Modulation of signal transduction by tea catechins and related phytochemicals

Mutation Research 591 (2005) 147–160

Review

Modulation of signal transduction by tea catechins and related phytochemicals Masahito Shimizu, I. Bernard Weinstein ∗ Herbert Irving Comprehensive Cancer Center and Department of Medicine, Columbia University Medical Center, HHSC-1509, 701 West 168 Street, NY 10032-2704, USA Received 10 January 2005; received in revised form 1 April 2005; accepted 12 April 2005 Available online 29 June 2005

Abstract Epidemiologic studies in human populations and experimental studies in rodents provide evidence that green tea and its constituents can inhibit both the development and growth of tumors at a variety of tissue sites. In addition, EGCG, a major biologically active component of green tea, inhibits growth and induces apoptosis in a variety of cancer cell lines. The purpose of this paper is to review evidence that these effects are mediated, at least in part, through inhibition of the activity of specific receptor tyrosine kinases (RTKs) and related downstream pathways of signal transduction. We also review evidence indicating that the antitumor effects of the related polyphenolic phytochemicals resveratrol, genistein, curcumin, and capsaicin are exerted via similar mechanisms. Some of these agents (EGCG, genistein, and curcumin) appear to directly target specific RTKs, and all of these compounds cause inhibition of the activity of the transcription factors AP-1 and NF-␬B, thus inhibiting cell proliferation and enhancing apoptosis. Critical areas of future investigation include: (1) identification of the direct molecular target(s) of EGCG and related polyphenolic compounds in cells; (2) the in vivo metabolism and bioavailability of these compounds; (3) the ancillary effects of these compounds on tumor–stromal interactions; (4) the development of synergistic combinations with other antitumor agents to enhance efficacy in cancer prevention and therapy, and also minimize potential toxicities. © 2005 Elsevier B.V. All rights reserved. Keywords: Phytochemicals; EGCG; Cell signaling pathway; RTK

Abbreviations: EGFR, epidermal growth factor receptor; RTK, receptor tyrosine kinase; EGCG, (−)-epigallocatechin-3-gallate; EGC, (−)-epigallocatechin; ECG, (−)-epicatechin-gallate; EC, (−)-epicatechin; TGF␣, transforming growth factor ␣; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; HRG, heregulin; VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; Grb2, growth factor receptor bound protein 2; Shc, src homology; Sos, son of sevenless; JNK, c-Jun N-terminal kinase; PLC␥, phospholipase C ␥; PKC, protein kinase C; IP3 , inositol triphosphate; DAG, diacylglycerol; PI3K, phosphatidylinositol 3-kinase; AP-1, activator protein-1; TRE, TPA responsive element; NF-␬B, nuclear factor-␬B; IKK, I␬B kinase; NIK, NF-␬B inducing kinase; FKHR, forkhead transcription factor; TPA, 12-O-tetradecanoylphobal 13acetate; UV, ultraviolet; COX-2, cyclooxygenase-2; MMP, matrix metalloproteinase; HNSCC, head and neck squamous cell carcinoma; TRAMP, transgenic adenocarcinoma of mouse prostate; IGF-1, insulin-like growth factor-1; CDK, cyclin dependent kinase ∗ Corresponding author. Tel.: +212 305 6921; fax: +212 305 6889. E-mail address: [email protected] (I.B. Weinstein). 0027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2005.04.010

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Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell signaling pathways and transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Membrane-associated RTKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ras/MAPK pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. PI3/Akt pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. The transcription factors AP-1 and NF-␬B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochemicals and cell signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. EGCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Resveratrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Genistein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Curcumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Epidemiologic studies provide rather consistent evidence that a diet that has a high content of fruits and vegetables reduces the risk for several types of cancer [1]. However, the specific components of these foodstuffs that exert this protective effect and the precise mechanisms by which they exert these effects are not known with certainty. As the same time, a number of specific phytochemicals (plant-derived chemicals) have been identified that demonstrate antitumor effects in various experimental systems. Several mechanisms have been implicated for specific compounds including: (1) antioxidant activity and/or trapping of oxygen radicals and other highly reactive compounds (i.e., vitamins A, E, C, selenium, and various thiol compounds); (2) induction of drug metabolizing and detoxifying enzymes isothiocyanates, etc. [2,3]. Recent studies have emphasized a third mechanism, namely the remarkable ability of several phytochemicals to modify the activities of various receptor tyrosine kinases (RTKs) and specific pathways of signal transduction, thereby altering the expression of genes involved in cell proliferation, angiogenesis, and apoptosis [4–13]. The purpose of this article is to review the latter subject emphasizing recent studies from our own and other laboratories on the compound EGCG (see Fig. 1) which appears to be the major biologically active component in green tea with respect to antitumor activity. In addition, we will briefly review the effects on similar pathways of signal transduction exerted by the phytochemicals resveratrol, genistein,

