Amelioration of Radiation-induced Fibrosis

Abstract

Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. Transforming growth factor-␤ (TGF-␤) has been proposed to be critical in tissue repair mechanisms resulting from radiation injury. Previously, we showed that interruption of TGF-␤ 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-␤ 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-␤ type II receptor (T␤RII). Attenuation of T␤RII levels was noted as early as 1 h and down-regulation persisted for 24 h. Halofuginone blocked TGF-␤-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 g/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-␤ signal pathway and show that halofuginone may lessen radiation-induced fibrosis in humans.

Halofuginone inhibits tumor growth in the polyoma middle T antigen mouse via a thrombospondin-1 independent mechanism

“…66 Although the antibodies used in our study cannot distinguish between phosphorylated Smad 2 and 3, a previous study demonstrated that halofuginone specifically inhibited Smad 3 but not Smad 2 phosphorylation. 14,70 The data suggest that halofuginone inhibits TGF-β dependent synthesis of VEGF and collagen and this can explain the halofuginone-dependent reduction in vessel number in the TSP-1 null mice. Thus, in our model, the inhibition of angiogenesis by halofuginone probably depends more upon the downregulation of the pro-angiogenic side of the angiogenic balance, rather than the upregulation of anti-angiogenic activity.…”

“…In most animal models of fibrosis, regardless of the tissue, halofuginone had a minimal effect on collagen content in the control, nonfibrotic animals, whereas it exhibited a profound inhibitory effect in the fibrotic organs. 2,6,12,14,16 These results suggest separate mechanisms for the maintenance of the usual low level of expression of collagen type I genes and the overexpression induced by fibrogenic stimuli such as TGFβ, an aggressive and a rapid process.…”

“…These models included mice afflicted with cGvHD and tight skin (Tsk+) mice, 5,6 rats with pulmonary fibrosis, 7 rats that had developed adhesions at various locations, [8][9][10] rats with liver fibrosis, [11][12][13] and mice with radiation-induced fibrosis. 14 Clinical efficacy was demonstrated by topical dermal halofuginone administration in a cGvHD patient 15 and in a scleroderma phase II clinical study. 16 In light of the involvement of the extracellular matrix (ECM) in general and of collagen type I in particular in angiogenesis, 17,18 the efficacy of halofuginone in inhibiting angiogenesis and tumor growth was evaluated.…”

“…26 Several groups have shown that halofuginone blocks TGF-β mediated collagen synthesis by decreasing activation of Smad 3 through c-Jun 27,28 and increasing expression of Smad 7, an inhibitor of Smad 2/3 activation. 14 One of the matrix proteins involved in fibrosis, angiogenesis and tumorigenesis is thrombospondin-1 (TSP-1). 29,30 TSP-1 is a 450kDa homotrimeric extracellular matrix protein expressed by both normal and tumor cells and has been shown to inhibit tumorigenesis, angiogenesis and experimental metastasis.…”

The pleiotropic roles of transforming growth factor beta in homeostasis and carcinogenesis of endocrine organs

“…Russo and colleagues have used a small molecular weight molecule, halofuginone, to block TGF-b signaling using a connective tissue radiation damage model (Xavier et al 2004). Halofuginone inhibits the TGF-b signaling pathway by elevating inhibitory Smad7, blocking formation of phosphoSmad2 and phospho-Smad3 and decreasing cytosolic and membrane TGF-b type II receptor.…”