The development of genetic oncology has demonstrated that damage to numerous regulatory genes may result in induction of invasive and metastatic cancer. It has been established that the pathological processes of multistep carcinogenesis comprises initiation, promotion, and progression.7 The natural history of carcinogen-esis and cancer development provides a strong rationale for a preventive approach to the control of this disease and leads one to consider the possibility of active pharmacological intervention to arrest or reverse the processes of carcinogenesis before invasion and metastasis occur. The inhibitory effects of tea against car-cinogenesis have been attributed to the biologic activities of the polyphenolic catechins in the tea. However, the biochemical and molecular mechanisms of cancer chemoprevention by tea catechins are not fully elucidated. Some of the recent developments in our laboratory and others are discussed in the following.
Tea is particularly rich in polyphenols, including catechins, theaflavins, and thearubigins, which are thought to contribute to the health benefits of tea. Tea polyphenols act as antioxidants in vitro by scavenging reactive oxygen and nitrogen species and chelating redox-active transition metal ions. They may also function indirectly as antioxidants through inhibition of the redox sensitive transcription factors, NFkB, and AP-1; inhibition of pro-oxidant enzymes such as inducible nitric oxide synthase (iNOS), lipoxygenase, cyclooxygenase, and xan-thine oxidase; and induction of phase II and antioxidant enzymes, such as glu-tathione S-transferases and superoxide dismutases. The fact that catechins are rapidly and extensively metabolized emphasizes the importance of demonstrating their antioxidant activity in vivo.39
Tea catechins show remarkable antioxidative effects in various systems. Tea catechins are strong scavengers against superoxide, hydrogen peroxide, hydroxyl radicals, nitric oxide, and peroxynitrite produced by various chemicals and biological systems. Chen and Ho40 showed that the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging ability of various tea polyphenols was proportional to the number of -OH groups in the catechins or theaflavins. Recent studies show that catechins in green tea are highly active in reducing the amount of oxidative damage sustained by DNA through hydroxyl radical (OH) attack. Catechins when compared with other classes of flavonoids are found to be very active in reducing the amount of strand breakage and residual base damage by a mechanism other than direct scavenging of hydroxyl radicals before they react with DNA.41 Pulse radiolysis data support the mechanism of electron transfer (or H-transfer) from catechins to radical sites on DNA.42 These results support an antioxidant role of catechins in their direct interaction with DNA radicals.
The inhibitory effects of tea polyphenols on xanthine oxidase (XO) were investigated.43 Catechins and theaflavins inhibit XO to produce uric acid and also act as scavengers of superoxide. The antioxidative activity of tea catechins is due not only to their ability to scavenge superoxides, but also to their ability to block XO and related oxidative signal transducers.43 It has been demonstrated that tea or tea catechins inhibit Cu2+-mediated LDL oxidation in vitro44 and induction of atherogenesis in mice.39 To determine whether tea catechins act as effective antioxidants in vivo, future studies in animals and humans should employ sensitive and specific biomarkers of oxidative damage to DNA, proteins, and lipids.
Biologically important ROS that can damage DNA and, thereby, alter gene expression in cell growth and differentiation include the hydroxyl radical, superoxide, peroxyradical, singlet oxygen, peroxynitrite, and hydrogen peroxide.42 As oxidative DNA damage is considered to be a pathogenic event in the induction of many cancers,45 a reduction in the rate of such damage by catechins acting as antioxidants may lead to a reduced risk of cancer.
