| Budget Amount *help |
¥22,750,000 (Direct Cost: ¥17,500,000、Indirect Cost: ¥5,250,000)
Fiscal Year 2011: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000)
Fiscal Year 2010: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2009: ¥11,570,000 (Direct Cost: ¥8,900,000、Indirect Cost: ¥2,670,000)
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| Research Abstract |
Thiocarbamoylation by electrophilic dietary isothiocyanates(ITCs) is necessary in order for them to exert cancer chemopreventive activities. In this study, we developed a novel method to identify ITCs-targeted molecules using two well-studied ITCs, benzyl ITC(BITC) and phenethyl ITC(PEITC). The principle of this method is based on identifying a pattern of difference between BITC and PEITC, since they show similar chemical and biological behaviors. For the method validation, dithiothreitol-reduced bovine insulin having free thiols as a model molecule was incubated with either BITC or PEITC, and then digested with endoprotease Glu-C. The generated peptides were analyzed by UPLC-TOF/MS and LC-Q-TOF/MS. Three peptides, NYCN, FVNQHLCGSHLVE and ALYLVCGE, were identified to be bound with BITC or PEITC on their cysteine residues. Each set of peptides bound with either BITC or PEITC showed retention times(RT_<BITC><RT_<PEITC>) by reverse-phase column chromatography with a difference of molecular mass(Δ14.01565). On the basis of these findings, computational mathematical schemes were constructed to extract sets of MS ions satisfying the abovecriteria. Application of the developed method to an extract of ITCs-treated human colon cancer HCT 116 cells, thiocarbamoylation of cysteine residues of glutathione and the N-terminal proline residues of PMFIVNTNVPR from macrophage migration inhibitory factor were successfully identified as the intracellular targets of ITCs. Moreover, the method also detected the thiocarbamoylated conjugates of ITCs with intracellular free cysteine and lysine. This novel developed method should be useful for identifying new target molecules which bind with cancer chemopreventive ITCs.
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