(The FASEB Journal.
© 2003 FASEB
NUS Graduate School of Integrative Sciences and Engineering and Department of Physiology, Faculty of Medicine, National University of Singapore, Singapore 117597
1Correspondence: National University Medical Institutes, National University of Singapore, MD9, #03-06, Singapore 117597. E-mail: email@example.com
WITH THE RAPID advances made over the last two decades in biomedical research, there has been an unprecedented interest in unraveling the magical properties of some commonly used natural products. Consequently, a wide variety of natural products are under scrutiny for their clinical potential, both in terms of disease prevention and treatment. Among the compounds under investigation is a family of polymers given the name viniferin. These compounds elicit strong anti-fungal properties and are therefore included under the broad class of plant antibiotics known as phytoalexins (1) . One remarkable compound in this list is resveratrol (RSV), a major active ingredient of stilbene phytoalexins, first isolated from the roots of the oriental medicinal plant Polygonum Capsidatum (Ko-jo-kon in Japanese) (2) . Observations that this compound was an active ingredient of a folk plant known for its remedial effects against a host of human afflictions (2 , 3) and that it was synthesized by leaf tissue in response to fungal infection of grapevines (Vitis vinifera) (4) provided the impetus for the increase in activity surrounding RSV in the field of biomedical research. The relatively high concentration of RSV in wine (5 ) and its documented cardioprotective effect (6) form the basis for the so-called “French paradox” (7) . Most of the initial work on RSV was centered around its effects on metabolic pathways regulating cardiovascular biology, such as lipid metabolism and platelet function; however, since the reported cancer chemopreventive activity of RSV in animal models of carcinogenesis (8) , recent investigations have been directed at understanding the molecular mechanism(s) of its diverse biological effects. As a result, the positive or negative effects of RSV on some important physiological pathways have been proposed as possible mechanisms for its observed cancer chemopreventive, cardioprotective, and neuroprotective activities. These include suppression of cellular proliferation via inhibition of key steps in the signal transduction pathways (9 10 11 12) and cyclin-dependent kinases (cdks) (13) , promotion of cellular differentiation (14) , scavenging/suppression of intracellular reactive oxygen intermediates (ROI) (15) , induction of apoptotic cell death through activation of mitochondria-dependent or -independent pathways (16 17 18) , anti-inflammatory activity via down-regulation of proinflammatory cytokines (19 , 20) , and inhibition of androgen receptor function and estrogenic activity (21 , 22) . This review is intended to provide the reader with an appreciation of the diverse biological effects of this remarkable compound, which could have tremendous potential as a chemopreventive and/or chemotherapeutic agent in clinical medicine.
OCCURRENCE, SYNTHESIS, AND CHEMISTRY OF RSV
Polygonum Capsidatum, root extracts of which have been used extensively in oriental folk medicine, is one of the richest sources of RSV (2 , 3) . Its occurrence has also been documented in trees, including eucalyptus and spruce, in a few flowering plants, such as Veratrum grandiflorum and Veratrum formosanum (two species of lilly), in peanuts and groundnuts, and in grapevines (1) . Since the first reported detection of trans-RSV in grapevines (Vitis vinifera) in 1976 (4) and later in wine in 1992 (5) , most of the work had focused on RSV in grapevines. This was mainly because compounds found in grapevines were implicated in epidemiological data demonstrating an inverse correlation between red wine consumption and incidence of cardiovascular disease—the “French paradox” (7) . Phytoalexins such as RSV are not present in healthy vine leaves or berries, but are abundant in mature vine wood, and their synthesis is stimulated by exposure to ultraviolet (UV) rays (23) . Oxidative dimerization of RSV leads to the formation of viniferins that could be reproduced by exposure of the parent compound to horseradish peroxidase–hydrogen peroxide system in vitro (24) . The synthetic potential is highest just before the grapes reach maturity, and is low in buds, flowers, and mature fruits; resistant species produce significantly higher amounts than susceptible ones (25) . Higher amounts are also observed in healthy areas around necrotic lesions following fungal infection of grape berry skins, and this seems to be an inherent protective mechanism to limit the spread. Studies conducted in the wine-producing French area of Burgundy demonstrated that the stimulus for RSV synthesis in healthy berries away from the site of infection involved a chemical signal generated by the pathogen or the host for regulating the activity of stilbene synthase, the terminal enzyme in the biosynthesis of RSV (26) . This results in accumulation of RSV around the infected area; however, 48–72 h later the activity of the regulatory enzyme stilbene oxidase is induced leading to oxidative degradation of RSV (27) .
The determination of RSV content has not been restricted to plant components such as leaves, skins, and petals, but also involves wines and grape juices. Initial attempts failed due to the absence of good methodology to recover RSV and prevent its oxidative degradation; however, more recent application of HPLC and GC-MS to the detection of RSV in wines has shown that both cis and trans isomers are present in wines, with the latter found in significantly higher concentration (28 , 29) . Generally, white wines contain 1–5% of the RSV content present in most red wines, and the highest concentration of trans-RSV has been reported in wines prepared from Pinot noir grapes (averaging 5.13 mg/L) (28) .
