Purification and Characterization of Antitumor Component from Guangxi Agkitrodon Acutus Venom

objective to purify an antitumorr component from Guangxi Agkitrodon acutus venom,exploring its possible mechanisms of antitumor function.Methods To extract crude venon through Sephadex G-75 gel filtration,ultrafiltration and DEAE Sepharose CL-6B ionexchange to acquire a small petide named frction K.SDS-polyacrylamid gel electrphoresis was used to identify the purity and measure moleculat weight,the inhibitory effects of fraction K against human cancer cells in vitro was mesured by MTT and Clony-forming assay and IC₅₀was calculated,the morphological changes were determined by means of H.E dyeing and electron microscopy respectively.cells apoptosis induction of fraction K was detected by acridine orange and ethidium bromide flurescence staining methods（AO-EB）and flow cytometry（FCM） through Annexin V-FITC/PI assay.Mitotic block induced by fraction K was investigated by PI staining assay.Cell adhesion assay was used to observe the effect of fraction K on the adhesion of A2780 cells to fibronectin（FN） Anti-angiogenesis effect was evaluated by investigating the effect of fraction K on the prolifilation and adhesion of endothelial cell line ECV304 in vitro and by chick embryo chorioallantoic membrane（CAM）assy in vivo.Results The molecular weight of fracton K was 7862D.Fraction K displayed marked inhibitory ability on human ovary cancer cell line A2780 and human gastiric canrcinoma cell line SGC-7901,IC₅₀of fraction K to A2780 in 12h,24h and 48h were 1.36μg/ml、0.814μg/ml and 0.566μg/ml,IC₅₀of fraction K to SGC-7901 in 12h、24h and 48h were 8.38μg/ml、3.21μg/ml、1.38μg/ml.The inhibitory rate of Clone-forming of A2780 ranged from 24%to 87%at the dose ranging from 0.2μg/ml to 1.2μg/ml.The dose-effect and time-effect relationship were described.Under microscope,characteristic morphology typical for apoptosis was observed at the dose of 1.00μg/ml for 24h including cells shrinkage,vacuolus in cytoplasm,pyknosis,aniso chromatin,chromatin margination and complete nuclear membrance were observed.Compared with the blank control,the amount of apoptotic A2780 increased when incubated with a concentration of 0.80μg/ml for 48h observed by AO/EB fluorescence staining.The apoptosis was detected by flow cytometry as well,the early apoptisis ratio progressing raised in a certain does range,when up to 0.8μg/ml,the lately apoptosis and necrosis increased greatly,while the early apoptosis was not obviouse.Cells in G₀/G₁ phase decreased,cells in G₂/M phase increased,the cell cycle was arrested in G₂/M phase.Fraction K could inhibit the adhesion of A2780 to fibronectin（FN）in does-dependent manner.The proliferation of endothelial cell line ECV304 was inhibited significantly by fraction K with IC₅₀of 24h being 2.82μg/ml.Fraction K inhibited endothelial cell adhesion to FN and tubule formation in CAM,and the does-dependent relationship was obviously.Conclusion The disintegrin-character component successful purified from Guangxi Agkitrodon acutus venom had a high ability on cancer cells growth inhibition in vitro,arrested cell cycle in G₂/M phase,induced cell apoptosis,anti-cells-adhesion to FN and anti-angiogenesis.