Cancer prevention – the potential for diet to modulate molecular signalling

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Cancer prevention – the potential for diet to modulate molecular signalling Margaret M. Manson Cancer Biomarkers and Prevention Group, Departments of Biochemistry and Oncology, Biocentre, University of Leicester, University Road, Leicester LE1 7RH, UK

As our understanding of the development of cancer and the complex signalling mechanisms involved improves, we are beginning to appreciate the enormous potential for intervention strategies that prevent or slow down the disease process. Although much research is currently aimed at developing drugs to target key molecules in tumour cells that are responsible for their proliferation and survival, dietary constituents also have potential as anti-cancer agents. Our goal should be not only to identify carcinogenic changes as early as possible and to intervene effectively long before lifethreatening tumours develop, but also to understand how a balanced, healthy diet can contribute to reduced incidence, as epidemiology so tantalizingly suggests. Hardly a week goes by without a news item about whether particular food items are good or bad for health. To date, the negative reports probably outweigh the positive, but there is a trend towards reporting the beneficial side of the story. For example, in 1997, a comprehensive international review [1] concluded that there was convincing evidence that consumption of fruit and vegetables decreases the risk of cancers of the mouth, pharynx, oesophagus, lung, stomach, colon and rectum. This report also concluded that eating fruit and vegetables probably also reduces the risk of cancers of the larynx, pancreas, breast and bladder. As a consequence, it was suggested that the adoption of ‘recommended diets together with maintenance of physical activity and appropriate body mass, could in time reduce cancer incidence by 30 – 40%’. Hence, the dietary advice to individuals was to ‘choose predominantly plant-based diets rich in a variety of vegetables and fruit, pulses (legumes) and minimally processed starchy staple foods.’ The realization that diet might have such a dramatic effect on health has come from a combination of epidemiology and experimental research. The examination of variations in cancer incidence between different countries, and in successive generations of people who migrated from one part of the world to another, has indicated that cancer is largely environmentally determined, with diet being a major variable. Over several decades, many studies using animal models of chemical carcinogenesis have shown that a wide range of dietary constituents have cancer chemopreventive potential [2] and, in the past few years, a number of reports have elucidated the cellular Corresponding author: Margaret M. Manson ([email protected]).

mechanisms by which prevention or suppression of cancer might occur. Edible plant matter, such as fruit, vegetables, cereals and pulses, contains many microconstituents that are now recognized as being biologically active (Table 1), in addition to vitamins and minerals. However, assessing the true impact of such constituents on human health is difficult. For example, in many cases the exact composition of foods and the bioavailability of active molecules is not known. Furthermore, the content of beneficial molecules varies with genetic strain, growth and storage conditions of the plant, and its preparation as food, making it difficult to predict intake accurately. Clinical trials that attempt to correlate food intake with prevention of a particular cancer are difficult to perform, owing to the cost, the length of time required, and the number of participants that would be necessary to assess effect with the appropriate statistical significance. Thus, most direct evidence of a beneficial effect of particular food items, or individual agents derived from them, has come from animal models. However, as in vitro studies begin to identify the molecular targets within cancer cells that are modulated by dietary constituents, smaller clinical trials, in which food intake is correlated with effects on appropriate biomarkers, become more feasible. The potential for identifying such biomarkers by understanding the molecular interactions of food components with cellular signalling pathways is the subject of this review. Intervention in the transformation process The process by which a normal, healthy cell becomes a tumour involves multiple steps over an extended period of time, as described for colon cancer by Fearon and Vogelstein [3]. To achieve full malignancy, cells must acquire certain transforming characteristics [4], including (1) self-sufficiency in growth signalling and limitless replicative potential, (2) becoming unresponsive to antiproliferative signals, (3) evading apoptosis, (4) inducing and sustaining angiogenesis, and (5) acquiring the ability to invade and metastasize. This sequence of events presents many opportunities for intervention, with the aim of preventing, slowing down or reversing the tranformation process (Fig. 1). Ideally, chemoprevention would halt the carcinogenic process at an early stage, perhaps even preventing the formation of preneoplastic lesions. In reality, individuals are continuously exposed to DNA-damaging, cancer-initiating events and, hence, any