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curcumin, and capsaicin (Fig. 1), compounds which have also been demonstrated to exert antitumor effects in various experimental systems. During the multistage process of carcinogenesis several alterations occur in cell signaling pathways that modulate gene expression, cell cycle progression, proliferation, cell motility, metabolism, and apoptosis. One of the central components of the complex intracellular network of signal transduction is the MAPK family of serine/threonine protein kinases [14,15]. Abnormalities in MAPK pathways and/or the related downstream transcription factors can cause uncontrolled cell replication and malignant cell transformation. Therefore, inhibition of these pathways may provide an effective strategy for the prevention and treatment of cancer [16]. In the next section of this paper we briefly review major aspects of some of these signaling pathways, and in the subsequent section we describe some of the effects of the phytochemicals listed in Fig. 1 on these pathways.

2. Cell signaling pathways and transcription factors 2.1. Membrane-associated RTKs The activation of membrane-associated RTKs located at the cell surface by specific ligands (growth factors and cytokines) plays an important role in the control of many fundamental cellular processes

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Fig. 1. Chemical structures of representative phytochemicals that display anti-cancer effects.

[17,18]. The EGFR (erbB1), HER2 (neu/erbB2), HER3 (erbB3), and HER4 (erbB4) proteins belong to subclass I (erbB) of the RTK superfamily [18]. The EGFR is activated by TGF␣, EGF, and other ligands, but no specific ligand for HER2 has been identified [18]. Ligand binding results in receptor homo- and hetero-dimerization, activation of tyrosine kinase activity, autophosphorylation of tyrosine residues, and phosphorylation of downstream targets. HER2 is the preferred heterodimerization partner for the other members of the erbB family of RTKs [18]. 2.2. Ras/MAPK pathway Fig. 2 summarizes some of the major signaling pathways that lie downstream of the erbB family of

membrane-associated RTKs. We should emphasize that this scheme is highly simplified but is useful to illustrate general principles. The Ras/MAPK pathway can be activated in response to a wide variety of extracellular stimuli. Activation of various RTKs by their specific ligands can play a critical role in the stimulation of this pathway [14,15,17–20]. In this cascade, activation of a RTK activates adaptor proteins (Grb2, Shc, Sos) which then activate Ras. Ras then interacts with and activates the serine/thereonine protein kinase Raf1, which in turn phosphorylates and activates MEK1/2 (MAP kinase kinase; MAPKK) on two distinct serine residues [21–23]. Activated MEK1/2 then phosphorylates ERK1 (p44MAPK ) and ERK2 (p42MAPK ) on both a tyrosine and a threonine residue [24]. In addition to the ERK1/2 pathway, the JNK1/2/3 and p38␣/␤/␥ path-