On the contrary, recent studies have demonstrated the pro-oxidant effects of green tea catechins.46,47 Green tea catechins enhanced colon carcinogenesis in rats.48 Experiments using 32P-labeled DNA fragments obtained from human cancer-related genes showed that catechins induced DNA damage in the presence of metals such as Cu(II) and Fe(III) complexes.46 It is concluded that EGCG can induce hydrogen peroxide generation and subsequent damage to isolated and cellular DNA, and that oxidative DNA damage may mediate the potential carci-nogenicity of EGCG.46 EGCG (12.5 to 50 \xM) decreased the viability of Jurkat cells and caused concomitant increase in cellular caspase 3 activity. Catalase and Fe(II) chelating reagent O-phenanthroline suppressed the EGCG effects, indicating involvements of both hydrogen peroxide and Fe(II) in the mechanism. Unexpectedly, ECG, which has Fe(III)-reducing potency comparable to EGCG, failed to decrease viability of Jurkat cells, while EGC, which has low capacity to reduce Fe(III), showed a cytotoxic effect similar to EGCG. These results suggest that a mechanism other than Fe(III) reduction plays a role in catechin-mediated Jurkat cell death. EGCG causes an elevation of hydrogen peroxide levels (a pro-oxidant effect) in Jurkat cell culture, in cell-free culture medium, and sodium phosphate buffer. Catechins with higher ability to produce hydrogen peroxide were more cytotoxic to Jurkat cells. Hydrogen peroxide itself exerted Fe(II)-dependent cyto-toxicity. Among human and normal cell lines tested, cells exhibiting lower hydrogen peroxide-eliminating activity were more sensitive to EGCG. It is proposed that cytotoxicity of catechins in certain tumor cells is due to their ability to produce hydrogen peroxide and that the resulting increase in hydrogen peroxide levels triggers Fe(II)-dependent formation of highly toxic hydroxyl radicals, which in turn induces apoptosis in the target cells.47
We have examined the apoptotic inducing effects of EGCG, theaflavins, and theasinensin A (from oolong tea) in the human cancer cell lines histolytic lymphoma U937 and acute T-cell leukemia Jurkat cells.49 The action mechanisms of tea polyphenols induced apoptosis as determined by annexin V apoptosis assay; DNA fragmentation and caspase activation were further investigated. Loss of membrane potential and ROS generation were also detected by flow cytometry. Treatment with tea polyphenols caused rapid induction of caspase-3, but not caspase-1, activity and stimulated proteolytic cleavage of poly(ADP-ribose)-poly-merase (PARP).49
Recent studies on apoptosis and cell cycle arrest in cancer cells by in vivo metabolites of teas have been described.50 The tea extracts from green, oolong, and black teas were prepared. The rat sera obtained after oral intubation of the prepared tea extracts, and tea polyphenolic compounds EGCG, EGC, ECG, and theaflavins were used in the related tests. All these tea samples significantly inhibited the proliferation of a rat hepatoma cell line (AH 109A) and murine B16 melanoma cells, but not normal rat mesothelial (M) cells.
EGCG was found to inhibit the growth of the transformed W138VA cells, but not of their normal counterparts. A similar growth inhibitory effect of EGCG between human colorectal cancer (Caco-2) cells, breast cancer (Hs578T) cells, and their respective normal counterparts were observed.51 EGCG treatment also induced apoptosis, and enhanced serum-induced expression of c-fos and c-myc genes in transformed W138VA cells, but not in the normal W138 cells. EGCG and other catechins inhibit growth of human lung cancer (PC-9) cells with a G2/M phase arrest of the cell cycle.52
17.6.4 Inhibition of Cellular Proliferation and Tumor Progression through Suppressing EGFR Signaling
Multiple data have demonstrated the pivotal role of mitogenic signal transduction in controlling the tumor proliferation.335 The induction of ornithine decarboxylase (ODC), PKC, protein kinase activities, and oxidative stress by TPA is believed to be closely related to the tumor promotion activity of this compound.753 Topical application of green tea catechins to mouse skin was found to inhibit TPA-caused induction of ODC activity in a dose-dependent manner.54 Our studies have demonstrated that EGCG and theaflavins inhibited TPA-induced transformation, PKC activation, and AP-1 binding activities in mouse fibroblast cells.5556 We have investigated the effects of EGCG on the proliferation of human epidermoid cancer cell line A431.9 EGCG strongly inhibited the DNA synthesis and protein tyrosine kinase activities of epidermal growth factor (EGF)-receptor, platelet derived growth factor (PDGF)-receptor, and fibroblast growth factor (FGF)-receptor. In an in vivo assay, EGCG could reduce the auto-phosphorylation level of EGF-receptor by EGF. EGCG inhibited the EGF-stimulated increase in phosphoty-rosine level in A431 cells. EGCG also blocked EGF-binding to its receptor. These results suggested that the inhibition of proliferation and suppression of the EGF signaling by EGCG might mainly mediate dose-dependent blocking of ligand binding to its receptor, and subsequently through inhibition of EGF-receptor kinase activity and its signaling.9
