POLYPHENOLS AND ERBB RECEPTORS SIGNALING PATHWAY IN CANCER CELLS

Among the different mechanisms explaining the anti-cancer effects of polyphenols, the modulation of ErbB receptors signaling cascades is one of the most important. Several studies have shown that a variety of polyphenols exhibits the ability to inhibit EGFR, ErbB2/neu and ErbB3 pathways both in vitro and in vivo.

In this regard, many investigations have demonstrated that the green tea polyphenol epigallocatechin-3 gallate (EGCG) is one of the most promising natural compounds that can be used to inhibit ErbB receptors downstream signaling in several types of tumors.  Pianetti et al. reported that EGCG was able to counteract the growth of ErbB2/neu-overexpressing mouse mammary tumor NF639 and SMF cell lines. EGCG reduced the basal phosphorylation and constitutive activation of ErbB2/neu and inhibited the PI3K, Akt kinase and NF-κB signaling pathway (68). In addition, a model for demonstrating the anti-tumor activity of EGCG at multiple steps in ErbB2- or/and ErbB3-overexpressing breast cancer cells has been proposed by Pan et al. The authors suggested that EGCG might interfere with heterodimerization and tyrosine phosphorylation of ErbB2-ErbB3 because it competes with Heregulin-β1, an ErbB3 ligand (69). Masuda et al. examined the anti-tumor activity of EGCG on human head and neck squamous cell carcinoma (HNSCC) and human breast cancer cell lines in which EGFR was constitutively activated. EGCG inhibited the activity of EGFR and blocked the activity of Stat3 and Akt in both cell lines (70). The same authors also examined the anti-tumor activity of EGCG on HNSCC and human breast cancer cell lines in which ErbB2 was constitutively activated. They reported that EGCG inhibited phosphorylation of ErbB2 in both cell lines, determining the inhibition of Stat3, c-fos and cyclin D1 promoter activity, and a reduction of cyclin D1 and Bcl-x_L protein levels (71). EGCG potentiated the anti-metastatic effects of gefitinib, a tyrosine kinase inhibitor, on CAL-27 human oral squamous carcinoma cells. Combined treatment with EGCG and gefitinib enhanced the suppression of phosphorylation of EGFR and suppressed the phosphorylation of ERK, JNK, p38 and Akt (72).  EGCG was also able to arrest growth and revert the transformed phenotype of ErbB2-overexpressing human breast cancer BT-474 cells resistant to trastuzumab, a humanized monoclonal antibody used for immunotherapy of ErbB2-overexpressing tumors. Resistance to trastuzumab is due to activation of Akt signaling cascade and loss of CDK inhibitor p27^Kip1 expression. Inhibition of proliferation of these cells by EGCG was mediated by its capacity to reduce Akt activity that determines an increase of FOXO-3a and p27^Kip1 levels. In agreement with these results EGCG reverted resistance to trastuzumab (73). In another study, the ErbB2/neu-transformed breast cancer cell line NF639 resistant to EGCG has been isolated. These cells showed enhanced activation of Akt and NF-κB, elevated MAPK signaling and a reduction of tyrosine phosphorylation of the ErbB2/neu receptor, that were responsible for invasive phenotype. Surprisingly, the authors observed that the treatment with EGCG in combination with dexamethasone, a potent inhibitor of NF-κB, or in combination with U0216, a MAPK inhibitor, blocked the growth and reverted the invasive phenotype of NF639 cells, suggesting that combinatorial treatments were more efficient than treatment with EGCG alone to enhance the potency of therapy against ErbB2/neu-overexpressing tumors (74). EGCG showed similar anti-tumor effects also in human esophageal, colon cancer and non-small cell lung cancer (NSCLC) cells. Hou et al. observed that EGCG inhibited the phosphorylation of EGFR in esophageal squamous carcinoma KYSE150 cells and in epidermoid squamous carcinoma A431 cells (75).  EGCG was able to decrease the phosphorylation of ErbB3, EGFR and ErbB2, thus inhibiting their downstream pathways, and to reduce the cellular levels of these proteins in the colon carcinoma cell line SW837, that displayed high levels and a constitutive activation of ErbB3 (76). Adachi et al. showed that EGCG inhibits the activation of EGFR and causes its internalization or degradation by altering plasma membrane organization in human colon cancer cells (SW480) (77,78). They also demonstrated that EGCG (50 µM) caused phosphorylation of EGFR at Ser^1046/1047, a site that is critical for its down-regulation, through activation of p38 kinase (79). Milligan et al. reported that EGCG in combination with erlotinib, an antagonist of EGFR, inhibited growth of erlotinib-sensitive and erlotinib-resistant NSCLC cell lines more strongly than either agent alone, blocking EGFR phosphorylation and affecting EGFR downstream pathways. In particular, EGCG inhibited phosphorylation of Akt and ERK1/2 (80). Similarly, genistein enhanced the inhibitory effects of erlotinib and gefitinib on EGFR phosphorylation in NSCLC cell lines, causing the suppression of EGFR and Akt expression as well as NF-κB inactivation (81).