The chemical structure of RSV has a striking similarity to the synthetic estrogen diethylstilbestrol. Two phenol rings are linked by a styrene double bond to generate 3,4′,5-trihydroxystilbene (Fig. 1 ). RSV exists as cis and trans isomeric forms, with trans to cis isomerization facilitated by UV exposure. Trans-RSV (MW=228) is commercially available and is relatively stable if protected from high pH and light (1) . The difference in absorption maxima (307 nm for the trans and 288 nm for the cis isomer) allows for their separation and detection by HPLC using a C18 reverse phase column (30) . More recently, gas chromatography has been used to measure concentrations of cis and trans isomers in plasma and cells (29) .
Figure 1. Structure of resveratrol (3,4′,5 trihydroxystilbene.
The terminal enzyme involved in RSV synthesis, stilbene synthase, normally is not active or expressed in nonstressed seedlings; however, within 6 h of exposure to UV radiation or fungal infection, enzyme activity is induced and peaks by 30 h. Soluble ß-glucans present in bacterial cell walls are potent inducers of stilbene synthase, and reports suggest that the presence of Ca2+ seems critical for the enzyme activity (31) . The potential of grapevines to synthesize RSV and viniferins (oxidation product of RSV) has been shown to provide resistance against a variety of plant diseases. Gene transfer of stilbene synthase into plants lacking this activity resulted in rapid induction of the enzyme and accumulation of RSV upon UV irradiation and conferred resistance to microbial infections (32 , 33) .
FROM WINE TO MAMMALIAN BIOLOGY
The diverse beneficial effects of extracts prepared from the plant Polygonum Capsidatum against a host of disease conditions had been documented decades before the actual identification of RSV as a potent ingredient of this folk medicine. Since this reported observation in the mid-1970s, much work went into the actual synthesis in plants and the factors that promoted expression of RSV-producing enzymes. Subsequent identification of RSV as a major phenolic constituent of wines and its association with decreased cardiovascular disease risk in moderate wine consumers have resulted in a tremendous increase in interest in elucidating the effects of this polyphenolic compound and its derivatives on human health and disease. Consequently, the diverse biological activities and potential beneficial effects of RSV have come to the fore over the past 5 years. In the following sections, an overview of the effects of RSV on biological systems and their potential relevance to disease prevention and treatment will be provided.
Antioxidant activity of RSV: potential cardio- and neuroprotective effects
Normal cellular metabolism generates reactive oxygen intermediates (ROI) such as superoxide (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH·). Excessive accumulation of ROI is kept in check by the cellular antioxidant defenses comprising a number of intracellular enzymes, such as glutathione (GSH), superoxide dismutase (SOD), and catalase (34) . A defect in the cells’ inherent ability to counteract the production of ROI results in their abnormal accumulation, a state commonly referred to as “oxidative stress.” Exposure of cellular macromolecules (lipids, proteins, and nucleic acids) to ROI results in their oxidative modifications with deleterious potential (35) . A good example is the oxidative modification of low density lipoproteins (LDL) (36) implicated in atherosclerosis and increased incidence of coronary artery disease. Whereas the endocytosis of normal LDL is tightly regulated, oxidized LDL is taken up by a nonregulated scavenger receptor system resulting in an abnormal accumulation of LDL in monocytic subendothelial cells (foam cells) (37) . Oxidized lipoproteins provide a permissive environment for atheroma formation and platelet aggregation, thereby fueling the process of atherosclerosis (37) . Phenolic compounds present in red wine elicit antioxidant activity and prevent LDL oxidation (38) . In line with this, epidemiological studies have linked moderate intake of wine with an appreciable decrease in the risk of coronary artery disease, particularly in regions of France where the diet is rich in fat (39) . There is evidence to support that RSV is a potent inhibitor of the oxidation of polyunsaturated fatty acids (PUFA) found in LDL that play a major role in atherosclerosis (40) . As a matter of fact, RSV was shown to be more potent than flavonoids in preventing copper-catalyzed oxidation, and as LDL has high affinity for copper this copper chelating activity prevents oxidative modification of LDL (41) . Further evidence for the protective effect of RSV on lipid accumulation was reported in human hepatocarcinoma HepG2 cells, which elicit most of the normal liver parenchymal functions (1 , 42) . Addition of RSV to the culture medium resulted in a dose-dependent decrease in the intracellular concentration of Apo B and a significant reduction in the rate of secretion of cholesterol esters and triglycerides. The latter is an indication of fewer VLDL and therefore lower LDL production. Although these observations provide evidence for anti-lipogenic and an atherosclerosis inhibitory effect of RSV, in vivo data fall short of corroborating these findings; studies using hyperlipidemic rabbits fed on RSV showed no decreases in serum cholesterol levels or atherosclerotic lesions (43) .
