Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone

J Biol Chem. 2004 Apr 9;279(15):15167-76. doi: 10.1074/jbc.M309798200. Epub 2004 Jan 19.


Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-beta (TGF-beta) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-beta signaling by deletion of Smad3 results in resistance to radiation-induced injury. In the current study, a small molecular weight molecule, halofuginone (100 nm), is demonstrated by reporter assays to inhibit the TGF-beta signaling pathway, by Northern blotting to elevate inhibitory Smad7 expression within 15 min, and by Western blotting to inhibit formation of phospho-Smad2 and phospho-Smad3 and to decrease cytosolic and membrane TGF-beta type II receptor (TbetaRII). Attenuation of TbetaRII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-beta-induced delocalization of tight junction ZO-1, a marker of epidermal mesenchymal transition, in NMuMg mammary epithelial cells and suggest halofuginone may have in vivo anti-fibrogenesis characteristics. After documenting the in vitro cellular effects, halofuginone (intraperitoneum injection of 1, 2.5, or 5 microg/mouse/day) efficacy was assessed using ionizing radiation-induced (single dose, 35 or 45 Gy) hind leg contraction in C3H/Hen mice. Halofuginone treatment alone exerted no toxicity but significantly lessened radiation-induced fibrosis. The effectiveness of radiation treatment (2 gray/day for 5 days) of squamous cell carcinoma (SCC) tumors grown in C3H/Hen was not affected by halofuginone. The results detail the molecular effects of halofuginone on the TGF-beta signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.

MeSH terms

  • Animals
  • Blotting, Northern
  • Blotting, Western
  • COS Cells
  • Carcinoma, Squamous Cell / drug therapy
  • Cell Line
  • Cell Line, Tumor
  • Cells, Cultured
  • DNA-Binding Proteins / metabolism
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Fibrosis / metabolism
  • Gene Deletion
  • Genes, Reporter
  • Humans
  • Immunoblotting
  • MAP Kinase Signaling System
  • Mice
  • Mice, Inbred C3H
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Piperidines
  • Plasmids / metabolism
  • Protein Synthesis Inhibitors / therapeutic use*
  • Quinazolines / therapeutic use*
  • Quinazolinones
  • Radiation Pneumonitis / drug therapy*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Smad3 Protein
  • Time Factors
  • Trans-Activators / metabolism
  • Transforming Growth Factor beta / metabolism*
  • Transforming Growth Factor beta1


  • DNA-Binding Proteins
  • Piperidines
  • Protein Synthesis Inhibitors
  • Quinazolines
  • Quinazolinones
  • SMAD3 protein, human
  • Smad3 Protein
  • Smad3 protein, mouse
  • TGFB1 protein, human
  • Tgfb1 protein, mouse
  • Trans-Activators
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • halofuginone