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Table 1. Examples of bioactive dietary compounds

a

Food source

Class of compound

Chemical

Cruciferous vegetables

Isothiocyanate

Cruciferous vegetables Cruciferous vegetables Onions, garlic, scallions, chives

Dithiolthione Glycosinolate Allium compound

Citrus fruit (peel) Citrus fruit Berries, tomatoes, potatoes, broad beans, broccoli, squash, onions Radish, horse radish, kale, endive Tea, chocolate

Terpenoid Flavonoid Flavonoid

Benzyl isothiocyanate, phenethyl isothiocyanate, sulforaphane Oltipraza Indole-3-carbinol, 3,30 -diindoylmethane, indole-3-acetonitrile Diallyl sulphide Allylmethyl trisulphide D -Limonene, perillyl alcohol, geraniol, menthol, carvone Tangeretin, nobiletin, rutin Quercetin

Flavonoid Polyphenol

Grapes Turmeric Strawberries, raspberries, blackberries, walnuts, pecans Cereals, pulses (millet, sorghum, soya beans) Orange vegetables and fruit Tomatoes Tea, coffee, cola, cacao (cocoa and chocolate)

Polyphenol Polyphenol Polyphenol Isoflavone Carotenoid Carotenoid Methylxanthines

Kaempferol Epigallocatechin gallate, epigallocatechin, epicatechin, catechin Resveratrol Curcumin Caffeic acid, ferulic acid, ellagic acid Genistein a- and b-carotene Lycopene Caffeine, theophylline, theobromine

Oltipraz is not a dietary agent, but is the most commonly used chemopreventive molecule in this class.

means of delaying the progression to full life-threatening malignancy is welcome. In vitro studies elucidating the mechanisms of action of putative chemopreventive agents suggest that they have the potential to modify many of the acquired characteristics,

Normal cell Blocking agents • ROS scavenging • Alter carcinogen metabolism Initiated cell Blocking agents • Prevent further DNA damage Suppressing agents • Induce apoptosis Preneoplasia Blocking agents • Prevent further DNA damage Suppressing agents • Induce cell-cycle arrest • Induce apoptosis • Inhibit angiogenesis Tumour Blocking agents • Prevent futher DNA damage Suppressing agents • Inhibit angiogenesis • Inhibit invasion Metastasis TRENDS in Molecular Medicine

Fig. 1. Potential for intervention in carcinogenesis. Chemopreventive agents can exert blocking or suppressing effects on different stages of the carcinogenic process. Blocking mechanisms prevent damage to DNA, whereas suppression slows down or inhibits the growth of transformed cells or new blood vessels. Abbreviation: ROS, reactive oxygen species. http://tmm.trends.com

described by Hanahan and Weinberg [4], that are required for tumour promotion and progression. In addition, many of these agents have the ability to block the initiation of carcinogenesis [2]. Previously, blocking mechanisms (which prevent damage to DNA) and suppression mechanisms (which slow down or inhibit the growth of transformed cells or new blood vessels) were considered separately, and most research concentrated on ways of blocking carcinogenesis. However, recent studies indicate that many of the same signalling pathways might be involved at different stages of prevention. The effects of particular dietary agents are likely to be dependent on tissue or cell type, could differ at high and low doses, and might not all be physiologically relevant. However, several potential mechanisms by which they might mediate their effects are frequently described: (1) altered signalling through the mitogen-activated protein kinases (MAPKs) [extracellular-signal-regulated kinase (ERK), JUN N-terminal kinase (JNK) and p38] (Fig. 2), or phosphoinositide-3-kinase (PI3K) and protein kinase B (PKB) (Fig. 3); (2) modified activity of transcription factors, such as activator protein-1 (AP-1) (Fig. 2) and nuclear factor kB (NF-kB) (Fig. 3); and (3) modulation of key molecules involved in control of the cell cycle [cyclindependent kinases (CDKs) and their inhibitors] (Fig. 4) or apoptosis (members of the Bcl-2 family) (Fig. 3). Blocking initiation or other DNA-damaging events in carcinogenesis The most effective way of preventing cancer is to block its initiation by preventing the DNA-damage that results from reactive oxygen species (ROS) or carcinogens. For example, ROS can be directly scavenged, and the metabolism of procarcinogenic molecules can be altered so that either they are not converted to carcinogenic species by phase-I drug-metabolizing enzymes (particularly cytochrome P450), or they are safely removed from the cell by a secondary line of defence that involves phaseII conjugating enzymes [e.g. glutathione-S-transferases (GST), glucuronidases and sulphotransferases]. Many

