Chemoprevention of urinary bladder carcinogenesis by flavokawain A

描述(由申请人提供)： 膀胱癌是一个主要的公共卫生负担。每个膀胱癌患者从确诊到死亡的成本是所有癌症中最高的(在美国，每个患者的成本为9.6万美元至18.7万美元)。我们的长期目标是开发新的化学预防药物来减轻膀胱癌的负担。由于接触致癌物是膀胱癌的主要危险因素，而植物食品中含有的许多潜在保护化合物集中在尿液中，因此植物中的生物活性物质可能在膀胱癌的化学预防中发挥作用。黄腐素A是从卡瓦根萃取物中鉴定出的主要查尔酮。黄卡旺A的前景基于以下数据：1)一项流行病学研究报告称，尽管南太平洋群岛的人口中存在许多吸烟者，但高卡瓦消费量与较低的癌症发病率相关；2)体外细胞培养研究表明，卡瓦提取物中的黄酮类而不是卡瓦拉酮对低级别和高级别的人膀胱癌细胞显示出强大的抗增殖和凋亡作用；3)黄卡旺A抑制浅表性和浸润性膀胱癌异种移植模型中的肿瘤生长，没有任何明显的副作用；4)与其他一些查尔酮类似，黄酮鸟苷A的化学结构使我们预测它不会对DNA造成直接损伤，可以与β-微管蛋白结合，并诱导II相酶来防止致癌。我们推测黄酮胺A通过促进细胞周期调节和提高细胞凋亡机制的敏感性而对膀胱上皮细胞具有化学预防作用。将使用三个小鼠模型来验证这一假设：致癌物[4-羟基丁基(丁基)亚硝胺(OH-BBN)]诱导的小鼠膀胱癌变模型，以及两个自发的小鼠膀胱转基因致癌模型，每个模型都将解决不同的问题。在小鼠体内的OH-BBN模型将被用来检验黄酮胺A在一级预防中的效用；用于预防那些具有吸烟和工业接触等危险因素的人首次发生膀胱癌。每一种转基因模型都会诱导晚期膀胱癌的一种单独的前驱状态：要么是乳头状瘤，要么是原位癌，就像临床膀胱癌的早期阶段一样。在这些转基因模型中检测黄酮胺A将提供有关其在二级预防中的有效性的信息，这将在临床上解决预防膀胱癌复发和进展的问题。我们的微阵列分析已经确定了几个潜在的转录靶点(即BIM、BID、Survivin、Plk1、14-3-3Gamma和SKP-2)。我们将通过检测它们在这些受试动物模型中的活体表达，通过使用RNA干扰和过表达载体来确定它们在体外对黄曲霉毒素A作用的贡献，以及通过研究这些靶点的下游事件来验证这些靶点。总而言之，这些研究将为黄酮素A作为化学预防和/或化疗药物的前景提供一个坚定的答案。 尽管膀胱癌是化学预防的理想候选药物，但目前可用的化学预防药物的数量有限。我们将在致癌物[4-羟基丁基(丁基)亚硝胺(OH-BBN)]诱导的小鼠膀胱癌模型和两种新的膀胱转基因小鼠模型中检测新型药物黄酮胺A的化学预防活性，这两种模型均可诱导晚期膀胱癌的单独前体状态：乳头状移行细胞癌或原位癌。此外，我们将验证潜在的转录靶点(即BIM、BID、Survivin、Plk1、14-3-3Gamma和SKP-2)在这些受试动物模型中的体内表达，并通过使用RNA干扰和过表达载体来确定它们在体外对黄酮亚胺A作用的贡献。

DESCRIPTION (provided by applicant): Bladder cancer represents a major public health burden. The cost per bladder cancer patient from diagnosis to death is the highest among all cancers ($96,000 to $187, 000 per patient in the US). Our long-term goal is to develop new chemopreventive agents to reduce the burden of bladder cancer. Because exposure to carcinogens is the major risk factor for bladder cancer and many potentially protective compounds contained in plant-derived food are concentrated in the urine, it is plausible that bioactive agents in plants may play a role in bladder cancer chemoprevention. Flavokawain A is the predominant chalcone identified from kava root extract. The promise of flavokawain A is based on the following data : 1) an epidemiological study reported that high kava consumption correlated with lower incidences of cancers despite the presence of many smokers in the populations of the South Pacific Islands; 2) In vitro cell culture studies showed that flavokawains but not kavalactones in kava extracts exhibited strong antiproliferative and apoptotic effects on human bladder cancer cells characterized as low- and high- grades; 3) Flavokawain A inhibited tumor growth in xenograft models of superficial and invasive bladder cancer without any noticeable side effects; and 4) The chemical structure of flavokawain A, similar to some other chalcones, leads us to predict that it would cause no direct damage to DNA, bind to beta-tubulin, and induce phase II enzymes to protect against carcinogenesis. We hypothesize flavokawain A has chemopreventive effects on bladder epithelium via enhancing cell cycle regulation and increasing the sensitivity of apoptotic mechanisms. Three mouse models will be used to test this hypothesis: a carcinogen [4-hydroxybutyl (butyl) nitrosamine (OH-BBN)]-induced carcinogenesis model in mice bladders, and two spontaneous mouse bladder transgenic carcinogenesis models, each of which will address distinct issues. The OH-BBN model in mice will be used to examine flavokawain A's utility in primary prevention; for preventing the first occurrence of bladder cancer in those with risk factors such as cigarette smoking and industrial exposures. The transgenic models each induce a separate precursor state to advanced bladder cancer: either papillary tumors or carcinoma in situ, as seen in early stages of clinical bladder cancer. Examining flavokawain A in these transgenic models will provide information about its usefulness in secondary prevention, which clinically will address preventing bladder cancer recurrence and progression. Our microarray analysis has identified a few potential transcriptional targets (i.e. BIM, BID, survivin, Plk1, 14-3-3gamma, and SKP-2) for flavokawain A when inducing apoptosis and cell cycle arrests. We will validate these targets by examining their expression in vivo in these tested animal models and by using RNA interference and overexpression vectors to determine their contributions to flavokawain A's action in vitro, as well as by studying the down-stream events of these targets. Together, these studies will provide a firm answer to the promise of flavokawin A as a chemoprventative and/or chemotherapeutic agent.Project Narrative Although bladder cancer is an ideal candidate for chemoprevention, the number of currently available chemopreventive agents is limited. We will examine the chemopreventive activities of a novel agent, flavokawain A, in a carcinogen [4-hydroxybutyl (butyl) nitrosamine (OH-BBN)]-induced mouse bladder carcinogenesis model, and two novel bladder transgenic mouse models each which induces a separate precursor state to advanced bladder cancer: either papillary transitional cell carcinoma or carcinoma in situ. In addition, we will validate potential transcriptional targets (i.e., BIM, BID, survivin, Plk1, 14-3-3gamma, and SKP-2) for flavokawain A-induced apoptosis and cell cycle arrests by examining their expression in vivo in these tested animal models and by using RNA interference and overexpression vectors to determine their contributions to flavokawain A's action in vitro.