Mechanisms By Which Flavonoids Inhibit Tumor Metastasis

Cancer mortality is primarily due to metastasis. In order for cancer cells to metastasize they must be able to degrade the basement membrane (BM), traverse the extracellular matrix (ECM), enter the lymphatic or vascular system, adhere to a distant site, and then invade the local tissue. BM and ECM are composed of several proteins with type IV collagen the major structural component. Penetration of BM and ECM by cancer cells through proteolytic degradation has been suggested to be the first step in invasion and metastasis. One of the important groups of proteolytic enzymes involving tumor invasion/metastasis is matrix metalloproteinases (MMPs). Another proteolytic enzyme is urokinase-type plasminogen activator (uPA). Besides proteolytic enzymes, tumor angiogenesis and cancer cell adhesion to ECM also play critical roles in cancer metastasis. Understanding the basic principles by which flavonoids inhibit tumor invasion and metastasis may lead to the development of new therapeutic strategies, in addition to supporting the role of flavonoids as cancer chemopreventive agents. Available evidence suggests that flavonoids may exert their anti-invasion and antimetastasis activities by modulating some of these critical events. In this section, the evidence of flavonoids on modulating the activities of proteolytic enzymes, angiogenesis, and adhesion is discussed.

16.3.1 Flavonoids and Angiogenesis

Angiogenesis, the formation of new blood vessels by sprouting from preexisting endothelium, is a critical event for tumor growth and metastasis. Angiogenesis is a complex process involving a series of cellular events other than proliferation. Formation of new capillaries begins with a localized breakdown of the basement membrane of the parent vessel via the finely tuned elaboration of proteolytic enzymes and their inhibitors, followed by migration of endothelial cells and invasion to the surrounding ECM. The proteolytic degradation of ECM components by capillary endothelial cells is one of the key prerequisites of the angio-genic process. In tumors angiogenesis is persistently upregulated. Although the mechanisms leading to persistent pathological angiogenesis are still unclear, resulting evidence indicates that it is due to an imbalance between angiogenic factors and inhibitors.59

Available evidence has demonstrated that one of the mechanisms by which flavonoids inhibit cancer cell invasion and metastasis is via inhibition of angio-genesis. Among flavonoids, EGCG and genistein have been shown to be potent anti-angiogenic compounds, and may be responsible, at least in part, to the antigrowth and antimetastasis effects of tea and soy products, respectively. On the other hand, there is no sufficient evidence to demonstrate whether apigenin or quercetin has anti-angiogenesis activity. Therefore in this section, we focus on the experimental evidence on anti-angiogenesis activities of tea polyphenols and soy isoflavones. Effects of Tea Polyphenols on Angiogenesis

Because the angiogenesis process involves a series of cellular events, a series of in vitro and in vivo methods is commonly used to identify potential anti-angio-genic agents. These assays include in vitro proliferation, migration, invasion, and tube formation of endothelial cells, in vivo angiogenesis assays, and in vivo tumor growth inhibition associated with modulation of markers for angiogenesis. The anti-angiogenesis activity of tea polyphenols, especially EGCG, has been evaluated by using these in vitro and in vivo angiogenesis assays. EGCG significantly inhibited the endothelial cell proliferation,60 migration,61 and invasion62 in vitro, suppressed endothelial cell tube formation61,62 in vitro, and inhibited angiogenesis in vivo.62 Further tumor growth inhibition studies showed that EGCG inhibited the growth of both colon 26 NL17 carcinoma and Meth A sarcoma, in part through the inhibition of angiogenesis.62 In addition, drinking green tea also significantly inhibited angiogenesis in vivo.60

Several in vitro studies also determined whether the inhibitory effect of EGCG on invasion of endothelial cells is via downregulation of MMP, and found that EGCG inhibited MMP activities in endothelial cells in vitro.61,63,64 It suggests the possibility that tea polyphenols suppress angiogenesis and metastasis in part via inhibition of MMP activities in endothelial cells. Effects of Soy Isoflavones on Angiogenesis

Among soy isoflavones, genistein has been shown to be a potent anti-angiogenic isoflavone. The anti-angiogenic ability of soy isoflavones was first identified by Fotsis and co-workers.65 They fractionated the urine samples of human subjects consuming a diet rich in plant products and found that fractions containing soy isoflavones and metabolites (genistein, daidzein, O-desmethylangolensin, or equol) inhibited basic fibroblast growth factor-stimulated proliferation of bovine brain-derived capillary endothelial cells (BBCE).65 Further studies indicated that pure genistein had a potent and dose-dependent inhibitory effect on proliferation of BBCE at IC50 of 5 |M.66 Genistein also inhibited the proliferation of other vascular endothelial cells such as that derived from bovine adrenal cortex, and aorta.66

