Abstract
Background5FU can be converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through two pathways: the orotate phosphoribosyl transferase–ribonucleotide reductase (OPRT–RR) pathway and the thymidine phosphorylase–thymidine kinase (TP–TK) pathway. We investigated the mechanism underlying 5FU-resistance, focusing on the changes in the 5FU metabolisms.MethodsMKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU or fluoro-deoxyuridine (FdU) in combination with hydroxyurea (HU) or tipiracil (TPI). The amount of FdUMP was determined by the density of the upper band of thymidylate synthase on Western blotting.ResultsThe MKN45/F2R cells exhibited 5FU resistance (37.1-fold) and showed decreased OPRT and increased TP levels. In both cells, the FdUMP after treatment with 5FU was decreased when RR was inhibited by HU but not when TP was inhibited by TPI. A metabolome analysis revealed the loss of intracellular deoxyribose 1-phosphate (dR1P) in both cells, indicating that FdUMP was synthesized from 5FU only through the OPRT–RR pathway because of the loss of dR1P. After the knockdown of TK, the FdUMP after treatment with FdU was decreased in MKN45 cells. However, it was not changed in MKN45/F2R cells. Furthermore, TP inhibition caused an increase in FdUMP after treatment with 5FU or FdU and reversed the 5FU resistance in MKN45/F2R cells, indicating that FdUMP was reduced through the TP–TK pathway in MKN45/F2R cells.ConclusionsIn MKN45/F2R cells, the reduction of FdUMP through the TP–TK pathway caused 5FU resistance, and the inhibition of TP reversed the resistance to 5FU, suggesting that the combination of 5FU and TPI is a promising cancer therapy.
Highlights
Gastric cancer remains a major cause of cancer death worldwide [1], as many patients with gastric cancer are still diagnosed only at late stages, and recurrent tumors are often detected even after curative surgery
MKN45/F2R cells that had been previously established as 5FU-resistant cells showed an IC50 of 50.4 ± 2.52 μM, which represented a 37.1-fold increased resistance compared with parental MKN45 (IC50: 1.36 ± 0.24 μM)
After 5FU treatment, both cell lines exhibited upper bands of thymidine synthase (TS) on a Western blot analysis (Fig. 1d), which represents TS in ternary complexes composed of TS, CH2THF and fluoro-deoxyuridine monophosphate (FdUMP); the density of the upper band is correlated with the intracellular concentration of FdUMP [16]
Summary
Gastric cancer remains a major cause of cancer death worldwide [1], as many patients with gastric cancer are still diagnosed only at late stages, and recurrent tumors are often detected even after curative surgery. Analyses of the gene expression in patients enrolled in the ACTS-GC trial, which investigated the efficacy of S-1 (5FU derivatives) for adjuvant therapy, revealed that increased TS was, if anything, related to a good prognosis, and that the expressions of OPRT and TP were not related to the prognosis at all [14] These findings suggest that there may be unknown mechanisms of action involved in the development of 5FU resistance. Conclusions In MKN45/F2R cells, the reduction of FdUMP through the TP–TK pathway caused 5FU resistance, and the inhibition of TP reversed the resistance to 5FU, suggesting that the combination of 5FU and TPI is a promising cancer therapy
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