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Isolation and characterization of Fuantai-03 fromDasyatis akajei and its inhibitory effects onmigration and proliferation of human umbilical vein endothelial cells, and angiogenesis and tumor-induced angiogenesis

Rundi Ma, Lijian Yu, Weiming Su, Mingneng Liao, Laizhen Huang, Haiying Xu, Tingxi Yu

A novel inhibitor of angiogenesis (Fuantai-03, FAT-03) was isolated and characterized fromDasyatis akajei, SDS-PAGE analysis and silver staining determined the purified FAT-03 as a single band at 43.0 kDa. Its puritywas also confirmed by the finding of a unique NH2-terminal amino acid sequence. To determine whether this proteinwas capable of inhibiting angiogenesis in vitro and in vivo, migration assay was performed using a Transwell model with polycarbonate membrane; the effect of FAT-03 on the growth of human umbilical vein endothelial cells (HUVECs) wasmeasured by MTT; apoptotic induction of HUVECs by rFAT-03 was determined by fluorescencemicroscopy and flowcytometry; the effects of FAT- 03 on angiogenesis in vivo were assayed in chick chorioallantoic membrane (CAM) and Lewis lung carcinoma (LLC). Immunohistochemistry assay was used to analyzed the effect of FAT-03 on intratumoral microvessel density (iMVD). The results showed that the eluted chromatographic fractions at the accorded molecular weght demonstrated an anti-angiogenic activity. This study is the first demonstration that both the cartilage and soft tissues of Dasyat akajei can produce a inhibitor of angiogenesis having anMr of 43.0 kDa.