A study by Fridrich and co-workers investigated the inhibitory properties of EGCG, the anthocyanin (ACN) aglycon delphinidin and the flavonol quercetin on EGFR and ErbB2 receptor activities. Delphinidin was able to suppress EGFR phosphorylation in a human colon carcinoma cell line (HT29) (IC₅₀: 54±11 µM) and in a human vulva carcinoma cell line (A431) (IC₅₀: 71±32 µM). Delphinidin was also able to down-regulate the phosphorylation of ErbB2 in A431 cells (IC₅₀= 60±21 µM) and to inhibit the activity of the downstream targets ERK1/2. In addition, the flavonol quercetin was more effective than delphinidin in inhibiting EGFR phosphorylation (IC₅₀: 0.6±0.1 µM) and in down-regulating ERK1/2 activities in HT29 cells. Conversely in A431 cells, quercetin had a marginal effect in inhibiting the activity of ErbB2 receptor (IC₅₀: ≥150 µM). Finally, in A431 cells EGCG exhibited significant inhibitory properties on the phosphorylation of ErbB2 only at high concentrations (IC₅₀: ≥150 µM). The authors concluded that quercetin is most effective against the EGFR, whereas delphinidin exhibits preference towards the ErbB2 receptor (82). The anti-cancer properties of delphinidin, and in particular its effect on EGFR signaling pathway, have been studied by Afaq et al. Delphinidin (5-40 µM) inhibited the phosphorylation of EGFR and of its downstream targets Akt, ERK1/2, JNK1/2/p38 in EGFR-positive AU-565 and immortalized MCF-10A breast cancer cells in a concentration-dependent manner. Delphinidin was also able to inhibit EGF-induced auto-phosphorylation of EGFR in the same cell lines (83). Another report by Teller et al., examined the ability of delphinidin in inhibiting the kinase activity of EGFR, ErbB2, VEGFR-2/3 and IGF-1R. Delphinidin was able to inhibit the protein tyrosine kinase activity of all receptors at concentrations ≥5 µM in a cell-free test system. Delphinidin was able to decrease the ligand-induced phosphorylation of EGFR and ErbB2 receptors in A431 cells (IC₅₀:72±32 µM for EGFR, IC₅₀: 51±23 µM for ErbB2) (84). In a recent report by Ozbay et al., the authors employed delphinidin to determine the in vitro properties of this ACN on the growth of seven established breast cancer cell lines of varying molecular subtypes including ErbB2-overexpressing, ER-positive, and triple negative cells. Delphinidin (12.5-100 µg/mL) was able to inhibit proliferation of HCC1806, MDA-MB-231, MDA-MB-468, SKBR3, MDA-MB-453, BT-474 and MCF-7 breast cancer cells. In particular, delphinidin induced the highest level of apoptosis (4- to 6-fold) in the ErbB2-overexpressing lines SKBR3 and BT-474 as compared to (2-fold) the triple negative lines HCC1806 and MDA-MB-231. In addition, delphinidin suppressed ErbB2 signaling in SKBR3 cells, which overexpress ErbB2, through the down-regulation of the phosphorylation of ErbB2 and the downstream targets ERK1/2 and Akt. Inhibition of ERK1/2 by delphinidin was also observed in HCC1806 and MDA-MB-468 cells. Thus, the authors suggested that delphinidin is an inhibitor of ErbB2/MAPK signaling (85).

