Dietary-feeding of grape seed extract prevents azoxymethane-induced colonic aberrant crypt foci formation in fischer 344 rats

Abstract

Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six-week-old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre- and post-AOM or only post-AOM experimental protocols. At 16 wk of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis, and molecular analyses by immunohistochemistry. GSE-feeding caused strong chemopreventive efficacy against AOM-induced ACF formation in terms of up to 60% (P < 0.001) reduction in number of ACF and 66% (P < 0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE-feeding inhibited AOM-induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX-2, iNOS, and survivin levels. Additional studies showed that GSE-feeding also decreased AOM-caused increase in beta-catenin and NF-kappaB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of beta-catenin and NF-kappaB signaling, and suggest its potential usefulness for the prevention of human CRC.

Fig. 1

Dietary GSE feeding inhibits cell proliferation and induces apoptosis in colon tissues from AOM-treated rats. (A) Experimental design for AOM and GSE treatments as described in Materials and Methods. Groups 3 and 4 represent protocol 1 (pre & post-AOM), and groups 5 and 6 represent protocol 2 (post-AOM) for GSE treatments. Representative photographs for IHC staining of PCNA (B) and TUNEL (C) positive cells in AOM alone and GSE + AOM treated groups, respectively, are shown at 400x magnification. The percentages of PCNA (B) and TUNEL (C) positive cells assessed by quantification of IHC stained rat colonic epithelium are calculated from 5 randomly selected fields from each tissue sample in each group as detailed in Methods, and represent mean ± SE value from 7 rats in each group. Representative colonic tissues from each group were also analyzed by immunoblotting for PCNA expression. Values of band intensity were adjusted with β-actin. AOM, azoxymethane; GSE, grape seed extract.

Fig. 2

Dietary GSE feeding suppresses cyclin D1 and survivin expression in colon tissues of AOM-treated rats. Representative photographs for IHC staining of cyclin D1 (A) and survivin (C) positive cells in AOM alone and GSE + AOM treated groups, respectively, are shown at 400x magnification. The percentages of cyclin D1 (B) and survivin (D) positive cells assessed by quantification of IHC stained rat colonic epithelium are calculated from 5 randomly selected fields from each tissue sample in each group as detailed in Methods, and represent mean ± SE value from 7 rats in each group. Representative colonic tissues from each group were also analyzed by immunoblotting for cyclin D1 and survivin expression. Values of band intensity were adjusted with β-actin. AOM, azoxymethane; GSE, grape seed extract.

Fig. 3

Dietary GSE feeding decreases the protein levels of iNOS and COX-2 in colon tissues from AOM-exposed rats. Representative photographs for IHC staining of iNOS (A) and COX-2 (C) positive cells in AOM alone and GSE + AOM-treated groups, respectively, are shown at 400x magnification. The percentages of iNOS (B) and COX-2 (D) positive cells assessed by quantification of IHC stained rat colonic epithelium are calculated from 5 randomly selected fields from each tissue sample in each group as detailed in Methods, and represent mean ± SE value from 7 rats in each group. Representative colonic tissues from each group were also analyzed by immunoblotting for iNOS and COX-2 expression. Values of band intensity were adjusted with β-actin. AOM, azoxymethane; GSE, grape seed extract; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2.

Fig. 4

Dietary GSE feeding decreases the expressions of β-catenin in colon tissues from AOM-treated rats. The positivity of β-catenin in cytoplasm (A), the percentage of nuclear β-catenin positive cells (B), and the percentage of NF-κB(p65) positive cells (C) were assessed by quantification of IHC stained rat colonic epithelium are calculated from 5 randomly selected fields from each tissue sample in each group as detailed in Methods, and represent mean ± SE value from 7 rats in each group. AOM, azoxymethane; GSE, grape seed extract.

Fig. 5

Proposed mechanism of GSE efficacy against AOM-induced colonic ACF formation in F344 rats. GSE suppresses β-catenin and NF-κB signaling and thereby inhibits cell proliferation and induces apoptosis together with down-regulation of cyclin D1, survivin, iNOS and COX-2 during AOM-induced colonic ACF formation in F344 rats. These molecular alterations by GSE could be, in part, underlying mechanisms by which it prevents AOM-induced colonic ACF formation. AOM, azoxymethane; GSE; grape seed extract, ACF, aberrant crypt foci; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2.