ВЫВОДЫ И БУДУЩИЕ НАПРАВЛЕНИЯ

2. INTRODUCTION

Tea represents the most widely consumed beverage in the world next to water, and is grown in at least 30 countries around the world. It is distinguished by the presence of catechins, a subgroup of polyphenols, known for their powerful antioxidant properties. Significant data amassed from many laboratories around the world provide convincing evidence that tea consumption has a preventive effect on carcinogenesis. A typical cup of brewed green tea contains in the extract 30-40% catechins. After consumption, tea polyphenols can be detected in both intact forms and as metabolites that could reach sub-micromolar concentrations in blood plasma (4). Absorption appears to take place in the small intestine, with intact metabolites reaching the colon, where they undergo extensive bacterial degradation (5).

Only a number of studies have examined the bioavailability and biotransformation of tea polyphenols and other catechins. In some cases, biotransformative pathways result in metabolites that are more active than the parent drugs (6). It is increasingly important to determine plasma and tissue levels of catechin metabolites and their biological significance to gain insight into therapeutic and preventative effects. Thus understanding the full array of mechanisms of tea polyphenols can facilitate the design of improved strategies for preventing and treating cancer.

In addition, a better understanding of the mechanisms of action will provide a rationale for the clinical development of tea polyphenol alone or in combination strategies. The initial mechanism-based studies using combinational and molecular cancer therapies focused on inhibition of mutagenesis (7), angiogenesis (8), and cell proliferation (9). It has been found that in human plasma the maximum concentration of the green tea supplement would reach upon 4,400 pmol/ml (10). This concentration of EGCG would be sufficient to exert antioxidant and other biological activities in the bloodstream. With this known many health products including beverages, foods, health care products, and cosmetics are now supplemented with the extracts of green tea.

In the present review, we will thus discuss the recent advances of tea polyphenols and their applications in the prevention and treatment of human cancers. Much emphasis is given to their defined molecular targets and their mechanisms of action. We also discuss previously conducted and ongoing clinical trials of polyphenolic compounds in different cancer types.

3. TEA POLYPHENOLS

3.1. History and tea ingredients

Tea is very rich in polyphenolic constituents which have high anti-inflammatory, antioxidant, and antimutagenic properties in various biological systems. Tea contains large amounts of various flavanoids, which are characterized by having the benzopyrane skeleton, with the pyrane ring bearing at least one aromatic ring. A major class of flavanoids is catechins, which include epicatechin (EC), epigallocaetchin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG) (11) (12) (Figure 1). EGCG is the most abundant of the catechins, and accounts for 50-75% of the total amount of catechins. Other catechins, such as catechin gallate, gallocatechin, gallocatechin galllate, epigallocatechin digallate, methylepicatechin and methyl EGC are present in smaller quantities. Flavanols, including quercetin, kaempferol, myricetin, and their glycosides are also present in tea. One bag of green tea contain between 80 and 100 mgs of the polyphenols with EGCG accounting for about 25 to 30 milligrams. Other ingredients in green tea include threonine, which constitute about 4-6% weight of dried tea and is responsible for its characteristics flavor. Catechins are present in higher quantities in green tea as compared to black and oolong tea, because of the different methods of leave processing after harvesting.

The manufacturing of black tea involves crushing of the leaves to promote oxidation and subsequent condensation of tea polyphenols in a process known as fermentation, which leads to the formation of theaflavins (TFs) and thearubigins. A benzotropolone ring structure is present in the flavins and is responsible for its unique black tea taste and bright red-orange color. Thearubigins, which have higher molecular weight, are poorly characterized chemically and biochemically.

3.2. Pharmacokinetics and bioavailability of green tea

Fewer studies have been conducted on the biological activities of the metabolites of green tea polyphenols. Under in vitro conditions, EGCG is biologically more active in cancer cells towards growth inhibition as compared to their metabolites. The polyphenolic structure of EGCG also makes them good hydrogen donors, allowing EGCG to bind tightly to proteins and nucleic acids. Recently, EGCG has been shown to bind strongly to Bcl-2 protein, laminin receptor, vimentin and proteasome, contributing to its anticancer activities (19-20). These studies may provide important insights into the design of future strategies aiming towards the development of green tea extracts (GTE) or green tea polyphenols (GTP) as a better chemopreventive agent.

