A PPAR-gamma agonist protects from radiation-induced intestinal toxicity

Abstract

Objective: Because of its anti-inflammatory, anti-fibrotic, anti-apoptotic and anti-neoplastic properties, the PPAR-γ agonist rosiglitazone is an interesting drug for investigating for use in the prevention and treatment of radiation-induced intestinal damage. We aimed to evaluate the radioprotective effect of rosiglitazone in a murine model of acute intestinal damage, assessing whether radioprotection is selective for normal tissues or also occurs in tumour cells.

Methods: Mice were total-body irradiated (12 Gy), with or without rosiglitazone (5 mg/kg/day). After 24 and 72 hours, mice were sacrificed and the jejunum was collected. HT-29 human colon cancer cells were irradiated with a single dose of 2 (1000 cells), 4 (1500 cells) or 6 (2000 cells) Gy, with or without adding rosiglitazone (20 µM) 1 hour before irradiation. HT-29-xenografted CD1 mice were irradiated (16 Gy) with or without rosiglitazone; tumour volumes were measured for 33 days.

Results: Rosiglitazone markedly reduced histological signs of altered bowel structures, that is, villi shortening, submucosal thickening, necrotic changes in crypts, oedema, apoptosis, and inflammatory infiltrate induced by irradiation. Rosiglitazone significantly decreased p-NF-kB p65 phosphorylation and TGFβ protein expression at 24 and 72 hours post-irradiation and significantly decreased gene expression of Collagen1, Mmp13, Tnfα and Bax at 24 hours and p53 at 72 hours post-irradiation. Rosiglitazone reduced HT-29 clonogenic survival, but only produced a slight reduction of xenograft tumour growth.

Conclusion: Rosiglitazone exerts a protective effect on normal tissues and reduces alterations in bowel structures and inflammation in a radiation-induced bowel toxicity model, without interfering with the radiation effect on HT-29 cancer cells. PPAR-γ agonists should be further investigated for their application in abdominal and pelvic irradiation.

Keywords: PPAR-gamma agonists; Rosiglitazone; bowel; radiation-induced toxicity; radioprotection.

Figure 1.

Rosiglitazone reduced histological signs of radiation-induced intestinal damage. Representative full-thickness images of jejunal wall of mice treated with saline (control), irradiation (IR, 12 Gy) or IR + RGZ (5 mg/kg) after 24 and 72 hours. Bars = 100 µm.

Figure 2.

Rosiglitazone decreased radiation-induced apoptosis in bowel tissues. a. Representative images of TUNEL-stained jejunum sections from mice treated with saline (control), irradiation (IR, 12 Gy) or IR + RGZ (5 mg/kg), after 24 and 72 hours. Bars = 100 µm. b. Average number of TUNEL-positive cells per field at 100 × magnification. Data, measured as fold increase in expression compared to control (taken as 1), are shown as the mean ± SEM. *p < 0.05 vs. control, #p < 0.05 vs. IR.

Figure 3.

Rosiglitazone decreased overexpression of radiation-induced fibrosis/inflammatory marker proteins in bowel tissues. a. Representative Western blot analysis of p-NF-kB p65 and TGF-β proteins in jejunum tissue homogenates from mice treated with saline (control), irradiation (IR, 12 Gy) or IR + RGZ (5 mg/kg), after 24 and 72 hours. GAPDH was used as a loading control. b. Densitometric analysis of Western blot bands, normalized for GAPDH and reported as percentage versus the respective control, set at 100. Data are shown as the mean ± SEM. *p < 0.05 vs. control, #p < 0.05 vs. IR.

Figure 4.

Rosiglitazone decreased the radiation-induced gene expression of fibrosis, inflammatory and apoptosis markers in bowel tissues. mRNA expression of Bax, Col1, Mmp13 and Tnfα, p53 in jejunum tissues of mice treated with saline (control), irradiation (IR, 12 Gy) or IR + RGZ (5 mg/kg) after 24 and 72 hours. Data, measured as fold increase in expression compared with control (taken as 1), are shown as mean ± SEM. *p < 0.05 vs. control, #p < 0.05 vs. IR.

Figure 5.

Rosiglitazone significantly reduced clonogenic survival of HT-29 cancer cells following irradiation with increasing radiation doses. Cells, pre-treated or not with RGZ (20 µM), were irradiated with 0–6 Gy. After 12–15 days, cells were stained and colonies over 50 cells counted. Data are shown as mean ± SEM, corrected by PE. PE was normalized to the respective control value (RGZ control in combined treatments) for each single experiment. *p < 0.05 vs. control.

Figure 6.

Rosiglitazone did not protect the tumour from irradiation. When HT-29 tumour xenograft diameter reached 5 mm, mice were treated with vehicle (asterisks), RGZ (5 mg/kg, black squares), 16 Gy irradiation (IR, black triangles) or IR + RGZ (white squares). Tumour volume was measured twice a week for 33 days. Data are mean ± SEM. *p < 0.05 vs. control, #p < 0.05 vs. RGZ.