Effect About The Synthetic Inhibitor Of Urokinase Plasminogen Activator On The Invasion And Metastasis And Apoptosis Of Human Cervical Cancer Cell In Vitro

Objective: To investigate the effects of Amiloride which is the synthetic inhibitor of urokinase plasminogen activator on the invasion,metastasis and apoptosis of Human Cervical Cancer Cell in Vitro and its possible mechanism.Methods: Culture human cervical cancer cells Hela in Vitro, and detecte the effects of Amiloride on the expressions of Matriptase,uPA,uPAR and MMP-2 mRNAs by reverse transcription polymerase chain reaction (RT-PCR); Test the effect of Amiloride on migration of Hela cells by scratch assay; Test the effect of Amiloride on the invasion of Hela cells by millicell chamber artificial reconstituted basement membrane invasion assay; Detecte the effect of Amiloride on apoptosis of Hela cells by flow cytometry analysis.Results:1.Human cervical carcinoma Hela cell lines with high expression of Matriptase, uPA, uPAR and MMP-2 mRNAs.2.After 24 hours incubation with various concentrations of Amiloride, 50μmol/L of Amiloride could cause the significantly decrease of the expressions of uPA,MMP-2 mRNAs in dose-dependent manner in comparison with the control group(P<0.05); various concentrations of Amiloride do no effect on the expressions of Matriptase and uPAR mRNAs (P﹥0.05); After incubation with 150μmol/L Amiloride in different time-points, the expression of uPA mRNA decreased obviously in comparison with the control group after 2 hours P<0.01), when the expression of MMP-2 mRNA decreased obviously after 4 hours(P<0.05), there was obvious time-dependent manner between Amiloride and uPA,MMP-2 mRNA.3.The 6,12,24hour'migration distance of Hela cells after 50,100,150μmol/L Amiloride incubation were significantly lower than the control group (F=56.893, 360.000,360.038,all P<0.01), and the migration was positively correlated with the uPA mRNA expression levels(r=0.942, P<0.01).4.The number of Hela cells that penetrated the Matrigel after 50,100,150μmol/L Amiloride incubation decreased obviously in comparison with control group (F=226.95,P<0.01),and the invassiveness was positively correlated with the uPA mRNA expression levels(r=0.969, P<0.01).5.The early apoptosis rate of Hela cells after 50,100,150μmol/L Amiloride incubation 24,48 hours increased obviously in comparison with control group (F=59.895,472.428,all P<0.01), and the apoptosis rate was negatively correlated with the uPA mRNA expression levels(r=﹣0.941, P<0.01).Conclusions:1.The low concentration of Amiloride could specificly inhibit the expression of uPA mRNA of human cervical carcinoma Hela cell lines in a concentration - time dependence, what could cause decrease of the expression of MMP-2 mRNA which was the uPA system's downstream factor, but do no effect to uPAR and Matriptase which was the uPA system's upstream regulator.2.uPA may be a gene associated with the invasion and metastasis of human cervical cancer, Amiloride can suppress the invasion and metastasis of Hela cells in vitro by down-regulation the uPA mRNA expression.3. uPA may be a gene associated with cell apoptosis, Amiloride can induce apoptosis of Hela cells in vitro by down-regulation the uPA mRNA expression, suggesting that deserves further study about its anti-tumor effect.