Anticancer

Curr. Med. Chem.
- Anti-Cancer Agents, 2002, 2, 441-463
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Green Tea Catechins as Novel Antitumor and Antiangiogenic Compounds

Michel Demeule, Jonathan Michaud-Levesque, Borhane Annabi, Denis Gingras, Dominique Boivin, Julie Jodoin, Sylvie Lamy, Yanick Bertrand, and Richard Bliveau*
Laboratoire de Mdecine Molculaire, UQAM-Hpital Sainte-Justine, Montral, Canada
Abstract: The concept of cancer prevention by use of naturally occuring substances that could be included in the diet is under investigation as a practical approach towards reducing cancer incidence, and therefore the mortality and morbidity associated with this disease. Tea, which is the most popularly consumed beverage aside from water, has been particularly associated with decreased risk of various proliferative diseases such as cancer and atherosclerosis in humans. Various studies have provided evidence that polyphenols are the strongest biologically active agents in green tea. Green tea polyphenols (GTPs) mainly consist of catechins (3-flavanols), of which (-)-epigallocatechin gallate is the most abundant and the most extensively studied. Recent observations have raised the possibility that green tea catechins, in addition to their antioxidative properties, also affect the molecular mechanisms involved in angiogenesis, extracellular matrix degradation, regulation of cell death and multidrug resistance. This article will review the effects and the biological activities of green tea catechins in relation to these mechanisms, each of which plays a crucial role in the development of cancer in humans. The extraction of polyphenols from green tea, as well as their bioavailability, are also discussed since these two important parameters affect blood and tissue levels of the GTPs and consequently their biological activities. In addition, general perspectives on the application of dietary GTPs as novel antiangiogenic and antitumor compounds are also presented.
INTRODUCTION The benefits of green tea in increasing alertness and staving off disease have been known for centuries. Most historians and botanists now believe that tea plant culture first began in China and was most likely brought to India, Korea, Sri Lanka and Japan; the human use of green tea is believed to have originated more than 5000 years ago [1, 2]. Various legends exist regarding the origins of tea. One of them is the story where the Emperor Sh'eng Nung took the first sip of tea about 2737 B.C. [2]. The legend holds that the emperor was relaxing outdoors under a tree for a royal nap listening to the perky bubbling of a small cauldron of water over a nearby fire. A gentle breeze wafted through the nearby trees, carrying with it a few leaves, which casually dropped into the boiling water. A less curious person would simply have thrown out the leaves or considered them a nuisance, but the emperor was intrigued by the aroma that resulted from this unexpected marriage of leaf and water. Ever game, he scooped a little of the leaf-spotted water into a small cup, and dared a sip then another and another. Soon he sent his servant to fetch more leaves, and a new beverage was born from the tree we now know as Camellia sinensis. The beneficial detoxification effects of tea leaves was first reported in a Chinese book on pharmaceutical plants written about 200 B.C. [3]. Tea was first considered as a
*Address correspondence to these authors at the Laboratoire de Mdecine Molculaire, Universit du Qubec Montral-Hpital SainteJustine, C.P. 8888, Succursale Centre-ville, Montral, Qubec, Canada H3C 3P8; Tel: (514) 987-3000, Ext. 8551; Fax: (514) 987-0246; E-mail: oncomol@nobel.si.uqam.ca
medicine until the Tang dynasty (A.D. 618-907), when tea was finally recognized and accepted as a beverage. For centuries, until the Ming dynasty (A.D. 1368-1644), green tea was the only type of tea produced in its homeland. In the early 8th century, green tea was transferred to Japan from China for medicinal use and historical records cite the existence of a powdered tea called hiki-cha (a very rough powdered tea, similar to the matcha green tea), which was bestowed upon priests summoned to the court of Japanese Emperor Shomu about A.D. 729. Later, in the 15th century, Japan ennobled tea into a religion of estheticism teaism [4]. Since then more than twenty schools of tea ceremony or tea cult have been developed, and the ceremonies themselves differ from very simple to hours-long elaborate performances [2]. Already in A.D. 1211, a renowned Zen Buddhist priest and teacher (Eisai, Kitcha Yojoki) said that tea is a miraculous medicine for the maintenance of health, an extraordinary power to prolong life and anywhere a person cultivates tea, long life will follow [2]. More recently pharmacologists, chemists, physicians, nutritionists and others in the field of science are recognizing the beneficial properties associated with daily consumption of green tea. Of particular interest are studies reporting that green tea reduces the risk of cancer, one of the major causes of mortality throughout the world [5, 6]. Like other chronic diseases, the main underlying causes of cancer are environmental in nature. Humans are exposed to a variety of carcinogens including tobacco smoke, alcoholic drinks, industrial carcinogens, aflatoxins, heterocyclic amines, Nnitroso compounds and polycyclic aromatic hydrocarbons. Dietary intervention is one way of influencing carcinogen metabolism. A wide variety of bioactive compounds and
2002 Bentham Science Publishers Ltd.
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their derivatives have been shown to inhibit carcinogenesis. In this context, green tea has been examined with increasing interest since a large body of evidence suggests that certain of its components have pronounced anti-cancer properties [7, 8, 9]. Worldwide, tea is a most popular beverage, second only to drinking water, its liquor is like the sweetest dew from heaven (Lu Yu A.D. 780) [2]. The tea plant is cultivated in more than 20 countries in Asia, Africa and South America and three main varieties of tea presently reach the market [10]. Black tea is the most prevalent (80%) and is the most popular tea in western countries. Oolong tea is less frequently consumed (about 1%) whereas green tea is most popular in China, Japan, North Africa, and the Middle East and represents about 20% of worldwide tea consumption [10, 11]. More recently, it has been suggested that green tea has antimutagenic and anticarcinogenic properties and, based on various reports, it has become clear that green tea is an effective chemopreventive agent for various types of cancer [12-15]. The components of green tea, such as polyphenols, caffeine, theanine and vitamins, give the specific taste and flavor and are the main reasons for which it is now one of the most popular drinks. Among all green tea components, several epicatechin derivatives (polyphenols) were reported to be effective chemopreventive agents against the initiation, promotion, and progression stages of multistage carcinogenesis [16]. This review describes general aspects of green tea catechins and their effects on crucial biological targets involved in the development of cancer. EPIDEMIOLOGICAL STUDIES ON THE RELATIONSHIP BETWEEN GREEN TEA POLYPHENOLS AND CANCER The major indication for green tea as a potential anticancer agent is the difference in cancer risk factor between Japanese and Westerners [17]. Many epidemiological studies have been conducted to examine the association between daily green tea consumption and cancer prevention. In some studies, green tea had protective effects on stomach [13, 1820], breast [21], bladder [22] and skin cancers [16]. Some other studies on colon, rectal, pancreas, oesophageal and lung cancers were, however, not conclusive [22]. As for lung cancer, a recent study established that the consumption of green tea was associated with a reduced risk of cancer among non-smoking women [23]. One possible explanation of these contradictory conclusions is that green tea consumption is not yet standardized. In addition, etiological factors as diverse as demographic characteristics, occupational history, medical history, family history, smoking, alcohol drinking, regular physical exercise, reproductive history, bowels habits, dietary habits and the differences in molecular mechanisms involved in various cancer types need to be controlled [13, 18]. Moreover, in recent animal studies in which experimental conditions were more controlled, GTPs suppressed the formation and growth of human cancer, including skin [24-26], lung [27], liver [28], and esophagus
[29] cancers. Other factors that could improve future studies are standardization of the quantity and the type of green tea catechin intake [30]. In this respect, during a phase I clinical trial, the maximum amount of tea that a cancer patient can consume was determined to avoid side effects related to caffeine, such as heartburn or sleeping difficulties. The dose recommended is 1.0 g/m2 of GTPs (equivalent to 7 or 8 japanese cups (120 ml) 3 times a day). Analyses of plasma from patients revealed an EGC6 concentration between 35 and 255 ng/ml (0.07-0.45 M) with no accumulation in various tissues over an 8 week period [31]. GENERAL ASPECTS OF GREEN TEA i. General Chemical Composition of Tea Leaves The tea plant is taxonomically classified as Camellia sinensis of the family of Theaceae, and is roughly divided into two varieties, assamica and sinensis. Like many crude extracts from natural products, green tea is a complex mixture of many substances. In general, the main substances found in green tea are caffeine (2-4%), amino acids (4%), lignin (6.5%), organic acids (1.5%), protein (15%), chlorophyll (0.5%) and the polyphenols (25-35%) [32, 33]. The yellowish green color of the unoxidized extracts is attributed to the chlorophyll content. A cup of green tea contains about 300 to 400 mg of polyphenols, which are essentially colorless. Of the polyphenols, epigallocatechin gallate (EGCG) and epigallocatechin (EGC) are the most important and it is estimated that a typical cup of green tea contains 10 to 30 mg of EGCG. Tea also contains flavonols, mainly quercetin, kaempferol, myricetin, and their glycosides. In black tea, the oxidation of polyphenols during processing leads to the formation of catechins and gallic acid complexes such as theaflavins, theaflavinic acids, thearubigins or theasinensis and proanthocyanidin polymers [32, 33]. Methylxanthines, caffeine and a small amount of theophylline and theobromine are also present [33]. Tea contains many amino acids but theanine, specific to the tea plant, is the most abundant, accounting for 50% of the amino acid content. Amino acid degradation is involved in the production of the tea aroma [32]. An important role in the development of the tea aroma is also imputed to chlorophyll, carotenoids, lipids and volatile compounds [33]. The volatile fractions of tea leaves have been studied in detail and more than 600 different molecules have been isolated [33-36]. These include terpenoids and degradation products of amino acids, carotenoids and linoleic acid [33]. Tea also contains carbohydrates, vitamins E, K, A and a low level of B vitamins. Vitamin C is present only in green tea since it is decomposed during the fermentation process of oolong and black teas. Tea also provides useful amounts of potassium, manganese and fluoride ions to the diet [37]. ii. Structures of Catechins Polyphenols are produced as secondary metabolites in higher plants. They are divided into two major groups: proanthocyanidins and the polyesters based on gallic acid
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and/or hexahydroxydiphenic acid and their derivatives [38]. The catechins shown in Fig. (1) are naturally occurring flavan-3-ols that are found in many foods of plant origin including fruits [39], wine [40], beer [41], chocolate [42] and