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Stimulus

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Growth factors, mitogens, ROS, phase-II-enzyme inducers

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Stress (ROS), inflammatory cytokines, anisomycin, phase-II-enzyme inducers

RAS

GFR

RAF

MLKs, TAK, ASK1

MEKK1/4, MLKs, ASK1, TAK

MAPKK

MEK1/2

MEK3/6

MEK4/7

MAPK

ERK1/2

p38α/β/γ

JNK1/2/3

Elk-1, SAP-1, MNK1/2, FOS, MSK1, Nrf2

ATF-2, Elk-1, MSK1, SAP-1, MAPKAPK2/3

JUN, ATF-2, Elk-1, Nrf2

MAPKKK

Target genes

Biological response

Modulation of AP-1, growth, development, differentiation, induction of phase-II enzymes

Modulation of AP-1, apoptosis, inflammation, differentiation, inhibition or induction of phase-II enzymes TRENDS in Molecular Medicine

Fig. 2. Mitogen-activated protein kinase (MAPK) cascades. Signalling through the ubiquitously expressed MAPK cascades leads to altered activity of target genes, including several transcription factors, and a range of biological responses. In tumour cells, key molecules, such as growth factor receptor (GFR), RAS, extracellular-signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK) are often upregulated leading to constitutive activation of the pathways in which they are involved. Depending on the circumstances, dietary compounds such as curcumin, epigallocatechin gallate (EGCG), resveratrol and indole-3-carbinol (I3C) can up- or down-regulate signalling to induce stress-response genes, or growth arrest and apoptosis. Abbreviations: ASK1, apoptosis-signal-regulating kinase 1; ATF, activating transcription factor; MAPKAPK, MAPK activated protein kinase; MEK, MAPK/ERK kinase; MLK, mixed-lineage kinase; MNK, MAPK-interacting kinase; MSK, mitogen- and stress-activated kinase; Nrf, nuclear-factor-E2-related factor; ROS, reactive oxygen species; SAP, stress-activated protein; TAK, transforming-growth-factor-b-activated kinase.

dietary agents possess antioxidant properties and/or the ability to induce the protective enzymes described above [5,6]. Carcinogenesis is a multistep process requiring several genetic modifications and, hence, blocking mechanisms could also be effective in slowing down the later stages of cancer progression (Fig. 1) and in preventing the development of second primary tumours following removal of the initial cancer. ROS modify the redox status of the cell and, therefore, the cysteine residues of proteins. This affects protein conformation and function, and transcription-factor– DNA binding, resulting in altered gene expression. The generation of ROS is inevitable in aerobic organisms, and cells maintain their redox potential with low-molecular-weight antioxidants, such as glutathione, and with enzymes, like catalase and superoxide dismutase. When the balance is perturbed by changes in the environment, cells mount an oxidative-stress response, involving altered expression of the genes that code for regulatory transcription factors, drug-metabolizing and antioxidant enzymes, and structural proteins, through modulation of signal-transduction cascades [7]. Paradoxically, some dietary compounds, such as ascorbic acid in fruits, epigallocatechin gallate (EGCG) http://tmm.trends.com

in green tea and curcumin in turmeric, act as both oxidants and antioxidants [8]. An antioxidant response element (ARE) (50 -A/G TGA C/T NNNGC A/G-30 ), first characterized by Pickett and coworkers [9], is found in the promoter regions of several drug-metabolizing enzymes, as well as of antioxidant enzymes [5,6] (Fig. 5). Induction through the ARE occurs in response to a variety of chemopreventive chemicals that can generate pro-oxidant conditions within the cell. There is accumulating evidence suggesting that the ARE responds, both positively and negatively, to stress-induced signalling via MAPKs or PI3K, and binds transcription factors of the basic-region-leucine-zipper (bZIP) family, in particular nuclear-factor-E2-related factor 2 (Nrf2) (Fig. 5) and the small Maf proteins [6,10]. Mice in which the Nrf2 gene is disrupted are deficient in the induction of GST, g-glutamylcysteinyl synthetase and NAD(P)H:quinone oxidoreductase by phenolic antioxidants, dithiolthiones or isothiocyanates [11,12]. In its inactive state, Nrf2 is sequestered in the cytoplasm by a protein known as Kelchlike ECH-associated protein 1 (Keap-1) (reviewed in [6]). Although several signalling pathways have been implicated in the activation of the ARE, it is still unclear exactly

