Black Raspberry Components Inhibit Proliferation, Induce Apoptosis, and Modulate Gene Expression in Rat Oesophageal Epithelial Cells

We have shown that a diet containing freeze-dried black raspberries (BRB) inhibits the development of chemically induced cancer in the rat esophagus. To provide insights into possible mechanisms by which BRB inhibit esophageal carcinogenesis, we evaluated an ethanol (EtOH) extract of BRB, and two component anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O−rutinoside) in BRB, for their effects on growth, apoptosis, and gene expression in rat esophageal epithelial cell lines. The EtOH extract and both anthocyanins selectively caused significant growth inhibition and induction of apoptosis in a highly tumorigenic cell line (RE-149 DHD) but not in a weakly tumorigenic line (RE-149). The uptake of anthocyanins from the EtOH extract into RE-149 DHD cells far exceeded their uptake intoRE-149 cells,which may have accounted for the selective effects of the extract on growth and apoptosis of RE-149 DHD cells. The growth inhibitory and proapoptotic effects were enhanced by the daily addition of the EtOH extract and the anthocyanins to the medium. Interestingly, the EtOH extract did not alter cyclooxygenase-2 (COX-2) and nitric oxide synthase (i-NOS) expression in RE-149 DHD cells, whereas both anthocyanins down-regulated the expressions of these genes. This differential effect may have been related to the relative amounts of anthocyanins in the extract vs.when theywere added individually to the medium. We conclude that the selective effects of the EtOHextract on growth and apoptosis of highly tumorigenic rat esophageal epithelial cells in vitro may be due to preferential uptake and retention of its component anthocyanins, and this may also be responsible for the greater inhibitory effects of freeze-dried whole berries on tumor cells in vivo.
Steven J. Schwartz and Gary D. Stoner. Nutrition and Cancer, 61(6) Pp. 816–26. DOI: 10.1080/01635580903285148

Piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene) is a stilbenoid, a type of phenolic compound. It is a metabolite of resveratrol found in red wine.

Piceatannol, a Natural Analog of Resveratrol, Inhibits Progression through the S Phase of the Cell Cycle in Colorectal Cancer Cell Lines.

Piceatannol, a naturally occurring analog of resveratrol, was previously identified as the active ingredient in herbal preparations in folk medicine and as an inhibitor of p72Syk. We studied the effects of piceatannol on growth, proliferation, differentiation and cell cycle distribution profile of the human colon carcinoma cell line Caco-2. Growth of Caco-2 and HCT-116 cells was analyzed by crystal violet assay, which demonstrated dose- and time-dependent decreases in cell numbers. Treatment of Caco-2 cells with piceatannol reduced proliferation rate. No effect on differentiation was observed. Determination of cell cycle distribution by flow cytometry revealed an accumulation of cells in the S phase. Immunoblotting demonstrated that cyclin-dependent kinases (cdk) 2 and 6, as well as cdc2 were expressed at steady-state levels, whereas cyclin D1, cyclin B1 and cdk 4 were downregulated. The abundance of p27Kip1 was also reduced, whereas the protein level of cyclin E was enhanced. Cyclin A levels were enhanced only at concentrations up to 100 µmol/L. These changes also were observed in studies with HCT-116 cells. On the basis of our findings, piceatannol can be considered to be a promising chemopreventive or anticancer agent.
Freya Wolter, Antje Clausnitzer, Bora Akoglu and Jürgen Stein. J. Nutr. February 1, 2002 vol. 132 no. 2 298-302

Piceatannol Inhibits TNF-Induced NF-κB Activation and NF-κB-Mediated Gene Expression Through Suppression of IκBα Kinase and p65 Phosphorylation

Piceatannol is an anti-inflammatory, immunomodulatory, and anti-proliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Previously, we have shown that resveratrol suppresses the activation of the nuclear transcription factor NF-κB. Piceatannol, previously reported as a selective inhibitor of protein tyrosine kinase Syk, is structurally homologous to resveratrol. Whether piceatannol can also suppress NF-κB activation was investigated. The treatment of human myeloid cells with piceatannol suppressed TNF-induced DNA binding activity of NF-κB. In contrast, stilbene or rhaponticin (another analog of piceatannol) had no effect, suggesting the critical role of hydroxyl groups. The effect of piceatannol was not restricted to myeloid cells, as TNF-induced NF-κB activation was also suppressed in lymphocyte and epithelial cells. Piceatannol also inhibited NF-κB activated by H2O2, PMA, LPS, okadaic acid, and ceramide. Piceatannol abrogated the expression of TNF-induced NF-κB-dependent reporter gene and of matrix metalloprotease-9, cyclooxygenase-2, and cyclin D1. When examined for the mechanism, we found that piceatannol inhibited TNF-induced IκBα phosphorylation, p65 phosphorylation, p65 nuclear translocation, and IκBα kinase activation, but had no significant effect on IκBα degradation. Piceatannol inhibited NF-κB in cells with deleted Syk, indicating the lack of involvement of this kinase. Overall, our results clearly demonstrate that hydroxyl groups of stilbenes are critical and that piceatannol, a tetrahydroxystilbene, suppresses NF-κB activation induced by various inflammatory agents through inhibition of IκBα kinase and p65 phosphorylation.
Kazuhiro Ashikawa, Sekhar Majumdar, Sanjeev Banerjee, Alok C. Bharti, Shishir Shishodia and Bharat B. Aggarwal. The Journal of Immunology December 1, 2002 vol. 169 no. 11 Pp.6490-7

