Targeting Signaling to Prevent Colon Cancer

描述(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)。在这项拟议的研究中，我们将分析Withaferin-A(WA)的作用机制，Withaferin-A是一种基于CAM的草药，在非洲和亚洲国家广泛用于治疗包括癌症在内的各种疾病。我们的初步体外数据表明，WA通过下调裂解的NOTCH1、PACK、pS6K、p4E-BP1和Bcl-2，同时促进caspase-3的激活和PARP的裂解来靶向结肠癌细胞。基于这些结果，我们推测Wa将有效地抑制结肠癌的生长，因为它能够失活Notch-1/Akt/mTOR介导的促生存信号，并诱导结肠癌细胞凋亡。为了解决我们的假设，我们建议：研究Wa介导的Notch-1/Akt/mTor抑制结肠癌的机制(目标1)，并确定Wa的体内疗效及其对过度表达Akt或mTor的结肠癌移植瘤的化疗增敏作用(Aim 2)。在我们的体外研究中，我们将使用RT-PCR、Western blotting、siRNA策略、免疫沉淀、细胞存活率检测、细胞凋亡检测、激酶检测、瞬时转染、基因转录检测、药理阻断和免疫细胞化学来阐明WA对Notch-1/Akt/mTOR信号轴的影响。在我们的体内研究中，结肠癌细胞将稳定地转染有成分活性的Akt或mTOR。利用这些转基因细胞在裸鼠体内形成异种移植瘤，并研究其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疗增敏作用，我们将用Wa和亚致死剂量的5-FU(5-FU)治疗小鼠。在研究结束后，将对肿瘤切片进行光镜、组织病理学和免疫组织化学(Notch-1、Akt、mTor、Cyclin D1、PAR-4和BID)分析。此外，还将采用高效液相色谱法测定裸鼠模型中的血药浓度。我们的长期目标是鼓励在临床环境中使用CAM，在临床环境中，这些药物可以最好地利用其化学预防和化疗特性。我们的初步数据表明，WA就是这样一种化合物，是研究其对结肠癌细胞的临床效力的可行候选者。 公共卫生相关性：结直肠癌是美国男性和女性中第三常被诊断的癌症，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)可能提供传统医学尚未探索的新颖和有效的治疗方法。关于膳食补充剂Withaferin-A(Withaferin-A)的拟议研究将为Withaferin-A抗结肠癌的作用机制提供分子水平的见解，并阐明Withaferin-A在化学预防和/或化学治疗结肠癌方面的临床潜力。

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.