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

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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.