Ameliorating effects of bortezomib, a proteasome inhibitor, on development of dextran sulfate sodium-induced murine colitis

Abstract

We examined the effect of bortezomib, a proteasome inhibitor, on the development of dextran sulfate sodium (DSS)-induced colitis in mice. DSS-colitis was induced by the administration of 3% DSS in water in C57BL/6J mice. Bortezomib was intraperitoneally administered daily for 9 days from the start of DSS. Ubiquitination of IκBα was evaluated by immunoblot. Bortezomib significantly ameliorated DSS-induced body weight loss and reduced the disease activity. The translocation of NF-κBp65 into the nucleus was markedly suppressed in the DSS + bortezomib group compared to the DSS group, but this difference was not detected in submucosal tissue. Ubiquitinated IκBα in the cytoplasm of colon epithelial cells was increased in the DSS + bortezomib group compared to the DSS group. In HT-29 cells, bortezomib blocked tumor necrosis factor-α (TNF-α)-induced nuclear translocation of NF-κB and this was accompanied by an increase in ubiquitinated IκBα in the cytoplasm. The mRNA expression of inflammatory mediators in colonic epithelial cells was significantly reduced by the treatment of bortezomib. Bortezomib inhibited the nuclear translocation of NF-κB in colonic epithelial cells by suppressing the degradation of IκBα and contributed to an improvement in DSS colitis. Our study suggests that bortezomib may be a new treatment option for IBD.

Keywords: IBD; NF-κB; proteasome inhibitor.

Fig. 1

The effect of bortezomib on DSS colitis. (A) Body weight, (B) disease activity index, (C) representative photographs of the colon, and (D) colonic weight/length on day 9. All data are means ± SEM (n = 15/group). Values not sharing a letter denote significant differences (p<0.05). BTZ, bortezomib.

Fig. 2

Histological evaluation of disease activity. (A) Picture on day 9 (original magnification ×100) and (B) histological score. All data are means ± SEM (n = 10/group). Values not sharing a letter denote significant differences (p<0.05). BTZ, bortezomib.

Fig. 3

The effect of bortezomib on NF-κB activation. (A) Immunoblot for NF-κBp65 in the nuclear protein isolated from colonic epithelial cells and submucosal tissues in DSS-colitis mice. Representative picture of four independent experiments. (B) Immunohistochemical staining for NF-κBp65 in the colon sections (original magnification ×400). NF-κBp65 was detected in the nucleus of the epithelial cells in the DSS group, and this was blocked in the DSS plus bortezomib group. (C) Cytoplasmic protein extracted from isolated colonic epithelial cells were immunoprecipitated for IκBα and then analyzed by immunoblot using antibody against ubiquitin. Representative picture of 4 independent experiments. BTZ, bortezomib.

Fig. 4

The effect of bortezomib on NF-κB activation in vitro. (A) Nuclear protein extracted from HT-29 cell line treated with bortezomib for 12 h and subjected to immunoblot. The translocation of NF-κB was suppressed by the administration of bortezomib. Lamin A/C was used as a loading control. The picture is representative of four independent experiments. (B) Cytoplasmic protein extracted from HT-29 cell line were immunoprecipitated for IκBα and then analyzed by immunoblot using antibody against ubiquitin. The picture is representative of four independent experiments. BTZ, bortezomib.

Fig. 5

The effect of bortezomib on the expression of proinflammatory cytokines. Mucosal mRNA expression of proinflammatory cytokines. The expressed cytokine mRNA was converted to a value relative to β-actin mRNA and revealed as a relative increase to the results for control mice. The data are expressed as means ± SEM (n = 10 mice/group). Values not sharing a letter denote significant differences (p<0.05). BTZ, bortezomib.

Fig. 6

The effect of bortezomib on the proliferation of colonic epithelial cells. (A) Immunohistochemical staining for Ki67 in the colon sections (original magnification ×200). Ki67 was detected in the nucleus of the crypt. Representative picture of 4 independent experiments. (B) Percentage of Ki-67-positive cells per crypt. The data are expressed as means ± SEM (n = 10/group). Values not sharing a letter denote significant differences (p<0.05). BTZ, bortezomib.