The Effects of Grape Seed Extract Against Lung Cancer

描述（由申请人提供）： 葡萄籽提取物（GSE）被广泛用于促进心血管健康，含有高水平的原花青素，具有强大的抗氧化特性。在初步的体外研究中，我们确定了多种GSE介导的抗肿瘤机制，包括通过下调特定microRNA（miRNA）诱导细胞凋亡和抑制细胞增殖。我们现在的目标是转化这些体外研究结果，并为未来的临床试验奠定基础。与大豆磷脂复合成磷脂复合物以提高生物利用度的标准化GSE制剂（Leucoselect）可在柜台购买。Leucoselect已被证明可以提高重度吸烟者血浆的总抗氧化能力，降低LDL对氧化应激的敏感性，但Leucoselect对肺癌的潜力从未被评估过。因此，我们假设口服Leucoselect将产生抗肿瘤作用， 有利地调节与肺癌相关的机制。提出了三个具体目标：目标#1：确定Leucoselect在小鼠模型中的最大耐受剂量（MTD）、药代动力学（PK）/药效学（PD）和抗癌作用。将在裸小鼠中经口灌胃给予不同剂量的leucoselect进行剂量范围探索研究。将获得血液和肺样本，以确定葡萄籽原花青素（GSP）和代谢物的PK，作为GSE生物利用度的替代标志物（全身和肺部），并确定2周治疗期间的MTD。目标1.1：使用快速冷冻小鼠肺切片与人肺肿瘤细胞的新型共培养系统，测定口服Leucoselect在靶器官部位的生物活性。将通过比较与人非小细胞肺癌（NSCLC）细胞系A549和支气管癌前病变细胞系1198共培养的对照与Leucoselect处理小鼠的肺组织切片，评估肺中的生物活性。不同GSE剂量对共培养的A549和1198细胞中增殖和凋亡的影响将与GSP和代谢物水平相关。目的#2：确定Leucoselect对肺癌异种移植模型的抗癌作用。基于MTD，将通过经口灌胃给予携带皮下NSCLC异种移植物的小鼠不同剂量的Leucoselect长达8周，并从每个治疗组连续收集血浆、肺组织和肿瘤异种移植物。抗癌作用将通过肿瘤生长延迟或达到最大肿瘤体积的时间、增殖（Ki-67）和凋亡（裂解的半胱天冬酶3）指数确定，并与血浆、肺组织和异种移植肿瘤样本中的GSP水平相关。目的#2.1：进一步表征Leucoselect在异种移植模型中对肺癌的机制作用并使其相关。GSE的机制作用将通过以下调节来测量：1）考克斯-2表达和PGE 2产生，和2）炎症的其它标志物：15（S）-羟基-二十碳四烯酸（15-HETE），白细胞介素（IL）-6，IL-10，和3）mir-19 a，-19 b和-106 b水平，4）癌症相关的途径特异性基因表达谱（GEP），5）microRNA表达，以及6）各种样本类型中常见的癌变生物标志物，例如p53、p27、p21和p16。目标3：验证和表征mir-19 a、mir-19 b和mir-106 b在介导Leucoselect抗肿瘤作用中的作用，并检查血浆mir-19 a、mir-19 b、mir-106 b作为SEBM用于治疗监测的潜力。在我们的初步体外研究中，我们已经确定了3个oncomir：mir-19 a，mir-19 b和mir-106 b在NSCLC和1198细胞系中通过GSE显著下调。这些miRNAs是被鉴定为肺癌发展的预测因子的主要血浆miRNAs之一。因此，我们将测量从异种移植模型获得的血浆中的这些miRNA水平，以确定它们作为替代终点生物标志物（SEBM）的效用。影响：该研究的结果将为GSE对抗肺癌的可行性和机制效应提供重要见解，有助于识别SEBM并为不久的将来的临床试验铺平道路。管理和预防的潜在收益 肺癌是巨大的。

DESCRIPTION (provided by applicant): Widely used to promote cardiovascular health, grape seed extract (GSE) contains high levels of proanthocyanidins with strong antioxidant properties. In preliminary in vitro studies, we identifie a variety of GSE-mediated anti-neoplastic mechanisms, including induction of apoptosis and inhibition of cell proliferation via down-regulation of specific microRNA (miRNA). We now aim to translate these in vitro findings and set the stage for future clinical trial. A standardized GSE preparation complexed with soy phospholipids into phytosomes to improve bioavailability (Leucoselect) is available over the counter. Leucoselect has been shown to improve the total antioxidant capacity of plasma and reduce LDL susceptibility to oxidative stress in heavy smokers, but the potential of Leucoselect against lung cancer has never been evaluated. We therefore hypothesize that oral administration of Leucoselect will exert antineoplastic effects and favorably modulate mechanisms associated with lung cancer. Three specific aims are proposed: Aim #1: To determine the maximum tolerated dose (MTD), pharmacokinetics (PK)/ pharmacodynamics (PD), and anti-cancer effects of Leucoselect in mouse models. A dose range finding study with leucoselect will be conducted in nude mice given varying doses via oral gavage. Blood and lung samples will