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TNFα, IL-1β, ROS

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Insulin, growth factors, ROS

p110 PI3K p85

RAS

NIK

PIP2 PTEN PIP3

IKK NF-κB

Nrf2

IκB

PDK-1 (and -2?) PKB

NF-κB

+

IκB

P Bad Caspase 9 Bcl-2, Bcl-XL

Cyclin D1, MYC, IκBα, MMPs

FKHR

GSK3β

COX2, IAPs

p53

BRCA1

Nuclear p21, p27

DNA repair

Bax

Apoptosis

Cell-cycle arrest TRENDS in Molecular Medicine

Fig. 3. Cell-survival signalling through nuclear factor kB (NF-kB), or phosphoinositide-3-kinase (PI3K) and protein kinase B (PKB). Upon activation by tumour necrosis factor (TNF) or other stimuli, the inhibitor of NF-kB (IkB) is phosphorylated, releasing NF-kB. NF-kB is then translocated to the nucleus where it upregulates many genes, including some that are involved in cell proliferation or inhibition of apoptosis. Similarly, stimulation of PI3K, followed by phosphorylation of phosphatidylinositol bisphosphate (PIP2) to the trisphosphate (PIP3) and activation of PKB, leads to the phosphorylation and inactivation of many genes that would otherwise induce apoptosis. The tumoursuppressor PTEN is a negative regulator of this pathway. In tumour cells, proteins such as RAS, IkB kinase (IKK), NF-kB, PI3K and PKB are frequently overexpressed, whereas PTEN is often absent. Different chemopreventive agents can suppress the activity of IKK, PI3K and/or PKB, inhibit the translocation of NF-kB to the nucleus, or upregulate PTEN. Abbreviations: Bcl, B-cell leukaemia; BRCA, breast-cancer-susceptibility gene; COX, cyclo-oxygenase; FKHR, Forkhead in rhabdomyosarcoma; GSK, glycogen-synthase kinase; IAP, inhibitor of apoptosis; IkB, inhibitor of NF-kB; IL-1b, interleukin-1b; MMP, matrix metalloproteinase; NIK, NF-kB-inducing kinase; Nrf2, nuclearfactor-E2-related factor; PDK, phosphoinositide-dependent kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; ROS, reactive oxygen species.

Inhibitory stimuli

p15ink

p16ink

P

P P P

Rb

P P

CDK4/6 Cyclin D

Proliferative stimuli

Rb

E2F DP-1

Transcription inhibited

Cyclin E CDK2 DP-1

p21cip

E2F

Activation of S-phase genes

p27kip Inhibitory stimuli TRENDS in Molecular Medicine

Fig. 4. Cell-cycle arrest in G1. Hypophosphorylated retinoblastoma (Rb) protein suppresses transcription of the genes necessary for entry into S phase by associating with the transcription factors, DP-1 and E2F. However, in the presence of proliferative stimuli, such as growth factors, complexes of cyclin and cyclin-dependent kinases (CDKs) phosphorylate Rb. This causes dissociation of Rb from the transcription-factor complex, allowing transcription to occur. Factors that inhibit the G1 –S transition include p53 (which upregulates p21), transforming growth factor b (TGFb) (which upregulates p15, p16, p21 and p27), contact inhibition (which induces p21 and p27) and growth-factor withdrawal [which activates glycogen-synthase kinase 3b (GSK3b) leading to degradation of cyclin D1]. In tumour cells, cyclins are often overexpressed, p21 and p27 are sometimes excluded from the nucleus rendering them inactive, or Rb can be lost. Chemopreventive agents, such as indole-3-carbinol and epigallocatechin gallate, can downregulate the expression or activity of CDKs and cyclins, and upregulate CDK inhibitors such as p21 and p27, thereby causing cell-cycle arrest in G1. http://tmm.trends.com

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Chemopreventive chemicals

ERK, JNK, p38, PKC, PI3K Nrf2 Keap1

P

Nrf2 ARE

bZip Enzyme induction

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process that often progresses slowly in the early stages and, hence, there is great potential for arresting or decelerating its development. Because tumours result from an imbalance between proliferative and apoptotic processes, any mechanism that halts or slows down inappropriate cell division, or that induces damaged cells to undergo apoptosis, is potentially useful [21– 25]. Thus, effective chemopreventive agents might target molecules that regulate the cell cycle, apoptosis or cellular senescence. A key feature would be the ability to compromise tumour cells but not normal cells. At later stages of tumourigenesis, a means of inhibiting angiogenesis (the development of new blood vessels required to support tumour growth) or preventing transformed cells escaping from their original location to invade surrounding tissue, would also be beneficial; long-term survival is more likely if the cancer remains localized.

