Effect of mutant Endostatin and Kringle 5 fusion protein on Tumor angiogenesis

Angiogenesis is important for the growth and metastasis of tumors. Endostatin is an endogenous inhibitor of angiogenesis and is shown to inhibit bFGF and VEGF induced signaling in endothelial cells. Recent evidence also indicates that it binds to alphanu and alpha5 group of integrins expressed endothelial cells and inhibits downstream signaling. Kringle 5, a potent anti-angiogenic molecule causes apoptosis of endothelial cells by associating with glucose-regulated protein 78 (GRP78). Previous studies from our laboratory have shown that a mutant (P125A) form of Endostatin (derived from collagen type XVIII) and Kringle 5 fragment of plasminogen can inhibit angiogenesis by inducing autophagy. P125A-endostatin and Kringle 5 interfere with distinct signaling pathways in endothelial cells. We hypothesized that a chimeric protein made of P125A-endostatin and Kringle 5 will have better anti-angiogenic activities. To test this hypothesis, we constructed a fusion protein consisting of P125A-endostatin and Kringle 5 (E-K5). Recombinant fusion protein was expressed in yeast and purified. E-K5 was found to inhibit proliferating endothelial cells and effectively blocked tumor induced angiogenesis. Anti-proliferative activity of E-K5 was linked to VEGF receptor and alphanubeta3 integrin-mediated signaling pathways. These studies establish the therapeutic potential of E-K5 as a potent anti-angiogenic molecule.