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J. Agric. Food Chem. 2006, 54, 980−985
Pomegranate Juice, Total Pomegranate Ellagitannins, and Punicalagin Suppress Inflammatory Cell Signaling in Colon Cancer Cells LYNN S. ADAMS,*,† NAVINDRA P. SEERAM,† BHARAT B. AGGARWAL,‡ YASUNARI TAKADA,‡ DANIEL SAND,† AND DAVID HEBER† Center for Human Nutrition, David Geffen School of Medicine, University of California, Los Angeles, California 90095, and Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Phytochemicals from fruits such as the pomegranate (Punica granatum L) may inhibit cancer cell proliferation and apoptosis through the modulation of cellular transcription factors and signaling proteins. In previous studies, pomegranate juice (PJ) and its ellagitannins inhibited proliferation and induced apoptosis in HT-29 colon cancer cells. The present study examined the effects of PJ on inflammatory cell signaling proteins in the HT-29 human colon cancer cell line. At a concentration of 50 mg/L PJ significantly suppressed TNFR-induced COX-2 protein expression by 79% (SE ) 0.042), total pomegranate tannin extract (TPT) 55% (SE ) 0.049), and punicalagin 48% (SE ) 0.022). Additionally, PJ reduced phosphorylation of the p65 subunit and binding to the NFκB response element 6.4-fold. TPT suppressed NFκB binding 10-fold, punicalagin 3.6-fold, whereas ellagic acid (EA) (another pomegranate polyphenol) was ineffective. PJ also abolished TNFR-induced AKT activation, needed for NFκB activity. Therefore, the polyphenolic phytochemicals in the pomegranate can play an important role in the modulation of inflammatory cell signaling in colon cancer cells. KEYWORDS: Cancer; pomegranate; AKT; COX-2; NFKB
INTRODUCTION
Cellular signaling activated by proinflammatory conditions can lead to the initiation and progression of cancer by inducing DNA damage and epigenetic changes, increased sensitivity to growth factors and abnormalities in the expression and activity of transcription factors that control cell cycle progression and survival (1). Specifically, chronic inflammation of the colon has been shown to increase the risk for colon cancer (2, 3) making inflammatory signaling pathways a target for cancer prevention and treatment. Epidemiological studies suggest that the consumption of a diet rich in phytochemicals can reduce the risk of cancer (4). Fruits and vegetables, which contain a diverse range of phytochemicals, are suggested to have properties important to the prevention of cancer including antioxidant, antiinflammatory, and antiproliferative activities as well as modulatory effects on subcellular signaling pathways, induction of cell cycle arrest, and apoptosis (5-7). Pomegranate (Punica granatum L.) fruits are widely consumed fresh and in processed forms as juice, jam, and wine (8). Commercial pomegranate juice (PJ) shows potent antioxidant and antiatherosclerotic properties attributed to its high * To whom correspondence should be addressed. Phone: (310) 8249632. Fax: (310) 206-5264. E-mail:
[email protected]. † University of California, Los Angeles. ‡ The University of Texas M. D. Anderson Cancer Center.
