Dietary feeding of grape seed extract prevents intestinal tumorigenesis in APCmin/+ mice

Abstract

Chemopreventive effects and associated mechanisms of grape seed extract (GSE) against intestinal/colon cancer development are largely unknown. Herein, we investigated GSE efficacy against intestinal tumorigenesis in APC(min/+) mice. Female APC(min/+) mice were fed control or 0.5% GSE (wt/wt) mixed AIN-76A diet for 6 weeks. At the end of the experiment, GSE feeding decreased the total number of intestinal polyps by 40%. The decrease in polyp formation in the small intestine was 42%, which was mostly in its middle (51%) and distal (49%) portions compared with the proximal one. GSE also decreased polyp growth where the number of polyps of 1 to 2 mm in size decreased by 42% and greater than 2 mm in size by 71%, without any significant change in polyps less than 1 mm in size. Immunohistochemical analyses of small intestinal tissue samples revealed a decrease (80%-86%) in cell proliferation and an increase (four- to eight-fold) in apoptosis. GSE feeding also showed decreased protein levels of cyclooxygenase-2 (COX-2) (56%-64%), inducible nitric oxide synthase (iNOS) (58%-60%), and beta-catenin (43%-59%) but an increased Cip1/p21-positive cells (1.9- to 2.6-fold). GSE also decreased cyclin D1 and c-Myc protein levels in small intestine. Together, these findings show the chemopreventive potential of GSE against intestinal polyp formation and growth in APC(min/+) mice, which was accompanied with reduced cell proliferation and increased apoptosis together with down-regulation in COX-2, iNOS, beta-catenin, cyclin D1, and c-Myc expression, but increased Cip1/p21. In conclusion, the present study suggests potential usefulness of GSE for the chemoprevention of human intestinal/colorectal cancer.

Figure 1

Effect of dietary feeding of GSE on intestinal tumorigenesis in APC^min/+ mice. Dietary feeding of GSE in AIN-76A diet (A) does not affect body weight gain in APC^min/+ mice and (B) inhibits intestinal polyp formation (C) with the most prominent effect in middle and distal regions of small intestine. (D) GSE also decreases the number of larger polyps (>1 mm) indicating its growth-inhibitory effect on polyps in APC^min/+ mice.

Figure 2

Modulatory effects of dietary GSE on cell proliferation and apoptosis in intestinal epithelium of APC^min/+ mouse. The percent of (A) PCNA-positive cells and (B) TUNEL-positive cells assessed by quantification of immunohistochemically stained mouse intestinal epithelium in 10 randomly selected fields from each tissue sample are represented. Data are shown as mean ± SE of eight samples in each group.

Figure 3

Effects of dietary GSE on COX-2 and iNOS expression levels in intestinal tissues of APC^min/+ mice. Representative photographs for immunohistochemical staining of (A) COX-2 and (C) iNOS-stained cells in APC^min/+ mouse control and GSE-treated groups, respectively, are shown at 400x magnifications. (B) COX-2 and (D) iNOS expression levels were assessed by the quantification of the specific immunostaining of mouse intestinal epithelium in 10 randomly selected fields as described in Materials and Methods. Data are shown as mean ± SE of eight samples in each group.

Figure 4

Effect of dietary GSE on β-catenin and Cip1/p21 expression levels in intestinal tissue of APC^min/+ mice. Representative photographs for immunohistochemical staining of β-catenin positivity (A) in APC^min/+ mouse control and GSE-treated groups are shown at 400x magnifications. (B) Immunopositivity of β-catenin and (C) Cip1/p21-positive cells were assessed by quantification of immunohistochemically stained mouse intestinal epithelium in 10 randomly selected fields as described in Materials and Methods. Data are shown as mean ± SE of eight samples in each group.

Figure 5

Effect of dietary GSE cyclin D1 and c-Myc expression levels in intestinal tissue of APC^min/+ mice. (A) Cyclin D1-positive cells and (B) immunopositivity of c-Myc were assessed by quantification of immunohistochemically stained mouse intestinal epithelium in 10 randomly selected fields as described in Materials and Methods. Data are shown as mean ± SE of eight samples in each group.