Research of new recombinant anticancer drug against epidermal growth factor receptor as a molecular target

以表皮生长因子受体为分子靶点的新型重组抗癌药物研究

• 批准号: 13671338
• 负责人: OZAWA Soji
• 金额: $ 2.3万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671338/
• 关键词: EGF; FGF; new anticancer drug; 新しい融合蛋白質; 受容体; 上皮増殖因子受容体

Study 1 : To minimize the side effects, we made a new anticancer drug which consisted of only physiologically active materials. We have made a conjugate of epidermal growth factor (EGF) and eosinophil cationic protein (ECP) and studied its cytotoxic effects on cancer cells. EGF and ECP were conjugated with 2-iminothiolane and SPDP. Cytotoxic effects of the conjugate were determined on BT-20 cells which overexpressed EGFR and H69 cells which did not expressed EGFR using a MTT assay. The conjugate killed BT-20 cells dose-dependently, but it did not kill H69 cells. Additional EGF bloked the cytotxic effects. These results suggested that the new anticancer drug which consisted of only physiologically active materials is promising and useful as a targeting tool.Study 2 : We fused a pancreatic-type ribonuclease (RNase) gene to human basic fibroblast growth factor (bFGF), and studied the inhibitory effect of the fused protein (CL-RFN89) on angiogenesis in vitro and in vivo. CL-RFN89 binding was assayed using a fluorescence labeled method. Inhibitory effects on in vitro angiogenesis were evaluated by an assay using a collagen gel system. Inhibitory effects on in vivo angiogenesis were evaluated by an mouse dorsal air sac assay. Fluorescence labeled CL-RFN89 was detected on cell membranes and its binding was inhibited by excess amount of FGF. Tube-formation of HUVEC was inhibited by CL-RFN89 dose-dependently and 53% inhibition was shown in 2uM of CL-RFN89. As for in vivo effects, 0.8 new vessels were observed in 2uM of CL-RFN89, but 4.4 new vessels were observed in none of CL-RFN89. These results suggested that CL-RFN89 bound to cell thgrough FGF receptor and that CL-RFN89 had anti-angiogenetic effects both in vitro and in vivo.

研究1：为了减少副作用，我们研制了一种只含有生理活性物质的抗癌新药。我们将表皮生长因子（EGF）与嗜酸性粒细胞阳离子蛋白（ECP）偶联，并研究其对癌细胞的细胞毒作用。EGF和ECP与2-亚氨基硫杂环戊烷和SPDP缀合。使用MTT测定法测定缀合物对过表达EGFR的BT-20细胞和不表达EGFR的H69细胞的细胞毒性作用。偶联物剂量依赖性地杀死BT-20细胞，但不杀死H69细胞。EGF对细胞增殖的影响。研究二：将胰腺型核糖核酸酶（RNase）基因与人碱性成纤维细胞生长因子（bFGF）融合，研究融合蛋白CL-RFN 89在体内外对血管生成的抑制作用。使用荧光标记方法测定CL-RFN 89结合。通过使用胶原凝胶系统的测定来评价对体外血管生成的抑制作用。通过小鼠背侧气囊试验评价对体内血管生成的抑制作用。在细胞膜上检测到荧光标记的CL-RFN 89，其结合被过量的FGF抑制。CL-RFN 89剂量依赖性地抑制HUVEC的管形成，并且在2uM的CL-RFN 89中显示53%的抑制。至于体内效应，在2uM的CL-RFN 89中观察到0.8个新血管，但在CL-RFN 89中均未观察到4.4个新血管。这些结果表明，CL-RFN 89通过FGF受体与细胞结合，CL-RFN 89在体外和体内均具有抗血管生成作用。