The Inhibition Of Carcinoma Cell Growth Induced By N3-o-toluyl-fluorouracil And Analysis Of Biotransformation

Background:N₃-o-toluyl-fluorouracil(TFU) is the derivative of atofluding, N₁-acetyl-N₃-o-toluyl-fluorouracil,and the prodrug of 5-FU.In this study,the growth inhibition of human hepatocellular carcinoma cells was measured and the biotransformation of TFU was analyzed by using High-performance liquid chromatography(HPLC) in vitro and in vitro.Methods:In vitro studies,the growth of human hepatocellular carcinoma cells SMMC-7721 and PLC/PRF/5 was measured in absence and in the present of liver microsomal enzymes and the efficacy of TFU were evaluated by using 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) assay.In vivo study,the S180 mouse sarcoma cells with ascites were harvested,and inoculated subcutaneously into the right armpit region of Kunming mice.The effects of TFU on the transplantable sarcinoma S180 in mice were examined after 10 to 14 days oral administration.In study of biotransformation of TFU,the measurement method of TFU and 5-FU were established using HPLC analysis.The levels of TFU and 5-FU in the medium in vitro,and in the serum and various of the tissue in treated mice were measured.Results:Human hepatocellular carcinoma cells SMMC-7721 were treated with TFU (5.0μg·mL^-1-200μg·mL^-1) for up to 120 h and then the rates of cell growth inhibition were evaluated based on the viable cell number as estimated by MTT assay. Incubation with TFU weakly prevented SMMC-7721 cell proliferation,although the significant differences were seen in the higher concentrations.Next,cells were incubated with TFU in the presence of liver microsomal enzymes and the cell proliferation was examined.Proliferation of SMMC-7721 cells was drastically suppressed in the presence of liver microsomal enzymes.The inhibition rates were significantly promoted in all concentrations(0.05—300μg·mL^-1) and incubation time (24 to 120 h).The gains of inhibitory rates were from 27.8 to 41.9%and the tumour growth inhibition was dose-dependent.The effects of TFU on tumor cell growth were then examined in the transplantable sarcinoma S180 in mice.TFU delayed the growth of sarcinoma S180 by 38.99%,51.83%and 60.68%after 10-12 days of oral administration with 50,100 and 200 mg/kg.The tumour growth inhibition was dose-dependent.The levels of 5-FU and TFU in the medium of the cell culture were analyzed by HPLC method every 24 hours for five consecutive days and the inhibition of cell growth was compared and the sustaining release of 5-FU was analyzed.HPLC analysis showed that TFU might transform into 5-FU by hydrolyzing in the presence of liver microsomal enzymes.The cell growth inhibition of SMMC-7721 may related to the sustaining release of 5-FU from TFU.We then measured the serum and various organs of mice after 10-12 days of oral administration with TFU using HPLC.The results showed that the level of 5-FU was measured in the tissuse of liver and the transplantable sarcinoma S180 of mice.5-FU was measured in the sissues of lung and serum in the treated mice although the levels were lowered than 0.05·mL^-1.5-FU were not measured in other tissue of organs.Conclusion:These results suggest that treatment of TFU in the presence of liver microsomal enzymes induced the enhancement of growth inhibition in the human hepatocellular carcinoma cells.The growth inhibition of TFU was confirmed in the transplantable sarcinoma S180 in mice.The mechanism of TFU action might via sustaining release of 5-FU by hydrolyzing in the presence of liver microsomal enzymes.This suggests that TFU may be a promising pro-drug of 5-FU for cancer treatments.