3.3. Chemical properties of tea constituents

3.3.1. Antioxidant nature of green tea polyphenols (GTPs)

Emerging evidence has placed considerable emphasis on the antioxidative properties of tea polyphenols. Tea polyphenols prevent the formation of reactive oxygen species (ROS) and are strong metal ion chelators. The strong antioxidant nature of tea polyphenols is attributed to their polyphenolic structure (21). The B ring consisting of vicinal dihydroxy or trihydroxy groups in GTPs is the preferred site for antioxidation. The free radical scavenging activities of catechins have been well studied. ROS such as superoxide radical, singlet oxygen, hydroxyl ROS, nitric oxide, nitrogen dioxide, and peroxynitrite are found to be precluded by the scavenging ability of tea polyphenols. EGCG induced inhibition of soybean lipoxygenase served as the earliest experimental evidence for its antioxidative properties (22). It was later reported that EGCG could inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative damage in Hela cells, prevented Cu⁺²-mediated oxidation of low density lipoprotein (LDL), decreased tert-butyl hyperperoxide-induced lipid peroxidation, and blocked the generation of ROS from NADPH-cytochrome P450-mediated oxidation (23). Sato et al reported the structural characteristics for superoxide anion radical-scavenging and productive activities of green tea polyphenols. In the polyphenols with the pyrogallol-type B-ring and/or galloyl group, electron-withdrawing substituents (carbonyl and ketal carbons) and/or intramolecular hydrogen bonding constituted structural characteristics against the autoxidation reaction. Though the O₂^.-productive activity partially counteracted O₂^.-scavenging activity, such structural characteristics appeared to enhance the scavenging activity (24). Neshchadin et al used CIDNP spectroscopy (CIDNP=chemically induced dynamic nuclear polarization) to provide a deeper understanding of the antioxidant mechanism and model hydrogen abstraction reactions with four catechin-based polyphenols: catechin (C), gallocatechin (GC), epigallocatechin (EGC), and epigallocatechin gallate (EGCG). They reported that hydrogen abstraction is an essentially stochastic process with a slight preference for the B rings in the catechin-based polyphenols (25). Luczajet et al reported the action of polyphenols against oxidative stress formation in endothelial cells. Endothelial cells pretreated with 100 mM tert-butyl hydroperoxide (t-BHP) for oxidative stress formation, subjected to EGCG, TFs and black tea extract, were partially protected against oxidation of t-BHP causing statistically significant increase in GSH-Px activity, GSH and tryptophan level and a decrease in MDA and dityrosine levels in comparison with human vein endothelial cells (HUVEC) pretreated with t-BHP group (26). Supplementation of GTP and black tea polyphenols (BTP) as a sole source of drinking solution to Winstar rats leads to scavenging of ROS by 72% and 69%, respectively (27). The presence of the vicinyl hydroxy groups also makes them more susceptible to air oxidation under alkaline and neutral pH.

In addition to their reduction potential and antioxidant abilities, GTPs can actively trap the reactive carbonyl species (RCS) (28). Extremely reactive RCS such as glyoxal (GO), methylglyoxal (MG), and 3-deoxyglucosone (3-DG), generated from the Maillard reaction, readily modify lysine, arginine, and cysteine residues of proteins, forming advanced glycation end products (AGEs). AGEs are associated with chronic and age-related diseases such as diabetes and diabetes complications, confirmed by several epidemiological and large prospective clinical studies. The binding site for RCS trapping is the A ring of the catechins, whereas the preferred sites for antioxidant activity in the B ring. (29).

Polyphenolic structures in GTPs favors the formation of strong H-bonds. These H-bonds mediate the biochemical effects of GTPs by enabling them to bind strongly with proteins and nucleic acids. Recent studies using NMR spectroscopy revealed that GTPs could directly bind to the BH3 domain of anti-apoptotic BCl-2 family of proteins (30) and mediate their cancer preventive effects by induction of apoptosis. Molecular modeling studies showed that EGCG binds and inhibits the proteasome (31) and urokinase plasminogen activator (uPA), a matrix remodeling protein thereby preventing tumor metastasis (32). On the other hand the large hydration shell affects their absorbability.

3.3.2. EGCG structure and activity

EGCG is a major constituent of green tea and various studies have demonstrated that EGCG can inhibit carcinogenesis and prevent metastasis in established tumors. The polyphenolic structure of EGCG consists of 4 rings, A, B, C and D (Figure 1). A and C rings constitute the benzopyran ring. This benzopyran ring has a phenyl group at C2 and gallate group at C3 positions. The B ring of EGCG has vicinal 3,4,5-trihydroxy groups, and the D ring galloyl moiety in EGCG is in the form of an ester at C3. The presence of this ester carbon makes EGCG highly susceptible to nucleophilic attack (Figure 1).

The precise molecular targets and the exact anti-cancer mechanism of EGCG are not clearly defined. However, convincing evidence suggests the proteasome is one of the molecular targets for EGCG. Regulated proteolysis via the proteasome pathway is important for the integrity of intracellular biochemical events. Inhibition of the ubiquitin-proteasome pathway (UPP) has emerged as a promising strategy in the prevention and treatment of human cancer. It was found that EGCG could potently and specifically inhibit the chymotrypsin-like activity of the proteasome both in vitro and in cultured tumor cells (31). The A-ring of (−)-EGCG-containing two hydroxyl groups at C5 and C7 positions facilitates binding to Lys 32 and Ala 146 in the hydrophobic S1 pocket of the Beta5-subunit of proteasome. Irreversible transfer of the gallate moiety from EGCG to the hydroxy oxygen of Thr 1 results in the inhibition of proteasome chymortypsin-like activity. In silico docking studies predict that electrophilic carbonyl carbon of (−)-EGCG is oriented in a suitable position for nucleophillic attack by the hydroxyl group of N-terminal Thr 1 of the Beta5-subunit of the proteasome, thus inhibiting the proteasomal chymotrypsin-like activity (33). The hydroxyl groups on D-ring forms hydrogen bonds with Gly 47 and Ser 131 of the proteasome which further augments the binding stability of (−)-EGCG to the proteasome (34).

