5FU resistance caused by reduced fluoro-deoxyuridine monophosphate and its reversal using deoxyuridine
5FU resistance caused by reduced fluoro-deoxyuridine monophosphate and its reversal using deoxyuridine
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DOI：
10.3892/ol.2017.6512
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发表时间：
2017-09-01
期刊：
影响因子：
2.9
通讯作者：
Yoshida, Kazuhiro
中科院分区：
文献类型：
--
作者：
Mori, Ryutaro;Futamura, Manabu;Yoshida, Kazuhiro
探讨了5-氟尿嘧啶(5FU)耐药的机制，重点研究了与氟代脱氧尿苷单磷酸(FdUMP)形成的胸苷合成酶(TS)三元复合体的水平。对MKN45和5FU耐药的MKN45/F2R细胞用5FU和氟代脱氧尿苷(FDU)联合脱氧尿苷(DU)和胸腺嘧啶核苷(DT)处理。用免疫印迹法检测三元复合体的表达水平，用四甲基偶氮唑盐比色法计算细胞存活率。与MKN45细胞相比，MKN45/F2R细胞表现出5FU抗性(是MKN45细胞的56.2倍)，并表现出OPRT降低和TS水平增加，需要更高浓度的5FU来诱导三元复合体的形成。转染针对OPRT的小干扰RNA后，MKN45对5FU的抗性增加，5FU处理后的三元复合体形成减少，表明OPRT的减少导致了5FU抗性的增加。然而，MKN45/F2R对FDU也表现出抗性，在没有OPRT的情况下，FDU可以转化为FdUMP，并且FDU处理后三元复合体的形成减少，表明5FU抗性细胞具有减少细胞内FdUMP的能力。在MKN45/F2R细胞中加入DU和胸腺嘧啶核苷dT可促进三元复合体的形成并逆转对5FU的耐药性，而dT则抑制另一种TS抑制剂雷替曲塞的作用。这些结果表明，耐5FU的细胞具有减少细胞内FdUMP的能力，而不考虑OPRT的降低，从而导致对5FU的抗性。这种抗性随后被DT或DU处理抑制。
The mechanism of 5-fluorouracil (5FU) resistance was investigated, focusing on the level of thymidylate synthase (TS) ternary complex formed with fluoro-deoxyuridine mono phosphate (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.