Department of Surgical Oncology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu, 501-1194, Japan.
Gastric Cancer. 2019 May;22(3):497-505. doi: 10.1007/s10120-018-0881-3. Epub 2018 Oct 1.
5FU can be converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through two pathways: the orotate phosphoribosyl transferase-ribonucleotide reductase (OPRT-RR) pathway and the thymidine phosphorylase-thymidine kinase (TP-TK) pathway. We investigated the mechanism underlying 5FU-resistance, focusing on the changes in the 5FU metabolisms.
MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU or fluoro-deoxyuridine (FdU) in combination with hydroxyurea (HU) or tipiracil (TPI). The amount of FdUMP was determined by the density of the upper band of thymidylate synthase on Western blotting.
The MKN45/F2R cells exhibited 5FU resistance (37.1-fold) and showed decreased OPRT and increased TP levels. In both cells, the FdUMP after treatment with 5FU was decreased when RR was inhibited by HU but not when TP was inhibited by TPI. A metabolome analysis revealed the loss of intracellular deoxyribose 1-phosphate (dR1P) in both cells, indicating that FdUMP was synthesized from 5FU only through the OPRT-RR pathway because of the loss of dR1P. After the knockdown of TK, the FdUMP after treatment with FdU was decreased in MKN45 cells. However, it was not changed in MKN45/F2R cells. Furthermore, TP inhibition caused an increase in FdUMP after treatment with 5FU or FdU and reversed the 5FU resistance in MKN45/F2R cells, indicating that FdUMP was reduced through the TP-TK pathway in MKN45/F2R cells.
In MKN45/F2R cells, the reduction of FdUMP through the TP-TK pathway caused 5FU resistance, and the inhibition of TP reversed the resistance to 5FU, suggesting that the combination of 5FU and TPI is a promising cancer therapy.
5FU 可以通过两条途径转化为其活性代谢物氟脱氧尿苷单磷酸（FdUMP）：乳清酸磷酸核糖基转移酶-核糖核苷酸还原酶（OPRT-RR）途径和胸苷磷酸化酶-胸苷激酶（TP-TK）途径。我们研究了 5FU 耐药的机制，重点关注 5FU 代谢的变化。
用 5FU 或氟脱氧尿苷（FdU）联合羟基脲（HU）或替比嘧啶（TPI）处理 MKN45 和 5FU 耐药的 MKN45/F2R 细胞。通过 Western blot 上胸苷酸合成酶上带的密度来测定 FdUMP 的量。
MKN45/F2R 细胞表现出 5FU 耐药性（37.1 倍），并显示 OPRT 减少和 TP 增加。在这两种细胞中，RR 被 HU 抑制时，5FU 处理后的 FdUMP 减少，但 TP 被 TPI 抑制时则不减少。代谢组学分析显示两种细胞内脱氧核糖 1-磷酸（dR1P）丢失，表明由于 dR1P 的丢失，FdUMP 仅通过 OPRT-RR 途径从 5FU 合成。TK 敲低后，MKN45 细胞中 FdU 处理后的 FdUMP 减少，但 MKN45/F2R 细胞中则没有变化。此外，TP 抑制导致 5FU 或 FdU 处理后的 FdUMP 增加，并逆转 MKN45/F2R 细胞中的 5FU 耐药性，表明 MKN45/F2R 细胞中 FdUMP 通过 TP-TK 途径减少。
在 MKN45/F2R 细胞中，通过 TP-TK 途径减少 FdUMP 导致 5FU 耐药，TP 抑制逆转了对 5FU 的耐药性，表明 5FU 与 TPI 的联合应用是一种有前途的癌症治疗方法。