Through its inhibitory effect on membrane lipid peroxidation, RSV has also been shown to reduce the toxic effects of ROI in living cells. For example, rat adrenal pheochromocytoma cells (PC12) exposed to ethanol-induced oxidative death were remarkably protected in the presence of RSV (44) . In similar experiments the death inhibitory activity was attributed to the ability of RSV to block internalization of oxidized lipoproteins. In addition, oxidized lipoprotein-induced cell death was inhibited in neuronal cells in the presence of RSV, indicating neuroprotective activity (45) . The antioxidant activity of RSV has also been shown to inhibit proliferation of hepatic stellate cells (46) , a major player in the development of liver fibrosis, suggesting a hepatoprotective effect. Further corroborating the antioxidant activity of RSV are data demonstrating significant inhibition of phorbol ester (PMA) -induced intracellular ROI production (47) . As phorbol esters are potent tumor promoters, this inhibitory activity of RSV could prevent the development of a pro-oxidant milieu that favors carcinogenesis (48) . However, more recent data and our unpublished results provide interesting insight into the effect of this compound on intracellular redox state. These data seem to support both anti- and pro-oxidant activity of RSV, depending on the concentration of RSV and the cell type. For example, in human leukemia cells RSV induces an increase in intracellular ROI, whereas in prostate cancer cells a dose-dependent decrease in intracellular ROI (in particular, O2-) is observed (Y. Li and S. Pervaiz, unpublished results).
In addition to the antioxidant property of RSV, two other biological effects of RSV support its cardioprotective ability. First, wine phenolics such as RSV have been shown to modulate the production of nitric oxide (NO) from vascular endothelium, a nitrogen species involved in inflammatory responses (49 , 50) . Increased levels of NO can cause vascular damage, thereby contributing to the development of atheromatous plaques. Second, RSV has been shown to inhibit platelet aggregation, another major contributor in the process of atherosclerosis (51 , 52) . Platelets stick to the endothelial surface of blood vessels; they can activate the process of thrombus formation and their aggregation could set into motion the process of vascular occlusion. Platelets have also been linked to the synthesis of eicosanoids from arachidonic acid that contributes to platelet adhesion (53) . A dose-dependent decrease in platelet aggregation has been demonstrated with RSV, lending further support to its preventive activity against coronary artery disease. This has been linked to the ability of RSV to inhibit eicosanoid synthesis (1) (discussed in more detail later).
The observations presented in the preceding section are further supported by human in vivo studies demonstrating increased antioxidant activity in blood of moderate red wine consumers (38) ; however, more needs to be done to establish that the reported beneficial effects on human cardiovascular, neurological, and hepatic systems are indeed a function of RSV.
Effect of RSV on inflammatory response modifiers: immunomodulation
Among the various beneficial effects of the extract prepared from the roots of Polygonum Capsidatum (the plant from which RSV was first isolated) were its “magical: effects on allergic and inflammatory diseases. Invariably, these pathological states are the result of an increase in activity of leukocytes that spit out an excess of biological response modifiers. The two enzyme systems involved in the synthesis of proinflammatory mediators such as 5-HETE (5-hydroxy-6,8,11,14-eicosatetraenoic acid), thromboxane A2, prostaglandins (PG), and HTT (12-hydroxy-5,8,10-heptadecatrienoic acid) are the cyclooxygenase (COX) and the lipoxygenase pathways (54) . COX-1 (also referred to as PGH synthase) is constitutively expressed, whereas an inducible COX-2 is also expressed constitutively in certain regions of the brain, kidneys, and cancerous tissues. COX-2 activity, usually undetected in normal tissues, generates proinflammatory substances by the oxygenation of arachidonic acid to PGs (PGD2 and PGE2) (54) . In addition, COX-2 can catalyze the formation of chemotactic substances such as HHT and thromboxane A2 from PGH2 via the thromboxane synthetase (55) . This chemotactic activity can in turn lead to platelet aggregation. The leukocyte lipoxygenase is known to catalyze the initial reaction that leads to the formation of 5-HETE, a stable derivative of the peroxy form 5-HPETE (56) . These substances have high chemotactic activity and are potent inducers of histamine release from basophils. The PGs have been implicated in promoting cell proliferation, suppressing immune surveillance, and stimulating tumorigenesis (57) . Due to these deleterious effects of PGs and other inflammatory substances generated by COX and lipoxygenase pathways, identification of molecules with the potential to inhibit these pathways has been a major focus of biomedical research. The effects of stilbene (in particular, RSV) on COX and leukocyte lipoxygenase pathways have produced interesting results. RSV was found to inhibit the 5-lipoxegenase product 5-HETE and the COX products HHT and thromboxane B2 (58) . Based on these results, the researchers further established that this inhibitory activity was directly responsible for the anti-platelet aggregation induced by RSV. The COX and lipoxygenase inhibitory activities of RSV have also been reported to account for its protective effect against oxidative stress-induced death of human erythroleukemia K562 cells (59) . The mechanism of this death inhibitory activity of RSV involved inhibition of H2O2-induced increases in PGE2 (product of COX activity) and leukotriene B4 (product of lipoxygenase activity) concentrations. This inhibitory effect on COX and lipoxygenase activities has been proposed as a possible mechanism for the anti-tumor activity of RSV; however, our studies (in HL60 leukemia and T47D breast carcinoma cells) and recent data from other groups provide evidence that the anti-tumor activity may be due in part to its ability to trigger apoptotic death in tumor cells (16 , 17 , 60 61 62 63) . Nevertheless, given the role that inflammatory mediators play in the induction and promotion of carcinogenesis, it is plausible that these activities may both play a role in cancer chemoprevention.
