Development of Anti-tumor Drugs by Targetting Tumor Specific Isozyme (Type II) of Hexokinase

靶向己糖激酶肿瘤特异性同工酶（II型）开发抗肿瘤药物

• 批准号: 05557112
• 负责人: TERADA Hiroshi
• 金额: $ 7.55万
• 依托单位: University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05557112/
• 关键词: Hexokinase; Tumor cells; Anti-tumor drug; Sugar metabolism; がん細胞株; 遺伝子構造

We found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat tumor cell lines. In this study, we tried to establish a new strategy for the development of anti-tumor drugs and obtained following results.1)We isolated the cDNA fragment encoding human type II hexokinase and analyzed its transcript level by Northern blotting. As a result, we found that the transcript level of the type II hexokinase is elevated not only in rat hepatoma cell line, but also in human tumor cell line, HepG2.2)We isolated the gene encoding the type II hexokinase and characterized its structual features. As a result, we found a region which seems to be responsible for the enhanced expression of this gene in tumor cells.3)We think that hexokinase increases its activity by receving ATP from the ADP/ATP carrier exist in the inner mitochondrial membrane. Thus, we exaimed structure and function of this carrier by measuring the reactivities of cystein residues exist in this carrier under the various conditions and succeeded to reveal the transport mechanism at a molecular level.4)To find candidates of anti-tumor drug, we tried to establish the screening system of the specific inhibitor of the type II hexokinase. To obatin certain amount of this isozyme, we estabished a over-expression system of this enzyme in E.coli.

我们发现，在大鼠肿瘤细胞系中，II型己糖激酶的稳态转录水平特异性地显著升高。本研究旨在为抗肿瘤药物的开发提供一种新的思路，主要研究结果如下：1）从大肠杆菌中克隆了编码人II型己糖激酶的cDNA片段，并通过北方印迹分析了其转录水平。结果发现，Ⅱ型己糖激酶不仅在大鼠肝癌细胞系中转录水平升高，而且在人肝癌细胞系HepG 2中转录水平也升高。3）我们认为己糖激酶是通过从线粒体内膜上的ADP/ATP载体上接受ATP而提高其活性的。因此，我们通过测定该载体中存在的半胱氨酸残基在不同条件下的反应性来阐明该载体的结构和功能，并成功地从分子水平上揭示了其转运机制。4）为了寻找抗肿瘤药物候选物，我们尝试建立了II型己糖激酶特异性抑制剂的筛选体系。为了获得一定量的该同工酶，我们建立了该酶在大肠杆菌中的高效表达系统。

项目成果

Y. Shinohara et al.: "Steady State Transcript Levels of the Type II Hexokinase and Type 1 Glucose Transporter in Human Tumor Cell Lines" Cancer Lett.82. 27-32 (1994)

Y. Shinohara 等人：“人类肿瘤细胞系中 II 型己糖​​激酶和 1 型葡萄糖转运蛋白的稳态转录水平”Cancer Lett.82。

Y.Shinohara and H.Terada: "Evolution of the Mammalian Hexokinase Gene by Duplacation and Fusion of the Glucokinase gene" Seikagaku. 67. 137-141 (1995)

Y.Shinohara 和 H.Terada：“通过葡萄糖激酶基因的复制和融合来进化哺乳动物己糖激酶基因”Seikagaku。

E.Majima et al.: "Importance of Loops of Mitochondrial ADP/ATP Carrier for Its Transport Activity Deduced form Reactivities of Its Cysteine Residues with the Sulfhydryl Reagent Eosin-5-Maleimide" Biochemistry. 33. 9530-9536 (1994)

E.Majima 等人：“线粒体 ADP/ATP 载体环对其转运活性的重要性，由其半胱氨酸残基与硫氢基试剂 Eosin-5-Maleimide 的反应性推导出来”生物化学。

J.Ichihara et al.: "Nucleotide Sequence of the 5'-Flanking Region of the Rat Type II Hexokinase Gene" Biochim.Biophys.Acta. (印刷中).

J. Ichihara 等人：“大鼠 II 型己糖​​激酶基因 5-侧翼区域的核苷酸序列”Biochim.Biophys.Acta。

島厚志、篠原康雄、寺田弘: "ミトコンドリア溶質輸送坦体ファミリーの遺伝子構造" 生体の科学. 45. 679-685 (1994)

Atsushi Shima、Yasuo Shinohara、Hiroshi Terada：“线粒体溶质转运载体家族的基因结构”生物科学 45. 679-685 (1994)。