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.
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.
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