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Fig. 5. Induction of stress-response genes by chemopreventive chemicals. Signalling through the mitogen-activated-protein-kinase pathways [extracellular-signalregulated kinase (ERK), JUN N-terminal kinase (JNK) and p38], protein kinase C (PKC) or phosphoinositide-3-kinase (PI3K) leads phosphorylation of transcription factors of the basic-leucine-zipper (bZIP) family, particularly nuclear-factor-E2related factor (Nrf2). Phosphorylation releases Nrf2, which is normally sequestered in the cytoplasm by a protein known as Kelch-like ECH-associated protein 1 (Keap1). Nrf2 stimulates the expression of stress-response genes that have a promoter containing one or more antioxidant response elements (AREs).

how Nrf2 is released from the cytoplasm to the nucleus. Protein-kinase-C-mediated phosphorylation of Nrf2 in HepG2 cells by phorbol 12-myristate 13-acetate (PMA), tert-butylhydroquinone and b-naphthoflavone has been proposed to be crucial for nuclear translocation of Nrf2 [13]. In the same cell type, activation of the ERK or JNK, but not the p38, MAPK pathways stimulates ARE transcriptional activation [14,15]. Interestingly, p38 was shown to negatively regulate this response element in hepatoma cells [16]. However, in neuroblastoma cells, it has been suggested that signalling through PI3K (independently of PKB) is responsible for activation of the ARE by Nrf2 [17]. In HepG2 cells, overexpression of MAPK/ERK-kinase kinase 1 (MEKK1), transforming-growth-factor-b-activated kinase (TAK) or apoptosis-signal-regulating kinase 1 (ASK1), which all signal through JNK, leads to Nrf2dependent activation of the ARE [15]. The kinase activity of ASK1 can be inhibited by binding of the redox regulatory protein, thioredoxin [18]. ROS relieve this inhibition by inducing dimerization of thioredoxin, an effect that can be abolished by free-radical scavengers [19]. To date, most studies of gene regulation via the ARE have used synthetic chemicals as inducers, but several dietary agents, such as EGCG and sulforaphane, have been proposed to have a similar capability [20]. It is likely that many other dietary agents that induce stress-response genes, including phaseII drug-metabolizing enzymes, do so via these signalling cascades. However, some, such as indole-3-carbinol (I3C), might work preferentially through a different response element, known as the xenobiotic response element [6]. Suppressing transformation It is unlikely that it will ever be possible to prevent all initiating events. However, carcinogenesis is a multistep http://tmm.trends.com

Pathways involved in proliferation and survival Signalling through MAPKs, PI3K and PKB, AP-1 and NF-kB favours cell proliferation and survival. Several key molecules in these pathways (e.g. growth-factor receptors, RAS, PI3K, PKB and NF-kB) are overexpressed or constitutively upregulated in many types of cancer, suggesting that their inhibition or downregulation might induce tumour cells to undergo cell-cycle arrest or apoptosis. Many dietary compounds, when tested in vitro, have proved effective in suppressing tumourigenic signalling. For example, curcumin has been shown to inhibit the phosphorylation of epidermal-growth-factor (EGF)-induced EGF receptor (EGFR) and ERK [26], anisomycin-induced activation of JNK [26,27], basal phosphorylation of PKB [26], and the activity of transcription factors AP-1 and NF-kB [28– 30]. In response to various stimuli, including interleukin-1b and tumour necrosis factor a (TNFa), curcumin prevents phosphorylation and degradation of the inhibitor of NF-kB (IkB) by inhibiting the pathway at or above the level of the IkB kinases (IKKs) (Fig. 3). This results in reduced translocation of NF-kB to the nucleus [31,32]. EGCG can inhibit or activate signalling through the EGFR family and ERK depending on circumstances [33,34]. For example, in vascular smooth-muscle cells it inhibits the activation of ERKs (and the induction of FOS expression) that is mediated by the platelet-derived growth factor and HER2 (also known as Neu) receptors, but not by EGFR [35]. Furthermore, in various cell types, EGCG leads to inhibition of the phosphorylation and activation of PI3K [35,36], and inhibition of AP-1 activity and NF-kB– DNA binding [37 – 39]. However, in rat hepatoma cells, EGCG acts as a pro-oxidant and mimics many of the effects of insulin, including activation of PI3K and repression of glucose production, possibly by regulating protein tyrosine phosphatases through modulation of the redox status of the cell [8]. I3C inhibits signalling through PKB and the binding of NF-kB to DNA [40]. Sulforaphane also inhibits NF-kB – DNA binding, without affecting its translocation to the nucleus or the phosphorylation of IkB [41]. Caffeine and theophylline inhibit the lipid-kinase activity of PI3K and, hence, the phosphorylation of PKB, in response to insulin