content of polyphenols including ellagitannins, ellagic acid, and other flavonoids (quercetin, kaempferol, and luteolin glycosides) (9-13). The most abundant of these polyphenols is punicalagin, an ellagitannin implicated as the bioactive constituent responsible for >50% of the juice’s potent antioxidant activity. Punicalagin is abundant in the fruit husk and during processing is extracted into PJ in significant quantities reaching levels of >2 g/L juice (14). The cyclooxygenase (COX) enzymes (COX-1 and COX-2) are expressed in a variety of cell lines and are responsible for the conversion of free fatty acids to prostanoids. COX-1 produces prostanoids that aid in the regulation of normal tissue homeostasis, whereas COX-2 expression results in prostanoids that induce inflammation. For this reason, COX-2 overexpression has been implicated in cancer initiation and progression. Solid tumors tend to produce high levels of prostanoids and exhibit increased levels of COX-2 expression (1). Tumor cells that do not express COX-2 proliferate more slowly in vivo (15), and COX-2 specific inhibitors decrease the proliferation of a number of cancer cell lines in vitro (16-19). COX-2 also inhibits apoptosis, which can aid in tumor cell survival (2022). In vivo, COX-2 inhibitors have shown effectiveness in the inhibition of tumor production in mice and in clinical trials with familial adenomatous polyposis (FAP) patients (23, 24). The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappa-B (NFκB) pathway positively
10.1021/jf052005r CCC: $33.50 © 2006 American Chemical Society Published on Web 01/13/2006
J. Agric. Food Chem., Vol. 54, No. 3, 2006
Pomegranate Juice and Inflammatory Cell Signaling Proteins affects COX-2 expression. PI3K directly activates AKT, which in turn phosphorylates and activates the IκB kinase (IKK) leading to NFκB activation. PI3K activity has been associated with colon cancer where phosphatase and tensin homologue gene (PTEN) mutations occur, as PTEN inhibits PI3K (25). Increased PI3K activity has been found in colon cancer cell specimens (26) and adenocarcinoma cell lines (27). In addition, inhibition of PI3K in colon and ovarian cancer cell lines leads to inhibition of cell proliferation (28). In HT-29 cells, the activation of NFκB by TNFR resulted in an increase in COX-2 protein expression, an effect that was inhibited by nonsteroidal antiinflammatory drugs (NSAIDS) (29). Consistent with the finding that the COX-2 promoter contains two NFκB binding sites (30), TNFR-regulated COX-2 protein expression in HT29 colon cancer cells was found to be dependent upon NFκB activation (31). Little information is available on the effects of pomegranate extracts on the cellular signaling pathways involved in cancer initiation and progression. The purpose of this study was to evaluate the effect of PJ, total pomegranate tannins (TPT), purified punicalagin, and ellagic acid (EA) on COX-2 and the signaling pathways leading to its expression in HT-29 colon cancer cell lines. Our results will further our understanding of how natural products may modulate cancer cell signaling and indicate possibilities for future investigation. MATERIALS AND METHODS Chemicals. NS398 was obtained from Sigma-Aldrich Co. (St. Louis, MO), wortmannin, the AKT kinase assay kit, phospho-p65 NFκB, total p65 NFκB, and antirabbit and antimouse antibodies were purchased from Cell Signaling Technology (Beverly, MA), and the COX-2 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). TNFR and the cell titer glow assay were obtained from Promega Corp. (Madison, WI). McCoy’s 5A medium was purchased from American Type Culture Collection (ATCC) (Manassas, VA), phosphate-buffered solution (PBS) was purchased from Invitrogen (Carlsbad, CA), and western blotting supplies were obtained from Biorad Laboratories (Hercules, CA). Pomegranate Extract. Pomegranate juice (POM Wonderful LLC, Los Angeles, CA) is commercially available for human consumption and was used in concentrate form (contains 1.74 g/L punicalagin). Ellagitannins were purified from fruit husk as previously reported and analyzed for purity by HPLC and liquid chromatography electrospray ionization mass spectroscopy (LC-ESI/MS) (14). Concentrations of punicalagin and TPT were normalized to deliver equivalent amounts as those found in PJ. Cell Culture. HT-29 colon cancer cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and were cultured in McCoy’s 5A medium containing 10% FBS and 1% penicillin/streptomycin. Human myeloid KBM-5 cells were originally obtained from ATCC and were cultured in Iscove’s modified Dulbecco’s medium with 15% FBS. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and maintained in the linear stage of growth. Cell Viability Assay. Proliferation was measured using the CellTiterGlo luminescent cell viability assay (technical bulletin no. 288, Promega, Madison, WI). HT-29 cells were plated in 96-well plates at a density of 10 000 cells/well and incubated for 24 h. Cells were treated with either 100 µL of control media, vehicle (