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Fig. 2. A simplified scheme indicating how activation of the erbB subfamily of RTKs induces pathways of signal transduction that lead to activation of the transcription factors AP-1 and NF-␬B. RTKs, like EGFR, are activated by specific ligands, thus leading to activation of their intrinsic tyrosine kinase and autophosphorylation of tyrosine residues. These activated RTKs then phosphorylate several downstream molecules, thus activating several signaling pathways. Activation of the small G protein Ras and effector proteins, such as Raf-1 and PI3K, stimulates several intracellular processes. Thus, activated Raf-1 stimulates the MAPKK, MEK1/2, and MEK1/2 cascade which then phosphorylates the MAPK protein ERK1/2. The other MAPKs JNK1/2/3 and p38␣/␤/␥ are mainly activated by various stress events and cytokine stimuli. Once activated, MAPKs can activate a variety of transcription factors, including ELK and c-Jun. The binding of AP-1, a dimeric complex that comprises members of the Jun and Fos families of transcription factors, to the TRE DNA sequence in various gene promoters, activates the expression of target genes, such as cyclin D1. PI3Ks are heterodimeric lipid kinases that are composed of a regulatory (p85) and a catalytic (p110) subunit. Among the RTKs, HER3 is the most efficient activator of PI3K because this receptor contains multiple binding sites for p85. Activation of PI3K causes the synthesis of the lipid PIP3 , which activates downstream pathways that involve Akt. Akt enhances cell survival because it possesses multiple anti-apoptotic effects. Akt and NIK play roles in phosphorylation and activation of the kinase IKK. Activated IKK phosphorylates I␬B, which triggers ubiquitinylation (Ub) and subsequent degradation of I␬B. The loss of I␬B releases rel from an inactive complex, which then translocates from the cytoplasm to the nucleus where it can activate the transcription of target genes. NIK, a MAPKKK, also enhances NF-␬B activity through activation of MEK1/2 and ERK1/2. Another MAPKKK, MEKK1, affects the activation of NF-␬B via phosphorylation and activation of IKK␤. For additional details see references and text. Proteins that appear to be direct cellular targets for the action of EGCG are indicated with the symbol ‘*’. They include EGFR, ERK1/2, Akt, and IKK. Other RTKs and signaling molecules may also be critical targets. These multiple effects of EGCG lead to inhibition of the transcriptional activities of AP-1 and NF-␬B and other impairments in signaling and gene expression, thus resulting in inhibition of cell proliferation and apoptosis.

ways are distinct but parallel MAPK cascades in mammalian cells [14,15]. Once activated, MAPKs (ERK, JNK, and p38) can activate a variety of transcription factors, including ELK and c-Jun, a component of AP1, thus leading to changes in the expression of genes

that play critical roles in cell proliferation, migration, and apoptosis [14,15]. Activated erbB proteins can phosphorylate and thereby activate PLC␥, thus releasing DAG and IP3 which lead to the activation of PKCs. Specific PKCs modulate MAPK pathways and other

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cellular functions [25]. Because dysregulation of these MAPKs pathways is frequently seen in a variety of human cancers, modulation of these MAPK pathways may provide novel strategies for the prevention or treatment of cancer [16]. 2.3. PI3/Akt pathway PI3K is another important effector of the erbB RTKs. It appears to play a critical role in cell survival rather that cell proliferation [26]. When PI3K is activated by these RTKs, it synthesizes the second messenger PIP3 , which is necessary for recruitment to the membrane fraction and phosphorylation of the serine/threonine kinase Akt [27]. Indeed, the strong prosurvival signaling mediated by PI3K is largely due to its ability to activate Akt [28]. Akt directly phosphorylates several components of the cell-death machinery, such as the pro-apoptotic protein Bad [29], thus enhancing the anti-apoptotic function of Bcl-xL Akt also phosphorylates and thereby inhibits the catalytic activity of the pro-death protease caspase-9 [30]. In addition, Akt can enhance cell survival by activating the transcription factor NF-␬B [31], which promotes survival in response to several apoptotic stimuli. Various components of the PI3K/Akt pathway are dysregulated in a wide variety of human cancers. Therefore, targeting this pathway may also be an effective strategy in cancer prevention and therapy [32]. 2.4. The transcription factors AP-1 and NF-κB The transcription factors AP-1 and NF-␬B are critical downstream effectors of the Ras/MAPK and PI3K/Akt signaling pathways, respectively. AP-1 is a dimeric complex that comprises members of the JUN and FOS families. Binding of the AP-1 complex to the TRE sequence present in the promoter region of several genes is induced by growth factors, cytokines, and oncoproteins, that play roles in cell proliferation, survival, differentiation, and transformation [33]. Indeed, the functional activation of AP-1 is associated with both tumor promotion and malignant transformation [34–37]. NF-␬B is a sequence specific transcription factor that is known to be involved in the inflammatory and innate immune responses [38,39]. NF-␬B is sequestered in the cytoplasm in an inactive form through interaction with I␬B. Phosphorylation of I␬B