be obtained to determine PK of grape seed procyanidin (GSP) and metabolites as surrogate markers of bioavailability of GSE (systemically and in the lungs), and to establish the MTD over 2 weeks treatment duration. Aim #1.1: To determine the bioactivity of oral Leucoselect at the targeted organ site using a novel co-culture system of snap frozen mouse lung sections with human lung neoplastic cells. Bioactivity in the lungs will be assessed by comparing lung tissue sections from control vs. Leucoselect treated mice co-cultured with the human nonsmall cell lung cancer (NSCLC) cell line, A549, and the bronchial premaligant cell line, 1198. The effects of varying GSE doses on proliferation and apoptosis in co-cultured A549 and 1198 cells will be correlated to GSP and metabolites levels. Aim #2: To determine the anti-caner effects of Leucoselect on lung cancer xenograft models. Varying doses of Leucoselect based on MTD will be administered via oral gavage to mice bearing subcutaneous NSCLC xenografts for up to 8 weeks, with serial collections of plasma, lung tissues and tumor xenografts from each treatment group. The anticancer effects will be determined by tumor growth delay or time to reach maximum tumor volume, proliferation (Ki-67) and apoptotic (cleaved caspase 3) indices, and correlated to GSP levels in plasma, lung tissue, and xenograft tumor samples. Aim #2.1: To further characterize and correlate the mechanistic effects of Leucoselect against lung cancer in xenograft models. The mechanistic effects of GSE will be measured by modulations of: 1) COX-2 expression and PGE2 production, and 2) Additional markers of inflammations: 15(S)-hydroxy-eicosatetraenoic acid (15-HETE), interleukin (IL)-6, IL-10, and 3) mir-19a, -19b and -106b levels, 4) cancer relevant, pathway specific gene expression profiles (GEP), 5) microRNA expression, and 6) common biomarkers of cancerization such as p53, p27, p21 and p16, in various sample types. Aim #3: To validate and characterize the roles of mir-19a, mir-19b, and mir-106b in mediating the anti- neoplastic effects of Leucoselect and examine the potential of plasma mir-19a, mir-19b, mir-106b as SEBM for therapeutic monitoring. In our preliminary in vitro studies, we have identified significant down-regulation of 3 oncomirs: mir-19a, mir-19b, and mir-106b in NSCLC and 1198 cell lines by GSE. These miRNAs are among the major plasma miRNAs identified to be predictors of lung cancer development. Therefore, we will measure these miRNA levels in plasma obtained from xenograft models to determine their utility as surrogate endpoint biomarkers (SEBM). IMPACT: Findings from the study will provide important insights into the feasibility and mechanistic effects of GSE against lung cancer, help identify SEBM and pave the way for clinical trials in the near future. The potential gain for the management and prevention of lung cancer is enormous.