(abundantly) in tea [43-46]. These flavan-3-ols are classified as C15 compounds and their derivatives possess two phenolic nuclei (A ring and B ring) connected by three carbon units (C-2, C-3 and C-4). The flavanol structure of
Fig. (1). Catechin structures. (A) General structure of catechins. (B) Major catechins, epimers at the C-2 position and methylated catechin derivatives found in tea.
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catechins (3, 3', 4', 5, 7-pentahydroxyflavan) contains two assymmetric carbon atoms at C-2 and C-3. Catechins are synthesized in tea leaves through metabolism of malonic acid and shikimic acid. Gallic acid is derived from an intermediary product produced in the shikimic acid metabolic pathway. The major polyphenols in green tea, which normally comprise more than 75% of all tea polyphenols, are (-)epicatechin (EC), (-)-epigallocatechin (EGC), (-)epicatechin-3-O-gallate (ECG) and (-)-epigallocatechin-3-Ogallate (EGCG). The family of GTPs also includes four epimers of the major polyphenols at the C-2 position and four catechin derivatives methylated at the 3'-O-position of the gallic acid moiety. Finally, two other catechins have been identified in green tea: (-)-epiafzelechin (EZ) and (-)epiafzelechin gallate (EZG) [45]. iii. Catechin Content in Green Tea Many chemical, physicochemical and biochemical studies of green tea polyphenols have been performed over
Table 1.
the past few decades. High performance liquid chromatography (HPLC) has been the most useful approach for determination of the catechins and caffeine in aqueous and biological samples [43, 47-49]. Other chromatographic methods have also been used such as micellar electrokinetic chromatography [50, 51]. UV or fluorescence [52], diodearray [53] and electrochemical [54] detection have been widely used in HPLC analyses. However, the most powerful techniques for the determination of different compounds, owing to efficiency in analyte identification and quantification, used mass spectrometry (MS) technology, [55], tandem mass spectrometry (MS/MS) [56], atmospheric pressure chemical ionisation-mass spectrometry (ACPI-MS) [45] and electrospray MS [57] coupled to an HPLC. Most of these analytical methods are sensitive and appropriate for the simultaneous determination of various biologically active catechins in tea infusions. Table 1 shows an overview of various studies on the polyphenol content present in black, green and oolong teas. In green tea (-)EGCG content is the highest followed by (-)-EGC > (-)-ECG > (-)-EC. Other catechins ((-)-GC, (-)-CG and (+)-C) are usually found in trace amounts, indicating that they are
Catechin Concentration in Various Tea Leaves. Results Taken from Literature [46, 50, 57]
Dry weight of tea leaves (%)
Tea variety EGCG I. Black Tea Red Rosea Orange (Tetley)a Darjeeling tea II. Green Tea China green tea Leaves Crushed Lung Chin Jasmine
a
EGC
ECG
EC
GC
CG
0.62 0.43 4.5
0.067 0.078 1.37
0.38 0.33 1.42
0.13 0.091 0.19
nd nd nd
nd nd nd
0.033 0.035 nd
5.20 9.9 3.15 3.15 8.21 8.24 10.09 7.96 7.24
2.30 9.61 1.33 0.94 6.19 2.39 3.82 4.11 4.71
1.45 2.9 0.97 1.45 2.01 4.43 7.43 7.83 3.84
1.00 1.5 0.77 1.15 0.68 0.81 2.01 1.88 1.67
nd nd nd nd nd 0.31 0.17 0.60 0.67
nd nd nd nd nd 0.10 0.05 0.19 0.08
0.10 nd 0.088 0.23 nd 0.44 0.79 0.76 0.98
Darjeeling tea Inzatsu Benifuuki Gokou Yutakamidori III. Oolong Tea Tie Guan Yin Tong ting B Tong ting D
a
2.80 6.58 2.22
0.89 6.12 1.90
0.62 0.82 0.37
0.54 0.79 0.33
nd 0.36 0.08
nd 0.01 *
0.065 0.07 0.03
: Commercial blended teas (teas blended with fruits, flowers and/or spices) *: Trace nd: not determined
minor components of tea leaves. The content of green tea polyphenols varies strongly depending on the tea vintage (species of tea plant, the soil for tea cultivation, the method of processing tea leaves for manufacturing tea material and the season for harvesting) (Tables 1 and 2). The tea leaves harvested in early summer are usually superior in quality to those harvested in later seasons [3, 58]. However, the contents of polyphenols, theanine and caffeine are higher in summer than in spring (Table 2). One of the most important processes in tea manufacturing is fermentation; the conversion of tannin by polyphenol oxidase in the tea leaves. The fermentability (activity of polyphenol oxidase) in young tea leaves varies greatly depending on the kind of cultivars (cultivated varieties). Processed tea leaves are classified into three types according to the degree of fermentation, unfermented, semifermented and fully-fermented tea leaves, and they are respectively called green tea, oolong tea and black tea. A distinctive feature of green tea processing is that the leaves are never subjected to fermentation; in fact, the enzymes of the leaves are inactivated by steam. Since the steaming treatment protects catechins against degradation, the content of GTPs in green tea is much higher than in fermented teas [58]. Almost all of the tea plants grown in Japan are of var. sinensis and utilized for green tea preparation.
iv. Optimization of Catechin Extraction and Stability Optimization of Extraction To increase the beneficial effects of tea, it is important to optimize the extraction of EGCG, the polyphenolic antioxidant believed to be responsible for most of the cancer chemopreventive properties. [7, 54, 58]. We investigated the time-dependent extraction of EGCG with hot water (90OC). The majority of the EGCG content of green tea leaves was extracted after 6 to 8 min of infusion (Fig. 2A). Furthermore, the pH of the extraction water (between 3.0 and 10.0), the ionic strength (between 0 and 1 M NaCl) and the sample matrix were not found to alter the amount of EGCG extracted from the tea leaves [57]. As shown in Fig. (2B), there is a large variability in the EGCG content between different tea leaves. These results suggest that the vintage of tea and the length of the infusion are the factors which most affect the bioavailibility of EGCG. More recently, it was shown that epimerization at the C-2 position occurs during the infusion of tea with hot water [54]. Extracting the catechins from tea powder with H2Oacetonitrile (1:1) at 30OC for 40 min prevents this phenomena, leading to a 30% increase in the concentration of EGCG and EGC. These results suggest that most of the epimers at the C-2 position of catechins detected in tea
Table 2.
Content of Several Characteristic Chemical Components in Leaves of Genus Camellia. Results Taken from Literature are Expressed as Dry Weight of Tea Leaves (%) [3, 58]
Polyphenols Species C I. Thea C. sinensis var. sinensis (spring) var. sinensis (summer) var. assamica C. taliensis II. Camellia C. japonica var. japonica var. decumbens C. pitardii III. Paracamellia C. sasanqua C. oleifera
*: trace
Theanine EC EGC ECG EGCG
Caffeine
* 0.07 0.02 *
1.50 1.50 1.44 0.58
4.00 3.70 0.35 0.8
2.80 4.10 3.35 1.9
8.80 12.20 12.10 6.84
0.002 0.4 1.43 0.27
3.07 3.48 2.44 2.54
0.25 2.04 0.25
4.81 3.57 6.64
0 0 0.46
0 0 0
0 0 0
0 0 0
0 0 0
0 0.19
0.02 0
0 0
0 0
0 0
0 0
0 0
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Fig. (2). EGCG extraction from green tea leaves. (A) Tea was prepared by pouring 100 ml of 90OC water over 1.0 g of green tea leaves and stirring for 20 min. An aliquot of the extract was collected at the indicated time and analysed by RP-HPLC coupled with an UV detector. Solvent elution was performed at 1.0 ml/min and the mobile phase was composed of acetonitrile:ethyl acetate:H3PO4 0.05% (12:0.6:90). The detection wavelength was 280 nm and the EGCG concentration was determined by a calibration curve of pure EGCG. (B) EGCG concentration was measured in different teas. The teas were prepared by pouring 100 ml of 90OC water over 1.0 g of green tea leaves and stirring. An aliquot of the extract was collected after 10 minutes and analysed by RP-HPLC. The quality grade of the tea is referred to as: 1st, 2nd and 3rd. Pilo Chun is a Japanese emperors tea. (C) EGCG stability was evaluated in aqueous solution. The tea was infused for 10 min with hot water (90OC). Aliquots of the extract were collected and stored at 20OC (m), 4 OC (l), 25 OC ( ) and 40 OC (x). After the indicated time, these extracts were analysed by RP-HPLC.
leaves might be produced by the steam treatment used during green tea production. The epimerization of EGCG to GCG was also observed when green tea catechin and EGCG were autoclaved at 120OC for 20 min [59]. In fact, the relatively high amount of GCG found in some tea drinks is most likely due to epimerization of EGCG during autoclaving. Stability of Green Tea Catechins The stability of green tea catechins (GTCs) as a mixture was examined under various processing conditions [59]. This study demonstrated that lyophilized GTCs were stable at room temperature. However, when brewed at 98OC for 7 h, GTCs were degraded by 20%. GTCs in aqueous solution are more stable when they are stored at low pH. The addition of citric acid and ascorbic acid protects GTCs from degradation in commercially available soft drinks. This suggests that other ingredients used in the production of tea drinks may interact with GTCs and affect their stability. Thus, when canned and bottled tea drinks are produced, stored, and transported, the degradation of GTCs must be taken into consideration. We investigated the stability of EGCG following its extraction by hot water. EGCG was very unstable in aqueous solution (Fig. 2C); the half-life of this catechin at 20O, 4O, 25O and 40OC is respectively 25, 8, 3.5 and 2 days. EGCG diluted in an organic solvent such as ethanol remains more stable. EGCG stability was also determined when administered topically to human and mouse skin [60]. The stability of EGCG in hydrophilic ointment is dependent on time, temperature and degree of oxidation. For example, 10% of the EGCG is lost after 2 days at 37OC, but the same formulation supplemented with 0.1% butylated hydroxytoluene (BHT) has a significant longer stability with over 90% of EGCG remaining after 130 days at 37OC. Glycerin-based vehicles containing BHT were also shown to increase EGCG stability with 90% of the EGCG remaining after 76 days at 50C [61]. These results suggest that EGCG is susceptible to oxidation but, if supplemented with BHT, the hydrophilic ointment formulation could potentially be used in clinical trials of skin cancer prevention. v. Bioavailability, Metabolism, Distribution, Excretion and Biological Interactions of Catechins Bioavailibility and Metabolism It is important to consider the bioavailability of green tea flavanoids including absorption, distribution, metabolism and elimination, to get a comprehensive understanding of their possible impact on living organisms. Until recently, polyphenolic compounds were considered as antinutrients causing adverse effects by reducing the digestibility of proteins, and it was commonly held that polyphenols were poorly assimilated [62]. Unfortunately, the metabolism of tea polyphenols has still not been completely elucidated. Recently, the bioavailability and distribution of polyphenolic compounds after tea consumption in laboratory animals and humans has been investigated using sensitive and accurate methods [63-65]. The absorption, distribution and elimination of EGCG, EGC and EC in rats after