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A study by Xu and co-workers investigated the effect of the ACN cyanidin-3-glucoside (C3G) on ethanol-mediated migration/invasion of breast cancer cells expressing high levels of ErbB2. MCF-7 cells overexpressing ErbB2 (MCF-7^ErbB2), MDA-MB-231 and BT-474 breast cancer cells were treated with ethanol, in presence or absence of C3G for 48h. It was found that ethanol increased migration/invasion of breast cancer cells, but the treatment with C3G (10-40 µM) was able to inhibit ethanol effects in a dose-dependent manner in MCF-7^ErbB2 and MDA-MB-231 cells. The effect of C3G on BT-474 breast cancer cells was not dose-dependent (86). Even flavones, such as apigenin and luteolin, displayed inhibitory activity against ErbB receptors. Masuelli et al. demonstrated that apigenin reduces ligand-induced phosphorylation of EGFR and ErbB2 and impairs their downstream signaling in HNSCC (12). In the ER-negative MDA-MB-231 breast cancer cells, luteolin exerted its inhibitory activity on cell proliferation through the suppression of EGFR phosphorylation, leading to the inactivation of the MAPK and PI3K/Akt pathways. In addition, a diet supplementation with luteolin determined a strong reduction of tumor volume in female athymic nude mice implanted with MDA-MB-231 cells (87).

The inhibitory properties of another plant polyphenol, tannic acid, on EGFR tyrosine kinase has been investigated in a study by Yang et al. The authors demonstrated that tannic acid was able to strongly inhibit tyrosine kinase activity of EGFR in vitro (IC₅₀= 323 nM). In addition, tannic acid inhibited the EGF-induced EGFR auto-phosphorylation in Swiss 3T3 mouse fibroblasts in a concentration-dependent manner. Tannic acid was also able to inhibit the growth of HepG2 cells previously stimulated with EGF. The authors also investigated the interaction of tannic acid with EGFR in a molecular modeling study. They suggested that tannic acid could be docked into the ATP binding pockets of EGFR, since the inhibition of EGFR kinase activity was found to be ATP-competitive (88).

Another report by Katdare et al. investigated the effects of soy isoflavone genistein against breast cancer. Genistein was able to inhibit proliferation of the human breast epithelial cell line 184-B5 overexpressing ErbB2/neu and this effect was due to the inhibition of the tyrosine kinase activity of the receptor (89). Moreover, genistein induced the suppression of BT-474 human breast cancer cells growth by affecting ErbB2 phosphorylation and protein expression (90).

The effect of oak ellagitannins on EGFR has been studied by Fridrich et al. in human colon carcinoma cells. The aglycones castalagin (IC₅₀: 11.1±1.8 μM) and vescalagin (IC₅₀: 22.4±2.6 μM) and the C-glycosides roburin E (IC₅₀: 16.7±1.4 µM) and grandinin (IC₅₀: 28.9±2.6 µM) inhibited the growth of HT29 colon cancer cells. Besides, these ellagitannins were able to inhibit the tyrosine kinase activity of EGFR in nanomolar concentration. Finally, grandinin (IC₅₀: 35±0.3 μM) and castalagin (IC₅₀~ 10 µM) also inhibited the auto-phosphorylation of EGFR in HT29 colon carcinoma cells (91).

