Cyclooxygenases catalyze the formation of prostaglandins from arachidonic acid. COX-1 is expressed constitutively as a housekeeping gene in most normal tissues, while COX-2 expression is induced by pro-inflammatory and other mitogenic signals. Aberrant COX-2 upregulation is a common feature of cancer cells and potentially preneoplastic conditions such as tissue inflammation. COX-2 has been causally linked to epithelial tumorigenesis and is under investigation as a target for cancer prevention and therapy (reviewed in Reference 33). COX-catalyzed conversion of arachidonic acid to prostaglandin involves two sequential reactions at spatially distinct active sites, i.e., a cyclooxygenase reaction followed by a hydroperoxidase reaction. In assessing resveratrol as a cancer preventive agent, the Pezzuto team reported that resveratrol inhibited the cyclooxygenase and hydroperoxidase activities of COX-1 but exhibited much weaker effects against COX-2 activity.3 More recently, a detailed mechanistic analysis established that resveratrol is a mechanism-based inactivator of COX-1 peroxidase catalysis. Resveratrol was oxidized at the peroxidase active site in a manner dependent on the presence of the substrate hydrogen peroxide, with concomitant inactivation of COX-1 peroxidase and cyclooxygenase activities. The authors hypothesized that inactivation entailed generation of resveratrol and protein radical species. COX-2 was more efficient than COX-1 in catalyzing peroxide-dependent resveratrol oxidation but was not inactivated by the polyphenol.34 Interestingly, COX-1 inactivation by resveratrol is distinct from classical nonsteroidal anti-inflammatory drug (NSAID) action, in that classical NSAIDs target the cyclooxygenase active site of COX isoforms.
While resveratrol potently inhibits COX-1 but not COX-2, it suppresses the expression of COX-2 but not COX-1. This is illustrated by the results of a study employing an experimental model of ulcerative colitis, which is a nonspecific inflammatory disorder of the colonic mucosa and submucosa. The model involves intracolonic administration of trinitrobenzenesulfonic acid (TNBS) in rats, thus effecting colonic inflammation and injury. Resveratrol attenuated TNBS-induced colonic inflammation and injury, and this was associated with suppression of TNBS-induced COX-2 expression and no change in COX-1 expression in the colonic epithelial cells.35 Similarly, the chemoprotective effects of resveratrol on ultraviolet B-damaged mouse skin were associated with attenuated COX-2 expression.18 Consistent with these in vivo models, resveratrol has been shown to inhibit TGFa-stimulated and basal human COX-2 promoter activity in a reporter gene assay in human colon cancer DLD-1 cells.36 In summary, studies of resveratrol effects on COX-1 and COX-2 indicate that COX-1 inhibition may reinforce the anti-inflammatory effects of COX-2 suppression by resveratrol, suggesting COX isoforms as important targets in the cancer preventive action of stilbenes.
18.8 MODULATION OF ESTROGEN-DEPENDENT GENE REGULATION BY RESVERATROL
The structural similarity between resveratrol and the synthetic estrogen diethyl-stilbestrol provided the first clue that resveratrol may function as a phytoestrogen. Estrogen-receptor (ER) agonists bind to the receptor and induce ER binding to estrogen response elements (EREs) in the promoters of target genes with the effect of promoter activation. Phytoestrogens are dietary nonsteroidal agents with ER agonist or mixed ER agonist/antagonist activity. In the first definitive analysis of resveratrol effects on the transcriptional activity of the ER, Jameson's group37 established that resveratrol competitively bound ER expressed by MCF7 cells, and activated an ERE-containing promoter-reporter gene construct in MCF7 cells but not a derived construct with the ERE deleted. Furthermore, resveratrol-induced activation of the ERE-containing promoter was abrogated by the pure antiestrogen ICI 182780, indicating that transcriptional activation entailed res-veratrol binding to the ER and thus identifying resveratrol as a phytoestrogen.37 Studies in a human endometrial cancer cell line, Ishikawa cells, established that resveratrol can also function as an ER antagonist. Resveratrol antagonized estrogen-induced expression of the endogenous genes alkaline phosphatase and progesterone receptor, and the polyphenol also antagonized estrogen-induced activation of an ERE-containing promoter-reporter construct. Furthermore, res-veratrol showed no ER agonist activity in these experiments when tested in the absence of estrogen.38 Collectively, the studies in MCF7 and Ishikawa cells established resveratrol as a mixed ER agonist/antagonist. Recently, an analysis of ER-negative human breast cancer MDA-MB-231 cells stably transfected with human ERa established that resveratrol acts as an ERa agonist in the cells to induce expression of an endogenous ER target gene, transforming growth factor-a (TGFa). Resveratrol induction of the TGFa gene was blocked by the pure antiestrogen ICI 182780, and resveratrol did not antagonize TGFa induction by estrogen.39 While phytoestrogens are widely viewed as healthful and cardiopro-tective, the mixed ER agonist/antagonist activity of resveratrol suggests potential for increased cancer risk, a possibility that warrants caution in the implementation of dietary stilbenes as cancer preventive agents or health-promoting dietary supplements in women.