administration of decaffeinated green tea (DGT) and pure EGCG were studied [63]. Following intravenous injection of DGT (25 mg/kg), the plasma concentration-time curves of EGCG, EGC and EC were fitted to a two-compartment model. The -elimination half-lives (t1/2) were 212, 45 and 41 min for EGCG, EGC and EC, respectively; the clearances were 2.0, 7.0 and 13.9 mlmin/kg and the apparent distribution volumes (Vd) were 1.5, 2.1 and 3.6 dl/kg. When pure EGCG was given, a shorter t1/2, a larger clearance and a larger Vd were observed for EGCG. This suggested that other components in DGT could affect the plasma concentration and elimination of EGCG. While pure EGCG is poorly absorbed, EGCG glycosides show moderate to rapid absorption in man probably due to active glucose transport in the small intestines [64]. After intragastric administration of DGT (200 mg/kg), ~13.7% of EGC and ~31.2% of EC were present in the plasma, but only 0.1% of EGCG was bioavailable. After intravenous administration of DGT (25 mg/kg), the level of EGCG was found to be highest in the intestines. The intestinal EGCG level declined with a t1/2 of 29 min. The liver and lung levels of EGCG, EGC and EC were generally lower than in the intestine or in the kidney. These distribution results suggest that EGCG is mainly excreted through the bile, and that EGC and EC are excreted through both the bile and urine. A study was conducted in 18 individuals who were given different amounts of green tea, and the plasma concentration and urinary excretion of tea catechins were measured over time [65]. At all the concentrations tested, the half-life of EGCG was found to be longer than the half-life of EGC or EC. EGC and EC, but not EGCG, were excreted in the urine. It was also demonstrated that 90% of the total urinary EGC and EC were excreted within 8 hours. When the tea dosage was increased, the amount of EGC and EC excretion also increased, but no clear dose-response relationship was observed. This study provided basic pharmacokinetic parameters of GTPs in humans, which may be used to estimate the levels of these compounds after drinking green tea. Recently, a study was conducted to determine the pharmacokinetic parameters of pure EGCG administered topically to human and mouse skin [60]. Topical application of EGCG in hydrophilic ointment to human or mouse skin resulted in substantial intradermal uptake of up to 1-20% of the applied dose. However, transdermal penetration was observed only in mouse skin indicating that EGCG does not enter into the systemic circulation in human. This study concluded that topical application of EGCG in a hydrophilic ointment achieved a high concentration in skin but negligible systemic availability. Distribution of EGCG The distribution of [3H]-EGCG in mouse organs following direct administration into the stomach was studied [66]. Significant radioactivity was found in the digestive tract, liver, lung, pancreas, mammary gland and skin, as well as other organs such as brain, kidneys, uterus, ovary and testes. Within 24h, 6.6% (females) and 6.4% (males) of total administered radioactivity was excreted in the urine, while 37.7 and 33.1% were measured in feces. In addition, it was found that a second administration of [3H]-EGCG enhanced tissue levels. This result suggests that frequent consumption
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of green tea enables the body to maintain a high level of tea polyphenols. Excretion of Metabolites A study was conducted to detect catechin metabolites in the urine and plasma of human volunteers after ingestion of green tea [67]. These metabolites were identified as (-)-5-(3, 4, 5-trihydroxyphenyl)--valerolactone (M4) and (-)-5-(3, 4-dihydroxyphenyl)--valerolactone (M6). Both metabolites appeared to be produced by intestinal microorganisms, with EGC and EC as the precursors of M4 and M6, respectively. They were also detected in the plasma, exhibiting kinetics similar to those of the urinary metabolites, and in the feces. Catechin metabolites such as 4-hydroxybenzoic acid, 3,4dihydroxybenzoic acid, 3-methoxy-4-hydroxy-hippuric acid and vanillic acid have also been detected in urine [68]. Catechins pass through glucuronidation, sulfation and Omethylation in the liver of animals and humans [62, 69]. Omethylated derivatives have been detected in the plasma and are excreted in bile and urine. Conjugates can move from bile to the small intestine where they are deconjugated and cleaved by microorganisms to simple phenolic acids and lactones which are then absorbed via the intestinal mucosa [62]. Likewise, bacteria in the colon cleave the ring, producing valerolactones as well as phenylpropionic and benzoic acids. Biological Interactions of Catechins Polyphenols interfere with the absorption of other compounds present in the diet. Polyphenols have a strong affinity for proteins via the various phenolic groups, particularly when proteins have a high proline content as in caseins, gelatin and salivary proteins. However, the addition of milk to green or black tea does not affect the polyphenol concentration in plasma, indicating that milk does not reduce tea polyphenol bioavailability [70]. The large, flexible, poorly water-soluble polyphenols have the most effective protein-binding capacity [69]. The protein-binding capacity of polyphenols may reduce the digestibility of alimentary proteins and increase faecal nitrogen excretion in humans in a manner similar to that observed in herbivorous animals [69], even though the formation of protein-polyphenol complexes is limited to molecules accessible to soluble proteins. In rats, the excreted nitrogen comes from endogenous proteins rather than from dietary protein intake [62]. Tea flavonoids have a strong affinity for transition metal ions and form insoluble complexes with iron [71]. This binding in the gastrointestinal tract strongly inhibits iron absorption and reduces the bioavailability of non-heme iron. This phenomenon can be overcome by the presence of ascorbic acid. No adverse effect of green tea has been observed in a population eating a varied diet, but anemia was more prevalent in populations with a poor iron intake [69]. This finding has important implications mainly for people following a vegetarian diet. Vegetarians might be advised to drink tea between meals rather than during meals because the iron from plant sources is less available and its binding by tea could further reduce the amount of available iron in their diet. Inhibition in the absorption of zinc has been observed in rats [69]. Polyphenols can also interfere with the bioavailibility of sodium and aluminum but not with
manganese, calcium or magnesium [62]. Protection against cardiovascular diseases, atherosclerosis, cancer, gene mutations, bacterial growth and diabetes are growing concerns. Knowledge about the digestion, absorption and metabolism of tea by humans is not well documented and more research needs to be done. BIOLOGICAL CATECHINS ACTIVITIES OF GREEN TEA
The current view of the cellular and molecular events involved in the development of cancer identify six major alterations in cell physiology that collectively dictate malignant growth: self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, tissue invasion, and sustained angiogenesis [72]. All of these processes are highly complex but, in each case, several key regulatory mechanisms governing the development of malignancy have been unveiled, providing novel drug targets amenable to therapeutic intervention [72]. For these reasons, many studies have been conducted on the effects of green tea catechins on these processes. ANTITUMOR ACTIVITIES i. Antioxidant Properties Various agents that cause cancer, such as ionizing radiation, ultraviolet light, tobacco smoke, ozone and oxides of nitrogen in polluted air, induce DNA damage [73]. The cell damage induced by such agents is mainly caused by their production of free radicals. Under normal conditions, defense systems including specific enzymes and small molecules such as glutathione and uric acid work efficiently and balance the oxidants that are produced in the body during various metabolic activities. However, a higher amount of antioxidant intake is required when oxidant stress is increased [74]. The most important antioxidant enzymes are superoxide dismutase, catalase, and peroxidases. Polyphenols found in green tea have a greater antioxidant activity than do either vitamins C or E and are believed to be suitable for protection against reactive oxygen species (ROS) and their associated pathologies [75]. The reactivity of oxygen species varies from high for hydroxyl radical (HO), ferryl ion (Fe(II)) or copper ion (Cu2+), to lower reactivity for superoxide (O 2), peroxyl radical (ROO), hydrogen peroxide (H2O2), singlet molecular oxygen (1O-2), hypochlorous acid - (HOCl), plasma nitrite (NO 2), peroxynitrite (ONOO ) and alkoxyl radicals (RO) [75, 76]. GTPs, and -especially the gallic acid moeity are known to scavenge O 2, HO and ROO [78, 79]. It has been reported that the action of GTPs on DNA protection combines fastreaction, H-atom transfer, between GTPs and ROS [80]. Reactive oxygen species play an important role in carcinogenesis through damaging DNA, altering gene expression, or affecting cell growth and differentiation [81, 82]. The actions of GTPs affect a variety of cells and body compartments. In human plasma, the total antioxidant capacities of plasma increase as a function of green tea
consumption [83]. In human red blood cells, GTPs also protect the cell against lipid peroxidation [84]. In skin, GTPs inhibit skin cancer induced by solar ultraviolet radiation when applied topically or orally to mice [85, 86]. In rat hepatocytes, GTPs are most effective at inhibiting the 12-Otetradecanoyl phorbol 13-acetate induced NO generation involved in tissue damage and inflammation [87]. Other studies, in vitro, showed that catechins, especially EGCG, ameliorate the free radical damage sustained by DNA via a reduction in both prompt DNA single-strand breaks and in residual damage to the DNA base [80, 88]. Emphasis has been placed on understanding the molecular mechanisms involved in the antioxidant properties of green tea. An interesting feature of GTPs is their ability to perform their actions at different levels of the cell. For example, in the intracellular compartment, GTPs inhibited 12-O-tetradecanoylphorbol-13-acetate induced hydrogen peroxide formation in mouse epidermis [89] and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced 8hydroxydeoxyguanosine formation in mouse lung [90, 91]. NNK, a tobacco-specific nitrosamine, induces lung adenomas in mice following a single intraperitoneal (i.p.) injection. The critical events for NNK-induced lung tumorigenesis in mice are thought to involve O(6)methylguanine (O(6)MeG) adduct formation, GC-->AT transitional mispairing, and activation of the K-ras protooncogene [92]. GTPs are also thought to be responsible for decreased susceptibility to UVB-induced tumorigenesis. UVB induces H2O2 production and EGCG scavenges the H2O2 which would otherwise lead to UVB-induced phosphorylation of proteins in the Ras pathway [93]. In the extracellular compartment, EGCG lowers the plasma concentration of phosphatidylcholine hydroperoxide (PCOOH). PCOOH is the predominant membrane lipid hydroperoxide in human plasma. The reactivity of EGCG with ROS and its chelating activity with Cu2+ would be important for the reduction of PCOOH [94, 95]. In the plasma membrane compartment, EGCG also inhibits anion transport caused by deoxy-oxy cycling lipid peroxidation [84]. EGCG was also shown to inhibit the production of ONOO- and lipid peroxidation by suppressing the induction of iNOS [96, 97]. In summary, GTPs can scavenge ROS in all cellular compartments, in a variety of cells and in different body compartments before they have time to cause damage. ii. Effect of Green Tea Catechins on Signal Transduction Pathways Initial mechanistic attempts to understand the biological effects of GTPS focused not only on their antioxidant properties but also on their prevention of the mutagenicity and genotoxicity of certain chemicals [10]. Other research on GTPs evaluated their effects on tumor initiation and promotion as well as on detoxification enzymes and on trapping the activated metabolites of carcinogens. Various reports have identified molecular mechanisms that could be targets for GTPs [6, 10]. Mutations in DNA can result in the formation of neoplastic cells [98]. The signal transduction cascade, including the factors NFB (nuclear factor kappa B) and AP-
