ANGIOCIDIN, A NEW ANGIOGENESIS INHIBITOR

血管抑制素，一种新的血管生成抑制剂

• 批准号: 6514791
• 负责人: GEORGE Paul TUSZYNSKI
• 金额: $ 24.89万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514791
• 关键词: SCID mouse; affinity chromatography; angiogenesis inhibitors; breast neoplasms; cell adhesion; cell proliferation; enzyme linked immunosorbent assay; extracellular matrix; growth factor; human tissue; immunocytochemistry; lung neoplasms; neoplasm /cancer chemotherapy; neoplasm /cancer invasiveness; nonhuman therapy evaluation; protein protein interaction; recombinant proteins; thrombospondins; vascular endothelium

We have cloned a novel high affinity thrombospondin-1 (TSP-1) binding protein from a prostate cancer cell library. The protein, termed angiocidin, inhibited endothelial cell viability and tube formation in vitro and prevented the growth of tumors in vivo. When injected intravenously into mice bearing Lewis Lung carcinoma, the protein inhibited the growth of the tumor by more than 500% as compared to the buffer control. As shown by immunohistochemical staining of human breast tumors angiocidin localized to malignant ductal epithelium while no significant staining of epithelium in normal and benign tissues was observed. In this proposal we will test the hypothesis that angiocidin exerts its anti-angiogenic activity by competing with TSP-1, its high affinity ligand, and regulating expression of other endothelial proteins important in angiogenesis. We will establish which steps in the angiogenic process are inhibited by angiocidin, what structural determinants of angiocidin mediate this activity, and what other proteins may be binding and regulating angiocidin activity. Our goal is to understand how angiocidin blocks angiogenesis. Cur long range goal is to develop angiocidin as a treatment for cancer. These goals will be accomplished by the following aims. In aim 1, we will establish the capacity of angiocidin to modulate the following TSP-1 dependent biological steps important in angiogenesis: a) endothelial cell adhesion to extracellular matrix b) growth factor dependent endothelial cell proliferation c) growth factor dependent endothelial cell invasion. In aim 2 we will map the structural domains of angiocidin that mediate its anti- angiogenic activity and TSP-1 binding activity with synthetic peptides and monoclonal antibodies whose binding epitopes will be mapped using peptide phage display. In aim 3 we will determine the molecular basis of the anti-angiogenic activity of angiocidin by establishing that endothelial cells bind angiocidin through cell surface receptors, which will be identified by affinity chromatography and molecular cloning. To further unravel the anti-angiogenic activity of angiocidin, genes regulated by angiocidin will be identified by cDNA expression array analysis. Finally in aim 4 the in vivo activity of angiocidin in preventing tumor growth as well as its in vivo distribution will be evaluated in a syngeneic mouse and orthotopic xenograft mouse model of tumor growth. These models will be used to show that injected angiocidin as well as angiocidin produced by transfected tumor cells decreases tumor microvessel density. The role of TSP-1 in the activity of angiocidin will be further evaluated in tumor bearing TSP-1 null mice. The experiments outlined above should advance the field of angiogenesis research by filling the gap in our understanding of how angiogenesis is regulated by molecules like angiocidin and its ligand TSP- 1. Finally, these experiments should provide the basis for the development of angiocidin as an anti-cancer therapeutic.

我们从前列腺癌细胞库中克隆了一种新的高亲和力凝血蛋白-1 （TSP-1）结合蛋白。这种蛋白被称为“血管杀虫素”，在体外抑制内皮细胞活力和管的形成，并在体内阻止肿瘤的生长。当将该蛋白静脉注射到患有Lewis肺癌的小鼠体内时，与缓冲对照相比，该蛋白抑制肿瘤生长的幅度超过500%。免疫组化染色显示，人乳腺肿瘤的血管杀虫素定位于恶性导管上皮，而正常组织和良性组织的上皮未见明显染色。在这一提议中，我们将验证一个假设，即血管杀素通过与高亲和力配体TSP-1竞争，并调节血管生成中重要的其他内皮蛋白的表达，来发挥其抗血管生成活性。我们将确定血管生成过程中的哪些步骤被抗血管毒素抑制，抗血管毒素的哪些结构决定因素介导这种活性，以及哪些其他蛋白质可能结合并调节抗血管毒素的活性。我们的目标是了解血管杀菌素是如何阻止血管生成的。我们的长远目标是开发出一种用于治疗癌症的血管杀菌素。这些目标将通过以下目标来实现。在目标1中，我们将建立血管杀虫素调节以下在血管生成中重要的TSP-1依赖性生物步骤的能力：a)内皮细胞与细胞外基质的粘附；b)生长因子依赖性内皮细胞增殖；c)生长因子依赖性内皮细胞侵袭。在目标2中，我们将绘制介导其抗血管生成活性和TSP-1结合活性与合成肽和单克隆抗体的结构域，其结合表位将使用肽噬菌体显示来绘制。在目标3中，我们将通过建立内皮细胞通过细胞表面受体结合血管杀素来确定血管杀素抗血管生成活性的分子基础，该受体将通过亲和层析和分子克隆进行鉴定。为了进一步揭示抗血管生成活性，我们将通过cDNA表达阵列分析鉴定抗血管生成素调控的基因。最后，在目的4中，我们将在同基因小鼠和同种异种移植小鼠肿瘤生长模型中评估抗血管毒素在体内阻止肿瘤生长的活性及其在体内的分布。这些模型将用于证明注射的抗血管毒素以及由转染的肿瘤细胞产生的抗血管毒素会降低肿瘤微血管密度。TSP-1对抗血管毒素活性的影响将在TSP-1缺失小鼠中进一步研究。上述实验应该通过填补我们对血管生成如何由血管杀虫素及其配体TSP- 1等分子调节的理解的空白来推进血管生成研究领域。最后，这些实验可为开发抗肿瘤药物抗血管毒素提供依据。