The anti-inflammatory activity of RSV has been demonstrated in a rat model of carrageenan-induced paw edema (64) . RSV inhibited both acute and chronic phases of this inflammatory process, with an activity greater than that of indomethacin or phenylbutazone. This effect was attributed to the impairment of PG synthesis via selective inhibition of COX-1. Similarly, preincubation with RSV decreased arachidonic acid release and COX-2 induction in mouse peritoneal macrophages stimulated with tumor promoter PMA, ROI, or lipopolysaccharides (LPS) (65) . Gene transfer experiments using a reporter construct containing COX-2 luciferase confirmed that an RSV-mediated decrease in COX-2 activity was indeed due to its inhibitory effect on protein kinase C (PKC)-driven activation of COX-2 transcription (66) . A more detailed account of the effect of RSV on gene transcription and transcriptional factors will be presented in a later section.
The effect of RSV on macrophages and polymorphonuclear cells (PMN) has also been evaluated. These cells are the major players in the body’s response to immunogenic challenges, and a biological response modifier secreted from these cells could contribute to the development of disease states such as allergy and inflammation (67) . A classical model of macrophage activation is bacterial LPS. Under normal physiological settings this activation leads to a moderate increase in iNOS activity resulting in NO production that has bactericidal effects. However, abnormally high concentrations of NO and its derivatives peroxynitrite or nitrogen dioxide give rise to inflammation and have been shown to contribute to the process of carcinogenesis (68) . Exposure of RAW 264.7 macrophage cells to LPS resulted in the induction of iNOS and the resultant release of nitrite into the culture medium (20 , 65) . Preincubation of cells with RSV resulted in a dose-dependent inhibition of iNOS induction, and decreases in the steady-state levels of iNOS mRNA and protein. Contrarily, in cultured bovine pulmonary artery endothelial cells RSV treatment resulted in an increase in NO production through the induction of NOS activity (69) . In this model, the vasodilatory activity of NO has been proposed as a possible mechanism for the prevention of initiation of atherosclerosis. The effect of RSV on PMN-induced proinflammatory signals has also been investigated. In these series of experiments, PMN were stimulated by exposure to formyl methionyl leucyl phenylalanine (fMLP), the complement fragment C5a, the Ca2+ ionophore A23187, or a monoclonal antibody to the ß2-integrin Mac-1, and the effect on ROI production, neutrophil degranulation, and the cell surface expression of Mac-1 were assessed (19) . Enhanced ROI generation by PMN can result in membrane lipid peroxidation, endothelial damage, and increased vascular permeability. Degranulation of neutrophils can result in the release of enzymes such as elastase and ß-glucuronidase that have also been linked to endothelial damage and subendothelial smooth muscle proliferation (67 , 70) . A third major contributor to endothelial injury is the increase in cell surface expression of adhesion molecules of the ß-2 integrin family such as CD11a/CD18 (LFA1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150/95) (71) . RSV remarkably inhibited ROS production, release of elastase, and ß-glucuronidase from neutrophil granules and the cell surface expression of the ß2 integrin MAC-1 upon PMN stimulation. These results strongly indicate that RSV elicits inhibitory effect at all physiological phases of the inflammatory response, i.e., from the initial recruitment of PMN to their activation and the subsequent release of inflammatory mediators. As the inflammatory response is a critical common denominator in the development of many systemic disorders, such as atherosclerosis and carcinogenesis, the strong anti-inflammatory activity of RSV could have tremendous clinical implications.
RSV and transcription factors: regulation of gene expression
So far we have discussed the biological effects of RSV on a variety of critical biochemical pathways involved in normal cellular physiology. This includes a direct or indirect effect on gene expression, a process controlled by a class of proteins called transcription factors. The upstream signals to trigger nuclear localization (for those transcription factors that are normally extranuclear) and DNA binding of these transcription factors vary depending on the stimulus, cell type, and the intended response. One of the most striking biological activities of polyphenolics such as RSV is their remarkable anti-inflammatory potential (as described in the preceding section). Due to this association, there has been a lot of interest in investigating the effect(s) of RSV and its derivatives on transcription factors that regulate the expression of inflammatory mediators, such as tumor necrosis factor α (TNF α), interleukin 1 (IL-1), IL-6, and iNOS (15 , 20) . These include C/EBP, fos/jun, AP-1, and the Rel family of transcription factors—in particular, NF-κB.