17.6.5 Inhibition of iNOS through Downregulating NFkB Activation
Nitric oxide (NO) is a small bioactive molecule that plays an important role in inflammation and multistep carcinogenesis. We have investigated the effects of tea polyphenols on the induction of iNOS in thioglycolate-elicited and lipopolysaccharide (LPS)-activated peritoneal macrophage.857 Gallic acid, EGC, EGCG, and theaflavins have found to inhibit nitrite production, iNOS protein, and mRNA in activated macrophages. Western blot, reverse transcriptase-poly-merase chain reaction (RT-PCR), and Northern blot analyses demonstrated that significantly reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS were expressed in LPS-activated macrophages with EGCG or theaflavins compared with those without tea polyphenols. Electrophoretic mobility shift assay (EMSA) indicated that EGCG blocked the activation of NFkB, a transcription factor necessary for iNOS induction.8 EGCG and theaflavins also blocked the disappearance of inhibitor IkB from the cytosolic fraction.57 These results suggest that EGCG and theaflavins decrease the activity and protein levels of iNOS by reducing the expression of iNOS mRNA and the reduction could occur through prevention of the binding of NFkB to the iNOS promoter, thereby inhibiting the induction of iNOS expression.8
17.6.6 Inhibition of Tumor Promotion and Cell Transformation through Suppressing AP-1 Activation
A number of studies have suggested that the activation of AP-1 plays an important role in tumor promotion; the downregulation of this transcription factor is now thought to be a general therapeutic strategy against cancer.58 59 Dong et al.60 investigated the anticancer-promoting effects of EGCG and theaflavins. Both were found to inhibit EGF- or TPA-induced cell transformation as well as AP-1-dependent transcriptional activity and DNA-binding activity. This study further showed that the inhibition of AP-1 activation occurs via the inhibition of a c-Jun NH2-terminal kinase (JNK)-dependent pathway.60 EGCG and theaflavins inhibited TPA-induced NFkB activity in a concentration-dependent manner. These tea polyphenols blocked TPA-induced phosphorylation of IKBa at Ser-32 in the same concentration range. These results suggest that inhibition of NFkB activation is also important in accounting for the antitumor promotion effects of EGCG and theaflavins.56-57-61
17.6.7 Suppression of Fatty Acid Synthase Expression by Catechins and Theaflavins
Fatty acid synthase (FAS) is a key enzyme in lipogenesis. FAS is highly expressed in the proliferative normal tissues and malignant tumors, is overexpressed in the malignant human breast carcinoma MCF-7 cells, and its expression is further enhanced by EGF. The EGF-induced expression of FAS was inhibited by green and black tea extracts. The expression of FAS was also suppressed by the catechins and theaflavins at both protein and mRNA levels that may lead to the inhibition of cell lipogenesis and proliferation.62 Both EGCG and theaflavin-3,3'-digallate inhibit the activation of AKt and block the binding of SP-1 to its target site at transcription promoter region. Furthermore, the EGF-induced biosynthesis of lipids and cell proliferation were significantly suppressed by EGCG and theaflavins.9-60 These findings suggest that tea polyphenols suppress FAS expression by downregulating the EGF-receptor/PI3K/AKt/Sp-1 signal transduction pathway; and tea and tea polyphenols might induce hypolipidemic and antiproliferative effects by suppressing FAS.62
Several reports have demonstrated that EGCG is a natural inhibitor of FAS in vitro. EGCG is an inhibitor of FAS from chicken liver.63 The marked inhibition of ketoacyl reduction shows that the inhibition is related to P-ketoacyl reductase of FAS. The observable protection of NADPH and competitive inhibition of NADPH for ketoacyl reduction indicate that EGCG may compete with NADPH for the same binding site.63 The analogs of galloyl moiety without the catechin skeleton such as propyl gallate also showed obvious slow-binding inhibition, whereas the green tea ungallated catechin did not.
