The Impact Of Interferon-α2a On Thymidine Phosphorylase (TP) Expression In Human Colon Carcinoma Cells.

Background Thymidine phosphorylase (TP) is a nucleoside metabolism enzyme,it is a homodimer of 45 kDa subunits,and platelet-derived endothelial-cell growth factor(PD-ECGF) and TP were confirmed as the same molecule,both of them showed significant angiogenic activity in vivo but no mitogenic activity in vitro. TP is regarded as important in the maintenance of a balanced deoxyribonucleoside triphosphate (dNTP) pool for DNA replication and repair. It was shown to be associated with the anti-apoptosis mechanism,because TP expression is very low in healthy human tissues, overexpression of the enzyme in cancer tissues has been associated with the promotion of tumorigenesis. Selective overexpression of TP in human cancer tissues has been investigated from various aspects.Initially, research on nucleoside metabolism enzymes showed that TP expression is frequently raised in a wide range of human cancer tissues .Expression is tumour dependent, and depends on the presence of other nucleoside metabolism enzymes such as thymidylate synthase. The prodrug or derivatives, such as 5'-deoxy-5-fluorouridine(5'-DFUR) and capecitabine (trade-name is Xeloda),of 5-fluorouracil(5-FU) must be activated and converted to 5-FU by TP in tissues,then both of them inhibit the synthesis of DNA and play a selective inhibition of the carcinoma cells.So it is the metabolic rate-limiting enzyme of 5'-DFUR and capecitabine. Several cytokines such as tumour necrosis factor (TNF)α,interleukin 1,and interferon can upregulate TP expression in malignant cells. Hypoxia or hypoglycaemic conditions could stimulate the expression of TP,which indicates that the enzyme is a product of inflammation or microenvironmental stress. TP-related cancer treatment is unique because there are already TP-targeting treatments such as fluorouracil and its derivatives. The combination of TP-inducible therapy with TP-targeting treatments of 5'-DFUR could enhance the overall effectiveness of therapy and result in a synergistic antitumour effect,and provide favourable effects for response rates,time to progression,and survival for patients with metastatic cancer. In this research we will detect the impact of interferon-α2a on thymidine phosphorylase (TP) expression levels in the human colon carcinoma cells,and provide experimental evidence for the clinical application of IFN-α2a combined with 5'-DFUR or capecitabine in the treatment of colon cancer. Since TP-targeting treatment has been developed in parallel with analyses of biological functions and translational implications,these therapies might become a specific chemotherapy method.Objective To detect the impact of interferon-α2a on thymidine phosphorylase (TP) expression levels in the human colon carcinoma cells.Methods MTT assay was used to determine the impaction of IFN-α2a on human colon carcinoma cells of the lines Lovo and SW480 in vitro, and to compare the cytotoxic effect of 5-FU or 5'-DFUR combined with IFN-α2a on the two cell lines.The cells were cultured and added with IFN-α2a of different doses, the method of Trizol was used to extracted the total RNA of cells, and then detected the quality and concentration of the total RNA. Fluorescence quantitative PCR was carried out on detection of the expression levels of cell TPmRNA of different groups. Results 1. The Lovo and SW480 cell lines were cultured and added with IFN-α2a of different doses, the survival ratio of cell lines between control group(IFN‐α2a0U/ml)and experimental groups(IFN-α2a by 2-fold serial dilutions were 320~1.25U/ml) did not change significantly, no significant difference between the groups(P>0.05);Before and after the combination of IFN-α2a , the toxic effects of 5-FU on Lovo and SW480 cell lines , namely the IC50 value of 5-FU had no significant changes, the difference was not statistically significant(P>0.05); the toxic effects of 5'-DFUR on the cells, that is, the IC50 value of 5'-DFUR decreased, and the difference was statistically significant(P<0.05).2.IFN-α2a increased the expression levels of TP in cell lines Lovo and SW480.Compared with the un-treated cells(IFN -α2a 0U/ml), the impact of 50 U/ml IFN-α2a doses on TP expression levels were no significant differences in both cell lines(P>0.05);IFN-α2a of the doses of 500U/ml and 5000U/ml significantly up-regulate the TP expression of the Lovo cells(P≤0.01); there were no significant differences in the TP expression of SW480 cells between the groups of 0U/ml and 500U/ml IFN-α2a doses(P>0.05),but IFN-α2a of the doses of 5000U/ml can significantly up-regulate the TP expression of SW480 cells(P=0.009). Within each cell lines, TP expression levels were not significantly different among experimental groups.Conclusion 1. IFN-α2a alone had no significant inhibitory effect on colon carcinoma cells in vitro. The combination of IFN-α2a did not improve the cytotoxic effect of 5-FU on the two cell lines, but can help enhance the inhibitory and cytotoxic effect of 5'-DFUR on them.2. TP were expressed in colon carcinoma cells of the lines Lovo and SW480. In a dose-dependent manner, IFN-α2a of certain doses up-regulate the TP expression levels in the human colon carcinoma cells lines Lovo and SW480.That is, only a certain concentration of IFN-α2a have a regulatory role. And after the dose range to achieve this effect, the dose of IFN-α2a have no significant effect on the regulation of TP expression level in the cells.