The number of hydroxyl groups on the B-ring and D-ring influences the potency of EGCG for proteasome inhibition. Thus analogs involving modifications of the A ring and B ring of EGCG as proteasome inhibitors has been examined (35). It was found that in the B ring, a decrease in the number of OH groups led to decreased potency. However, the addition of a hydrophobic benzyl group in the C8 position had no significant affect on the potency.

The D-ring (galloyl group) composed of one benzoyl and three phenoxy groups in EGCG shows significant antiproliferative activity in PC-9 human lung cancer cells (36). The hydroxyl groups of the galloyl moiety were also discovered to be essential for cell growth inhibition, indicating that the galloyl group greatly contributes to its activities. There was a strong antiproliferative effect by alkyl gallate and gallamide derivatives towards human leukemia HL-60 cells through inducing apoptosis (37). Various bisgallate and bisgallamide derivatives were synthesized and tested for antiproliferative activity towards HL-60 cells. In gallamide derivatives having a short alkyl chain, the additional galloyl group enhanced the antiproliferative activity but in gallate derivatives, the addition of a galloyl group had no effect on the antiproliferative activity.

3.3.3. Stability

EGCG is less stable in neutral and alkaline medium because the hydroxyl groups on the phenyl ring are attacked by the basic medium leading to the formation of a more active phenoxide anion. Autooxidation of EGCG results in the generation of superoxide anion and hydrogen peroxide. This can be prevented in cell culture which can be stabilized by the addition of superoxide dismutase (38). Many of the reported activities could thus be attributed to the production of ROS. For example induction of apoptosis reported in H661 lung cancer cells by EGCG could be blocked by the presence of catalase (39). Recently Smith et al (40) investigated the ability of one preformulation method to improve the oral bioavailability of EGCG and found that forming nanolipidic EGCG particles improves oral bioavailability in vivo by more than two-fold over free EGCG. When EGCG was encapsulated in polylactic acid (PLA)-polyethylene glycol (PEG) nanoparticles, the resulting nano-EGCG was reported to retain its biological efficacy when compared to non-encapsulated EGCG, with over 10-fold dose advantage both in cell culture system and in vivo settings in athymic nude mice implanted with human prostate cancer cells (41).

3.3.4. Effect of substitutions on different rings

As mentioned, EGCG is very unstable in neutral or alkaline medium, and this instability leads to a low bioavailability. To enhance the stability of EGCG, peracetate protection groups were introduced on the reactive hydroxyls of EGCG. Analogs involving protected OH-groups called Pro-EGCG(1) (Figure 2) show a higher level of potency compared to natural –(−)EGCG. As expected, these prodrugs fail to show any inhibitory activity toward purified 20S proteasome, but show increased proteasome-inhibitory activity in intact leukemic cells compared to its parent compound. To improve the stability and potency of (−)-EGCG, OH groups from D-ring were replaced with one or two fluorines. The novel fluoro-substituted (−)-EGCG analogues were named F-EGCGs (42). Prodrug of fluoro-substituted (−)-EGCG at meta-position on the phenyl ring (Pro-F-EGCG2) or difluoro-substituted (−)-EGCG at both meta- and para-positions on the phenyl ring (Pro-F-EGCG4, Figure 2) had similar or even more inhibitory potency as Pro-EGCG (1) to induce apoptosis in cultured human breast cancer cells.

Analogs of (−)-EGCG containing a para-amino group on the D-ring (p-NH₂EGCG, Figure 2) in place of the hydroxyl groups were tested for their proteasome-inhibitory activities (43). The O-acetylated (−)-EGCG analogs possessing a p-NH2 [or p-NHBoc (Boc; tert-butoxycarbonyl)] in the D-ring (9) act as novel tumor cellular proteasome inhibitors. Their potency was similar to natural (−)-EGCG and (−)-EGCG peracetate (1). Inactivating processes such as methylation render EGCG inactive. Methylation of (−)-EGCG occurs by catechol-O-methyltransferase (COMT), an enzyme widely distributed throughout the body. Synthetic analogues were synthesized with no hydroxyl groups at C5 & C7 of the A ring in EGCG (44). In MDA-MB-231 breast cancer cells with high COMT pro-drug 8 of synthetic analog 7 was more active than the prodrugs of EGCG and analog 5 because it is not a substrate of COMT (Figure 2).

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