Research of recombinant human RNase fused protein on angiogenesis and tumor growth

重组人RNA酶融合蛋白对血管生成和肿瘤生长的影响研究

• 批准号: 11671272
• 负责人: OZAWA Soji
• 金额: $ 2.5万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671272/
• 关键词: 食道癌; 生理活性物質; 血管新生阻害剤; VEGF; RNase; 血管新生; FGF

Study 1 : To minimize the side effects, we made a new angiogenic inhibitor which consists of only physiologically active materials. We have fused a pancreatic-type ribonuclease (RNase) gene to human basic fibroblast growth factor (bFGF), and studied the inhibitory effect of the fused protein on angiogenesis and tumor growth in vitro and in vivo. Inhibitory effects on in vitro angiogenesis were evaluated by an assay using fragments of human placental blood vessels (Brown, 1996). In an in vitro study, angiogenesis index calculated in wells with the fused protein was 9% of the control and the fused protein inhibited angiogenesis dose dependently. In an in vivo study, A431 cells were injected into the subcutaneous layer of SCID mice and 1.45 mg of the fused protein was injected around the tumor every day for 3 weeks. The estimated tumor weight in the fused protein injection group (n=4, 474+/-73 mg) was lighter than that in the control group (1161+/-220 mg)(P=0.04). Effects of the fuse prot … More ein on tumor growth was also observed. These results suggest that the RNase-FGF fused protein is a candidate for a new angiogenic inhibitor.Study 2 : Human lymphocytes isolated from healthy histoincompatible donors were used in mixed lymphocyte cultures or were stimulated with phytohemaggulutinin (PHA) to promote IL-2R alfa expression. MJ, an HTLV-1 infected malignant T-cell line overexpressing IL-2Ralfa, and IL-2Ralfa-negative cell lines MOLT 4F and MT-1 were used as controls. Bovine RNaseA was chemically conjugated to 7G7B6, a monoclonal antibody to the alfa-chain of human IL-2 receptors, and several concentrations of the conjugates were added to the lymphocyte cultures. Inhibition of protein synthesis was measured as percent 3H-thymidine incorporation in 24 hours. 7G7B6-RNaseA dose-dependently inhibited protein synthesis in PHA-stimulated human lymphocytes at an IC50 of 2 X 10-7M, whereas RNase alone and RNase plus antibody had no inhibitory effect. 7G7B6-RNaseA also dose-dependently inhibited human mixed lymphocyte reaction at an IC50 of 2 X 10-6M, whereas RNase alone caused no inhibition. The conjugate also inhibited protein synthesis in MJ cells, a cell line that is infected with HTLV-1 and overexpresses the high-affinity IL-2 receptor, at an IC50 of 5 X 10-7M.However the conjugate had no inhibitory effect on IL-2 receptor non-expressing human T-cell lymphoblastic leukemia cell lines MOLT4F or MT-1. 7G7B6-RNaseA can inhibit protein synthesis in antigen-or mitogen-stimulated lymphocytes overexpressing high-affinity IL-2 receptors, and it may be useful as a safer therapy than conventional chemotherapies or immunotoxins for transplant rejection, certain lymphocyte malignancies, and other IL-2R-associated diseases, because it is composed of a mammalian cytotoxic enzyme. Less

研究一：为了减少副作用，我们研制了一种新的血管生成抑制剂，它只含有生理活性物质。我们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，研究了融合蛋白对血管生成和肿瘤生长的抑制作用。通过使用人胎盘血管碎片的试验评价了对体外血管生成的抑制作用（Brown，1996）。在体外研究中，在具有融合蛋白的威尔斯孔中计算的血管生成指数是对照的9%，并且融合蛋白剂量依赖性地抑制血管生成。在体内研究中，将A431细胞注射到SCID小鼠的皮下层中，并且每天在肿瘤周围注射1.45mg融合蛋白，持续3周。融合蛋白注射组的估计肿瘤重量（n=4，474+/-73 mg）比对照组（1161+/-220 mg）轻（P=0.04）。保险丝保护的影响 ...更多信息 还观察了Ein对肿瘤生长的影响。这些结果表明，RNase-FGF融合蛋白是一个新的血管生成inhibitors.Study 2的候选人：人淋巴细胞分离健康histocompatible捐助者用于混合淋巴细胞培养或刺激植物血凝素（PHA），以促进IL-2 R α的表达。MJ，一种过表达IL-2 Ralfa的HTLV-1感染的恶性T细胞系，和IL-2 Ralfa阴性细胞系MOLT 4F和MT-1用作对照。将牛RNaseA与7 G7 B6（一种针对人IL-2受体α链的单克隆抗体）化学结合，并将几种浓度的结合物加入淋巴细胞培养物中。蛋白质合成的抑制被测量为24小时内3 H-胸苷掺入的百分比。7 G7 B6-RNaseA剂量依赖性地抑制PHA刺激的人淋巴细胞中的蛋白质合成，IC 50为2 × 10- 7 M，而单独的RNase和RNase加抗体没有抑制作用。7 G7 B6-RNaseA也剂量依赖性地抑制人混合淋巴细胞反应，IC_（50）为2 × 10 ~（-6）M，而单独的RNase没有引起抑制。该偶联物还抑制MJ细胞（一种感染HTLV-1并过表达高亲和力IL-2受体的细胞系）中的蛋白质合成，IC 50为5 × 10- 7 M。然而，该偶联物对不表达IL-2受体的人T细胞淋巴母细胞白血病细胞系MOLT 4F或MT-1没有抑制作用。7 G7 B6-RNaseA可抑制抗原或促分裂原刺激的过表达高亲和力IL-2受体的淋巴细胞中的蛋白质合成，并且由于其由哺乳动物细胞毒性酶组成，因此其可用作比常规化疗或免疫毒素更安全的治疗，用于移植排斥、某些淋巴细胞恶性肿瘤和其它IL-2 R相关疾病。少

项目成果

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Suwa T等人：“抗EGFR抗体-核糖核酸酶缀合物对人类癌细胞的表皮生长因子受体依赖性细胞毒性作用”Anticancer Res.19(5B)。

Takeuchi H, et.al.: "Further evidence that altered p16/CDKN2 gene expression is associated with lymph node-metastasis in squamous cell carcinoma of the esophagus"Oncology Reports. (in press). (2001)

Takeuchi H 等人：“进一步证据表明 p16/CDKN2 基因表达的改变与食管鳞状细胞癌的淋巴结转移相关”《肿瘤学报告》。

Takeuchi H, et al.: "Further evidence that altered p16/CDKN2 gene expression is associated with lymph node metastasis in squamous cell carcinoma of the esophagus."Oncol Rep.. 8 (3). 627-32 (2001)

Takeuchi H 等人：“进一步的证据表明 p16/CDKN2 基因表达的改变与食管鳞状细胞癌的淋巴结转移有关。”Oncol Rep.. 8 (3)。

Yamamura, et.al.: "Immuno Suppressive and 〓n effect of a mammalian who nuclease targeting high-aff〓ty 〓uler leukio-2 receptors"Eur.J.Surg. (in press). (2001)

Yamamura 等人：“针对高亲和力 〓uler leukio-2 受体的哺乳动物的免疫抑制和效果”Eur.J.Surg。