5FU is a pyrimidine analogue that inhibits DNA synthesis. The present study investigated the influence of nucleosides and nucleotides on 5FU-resistance in gastric cancer cells.
MKN45 cells (parental cells) and 5FU-resistant MKN45/F2R cells (resistant cells) were treated with 5FU, fluorodeoxyuridine monophosphate (FdUMP), and trifluorothymidine (TFT), which are inhibitors of thymidylate synthase (TS). Deoxyuridine monophosphate (dUMP), thymidine monophosphate (dTMP) and thymidine (dT) were combined with these drugs. The expression levels of TS and orotate phosphoribosyltransferase (OPRT) were determined by Western blotting. The viability of the cell lines was calculated by an MTT assay.
The resistant cells exhibited resistance to 5FU (93-fold), FdUMP (45-fold) and TFT (79-fold) and expressed decreased OPRT (0.74-fold) and increased TS (2.4-fold) levels in comparison to the parental cells. While dUMP alone was not cytotoxic to either of the cells, it decreased the IC50 for 5FU (88.2 ÂµM to 31.2 ÂµM) and FdUMP (51.2 ÂµM to 5.6 ÂµM) in the resistant cells and increased the IC50 for TFT in the parental cells (0.6 ÂµM to 3.15 ÂµM). Low concentrations (<10 ÂµM) of dTMP and dT reversed the cytotoxicity of TFT, while high concentrations were cytotoxic. A Western blotting analysis revealed that dUMP enhanced the upper band of TS after treatment with 5FU and FdUMP, which presented an increase in the ternary complex composed of FdUMP, folic acid and TS.
The increase in the ternary complex after treatment with 5FU with dUMP in resistant cells suggests that dUMP increased the level of FdUMP that was metabolized from 5FU. Furthermore, treatment with FdUMP (instead of 5FU) revealed similar result, indicating that the resistant cells had the ability to reduce the amount of intracellular FdUMP and that dUMP inhibited this ability because the decrease in OPRT and the increase in TS cannot explain these results.
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All authors have declared no conflicts of interest.