| Objective: To study the inhibition of proliferation of LAAO purified from King Cobra venom on the cultured tumor cells which were tested by traditional assays—the inhibition of proliferation on the cultured tumor cells in vitro and ATP-Bioluminescence assay, and to explore its possible antitumor mechanism by induced- apoptosis on tumor cells .Methods: The primary culture and subculture of HUVECs were cultured by improving culture method of Jaffe A, and then verifying them; The inhibition of proliferation of LAAO was studied in different concentrations and different time on four cultured tumor cells: NACC,CNE2,TCA8113 and Bel7404 by MTT, resisted-colour trypan blue, clony-forming and ATP-Bioluminescence assay. Negative control is 0.02M pH 7.4 Tris-HCl and positive control is MMC, while HUVEC is normal cell control; The effect of LAAO induced-apoptosis on NACC cells was observed in different concentrations by inverted phase contrast microscope, H.E. dyeing, transmission electron microscopy and TUNEL assay. Results: The HUVECs arrayed like 'cobblestone' under inverted phase contrast microscope, and factor VIII related antigen in the cytoplasm of HUVECs showed positive reaction by immunohistochemistry and Weibel-Palade bodies were observed in the cytoplasm under transmission electron microscope, which were identified as HUVECs; The effect of cytotoxicity of LAAO was found on CNE2, NACC, TAC8113, Bel7404 and HUVEC cells and displayed fine concentration-dependent relationship. The correlation coefficients were 0.9758,0.9793,0.9976,0.9864,0.9361(p<0.05 ). When four tumor cells mentioned above were explored on LAAO for 24h, we can get their IC50:0.26U/ml,0.54U/ml,0.23U/ml and 0.24U/ml by MTT assay. But the IC50 of HUVEC cells was 5.59U/ml, and there were 21.50,10.35,24.30,23.29 times when compared with IC50 of four tumor cells. Their growth was inhibited significantly when four tumor cells mentioned above were explored on LAAO for 24h,48h and 96h, and displayed fine time-dependently and concentration- dependent relationship. When tumor cells were explored on LAAO for 24h, the correlated coefficients were 0.9986,0.9867,0.9916,0.9915 (P<0.05) and their IC50 were 0.31U/ml,0.64U/ml,0.36U/ml,0.30U/ml. The correlation coefficients (r) were 0.9973,0.9978,0.9858,0.9916 (P<0.05) and their IC50 were0.21U/ml, 0.32U/ml,0.26U/ml,0.20U/ml for 48h, while the correlation coefficients were 0.9925,0.9980,0.9950,0.9740 (P<0.05) and their IC50 were 0.10U/ml, 0.18U/ml, 0.11U/ml, 0.11U/ml for 96h. The inhibitory effect of LAAO on the growth of four tumor cells were more strengthen for 96h. In addition, the inhibitory effect of LAAO on the clony forming of NACC cells was significant and had fine concentration-dependent relationship. The correlation coefficients (r) was 0.9782(p<0.01) and its IC50 was 0.49U/ml; There was linear relationship between the log ATP standard and log intensity of fluorescence and between the log cell number and log intensity of fluorescence. Line equations were Y=2.805+0.8926X r=0.9993 (P<0.001),Y=1.1047+0.6376X r=0.9735(P<0.001) and CV was small for 3.1%. Their growth was inhibited significantly when four tumor cells were explored on LAAO for 24h,48h and 96h, and displayed fine time-dependent relationship and concentration-dependent relationship.Their IC50were 0.59U/ml, 0.26U/ml, 0.28U/ml,0.27U/ml in 24h, and it was not significant when compared with MTT assay(P>0.05). Their IC50 were 0.47U/ml,0.18U/ml,0.18 U/ml ,0.17 U/ml in 48h, and 0.21U/ml,0.10 U/ml,0.12 U/ml,0.11 U/ml in 96h. All CV were small for 3.5%. The cytotoxicity of LAAO on tumor cells in vitro was verified apoptosis not necrosis. Conclusion: The cultured cells were verified as human umbilical vein endothelial cells; LAAO of King Cobra venom can cause cytotoxicity and inhibitory effect on the growth of four tumor cells- NACC, CNE2, Bel7404and TCA8113, and there was at least eight times on concentration of LAAO when compared with IC50 of HUVEC; There was fine stability, repeatity and sensitivity on ATP-Bioluminescence assay when using on the cytotoxicity and... |
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