The proinflammatory, carcinogenic, and growth modulating effects of many compounds are mediated by NF-κB (72 , 73) ; therefore, considering the anti-inflammatory and growth inhibitory properties of RSV, a significant amount of work is under way to elucidate the effect of RSV on NF-κB activity. NF-κB comprises two proteins of 50 kDa and 65 kDa and, in its unstimulated form, resides in the cytosol in a complex with an inhibitory subunit IκB. Activation-induced phosphorylation of IκB results in its degradation, allowing NF-κB to enter the nucleus and activate gene transcription (74) . The effect of RSV on the activation of NF-κB induced by a variety of inflammatory agents was investigated in myeloid (U937), lymphoid (Jurkat), and epithelial (HeLa) cell lines. Results indicated that the presence of RSV prevented the activation of NF-κB triggered by exposure of these cell types to TNF, PMA, H2O2, LPS, okadaic acid, and ceramide (15) . Inhibition of TNF-induced activation by RSV was shown not only by electrophoretic gel mobility shift assays, but also confirmed by specific antibodies against the p50 and p65 subunits (of NF-κB) and by the ability of the cold DNA probe containing NF-κB to completely inhibit the shift in gel mobility. The mechanism of RSV-induced inhibition of NF-κB activity is distinctly different from that reported with chemical modifiers of NF-κB subunits that prevent its DNA binding activity, such as herbimycin A and caffeic acid phenylethyl ester; RSV does not affect DNA binding activity of NF-κB or other transcription factors like SP-1 or Oct-1. However, the report provides evidence to support inhibition of TNF-induced phosphorylation and nuclear localization of the p65 subunit by RSV. Alternatively, RSV has been shown to inhibit the phosphorylation (inactivation) and degradation of IκBα (75) , which sequesters NF-κB in the cytoplasm, although results to the contrary have also been reported (76) . The ability of RSV to inhibit NF-κB activation triggered by a diverse class of stimuli that engage different intracellular signaling complexes suggests that RSV interferes with the activation of this critical transcription factor at a step common to many stimuli and signal transduction pathways. An attractive candidate could be the intracellular level of ROI, as ROI are induced by most of the stimuli mentioned in the study. Considering that NF-κB is a pleiotropic transcription factor involved in diverse pathways ranging from endotoxin-induced inflammation to cell proliferation and oncogenesis to signals that turn on the cells’ suicide program, molecules such as RSV that interfere with the activation of NF-κB are of particular interest to biologists and clinicians. However, it must be pointed out that there is some controversy vis a vis the inhibitory activity of RSV on NF-κB. For example, in a macrophage model of LPS-induced inflammatory response RSV had not effect on NF-κB activation (20) , and the authors contended that RSV had a more selective action on genes activated by LPS independent of NF-κB.
Most agents that activate NF-κB also activate another transcription factor AP-1 (activator protein 1) (77) . RSV has been shown to inhibit TNF-induced activation of AP-1 (15) . The activation of AP-1 is mediated by JNK (c-Jun N-terminal protein kinase) and the upstream kinase MEK (mitogen-activated protein kinase kinase or MAPKK) (78) . TNF-induced activities of JNK and MEK were inhibited by RSV, thus providing a possible mechanism for AP-1 inhibition (15) . Among the proteins induced upon activation of NF-κB and AP-1 are iNOS and COX-2 (79 , 80) , two enzymes inhibited by RSV. Thus, it is possible that RSV inhibits iNOS and COX-2 via its inhibitory effect on these transcription factors. It is also plausible that the expression of other genes regulated by NF-κB or AP-1, such as matrix metalloproteinase 9 (MMP-9) and cell surface adhesion molecules ICAM-1 and VCAM-1 (81 , 82) , which have been implicated in carcinogenesis, may also be muted upon exposure to RSV. Consistent with this, a recent report has demonstrated that RSV suppresses carcinogenesis in rats via down-regulation of MMP9 (83) .
The expression/function of the androgen receptor (AR), a transcription factor belonging to the nuclear steroid hormone receptor family, is also inhibited by RSV in prostate cancer cells (22) . This transcription factor is an essential mediator of androgen action; it controls the transcription of androgen-inducible genes such as PSA (prostate specific antigen) and is implicated in the development of prostate cancer (84) . Exposure of LNCaP (prostate cancer cell line) cells to RSV resulted in inhibition of growth of this androgen-responsive cell line via pathways that involved a decrease in the expression and function of the AR; the transcriptional activity of PSA was dramatically reduced upon RSV treatment of the prostate cancer cell line (18 , 85) . RSV has been shown to possess both estrogen agonist and antagonist activities (21 , 86) . Estrogens act via binding to the estrogen receptor (ER), another member of the nuclear receptor superfamily. This ligand/receptor binding brings about transcriptional activation of estrogen responsive target genes (57) . In studies using human breast carcinoma cells (MCF-7), exposure to RSV resulted in activation of transcription of genes (transfected in MCF-7 cells) responsive to estrogen (87) . This activity was dependent on the presence of the estrogen response element (ERE) sequence and the type of ER (higher transcriptional activity with ERß than ERα). However, the reported superagonist activity of RSV in the presence of estradiol was contradicted by reports demonstrating anti-estrogenic activity of RSV; RSV suppressed estradiol-induced progesterone receptor expression (21) . In another breast cancer cell line, T47D (estrogen dependent), a low concentration of RSV (10 µM) resulted in an increase in cell proliferation and growth (87) whereas at relatively higher concentrations the cells underwent apoptotic death (16) . These opposing actions of RSV have given rise to some controversy with respect to the use of RSV or similar compounds as therapeutic agents against ER+ breast cancer cells. The estrogenic activity of RSV has also been demonstrated in ER+ pituitary cells, which undergo a significant increase in prolactin secretion (88) , and in MC3T3-E1 osteoblastic cells that respond to RSV by increasing alkaline phosphatase and hydroxylase activity (14) , indicating estrogenic and bone loss preventive effect. Despite this in vitro evidence suggesting strong estrogenic activity of RSV, in vivo studies using rat models have so far failed to corroborate the in vitro data (89 90 91) .