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by I␬B kinase (IKK) causes ubiquitination and degradation of I␬B, thus releasing NF-␬B which then translocates to the nucleus, where it binds to specific ␬B binding sites in the promoter regions of several genes [38,39]. Akt can exert a positive effect on NF-␬B function by phosphorylating and thereby activating IKK [31]. The phosphorylation and activation of IKK is also controlled by an NF-␬B-inducing kinase (NIK) [38,39], and there is cross talk between activation of the MEK/ERK pathway and the NIK/IKK/NF-␬B pathway [40]. The MAPKKK protein MEKK1, which plays a role in the JNK-mediated signaling pathway, also activates NF-␬B via activation of IKK␤ [41]. Dysregulation of the NF-␬B pathway plays an important role in the development of various types of cancer [42,43]. Activation of AP-1 and NF-␬B can act independently, and/or coordinately, to regulate the expression of specific target genes. For example, the promoter region of the cyclin D1 gene, a gene that plays a critical role in the G1 –S transition of the cell cycle and is frequently overexpressed in a variety of human cancers [44], contains binding sites for both AP-1 and NF␬B. Therefore, the activity of this gene is controlled by extracellular stimuli via activation of both of these transcription factors [45,46]. It is of interest that cyclin D1 expression is required for breast carcinogenesis following mammary-specific expression of the HER2 or H-ras oncogenes [47]. HER2 and/or H-ras also trigger signaling cascades that lead to NF-␬B activation in breast cancer [48,49]. These results suggest that activation of both AP-1 and NF-␬B plays a critical role in the process by which HER2 and/or H-ras can cause overexpression of the cyclin D1 gene, and thus promote carcinogenesis. It is of interest that several phytochemicals that have antitumor activity can suppress activation of AP-1 and NF-␬B, and inhibit the expression of cyclin D1 in cancer [5,50]. This and related subjects are discussed in greater detail in the next section of this article.

3. Phytochemicals and cell signaling pathways 3.1. EGCG Epidemiologic studies suggest that the consumption of tea, especially green tea, is linked to a decreased incidence of various cancers [51]. Numerous experimental studies in rodents have demonstrated that green tea or

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its constituents can inhibit carcinogenesis and also the growth of established cancers at various organ sites [52]. Green tea contains the catechins EGCG, EGC, ECG, and EC. EGCG is one of the major constituents, and it appears to be the most potent compound in tea with respect to inhibiting cell proliferation and inducing apoptosis in cancer cells [53,54]. Fig. 1 illustrates the chemical structure of EGCG. It has various anti-cancer effects, including inhibition of oxidative stress, inhibition of carcinogen-induced mutagenesis, induction of apoptosis, and inhibition of angiogenesis [52]. EGCG has been shown to inhibit malignant transformation induced by EGF or TPA in the mouse epidermal JB6 cell line. This effect may be mediated, at least in part, by inhibition of the activation of AP-1 or NF-␬B because EGCG inhibits AP-1-dependent and NF-␬B sequence-specific DNA binding activity in these cells [55,56]. Treatment of human epidermal keratinocytes with EGCG caused significant inhibition of UVB-mediated activation of IKK␣, degradation and phosphorylation of I␬B␣, and nuclear translocation of the NF-␬B/p65 transcription factor [57]. In Hras-transformed JB6 cells, EGCG also caused strong inhibition of cell growth, and inhibition of the MAPK signaling pathway, and AP-1 activity [58]. These results indicate that EGCG can inhibit both AP-1 and NF-␬B activities, and provide evidence that these effects play a role in the inhibition of both cell growth and malignant cell transformation by this compound. They do not, however, indicate the primary cellular target(s) of EGCG, a subject which is discussed later in this paper. A seminal study by Liang et al. [4] demonstrated that EGCG binds to and directly inhibits the tyrosine kinase activity of the EGFR in human A431 epidermoid carcinoma cells. We recently extended this finding and found that EGCG inhibits activation of the EGFR, and also HER2, and multiple downstream signaling pathways in human HNSCC and breast cancer cell lines [6,7]. Thus, EGCG inhibits activation of ERK, inhibits basal and TGF␣-stimulated c-fos and cyclin D1 promoter activity, and causes a decrease in cellular levels of the cyclin D1 and Bcl-xL proteins. This effect on cyclin D1 may explain why the treated cells were arrested in G1 and the effect on Bcl-xL may contribute to the apoptotic effect of EGCG [6,7]. We found that in HT29 human colon cancer cells EGCG also inhibits activation of Akt and ERK, inhibits transcriptional activity