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(Fig. 3) [42]. Resveratrol, a constituent of grape skins and of red wine, blocks NF-kB activation by several stimuli, and inhibits the activities of MAPK/ERK kinase (MEK) and JNK and the binding of AP-1 to DNA [43]. Between them, the transcription factors AP-1 and NF-kB contribute directly or indirectly to the regulation of many genes, including those encoding cyclin D1, MYC, cyclooxygenase 2, p53, p21, Bcl-XL, inhibitors of apoptosis (IAPs), matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) (Fig. 3). These are involved in proliferation, transformation, invasion and death [44 – 46] and, hence, the ability of chemopreventive agents to modify their activities could have far-reaching consequences. Cell-cycle arrest and apoptosis In many different cell types, a range of dietary constituents can induce cell-cycle arrest and/or apoptosis. Studies have identified key molecules, the expression of which might be altered to contribute to these effects. Furthermore, tumour cells seem to be more sensitive to these influences than normal cells. I3C was reported to induce G0 – G1 cell-cycle arrest in breast cells through downregulation of CDK6, which results in hypophosphorylation of the retinoblastoma protein (Rb) and prevention of the transcription of genes crucial for S-phase transition (Fig. 4). Upregulation of the CDK inhibitors p21 and p27 was also observed. I3C prevents binding of the transcription factor Sp1 to the promoter region of CDK6 [47] and, interestingly, EGCG has also been reported to disrupt Sp1 binding, in this case to the promoter region of the gene encoding the androgen receptor in prostate cells [48]. 3,30 -diindolylmethane (DIM), an acid condensation product of I3C, also causes G1 arrest in breast cells and reduces CDK2-mediated phosphorylation of Rb. However, in this instance, an increase in the expression of p21 was attributed to increased binding of Sp1 to the promoter of this gene [49], suggesting that I3C and DIM exert their transcriptional regulatory effects through distinct gene-specific mechanisms. In breast and prostate cell lines, I3C and DIM can also affect the ratio and cellular localization of the antiapoptotic proteins Bcl-2 and Bcl-XL and the pro-apoptotic protein Bax, producing conditions that favour apoptosis [50 – 52]. EGCG can induce arrest in the G1 phase of the cell cycle and has been reported to modulate expression of CDKs 2 and 4, as well as of the CDK inhibitors p21 and p27 [34,53]. Curcumin treatment, which results in G2 – M arrest in several cell types, causes a reduction in the expression of p53, p21, egr-1, MYC and Bcl-XL and an upregulation of Bax [54,55]. Treatment of A431 cells with resveratrol leads to G1 arrest and apoptosis, preceded by increased expression of p21 and decreased expression of cyclins D1, D2 and E and CDKs 2, 4 and 6 [56]. In vivo, resveratrol treatment of rats enhances Bax expression in aberrant crypt foci, with dowregulation of p21 in normal mucosa [57]. http://tmm.trends.com