In the first reported cancer chemopreventive activity in vivo, RSV was shown to inhibit tumor formation induced by the aryl hydrocarbon DMBA (7,12-dimethylbenzanthracene) (8) . The genotoxicity of aryl hydrocarbons is a function of their metabolic activation via binding to the aryl hydrocarbon receptor (AHR), a cytosolic protein that translocates to the nucleus upon ligand binding (92) . Once in the nucleus, AHR forms a heterodimer with the aryl hydrocarbon nuclear translocator, forming a transcription factor that initiates the transcription of a number of genes (93) . The best-characterized response to AHR activation is the increase in transcription of the CYP1A1 gene that encodes for the cytochrome P450 (CYP450) isozyme CYP1A1 (94) . CYP450 isozymes belong to a family of constitutive and inducible heme-containing enzymes involved in the metabolism of a wide variety of substances including carcinogens such as aromatic hydrocarbons and heterocyclic amines (95 , 96) . The metabolized active forms of carcinogens can subsequently interact with human DNA and cause mutations. Considering their carcinogenic potential, approaches aimed at directly inhibiting enzyme activity or upstream inhibition of the transcription of CYP450 genes have been proposed as probable strategies for cancer prevention (96) . The effect of RSV on AHR function and CYP1A1 transcription has been the focus of intense investigations. These studies provide ample evidence that RSV inhibits AH-induced CYP1A1 expression at the mRNA and protein levels (94 , 97 , 98) . In experiments done with DMBA, RSV inhibited the binding of the nuclear AHR to the xenobiotic response element of the CYP1A1 promoter without directly binding to the AHR (99) . Similar effects of RSV on other CYP450 isozymes, such as CYP1A2 and CYP3A4 have also been documented (100 , 101) . Considering that CYP450s are overexpressed in a variety of human tumors, the strong inhibitory effect of RSV and similar compounds could have tremendous implications for the prevention and treatment of cancer.
A discussion on transcription factors will not be complete without mentioning the tumor suppressor p53. p53 serves as the guardian of the genome by regulating the cell cycle (prevents progression through S phase) and activating the transcription of DNA repair enzymes such as GADD45, thereby preventing damaged DNA from being replicated (102) . In addition, p53 can activate the transcription of genes involved in the apoptotic pathway, thus ensuring deletion of damaged or unwanted cells (103) . Loss-of-function mutations of p53 are associated with an increased incidence of tumor formation (104) . The effect of RSV on intracellular p53 level and its transcriptional activity has been studied in cancerous and noncancerous cell lines. In one such study involving cultured bovine pulmonary artery endothelial cells, RSV was shown to induce accumulation of p53 with the resultant increase in transcription of the cyclin-dependent kinase (cdk) inhibitor p21Waf1/Cip1 and cell cycle arrest (69) . This prevents proliferation of endothelial cells, a factor that may explain the cardioprotective effect of red wine polyphenols. In a different model using mouse JB6, epidermal cell line RSV was shown to increase the transactivation of p53 activity by specifically activating phosphorylation at serine 15, mutation of which abrogates the apoptotic activity of p53 (105) . This was linked to an upstream activation of the MAP kinases, particularly ERKs (extracellular signal-regulated protein kinase) and p38 kinase. Similarly, gene knockout of p53 (p53-/-) in mouse fibroblasts resulted in complete resistance to RSV-induced death, further supporting the involvement of p53 in the biological activity of RSV (17) .
Regulation of cell proliferation and apoptosis: cancer chemopreventive activity
Since its reported cancer chemopreventive activity in a mouse model of carcinogenesis, there has been a flurry of papers reporting the effects of RSV on critical events that regulate cellular proliferation and growth. Biochemical pathways involved in differentiation, transformation, cell cycle regulation, and cell death induction have all been demonstrated as potential targets of RSV (57 , 101 , 106 , 107) . In the preceding sections describing the diverse signaling networks affected by RSV, I touched on some of the intricate pathways operational in carcinogenic transformation of cells. These include intracellular generation of ROI, activation of protein kinases, induction of enzymes that generate proinflammatory mediators such as COX and lipoxygenase, activation of transcription factors such as NF-κB, AP-1, CYP1A1, AHR, and p53, growth stimulation of estrogen responsive cells, etc. As remarkable as it seems, the regulatory/inhibitory effect of RSV on these signal transduction pathways has generated tremendous interest in its clinical chemopreventive and chemotherapeutic potential.