of the AP-1 and NF-␬B promoters, and causes activation of caspases 3 and 9 [5]. Other investigators found that EGCG inhibits HER2 receptor tyrosine phosphorylation in a mouse mammary tumor cell line and this was associated with inhibition of the PI3K/Akt kinase, and NF-␬B signaling pathways [9]. In addition, we found that in human HNSCC and breast cancer cell lines EGCG inhibits the constitutive activation of the transcription factor Stat3, which also lies downstream of EGFR [6–8]. Furthermore, we found that EGCG inhibits VEGF production in human HNSCC and breast cancer cells, apparently by inhibiting both the activation of Stat3 and NF-␬B in these cells [8]. This effect could contribute to the anti-angiogenic effects of EGCG. A detailed study in immortalized human cervical cells also found that EGCG inhibits activation of the EGFR and that this is associated with inhibition of activation of Akt and ERK [59]. Furthermore, there was reduced phosphorylation of the downstream substrates, p90RSK , FKHR, and BAD; increased levels of p53, p21CIP1 , and p27KIP1 ; reduced levels of cyclin E and CDK2 [59]. In H-ras-transformed mouse epidermal cells, treatment with EGCG caused a decrease in activation of ERK and MEK1, a decrease in the association of Raf-1 with MEK1, and inhibition of AP-1 activity [60]. In addition, in subcellular assays 10 ␮M EGCG directly inhibited phosphorylation of Elk-1 by phospho-ERK, apparently by interfering with binding of the Elk-1 protein substrate to the phospho-ERK kinase substrate [60]. Taken together, the effects on signaling molecules observed in the above described studies may explain why the treatment of cells with EGCG can arrest cells in G1 , inhibit cell proliferation, and induce apoptosis [5,6]. A recent study demonstrated that oral infusion of a green tea polyphenol mixture inhibited the development and progression of prostate cancer in a mouse transgenic model (TRAMP) of this disease. This was associated with reduction in levels of the growth factor IGF-1, decreased activation of Akt and ERK, and decreased levels of VEGF and MMPs 2 and 9 in the dorso-ventral prostate of these mice [61]. Since IGF-1 acts by binding to and activating the type I IGF receptor (IGF-1R), a member of the RTK family of receptors, these findings provide another example of the ability of plant polyphenolic compounds to inhibit RTK-related pathways of signal transduction.

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3.2. Resveratrol The polyphenolic compound resveratrol (Fig. 1) is present in a variety of plant species, including food products like grapes and peanuts, and it is also present in red wine and various herbs [62,63]. Previous investigators have demonstrated that it possesses anti-oxidant, anti-inflammatory, and antitumor activities. It can inhibit transcription of COX-2 and also directly inhibit the enzymatic activity of COX-2, and it can induce apoptosis. Thus, it can inhibit multiple cellular events that are associated with carcinogenesis and tumor growth [62,63]. In TPA-treated human mammary epithelial cells, resveratrol inhibited the transcription of COX-2 apparently by inhibiting PKC activation and the transcriptional activity of AP1 [64]. Pretreatment with resveratrol inhibited TPAand UV-light-induced activation of AP-1 and MAPKs (ERK2, JNK1, and p38) in HeLa cells, and these effects were associated with inhibition of PKC and c-Src tyrosine kinase activity [65]. Resveratrol also suppressed TNF-induced phosphorylation and nuclear translocation of the p65 subunit of NF-␬B, and NF␬B-dependent reporter gene transcription [66]. The suppression of NF-␬B activity by resveratrol coincided with inhibition of TNF-dependent AP-1 activation [66]. In human cervical cancer cells resveratrol significantly inhibited TPA-induced MMP-9 expression and activity, and it is known that MMP-9 expression is mediated by AP-1 and NF-␬B [67]. In the DMBA-induced rat mammary carcinogenesis model, dietary administration of resveratrol suppressed the incidence and multiplicity of mammary tumors, caused a decrease in the levels of COX-2 and MMP-9 expression, and suppressed NF-␬B activation in mammary tissues [68]. Resveratrol inhibited cell growth and EGF- and TPA-induced activation of ERK in androgen-independent prostate cancer cells [69]. The suppression of ERK activation by resveratrol correlated with inhibition of PKC␣ in these cells [69]. 3.3. Genistein Genistein (Fig. 1), the predominant isoflavone found in soy, has antitumor activity in rodent models and has been shown to inhibit the growth of various types of cancer cells through modulation of several signal transduction pathways [70]. Thus, genistein inhibited TPAinduced increases in c-fos expression, AP-1 activity,