The same authors explored the effect of procyanidins on EGFR in human colon carcinoma cells. In particular, they isolated from grape seed extracts the dimeric procyanidins B1 (PB1), B2 (PB2), B3 (PB3), B4 (PB4), the trimeric procyanidin C1 (PC1) and tetrameric procyanidin cinnamtannin A2 (PA2). The dimeric procyanidins were able to inhibit the protein tyrosine kinase activity of EGFR with IC₅₀ values of about 10 µM while the trimeric (PC1) and tetrameric (PA2) procyanidins inhibited the activity of EGFR with IC₅₀ values of 1.3±0.4 µM and 0.2± 0.06 µM, respectively. It was also found that PC1 was able to inhibit EGFR auto-phospohorylation in HT29 colon carcinoma cells with an IC₅₀ value of 35±15 µM, while PA2 affected the auto-phosphorylation of EGFR only at concentrations up to 50 µM (92).

Another plant polyphenol, quercetin, has been evaluated for its anti-tumor activity in breast cancer cells. In particular, a study by Jeong et al., investigated the effect of quercetin in inhibiting ErbB2 signaling pathway. The authors observed that quercetin induced the ubiquitinylation and down-regulation of ErbB2/neu in SKBR3 breast cancer cells. Quercetin treatment (100-200 µM) induced a decrease in ErbB2/neu protein level and a dephosphorylation of PI3K and Akt in this cell line. They also observed that quercetin was able to inhibit the tyrosine kinase activity of ErbB2/neu and suggested that quercetin could bind to ErbB2/neu and cause an alteration in its protein structure, which could lead to its degradation (93).

In studies conducted by Menendez et al. it has been determined the relationship between chemical structures of polyphenols derived from extra-virgin olive oil (EVOO) and their ability to inhibit phosphorylation of ErbB2 in MCF-10A breast cancer cells engineered to overexpress the wild type form of human ErbB2, and in human breast cancer SKBR3 and MCF-7 cell lines overexpressing ErbB2. The authors observed that among EVOO-derived polyphenols, lignans and secorroids, which have a complex chemical structure, were more effective than single phenols and phenol acids in suppressing ErbB2 phosphorylation and reducing ErbB2 protein expression in all cell lines, suggesting that the presence of more phenol rings was necessary to exert inhibitory effects on ErbB2 activity (94,95). The anti-cancer effects of EVOO-derived polyphenols were also tested on the human breast cancer JIMT-1 cell line, which overexpresses ErbB2 and has cross-resistance to multiple EGFR/ErbB2-targeted drugs, such as trastuzumab, gefitinib, erlotinib and lapatinib. Even in these cells, lignans and secorroids showed a strong ability in suppressing phosphorylation and biological activity of ErbB2 and in determining an inhibition of ErbB2 downstream signaling cascades. In particular, the treatment with EVOO-derived polyphenols resulted in a reduction of endogenous levels of phosphorylated Akt, MAPKs, Stat3 (96).

