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. 2017 Sep;14(3):3162-3168.
doi: 10.3892/ol.2017.6512. Epub 2017 Jun 30.

5FU resistance caused by reduced fluoro-deoxyuridine monophosphate and its reversal using deoxyuridine

Ryutaro Mori  1 , Manabu Futamura  1 , Toshiyuki Tanahashi  1 , Yoshihiro Tanaka  1 , Nobuhisha Matsuhashi  1 , Kazuya Yamaguchi  1 , Kazuhiro Yoshida  1
Affiliations

5FU resistance caused by reduced fluoro-deoxyuridine monophosphate and its reversal using deoxyuridine

Ryutaro Mori et al. Oncol Lett. 2017 Sep.

Abstract

The mechanism of 5-fluorouracil (5FU) resistance was investigated, focusing on the level of thymidylate synthase (TS) ternary complex formed with fluoro-deoxyuridine monophosphate (FdUMP). MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU and fluoro-deoxyuridine (FdU) in combination with deoxyuridine (dU) and thymidine (dT). Subsequently, the levels of ternary complex were determined by western blotting and the cell viability was calculated using an MTT assay. MKN45/F2R cells exhibited 5FU resistance (56.2-fold relative to MKN45 cells), and demonstrated decreased orotate phosphoribosyltransferase (OPRT) and increased TS levels, requiring a higher concentration of 5FU to induce ternary complex formation than MKN45 cells. Following transfection of small interfering RNA against OPRT, MKN45 exhibited increased resistance to 5FU and decreased ternary complex formation subsequent to treatment with 5FU, indicating that decreased OPRT led to increased 5FU resistance. However, MKN45/F2R also exhibited resistance to FdU, which can be converted to FdUMP without OPRT, and there was decreased ternary complex formation after treatment with FdU, indicating that the 5FU-resistant cells had the ability to decrease intracellular FdUMP. The addition of dU and thymidine dT to 5FU promoted the formation of ternary complexes and reversed 5FU resistance in MKN45/F2R cells, although dT inhibited the efficacy of raltitrexed (another TS inhibitor). These results suggested that 5FU-resistant cells had the ability to reduce intracellular FdUMP irrespective of decreased OPRT, which led to resistance to 5FU. This resistance was then inhibited by treatment with dT or dU.

Keywords: 5FU; drug resistance; fluorodeoxyuridylate; stomach neoplasms.

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Figures

Figure 1.
Figure 1.
Effect of 5FU, FdU and OPRT siRNA on the expression of TS and OPRT, and the viability of MKN45 and MKN45/F2R cells. (A) Western blot analysis of TS and OPRT protein expression following treatment with 5FU in MKN45 and MKN45/F2R cells. (B) Western blot analysis of TS and OPRT protein expression following transfection with OPRT siRNA and treatment with 5FU in MKN45 cells. (C) MTT assay for the effect of 5FU treatment on the relative proliferation of MKN45 cells, MKN45 cells transfected with OPRT siRNA and MKN45/F2R cells. (D) Western blot analysis for TS and OPRT protein expression following treatment with 5FU and FdU in MKN45 and MKN45/F2R cells. (E) MTT assay for the effect of 5FU and FdU treatment on the relative proliferation of MKN45 and MKN45/F2R cells. 5FU, 5-fluorouracil; FdU, fluoro-deoxyuridine; OPRT, orotate phosphoribosyltransferase; siRNA, small interfering RNA; TS, thymidylate synthase.
Figure 2.
Figure 2.
Combination of dT and dU with 5FU in MKN45 and MKN45/F2R cells. Western blot analysis for TS protein expression following treatment with (A) 5FU (1 µM) and FdU (1 µM) with/without dU (1,000 µM) and (B) 5FU (1 µM) and FdU (1 µM) with/without dT (1,000 µM). MTT assay for the relative proliferation of MKN45 and MKN45/F2R cells treated with (C) dU alone, (D) dU with 5FU, (E) dT alone and (F) dT with 5FU. dT, thymidine; dU, deoxyuridine; 5FU, 5-fluorouracil; TS, thymidylate synthase; FdU, fluoro-deoxyuridine.
Figure 3.
Figure 3.
Resistance to raltitrexed in 5-fluorouracil-resistant MKN45/F2R cells and the inhibition of raltitrexed by dT. MTT assay for the relative proliferation of MKN45 and MKN45/F2R cells treated with (A) raltitrexed, (B) dU with raltitrexed, and (C) dT. dT, thymidine; dU, deoxyuridine.
Figure 4.
Figure 4.
Enhancement of the efficacy of 5FU by LV. (A) Western blot analysis for TS protein expression following treatment with 5FU, LV, their combination or 5FU with dU in MKN45 and MKN45/F2R cells. MTT assay for the relative proliferation of MKN45 and MKN/F2R cells treated with (B) LV and (C) LV with 5FU in MKN45 and MKN45/F2R cells. 5FU, 5-fluorouracil; LV, leucovorin; TS, thymidylate synthase; dU, deoxyuridine.
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