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Angiogenesis and invasion Approximately thirty years ago, Judah Folkman proposed that because tumour growth and metastasis are dependent on angiogenesis, blocking this process could be a strategy for arresting tumour growth. Cells produce a range of pro- and anti-angiogenic factors, which counteract each other. However, in response to various stimuli, such as activation of oncogenes or inactivation of tumoursuppressor genes, the pro-angiogenic switch is thrown [58]. Although this stage is later than is desirable for chemoprevention, there is still potential for dietary agents to adjust the balance and switch off angiogenesis. For example, anti-angiogenic effects of EGCG and curcumin through downregulation of VEGF, basic fibroblast growth factor (bFGF) and MMPs have been reported [59,60]. It is not yet known whether this is entirely due to decreased AP-1 and NF-kB activities, or whether other signalling pathways are involved. Conclusions In this review, only some of the more commonly observed effects of a few dietary chemopreventative agents have been discussed in any detail. However, these examples illustrate the enormous potential of these compounds in the modulation of the carcinogenic process. These agents have been described as ‘dirty’ in comparison with chemotherapeutic drugs designed to interact with a specific target site. However, because there is a high degree of redundancy in many cellular pathways, the targeting of a single molecule might ultimately have little or no effect, resulting in the need for combination therapy. Thus, the pleiotropic effects of dietary chemopreventive agents might, in fact, contribute to their efficacy and might result from their interaction with something as fundamental as the redox potential within the cell. Most agents are non-toxic in vivo, even at high doses, and their mode of action can be viewed as returning normal regulation to the cell (which might mean elimination in the case of a tumour cell), rather than eliciting a more general cytotoxic effect. Clearly the response of a cell is determined by its environment at the time of exposure, with the net outcome being the result of integration from the whole signalling network. The promoter regions of genes contain response elements for multiple transcription factors, which allows both positive and negative regulation. Thus, induction of gene expression will involve the simultaneous and parallel activation of multiple kinases, the phosphorylation of more than one transcription factor and the delivery of positive signals to the gene through several response elements. Removal of negative signals, including dephosphorylation, could also be required. Sometimes, the same compound has been shown to exert opposing effects at high and low doses or in different cell types. For example, EGCG can be pro- or anti-oxidant, and can activate or inhibit phosphorylation of ERKs. Furthermore, Kong et al. [20] have suggested that compounds that induce a battery of protective genes through the ARE at low concentrations can induce apoptosis at higher concentrations. Quantitative regulation of transcription allows extracellular factors to elicit

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different responses at different concentrations. Although it is unlikely that different concentrations produce completely distinct signalling responses, the strength and duration of signals could be affected [61]. For example, FOS has been described as a sensor for ERK signal duration because FOS is unstable when activation is transient, whereas sustained signalling leads to phosphorylation and stabilization of FOS. Thus, signal duration can alter the functional activity of the immediate early gene product, which in turn controls biological outcome [62]. The previously sharp distinction between blocking and suppressing mechanisms of cancer chemopreventive action is now blurred, owing to recent data suggesting that many effects of these agents are related to their ability alter the redox state of the cell, leading to up- or downregulation of key signalling pathways. These modify the regulation of immediate early genes, such as JUN, FOS and Nrf2, as well as the transcription factor NF-kB. Significant progress has recently been made in our understanding of how dietary agents can affect cell biochemistry. The challenge for the future is to harness this knowledge for effective chemoprevention, not only of cancer, but of cardiovascular and other chronic diseases. This will require extrapolation of in vitro monoculture data to more complex multicellular models, to determine the true physiological effects of these agents and the most informative biomarkers of their clinical efficacy. References 1 World Cancer Research Fund/American Institute for Cancer Research, (1997) Food, Nutrition and the Prevention of Cancer: A Global Perspective, American Institute for Cancer Research, Washington DC, USA 2 Wattenberg, L.W. (1985) Chemoprevention of cancer. Cancer Res. 45, 1–8 3 Fearon, E.R. and Vogelstein, B. (1990) A genetic model for colorectal carcinogenesis. Cell 61, 759– 767 4 Hanahan, D. and Weinberg, R.A. (2000) The hallmarks of cancer. Cell 100, 57 – 70 5 Hayes, J.D. et al. (1999) Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress. Biochem. Soc. Symp. 64, 141 – 168 6 Hayes, J.D. and McMahon, M. (2001) Molecular basis for the contribution of the antioxidant responsive element to cancer chemoprevention. Cancer Lett. 174, 103– 113 7 Dalton, T.P. et al. (1999) Regulation of gene expression by reactive oxygen. Annu. Rev. Pharmacol. Toxicol. 39, 67 – 101 8 Waltner-Law, M.E. et al. (2002) Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J. Biol. Chem. 277, 34933 – 34940 9 Rushmore, T.H. et al. (1991) The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. J. Biol. Chem. 266, 11632 – 11639 10 Itoh, K. et al. (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun. 236, 313 – 322 11 McMahon, M. et al. (2001) The cap ‘n’ collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res. 61, 3299– 3307 12 Kwak, M.-K. et al. (2002) Enhanced expression of the transcription factor Nrf2 by cancer chemopreventive agents: role of antioxidant http://tmm.trends.com

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