Another polyphenol that exerts a potent effect on EGFR/ErbB2/ERbB3 activity, influencing its downstream cascade signaling, is curcumin. A study by Hong et al. demonstrated that curcumin was able to inhibit ErbB2 tyrosine kinase activity, by depleting ErbB2 protein. Curcumin down-regulated ErbB2 protein by disrupting its binding with the molecular chaperone GRP94 in the endoplasmic reticulum (97). A recent report investigated the molecular mechanism underlying curcumin-induced ErbB2 depletion. Curcumin induced association of the ubiquitin ligase CHIP with ErbB2 and the subsequent ubiquitination of ErbB2. The kinase domain of ErbB2 was required for curcumin-induced ErbB2 ubiquitination and degradation (98). Squires et al. investigated the ability of curcumin to modulate EGFR-mediated signaling pathways in human breast cancer MDA-MB-468 cells and observed that curcumin inhibited EGFR phosphorylation, leading to a suppression of c-fos expression, and ERK activity (99). Curcumin was also shown to induce apoptosis of triple-negative breast cancer MDA-MB-231 cells, an aggressive breast cancer phenotype in which expression of the estrogen receptor (ER), progesterone receptor (PR) and ErbB2 is lacking. After treatment with curcumin the expression of phosporylated forms of EGFR and ERK1/2 was significantly decreased (100). In addition, the growth of MDA-MB-231 cells was also suppressed in vitro and in vivo using a combinatorial treatment with curcumin and EGCG. After treatment, changes in the expression of proteins involved in cell proliferation were analyzed and a significant reduction of EGFR, phospho-EGFR, Akt and phospho-Akt levels was detected. Furthermore, athymic nude female mice implanted with MDA-MB-231 cells and treated with curcumin plus EGCG showed a 49% reduction of tumor volume when compared with untreated mice (101).  Sun et al. demonstrated that curcumin inhibited cell proliferation and induced cell cycle arrest at G1 phase (30 µM) and apoptosis (50 µM) in MDA-MB-231 cancer cells overexpressing ErbB2. It has also been observed that curcumin inhibited the phosphorylation of Akt and MAPKs. Moreover, curcumin increased expression levels of p27, by down-regulating Skp2 and ErbB2 (102). Another report by Lai et al. analyzed the effect of curcumin in the treatment of ErbB2-overexpressing breast cancer and its interaction with herceptin. Curcumin affected the growth of cancer cells and induced a dose-dependent down-regulation of ErbB2 oncoprotein, phospho-Akt, phospho-MAPK, and NF-κB in both BT-474 and herceptin-resistant SKBR3 cells (103). The ability of curcumin to inhibit EGFR downstream signaling was also reported in tumors affecting the gastro-intestinal tract. It has been reported that curcumin suppressed expression of ErbB2 and down-regulated expression of cyclin D1 in gastric cancer cells, determining an arrest of proliferation and invasion of these cells (104).  Majumdar et al. described the synergistic effect of curcumin and resveratrol on proliferation of colon cancer HCT-116 cells both in vitro and in vivo. These two polyphenols resulted in the inhibition of the constitutive activation of EGFR and IGF-1R in vitro and in reduction of tumor growth and induction of apoptosis together with attenuation of NF-кB in vivo, in SCID mice transplanted with HCT-116 cells (105). Moreover, curcumin was demonstrated to enhance the anti-tumor effects of biological and chemical drugs used in the treatment of colon cancer. For example, it has been demonstrated that curcumin enhanced inhibition of growth and transformation mediated by dasatinib, an inhibitor of Src family kinases (SFK), in several types of colon cancer cells. Colon cancer cells treated with curcumin in combination with dasatinib displayed reduction of EGFR, ErbB2 and ErbB3 levels as well as a strong suppression of c-Src and IGF-1R phosphorylation. In addition, the combination therapy decreased the phosphorylated forms of Akt and ERKs and caused a strong attenuation of the DNA binding activity of NF-κB. Finally, the anti-proliferative activity of curcumin plus gefitinib has been detected in vivo using C57BL/6J-APCMin+/− mice which develop spontaneous intestinal adenoma. Mice treated with the combined therapy showed a significant regression of intestinal tumors (90-99%), an event associated with the induction of apoptosis by curcumin and gefitinib (106). Curcumin has also shown the ability to enhance the effects of 5-fluorouracil and oxaliplatin therapy (FOLFOX) in inhibiting growth of HCT-116 and HT29 colon cancer cells. These events were associated to the decreased expression and activation of EGFR, ErbB2, ErbB3 and IGF-1R that led to inhibition of Akt phosphorylation (107,108).  Inhibitory effects on EGFR activation and expression were also exerted by curcumin in other types of tumor, such as bladder, prostate and lung tumors. Chadalapaka et al. demonstrated that curcumin in association with betulinic acid caused the decrease of EGFR levels and the suppression of phosphorylation of Akt in KU7 and 253JB-V human bladder cancer cells (109). Similar effects were detected in human LNCaP and C4-2B prostate cancer cells in which curcumin determined a down-regulation of EGFR and ErbB2 expression (110). In addition, human prostate cancer PC-3 cells treated with curcumin in combination with β-phenylethyl isothiocyanate showed a drastic suppression of EGFR phosphorylation that resulted in the prevention of Akt and PI3K activation and in suppression of NF-κB pathway through attenuation of IкB-α phosphorylation. The inhibition of multiple pathways associated with EGFR activity promoted the activation of apoptosis and the arrest of proliferation of these cells (111). Lev-Ari and co-workers assessed the effects of curcumin on pancreatic and lung adenocarcinoma cells. Cell lines co-expressing COX-2 and EGFR (PC-14 and p34, respectively) and cells expressing EGFR but deficient in COX-2 (H1299 and Panc-1, respectively) were treated with curcumin (0-50 µM for 72h). Curcumin inhibited cell survival and induced apoptosis of all cell lines, but the effect was higher in PC-14 and p34 cells, which express COX-2. Moreover, in these two cell lines, curcumin demonstrated to decrease COX-2, EGFR and phospho-ERK1/2 expression in a dose dependent-manner (112). The effects of curcumin in human lung adenocarcinoma cells were also investigated by Lee et al. The results of this study showed that curcumin potentiates the anti-cancer effects of gefitinib in vitro employing CL1-5, A549 and H1975 cells and in xenograft mouse models. Curcumin exerted these effects through the inhibition of proliferation, and EGFR phosphorylation, and the induction of EGFR ubiquitination and apoptosis (113). Finally, curcumin induced apoptosis of rhabdomyosarcoma and osteosarcoma cells and strongly reduced Akt expression levels as well as phospho-ERK levels in malignant rhabdoid SJ-RH4 cells (16).

