Mechanistic Studies on Lung Cancer Prevention by Tea Polyphenols

描述（由申请人提供）：我们的长期目标是了解茶叶成分的癌症预防作用及其在人类癌症预防中的应用。茶多酚的防癌作用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(-)-表没食子儿茶素-3-没食子酸酯（EGCG）预防肺癌的机制，EGCG是主要的绿茶多酚。我们的目标是建立一个综合的体外和体内实验系统来验证我们的中心假设，即EGCG通过与对癌变至关重要的特定靶分子结合来抑制肺癌的发生，这种结合触发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。为了有效地验证我们的假设，我们计划同时使用体外和体内实验系统。具体目的如下：1。建立肺癌细胞系的体内（异种移植和异体移植）和体外实验系统并进行比较，研究EGCG的作用机制。肺癌细胞系H1299（人）和CL13和CL30（小鼠）将在培养和异种移植/同种异体移植中进行研究。EGCG治疗对肿瘤生长、凋亡、血管生成及相关分子变化（如ERK1/2、AKT、ASK1、JNK、VEGF和氧化应激参数）的影响将在体内和体外进行比较。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，使用亲和色谱法和质谱法。假设的EGCG靶蛋白在介导EGCG作用中的功能作用将通过在工程细胞和在Aim 1中建立的异种移植/同种异体移植模型中进行的功能丧失或获得实验来研究。3. 阐明EGCG在4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）诱导的a /J小鼠肺癌模型中的防癌机制。腺瘤小鼠将接受长期或短期的EGCG治疗。与抑制肿瘤进展相关的细胞和分子变化将通过免疫组织化学、Western blots和其他方法进行研究。我们将测试基于目标1和目标2中关于假定的EGCG分子靶点和相关分子事件的结果所提出的特定假设。更好地了解EGCG预防癌症作用的机制和剂量-反应关系将有助于我们设计未来的人类肺癌预防试验，例如，在戒烟者中。

DESCRIPTION (provided by applicant): 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. Project Narrative: Green tea has been shown to have cancer preventive activity in animal models. This project aims to study the lung cancer preventive mechanisms of the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG). The results will help us to design future lung cancer prevention trials in humans and to assess the effects of tea consumption on human lung cancer.