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and ERK activity in human breast cancer cells [71]. In prostate cancer cells, genistein also reduced the phosphorylation of I␬B and blocked nuclear translocation of NF-␬B, thus inhibiting DNA binding and NF-␬B activation [72]. Recent studies indicate that the inhibition of NF-␬B activation by genistein in prostate [73] and breast cancer [74] cells is mediated via inhibition of the Akt signaling pathway, when this pathway is induced by treating the cells with EGF. Genistein inhibited the phosphorylation of Akt induced by HRG-␤1, a ligand for HER3 and HER4, in MCF-7 breast cancer cells [10]. In the TRAMP transgenic mouse model of adenocarcinoma of the prostate, administration of genistein in the diet significantly down-regulated activation of the RTKs EGFR and IGF-1R, and their downstream effector ERK, thus inhibiting cell proliferation [11]. As discussed above, green tea polyphenols also inhibit similar signaling pathways and tumorigenesis in the TRAMP model [61]. 3.4. Curcumin It is widely accepted that curcumin (Fig. 1), a yellow pigment found in the rhizome of the spice turmeric, has potent cancer chemopreventive activity in rodent models of several types of cancer [75]. One of the most important effects of curcumin is its anti-inflammatory property. In human myeloid cells, curcumin is a potent inhibitor of NF-␬B activation in response to treatment with TNF, TPA, and H2 O2 [76]. AP-1 binding factors were also found to be down-modulated by curcumin in these cells. Evidence was obtained that suppression of NF-␬B activation by curcumin in human cancer cells is due to inhibition of the phosphorylation and subsequent degradation of I␬B, thus abrogating nuclear translocation of NF-␬B/p65 [76]. Curcumin also suppressed NF-␬B activation by inhibiting the upstream kinases that induce the phosphorylation of I␬B, NIK, and IKK, thus inhibiting induction of COX-2 in human colon epithelial cells [77]. Curcumin also inhibited COX-2 expression in HT29 colon cancer cells [78]. These effects were associated with inhibition of expression of cyclin D1 [79]. When curcumin was topically applied prior to TPA to the dorsal skin of female ICR mice, it significantly attenuated the TPA-induced activation of both NF-␬B and AP-1 in the skin of these mice [80]. TPA-induced binding of NF-␬B and AP1 to their respective DNA binding sites was inhibited

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by curcumin in HL-60 promyelocytic leukemia cells [81]. Curcumin has also been identified as a RTK inhibitor, since it caused a dose- and time-dependent inhibition of EGFR tyrosine phosphorylation in EGFstimulated NIH3T3 cells [12]. Recent studies indicate that in human breast cancer cells curcumin inhibited EGF-stimulated phosphorylation of the EGFR and also inhibited phosphorylation of ERK, as well as ERK activity, and caused a decrease in levels of c-fos [13]. Curcumin also inhibited the basal level of phosphorylation of Akt in these cells [13]. 3.5. Capsaicin In addition to curcumin, other phytochemicals derived from spices also possess antitumor-related activities. Thus, in mouse epidermis, capsaicin (Fig. 1), a pungent component of hot chili pepper, inhibited TPA-induced activation of NF-␬B by blocking degradation of I␬B and subsequent nuclear translocation of NF-␬B/p65 [82]. It also inhibited TPA-induced activation of AP-1 in the dorsal skin of female ICR mice [82]. TPA-stimulated activation of NF-␬B and AP-1 activity were also suppressed by capsaicin in human promyelocytic leukemia cells [83]. Capsaicin inhibited TNF␣- and TPA-induced binding of AP-1 and NF␬B to their specific binding sites on DNA in human chronic myelogenous leukemia cells [84]. Capsaicin selectively induced apoptosis in H-ras-transformed human breast epithelial cells. This was accompanied by marked activation of JNK1 and p38, and deactivation of ERK [85]. A recent study indicated that in human vascular endothelial cells capsaicin inhibited VEGFinduced p38 MAPK and Akt activation, thus inhibiting VEGF-stimulated angiogenesis [86].

