Mechanism of acquired 5FU resistance and strategy for overcoming 5FU resistance focusing on 5FU metabolism in colon cancer cell lines

Abstract

Fluorouracil (5FU) is converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through the orotate phosphoribosyl transferase (OPRT)-ribonucleotide reductase (RR) pathway and thymidine phosphatase (TP)-thymidine kinase (TK) pathway and inhibits thymidylate synthase (TS), leading to inhibition of thymidine monophosphate (dTMP) synthesis through a de novo pathway. We investigated the mechanism of 5FU resistance and strategies to overcome it by focusing on 5FU metabolism. Colon cancer cell lines SW48 and LS174T and 5FU-resistant cell lines SW48/5FUR and LS174T/5FUR were used. FdUMP amount was measured by western blotting. The FdUMP synthetic pathway was investigated by combining TP inhibitor (tipiracil hydrochloride; TPI) or RR inhibitor (hydroxyurea; HU) with 5FU. Drug cytotoxicity was observed by crystal violet staining assay. FdUMP was synthesized through the OPRT-RR pathway in SW48 cells but was scarcely synthesized through either the OPRT-RR or TP-TK pathway in SW48/5FUR cells. FdUMP amount in SW48/5FUR cells was reduced by 87% vs. SW48 cells. Expression levels of OPRT and TP were lower in SW48/5FUR when compared with these levels in the SW48 cells, indicating decreased synthesis of FdUMP-led 5FU resistance. These results indicated that fluoro-deoxyuridine (FdU) rather than 5FU promotes FdUMP synthesis and overcomes 5FU resistance. Contrastingly, FdUMP was synthesized through the OPRT-RR and TP-TK pathways in LS174T cells but mainly through the TP-TK pathway in LS174T/5FUR cells. FdUMP amount was similar in LS174T/5FUR vs. the LS174T cells. OPRT and RR expression was lower and TK expression was higher in LS174T/5FUR vs. the LS174T cells, indicating that dTMP synthesis increased through the salvage pathway, thus leading to 5FU resistance. LS174T/5FUR cells also showed cross-resistance to FdU and TS inhibitor, suggesting that nucleoside analogs such as trifluoro-thymidine should be used to overcome 5FU resistance in these cells. 5FU metabolism and mechanisms of 5FU resistance are different in each cell line. Both synthesized FdUMP amount and FdUMP sensitivity should be considered in 5FU-resistant cells.

5-fluorouracil treatment of patient-derived scaffolds from colorectal cancer reveal clinically critical information

“…The observation that cancer cells cultured in colorectal PDS were less sensitive to 5FU treatment compared to standard adherent cultures are in line with earlier published data [14,23]. Resistance to 5FU can develop through multiple and diverse mechanisms, including alterations of different pathways of 5FU metabolism [24] leading to perturbations of different cellular functions, such as apoptosis or cell cycle, with a significant regulatory role played by the tumor microenvironment [6]. In the present study, the reduced sensitivity to 5FU observed in PDS cultures did not seem to be related to reduced cell death, but rather to a reduced efficiency of 5FU in arresting cell cycle.…”

The mechanism underlying resistance to 5‑fluorouracil and its reversal by the inhibition of thymidine phosphorylase in breast cancer cells

“…In the present study, in the 5-FU-resistant MKN45/F2R cells established at our institution, resistance to 5-FU was almost completely reversed by the inhibition of TP like MCF-7/5FUR cells, although sensitivity to 5FU was not changed by the inhibition of TP in parental MKN45 cells, unlike MCF-7 cells (20,21). In contrast, the resistance to 5-FU in 5-FU-resistant SW48 and LS174T colon cancer cells, which were also established in our institution, was not reversed by the inhibition of TP although FdUMP in these cells was synthesized through the TP-TK pathway, like the MDA-MB-231/5FUR cells in the present study (22). To our knowledge, this is the first report describing the enhancement of the efficacy of 5-FU in parental cells and the reversal of resistance to 5-FU in cells in which FdUMP was synthesized through the TP-TK pathway.…”

Trapping of 5-Fluorodeoxyuridine Monophosphate by Thymidylate Synthase Confers Resistance to 5-Fluorouracil

“…Furthermore, the accumulation of the FdUMP-TS protein after 5-FU for 24 h was dramatically increased in HCT116R F10 cells (1.8–3.0-fold higher) compared with HCT116 cells transfected with nonsilencing siRNA or TYMS -targeted siRNA. It is known that the FdUMP-TS protein band, indicating the FdUMP-covalent form, represents TS in ternary complexes and is correlated with the intracellular concentration of FdUMP. − Similarly, the storage of active free-TS protein after 5-FU for 24 h was significantly increased in HCT116R F10 cells (2.5–2.9-fold higher) compared with HCT116 cells after transfection of nonsilencing siRNA or TYMS -targeted siRNA. Notably, the expression of free-TS protein in 5-FU-resistant HCT11R F10 cells was decreased (19% at 24 h and 26% at 48 h in NSsi-transfected cells; 23% at 24 h and 23% at 48 h in TSsi-transfected cells) by 5-FU treatment compared with no treatment after transfection of TYMS -targeted siRNA or nonsilencing siRNA, respectively.…”