The effects of genistein on angiogenic factors were determined to elucidate the mechanisms by which genistein might inhibit angiogenesis. Tumor cells produce angiogenic factors that include platelet-derived growth factor, vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and platelet-derived endothelial cell growth factor, to stimulate neovascularization. Among angiogenic factors, VEGF, an endothelial cell-specific mitogen and a vascular permeability, plays an important role in tumor angiogenesis. Genistein inhibited angiogenesis by reducing VEGF levels via post-transcriptional regulation of VEGF expression67 in vitro. Genistein also inhibited VEGF-promoted endothelial cell proliferation6868» and basic fibroblast growth factor-mediated vascular endothelial cell proliferation69 in vitro.

Besides its anti-angiogenic activity in vitro, genistein has shown anti-angio-genesis activity in vivo. Wietrzyk and co-workers70 measured the volume of blood present in tumor tissue as a marker of angiogenesis, and found that genistein treatment reduced tumor blood supply by 35%. We studied the anti-angiogenic activities of both genistein and genistein-enriched SPC in a series of animal tumor models, and found that their antigrowth and anti-angiogenesis effects on bladder tumors and prostate tumors were associated with significant inhibition of tumor microvessel density, a marker of angiogenesis in vivo.12 30 71 72 Although genistein and SPC did not significantly reduce angiogenesis in MCF-7 tumors, the combination of SPC with tea significantly reduced angiogenesis,73 which suggests that one possible mechanism by which soy and tea synergistically suppress estrogen-dependent breast tumors may be via interactions that impede tumor angiogenesis. We also determined the molecular targets that may be responsible for anti-angiogenesis activity of soy components and found that the expression of basic fibroblast growth factor, not VEGF, was slightly but significantly down-regulated by genistein in vivo.30

16.3.2 Flavonoids and Proteolytic Enzymes

MMPs are naturally occurring, Zn2+-dependent, endopeptidases, involved in normal turnover of connective tissue matrix, as well as in certain disease processes such as cancer cell invasion and metastasis. MMPs are secreted in proenzyme forms and activated extracellularly. The activated forms of MMPs can be inhibited by tissue inhibitors of metalloproteinases (TIMPs). uPA is a serine proteinase that functions in conversion of the circulating zymogen plasminogen to the active serine proteinase plasmin. The biological activity of uPA is regulated by a functional interplay between the proteinase, its receptor, uPA receptor (uPAR), and the uPA inhibitor 1 (PAI-1).

One of the most important aspects for tumor metastasis is via its invasion through the ECM. MMPs produced by tumor and stromal cells are believed to play a key role in the degradation of ECM instrumental to invasion. Both in vitro and in vivo studies have suggested that one of the mechanisms by which fla-vonoids inhibit tumor metastasis may be via inhibition of MMP activity. Among these flavonoids, tea polyphenol EGCG and soy isoflavone genistein have been shown to be potent inhibitors of MMPs. Although flavonoids apigenin and quer-cetin have anti-invasion and antimetastasis activities, their effects on MMPs have not been adequately studied. On the other hand, the effects of flavonoids on uPA activity have not been adequately studied and experimental results have been insufficient for discussion. This part of the chapter will primarily focus on modulation of MMP activities by tea polyphenols and soy isoflavones. Effects of Tea Polyphenols on MMP Activities

A series of in vitro studies has been conducted to determine the effects of tea polyphenols, especially EGCG on the expression and activity of MMP. The MMP expression is usually determined by RNA and/or protein levels, whereas its activity is measured by zymography. Tea catechin significantly inhibited B16-F10 melanoma cell invasion in vitro by inhibition of the activities of MMP-2 and MMP-9.9 EGCG significantly inhibited the invasion of B16-F3m melanoma cells and MMP-9 activity,8 inhibited invasion of TRAMP-C1 prostate cancer cells in vitro and downregulation of MMP-2 activity,14 and inhibited the gastric cancer cell invasiveness and MMP-9 expression.19 EGCG, theaflavin, and theaflavin digallate significantly inhibited invasion of mouse Lewis lung carcinoma LL2-Lu3 cells in vitro in part by inhibition of MMP activities.15 ECG, EGCG, and theaflavin significantly suppressed the invasion of fibrosarcoma HT1080 cells and the expression and activity of MMP-2 and MMP-9.18

