5-Aza-2-deoxycytidine Enhances the Sensitivity of 5-Fluorouracil by Demethylation of the Thymidine Phosphorylase Promoter

Anticancer Res. 2019 Aug;39(8):4129-4136. doi: 10.21873/anticanres.13571.


Background/aim: 5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP).

Materials and methods: The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively.

Results: Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU.

Conclusion: Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.

Keywords: 5-aza-2-deoxycytidine; 5-fluorouracil; methylation; thymidine phosphorylase.

MeSH terms

  • Binding Sites / drug effects
  • Carcinoma, Squamous Cell / drug therapy*
  • Carcinoma, Squamous Cell / genetics
  • Carcinoma, Squamous Cell / pathology
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • DNA Methylation / drug effects
  • DNA Methylation / genetics*
  • DNA-Binding Proteins / genetics
  • Decitabine / metabolism
  • Drug Resistance, Neoplasm / genetics*
  • Fluorouracil / pharmacology
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Silencing
  • Humans
  • Promoter Regions, Genetic / drug effects
  • RNA, Messenger / genetics
  • Sp1 Transcription Factor / genetics*
  • Thymidine Phosphorylase / chemistry
  • Thymidine Phosphorylase / genetics*


  • DNA-Binding Proteins
  • RNA, Messenger
  • Sp1 Transcription Factor
  • SP1 protein, human
  • Decitabine
  • Thymidine Phosphorylase
  • Fluorouracil