Folic Acid-Metabolizing Enzymes Regulate the Antitumor Effect of 5-Fluoro-2'-Deoxyuridine in Colorectal Cancer Cell Lines.

Hiroshi Tsukihara,Teiji Takechi,Kenta Tsunekuni,Bing Xu

doi:10.1371/journal.pone.0163961

Abstract

In colorectal cancer chemotherapy, the current standard of care includes combination therapy with 5-fluorouracil (5-FU) and leucovorin (LV). However, the factors that determine the LV-mediated enhancement of 5-FU antitumor activity are not fully understood. Therefore, we investigated the roles of thymidine synthase (TYMS), folate receptor 1 (FOLR1), dihydrofolate reductase (DHFR), phosphoribosylglycinamide formyltransferase (GART), methylenetetrahydrofolate dehydrogenase (MTHFD1), and methylenetetrahydrofolate reductase (MTHFR) in LV-mediated enhancement of 5-fluoro-2′-deoxyuridine (FdUrd) cytotoxicity in vitro as a model of 5-FU antitumor activity. These genes were downregulated in DLD-1 and HCT116 human colorectal cancer cells by using small-interfering RNA. Reduced expression of TYMS mRNA significantly increased FdUrd cytotoxicity by 100- and 8.3-fold in DLD-1 and HCT116 cells, respectively. In contrast, reducing the expression of FOLR1, DHFR, GART, MTHFD1, and MTHFR decreased FdUrd cytotoxicity by 2.13- to 12.91-fold in DLD-1 cells and by 3.52- to 10.36-fold in HCT116 cells. These results demonstrate that folate metabolism is important for the efficacy of FdUrd. Overall, the results indicate that it is important to clarify the relationship between folate metabolism-related molecules and 5-FU treatment in order to improve predictions of the effectiveness of 5-FU and LV combination therapy.

Highlights

Worldwide, colorectal cancer (CRC) was the third most common cancer (9.7%) and the fourth leading cause of cancer-related deaths in 2012 [1]
Downregulation of gamma-glutamyl hydrolase (GGH) by siRNA increased the sensitivity of cells to 5-fluoro-20-deoxyuridine (FdUrd) when combined with LV [4]. These results suggest that expression of both folylpolyglutamate synthetase (FPGS) and GGH within tumor cells controls the ability of LV to enhance the antitumor activity of 5-FU by regulating folate levels
To examine the influence of thymidylate synthase (TYMS) expression on the cytotoxicity of FdUrd, we abolished the expression of TYMS in DLD-1 and HCT116 cells using siRNA (S1 Table)

Summary

Introduction

Colorectal cancer (CRC) was the third most common cancer (9.7%) and the fourth leading cause of cancer-related deaths in 2012 [1]. In CRC chemotherapy, the current standard of care includes combination treatment with 5-fluorouracil (5-FU) and leucovorin (LV). One of the anticancer mechanisms of 5-FU involves the inhibition of thymidylate synthase (TYMS) via formation of a ternary complex between TYMS, 5,10-methylenetetrahydrofolate (5,10-CH2FH4), and 5-fluoro-2’-deoxyuridine 5’-monophosphate (FdUMP), which is the active form of 5-FU (Fig 1). LV itself has no antitumor activity, it has been used to increase the intracellular concentration of 5,10-CH2FH4, thereby increasing the efficacy of 5-FU.

Methods

Results

Conclusion