Mechanism of tumor growth suppression, tumor regression and induction of dormancy by endostatin, new antiangiogenic inhibitor.

内皮抑素是来自构成血管基底膜的XVIII型胶原的片段，显示出强的抗血管生成活性和抑制肿瘤生长。内皮抑素作为抗癌药物在美国进行II期临床研究。然而，Endostain/胶原XVIII的功能机制仍然是未知的。本课题的研究目的是探讨内皮抑素抑制肿瘤生长、侵袭和转移的分子机制。同时，我们评估了内皮抑素在肿瘤动物模型中的疗效.为了分离内皮抑素受体，通过固定化内皮抑素的亲和柱制备结合级分。对该级分中的几个蛋白质进行了氨基酸测序，但我们没有获得任何数据。目前我们正在通过酶切制备多肽片段，并对这些片段进行氨基酸序列分析. Endostain对生长因子VEGF诱导的内皮细胞迁移有抑制作用。内皮抑素在VEGF诱导的信号转导通路中的作用尚不清楚。但我们发现内皮抑素本身对MAP激酶的磷酸化有微弱的诱导作用，并计划研究MAP激酶激活的竞争性.为了定位内皮抑制素的活性位点，制备具有氨基酸取代的突变体并与野生型进行比较。活性中心被成功地定位在分子表面区域。覆盖该区域的合成肽未能显示出相当的内染色剂活性。制备了覆盖略有不同区域的新肽，并检查了结构对活性贡献的可能性。在脑肿瘤动物模型中，重组内皮抑素使肿瘤体积缩小至四分之一。启动了利用编码内皮抑素的质粒DNA进行基因治疗的途径。

Endostatin is a fragment derived from type XVIII collagen which constitutes blood vessel basement membrane, showed strong antiangiogenic activity and suppressed tumor growth. The phase II clinical study of Endostatin is performing as anti-cancer drug in USA. However, functional mechanism of endostain/collagen XVIII is still unknown. Our research goal is to analyze molecular mechanisms of action of endostatin on suppression of tumor growth, invasion and metastasis. In parallel, we estimated the efficacy of endostatin in tumor animal model.1. For isolation of endostatin receptor, the binding fraction was prepared by edostatin-immobilized affinity column. A couple of proteins in the fraction were subjected to amino acid sequencing, but we cold not obtain any data. Now we are preparing peptide fragments by enzyme digestion and analyzing amino acid sequence of these fragments.2. Endostain showed inhibitory activity of migration of endothelial cell induced by growth factor, VEGF. It is not clear the affect of endostain in signal transduction passway induced by VEGF. But we found that endostatin itself induced weak phosphorylation of MAP kinase and planed to investigate the competition of MAP kinase activation.3. To localize the active site of endostatin, mutants having amino acid substitution were prepared and compared to wild type. The active site was successfully localized the region of molecular surface. The synthetic peptide covered the region failed to show comparable activity of endostain. The new peptides covering slightly different region are prepared and the possibility of structural contribution to activity is examined.4. In animal model that has brain tumor, the recombinant endostatin regress the tumor volume to one fourth. The approach of gene therapy using plasmid DNA coding endostain was launched.