1 (activator protein 1) is redox-regulated and is therefore sensitive to the oxidant/antioxidant status of the cell [99]. Elevated MAP kinase and AP-1 activities are involved in many disease processes such as inflammation, neoplastic transformation, cancer cell invasion, metastasis and angiogenesis [100]. Interestingly, AP1 transcription factors and AP1 factor-associated signal transduction are important targets of EGCG action [101-103]. AP1 proteins consist of homodimers of jun proteins and heterodimers of jun and fos factors [104]. It has been reported that EGCG produces specific changes in AP1 factor function in immortalized and transformed keratinocytes. These changes include an EGCGdependent reduction in phorbol ester-dependent mitogen activated protein kinase activity [102] and reduced AP1 factor level and activity [100, 101]. EGCG also inhibits ultraviolet light-associated activation of c-fos gene expression and the accumulation of c-fos protein [101]. EGCG stimulates a Ras, MEKK1, MEK3, p38 cascade to increase AP1 factor-dependent involucrin gene expression in normal human keratinocytes [105]. To examine the antitumor promotion effects of EGCG at the molecular level, a JB6 mouse epidermal cell line has been used as an in vitro model for tumor promotion studies [106]. Under these experimental conditions, EGCG inhibited EGF- or TPAinduced cell transformation as well as AP1-dependent transcriptional activity and DNA binding activity. These results showed that the inhibition of AP1 activation occurs through inhibition of a pathway dependent on c-Jun Nterminal kinase. Mitogen-activated protein kinases (MAPKs), which are members of a serine/threonine kinase family, are activated in response to many extracellular stimuli in order to transduce signals from the cell membrane to the nucleus. One welldefined MAPK subfamily consists of the extracellularregulated kinases ERK1 and ERK2, which are strongly activated by growth factors via the Ras/Raf/MAPK-kinase pathway [107, 108]. Another emerging group of MAPKs are the c-Jun N-terminal kinases (JNKs), also called stressactivated protein kinases (SAPK) [109]. JNKs can be induced by stimuli as diverse as UV irradiation, -irradiation, heat shock, certain protein synthesis inhibitors, cytokines, growth factors and T cell activators [108, 110]. A third group of MAPKs are the p38 MAPKs, which are also activated by stresses such as DNA-damaging agents. A GTP extract was shown to induce chloramphenicol acetyltransferase (CAT) activity in human hepatoma HepG2 cells transfected with a plasmid construct containing an antioxidant-responsive element (ARE) and a minimal glutathione S-transferase Ya promoter linked to the CAT reporter gene [110]. This result indicates that GTPs stimulate the transcription of Phase II detoxifying enzymes through the ARE. In addition, GTP treatment of HepG2 cells resulted in a significant activation of ERK2 and JNK-1. Furthermore, GTP treatment also increased mRNA levels of the immediate-early genes c-jun and c-fos. Comparing the activation of MAPK by different polyphenols in human hepatoma HepG2-C8 cells, it was demonstrated that only EGCG showed potent activation of all three MAPKs (ERK, JNK and p38) in a dose- and timedependent manner, whereas EGC preferentially activated ERK and p38 [111]. In another study, EGCG was shown to inhibit the activation of ERK1 and ERK2 in rat vascular smooth-muscle cells, as well as the induction of c-fos and
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egr-1 following the activation of PDGF-R [112]. However, in an H-ras transformed mouse epidermal cell line (30.7b Ras 12), EGCG decreased the levels of phosphorylated Erk1/2 and MEK1/2 [113]. In this study, EGCG seemed to inhibit the association of Raf-1 with MEK1 as well as the phosphorylation of Elk-1 by Erk1/2. The possible mechanisms of action underlying the antiproliferative effect of EGC on vascular smooth muscle cells (A7r5) were also investigated [114]. The activity of the serum-stimulated membranous protein tyrosine kinase (PTK) is inhibited by EGC. It was also found to reduce the levels of c-jun mRNA, phosphorylated JNK1, and JNK1-kinase activity. These results suggest that the antiproliferative effect of EGC on A7r5 cells may partly be mediated through inhibition of protein kinase activity, reducing c-jun mRNA expression and inhibiting JNK1 activation. Ultraviolet (UV) radiation has been identified as a complete skin carcinogen, possessing both tumor-initiating and tumor-promoting properties [115]. Early studies have demonstrated that topical application or ingestion of green tea polyphenols or EGCG act to inhibit tumor initiation and promotion by chemical carcinogens or UV light in mice [89, 116]. EGCG had an inhibitory effect on UVB-induced c-fos gene expression [102], p38 mitogen-activated protein kinase activation [102, 93] and phosphorylation of ERK1/2 and JNK [93]. In another study, EGCG inhibited UVB-induced activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effectors, Akt and p70 S6-K [117]. iii. Effect of Green Tea Catechins on Receptor Tyrosine Kinase Activities A wide variety of growth factors elicit mitogenic signals through autophosphorylation of their respective receptors. Autophosphorylation triggers the tyrosine phosphorylation of several substrate proteins, resulting in their selective interaction with the activated receptors [118] and activation of downstream signaling pathways that ultimately lead to gene transcription and to cell proliferation. Thus, classic growth factors such as PDGF- and EGF propagate their mitogenic signals through autophosphorylation of the PDGF -receptor (PDGF-R) and EGF receptor (EGF-R) on tyrosine residues. Under physiological conditions, the receptor tyrosine kinases, such as the PDGF-R and EGF-R, are at equilibrium between the inactive, unphosphorylated and the active, phosphorylated states. Enhanced activity of receptor tyrosine kinases (RTKs), including PDGF-R, EGF-R and VEGF-R, has been implicated as a contributing factor in the development of malignant proliferative diseases such as cancer [119, 120]. As enhanced activity of receptor tyrosine kinases has been implicated in the pathogenesis of many cancers (and other nonmalignant proliferative diseases such as atherosclerosis), inhibition of the intracellular signaling pathway of growth factors is crucial for preventing the development of cancer and cardiovascular disease [120]. For this reason, inhibition of growth factor receptordependent signal transduction as a means of blocking tumor growth has received considerable attention, leading to the development of several compounds that selectively inhibit distinct classes of receptors [120, 121]. However, the potential bioactivity of natural compounds, including green
tea catechins, for inhibiting RTK activities has not been studied in detail. EGF Receptor Overexpression of EGF receptor can produce a neoplastic phenotype in cells and is associated with a number of human tumors including breast cancer [122], squamous cell carcinoma of the lung and oral cavity [123], bladder carcinoma and esophageal cancer [124]. An inhibitory effect of green tea catechins on RTK activity was first suggested by studies performed on the EGF receptor [125]. In this study, the authors explored the inhibition of growth exhibited by EGCG on human epidermoid carcinoma A431 cells, which express high levels of epidermal growth factor (EGF) receptors. In these cells, EGCG significantly inhibited DNA synthesis. By in vitro kinase assay, the inhibitory activity of EGCG was shown to be more effective against receptor-type protein tyrosine kinases such as EGF-R, PDGF-R, and FGFR (IC 50 value of 0.5-1 g/ml) than towards nonreceptor-type protein tyrosine kinases, such as pp60v-src (IC50 > 10 g/ml). EGCG showed a more selective inhibition of EGF-R (IC50 = 0.5 g/ml) than of other receptor-type PTKs examined in this study. By contrast, EGCG scarcely inhibits serine- and threonine-specific protein kinases, such as protein kinases A and C, when present extracellularly at 20 g/ml. EGCG also inhibited the EGF-stimulated increase in phosphotyrosine levels in A431 cells [126]. In addition, EGCG and ECG blocked the binding of EGF to its receptor, resulting in the inhibition of EGF-R kinase activity. These results suggest that EGCG could block many signal transduction events originating from the cell surface and might be a useful compound for blocking RTK signaling. However, the effects of EGCG on EGF-R remain unclear since other studies failed to reproduce earlier observations in A172 glioblastomas [127] and in vascular smooth muscle cells from rat aorta [112]. PDGF Receptor PDGF and its receptors have been implicated in a number of cancer diseases including glioma [128], sarcoma [128], breast cancer [129], colon cancer [130] and melanoma [131], as well as in the pathophysiology of atherosclerosis. For this reason, inhibition of PDGF-R represents an interesting target for therapeutical intervention. Recent reports showed that several non-toxic compounds isolated from green tea are also able to act as PDGF-R tyrosine kinase inhibitors. Treatment of A172 glioblastoma cells with 50 M CG, ECG, EGCG and 25 M Tyrphostin 1296 resulted in inhibition of the PDGF-BB-induced tyrosine phosphorylation of PDGFR [1 27]. The PDGF-R downstream intracellular transduction pathway, involving tyrosine phosphorylation of phospholipase C-1 (PLC-1) and PI3K was also inhibited. Tyrphostin 1296 is a selective inhibitor of the tyrosine phosphorylation of PDGF-R [120]. In order to demonstrate whether there is a correlation between the inhibitory effects of CG, ECG, EGCG or Tyrphostin 1296 with tyrosine phosphorylation of PDGF-R and the transformation of A172 cells, spheroid formation of these cells in the presence and absence of CG, ECG, EGCG or Tyrphostin 1296 was examined. Spheroid formation was almost completely inhibited by 50 M ECG, CG and EGCG and by 25 M Tyrphostin 1296.
In another study from the same group [112], the treatment of vascular smooth muscle cells (VSMCs) from rat aorta with EGCG (50 M) resulted in an almost complete inhibition of the PDGF-BB-induced activation of the MAP kinase isoforms p44mapk/p42mapk. In contrast, EGCG (1-100 M) did not influence MAP kinase activation by EGF or by angiotensin II, a substance acting through a G-coupled receptor. The maximal effect of PDGF-BB on c-fos and egr1 mRNA expression was completely inhibited in EGCGtreated VSMCs, whereas the effect of EGF was not affected. Quantification of immunoprecipitated tyrosine-phosphorylated PDGF-R, PI3K, and phospholipase C-1 revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, these authors showed that spheroid formation of human glioblastoma cells (A172) and colony formation of sistransfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 M EGCG. Overall, these studies strongly suggest that green tea compounds may represent attractive new PDGF-R antagonists and may be useful for the treatment of diseases in which this receptor is involved [128, 132]. Although GTPs also affect the VEGF-R, these effects will be presented and discussed in the section below on the antiangiogenic properties of green tea. iv. Induction of Apoptosis and Cell Cycle Arrest by Green Tea Catechins The anticarcinogenic properties of green tea observed in animal models [133-136] have led investigators to test whether tea catechins could inhibit the growth of a variety of cancer cell lines and kill them by inducing programmed cell death (apoptosis). Suppression of apoptosis is thought to play a role in the development of tumors and in the acquisition of resistance to chemotherapy [72, 137]. Cancer cell and endothelial cell apoptosis is now regarded as a therapeutic target both for the discovery of new drugs and for the identification of cancer-preventive agents. Apoptotic cell death is a complex, tightly regulated process that involves drastic structural changes in cell morphology, including chromatin condensation, disassembly of the nuclear and cytoplasmic networks, DNA fragmentation and membrane blebbing [138, 139]. This results in fragmentation of the cell into apoptotic bodies that are rapidly phagocytosed by neighboring cells. The activation of a unique family of cysteine proteases named caspases plays a central role in the signaling and execution phases of apoptosis when induced by a variety of stimuli, including cytokines like TNF, radiation and chemotherapeutic drugs [140]. Caspases exist in cells as inactive zymogens that are activated by proteolytic cleavage upon appropriate apoptotic signals. Activated caspases selectively cleave critical target proteins, leading to dismantling of the cells architecture, signaling apparatus, and repair mechanisms and leading to subsequent internucleosomal DNA fragmentation [141]. GTPs and Apoptosis The first evidence that catechin compounds induce apoptosis was reported in 1996 [142]. The growth of human lymphoid Molt 4B cells was strongly inhibited by the catechins EGC and EGCG at 100 M and by other catechins,