18.9 INHIBITION OF PROTEIN KINASE C (PKC) ISOZYMES BY RESVERATROL
Protein kinase C (PKC) is a family of ten isozymes that play critical roles in the regulation of cell growth and survival. Most PKC isozymes are activated by phosphatidylserine-dependent mechanisms that involve binding of the stimulatory cofactors sn-1,2-diacylglycerol (DAG) and Ca2+ to the regulatory domain; PKCa, PKCP1, PKCP2, and PKCy are activated by Ca2+ and DAG, and PKCS, PKCe, PKC0, and PKCn are activated by DAG alone.40 The discovery that phorbol-ester tumor promoters, such as 12-O-tetradecanoyl phorbol-13-acetate (TPA), could substitute for DAG and activate PKC isozymes at nanomolar concentrations41 led to the consideration of PKC as a target for cancer prevention or therapy. Subsequent studies established that the PKC family includes oncogenic isozymes, e.g., PKCe,42 43 and tumor-suppressive isozymes, e.g., PKCS,44 indicating the need for isozyme-selective PKC targeting in designing strategies of cancer prevention and therapy. A study of resveratrol effects on purified PKC isozymes established that resveratrol is PKC inhibitory, but the inhibitory potency of the polyphenol was similar against the seven isozymes surveyed, which were the Ca2+, DAG-activated isozymes PKCa, PKCP1, PKCP2, and PKCy, the DAG-activated isozymes PKCS and PKCe, and the DAG-independent isozyme PKCZ. PKC inhibition by resveratrol entailed competition with ATP substrate.45
Protein kinase D (PKD) is a family of three isozymes, PKD1-3, that have in common with PKC a conserved DAG/TPA binding-site. However, PKD is not activated by DAG/TPA, and is instead activated downstream of PKC by phosphorylation at activation-loop residues serine 744/748. PKD is involved in the transmission of PKC-dependent cell proliferative and survival signals.46 Interestingly, cell survival signaling by PKD may involve suppression of the JNK signaling pathway through PKD-JNK complex formation.47 A comparison of the inhibitory effects of resveratrol against purified PKD-1 (formerly called PKC|j) vs. the PKC isozymes analyzed in Reference 45 determined that the polyphenol is somewhat more potent against PKD-1, and markedly so with respect to the kinase autophosphorylation reaction.48 This suggested that resveratrol inhibition of PKC might be reinforced in cells by coordinate inhibition of the PKC effector PKD. A recent analysis of growth-suppressive effects of resveratrol in the andro-gen-independent human prostate cancer cell line PC-3 established that resveratrol blocked TPA-induced PKCa activation without any effect on the other phorbol ester-activated protein kinases in the cells (PKD, PKCe).49 These findings are consistent with a recent report identifying PKCe as the endogenous activator of PKD.50 Furthermore, they reveal that, although an indiscriminate inhibitor of purified PKC/PKD species, resveratrol selectively inhibits a subset of these targets in cells. The strong suppression of DMBA (7,12-dimethyl-benz(a)anthracene)/TPA-induced papilloma formation achieved by resveratrol, when co-administered with TPA in the promotion phase of the two-stage mouse skin carcinogenesis model, offers evidence that, in at least some tissues, the sum total of resveratrol inhibitory effects against TPA-responsive PKC isozymes and PKD may be cancer preventive.3
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