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

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.