Abstract
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.
Highlights
Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation
It has been suggested that TGF- is the master switch cytokine, which once activated after radiation promotes a train of cellular events that result in radiation-induced fibrosis [7, 9]
Halofuginone Inhibits TGF--dependent Phosphorylation of Smad2 and Smad3—Because the Transforming growth factor- (TGF-)1 signaling pathway as assessed by the above reporters can be interrupted at many sites, we sought to determine whether Smad3 or Smad2 phosphorylation was inhibited, and if so, whether the inhibition is cell-type specific
Summary
Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Unlike a trauma-induced wound, wherein platelets and other cells from the circulation discharge into the injured tissue and repair is accomplished without fibrosis, high dose radiation induces cellular damage that results in cytokine and growth factor-mediated signaling that inevitably leads to dysfunctional repair and fibrosis, if not ulceration and necrosis [3,4,5]. Numerous cytokines such as tumor necrosis factor-␣, interleukin-1, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor- (TGF-) have been reported to be increased within irradiated tissue [6]. We showed that Smad3ex8/ex (referred to as Smad null) animals are less susceptible to radiation-induced injury [13] and have sought a small molecular weight compound to interfere with TGF-1
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