such as C, EC and ECG, at higher concentrations. EGC and EGCG, but not C, EC nor ECG, induced the degradation of genomic DNA into internucleosomal fragments (DNA ladder), a characteristic of cells undergoing apoptosis. EGCG has also been shown to inhibit growth and induce apoptosis of a variety of mammalian cell lines, including human carcinoma cells [143, 144], human leukemia cells [145], prostate cancer cells [146], human lung adenocarcinoma cells [147], and SV40 transformed human lung fibroblasts [148]. In one study, EGCG strongly inhibited the growth of two lung tumor cell lines (H661 and H1299) and EGCG-induced apoptosis in H661 cells was demonstrated by Annexin V and TUNEL assays [27]. Interestingly, EGCG treatment was also found to induce the formation of H2O2 in H661 cells and EGCG-induced apoptosis was completely inhibited by adding exogenous catalase to the cell culture. EGCGinduced H2O2 formation was observed in other cell lines where, again, destruction of peroxides by catalase inhibited EGCG-induced apoptosis [149]. In order to clarify whether growth inhibition and induction of apoptosis by GTPs are specific to cancer cells, one study compared the effects of EGCG on the growth of SV40-transformed lung fibroblast cells (WI38VA), a colorectal cancer cell line (Caco-2), a breast cancer cell line (Hs578T) with its effects on their normal counterparts [148]. EGCG treatment had a strong inhibitory effect on the growth of SV40-immortalized WI38VA cells but had little effect on normal WI38 cells. Similarly, EGCG was a more potent inhibitor of the growth of cancer cells Caco-2 and Hs578T than of their normal counterparts CCD-33Co and Hs578Bst, respectively. After 24h treatment with EGCG, the transformed WI38VA cells became massively apoptotic whereas only a small percentage of normal WI38 cells were undergoing apoptosis. Similarly, EGCG induced apoptosis in human carcinoma keratinocytes HaCaT and human epidermoid carcinoma (A431) cells but not in normal human epidermal keratinocytes (NHEK) [143, 150]. The induction of apoptosis by EGCG in the human lung cancer cell line PC-9 was synergistically enhanced by another tea catechin (EC) and by cancer-preventive agents such as sulindac and tamoxifen [151]. These results suggest that the green tea beverage, containing a mixture of polyphenols, might be better than EGCG alone for cancer prevention and that a combination of cancer-preventive agents might be more effective than EGCG alone. Since high concentrations of EGCG (75-100 M) were used to study the synergistic induction of apoptosis, it would be interesting to know whether EGCG also acts synergistically at the concentrations (0.1-1 M) found in the blood of tea drinkers [147]. EGCG-induced apoptosis in human chondrosarcoma cells (HTB-94) was shown to involve caspase-3 activity [152]. Pretreatment of cells with a broad spectrum inhibitor of caspases, or with a specific caspase-3 inhibitor, prevented EGCG-induced cleavage of poly(ADP-ribose) polymerase (PARP), a caspase-3 substrate, and EGCG-induced cell death.
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In a recent study, it was suggested that the binding of EGCG to the Fas receptor (also known as CD95 or APO-1) might trigger Fas-mediated apoptosis in human monocytic leukemia U937 cells [153]. Treatment of U937 cells with high concentrations of EGCG (50-400 M) resulted in the processing of procaspase-8, induction of caspase-8 activity (as measured with an exogenous substrate) and formation of a DNA ladder. DNA fragmentation was not observed when the caspase-8 inhibitor Ac-IETD-CHO was co-incubated with EGCG, suggesting the involvement of the deathreceptor-associated caspase-8 in the apoptotic cell death. Affinity chromatography, using EGCG immobilized on agarose, showed that the Fas receptor was retained on the column after loading a U937 cell extract. This study suggested direct interaction between EGCG and the Fas receptor; however, the exact nature of this molecular interaction remains to be established. Although it is now well established that GTPs induce apoptosis in several cancer cell lines, the significance of this activity in relation to an anticarcinogenic effect of green tea is unclear. It is particularly difficult to reconcile data obtained from tissue culture with that from animal models because apoptosis is only observed in most cancer cell lines when using high concentrations of GTPs, 10 to 100 times higher than the concentration found in the blood of treated animals or tea drinkers. Future studies which use lower concentrations of GTPs are needed in order to better assess the contribution of GTP-induced apoptosis to the beneficial effects of tea consumption. The molecular targets and the precise mechanism of action of the GTPs involved in the induction of apoptosis are still unknown and this area of research will continue to be a focus of future investigations. GTPs and Cell Cycle Arrest EGCG treatment has been shown to result in a G0/G1phase cell cycle arrest and in apoptosis of human epidermoid carcinoma (A431) cells [143] and human prostate carcinoma (LNCaP) cells [154]. EGCG-induced cell cycle arrest and apoptosis have been studied in greater detail in A431 cells and appears to involve modulation of cycle kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery [155] and the inhibition of NFB [155]. EGCG did not inhibit growth of normal human epidermal keratinocytes (NHEK) nor did it induce in them cell cycle arrest or apoptosis. Interestingly, EGCG-induced inhibition of NFB in NHEK occurred only at higher concentrations of EGCG (40 and 80 M); this contrasts to A431 cells where a strong inhibition of NFB occurred at 10 M EGCG. How EGCG inhibits NFB and how this is related to cell cycle deregulation and apoptosis remains unknown. v. Reversal of Multidrug Resistance The development of resistance to multiple drugs is another serious problem in cancer chemotherapy and has been under intensive investigation by numerous research groups. Many tumors that initially respond to chemotherapy subsequently develop a multidrug resistance (MDR) phenotype associated with the expression of the human MDR1 gene product, P-glycoprotein (P-gp). Multidrug
resistance mediated by the drug-efflux pump P-gp is one mechanism used by tumor cells to survive chemotherapeutic drugs [156]. Recent observations have challenged the notion that P-gp has evolved merely to mediate the efflux of xenotoxins out of healthy cells and raised the possibility that P-gp and related transporters might play a fundamental role in regulating cell differentiation, proliferation and survival [157]. P-gp acts as an efflux pump that extrudes many chemotherapeutic agents out of the cells, decreasing their intracellular concentration. This transporter exports a wide variety of structurally unrelated compounds including anticancer agents such as vinka alkaloids, epipodophyllotoxins, antibiotics and antracyclines. Molecules other than cancer agents are also reported to be transported by P-gp. For example, cytokines, bilirubin, opioids and steroids are substrates of P-gp [158-160]. The development of non-toxic P-gp modulators that block its transport activity has been a pharmaceutical challenge, in order to reverse the MDR phenotype. Such P-gp modulators, when co-administered with chemotherapeutic agents such as cyclosporine A, enhance intracellular accumulation of the cytotoxic drugs within tumor cells [161]. P-gp is normally expressed in tissues such as brain and testis at the blood-tissue barrier, in kidney, adrenal glands, lung, liver and intestines [162-164]. At the blood-brain barrier, P-gp plays an important role in brain protection. Its expression at the luminal side of endothelial cells in brain capillaries prevents the passage of many agents into the brain [165-167]. Furthermore, this protein is involved in detoxification of the organism by excreting toxic compounds from the blood into the bile, urine and gastrointestinal tract [165]. P-gp was also suggested to be involved in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum [168]. It was recently reported that P-gp is localized in caveolar microdomains in MDR cells and in the endothelial cells of the blood brain barrier[169, 170]. This particuliar localization could be useful for understanding the role of P-gp in drug elimination and transport. Considerable interest is growing in order to identify the targets of GTPs and to understand the molecular mechanisms affected by green tea and its major constituents. P-gp is frequently targeted in cancer treatment because this protein confers the MDR phenotype, by which cancer cells exposed to a single chemotherapeutic agent become simultaneously resistant to that drug as well as to drugs of unrelated structure or function. The effects of GTP catechins on P-gp function were recently investigated in order to verify whether they could be used as P-gp modulators [171]. The effects of GTPs on P-gp binding and transport activities were studied with CHRC5 cells, a multidrugresistant cell line that overexpresses P-gp. Photoaffinity labeling experiments using [125I]-iodoaryl azidoprazosin (IAAP), a P-gp substrate which can be cross-linked to P-gp, showed that GTPs compete with IAAP for the substrate binding site of P-gp and, consequently, photolabeling of Pgp with IAAP decreases in the presence of increasing concentrations of GTPs (Fig. 3A). Furthermore, these polyphenols inhibit the efflux of rhodamine 123, a P-gp
substrate, and increase its accumulation in CHRC5 cells (Fig. 3B). Thus, GTPs compete with other P-gp substrates for the binding and transport activities of P-gp, indicating that at least one component of these polyphenols could be carried by this transporter [171]. Each catechin present in green tea was studied in order to identify which are responsible for the inhibition of P-gp. Among the catechins present in GTPs, the three catechins gallate esters EGCG, ECG and CG inhibit both the binding and transport activities of P-gp, whereas EGCG is the most potent P-gp modulator (Fig. 3C and 3D). The proportions of EGCG, ECG and CG in polyphenols from green tea samples used in this study were 33, 4 and less than 1% (w/w), respectively. Thus, the effect of these polyphenols on P-gp was mainly caused by EGCG. Taken together, these results suggest that GTPs and particularly EGCG, which interacts with P-gp and inhibits its transport activity, could be used as
modulators of P-gp function. Moreover, P-gp transport inhibition by GTPs is a rapid and reversible process [171], in contrast to results seen with another P-gp substrate, PSC 833 [172]. Since P-gp has many physiological functions in normal tissues, inhibition of this multidrug transporter during cancer chemotherapy should be rapid and reversible in order to reduce undesirable side effects. Furthermore, EGCG has been reported to inhibit cell growth [88]. We observed that treatment of multidrugresistant CHRC5 cells with EGCG in combination with the chemotherapeutic agent vinblastine potentiates the toxicity of vinblastine. The IC50 of vinblastine for CHRC5 cells exposed to vinblastine in combination with EGCG was the same as the IC50 for vinblastine alone in AuxB1 cells, indicating that EGCG sensitizes CHRC5 cells, which overexpress P-gp, to levels found in non-MDR cells. Thus, by inhibiting P-gp, EGCG enhances vinblastine
