Study On The Relationship Among The NF-κB Activation, UPA Expression And Apoptosis Induced By 5-Fu In HCT116 Human Carcinama Cell Line

Objective:The purpose of this study was to investigate the efficacy of 5-fluorouracil(5-Fu) in inducing cytotoxity and apoptosis of colon carcinama cell line HCT116.The effect of pyrroledithiocarbomate(PDTC)combined with 5-Fu on the apoptosis and the activation of nuclear factor kappa B(NF-κB)was studied in HCT116 cell line.It was also observed that the fluctuation of urokinase-type plasminogen activator(uPA)according to the variation of the activity of NF-κB.Methods:The cytotoxity of 5-Fu or 5-Fu combined with PDTC to HCT116 cell line for 48 hours were detected by MTT method.The apoptotic rate was analysed by Annexin V-FITC. Fluctuation of NF-κB and uPA was detected by semi-quantitative immunohistochemistry.Results:1.We found that with the increase of dose of 5-Fu,the cytotoxity of 5-Fu on HCTll6 cell line was dramatically increased in a dose-dependent manners by MTT assay. There were significant difference between experimental and control groups(P＜0.01). Administration of 5-Fu combined with PDTC(luM)significantly improved cytotoxity of 5-Fu to HCT116 cells.There were significant difference between experimental and control groups (P＜0.001).2.Flow cytometry analysis revealed that there were no significant difference between control group and experimental group which HCTll6 cells were treated with luM 5-Fu or luM PDTC alone for 12 hour in apoptosis(P＞0.05).However,compared with control group,after 12 hour treatment of 10,100uM 5-Fu alone,there was significant difference in apoptosis (P＜0.05).There was a dose-dependent manner between 5-Fu and the apoptosis of HCT116 cells. luM PDTC increased significantly the apoptotic rate of HCT116 cells induced by 10 uM 5-Fu (P＜0.05).3.Our findings suggest that the level of NF-κB and uPA expression significantly increased in the treating group 10 uM 5-Fu alone,the positive unit of expression of NF-κB and uPA was dramatically increased in HCT116 cells by immunohistochemistry assay and image analysis system.There were significant difference between experimental and control groups (P＜0.05).After 24 hour treatment of 1uM PDTC alone,the level of NF-κB and uPA expression in this treatment group comparing with the control group had no significant difference(P＞0.05).Then,the level of NF-κB expression was decreased when HCT116 cells were pretreated with 1 uM PDTC for 1 hour before exposed to 10 uM 5-Fu for 24 hour.The level of uPA expression was so.PDTC could suppressed the activation of NF-κB induced by 5-fluorouracil.Compared with 10 uM 5-Fu alone group,There was significant difference in NF-κB and uPA expression(P＜0.05).Conclusion:1.PDTC evidently enhance 5-fluorouracil in inducing cytotoxicity in HCT116 cells,and thus inhibit the growth of cells.2.5-fluorouracil could induce apoptosis of HCT116 cells,activate NF-κB and up-regulate expression of uPA.3.PDTC could suppress the activation of NF-κB induced by 5-fluorouracil and decrease expression of uPA.4.PDTC could increase significantly the apoptotic rate of HCT116 cells induced by 5-fluorouracil and decrease expression of uPA.