Studies On Antitumor Activity Of L-Amino Acid Oxidase From Guangxi Cobra Venom In Vitro

Objective:To study the cytotoxic effects of L-amino acid oxidase(LAAO) purified from Guangxi cobra venom on the cultured tumor cells and study the mechanism of anti-tumor activity of LAAO from the relations of LAAO and H₂O₂,the angle of apoptosis and the effect of the tumorous cell cycle induced by LAAO.Methods:1.By using of DEAE-Sepharose CL-6B ion-exchange chromatography and CM-Sepharose CL-6B ion-exchange chromatography,a protein with L-amino acid oxdiase activity was purified from Guangxi cobra venom. SDS-polyacrylamid gel electrphoresis(SDS-PAGE)was used to analyze its purity and molecular weight under reducing and non-reducing conditions.2.The cytotoxic effect of LAAO was studied in different concentrations and different time on three cultured tumors:K562,Bel-7404,SGC7901 by MTT and resisted-colour trypan blue.3.Protective effects of catalase of K562 cells against cytotoxicity by LAAO or exogenous H₂O₂ were studied by MTT.4.The effect of LAAO induced,apoptosis on K562,Bel-7404 and SGC7901 cells was observed in different concentrations by inverted phase contrast microscope,H.E.dyeing,transmission electron microscopy and TUNEL assay.5.The effect of cell cycle of K562,Bel-7404 and SGC7901 cells induced by LAAO was observed by flow cytometric analysis. Results:1.The L-amino acid oxidase was purified from Guangxi cobra venom,its molecular weight is 55.2KD.2.The cytotoxic effect of LAAO was found on K562,Bel-7404,SGC7901 and LO2 cells and displayed fine concentration-dependent relationship.All of the correlation coefficients were greater than 0.95(p＜0.05).When three tumor cells and human normal hepatic cells mentioned above were explored on LAAO for 24h, we could get their IC₅₀:0.6U/ml,3.37U/ml,4.74U/ml and by MTT assay.But the IC₅₀of LO2 was 5.85U/ml,and there were 9.75,1.74,1.23 times compared with IC50 of three tumor cells,However,when four cells mentioned above were incubated with 5-Fu,the inhibition rate was 46.22%,42.99%,41.75%,46.45% respectively.To use resisted-colour trypan blue,we could learn following:their growth was inhibited significantly when three tumor cells mentioned above were incubated with LAAO for 24h,48h and 72h,and displayed fine time-dependently and concentration-dependent relationship.The inhibition of proliferation of LAAO was increased with the medicine function time prolonging.All of the correlation coefficients were greater than 0.95(p＜0.05).3.When LAAO(1.36U/ml)and H₂O₂(0.03mM)were respectively incubated with K562 cells,cytotoxicity was significant(p＜0.01),the effects of two medicines were equal(p＞0.05),and cell viability rate was 6.4%.The K562 cells were cultured with LAAO and catalase for 24h to get rid of the generated H₂O₂ in culture media,Nevertheless,the cell was not recovered fully and the viability rate was 78.49%.And the cell viability was recovered fully,when equal dosage of catalase and exogenous H₂O₂ were cultured with K562 cells.4.When low or high concentration of LAAO was cultured with K562, Bel-7404,SGC7901 cells for 24h,there were many typical apoptosis cells could be seen by morphological observation.The numbers of apoptosis cells was significant and had fine concentratin-dependent relationship.By TUNEL,we could observe every cells' apoptosis index(AI).The AI of low and high concentration of LAAO were 15.1%and 29.53%for K562,the AI of Bel-7404 cell were 29.34%and 54.66%;and that of SGC7901 cells were 17.07%and 30.57%.5.LAAO arrested K562 cells in G₀/G₁ phase of the cell cycle,and with the concentration of LAAO increasing,the proportion of K562 cells which arrested in G₀/G₁ phase increased;However,in Bel-7404,low concentration of LAAO arrested the cells in G₀/G₁ phase and S phase,high concentration of LAAO arrested the cells in S phase;And in SGC7901,compared with RPMI1640 group, the proportion of S phase cells increased in LAAO-treated groups,which appear to be dose-dependent.Conclusions:L-amino acid oxidase(LAAO)was purified from Guangxi cobra venom.In vitro,LAAO had obvious cytotoxicity in K562,Bel-7404, SGC7901 cells.And it had cytotoxic effect in LO2 cells,compared with the cytotoxicity in three tumor cells mentioned above,LAAO had better selectivity on K562 cells.But the positive control medicine——5-Fu had worse selectivity on tumor cells mentioned above.H₂O₂ catalysed by LAAO played a major role in the anti-tumor function of LAAO.The apoptosis induced by LAAO and inhibition of LAAO in the cell cycle in G₀/G₁ or S phase account for the mechanism of the anti-tumor activity of LAAO.