Targeting Signaling to Prevent Colon Cancer

描述（由申请人提供）：我们的研究重点是具有抗癌特性的补充和替代药物（CAM）。在拟议的研究中，我们将分析Withaferin-A（WA）的作用机制，这是一种在非洲和亚洲国家广泛使用的基于CAM的草药，用于治疗各种疾病，包括癌症。我们的初步体外数据表明，WA通过下调切割的NOTCH 1、pAkt、pS 6 K、p4 E-BP 1和Bcl- 2，同时促进caspase-3活化和PARP切割来靶向结肠癌细胞。基于这些结果，我们假设WA将有效地抑制结肠癌生长，由于其能够抑制Notch-1/Akt/mTOR介导的促存活信号传导，并诱导结肠癌细胞凋亡。为了解决我们的假设，我们提出：研究WA介导的Notch-1/Akt/mTOR抑制结肠癌的机制（目的1），并确定WA的体内疗效及其对过表达Akt或mTOR的结肠癌异种移植物的化学增敏作用（目的2）。对于我们的体外研究，我们将使用RT-PCR，Western Blotting，SiRNA策略，免疫沉淀，细胞活力测定，凋亡测定，激酶测定，瞬时转染，基因转录测定，药理学阻断和免疫细胞化学来阐明WA对Notch-1/Akt/mTOR信号传导轴的影响。对于我们的体内研究，结肠癌细胞将用组成型活性Akt或mTOR稳定转染。利用这些转染子，将在裸鼠中形成异种移植物，并将研究WA对荷瘤动物模型的影响。此外，为了确定WA对荷瘤动物模型的化学增敏作用，我们将用WA和亚致死剂量的5-氟尿嘧啶（5-FU）处理小鼠。研究结束后，将对肿瘤切片进行尸检、组织病理学和免疫组织化学（Notch-1、Akt、mTOR、细胞周期蛋白D1、Par-4和Bid）分析。此外，将进行HPLC以测定裸鼠模型中WA的血清浓度。我们的长期目标是鼓励在临床环境中使用CAM，这些药物可以最好地用于其化学预防和化疗特性。我们的初步数据表明WA是这样一种化合物，并且是研究其对结肠癌细胞的临床效力的可行候选物。 公共卫生相关性：结直肠癌是美国男性和女性中第三大最常诊断的癌症，尽管由于早期发现和辅助治疗，其死亡率有所下降，但一旦发生转移，这种疾病就无法治愈。补充和替代医学系统（CAM）可以提供传统医学尚未探索的新颖有效的治疗方法。拟对膳食补充剂Withaferin-A（WA）（来自睡茄的主要生物活性化合物）进行的研究，将在分子水平上深入了解Withaferin-A对结肠癌的作用机制，并阐明WA在结肠癌化学预防和/或化疗方面的临床潜力。

DESCRIPTION (provided by applicant): Our research focuses on complementary and alternative medicines (CAM) that possess anti- cancer properties. In the proposed study, we will analyze the mechanism of action of Withaferin-A (WA), a CAM based herbal used extensively in African and Asian countries for the treatment of various ailments, including cancer. Our preliminary in vitro data suggest WA targets colon cancer cells by down-regulating cleaved NOTCH1, pAkt, pS6K, p4E-BP1, and Bcl- 2, and simultaneously promoting caspase-3 activation and PARP cleavage. Based on these results, we hypothesize WA will effectively inhibit colon cancer growth due to its ability to inactivate Notch-1/Akt/mTOR-mediated pro-survival signaling, and induce apoptosis in colon cancer cells. To address our hypothesis, we propose: study the mechanism(s) of WA-mediated Notch-1/Akt/mTOR inhibition in colon cancer (Aim 1), and determine the in vivo efficacy of WA and its chemosensitization effect on colon cancer xenografts that over-express either Akt or mTOR (Aim 2). For our in vitro studies, we will use RT-PCR, Western Blotting, SiRNA strategies, immunoprecipitation, cell viability assays, apoptotic assays, kinase assays, transient transfection, gene transcription assays, pharmacological blocking, and immunocytochemistry to clarify the impact of WA on the Notch-1/Akt/mTOR signaling axis. For our in vivo studies, colon cancer cells will be stably transfected with either constitutively active Akt or mTOR. Using these transfectants, xenografts will be formed in nude mice, and the effect of WA on the tumor bearing animal models will be studied. Additionally, to determine the chemosensitization effect of WA on tumor bearing animal models, we will treat mice with WA and a sub-lethal dose of 5-Fluorouracil (5-FU). Necroscopy, histopathology, and immunohistochemical (Notch-1, Akt, mTOR, cyclin D1, Par-4 and Bid) analyses will be performed on the tumor sections following the termination of the study. Additionally, HPLC will be performed to determine serum concentrations of WA in the nude mice models. Our long term goal is to encourage the use of CAMs in a clinical environment, where these agents can be best used for their chemopreventive and chemotherapeutic properties. Our preliminary data indicate WA is one such compound, and is a viable candidate for investigating its clinical potency against colon cancer cells. PUBLIC HEALTH RELEVANCE: Colorectal cancer is the third most frequently diagnosed cancer in men and women in the United States, and, although its mortality rates have decreased due to early detection and adjuvant therapies, the disease is incurable once metastases develop. Complementary and alternative systems of medicine (CAM) may offer novel and effective therapies not yet explored by conventional medicine. The proposed study of the dietary supplement Withaferin-A (WA), the major bioactive compound from Withania somnifera), will provide molecular level insight into the mechanism of action of Withaferin-A against colon cancer, and clarify the clinical potential of WA with respect to the chemoprevention and/ or chemotherapy of colon cancer.