Mechanism of 5-fluorouracil required resistance in human hepatocellular carcinoma cell line Bel7402

AIM:TO investigate the resistance mechanism of 5-fluorouracil(5-FU)in Bel7402/5-FU cells which was establishedin our lab by in vitrocontinuous stepwise exposure of humanhepatocellular carcinoma(HCC)cell line Bel7402to 5-FU.METHODS:The expression of multidrug resistance-associated protein(MRP)and thymidylate synthase(TS)inBel7402cells was detected by immonocytochemistry.Thefluorescein(FLU)accumulation,an index of MRP functionalactivity,was determined by flow cytometry.The distributionof FLU was observed by confocal laser scanning microscope.The spectrofluorometry was used to show the intracelluarcontent of glutathione(GSH).Cell growth inhibition wasdetermined by MTT assay.The activity of glutathione S-transferases(GSTs)was determined by spectrophotometry.RESULTS:A higher expression of MRP in the Bel7402/5-FUcells was observed by using monoclonal mouse anti-MRPantibody,MRPr-1,in comparison with Bel7402cells.Bel7402/5-FU cells also showed a significant decrease of FLUaccumulation.FLU mainly accumulated in the nucleus witha high nuclear/cytoplasmic ratio in Bel7402cells,whereas therewas no difference of FLU accumulation between the nucleusand cytoplasm in Bel7402/5-FU cells.The intracellular GSHcontent in Bel7402/5-FU cells was almost 3 folds higher thanthat in Bel7402cells.Addition of D,L-buthione-S,R-sulfoximine(BSO)dose-dependently reduced the GSH content in Bel7402/5-FU cells,however,only a weak enhancement on thecytotoxicity of 5-FU and doxorubicin(Dox)to Bel7402/5-FU cellswas observed.Bel7402/5-FU cells also exhibited 29.1% highertotal GSTs activity than Bel7402cells.Immunocytochemicalstaining by using anti-TS monoclonal antibody TS 106 showedthat the level of TS in Bel7402/5-FU cells elevated markedly ascompared with Bel7402cells.CONCLUSION:The continuous exposure of Bel7402cells to5-FU led to overexpression of TS and MRP,as well asincreased intracellular GSH content and total GST activity.