Anti-inflammatory action of pterostilbene is mediated through the p38 mitogen-activated protein kinase pathway in colon cancer cells

Abstract

Oxidative/nitrosative stress and generation of proinflammatory cytokines are hallmarks of inflammation. Because chronic inflammation is implicated in several pathologic conditions in humans, including cancers of the colon, anti-inflammatory compounds may be useful chemopreventive agents against colon cancer. Stilbenes, such as resveratrol, have diverse pharmacologic activities, which include anti-inflammation, cancer prevention, a cholesterol-lowering effect, enhanced insulin sensitivity, and increased life span. We previously showed that pterostilbene (trans-3,5-dimethoxy-4'-hydroxystilbene), a structural analogue of resveratrol, is present in blueberries and that pterostilbene inhibited expression of certain inflammation-related genes in the colon and suppressed aberrant crypt foci formation in rats. Here, we examined molecular mechanisms of the action of pterostilbene in colon cancer. Pterostilbene reduced cell proliferation, down-regulated the expression of c-Myc and cyclin D1, and increased the level of cleaved poly(ADP-ribose) polymerase. A combination of cytokines (tumor necrosis factor-alpha, IFN-gamma, and bacterial endotoxin lipopolysaccharide) induced inflammation-related genes such as inducible nitric oxide synthase and cyclooxygenase-2, which was significantly suppressed by treatment with pterostilbene. We further identified upstream signaling pathways contributing to the anti-inflammatory activity of pterostilbene by investigating multiple signaling pathways, including nuclear factor-kappaB, Janus-activated kinase-signal transducer and activator of transcription, extracellular signal-regulated kinase, p38, c-Jun NH(2)-terminal kinase, and phosphatidylinositol 3-kinase. Cytokine induction of the p38-activating transcription factor 2 pathway was markedly inhibited by pterostilbene among the different mediators of signaling evaluated. By silencing the expression of the p38 alpha isoform, there was significant reduction in cytokine induction of inducible nitric oxide synthase and cyclooxygenase-2. Our data suggest that the p38 mitogen-activated protein kinase cascade is a key signal transduction pathway for eliciting the anti-inflammatory action of pterostilbene in cultured HT-29 colon cancer cells.

Figures

Fig. 1

Perostilbene inhibits [3H]thymidine incorporation into the DNA of HT-29 cells and modulates the level of proteins involved in cellular proliferation and apoptosis. A. Structure of pterostilbene. B. HT-29 cells were seeded on a 24-well plate (20,000 cells/well) in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were incubated with pterostilbene at different concentrations or with DMSO as the control vehicle for 1, 2 or 3 days. One μCi of tritium labeled thymidine ([3H]thymidine) per well was added and the amount of incorporated radioactivity in DNA was measured using a liquid scintillation spectrometer. The experiment was repeated twice, with each experiment done in duplicates. The data are represented as mean ± standard deviation. C. Experiments were set up and conducted similar to the conditions as mentioned for Fig. 1B, with the exception that HT-29 cells were treated with pterostilbene, resveratrol or DMSO as control for 3 days before [3H]thymidine incorporation. The experiment was repeated twice, with each experiment done in duplicates. The data are represented as mean ± standard deviation. D. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were treated with pterostilbene at a concentration of 50 μM. The cells were harvested after 9 or 18 h of incubation with pterostilbene, and protein samples were analyzed by Western blotting.

Fig. 2

Effect of cytokines and pterostilbene on the level of iNOS and COX-2 protein in HT-29 cells. A. iNOS and COX-2 proteins are induced differentially by various cytokines. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were treated with TNF-α, IFN-γ, LPS, and IL-1β (each at 10 ng/ml) either alone or in different combinations. The cells were harvested for protein measurements after 15 h, and the samples were immunoblotted to determine induction of iNOS and COX-2. β-Actin levels were used as the loading control. B. Comparison of pterostilbene and resveratrol. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were treated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) (labeled as cytokines or cyto) with or without pterostilbene (Ptero, 30 μM) or resveratrol (Res, 30 μM) for 15 h. The protein levels of iNOS and COX-2 were determined. C. iNOS and COX-2 induction by the cytokine mixture is time dependent: effect of pterostilbene. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were treated with the combination of TNF-α, IFN-γ and LPS, each at 10 ng/ml, for 9, 12 or 15 h in the presence or absence of pterostilbene (50 μM). The protein levels of iNOS and COX-2 were determined. D. Pterostilbene blocks the induction of iNOS and COX-2 in a dose-dependent manner. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were treated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) in the presence or absence of different concentrations of pterostilbene for 15 h. The protein levels of iNOS and COX-2 were determined by Western Blot analysis.

Fig. 3

Cytokine-induced formation of mRNA for iNOS, COX-2, IL-1β and TNF-α: effect of pterostilbene. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were treated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) alone or together with different concentrations of pterostilbene (μM) for 9 h. Total RNA was isolated and mRNA levels for iNOS, COX-2, TNF-α and IL-1β were measured using quantitative RT-PCR analysis as described in Materials and Methods. The data are represented as mean± standard deviation. Statistical significance was calculated by the Students t-test (n = 4, *** p <0.001).

Fig. 4

Evaluation of different cellular signaling pathways affected by pterostilbene in HT-29 cells. A. Pterostilbene showed little or no effects on the NF-κB and JAK-STAT pathways. B. Pterostilbene effectively blocks activation of p38 MAPK signaling. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were incubated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) and pterostilbene (30 μM) for 15 min. The cells were harvested and protein samples were immunoblotted for IκBα, p-STAT3, p-STAT1, p-ERK1/2, p-p38, p-JNK1/2, p-AKT and β-actin.

Fig. 5

Effect of pterostilbene on the p38-ATF2 pathway. A. Pterostilbene down-regulates the phosphorylation of upstream kinase of p38, MKK3/6. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were incubated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) in the presence or absence of pterostilbene (30 μM) for 15 min, and protein samples were immunoblotted for p-MKK3/6 and β-actin. B. Pterostilbene down-regulates the phosphorylation of downstream targets of p38. HT-29 cells (1.5 × 106 cells/ 100 mm dish) were incubated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) in the presence or absence of pterostilbene (30 μM) for 30 min, and protein samples were immunoblotted for p-p38, p-Elk1 and p-ATF2 and β-actin. C. Pterostilbene abolishes nuclear accumulation of p-p38 induced by cytokine mixture. HT-29 cells (30,000 per chamber in a 4-well chamber slide) were incubated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) and pterostilbene (30 μM) for 15 min. Green, staining for pp38; blue, nuclear staining by DAPI; magnification, 63X. D. Pterostilbene abolishes nuclear accumulation of p-ATF2 induced by cytokine mixture. HT-29 cells (30,000 per chamber in a 4-well chamber slide) were incubated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) and pterostilbene (30 μM) for 30 min. Green, staining for p-ATF2; blue, nuclear staining by DAPI; magnification, 63X.

Fig. 6

Effect of pterostilbene on the induction of iNOS and COX-2 on knocking down p38α and p38β. HT-29 cells (150,000 cells per well in 6-well plate) were transfected with siRNA against p38α and p38β and incubated in serum free Accell™ delivery medium for 72 h. Cells were then incubated with a mixture of TNF-α, IFN-γ and LPS (each at 10 ng/ml) in the presence or absence of pterostilbene (30 μM) for an additional 15 h. Cells were harvested for protein and the samples analyzed by Western blotting.