4. Discussion The above studies provide evidence that various phytochemicals can inhibit pathways of signal transduction and gene expression that play critical roles in carcinogenesis and tumor growth. In this review we focused mainly on the inhibitory effects of phytochemicals on the erbB family of RTKs and their downstream signaling pathways, and the inhibitory effects of these chemicals on the transcription factors AP-1 and NF-␬B. It is remarkable that despite the

diversity in their chemical structures (Fig. 1), the phytochemicals EGCG, resveratrol, genistein, curcumin, and capsaicin caused inhibition of the AP-1 transcription factor, which normally stimulates cell proliferation, as well as inhibition of the NF-␬B transcription factor, which normally enhances cell survival (Fig. 2). These combined effects appear to play an important role in the antitumor effects of these compounds, although other cellular effects of these compounds probably also play a role. This theme has been previously emphasized by Surh [50]. Another theme is that at least three of these phytochemicals, EGCG, genistein, and curcumin can inhibit activation of the EGFR and/or the HER2 receptor. Our laboratory has focused on this aspect with respect to the anti-proliferative aspects of EGCG [5–8]. We will now discuss possible mechanisms by which EGCG inhibits activation of the EGFR and downstream signaling pathways, using Fig. 2 as a framework for this discussion. Some of these mechanisms also appear to be relevant to the action of other phytochemicals shown in Fig. 1. The EGFR and other members of the erbB family of RTKs are activated by specific ligands [18]. Ligand binding results in receptor homo- and hetero-dimerization leading to kinase activation, phosphorylation of tyrosine residues in target molecules, activation of downstream signaling pathways, and increased expression of target genes that enhance cell proliferation and/or inhibit apoptosis [18]. It is known that EGCG can directly inhibit the binding of EGF, PDGF, and FGF to their respective receptors [4,87]. This could account for the inhibitory effects of EGCG on autophosphorylation of the EGFR and HER2 receptor in various types of cancer cells [4–7,9]. Time course studies in human colon cancer cells indicate that this effect occurs within 6 h, while inhibition of phosphorylation of the ERK and Akt proteins occurs after about 6–12 h [5]. These data suggest that EGCG initially inhibits the activation of RTKs at the cell surface and that this leads to subsequent inhibition of the activation of downstream effectors in the cytoplasm, including the ERK and Akt proteins [5]. On the other hand, there is evidence that EGCG can directly target intracellular signaling molecules. In extracts of immortalized human cervical cells a low concentration of EGCG (5 ␮M) directly inhibited the subcellular kinase activities of ERK and Akt [59]. In the same subcellular assays EGCG did not inhibit the kinase activities of p38 or JNK1/2, thus indicating the specificity of this

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effect [59]. In extracts of H-ras-transformed murine epithelial cells EGCG (10 ␮M) directly inhibited the phosphorylation of Elk-1 by phospho-ERK, possibly by binding to the proline-rich sequence on Elk-1 [60]. EGCG can also directly and specifically inhibit subcellular IKK activity in extracts of TNF␣-stimulated fetal rat intestinal epithelial cells [88]. Taken together, these findings suggest that the antitumor effects of EGCG and several other phytochemicals may be due to binding, probably with relatively low affinity, to multiple cellular targets. However, the precise identification of the direct and critical cellular target(s) of EGCG, and the other phytochemicals discussed in this paper, remains to be determined. This will probably require novel and highly sensitive methods. Of interest is a recent study that used surface plasmon resonance analysis to demonstrate that the 67 kDa laminin receptor can confer EGCG responsiveness to cancer cells at physiologically relevant concentrations of EGCG (0.1–1.0 ␮M) [89], but the underlying mechanism is not known. In addition to targeting specific RTKs and related downstream signaling pathways, EGCG can also target directly, or indirectly, other pathways that can play critical roles in tumorigenesis, including; inhibition of CDKs 2 and 4 activities as well as induction of p21CIP1 and p27KIP1 [90]; inhibition of telomerase activity [91]; inhibition of topoisomerases I and II [92]; inhibition of DNA methylation, thus derepressing gene expression [93]; inhibition of expression of the androgen receptor (AR) in prostate cancer cells, thus inhibiting the expression of AR-mediated pathways [94]. We should emphasize that in this review we have emphasized the role of the parent compounds on pathways of signal transduction. However, further studies are required to evaluate the possible roles of reactive oxygen species and/or metabolites of EGCG and other phytochemicals in exerting the various cellular and biochemical effects we have discussed [95,96]. A critical issue is whether specific phytochemicals can exert anti-cancer effects without causing undesirable side effects, i.e., whether the agent can selectively or preferentially inhibit pre-neoplastic or cancer cells without significantly affecting normal cells in the host. With respect to this aspect it is encouraging that in the numerous rodent studies with EGCG and other phytochemicals, anti-cancer effects were observed with little or no toxicity to the host animal [52,62,70,75]. A pos-

