Effect of thymidine phosphorylase cDNA transfection on the inhibition of human colon carcinoma cell line by 5'-deoxy-5-fluorouridine / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Enhanced anticancer effects of 5'-DFUR on colorectal cancer cell lines SW480 and LOVO by transfection with thymidine phosphorylase cDNA / 中华外科杂志

<p><b>OBJECTIVES</b>To study the change of ability to transform from 5'-deoxy-fluorouracil monophosphate (5'-DFUR) to fluorouracil (5-FU) in human colon cancer cell lines SW480 and LOVO which transfected with thymidine phosphorylase (TP) gene. And to discuss the anti-cancer activity of 5'-DFUR to SW480 and LOVO cells.</p><p><b>METHODS</b>TP cDNA were transfected into human colorectal cancer cell lines SW480 and LOVO with the lentiviral vector, pLenti6.3_MCS_IRES2-EGFP. The transfection efficiency was analyzed by flow cytometer, the mRNA expression of TP was detected by RT-PCR, and the TP protein expression was detected by Western blot, and the volumes of 5-FU converted from 5'-DFUR both in 2 cells and medium were detected by high performance liquid chromatography (HPLC). The 50% inhibitory concentration (IC50) of 5'-DFUR on these 2 colon cancer cell lines both wild type and TP-transfected cells were evaluated by MTT assay.</p><p><b>RESULTS</b>The colorectal cancer cell lines SW480 and LOVO transfected with human TP cDNA were monitored 5 generations, and the transfections efficiency rate wea about 95%. Compared with wild type cell SW480 and LOVO, the RQ values of mRNA expression of SW480-TP and LOVO-TP were (695 ± 171) folds (t = -7.00, P = 0.002) and (282 ± 87) folds (t = -5.61, P = 0.030), respectively. Also TP protein expression in SW480-TP and LOVO-TP were higher than their parent cells shown by Western blot. The volume of 5-FU converted from 5'-DFUR in the medium cultured SW480-TP and LOVO-TP were increased compared with their parent cells, respectively (t = 19.406-66.921, P < 0.01), whereas few of 5-FU was detected both in wild, and TP-transfected cells. After transfected with TP cDNA, the IC50 of 5'-DFUR on SW480-TP and LOVO-TP were (587 ± 17) µmol/L and (1088 ± 89) µmol/L respectively, and there were significantly less than their parent cells (t = -32.59 and -8.52, P < 0.01).</p><p><b>CONCLUSIONS</b>The stabilized transfections of SW480 and LOVO with higher TP expression could be built with lentiviral vector. Transfected TP cDNA into SW480 and LOVO, could improve the expression both of TP mRNA and TP protein, increase the volume of 5-FU converted from 5'-DFUR in medium, and result in an enhancement of anticancer effect on these 2 cells.</p>