Atomic orbital energy analyses suggest that the positive charge is more distinctly distributed on the carbon atom of ester bond of galloyl moiety of gallate catechins, and that gallated forms are more susceptible for a nucleophilic attack than other catechins.64 Thus, gallated catechins provide a nucleophilic target for the ketoacyl reductase of FAS that lead to the inhibition of the enzyme. EGCG also showed profound inhibition on the bacterial type II fatty acid synthase.65 EGCG and the related tea catechins potently inhibit both the FabG and FabI reductase steps in the fatty acid elongation cycle with IC50 values between 5 and 15 |M. The presence of the galloyl moiety was essential for activity.
Chemical inhibitors of FAS inhibit growth and induce apoptosis in several cancer cell lines in vitro66 and in tumor xenografts in vivo.61 EGCG inhibits FAS activity as efficiently as currently known synthetic inhibitors and selectively causes apoptosis in LNPaP cells but not in nontumoral fibroblasts. These findings establish EGCG as a potent natural inhibitor of FAS in intact cells and strengthen the molecular basis for the use of EGCG as a chemopreventive agent.68
17.6.8 Inhibition of Ubiquitin-Proteasome Pathway by Catechins
Many cell-cycle and cell-death regulators are identified as targets of the ubiq-uitin-proteasome-mediated degradation pathway. Proteasome inhibitors are able to induce tumor growth arrest, and tea consumption is correlated with cancer prevention.129 It has been demonstrated that ester bond-containing tea polyphenols such as EGCG potently and specifically inhibit the chymotrypsin-like activity of the proteasome in vitro at concentrations that are found in the serum of green tea drinkers.69 Atomic orbital energy analyses and high-performance liquid chromatography suggest that the carbon of the polyphenol ester bond is essential for targeting and thereby inhibiting the proteasome in cancer cells. This inhibition of the proteasome results in accumulation of two proteasome substrates p27/kip1 and iKBa, an inhibitor of NFkB, and this inhibition is followed by growth arrest in the G1 phase of the cell cycle. Tea polyphenols without an ester bond were inactive in this capacity. It is estimated that 20S proteasome inhibition is achieved via acylation of the catalytic N-terminal threonine on the proteasome's P5 subunit, wherein the A ring of (-)-EGCG acts as a tyrosine mimic to bind the hydrophobic S1 pocket of P5, which is the location of the chymotrypsin active site.69 Eight potential hydrogen bonds support docking of the complex. Synthetic (+)-EGCG was more potent to purified 20S proteasome than normal cis(-)-EGCG. Furthermore, compared with their simian virus-transformed counterparts, the parental normal human fibroblasts are much more resistant to EGCG-induced p27/kip1 protein accumulation and G1 arrest.69 This study suggests that protea-some is a cancer-related molecular target of tea polyphenols and that inhibition of proteasome activity by ester bond-containing polyphenols may contribute to the cancer chemopreventive effects of tea. It is interesting to note that the naturally occurring ester bond containing polyphenol pentagalloylglucose has been shown to be a strong inhibitor of 26S proteasome in our laboratory.70
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