Fig. (3). Inhibition of P-gp substrate binding and transport activities by green tea polyphenols. Photolabeling of P-gp with [125I]-iodoaryl azidoprazosin (IAAP) in CHRC5 membranes was performed in the presence of various concentration of GTPs (A) or 50 M of each major catechin (C). Briefly, CH RC5 membrane proteins were incubated with GTPs along with IAAP and then cross-linked with UV light. Proteins were resolved by gel electrophoresis and the levels of photolabeled P-gp were estimated by autoradiography and analysed by laser densitometry. Results are expressed as percentage of photolabeling in the absence of GTPs. Rhodamine accumulation was measured in the presence of various concentrations of GTPs (B) or 100 M of each major catechin (D). The accumulation of rhodamine 123, a P-gp substrate, was increased in CHRC5 cells in the presence of GTPs or pure catechins. Results are expressed as a percentage of rhodamine accumulation in the control cells.
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accumulation in CHRC5 cells and therefore increases its toxicity [171]. Other studies have also observed that EGCG enhances the antitumor activity induced by doxorubicin and adriamycin [173, 174]. Thus GTPs, and especially EGCG, could improve the efficacy of cancer treatment by increasing the accumulation of chemotherapeutic drugs in cancer cells by blocking P-gp function. Further in vivo studies will be required in order to verify that the same effects are observed in humans. It has been reported that the intestine weakly absorbs EGCG. Expressed in the epithelial cells of the intestine, P-gp could contribute to the low intestinal absorption of this catechin. By using Caco-2 cells, which form an intestinal monolayer, we have shown that EGCG interacts with P-gp in this intestinal model, suggesting that P-gp in the apical membranes of intestinal cells may reduce the absorption of EGCG by transporting this catechin back into the lumen of the intestine [171]. Interestingly, other compounds derived from natural products have already been reported to inhibit P-gp. Other natural compounds known to interact with P-gp include resveratrol, curcumin, genistein, caffeine, theanine, components of fruit juices such as quercetin, naringin and furanocoumarins from grapefruit and methoxyflavones from orange [171, 174-179]. Thus, during oral chemotherapy, the diet of patients may influence the efficacy of therapy using P-gp substrates, suggesting that cancer treatment planning
must take into account the diet of each patient. The anticancer properties of GTPs are summarized in Fig. (4). ANTIANGIOGENIC ACTIVITIES There is increasing evidence supporting the central role of angiogenesis in tumor growth and metastasis. Angiogenesis, the growth of new vasculature, is an absolute requirement for the maintenance and progression of most solid tumors. As a consequence, tremendous efforts have been made to identify antiangiogenic molecules with antitumor properties. This has led to the developement of a variety of molecules that are directed against critical aspects of angiogenesis such as cell adhesion, extracellular matrix degradation and the stimulation of endothelial cells by angiogenic cytokines or growth factors. This interest in targeting tumor-induced angiogenesis as a means of blocking tumor progression stems from several observations that tumor cells cannot grow significantly in the absence of blood vessels [180] and that molecules interfering with angiogenesis have potent antitumor properties in animal models [181]. Moreover, since endothelial cells are genetically stable, inhibitors specific to these cells should not induce resistance in tumors in contrast to cytotoxic compounds (antimitotic, antimetabolites and alkylating agents) for which resistance is commonly observed [182].
Fig. (4). Anticancer activities of green tea polyphenols. 1) Antioxidant properties of GTPs protect the cell against DNA damage by scavenging reactive oxygen species (ROS). 2) GTPs, mainly EGCG, modulate key players in various signal transduction pathways such as AP-1, MAPK, PI 3-K, p70S6-K and Akt. 3) GTPs reduce the activity of tyrosine kinase receptors (PDGF-R, EGF-R) which contribute to the malignant proliferation of tumor cells. 4) GTPs induce cell apoptosis in tumor cells. 5) GTPs reverse the MDR phenotype by blocking Pgp efflux of anticancer drugs.
Extensive studies on the cellular and molecular processes underlying angiogenesis have identified key events associated with tumor-induced neovascularization: (1) stimulation of endothelial cells by tumor-derived angiogenic cytokines, such as vascular endothelial growth factor (VEGF), resulting in increased endothelial cell proliferation and migration, (2) secretion of matrix-degrading enzymes, such as matrix metalloproteinases and plasminogen activators, resulting in digestion of the surrounding extracellular matrix (ECM), and (3) formation of a three dimensional capillary network in the vicinity of the tumor cells, allowing their sustained growth by providing oxygen and essential nutrients. These cellular and molecular steps all represent attractive antiangiogenic targets and have led to the identification and development of a variety of compounds targeting vessel formation, endothelial cell proliferation or migration [183]. i. Inhibition of Microvessel Formation The potential use of antiangiogenic molecules as inhibitors of tumor progression was first suggested by the identification of angiostatin, a plasminogen (Pgn) fragment, in the serum and urine of syngenic mice bearing Lewis lung carcinoma (LLC) [184]. The protein contains the first four triple loop disulfide-linked regions of Pgn known as kringle domains, and showed significant inhibitory activity towards endothelial cell functions [185]. Several other endogenous inhibitors of angiogenesis have subsequently been described which are fragments of abundant proteins and which gain the ability to inhibit endothelial cell function upon proteolytic cleavage. These include the plasminogen fragment kringle 5 [185], the collagen fragments endostatin [186], canstatin [187], and tumstatin [188], as well as fragments derived from fibronectin [189], prolactin [190], MMP-2 [191] and calreticulin [192], among others. These molecules inhibit endothelial cell proliferation, migration and capillary-like structure formation in vitro [185-192]. The antiangiogenic effect of green tea was first suggested by the observation that green tea extracts block neovascularization in the chick embryo neovascularisation assay [193]. This inhibitory effect of green tea extract was reproduced by EGCG, suggesting that this particular constituent may be responsible for the antiangiogenic effects of green tea but the mechanisms involved this remain for the most part unknown. ii. Inhibition of Tyrosine Phosporylation of VEGF Receptor VEGF receptors play a major role in tumor angiogenesis and thus represent attractive targets for the development of novel anticancer therapeutics. Recently, we reported that the antiangiogenic effects of green tea were correlated with the inhibitory activity of catechins towards VEGF receptors (Fig. 5) [194]. Addition of low, physiological concentrations (0.011 M) of EGCG, ECG and CG markedly inhibited the VEGF-dependent tyrosine phosphorylation of VEGF receptors [194], EGCG being the most potent inhibitory catechin. Interestingly, the effect of EGCG on VEGFR-2 phosphorylation was very rapid, as maximal inhibition of the
receptor was observed after only 30 minutes. Furthermore, the extent of VEGFR-2 inhibition induced by EGCG was comparable to that of Semaxanib (SU5416), a VEGFR-2 specific antagonist that is currently being developed for the treatment of solid tumors. Overall, these data suggest that the antiangiogenic effect of green tea catechins could be related to the inhibition of VEGF receptor activity and that these molecules could serve as lead compounds for the design of novel VEGF receptor antagonists. In this respect, it is interesting to note that the effect of green tea catechins on VEGF receptor function are correlated with a pronounced effect of these molecules on the formation of capillary-like structures by endothelial cells in Matrigel, a laminin-rich reconstituted basement membrane matrix [194]. Matrigel represents a rich source of many angiogenic factors that likely influence the response of the cells to form capillarylike structures [195]. Under these experimental conditions, the catechins disturbed the assembly of endothelial cells into tube-like structures. Since there is strong evidence that blocking VEGFR-2 activity limits the ability of most tumors to stimulate the formation of blood vessels [196], it is possible that EGCG, CG and ECG catechins may also ultimately disturb the assembly of endothelial cells into capillary-like structures by blocking VEGFR-2 activity. Futhermore, these catechins seem to be more inhibitory than SU5416, a specific VEGFR-2 inhibitor, possibly reflecting additional activity against other events in the angiogenesis process involving other tyrosine kinase receptors (e.g. PDGFR) and ECM degradation mediated by matrix metalloproteinases (see section 2.3). Interestingly, green tea catechins appear to be more inhibitory towards VEGF receptors than PDGF receptors. In the studies on PDGFR function, high concentrations of EGCG were needed to inhibit PDGF-R (IC50 = 20 M) in vascular smooth muscle cells [112] or in A172 glioblastoma cells [131]. The treatment of A172 cells with 1 to 20 M CG, ECG and EGCG had no effect on the tyrosine phosphorylation of PDGF-R. The inhibitory effect appeared at 50 M. By contrast, under our experimental conditions, physiological concentrations of EGCG (IC50 = 0.01 M) were sufficient to block VEGF receptor activity (Fig. 5B). Thus, according to these results, the green tea catechins seem to be more active against VEGFR than against PDGFR. In addition, when we investigated the effect of EGCG on the EGF receptor, no effect of catechins was observed on the EGF-induced increase in tyrosine-phosphorylated EGFR, as reported previously [131]. EGCG was also found to inhibit angiogenesis induced by colon cancer cells through the blockade of ERK-1 activation, ERK-2 activation and VEGF expression [197]. Blockade likely occurred at the transcriptional level since EGCG inhibited VEGF promoter activity, although high concentations of EGCG were required for significant inhibition. In vivo, daily intraperitoneal injections of EGCG (1.5 mg/day) inhibited tumor growth and diminished microvessel density, possibly through inhibition of tumor cell proliferation and induction of both tumor and endothelial cell apoptosis [197]. Taken together with the data on the inhibitory effect of catechins on VEGF receptors, these results emphasize the antiangiogenic effects of EGCG.
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Fig. (5). Inhibition of VEGF-induced tyrosine phosphorylation of VEGFR-2 in endothelial cells by green tea catechins. Bovine aortic endothelial cells were incubated in serum-free medium in the presence of 25 M of the major catechins for 24 hrs (A) or with various concentrations of EGCG (B). The medium was replaced with serum-free medium without catechins and endothelial cells were stimulated with 1 nM recombinant VEGF for 1 min. The extent of tyrosine phosphorylation was determined by immunoprecipitation of VEGFR-2 followed by immunoblotting with anti-Tyr(P) monoclonal antibody. The results indicate that EGCG strongly inhibits phosphorylation of VEGFR-2.
iii. Inhibition of Extracellular Matrix Degradation by Green Tea Catechins Matrix metalloproteinases (MMPs) represent a growing family of zinc-dependent proteases involved in matrix turnover during normal and pathological processes, and play a crucial role in several physiological processes involving interactions between cells and the ECM [198]. In this section, MMPs are presented as new molecular targets for GTCs. Evidence is also provided for a central role for membrane-type 1-MMP (MT1-MMP) in acquisition of the invasive properties of cancer cells; it may thus represent a direct target for green tea in the chemoprevention of cancer. Inhibition of Matrix Metalloproteinases Two particular members of the MMP family, gelatinases A and B (MMP-2 and MMP-9), seem to play an important role in tumor invasion and metastasis [198]. These two type IV collagenases are the dominant MMPs released by most epithelial and endothelial cells. They are involved in the turnover of basement membrane collagen under basal conditions and of other matrix proteins during angiogenesis, tissue remodeling, and repair [198]. Recently, it was reported
