Objective: To investigate the inhibiting impact of 5'-deoxy-5-fluorouridine (5'-DFUR) on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA.
Methods: TP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP, and the transfection efficiency was analyzed by flow cytometry. TP mRNA and protein expressions were detected by RT-PCR and Western blotting respectively. The IC50 of 5'-DFUR on TP-transfected LOVO and parental cell were evaluated by MTT assay. The volumes of 5-FU converted from 5'-DFUR in media, where TP-transfected and parental LOVO were cultured, were detected by HPLC.
Results: The stable transfectants passed 5 generations were obtained and the transfection rate was 95%. Compared with parental cell, the RQ values of mRNA expression in TP-transfected LOVO was (282.5±86.8) folds higher significantly (P<0.01), also the TP protein expression of TP-transfected LOVO was obviously up-regulated as compared to parental cells. The IC50 value of 5'-DFUR of TP-transfectants was (1087.7±89.1) μmol/L, less than (1607.3±56.8) μmol/L of parental cells significantly (P<0.01), while there was no significant difference between parental cells and vector-transfectants [(1699.5±38.7) μmol/L, P>0.05]. HPLC revealed that when medium was added with 0, 500, 1000, and 2000 μmol/L of 5'-DFUR respectively, 0, 2.10, 3.13, and 7.19 μmol/L of 5-FU was found in the parental cells culture, while 0, 22.16, 30.94 and 40.02 μmol/L of 5-FU was found in TP-transfectants culture, but no 5-FU was found in the vector-transfectants culture.
Conclusion: TP cDNA transfection into LOVO can up-regulate the TP mRNA and protein expressions, increase the 5-FU converted from 5'-DFUR, and enhance the cytotoxic effect of 5'-DFUR on the LOVO cells.