Study On Induction Of Apoptosis In Human Tumor SMMC-7721Cells And Skov3Cells In Vitro By Natrin From Guangxi Cobra Venom

Aim: To separate and purify natrin from Guangxi cobra venom by gelchromatography, ion-exchange column chromatography and FPLC; To observegrowth inhibitory effects and apoptosis inducing effects of natrin on humanhepatocarcinoma SMMC-7721cells and human ovarian carcinoma SKOV3cellsin vitro, and then study apoptosis inducing mechanism of natrin on humanhepatocarcinoma SMMC-7721cells.Methods:(1) To isolate and purify natrin from crude Guangxi cobra venomby Sephadex G50gel filtration, CM Sepharose CL-6B cation exchange columnand FPLC mono-S column chromatography, SDS-PAGE and Mass Spectrometrywas used to investigate the purity and the relative molecular mass of natrin.(2)The growth inhibition ratio of SMMC-7721cells and SKOV3cells afterexposure to different concentrations of natrin24h were measured using MTTassay.(3) Morphological changes of apoptosis was detected by AO/EB stainingand transmission electron microscopy.(4)The cell cycle and apoptosispercentage were detected using FCM.(5)After exposure to different concentrations of natrin24h, the changes of mRNA expression quantity ofapoptosis related gene bax, bcl-2, p53and caspase-3were analyzed by FQ-PCRmethod in human hepatocarcinoma SMMC-7721cells.Results:(1) The molecular mass of the high purity protein natrin isolatedfrom Guangxi cobra venom was calculated to24.937KD by mass spectrum.(2)The growth of human hepatocarcinoma SMMC-7721cells and human ovariancancer SKOV3cells were inhibited by the treatment of Natrin. With the increaseof drug concentration, the growth inhibitory rate also increased. IC50of24h was138.69mg.L-1and26.13mg.L-1respectively.(3)After treated by natrin, typicalmorphological changes of apoptosis of the two kinds of cells were observed byfluorescence microscope and transmission electron microscope.(4)With theincrease of drug concentration, apoptosis percentage performed by flowcytometry also increased.(5) Natrin showed the effects of S and G2/M cell cyclearrest.(6) Natrin can reduce the expression of bax, bcl-2, p53and caspase-3inhuman hepatocarcinoma SMMC-7721cells. With the increase of the dose, themRNA expression quantity of each gene decreases.Conclusion: Natrin separated from the Guangxi cobra venom can inhibitproliferation and induce apoptosis in human tumor SMMC-7721cells andSKOV3cells in vitro; Apoptosis inducing mechanisms may be associated withthe effects of S and G2/M cell cycle arrest and reducing mRNA expression ofbcl-2gene in SMMC-7721cells.