It should also be considered that several studies analyzed the direct effects of polyphenols on the MAPK signaling pathway without investigating their role on ErbB receptors, since these kinases represent the principal read out of the activation of tyrosine kinase receptors. Shin et al. studied the anti-proliferative activity of anthocyanins derived from Vitis coignetiae Pulliat on the human colon cancer cell line HCT-116. These flavonoids inhibited growth and induced apoptosis of these cells in a dose-dependent manner. Further, the authors found that apoptosis was associated with the activation of p38 kinase and the inactivation of Akt (114). Ho et al. investigated the effects of the anthocyanin peonidin 3-glucoside (P3G) in lung cancer cells. They found that P3G inhibited the invasion, motility and secretion of MMP-2, MMP-9, and urokinase-type plasminogen activator (u-PA) of these cells. These effects were partly due to the decrease of ERK1/2 phosphorylation and the inactivation of AP-1 (115).

Gopalakrishnan et al. focused their research in assessing the modulation of AP-1 protein and MAPK pathway by flavonoids. In particular, they found that quercetin, chrysin, genistein and kaempferol were able to induce AP-1 in human prostate cancer cells (PC-3). In addition, they demonstrated that kaempferol, apigenin, genistein and naringenin induced the phosphorylation of JNK and ERK in the same cells. They also demonstrated that the JNK pathway is involved in the induction of AP-1 by genistein, while the MEK pathway is involved in the induction of AP-1 by kaempferol (116).

The anti-cancer effect of apigenin (5-40 µM) on androgen‑responsive human prostate cancer LNCaP cells and androgen‑refractory PC‑3 cells was also investigated. Apigenin inhibited cell growth, arrested cell cycle in G0/G1 phase and decreased the phosphorylation of Rb protein in these cell lines. However, although apigenin increased phosphorylation of ERK1/2 and JNK1/2, it reduced phosphorylation of ELK-1 and c-fos protein expression. In addition, apigenin reduced expression of cyclin D1 as well as expression and phosphorylation of p38 kinase and PI3K/Akt (117). In another study evaluating the anti-cancer effects of apigenin on anti-estrogen-sensitive and -resistant breast cancer cells, a biphasic effect of apigenin was observed. In fact, at low concentrations, apigenin induced cell growth by activating ERα-mediated gene expression, while at high doses it inhibited cell growth through reduction of ERα protein levels and inhibition of the activities of multiple kinases involved in anti-estrogen resistance (p38, MAPK, PKA and PI3K/Akt) (118).