Piceatannol, a catechol-type polyphenol, inhibits phorbol ester-induced NF-κB activation and cyclooxygenase-2 expression in human breast epithelial cells: cysteine 179 of IKKβ as a potential target

There are multiple lines of evidence supporting that chronic inflammation is linked to carcinogenesis. Nuclear factor-κB (NF-κB), a major redox-sensitive transcription factor responsible for the induction of a wide array of pro-inflammatory genes, is frequently overactivated in many tumors. Moreover, constitutive activation of IκB kinase (IKK), a key regulator of NF-κB signaling, has been implicated in inflammation-associated tumorigenesis. Piceatannol (trans-3,4,3′,5′-tetrahydroxystilbene; PIC) derived from grapes, rhubarb and sugarcane exhibits immunosuppressive and antitumorigenic activities in several cell lines, but the underlying mechanisms are poorly understood. In the present study, we found that PIC inhibited migration and anchorage-independent growth of human mammary epithelial cells (MCF-10A) treated with the prototypic tumor promoter, 12-O-tetradecanoylphorbol-13-aceate (TPA). PIC treatment suppressed the TPA-induced activation of NF-κB and expression of cyclooxygenase-2 (COX-2) in MCF-10A cells. We speculate that an electrophilic quinone formed as a consequence of oxidation of PIC bearing the catechol moiety may directly interact with critical cysteine thiols of IKKβ, thereby inhibiting its catalytic activity. In support of this speculation, the reducing agent dithiothreitol abrogated the inhibitory effects of PIC on TPA-induced activation of NF-κB signaling and expression of COX-2. In addition, the inhibitory effects of PIC on NF-κB activation and COX-2 induction were blunted in cells expressing mutant IKKβ (C179A) in which cysteine 179 was replaced by alanine. In conclusion, our results show that direct modification of IKKβ by PIC, presumably at the cysteine 179 residue, blocks NF-κB activation signaling and COX-2 induction in TPA-treated MCF-10A cells and also migration and transformation of these cells.
Pil-Soon Son. Sin-Aye Park, Hye-Kyung Na, Dae-Myung Jue, Sanghee Kim and Young-Joon Surh. Carcinogenesis (2010) 31 (8): 1442-1449. doi: 10.1093/carcin/bgq099

The phytochemical piceatannol induces the loss of CBL and CBL-associated proteins

Piceatannol is a naturally occurring bioactive stilbene with documented antileukemic properties. It has been extensively used as a Syk-selective protein tyrosine kinase inhibitor for the study of various signaling pathways. Herein, we show that the hydroxystilbene, piceatannol, and related catechol ring-containing compounds are able to induce the loss of the Cbl family of proteins. Normal cellular Cbl-regulatory mechanisms were not involved in this process. Screening of a small library of piceatannol-like compounds indicated that aromaticity and a catechol ring were required for the induction of Cbl loss. Further examination of these two chemical properties showed that the oxidative conversion of the catechol ring of piceatannol into a highly reactive O-benzoquinone was the cause of piceatannol-induced Cbl loss. Characterization of the Cbl selectivity of piceatannol-induced protein loss revealed that this compound was also able to induce the functional loss of specific Cbl-associated proteins involved in signaling pathways commonly associated with cancer. This work uncovers a new, piceatannol-dependent effect and shows a novel way in which this phenomenon can be exploited to inhibit disease-associated signaling pathways. [Mol Cancer Ther 2009;8(3):602–14]
Alexander C. Klimowicz, Sabine A. Bisson, Karm Hans, Elizabeth M. Long, Henrik C. Hansen, & Stephen M. Robbins. Mol Cancer Ther March 2009 8; 602 doi: 10.1158/1535-7163.MCT-08-0891

Piceatannol Inhibits Phorbol Ester-Induced NF-B Activation and COX-2 Expression in Cultured Human Mammary Epithelial Cells

There are multiple lines of evidence supporting that inflammation is causally linked to carcinogenesis. Abnormal up-regulation of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the prostaglandin biosynthesis, has been implicated in carcinogenesis. Trans-3,4,3',5'-tetrahydroxystilbene (piceatannol), a naturally occurring hydroxylated stilbene with potent anti-inflammatory and antioxidative activities, has been shown to inhibit the proliferation of several cancer cells by inducing apoptosis or blocking cell cycle progression. In this study, we examined the effect of piceatannol on activation of the nuclear transcription factor NF-κB, one of the major transcription factors that regulate proinflammatory COX- 2 gene transcription, in human mammary epithelial (MCF-10A) cells treated with the tumor promoter 12-O-tetradecanoylphorbol- 13-acetate (TPA).When pretreated toMCF-10A cells, piceatannol markedly inhibited TPA-induced NF-κB DNA binding to a greater extent than resveratrol and oxyresveratrol, stilbene analogs structurally related to piceatannol. Piceatannol also inhibited TPAinduced phosphorylation and degradation of IκBα as well as nuclear translocation of the phosphorylated form of p65, the functionally active subunit of NF-κB. Likewise, TPA-induced expression of COX-2 was abrogated by piceatannol pretreatment. The thiol reducing agent dithiothreitol abolished the inhibitory effects of piceatannol on NF-κB DNA binding activity, suggesting that piceatannol may directly modify NF-kB
Dan Liu, Do-Hee Kim, and Jong-Min Park. Nutrition and Cancer, 61(6) Pp. 855–63. DOI: 10.1080/01635580903285080

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