that collagenases from mouse lung carcinoma cells are inhibited by green tea catechins and black tea theaflavins [199]. We have further investigated the effects of different biologically active components from natural products, including GTPs, resveratrol, genistein and organosulfur compounds from garlic on purified MMP-2, MMP-9 and MMP-12 activities [200]. MMP-12 is specifically expressed by macrophages and is associated with inflammatory processes [201]. GTPs caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or casein as substrates. The inhibition of purified MMP-2, MMP-9 and MMP-12 by GTPs was confirmed by zymography and was also observed against MMPs associated with various rat tissues and with human brain tumors (glioblastoma and pituitary tumors) [200]. The most potent inhibitors of these MMP activities were EGCG and ECG. While MMP-9 exhibited the highest affinity for GTPs, the IC 50 values obtained for MMP inhibition ranged between 0.3-28 M. Recently, it was proposed that the anti-cancer activity of EGCG was associated with the inhibition of urokinase, a key player in tumor invasion and metastasis, which is frequently expressed in human cancer [202]. Based on computer
molecular modeling, it was proposed that EGCG could effectively bind to urokinase. This was later confirmed by measuring the inhibition of urokinase activity by EGCG via spectrophotometric amidolytic assays. However, the concentration of EGCG needed to inhibit urokinase activity was much higher than that needed to inhibit MMPs, indicating that MMPs are much sensitive to GTP and thus represent a better target for these polyphenols [200, 203]. MT1-MMP-Mediated Events as New Targets for EGCG Action MMPs are secreted by cells as proenzymes that must be cleaved in order to become functional. Cleavage of the prodomains of MMPs is generally mediated by soluble MMPs or by serine proteases such as plasmin, plasma kallikrein and neutrophil elastase [198, 204]. In contrast to most MMPs, proMMP-2 possesses a propeptide that is not susceptible to proteolytic cleavage by serine proteinases [205] and it is now well established that part of its activation is distinct and involves another member of the MMP family, the membrane-type 1-MMP (MT1-MMP) [206]. Interestingly, the MT1-MMP-mediated events only recently received attention as regulators of cell invasion and morphogenesis, and were shown to be affected by GTPs [203, 207]. We have reported that GTPs, and particularly EGCG, potently inhibited the activation of proMMP-2 in glioblastoma cells and in MT1-MMP-transfected COS-7 cells [200, 208]. More recently, these results were extended by showing that EGCG interfered with proMMP-2 activation through transcriptional regulation of the MT1-MMP gene [207]. Since MT1-MMP is responsible for the activation of proMMP-2 at the cell surface, these results strongly suggest that EGCG may interfere with the activity of this enzyme by regulating intracellular MT1-MMP gene expression (Fig. 6). As previously reported, EGCG also inhibits activator protein 1 (AP-1) function, which has been associated with the invasive and metastatic characteristics of cancer cells [99, 209]. One class of genes that AP-1 regulates is MMPs, and increased MMP secretion has been associated with AP-1 activity in the MCF-7 breast cancer cell line [210]. This suggests that EGCG may have access to and interact with extracellular and intracellular proteins potentially involved in signal transduction pathways. Moreover, EGCG was demonstrated to specifically down-regulate the gene expression of MT1-MMP in glioma cells, but not that of MMP-2, indicating that the specific mechanisms underlying transcriptional regulation of MT1-MMP may impair initiation of the invasive phenotype of these cells. Intriguingly, although most MMP genes are known to be strongly regulated by AP-1 and AP-2 [209, 211], the consensus binding sites for these two transcription factors were absent from the MT1-MMP promoter region [212] and only AP-2 binding sites were found in the MMP-2 promoter region [213]. This implies that some unique MT1-MMP transcriptional control elements, which remain to be identified, exist and are involved in the EGCG inhibitory effect. Although MMP-2 gene expression was unaffected by GTCs, EGCG was shown to reduce proMMP-2 secretion very efficiently and rapidly through a mechanism affecting some vesicular trafficking pathway from the intracellular MMP-2 pool to the plasma membrane [207].
Specific Inhibitory effect of EGCG on MT1-MMP-Induced Cell Migration It is now well established that MT1-MMP enables and regulates cell invasion activity [214-216]. Accordingly, overexpression of MT1-, MT2-, and MT3-MMP in epithelial cells promoted the invasion of basement membranes whereas overexpression of a wide range of soluble MMPs did not [214]. To further establish the involvement of MT1-MMP in the inhibitory effect of EGCG, the effect of this catechin on the migratory potential of U-87 glioma cells was examined. Migration of U-87 cells was found to be specifically inhibited by EGCG but not by EGC [207]. In addition, EGCG was reported to suppress invasion of human HT-1080 fibrosarcoma cells [203, 217] and of mouse lung carcinoma LL2 cells [199]. COS-7 cells, which lack endogenous MT1MMP, had their cell migration increased only when they overexpressed MT1-MMP protein, and this migration was also specifically antagonized by EGCG [208]. This observation implies that EGCG may directly inhibit the MT1-MMP activity that is crucial for cell migration because
Fig. (6). EGCG has a dual effect on proMMP-2 activation and protein secretion. U-87 glioblastoma cells were serum-starved and treated for 18 hrs with EGCG in the presence or absence of Concanavalin-A (Con-A). (A) MMP-2 activity was assessed by gelatin zymography using the corresponding conditioned media whereas MT1-MMP was immunodetected in cell lysates by Western blots. (B) Total RNA was extracted from U-87 glioblastoma cells, some of which were treated with 25 M EGCG for 18 hrs in serum-deprived media, and then subjected to RT-PCR analysis of MMP-2 and MT1-MMP genes. The results indicate that EGCG inhibits the gelatinolytic activity of MMP-2 as well as its processing to an active form through a transciptional regulation of MT1-MMP gene.
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it did not affect the MT1-MMP transcript levels in COS-7 cells which were transiently transfected with MT1-MMP. Direct demonstration of such an interaction was also recently reported [203]. The inhibitory effect of EGCG observed on cellular migration thus suggests that this catechin may also bind and interact with specific intramolecular sites of proteins and specifically target MT1-MMP. Also supporting this hypothesis is the low IC50 value obtained for the EGCG effect on MT1-MMP, which is comparable to the EGCG plasma concentration (0.3 M) measured in humans after consumption of tea [193]. The possible inhibition of other membrane-type MMPs by EGCG is currently under investigation. In conclusion, the inhibition of MMPs as well as the activation of proMMP-2 by MT1-MMP located at the tumor cell surface are thought to represent crucial steps in tumor invasion and metastasis. Whether the mechanism of action of green tea can be generalized to other protease systems also deserves further investigation. One recent study demonstrated that EGCG potently inhibited the chymotrypsin-like activity of the proteasome in vitro (IC 50= 86-194 nM) [218]. Since the proteasome plays a critical role in the specific degradation of cellular proteins [219], allows tumor cell cycle progression as well as protects cells against apoptosis [220], inhibition of the proteasome activity by EGCG may contribute to the cancer-preventative effect of green tea. Taken together with numerous recent reports on
the inhibitory effects of these molecules on various aspects of cancer cell biology, these observations emphasize the pleiotropic anticancer properties of GTPs by targeting the angiogenic molecular mechanisms that are summarized in Fig. (7). PERSPECTIVES ON GREEN TEA CATECHINS AND CANCER Both epidemiological and laboratory studies have shown an inverse association between high intake of green tea and the development of various cancer types. Several recent findings indicate that the biological activities of various enzymes important in human carcinogenesis and involved in signal transduction pathways, angiogenesis, regulation of cell death and multidrug resistance, are affected by GTCs and especially by EGCG. Taken together, these studies indicate that EGCG targets multiple mediated cellular events in cancer cells and provides new mechanisms for the anticancer properties of green tea. From a traditional beverage associated with rituals, tea is now viewed as a healthy drink, a source of pharmacologically active molecules, an important member of the antioxidant food group, and a functional food endowed with beneficial health properties [8]. The beneficial biological activities of GTPs have raised the possibility for the food industry to produce new products in which these
Fig. (7). Antiangiogenic activities of green tea polyphenols. Several molecular mechanisms involved in angiogenesis are targeted by GTPs, particularly by EGCG, even at very low concentrations (<1 M). The activity of VEGFR-2, the expression of MT1-MMP and secretion of MMP-2 are inhibited by GTPs. Catechins also inhibit the activity of MMP-2, MMP-9 and MMP-12 as well as cell migration.
molecules have been added. For example, because of the synergy with other natural products, encapsulated polyphenols (green tea) with isoflavones (soybean), curcumin (turmeric), silymarin (milk thistle), flax lignans (flax) and selenium (yeast) are now available as a food supplement (PhytostatinTM) with anticancer properties. Clinical trials are currently being conducted to evaluate the efficacy of green tea extracts. A phase I study recently established the daily dose and safety of green tea as an anticancer agent and the National Cancer Institute cooperative group program is sponsoring a phase II study to determine the effectiveness and toxicity of green tea extracts with patients suffering from androgen-independent metastatic prostate cancer. Moreover, a phase III randomized study has started to determine the effect of a diet low in fat and high in soy, fruits, vegetables, green tea, vitamin E, and fiber on prostate-specific antigen (PSA) levels in patients with prostate cancer (http://www.nci.nih.gov/occam/ trials.html). If these trials and future studies show a benefit, the next step would be to convince Westerners to switch from their usual black tea to the Asian green variety. Thus a better understanding of the molecular events regulating the bioavailibility and the biological activities of GTCs may be useful in designing better chemopreventive strategies against cancer. As EGCG is a potent inhibitor of gelatinases and an orally available pharmacologic agent that may confer the antiangiogenic and antitumor activities associated with green tea, the clinical usefulness of these GTCs either as chemopreventive agents or for the design of structural analogs represents an interesting area for future research. ACKNOWLEDGMENTS This work has been supported by grants from the Fondation Charles-Bruneau of Hpital Ste-Justine, the Natural Sciences and Engineering Research Council of Canada and from the Cancer Research Society to R.B. REFERENCES
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Curr. Med. Chem.

