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5-Fluorouracil: mechanisms of action and clinical strategies

Key Points

  • The fluoropyrimidine 5-fluorouracil (5-FU) is an antimetabolite drug that is widely used for the treatment of cancer, particularly for colorectal cancer.

  • 5-FU exerts its anticancer effects through inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA.

  • Modulation strategies, such as co-treatment with leucovorin and methotrexate, have been developed to increase the anticancer activity of 5-FU.

  • Molecular biomarkers that predict tumour sensitivity to 5-FU have been identified, including mRNA and protein expression levels of TS.

  • DNA microarray analysis of 5-FU-responsive genes will greatly facilitate the identification of new biomarkers, novel therapeutic targets and the development of rational drug combinations.


5-Fluorouracil (5-FU) is widely used in the treatment of cancer. Over the past 20 years, increased understanding of the mechanism of action of 5-FU has led to the development of strategies that increase its anticancer activity. Despite these advances, drug resistance remains a significant limitation to the clinical use of 5-FU. Emerging technologies, such as DNA microarray profiling, have the potential to identify novel genes that are involved in mediating resistance to 5-FU. Such target genes might prove to be therapeutically valuable as new targets for chemotherapy, or as predictive biomarkers of response to 5-FU-based chemotherapy.

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Figure 1: 5-Fluorouracil metabolism.
Figure 2: Mechanism of thymidylate synthase inhibition by 5-fluorouracil.
Figure 3: Modulation of 5-fluorouracil activity.
Figure 4: Activation of p53 by 5-fluorouracil.


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This work was supported by Cancer Research UK; the Research and Development Office, Department of Health and Social Services, Northern Ireland; and the Ulster Cancer Foundation.

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Correspondence to Patrick G. Johnston.

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annexin II











folylpolyglutamate synthetase
















hereditary non-polyposis colon cancer



Antimetabolite drugs such as 5-fluorouracil that are fluorinated derivatives of pyrimidines.


An anticancer drug that inhibits DNA topoisomerase I. It is used in the treatment of advanced colorectal cancer.


A platinum-based DNA-damaging anticancer drug that is used in the treatment of advanced colorectal cancer.


A technique that allows global changes in gene expression to be assessed.


Molecular markers that predict tumour sensitivity to chemotherapy.


Family of essential vitamins that act as cofactors in one-carbon transfer reactions.


A stable complex that is formed between 5-fluorouracil, thymidylate synthase and 5,10-methylene tetrahydrofolate, and that blocks synthesis of thymidylate by the enzyme.


(Ribosomal RNA). The RNA component of ribosomes, which translate mRNA into protein.


(Transfer RNA). tRNAs bond with amino acids and transfer them to the ribosomes, where proteins are assembled according to the genetic code that is carried by mRNA.


(Small nuclear RNA). Small nuclear RNAs have key roles in the splicing of pre-mRNA into mature mRNA.


(Messenger RNA). RNA that serves as a template for protein synthesis.


Mature mRNAs have a homopolymer of adenosine residues (poly(A) tails) at their 3′-termini, which are important in regulating their stability and translation.


Addition of glutamate residues to folates by folylpolyglutamate synthase (FPGS), increasing their intracellular retention. Most folate-dependent enzymes have a higher affinity for the polyglutamate forms of their folate cofactors.


Occurrence of variant DNA sequences in a population at frequencies that are too high to be due to random mutations.


Microsatellite instability refers to variations in the numbers of repetitive di-, tri- and tetranucleotide repeats (called microsatellites) found in DNA.

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Longley, D., Harkin, D. & Johnston, P. 5-Fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 3, 330–338 (2003).

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