Bladder Cancer Chemoprevention with Allyl Isothiocyanate

描述(由申请人提供)： 本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是一种自然产生的植物化学物质。辣根粉末(HRP)是AITC的极好来源。现有数据有力地表明，AITC是一种非常有前途的膀胱癌预防药物。我们假设，AITC在体内通过尿液排泄选择性地传递到膀胱组织，一旦进入膀胱，AITC就通过靶向stathmin导致癌细胞有丝分裂停滞，有丝分裂停滞又通过Bcl-2失活启动caspase依赖和caspase非依赖的细胞死亡。初步研究表明：a)AITC在生理上可达到的浓度引起膀胱癌细胞有丝分裂停滞和凋亡，而对正常的人膀胱上皮细胞几乎没有毒性；b)stathmin是一种对有丝分裂至关重要的癌蛋白，在相当大比例的人膀胱癌中过表达，它被AITC下调，似乎是AITC的关键靶点；c)AITC导致抗凋亡的Bcl-2显著磷酸化，并激活caspase依赖和caspase非依赖的凋亡基因因子，但这些事件依赖于有丝分裂抑制；D)AITC在食用后几乎完全以N-乙酰半胱氨酸结合物(NAC-AITC)的形式排出并集中在尿液中，NAC-AITC是AITC的载体，大多数人的膀胱癌发生在直接接触尿液的上皮细胞中。目的1是验证stathmin作为AITC的关键靶点，并阐明AITC下调stathmin的分子机制。目的2研究AITC诱导Bcl2磷酸化的机制以及Bcl2磷酸化在AITC诱导的细胞死亡中的作用。AIMS 1和AIMS 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、免疫印迹和酶抑制等方法。将纯AITC的活性与NAC-AITC和HRP的活性进行比较。目的3通过检测AITC在大鼠体内的药代动力学、尿液排泄和组织蓄积，用LC/MS/MS测定样品中AITC及其代谢物的浓度，评价口服AITC对膀胱组织选择性给药的性质以及HRP是否是AITC的良好载体。目的4评价AITC的体内抗癌活性。将采用原位膀胱癌和皮下膀胱癌同时生长的大鼠模型。该模型不仅衡量AITC的抗膀胱癌疗效，还评估AITC是否对膀胱癌生长特别有效。此外，还将AITC与HRP的疗效进行比较。总体而言，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，并为未来这些药物的翻译研究提供关键信息。项目简介异硫氰酸烯丙酯(AITC)是一种自然产生的植物化学物质，在初步研究中显示出非常有希望的预防膀胱癌的化学潜力。此外，辣根粉末已被确定为AITC的极好来源。公共卫生相关性拟议的项目旨在利用临床前模型评估AITC和辣根粉末对膀胱癌的化学预防效果和作用机制，这有望为这些物质用于膀胱癌预防的潜在临床翻译奠定基础。

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.