The Effects of Grape Seed Extract Against Lung Cancer

描述(由申请人提供)： 葡萄籽提取物(GSE)广泛用于促进心血管健康，含有高水平的原花青素，具有很强的抗氧化性能。在初步的体外研究中，我们确认了GSE介导的多种抗肿瘤机制，包括通过下调特定的microRNA(MiRNA)诱导细胞凋亡和抑制细胞增殖。我们现在的目标是将这些体外发现转化为未来的临床试验。一种标准化的GSE制剂与大豆磷脂复合成植酸体以提高生物利用度(Leucoselect)在非处方药中可用。Leucoselect已被证明可以提高血浆总抗氧化能力，降低重度吸烟者对氧化应激的低密度脂蛋白敏感性，但Leucoselect抗肺癌的潜力从未被评估过。因此，我们假设，口服Leucoselect将发挥抗肿瘤作用和 有利地调节与肺癌相关的机制。提出了三个具体的目标：目的1：在小鼠模型中测定Leucoselect的最大耐受量(MTD)、药代动力学(PK)/药效学(PD)和抗癌作用。Leucoselect将在裸鼠身上进行剂量范围寻找研究，通过口服给予不同剂量。将采集血液和肺样本，以测定葡萄籽原花青素(GSP)及其代谢物的PK，作为GSE(全身和肺部)生物利用度的替代标志，并建立超过2周治疗期间的MTD。目的#1.1：利用一种新的小鼠肺组织冰冻切片与人肺肿瘤细胞共培养系统，测定口服Leucoselect在靶器官部位的生物活性。通过比较与人类非小细胞肺癌(NSCLC)细胞系A549和支气管癌前细胞系1198共培养的对照和Leucoselect处理组小鼠的肺组织切片，将评估肺中的生物活性。不同剂量的GSE对共培养的A549和1198细胞的增殖和凋亡的影响将与GSP和代谢产物水平有关。目的#2：研究亮氨酸对肺癌移植瘤模型的抗癌作用。基于MTD的不同剂量的Leucoselect将通过口服给携带皮下NSCLC异种移植的小鼠最多8周，从每个治疗组连续收集血浆、肺组织和肿瘤异种移植。抗肿瘤效果将取决于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caspase 3)指数，并与血浆、肺组织和移植瘤标本中GSP水平相关。目的#2.1：在异种移植模型中进一步表征和关联Leucoselect抗肺癌的机制效应。GSE的作用机制将通过调节：1)COX-2的表达和PGE_2的产生，2)其他炎症标志物：15(S)-羟基二十碳四烯酸(15-HETE)，白细胞介素6(IL)-6，IL-10和3)mir-19a，-19b和-106b的水平，4)癌症相关的途径特异性基因表达谱(GEP)，5)microRNA的表达，以及6)常见的癌变生物标志物，如p53，p27，p21和p16，在不同类型的样本中。目的#3：验证和表征mir-19a、mir-19b和mir-106b在Leucoselect抗肿瘤作用中的作用，并检测血浆mir-19a、mir-19b和mir-106b作为SEBM用于治疗监测的潜力。在我们的初步体外研究中，我们发现在NSCLC和1198细胞系中，GSE显著下调了3个Oncomir：MIR-19a、mir-19b和mir-106b的表达。这些miRNAs是被确认为肺癌发展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中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记物(SEBM)的有效性。影响：这项研究的结果将为GSE治疗肺癌的可行性和机制效应提供重要的见解，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.