The role of thymidylate synthase and dihydropyrimidine dehydrogenase in resistance to 5-fluorouracil in human lung cancer cells

Summary

The expressions of thymidylate synthase (TS) and intracellular metabolic enzymes have been reported to be associated with the sensitivity and/or resistance to 5-fluorouracil (5-FU). However, since the role of these enzymes in the mechanism of resistance to 5-FU has not been fully examined in lung cancer, in the present study we measured the expression levels of TS, dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyltransferase (OPRT) genes in lung cancer cell lines by real-time PCR, and the sensitivity to 5-FU using the MTS assay. The expression of DPD was significantly correlated with the concentration of 5-FU for 50% cell survival in 15 non-small-cell lung cancer (NSCLC) cell lines (p < 0.05), but the expressions of TS, TP, and OPRT were not. Treatment with 5-chloro-2,4-dihydroxypyridine, an inhibitor of DPD, altered the sensitivity to 5-FU in DPD-expressing RERF-LC-MT cells, indicating that modulation of DPD activity could increase the 5-FU sensitivity in lung cancer. In contrast, TS expression was dramatically higher in a 5-FU-resistant small-cell lung cancer cell line than in the parent cell line, whereas the expressions of DPD, TP, and OPRT genes were not markedly different. In order to examine the effect of other cytotoxic agents on TS and DPD expression, we compared the expressions of both genes between cisplatin-, paclitaxel-, gemcitabine-, or 7-ethyl-10-hydroxycamptothecin-resistant lung cancer cells and their respective parent cells, but found no differences between any pair of resistant subline and the corresponding parent cell line. Our results indicate that degradation of 5-FU due to DPD is an important determinant in 5-FU sensitivity, while induction of TS contributes to acquired resistance against 5-FU in lung cancer. Therefore, the expression levels of TS and DPD genes may be useful indicators of 5-FU activity in lung cancer.

Introduction

5-Fluorouracil (5-FU) and its derivatives are antimetabolite drugs that are widely used in cancer chemotherapy. The effects of 5-FU have been attributed to inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA. 5-FU enters the cells rapidly and is converted intracellularly by metabolic enzymes. Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme for converting 5-FU into an inactive metabolite, dihydrofluorouracil. In contrast, other first metabolic enzymes, namely orotate phosphoribosyltransferase (OPRT) and thymidine phosphorylase (TP), catalyze synthesis of active metabolites, resulting in disruption of the action of TS and DNA or RNA synthesis [1].

Lung cancer is one of the most common malignancies worldwide, and several randomized clinical trials and meta-analyses have demonstrated that survival in patients with advanced non-small-cell lung cancer (NSCLC) can be slightly but significantly prolonged with chemotherapy [2], [3]. Although 5-FU derivatives are not usually administered as a first-line chemotherapy, a recent study showed that postoperative oral administration of uracil-tegafur improves survival among patients with completely resected stage I lung adenocarcinomas [4]. Further, a novel oral fluorouracil S-1 was shown to exert promising effects against advanced NSCLC [5], [6]. These results indicate the effectiveness of 5-FU derivatives in NSCLC treatment.

Biomarkers for the prediction of the sensitivity and/or resistance to 5-FU have been identified previously, including TS and intracellular metabolic enzymes [1], [7], [8]. It was demonstrated that TS and DPD gene expression and/or activity are associated with the efficacy of 5-FU in NSCLC [9], [10], [11]. However, the role of TS and intracellular metabolic enzymes in the mechanism of resistance to 5-FU has not been fully examined in lung cancer. Therefore, we examined the expression levels of TS, DPD, TP, and OPRT genes to clarify the mechanism of sensitivity and resistance to 5-FU in lung cancer.