5FU resistance caused by reduced fluoro-deoxyuridine monophosphate and its reversal using deoxyuridine

Abstract

Abstract. The mechanism of 5-fluorouracil (5FU) resistance was investigated, focusing on the level of thymidylate synthase (TS) ternary complex formed with fluoro-deoxyuridine monophosphate (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. IntroductionDespite recent advances in the development of diagnostic tools for gastric cancer, many patients with gastric cancer continue to be diagnosed at a late stage, and even following curative surgery, recurrent tumors frequently occur. Therefore, gastric cancer remains a major cause for cancer-associated mortality worldwide (1) and the development of novel drug therapies for gastric cancer is important.5FU is currently a key drug for adjuvant therapy following curative surgery (2-4) and for the treatment of metastatic gastric cancer (5,6). Three mechanisms have been proposed for its action: Incorporation into RNA (7), incorporation into DNA (8) and the inhibition of thymidine synthase (TS) leading to the inhibition of DNA de novo synthesis by forming a ternary complex composed of TS, 5,10-methylenetetrahydrofolate (CH2THF) and fluoro-deoxyuridine monophosphate (FdUMP) (9). The first step in the activation of 5FU is the phosphorylation of 5FU by orotate phosphoribosyltransferase (OPRT), which metabolizes 5FU to 5-fluorouridine monophosphate (FUMP). FUMP is then metabolized into 5-fluorouridine triphosphate (FUTP), which can be incorporated into RNA and into 5-flurodeoxyuridine triphosphate (FdUTP), which can be incorporated into DNA (10). Although the incorporation into RNA and DNA is certainly an important aspect of the mechanism of action of 5FU,...

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

“…We previously elucidated the mechanism of acquired 5FU resistance by focusing on the changes in the expression levels of enzymes for 5FU metabolism in gastric cancer cell lines ( 12 , 13 ). In this study, we investigated the mechanisms of acquired 5FU resistance in colon cancer cell lines by investigating the changes in the related enzymes and the amount of synthesized FdUMP.…”

Cytotoxic response of 5-fluorouracil-resistant cells to gene- and cell-directed enzyme/prodrug treatment

“…Even though the monotherapy by raltitrexed is sometimes suggested for the patients with advanced colorectal cancer refractory to the 5-FU [30], our data show that the resistance to 5-FU was associated with the cross-resistance to this drug as well. Decreased OPRT and increased TS levels connected to the 5-FU resistance on a model of gastric cancer cell line were demonstrated [31]. We showed that the decreased OPRT expression potentially leading to inefficient 5-FU metabolic conversion might be partially compensated by the co-culture of the tumor cells with the MSC expressing CD::UPRT.…”

Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells

“…Importantly, it should be noted that total TS, the free form, the FdUMP-covalent form, and total TS was overexpressed in HCT116R F10 cells rather than in HCT116 cells ( Figure 6 a top panel and Figure 6 d). The upper band of TS, indicated FdUMP-covalent form, which represents TS in ternary complexes and is correlated with the intracellular concentration of FdUMP [ 12 , 13 , 14 ]. In addition, DPD protein levels were slightly decreased in HCT116R F10 cells than in parental HCT116 cells ( Figure 6 a second panel and Figure 6 e).…”