ANGIOCIDIN, A NEW ANGIOGENESIS INHIBITOR

血管抑制素，一种新的血管生成抑制剂

• 批准号: 6652404
• 负责人: GEORGE Paul TUSZYNSKI
• 金额: $ 24.98万
• 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/6652404
• 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血小板反应蛋白-1（TSP-1）结合蛋白。这种蛋白质被称为angiocidin，在体外抑制内皮细胞的活力和管的形成，并在体内阻止肿瘤的生长。当静脉注射到携带刘易斯肺癌的小鼠中时，与缓冲液对照相比，该蛋白质抑制肿瘤生长超过500%。免疫组化染色显示，血管杀菌素定位于恶性乳腺导管上皮，而在正常和良性组织中没有观察到显着的上皮染色。在这个建议中，我们将测试的假设，即angiocidin发挥其抗血管生成活性的竞争与TSP-1，其高亲和力的配体，并调节表达的其他内皮细胞蛋白在血管生成中的重要。 我们将确定血管生成过程中的哪些步骤被血管生成素抑制，血管生成素的哪些结构决定因素介导这种活性，以及其他哪些蛋白质可能结合和调节血管生成素活性。我们的目标是了解血管杀菌素如何阻断血管生成。目前的长期目标是开发血管杀素作为癌症的治疗方法。这些目标将通过以下目标实现。在目的1中，我们将确定血管杀菌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）内皮细胞粘附于细胞外基质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，我们将用合成肽和单克隆抗体定位血管杀菌素的结构域，所述合成肽和单克隆抗体介导其抗血管生成活性和TSP-1结合活性，所述合成肽和单克隆抗体的结合表位将使用肽噬菌体展示定位。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angiocidin来确定angiocidin的抗血管生成活性的分子基础，所述细胞表面受体将通过亲和层析和分子克隆来鉴定。为了进一步阐明angiocidin的抗血管生成活性，将通过cDNA表达阵列分析鉴定由angiocidin调控的基因。最后，在目标4中，将在肿瘤生长的同基因小鼠和原位异种移植小鼠模型中评价血管杀菌素在预防肿瘤生长中的体内活性以及其体内分布。这些模型将用于显示注射的血管杀素以及由转染的肿瘤细胞产生的血管杀素降低肿瘤微血管密度。TSP-1在杀血管素活性中的作用将在荷瘤TSP-1敲除小鼠中进一步评价。上述实验填补了我们对血管生成如何受血管生成素及其配体TSP- 1等分子调节的理解中的差距，从而推进了血管生成研究领域。最后，这些实验应该为血管杀菌素作为抗癌治疗剂的发展提供基础。