More in vitro studies have further determined how tea polyphenols modulate the activities of MMP-2 and MMP-9. The membrane type 1 matrix metallopro-teinase (MT1-MMP) is the receptor and the major activator of MMP-2. EGCG inhibited MMP-2 activity in part by significant inhibition of MT1-MMP activity.74,75 Other studies showed that EGCG inhibited MMP-9 expression through the suppression of MAP kinase via inhibition of the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2)75 and/or activator protein (AP)-1 activation.19

Despite promising data from the in vitro studies, few animal studies have determined the correlation between the antimetastasis effect of tea polyphenols and the inhibition of MMP activities. In one animal study, oral feeding of green tea polyphenols as the sole source of drinking fluid to TRAMP mice resulted in significant inhibition of the tumor metastasis and MMP-2 and MMP-9 expres-

sion.76 Effects of Soy Isoflavones on MMP Activities

Some in vitro studies have been conducted to determine the effects of soy isofla-vones, especially genistein, on the expression and/or activity of MMPs. Genistein inhibited both constitutive and EGF-stimulated invasion in ER-negative human breast carcinoma lines and the downregulation of MMP-9 and upregulation of TIMP-1 in vitro,28 inhibited invasion of HT1080 fibrosarcoma cells in vitro by suppression of conversion of latent forms of MMP-2 and MMP-9 into active forms and by increase of TIMP-1 expression,37 and inhibited invasion of head and neck cancer cells by downregulation of MMP-2 and MMP-9 secretion.40 Genistein and glycitein inhibited Jurkat cell invasion, in part through the down-regulation of MMP-13 activity and MMP-8 expression.4377 Genistein inhibited transforming growth factor-beta-1 (TGF-ß1)-stimulated cell migration and invasiveness of mouse transformed keratinocytes in vitro and increased uPA expression/secretion.41

In contrast to in vitro studies, few animal studies have determined the correlation between antimetastasis activities of soy isoflavones to their inhibition of MMPs. Although genistein inhibited invasion of oral squamous cell carcinoma cells in vitro,42 the animal study failed to show the significant effects of genistein supplementation on inhibition of tumor growth and metastasis, or the expression of MMP-2 and basic fibroblast growth factor.42

16.3.3 Flavonoids and Cancer Cell Adhesion

Altered cell adhesion ability is suggested to play a critical role in cancer cell migration, proliferation, invasion, and metastasis. The invasiveness of cancer cells strongly depends on their ability to migrate and to adhere to the ECM. Cell adhesion is primarily mediated by a class of proteins called integrins. Integrins are cell surface aß-heterodimeric glycoproteins. To date, 24 aß-heterodimers formed by 8 ß and 18 a subunits are identified.78 Integrins bind to various ECM proteins such as collagen, fibronectin, laminin, and vitronectin, and their binding activates important intracellular signaling pathways that regulate cell proliferation, migration, invasion, and metastasis. Experimental evidence suggests that one of the mechanisms by which flavonoids such as tea polyphenols and soy isoflavones inhibit cancer cell invasion and metastasis may be via modulation of the expression of integrins. Effects of Tea Polyphenols on Cancer Cell Adhesion

The effects of EGCG on modulation of integrins have been studied in several cancer cell types in vitro. EGCG inhibited invasion of the pediatric brain tumor-derived medulloblastoma cells in vitro, in part via modulation of specific cell surface integrins, notably upregulation of ß1 integrin.21 Laminin is a major gly-coprotein of basement membrane and plays significant roles during cancer cell invasion and metastases. Tea catechin inhibited laminin-promoted MO4 brain cancer cell attachment in vitro.79 EGCG also inhibited the adhesion of mouse melanoma B16 cells to laminin in vitro.80 Effects of Soy Isoflavones on Cancer Cell Adhesion

The effects of genistein on modulation of cancer cell adhesion are basically dependent on its protein tyrosine kinase activity. Protein tyrosine phosphorylation occurs as one of the earlier events in cancer-ECM interaction.59 Genistein, as a protein tyrosine kinase inhibitor,81 significantly inhibited invasion of B16-BL6

melanoma cells in vitro associated with suppression of the tyrosine phosphorylation of proteins located at the cell periphery when cells attached to and interacted with ECM.3233 Focal adhesion kinase (FAK) is a protein tyrosine kinase located at the focal adhesion sites of spreading cells. It is a pivotal element in the signaling cascade associated with cell-ECM interaction. It is suggested that suppression of adhesion-induced tyrosine phosphorylation of FAK may interrupt cancer cell-ECM interaction and subsequent invasion and metastasis potential of cancer cells. Genistein inhibited FAK activity in vitro.82

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