will use affinity chromatography and mass spectrometry in collaboration with Dr. Zigang Dong at the University of Minnesota. The functional roles of the putative EGCG target proteins in mediating the action of EGCG will be studied with loss or gain of function experiments in engineered cells and in a xenograft/allograft model established in Aim 1. 3. Elucidate the mechanisms of cancer prevention by EGCG in a 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK)-induced lung carcinogenesis model in A/J mice. Adenoma-bearing mice will receive long- or short-term treatment with EGCG. The cellular and molecular changes related to the inhibition of tumor progression will be investigated using immunohistochemistry, Western blots, and other approaches. We will test specific hypotheses developed based on results from Aims 1 and 2 concerning putative EGCG molecular targets and related molecular events. A better understanding of the mechanisms of the cancer preventive action of EGCG and the dose- response relationship will help us to design future lung cancer prevention trials in humans, for example, in ex-smokers.