Isolution Identification And Fuction Of Anti-tumor Activity Compontents Guangdong Cobra Venom (Naja Naja Atra)

Snake venom is a natural protein which is secreted by snakes.Venom is a mixtureconsisting of10¹5kinds of substances, wherein there are5¹5kinds of enzymes,3¹2kinds of non-enzymes and other types of small-molecular substances. The current extensiveresearched are cardiotoxin, neurotoxins, nerve growth factor, anticoagulant and procoagulanttoxins. Previous studies separated and purified the protein with analgesic activity. On the basisof previous research, this research can separate cardiotoxin from snake venom. The aim is tostudy of the active of cardiotoxin effect on tumor cells and the mechanism of cardiotoxin onA549cells.MethodsPortion of the material was removed by PAA. Used ion cation exchange UNO SphereTMSto obtain SVI3, then identified by SDS-PAGE and HPLC. MTT method was used fordetermination of SVI3in vitro cytotoxicity. Giemsa stain and Hoechst33258staining methodwere employed to examine the effects of SVI3on apoptpsis of A549cells. The apoptosis ratewas quantified by flow cytometry （FCM）. JC-1was used to observe the changes inmitochondrial membrane potential in A549cells. The spectrophotometer assay was used todetected Caspase-3and Caspase-9activity. The activity of Caspase-3and Caspase-9weredetected by the western blotting. S180mouse sarcoma model was used to checked SVI3invivo anti-tumor activity.Results1. After processing, chromatographically pure SVI3could be obtained. The results of theMTT assay showed that the different cytotoxic on A549cells have a different cell toxicity.And the IC₅₀of SVI3was0.770μg/mL.2. Some apoptotic bodies could be observed in the2μg/mL of SVI3by Giemsa staining andHoechst33258staining. The number of cells was significantly reduced and there was densestaining, nuclear pyknosis, chromatin condensation and marginalization when the SVI3concentrations reached3μg/mL. Analyzed by flow cytometry, SVI3induced apoptosis inA549cells in a dose-dependent manner. 3．SVI3could induce A549cell membrane mitochondrial membrane potential by the JC-1.Cytochrome C in the cytosol was detected simultaneously, which indicated the release ofCytochrome C from mitochondria to cytosol. The caspase-9was not activated initially,whereas the cleavage of caspase-3was detected at0.5h （CTX5μg/mL）. SVI30.5mg/kg/dayand1.0mg/kg/day intraperitoneally for eight days significantly reduced the weight of S180versus untreated control, respectively59.99%and85.45%.Conclusion1. An effective method can be established for the SVI3of snake venom. Aslo the purity ofSVI3is99%.2. The vitro experiments and vivo experiments show that SVI3could inhibit the tumor. Andthe results reveal that inhibition effect of SVI3is higher than the cyclophosphamide group.3. SVI3can not only through the mitochondrial pathway of apoptosis of A549cells, butalso there may be other ways to affect the caspase family involved in apoptosis.