| Background Thymidine phosphorylase (TP) is a key enzyme that contributes in themetabolic process of pyrimidine nucleotides. TP has a structure similar to that ofplatelet-derived endothelial cell growth factor (PD-ECGF); therefore, these twoconstituents are considered to be the same substance. TP promotes angiogenesis andhas important functions in growth, invasion, and metastasis of colorectal carcinoma.High expression of TP can selectively activate5’-deoxy-5-fluorouridine (5’-DFUR),capecitabine (CAP), and other drugs; then TP can converts these drugs into5-FU.Consequently, these drugs can have antitumor effects. Therefore, TP expressionstudies are important in angiogenesis, invasion, and metastasis of colorectalcarcinoma, as well as in chemotherapy. The concentration of TP in colorectalcarcinoma tissues is apparently higher than in the surrounding normal tissues.However, different findings exist on whether cancer cells or interstitial cells incarcinoma tissues express TP. Early studies have indicated that cancer cells coulddirectly express high concentration of TP, as verified in gastric and breast cancers. Inrecent years, studies have suggested that TP is mainly expressed by interstitial cells inthe tumor tissue, especially the tumor-associated macrophage (TAM) on the tumoredge. Our preliminary studies performed TP enzyme assays and protein quantitativedetections for40cases of colorectal carcinoma specimens;6colorectal carcinoma celllines, namely, LS174T, Clone A, Colo320, CX-1, Lovo, and MIPIO1; and2macrophagic systems of THP-1and U937. The results showed that the TP activity ofcolorectal carcinoma cell was evidently higher than that of the normal tissue. Thisfinding indicated that few TP proteins were expressed in colorectal carcinoma cells.The majority of cells that expressed TP activity were interstitial cells surrounding the cancer cells, especially the TAM. In fact, most results showed that TP activity waslikely expressed by TAM. On the other hand, transferred the IFN-α2a into LOVO andSW480colorectal carcinoma cells, and found that the levels of TP mRNA increased.This laboratory finding indicated that INF could adjust TP expression during or afterthe transcription process. Thus, the conversion efficiency of5’-DFUR to5-FUincreased, and the antitumor effects of5’-DFUR was enhanced.Lentiviral vectors are a type of viral vector system that remoulded from HIV-1. Itinfect both dividing and nondividing cells. Lentiviruses can be used to provide highlyeffective gene therapy as lentiviruses can change the expression of their target cell’sgene. They can be used for nondividing or terminally differentiated cells. Therefore agrowing number of scholars use lentiviral vectors to build the human colorectalcancer cell lines with the high expression of TP.Our experiment is transfecting TP cDNA with lentiviral vector into human coloncarcinoma cell line HT29and LS174T, studying the change between with the parentalcells, such as the transfection efficiency, the changes of IC50on these cells to5’-DFUR before and after transfection, and the volumes of converted5-FU either inthe medium contained a series of concentration of5’-DFUR or in2cultured cellslysate.Objective Thymidine phosphorylase (TP) cDNA was transfected into colorectalcancer cell lines HT29and LS174T with the lentiviral vector, then the anticancereffeciency of5’-deoxy-5-fluorouridine (5’-DFUR) and5-fluorouracil (5-FU) on these2cells both for parents cells and TP-transfected cells were evaluated.Methods TP cDNA were transfected into human colorectal cancer cell lines HT29and LS174T with the lentiviral vector pLenti6.3_MCS_IRES2-EGFP, and thetransfection efficiency was analyzed by flow cytometer. Then the TP proteinexpression and the relative quantitative analysis for TP mRNA in these2cells weredetected by immunohistochemistry, Western blot, and RT-PCR respectively. The50%inhibitory concentration (IC50) of5’-DFUR and5-FU on HT29, LS174T both parentscells and TP-transfected cells were evaluated by MTS assay. At last, the volumes ofconverted5-FU either in the medium contained a series of concentration of5’-DFUR, in which HT29/HT29-TP or LS174T/LS174T-TP were cultured, or in2cultured cellslysate, were detected by high performance liquid chromatography (HPLC).Results The HT29and LS174T transfected with human TP cDNA were monitored5generations, and the transfections efficiency was about95%. Immunohistochemicalstaining and Western blot showed that the TP protein expression in HT29-TP andLS174T-TP were obviously increased compared with their parents cells. The RQvalues of TP mRNA expression in HT29-TP and LS174T-TP were also8.45folds and2615.02folds higher than their parents cells, respectively (P<0.01). The IC50values of5’-DFUR on HT29-TP and LS174T-TP were14.33μmol/L and0.59μmol/L,respectively, significantly lower than their parents cells707.66μmol/L and239.20μmol/L (P<0.01). Also the IC50values of5-FU on HT29-TP and LS174T-TP LS174Twere5.42μmol/L±0.75μmol/L and4.41μmol/L±0.96μmol/L, significantly lowerthan their parents cells, which were14.19μmol/L±0.97μmol/L and16.42μmol/L±2.12μmol/L, respectively (P<0.01). The HPLC results show us that the5-FUvolumes detected from media contained series concentration of5’-DFUR forculturing HT29-TP and LS174T-TP were12.2~28.7folds, and13.1~23.6folds morethan their parents cells respectively (P<0.01). Otherwise, just a little of5-FU wasdetected in the2TP-transfected cells lysate, whereas no5-FU was detected in the2parents cells lysate.Conclusion Transfected TP cDNA into colorectal cancer cell line HT29andLS174T with lentiviral vector could improve the expression both of TP mRNA and TPprotein level, increase the volume of5-FU converted from5’-DFUR in medium, andresult in an enhancement of anticancer effect on these2cells. |
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