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sible explanation is that the roles of specific RTKs and the complex circuitry of signal transduction pathways in cancer cells differ sufficiently to provide differential responses to these compounds [97]. For example, Ahmad et al. [98] found that EGCG caused inhibition of cell growth and induction of apoptosis in human epidermoid carcinoma cells, but did not cause these effects in normal human epidermal keratinocytes. The authors suggested that this was due to the fact that EGCG inhibited activation of NF-␬B in the carcinoma but not in the normal cells [98]. We found that EGCG preferentially inhibited the growth of human colon cancer cells when compared to FHC normal human fetal colon cells, and that this was associated with constitutive activation of the EGFR and HER2 proteins in the colon cancer cells but not in the normal colon cells [5]. Other investigators found that a resveratrol derivative exerted a potent inhibitory effect on EGF-induced transformation of JB6 cells, but had a negligible cytotoxicity on normal non-transformed epidermal cells. This inhibitory effect was associated with inhibition of activation of the PI3K/Akt pathway induced by EGF [99]. Taken together, these results are encouraging. Hopefully further elucidation of the critical molecular targets of EGCG and the other phytochemicals discussed in this paper, and their complex effects on signaling pathways and gene expression, will lead to identification of which phytochemicals, or synthetic derivatives, are most likely to have potent antitumor effects and negligible toxicity to normal tissues. In this review we have emphasized the direct effects of EGCG and related phytochemicals on the proliferation of epithelial cells and tumor cells. However, there is evidence that in the intact animal some of these compounds might also exert effects at the level of tumor–stromal interactions. Thus, EGCG can directly inhibit the expression of extracellular proteases through the suppression of MAPK activation and inhibition of AP-1 and NF-␬B activities [100,101], and also directly inhibit the activities of extracellular proteases [102–104]. With respect to angiogenesis, we [8] and other investigators [105] found that EGCG can inhibit the expression of VEGF in tumor cells, and other studies indicate that EGCG and related phytochemicals can directly inhibit angiogenesis [106–108]. In a recent in vitro study employing human microvascular endothelial cells (HMVEC), Tang et al. [109] found that EGCG inhibited VEGF-induced tube formation, and

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obtained evidence that this effect is mediated, in part, by suppression of vascular-endothelial (VE)-cadherin tyrosine phosphorylation and inhibition of Akt activation. The direct target(s) of EGCG in this system are, however, not known. In recent years, there has been renewed interest in the role of inflammation in enhancing tumor development and growth [110,111]. It appears that activation of NF-␬B in macrophages plays a key role in this process [42,110,111]. As discussed above, EGCG inhibits NF-␬B activation in cancer cells. It is possible that the in vivo antitumor effects of EGCG may be augmented by a similar inhibition of NF-␬B in macrophages, and/or inhibition of other functions of macrophages that stimulate tumor growth. Green tea polyphenols have been shown to inhibit phorbol ester induced inflammation in mouse skin [112,113], and this inhibitory effect was also seen with black tea polyphenols [114,115]. However, the precise mechanism(s) is not known. Nor is it known whether these inhibitory effects on inflammation play a role in the ability of these polyphenolic compounds to inhibit tumor promotion on mouse skin. Finally, we should also emphasize that most of the above-described effects of EGCG and other phytochemicals in cell culture systems were obtained with relatively high concentrations of these chemicals. However, it is unlikely that these concentrations can be readily obtained in vivo. Thus, pharmacokinetic studies in humans indicate that the peak plasma concentration after administration of a single dose of EGCG is less than 1.0 ␮M [116,117]. Nevertheless, doses of green tea or doses of EGCG equivalent to those consumed by heavy tea drinkers have been shown to exert antitumor effects in rodents [52]. This may reflect the longer duration of exposure and/or tissue accumulation of these compounds in the intact animal. Indeed, we found that when colon cancer cells were treated for 96 h rather than 48 h, 1.0 ␮g/ml (about 2.2 ␮M) EGCG was sufficient to inhibit growth, inhibit activation of the EGFR and HER2 receptor, and induce apoptosis [5]. Furthermore, for the therapy of cancer, it may be efficacious to combine EGCG or other phytochemicals with chemotherapy agents, since in cell culture studies we found that low concentrations of EGCG (0.1–1.0 ␮g/ml) exerted synergistic growth inhibition of cancer cells when combined with 5-FU [6] or taxol [7]. EGCG has also been shown to act synergistically with sulindac [118], (−)-epicatechin [118],

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