Polypeptide From Shark Cartilage With Potent Anti-angiogenic Activity

Anti-angiogenesis as a promising anticancer strategy has led to the discovery of many natural and synthetic anticancer agents. Shark cartilage has been investigated as a source of anti-angiogenesis agents for more than 20 years. Numerous studies have confirmed that shark cartilage extract possesses anti-angiogenic activity. However, little is known about the exact composition of polypeptides. And also, there is very little information about the mode of action on anti-angiogenesis of polypeptides from shark cartilage.Using guanidine-HCl extraction, acetone precipitation, ultra-filtration and chromatography, a novel polypeptide with potent anti-angiogenic activity was purified from cartilage of the shark, Prionace glauca. N-terminal amino acid sequence analysis and SDS-PAGE revealed that the substance is a novel shark cartilage polypeptide with MW 15500 (designated name, PG155).Polypeptide PG155 inhibited vascular endothelial growth factor (VEGF) induced tube formation and migration of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner in vitro. Treatment with 200μg/ml PG155 did not affect the growth of Bovine Aortic Endothelial Cells (BAECs) as well as HUVECs. Also we tested if PG155 inhibited the proliferation of cancer cells, including human hepatoma Bel-7402 cells (Bel-7402), human oral epidermoid carcinoma KB cells (KB), human colon cancer HCT-18 cells (HCT-18), as well as human breast MCF7 cancer cells (MCF7), and no cytotoxic effects was found. Transwell experiment revealed that the polypeptide PG155 inhibited VEGF-induced migration of HUVECs. Exposure of HUVECs in 20μg/ml PG155 significantly decreased the density of migrated cells and the tube formation of HUVECs. Almost complete inhibition of cell migration and tube formation was found when HUVECs were treated with 40-80μg/ml PG155.Treatment of the zebrafish embryos with PG155 resulted in a significant reduction in vessel formation when introduced to zebrafish embryos prior to the onset of angiogenesis. Morphologic observation showed that PG155 markedly inhibited the growth of subintestinal vessels (SIVs) of zebrafish embroy. A higher dose resulted in almost complete inhibition of SIVs growth, as observed by endogenous alkaline phosphatase (EAP) staining assay. A dose-dependent effect was also found when treatment of zebrafish embroys with PG155 at different concentrations. 160μg/ml PG155 inhibited heart development of zebafish embryo.There is little information available concerning the new tumor angiogenesis inhibitors (TAIs) extracted from marine organism. Our observations indicated that shark cartilage may be as an important source of TAI. The new polypeptide, PG155, as a specific angiogenic inhibitor, may be developed as a novel kind of anti-tumor agent.