Depending on its concentration, RSV can either stimulate (as shown with ER+ breast cancer and pituitary cells) (14) or inhibit cell proliferation (16 , 107 , 108) . At the concentrations used in vitro, the effect generally is predominantly anti-proliferative as demonstrated in a variety of cancer cell lines (101) . The mechanism(s) for this growth inhibitory activity of RSV could be due to its ability to block ribonucleotide reductase, a complex enzyme that catalyzes the reduction of ribonucleotides into the corresponding deoxyribonucleotides (109) . Inhibitors of ribonucleotide reductases, such as gemcitabine (2′-difluoro-2′-deoxycytidine), have been in clinical use due to their inhibitory effect on DNA synthesis (110) . A second probable mechanism for the observed anti-proliferative activity of RSV could be its ability to inhibit DNA polymerase (111) or ornithine decarboxylase (112) , a key enzyme involved in polyamine synthesis that is increased in cancer growth. The antioxidant activity of RSV could be yet another mechanism for growth inhibition, as a slight pro-oxidant intracellular milieu, an invariable finding in cancer cells (48) , is a strong stimulus for proliferation. However, recent findings and our unpublished data (Y. Li and S. Pervaiz) provide evidence to suggest that RSV can both inhibit or stimulate intracellular ROI production depending on the cell type. These data may help to explain the conflicting results demonstrating pro- and anti-proliferative effects of RSV on mammalian cells.
Carcinogenesis is a process that involves dysregulated growth, an outcome of enhanced proliferation and resistance to apoptotic triggers. Regulation of growth and proliferation in untransformed cells is maintained via regulation of the cell cycle by the cell cycle checkpoint proteins: p53, Rb, p27, and the cdk inhibitor p21Waf1/Cip1. Any alteration in the normal functioning of these proteins allows the cells to undergo unabated cycling resulting in accumulation of DNA mutations, a prerequisite for carcinogenesis. A number of studies have now established that RSV inhibits cellular proliferation by inducing cell cycle arrest in the G1/S phase (101) . In CEM-C7H2 acute leukemia cells and MCF-7 breast cancer cells, exposure to RSV resulted in accumulation of cells in the S phase (107 , 113) . Exposure to RSV also resulted in a transient increase in the expression of G1/S regulators, such as cyclin D1, cdk4, and cyclin E, in MCF-7 cells (12) ; cyclins D1 and E are responsible for S phase entry. These cells ultimately undergo apoptotic death, unlike the breast cancer cell line MDA-MB-231, where cell cycle activation or up-regulation of p21, p53, or p27 did not occur and the mode of inhibition of cell proliferation was attributed to nonapoptotic death of the cell (12) . The compound was extremely effective against a highly invasive breast carcinoma cell line, MDA-MB-435 (114) . Similarly, RSV treatment has been shown to induce cell cycle arrest in human promyelocytic leukemia HL60 cells at the S/G2 phase transition and a subsequent increase in the number of cells in the G1/S phase, brought about by an increase in cyclins A and E and inactivation of cdc2 (13) . In another study using A431 epidermoid cancer cells, RSV decreased the levels of cyclins D1, D2, E, and cdk2 and cdk4/6 (60) . RSV has also been shown to induce changes in the cell cycle in prostate cancer LNCaP cells with a significant decrease in the levels of prostate specific antigen (PSA) (18) . Suppression of cell cycle progression through the S and G2 phase and a concomitant increase in the expression of p53 and p21Waf1/Cip1 have also been demonstrated in pulmonary epithelial cells upon exposure to RSV (69) . In contrast, RSV has been shown to stimulate proliferation and differentiation of MC3T3-E1 osteoblast cells as shown by an increase in DNA synthesis, alkaline phosphatase, and hydroxylase activity estrogenic effect (14) . Collectively, the effect of RSV on growth and cell cycle control proteins seems to vary between cell types.
