Bladder Cancer Chemoprevention with Allyl Isothiocyanate

描述（由申请人提供）： 该项目的重点是确定异硫氰酸烯丙酯（AITC）对膀胱癌的化学预防潜力。AITC是一种天然存在的植物化学物质。辣根根粉（HRP）是AITC的极好来源。现有数据强烈表明，AITC是一种非常有前途的膀胱癌预防剂。我们假设AITC通过体内尿液排泄选择性地递送到膀胱组织，并且一旦在膀胱中，AITC通过靶向stathmin引起癌细胞的有丝分裂阻滞，并且有丝分裂阻滞反过来通过Bcl-2失活启动半胱天冬酶依赖性和半胱天冬酶非依赖性细胞死亡。初步研究显示：B）Stathmin是一种对有丝分裂至关重要的癌蛋白，并且在相当大比例的人膀胱癌中过表达，其被AITC下调，并且似乎是关键的AITC靶标; c）AITC引起抗凋亡Bcl-2的显著磷酸化并激活半胱天冬酶依赖性和半胱天冬酶非依赖性促凋亡因子，但这些事件依赖于有丝分裂停滞; d）AITC在消耗后几乎完全排泄并作为N-乙酰半胱氨酸缀合物在尿中浓缩膀胱癌是由作为AITC载体的NAC-AITC（NAC-AITC）引起的，并且大多数人膀胱癌发生在直接暴露于尿液的上皮中。目的1是验证stathmin作为AITC的关键靶标，并阐明AITC下调stathmin的分子机制。目的二是研究AITC诱导Bcl-2磷酸化的机制，以及Bcl-2磷酸化在AITC诱导细胞死亡中的作用。目的1和2中提出的研究将在培养的膀胱癌细胞中进行，采用基因沉默、基因转染、免疫染色、免疫印迹和酶抑制的方法。将纯AITC的活性与NAC-AITC和HRP的活性进行比较。目的3是通过研究AITC在大鼠体内的药代动力学、尿中分布和组织蓄积，采用LC/MS/MS测定AITC及其代谢产物在大鼠体内的水平，评估口服给药的AITC选择性递送至膀胱组织的性质以及HRP是否是AITC的良好载体。目的4：评价AITC的体内抗肿瘤活性。将采用原位和皮下膀胱癌同时生长的大鼠模型。该模型不仅测量AITC的抗膀胱癌功效，还评估AITC是否对膀胱中生长的癌症特别有效。此外，AITC的功效将与HRP的功效进行比较。总的来说，拟议的研究将促进我们对AITC和HRP的膀胱癌化学预防潜力的理解，并为这些药物的未来预防研究提供关键信息。异硫氰酸烯丙酯（AITC）是一种天然存在的植物化学物质，在初步研究中显示出非常有希望的膀胱癌化学预防潜力。此外，辣根根粉已被确定为AITC的极好来源。公共卫生相关性拟议项目旨在使用临床前模型评估AITC和辣根根粉的膀胱癌化学预防功效和作用机制，预计将为这些物质用于膀胱癌预防的潜在临床转化奠定基础。

DESCRIPTION (provided by applicant): This project is focused on determining the chemopreventive potential of allyl isothiocyanate (AITC) against bladder cancer. AITC is a naturally occurring phytochemical. Horseradish root powder (HRP) is an excellent source of AITC. Available data strongly suggest that AITC is a highly promising agent for bladder cancer prevention. We hypothesize that AITC is selectively delivered to the bladder tissue through urinary excretion in vivo and that once in the bladder AITC causes mitotic arrest of cancer cells by targeting stathmin and that mitotic arrest in turn initiates both caspase-dependent and caspase-independent cell death through Bcl-2 inactivation. Preliminary studies show: a) AITC at physiologically achievable concentrations causes mitotic arrest and apoptosis in bladder cancer cells while showing little toxicity toward normal human bladder epithelial cells; b) stathmin, which is an oncoprotein critical for mitosis and is overexpressed in a significant percentage of human bladder cancers, is down regulated by AITC and appears to be the key AITC target; c) AITC causes marked phosphorylation of anti-apoptotic Bcl-2 and activates both caspase-dependent and caspase- independent apoptogenic factors, but these events depend on mitotic arrest; d) AITC after consumption is almost exclusively excreted and concentrated in the urine as N-acetylcysteine conjugate (NAC-AITC), which is a carrier of AITC, and the majority of human bladder cancers occur in the epithelium that is directly exposed to the urine. Aim 1 is to validate stathmin as a key AITC target and to elucidate the molecular mechanism by which AITC down regulates stathmin. Aim 2 is to determine the mechanim by which AITC causes Bcl-2 phosphorylation and the role of Bcl-2 phosphorylation in AITC-induced cell death. Studies proposed in Aims 1 and 2 will be performed in cultured bladder cancer cells, employing methodologies of gene silencing, gene transfection, immunostaining, immunoblotting and enzyme inhibition. The activity of pure AITC will be compared with that of NAC-AITC and HRP. Aim 3 is to assess the nature of selective delivery of orally dosed AITC to the bladder tissue and if HRP is a good vehicle for AITC, by examining the pharmacokinetics, urinary disposition and tissue accumulation of AITC in rats, using LC/MS/MS to determine the levels of AITC and its metabolites in the specimens. Aim 4 is to assess the anticancer activity of AITC in vivo. A rat model that presents simultaneous growth of both orthotopic and subcutaneous bladder cancers will be employed. This model not only measures the anti-bladder cancer efficacy of AITC but also assesses if AITC is particularly effective against cancer growing in the bladder. Moreover, the efficacy of AITC will be compared with that of HRP. Overall, the proposed studies will advance our understanding of the bladder cancer-chemopreventive potential of AITC and HRP and provide critical information for future translationation study of these agents. Project Narrative Allyl isothiocyanate (AITC) is a naturally occurring phytochemical and has shown a highly promising chemopreventive potential against bladder cancer in preliminary studies. Moreover, a horseradish root powder has been identified as an excellent source of AITC. PUBLIC HEALTH RELEVANCE The proposed project seeks to evaluate the bladder cancer chemopreventive efficacy and the mechanism of action of both AITC and horseradish root powder using preclinical models, which is expected to lay the groundwork for the potential clinical translation of these substances for bladder cancer prevention.