Department of Surgical Oncology, Gifu University Graduate School of Medicine, Gifu 501‑1194, Japan.
Oncol Rep. 2021 Apr;45(4). doi: 10.3892/or.2021.7978. Epub 2021 Mar 2.
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 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.
氟尿嘧啶（5FU）通过乳清酸磷酸核糖转移酶（OPRT）-核糖核苷酸还原酶（RR）途径和胸苷磷酸酶（TP）-胸苷激酶（TK）途径转化为其活性代谢物氟脱氧尿苷单磷酸（FdUMP），并抑制胸苷酸合成酶（TS），从而通过一条途径抑制胸苷单磷酸（dTMP）的合成。我们通过专注于 5FU 代谢来研究 5FU 耐药的机制和克服耐药的策略。使用结肠癌细胞系 SW48 和 LS174T 以及 5FU 耐药细胞系 SW48/5FUR 和 LS174T/5FUR。通过 Western blot 测定 FdUMP 量。通过将 TP 抑制剂（盐酸 tipiracil；TPI）或 RR 抑制剂（羟基脲；HU）与 5FU 结合，研究 FdUMP 合成途径。通过结晶紫染色法观察药物细胞毒性。SW48 细胞中通过 OPRT-RR 途径合成 FdUMP，但 SW48/5FUR 细胞中几乎不通过 OPRT-RR 或 TP-TK 途径合成 FdUMP。SW48/5FUR 细胞中的 FdUMP 量比 SW48 细胞减少了 87%。与 SW48 细胞相比，SW48/5FUR 细胞中的 OPRT 和 TP 表达水平较低，表明 FdUMP 合成减少导致 5FU 耐药。这些结果表明，氟脱氧尿苷（FdU）而不是 5FU 促进 FdUMP 合成并克服 5FU 耐药性。相反，LS174T 细胞中通过 OPRT-RR 和 TP-TK 途径合成 FdUMP，但 LS174T/5FUR 细胞中主要通过 TP-TK 途径合成 FdUMP。LS174T/5FUR 细胞中的 FdUMP 量与 LS174T 细胞相似。LS174T/5FUR 细胞中的 OPRT 和 RR 表达水平较低，TK 表达水平较高，表明通过补救途径增加了 dTMP 合成，从而导致 5FU 耐药性。LS174T/5FUR 细胞对 FdU 和 TS 抑制剂也表现出交叉耐药性，表明应使用三氟胸苷等核苷类似物来克服这些细胞中的 5FU 耐药性。5FU 代谢和 5FU 耐药的机制在每个细胞系中都不同。在 5FU 耐药细胞中，应同时考虑合成的 FdUMP 量和 FdUMP 敏感性。