Although some of the in vitro biological effects of RSV have not been corroborated in vivo, there is evidence to support the anti-proliferative and growth inhibitory activity in animal models of carcinogenesis. For instance, RSV-treated mice developed fewer tumors in response to DMBA and PMA (8) . Similar effects were observed in a rat tumor model (115) . Our group was the first to investigate the mechanism of the reported cancer chemopreventive activity of RSV. Using HL60 human leukemia and T47D breast carcinoma cells, we showed that the decrease in incidence of tumors in RSV-treated mice could be due to targeted killing of the tumor cells by RSV. Both cell lines exhibited classical hallmarks of apoptotic death (16 , 108) . Apoptosis is a programmed series of events triggered in response to death receptor ligation such as CD95/Apo1/Fas or TNF-r or exposure to genotoxic agents such as anti-cancer drugs or UV irradiation (116) . The commitment and execution phases of this death pathway depend on an intricate cross-talk between the caspase family of intracellular cystine proteases and amplification factors derived from the mitochondria (117 , 118) . In a classical death receptor model (CD95 ligation), early recruitment and activation of the initiator caspase-8 result in direct activation of downstream caspases, leading to the executioner caspase-3 activation and cellular disassembly, or can engage the mitochondrial death pathway to trigger the release of apoptogenic factors such as Cyt.C, apoptosis-inducing factor (AIF), and Smac/Diablo. Cyt.C can complex with cytosolic Apaf-1 (apoptosis protease-activating factor-1) and pro-caspase 9 in the presence of dATP to form the apoptosome (119) . Once assembled, this complex then activates caspase 9, which then engages caspase 3, resulting in DNA fragmentation and death. Caspase-8-dependent recruitment of the mitochondria is facilitated by the proapoptotic Bcl-2 family proteins Bax and Bid (120) , which can translocate to the mitochondria and trigger mitochondrial permeability transition by changing the conformation of the mitochondrial inner membrane pore (PT pore) (121) . Due to the critical role that mitochondria play in the execution of the apoptotic signal and the fact that the death inhibitory protein Bcl-2 was first isolated as a mitochondrial protein, they have become attractive candidates for the design of targeted therapies. Our initial findings showed that exposure of HL60 cells to RSV resulted in changes in the mitochondrial transmembrane potential and release of Cyt.C, suggesting a mitochondrial specific activity of RSV (16 , 108) . Since these observations, other reports have corroborated the mitochondrial-dependent apoptotic activity of RSV against a variety of tumor cell lines (122 123 124 125) . There are also reports indicating up-regulation of the proapoptotic protein Bax and down-regulation of Bcl-2 upon exposure to RSV (126) . However, a recent report (127) and our data (K. Ahmad and S. Pervaiz, unpublished results) seem to suggest that RSV-mediated apoptosis is independent of Bax, as gene knock out of Bax (Bax-/-) did not alter tumor cell sensitivity to RSV. We also showed that pretreatment of cells with relatively low doses of RSV can sensitize the cells to drug-induced apoptosis (108) , thereby suggesting a potential synergistic activity that could be of clinical relevance. However, our more recent findings indicate that RSV can facilitate or inhibit the death signal and that this effect is dependent on whether RSV elicits a pro- or antioxidant effect in the cell type. For example, exposure of HL60 and LNCaP cells to similar concentrations of RSV resulted in a distinctly different effect on the cellular redox state: RSV increase intracellular O2- in HL60 cells whereas a dose-dependent drop was observed in LNCaP cells (Y. Li and S. Pervaiz; unpublished results).
The involvement of p53 and p21 (as discussed earlier) has also been reported in the apoptotic response elicited by RSV. As p53 controls the transcription of a number of essential mediators of apoptosis such as CD95/Apo1/Fas, Bax, p21, etc., the p53 dependence of RSV-induced apoptosis is of particular importance. In our earlier report we showed that RSV-induced apoptosis in HL60 and T47D cells was mediated by up-regulation of CD95-CD95L interaction, thereby resulting in death of CD95 expressing cells (16) . A number of other apoptosis-inducing agents have been shown to sensitize tumor cells by enhancing the interaction between the CD95 receptor and its ligand (128) . However, CD95-independent death signaling has been demonstrated in other cell lines, too (113 , 129) . In a recent report, suppression of DMBA-induced mammary carcinogenesis by RSV in rats was linked to inhibition of COX-2 and MMP9 expression and the blocking of NF-κB activation (83) .
In contrast to its apoptosis-inducing activity, RSV has been shown to inhibit apoptosis in some systems. An earlier report indicated that RSV interfered with H2O2-induced apoptotic signal (59) . We have shown that H2O2 triggers apoptosis by decreasing intracellular O2- and cytosolic pH (130) , thus creating a permissive intracellular milieu for death execution (131) . A slight increase in intracellular O2- can inhibit receptor or drug-induced apoptosis via direct or indirect effect on caspase activation pathways (132 , 133) . Our recent data (K. Ahmad and S. Pervaiz; unpublished results) indicate that the inhibitory effect of RSV on H2O2-induced apoptosis is mediated by its pro-oxidant activity in HL60 cells (increase in intracellular O2-), which prevents a H2O2-mediated drop in intracellular O2- and cytosolic pH, creating a nonconducive environment for apoptotic execution.
The body of evidence presented here speaks volumes for the clinical potential of wine polyphenolics such as RSV and related compounds. Its relatively simple chemical structure enables RSV to interact with receptors and enzymes, giving rise to biological effects such as suppression of growth, induction of differentiation, inhibition of ROI production, cell cycle regulation, inhibition of lipid peroxidation, down-regulation of proinflammatory mediators, regulation of gene expression by affecting transcription factor activity, and up-regulation of death-inducing factors. These in vitro effects have been corroborated in some studies demonstrating the beneficial effects on cardiovascular, neurological, and hepatic systems; however, the most exciting in vivo data relate to its cancer chemopreventive and chemotherapeutic activity. By dint of diverse biological activity, RSV and related compounds have joined many other promising agents being investigated for their disease-preventive and therapeutic potential. Being a natural constituent of wine, fruits, and nuts and the fact that it has no untoward effects on normal cells or tissues, RSV is under preclinical scrutiny. The outcome of these studies will provide the definitive answer to the real clinical potential of this remarkable compound.
I would like to extend my sincere apologies to those whose invaluable contributions could have been inadvertently overlooked. I wish to acknowledge the National Medical Research Council and the Biomedical Research Council of Singapore and the Academic Research Fund, National University of Singapore for funding support.
Received for publication April 8, 2003. Accepted for publication June 26, 2003.
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