Dietary intake of pterostilbene, a constituent of blueberries, inhibits the beta-catenin/p65 downstream signaling pathway and colon carcinogenesis in rats

Abstract

Stilbenes are phytochemicals present in grapes, berries, peanuts and red wine. A widely studied stilbene, resveratrol (trans-3,5,4'-trihydroxystilbene), has been shown to exert antioxidant, anti-inflammatory, chemopreventive and antiaging effects in a number of biological systems. We reported earlier that pterostilbene (trans-3,5-dimethoxy-4'-hydroxystilbene), a structurally related stilbene found in blueberries, was effective in reducing the incidence and multiplicity of aberrant crypt foci formation in the colon of rats injected with azoxymethane (AOM). Our present study was to identify the chemopreventive potential of pterostilbene with colonic tumor formation as an end point and further to evaluate the mechanistic action of pterostilbene during colon carcinogenesis. F344 rats were given two AOM injections subcutaneously when they were 7 and 8 weeks old and continuously fed the control or 40 p.p.m. pterostilbene diet for 45 weeks. Overall analyses indicated that pterostilbene reduced colon tumor multiplicity of non-invasive adenocarcinomas, lowered proliferating cell nuclear antigen and downregulated the expression of beta-catenin and cyclin D1. Pterostilbene decreased mucosal levels of the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-4. Colon tumors from pterostilbene-fed animals showed reduced expression of inflammatory markers as well as nuclear staining for phospho-p65, a key molecule in the nuclear factor-kappaB pathway. In HT-29 cells, pterostilbene reduced the protein levels of beta-catenin, cyclin D1 and c-MYC, altered the cellular localization of beta-catenin and inhibited the phosphorylation of p65. Our data with pterostilbene in suppressing colon tumorigenesis, cell proliferation as well as key inflammatory markers in vivo and in vitro suggest the potential use of pterostilbene for colon cancer prevention.

Fig. 1.

(A) Structure of pterostilbene. (B) Hematoxylin and eosin staining (top panels), PCNA staining (middle panels), and PCNA counting (bottom panels) of the colon tumors. For PCNA counting, four independent sections of the colon per animal with three animals per group were stained, and ∼1000 cells were counted from each section in total. The PCNA labeling index (PI) was calculated as the [(number of positive cells)/(total number of epithelial cells)] × 100 for each field. These PI values for different colon sections from the animals belonging to same group were then averaged. Statistical significance of treatment between the groups was analyzed by Student’s t-test. * P < 0.05.

Fig. 2.

Pterostilbene inhibits the expression of β-catenin, cyclin D1, COX-2, iNOS and phospho-p65 protein in the colon tumors. The colon tumor sections were processed and incubated with the respective primary antibodies as described in the Materials and Methods. β-Catenin and cyclin D1 staining was high in the cytosol and also present in the nucleus to a lower extent. iNOS and COX-2 showed cytoplasmic staining, whereas nuclear staining was predominant with phospho-p65. n = 3 per group for each analysis. A representative section is shown. Image magnification, ×400.

Fig. 3.

Pterostilbene lowers the production of the inflammatory cytokines, TNF-α (A), IL-1β (B) and IL-4 (C), in the colonic mucosa. The mucosa samples were homogenized and assayed by enzyme-linked immunosorbent assay for the different cytokines, as described under Materials and Methods section. Colon mucosa samples were randomly selected from each group and cytokine levels were analyzed (n = 12 in control group, n = 6 in pterostilbene group). The mean ± SD values are shown. **P < 0.01, ***P < 0.005.

Fig. 4.

Pterostilbene downregulates the expression of β-catenin and its downstream targets, cyclin D1 and c-MYC, in HT-29 colon cancer cells. (A) Effect of pterostilbene on levels of β-catenin, cyclin D1 and c-MYC. HT-29 cells (1.5 × 10⁶ per 100 mm dish) were treated with pterostilbene (50 μM) for 30 min or 4 h. The cells were harvested for whole cell protein and samples were immunoblotted. (B) Action of pterostilbene on cellular localization of β-catenin. HT-29 cells (30 000 per chamber in a four-well chamber slide) were incubated with pterostilbene (50 μM) for 4 h. Green, staining for β-catenin; blue, nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI). (C) Effects of pterostilbene on Wnt/β-catenin signaling proteins in the presence of Wnt agonist. HT-29 cells were treated with a Wnt agonist (10 μM) with or without pterostilbene (50 μM) for 4 h. The whole-cell and nuclear proteins were harvested and immunoblotted for β-catenin, cyclin D1, c-MYC and β-actin. (D) Effects of pterostilbene on the nuclear localization of β-catenin induced by Wnt agonist. HT-29 cells were incubated with a Wnt agonist (10 μM) with or without pterostilbene (50 μM) for 4 h. Green, staining for β-catenin; blue, nuclear staining by DAPI. Quantification of western blots was done by ImagePro 6.2 program, and the numbers are given at the bottom of each western blot.

Fig. 5.

Effects of pterostilbene on the NF-κB pathway. (A) Pterostilbene lowers nuclear phospho-p65 levels in HT-29 cells. HT-29 cells (1.5 × 10⁶ cells per 100 mm dish) were incubated with a mixture of TNF-α, interferon (IFN)-γ and lipopolysaccharide (LPS) (each at 10 ng/ml) in the presence or absence of pterostilbene (30 μM) for 1 h, and cytosolic and nuclear protein fractions were collected and immunoblotted. (B) DNA binding of NF-κB is reduced by pterostilbene at 1 h. HT-29 cells were incubated with a mixture of TNF-α, IFN-γ and LPS together with pterostilbene (30 μM) for 1 or 4 h. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. (C) Phosphorylation of p65 mediated through the p38/MSK-1 pathway is inhibited by pterostilbene in HT-29 cells. HT-29 cells were incubated with a mixture of TNF-α, IFN-γ and LPS in the presence or absence of pterostilbene (30 μM), MSK-1 inhibitor (H-89, 10 μM) or p38 mitogen-activated protein kinase inhibitor (SB203580, 10 μM) for 1 h. The nuclear proteins were harvested and immunoblotted for phospho-MSK-1, phospho-p65 and p65. Quantification of western blots was done by ImagePro 6.2 program, and the numbers are given at the bottom of each western blot.