- Anti-Cancer Agents, 2002, 2, 441-463

Green Tea Catechins as Novel Antitumor and Antiangiogenic Compounds

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Green Tea Polyphenols and Cancer

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Curr. Med. Chem. Anti-Cancer Agents, 2002, Vol. 2, No. 4

0.62 0.43 4.5

0.067 0.078 1.37

0.38 0.33 1.42

0.13 0.091 0.19

5.20 9.9 3.15 3.15 8.21 8.24 10.09 7.96 7.24

2.30 9.61 1.33 0.94 6.19 2.39 3.82 4.11 4.71

1.45 2.9 0.97 1.45 2.01 4.43 7.43 7.83 3.84

1.00 1.5 0.77 1.15 0.68 0.81 2.01 1.88 1.67

nd nd nd nd nd 0.31 0.17 0.60 0.67

nd nd nd nd nd 0.10 0.05 0.19 0.08

0.10 nd 0.088 0.23 nd 0.44 0.79 0.76 0.98

2.80 6.58 2.22

0.89 6.12 1.90

0.62 0.82 0.37

0.54 0.79 0.33

0.065 0.07 0.03

Green Tea Polyphenols and Cancer

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Theanine EC EGC ECG EGCG

1.50 1.50 1.44 0.58

4.00 3.70 0.35 0.8

2.80 4.10 3.35 1.9

8.80 12.20 12.10 6.84

0.002 0.4 1.43 0.27

3.07 3.48 2.44 2.54

0.25 2.04 0.25

4.81 3.57 6.64

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