Purification And Characterization Of Bungarotoxin Cytotoxin And Experimental Study Of Its Anti-Tumor Effect

OBJECTIVE In oder to isolate and purify cytotoxin from the venom of Bungarus fasciatus and identify its anti-tumor activity.METHODS Low-temperature high-speed centrifugation was used to pretreat the venom of Bungarus Fascitus before chromatography, supernatant taken was gel filtrated by Sephadex G-75,and then collected each component. Each component was implemented red blood cell hemolysis test to identify cytotocity.Peaks that has Cytotoxic activity were gradient eluted by Ion-exchange column CM-Sepharose CL-6B,and then collected each component. The solution of each component was implemented toxicity test in mice and guinea pig hearts to identify cytotoxicity. Then the component with cytoxicity was implemented polyacrylamide gel electrophoresis experiments to determine protein content and purity. MTT assay was used to detect the anti-tumor effect of the active protein peaks in vitro.RESULTS The venom of Bungarus fasciatus was separated to four active protein peaks by Sephadex G-75, protein peaks II and III have severe hemolysis,which demonstrated their cytotoxicity.Then protein peaksⅡ,Ⅲwere gradient eluted by Ion exchange column CM-Sepharose CL-6B,and then collected ten components A,B,C,D,E,F,G,H, I,J,and each component was implemented acute toxicity test in mice and guinea pig isolated heart perfusion experiments.The results showed that protein peak components C and D could cause death in mice, and which could make guinea pig in the Langen-dorff heart contraction increased rapidly after a short decline, heart rate decreased, heart contracted, the last it arrested, which showed obvious cardiac toxicity.But other components have no significant effect on the heart.Protein peaks C and D were implemented polyacrylamide gel electrophoresis experiments to determine protein content and purity.The results showed it a single band. Molecular Weight was 7000～8000da.In vitro experiments protein peaks C and D strongly inhibited the growth of vitro gastric cancer cell line (MGC-803),cervical cancer cell line (Hela) and liver cancer cell lines (BEL-7402); and as the concentration increased, the rate of inhibition increased,and which was dose-dependent. When the dose were 5.0μg/ml and 10.0μg/ml,comparing the rate of inhibition of protein peaks C and D with the 5-Fu control group,there were significant difference respectively (p<0.01).In the same concentration,growth inhibition that the protein peak components C and D on three tumor cells compared with each other was no significant difference(P> 0.05).The IC50 of 24h inhibition that protein peak C component was on MGC-803 cells,Hela cells and BEL-7402 cells were respectively 0.857μg/ml,0.406μg/ml and 0.287μg/ml; and the IC50 of 24h inhibition that protein peak D component was on MGC-803 cells,Hela cells andBEL-7402 cells were respectively 0.502μg/ml,0.417μg/ml and 0.704μg/ml.CONCLUSIONS Applying the methods of gel filtration and Ion exchange to purify cytotoxin from the venom of Bungarus fasciatus, which has anti-tumor effect. This study confirmed that the venom of Bungarus fasciatus had anti-tumor cytotoxic effect, which would provide experimental basis for the clinical developing that anti-tumor agents of Bungarus fasciatus venom.