Chemical biology approach for investigating molecular mechanisms of isothiocyanates-inducing bioactivities

研究异硫氰酸盐诱导生物活性分子机制的化学生物学方法

• 批准号: 21680052
• 负责人: MIYOSHI Noriyuki
• 金额: $ 14.56万
• 依托单位: University of Shizuoka
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (A)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21680052/
• 关键词: 健康と食生活; 機能性食品; がん細胞増殖抑制; 質量分析

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.

亲电膳食异硫氰酸酯（ITCs）的硫代氨基甲酰化是必要的，以发挥癌症的化学预防活性。在这项研究中，我们开发了一种新的方法来识别ITCs靶向分子使用两个研究充分的ITCs，苄基ITC（BITC）和苯乙基ITC（PEITC）。这种方法的原理是基于识别BITC和PEITC之间的差异模式，因为它们显示出相似的化学和生物学行为。对于方法验证，将具有游离巯基的二硫苏糖醇还原牛胰岛素作为模型分子与BITC或PEITC孵育，然后用内切蛋白酶Glu-C消化。通过UPLC-TOF/MS和LC-Q-TOF/MS分析所产生的肽。鉴定出三种肽NYCN、FVNQHLCGSHLVE和BLVCGE在其半胱氨酸残基上与BITC或PEITC结合。与BITC或PEITC结合的每组肽通过反相柱色谱显示保留时间（RT_<BITC>&lt;RT_<PEITC>），具有分子量差（Δ14.01565）。在这些发现的基础上，构建了计算数学方案来提取满足搜索器标准的MS离子集。将所开发的方法应用于ITCs处理的人结肠癌HCT 116细胞的提取物，谷胱甘肽的半胱氨酸残基和来自巨噬细胞移动抑制因子的PMFIVNTNVPR的N-末端脯氨酸残基的硫代氨基甲酰化被成功地鉴定为ITCs的细胞内靶点。此外，该方法还检测了ITC与细胞内游离半胱氨酸和赖氨酸的硫代氨基甲酰化缀合物。这种新开发的方法应该是有用的，以确定新的目标分子结合癌症化学预防的ITCs。

项目成果

Inflammation Induced Carcinogenesis and Chemoprevention. Chemoprevention of Cancer and DNA Damage by Dietary Factors

炎症诱发的癌变和化学预防。

• 发表时间: 2009
• 作者: Ohshima H.;Miyoshi N.;et al.
• 通讯作者: et al.

大腸がんHCT116細胞におけるがん予防成分イソチオシアネートの標的タンパク質探索

结直肠癌HCT116细胞中防癌成分异硫氰酸酯的靶蛋白搜索

• 发表时间: 2011
• 作者: 米持巧;三好規之;中村宜督;大島寛史
• 通讯作者: 大島寛史

アセトアルデヒドを消去する食品及び食品成分の探索

寻找消除乙醛的食品和食品成分

• 发表时间: 2010
• 作者: 鈴木啓子;三好規之;糠谷東雄;大島寛史
• 通讯作者: 大島寛史

食べ物と健康の基礎実習

食品与健康基础培训

• 发表时间: 2010
• 作者: Matsumura M;Watanabe YY;Robinson PW;Miller PJO;Costa DP;Miyazaki N.;三好規之
• 通讯作者: 三好規之

Luteolin overcomes resistance to benzyl isothiocyanate-induced apoptosis in human colorectal cancer HCT-116 cells

• DOI: 10.38212/2224-6614.2122
• 发表时间: 2020-07
• 期刊: Journal of Food and Drug Analysis
• 影响因子: 3.6
• 作者: R. Sakai;Shintaro Yokobe;Naomi Abe;N. Miyoshi;Y. Murata;Yoshimasa Nakamura