The effects of flavanone and 2’-OH flavanone on MAPK signaling have been investigated by Hsiao et al. in lung cancer cells (A549). These compounds (10-50 µM) inhibited the phosphorylation of ERK1/2 and p38 kinase and the activation of NF-κB and AP-1, leading to a decreased expression of MMP-2 and u-PA (119).

The modulatory activity of quercetin on MAPK signaling was evaluated in several tumor cell lines. In the human hepatoma HepG2 cell line the treatment with quercetin induced a strong suppression of Akt and ERK1/2 phosphorylation but did not affect the expression of PI3K. In addition, quercetin inhibited NF-κB activation and upregulated the AP-1/JNK pathway. The modulation of these different signaling cascades induced apoptosis by direct activation of the intrinsic way (12,121). Conversely, quercetin arrested cell proliferation and induced apoptosis in A549 lung cancer cells, in a dose dependent manner, through the inactivation of Akt-1 and the enhancement of ERK-MEK1/2 phosphorylation (122).

Resveratrol, another polyphenol, was shown to impair MAPK signaling as well. Resveratrol induced growth inhibition and apoptosis by the transient activation of MAPK and inhibition of pS6 ribosomal protein expression in MDA-MB-231 breast cancer cells (123). This polyphenol exhibited modulatory activities on MAPK not only in breast cancer cells. In this regard, several studies reported the ability of resveratrol to affect MAPK signaling in different types of cancer. Parekh et al. demonstrated that resveratrol suppressed the growth of liver HepG2 cancer cells by down-regulating cyclin D1, p38 kinase, Akt and Pak1 expression and activity. In addition, the treatment with resveratrol increased phospho-ERK1/2 levels, inducing the activation of apoptosis (124). In human epidermoid carcinoma A431 cells, resveratrol exerted its anti-proliferative activity by inhibiting cyclin D1 and MEK1, ERK1/2 signaling and down-regulating c-Jun expression, which led to alteration of AP-1 activity and suppression of cell proliferation (125). In addition, combinatorial effects of resveratrol and black tea polyphenols on regression of tumor growth were reported by George et al. in BALB/c mice bearing skin tumors. The combinatorial treatment resulted in a significant regression of tumor volume and number and this phenomenon was associated with the reduction of MAPK (p38, ERK1/2 and JNK1/2) activity (126).

EGCG and theaflavins also inhibited the proliferation of DU145 and LNCaP prostate cancer cells by modulating PI3K and MAPK pathways. In this regard, EGCG and theaflavins decreased PI3K and phospho-Akt levels and enhanced ERK1/2 expression (127).

Impairment of PI3K and MAPK signaling pathways by curcumin has been demonstrated in a study by Sun et al. They showed that curcumin inhibited cell proliferation and induced cell cycle arrest in G1 phase (at 30 µM) and apoptosis (at 50 µM) in ErbB2/Skp2-over-expressing breast cancer cell lines (MDA-MB-231^ErbB2 cells). In addition, curcumin repressed the phosphorylation of ERK and Akt, without affecting JNK and p38 phosphorylation (128). The effects of the same polyphenol have been investigated in ovarian cancer cells as well. Curcumin induced p53-independent apoptosis through the down-regulation of Akt and phospho-Akt and the activation of p38 kinase, without affecting ERK1/2 activity in HEY cells (129).

Other studies have investigated the antiproliferative and differentiating activities of 5,7-dimethoxycoumarin on murine (B16) and human (A375) melanoma cell lines. The 5,7-dimethoxycoumarin reduced cell proliferation in a time- and dose-dependent manner by blocking cell cycle in G0/G1 phase. Furthermore, it was found for the first time that this compound inhibited the enzymatic activity of the activated MEK1/2, leading to a decrease of ERK1/2 phosphorylation (14,130). Effects of polyphenols on ErbB receptors signaling pathway in cancer cells are summarized in Table 1.