Effects Of INOS Inhibitor 1400W On Proliferation And VEGF Expression In Hepatocarcinoma Cell Line SMMC-7721

ObjectivesTo determine the effects of iNOS inhibitor 1400W on the proliferation of HCC SMMC-7721 and the expression of vascular endothelial growth factor (VEGF) in Hepatocarcinoma cell line SMMC-7721.Methods1. The cultured SMMC-7721 cells were treated with different concentrations of 1400W(12.5,25, 50, 100, 200,400μmol/L) for different time(24h,48h,72h). The effect of 1400W on the growth of SMMC-7721 cells was evaluated by methyl thiazolyl tetrazolium(MTT) assay. The apoptosis rate (AR)was quantified by flow cytometry (FCM). The levels of NO in the supernatants was measured with nitrate reducase method. The expression of iNOS and VEGF mRNA in SMMC-7721 cells were examined by Real-time polymerase chain reaction.2. The SMMC-7721 cells cultured in hypoxia (5%CO2,95%N2)were treated with different concentrations of 1400W(50, 100, 200μmol/L) for 24h. The levels of NO in the supernatants were determined by nitrate reducase method and the expression of iNOS and VEGF mRNA in SMMC-7721 cells were examined by Real-time polymerase chain reaction.Results1. Compared to control group , 1400W could inhibit the proliferation of SMMC-7721 cells significantly(P<0.05). All this effects of 1400W were in a dose-time - dependent manner. After treated with 50μmol/l, 100μmol/l, 200μmol/l, 400μmol/l 1400W for 72 hours,the inhibition rates were 16.42%,25.37%,31.34%,34.33%,respectively;2. After treated with 50μmol/l,100μmol/l,200μmol/l 1400W for 48 hours,the G0/G1 phase cells increased (P<0.05) while the S phase cells decreased(P<0.05),this effects of 1400W were in a dose-dependent manner,and 76.48%±0.14 of cells were arrested at G0/G1 phase after treated with 200μmol/l 1400W. and S phase cells were 18.34%±0. 20. Meanwhile,1400W could also induce the apoptosis of SMMC-7721 cells. After treated with 100μmol/l,200μmol/l 1400W for 48 hours,The apoptosis rate of SMMC-7721 cells were 4.52%±0.28,9.14%±0.31,respectively;3. Nitrate reducase method found that in normal cultured condition , compared to control group, the levels of NO in the SMMC-7721 cells supernatants were inhibited by 1400W(P<0.05),all this effects of 1400W were in a dose-time-dependent manner. After treated with 50μmol/l,100μmol/l,200μmol/l,400μmol/l 1400W for 72 hours,the levels of NO in the cells supernatants were 36.36±1.4,33.23±1.5,29.55±2.2,21.59±3.0μmol/l respectively. In hypoxia condition, SMMC-7721 cells were treated with 50, 100, 200μmol/l 1400W for 24h, the levels of NO in the cells supernatants were 28.77±2.1 , 21.63±3.0 ,17.23±2.4μmol/l, respectively. Compared to control group ,the levels of NO in the supernatants was decreased (P<0.01)in a dose-dependent manner;4. RT-PCR found : (1) in normal cultured condition , after treated with 0μmol/l,50μmol/l, 100μmol/l,200μmol/l 1400W for 48 hours, the expression of iNOSmRNA were 0.217±0.038,0.170±0.030,0.145±0.030,0.085±0.031,respectively; the expression of VEGFmRNA were 0.446±0.016,0.341±0.022,0.261±0.034,0.190±0.036, respectively. Compared to control group,1400W could down-regulate the expression of iNOSmRNA (P<0.05)and VEGFmRNA(P<0.05)in SMMC-7721 cells significantly. (2) in hypoxia condition, after treated with 0μmol/l ,50μmol/l ,100μmol/l,200μmol/l 1400W for 24 hours, the expression of iNOSmRNA were 0.246±0.009,0.212±0.009,0.169±0.012,0.136±0.006, respectively; the expression of VEGFmRNA were 0.598±0.040,0.429±0.038,0.309±0.022,0.249±0.014, respectively. Compared to control group,1400W could down-regulate the expression of iNOSmRNA (P<0.05)and VEGFmRNA(P<0.05)in SMMC-7721 cells significantly.ConclusionsiNOS inhibitor 1400W can inhibit the proliferation and induce the apoptosis of SMMC-7721 cells in vitro. The mechanism may be related to the inhibition of cell circle growth. These results indicate that 1400W can inhibit growth and metastasis of HCC. This study provided a preliminary experimental evidence for the screening of iNOS inhibitor analogues in clinic therapy of HCC.