Bitter Melon Component and Colon Cancer Prevention

描述（由申请人提供）：尽管在识别和切除结肠息肉方面做出了最好的筛查努力，但结肠癌仍然是美国和世界各地癌症相关发病率和死亡率的主要原因。此外，目前的治疗方法虽然能很好地去除大多数癌细胞，但还不够，因为它们会留下一些细胞。这是因为这些细胞可以重新出现并形成新的肿瘤，在许多情况下，由于转移，肿瘤可以在不同的器官中表现出来。使用源自水果和蔬菜的天然产品比使用合成制剂预防癌症的优势在于，它们促进人体健康，而且没有明显的副作用。在这方面，我们最近发现苦瓜提取物（BME）抑制DCLK1+细胞的生长。我们现在已经发现，这种提取物中的一种活性成分charantin是结肠癌生长的有效抑制剂。当前项目的目标是进一步表征charantin，并产生临床前数据，作为预防结肠癌的膳食剂。在初步研究中，我们已经确定charantin抑制DCLK1激酶和相关激酶CAMKIIa的活性，但不抑制CAMKIIb和CAMKIV。此外，charantin抑制DCLK1诱导的AKT1/PKB磷酸化。AKT1是一种丝氨酸-苏氨酸蛋白激酶，在两个关键位点Thr308和Ser473磷酸化之前，它是催化无活性的。多种药物如生长因子和细胞因子可通过磷脂酰肌醇3-激酶（PI3K）的作用迅速激活该蛋白。激活后，AKT1诱导GSK-3b磷酸化，同时抑制JNK的激活。我们已经确定charantin抑制AKT1激活并诱导JNK。此外，JNK诱导b-catenin磷酸化，这反过来又受到b-TRCP独立，SIAH1依赖的泛素-蛋白酶体降解途径的影响。我们还确定charantin在一种称为“肿瘤培养皿”（TiD）的新型培养方法中抑制结肠癌细胞的生长。在这个系统中，细胞生长在三维基质中，基质中含有正常肺细胞，包括正常上皮细胞、成纤维细胞和内皮细胞。该模型创建了一个接近活体的肿瘤微环境，包括细胞间接触、三维结构和不同细胞类型的影响。在该系统中观察到的选择性杀死癌细胞的结果表明，该化合物具有高度特异性和良好的效力。基于这些初步结果，我们建议进一步开发charantin作为膳食预防剂，并将其推向临床。在目的1中，我们提出确定PI3激酶在charantin对dclk1介导的AKT1激活的作用。在Aim 2中，我们建议确定charantin是否影响akt介导的-catenin激活。最后，在Aim 3中，我们将确定charantin是否诱导JNK1激酶和SIAH1诱导-catenin降解。我们将确定该化合物对DCLK1表达、Akt磷酸化和b-catenin降解的影响。这些研究也将有助于理解抑制b-连环蛋白信号传导的非规范途径，并为未来的临床研究确定新的生物标志物。

DESCRIPTION (provided by applicant): Despite the best screening efforts to identify and remove colon polyps, colon cancer remains a leading cause of cancer related morbidity and mortality, both in the US and around the world. Also, current therapeutics while good in removing most cancer cells are not adequate because they leave some cells behind. This is because these cells can reemerge and develop a fresh tumor, which in many cases can manifest in a different organ due to metastasis. The advantage of using natural products that originate from fruits and vegetables over synthetic agents for preventing cancer is that they promote human health without recognizable side effects. In this regard, we have recently shown that bitter melon extracts (BME) inhibit the growth of DCLK1+ cells. We have now discovered that charantin, an active ingredient in the extracts is a potent inhibitor of colon cancer growth. The goal of the current project is to further characterize charantin and generate preclinical data as a dietary agent for the prevention of colon cancer. In preliminary studies, we have determined that charantin inhibits DCLK1 kinase activity and that of a related kinase CAMKIIa but not that of CAMKIIb and CAMKIV. Furthermore, charantin inhibits DCLK1 induced phosphorylation of AKT1/PKB. AKT1 is a serine-threonine protein kinase that is catalytically inactive until it is phosphorylated at two critical sites, Thr308 and Ser473. Various agents such as growth factors, and cytokines can rapidly activate the protein through the actions of phosphatidylinositol 3-kinase (PI3K). Upon activation, AKT1 induces phosphorylation of GSK-3b and also inhibits JNK activation. We have determined that charantin suppresses AKT1 activation and induces JNK. Moreover, JNK induces b-catenin phosphorylation, which in turn is subjected to b-TRCP independent, SIAH1- dependent ubiquitin-proteasome degradation pathway. We have also determined that charantin inhibits the growth of colon cancer cells in a novel culture method termed "Tumor in a Dish" (TiD). In this system, cells are grown in a three dimensional matrix that contains normal lung cells including normal epithelial cells, fibroblasts and endothelial cells. The model creates a near-in vivo tumor microenvironment including cell-cell contact, 3D- architecture, and the influence of different cell types. The observed selective killing of cancer cells in this system suggests that the compounds are highly specific and have good potency. Based on these preliminary results, we propose to further develop charantin as a dietary prevention agent and move it towards the clinic. In aim 1, we propose to determine the role of PI3 kinase in charantin effect on DCLK1-mediated AKT1 activation. In Aim 2, we propose to determine whether charantin affects AKT-mediated �-catenin activation. Finally, in Aim 3, we will determine whether charantin induces JNK1 kinase and SIAH1 to induce �-catenin degradation. We will determine the effect of the compound on DCLK1 expression, and on Akt phosphorylation, and b-catenin degradation. These studies will also aid in understanding a non-canonical pathway to suppress b-catenin signaling and identify novel biomarkers for the future clinical studies.