Amelioration of radiation-induced fibrosis: inhibition of transforming growth factor-beta signaling by halofuginone.
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.
Xavier
Sandhya
S
Radiation Biology Branch, Nationa Institutes of Health, Bethesda, Maryland 20892, USA.
Piek
Ester
E
Fujii
Makiko
M
Javelaud
Delphine
D
Mauviel
Alain
A
Flanders
Kathy C
KC
Samuni
Ayelet M
AM
Felici
Angelina
A
Reiss
Michael
M
Yarkoni
Shai
S
Sowers
Anastasia
A
Mitchell
James B
JB
Roberts
Anita B
AB
Russo
Angelo
A
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005355:Fibrosis / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D020935:MAP Kinase Signaling System; D051379:Mice; D008809:Mice, Inbred C3H; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010880:Piperidines; D010957:Plasmids / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000627:therapeutic use*; D011799:Quinazolines / Q000627:therapeutic use*; D052999:Quinazolinones; D017564:Radiation Pneumonitis / Q000188:drug therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051900:Smad3 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
D004268:DNA-Binding Proteins; D010880:Piperidines; D011500:Protein Synthesis Inhibitors; D011799:Quinazolines; D052999:Quinazolinones; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C010176:halofuginone