Green tea polyphenol (-)-epigallocatechin-3-gallate induces neurorescue of long-term serum-deprived PC12 cells and promotes neurite outgrowth

Our previous studies have shown that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) prevents neuronal cell death caused by several neurotoxins. The present study sought to determine the neuroprotective effect of EGCG when it is administered after the induction of cell damage ('neurorescue'). In an attempt to imitate a progressive mode of death, PC12 cells were initially subjected to serum-starvation conditions for a period of 1 or 3 days before administration of EGCG (0.1-10 microM) for up to 3 days. In spite of the high percentage of cell death, single or repetitive administration of EGCG (1 microM) significantly attenuated cell death. The neurorescue effect of EGCG was abolished by pre-treatment with the protein kinase C inhibitor GF109203X (2.5 microM), suggesting the involvement of the protein kinase C pathway in neurorescue by the drug. This is consistent with the rapid (15 min) translocation of the protein kinase C alpha isoform to the cell membrane in response to EGCG. The correlative neurite outgrowth activity of EGCG on PC12 cells may also contribute to its neurorescue effect. The present findings suggest that EGCG may have a positive impact on aging and neurodegenerative diseases to retard or perhaps even reverse the accelerated rate of neuronal degeneration.     

Covalent modification of proteins by green tea polyphenol (-)-epigallocatechin-3-gallate through autoxidation

Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has various beneficial properties including chemopreventive, anticarcinogenic, and antioxidant actions. The interaction with proteins known as EGCG-binding targets may be related to the anticancer effects. However, the binding mechanisms for this activity remain poorly understood. Using mass spectrometry and chemical detection methods, we found that EGCG forms covalent adducts with cysteinyl thiol residues in proteins through autoxidation. To investigate the functional modulation caused by binding of EGCG, we examined the interaction between EGCG and a thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Concentration-dependent covalent binding of EGCG to GAPDH was found to be coupled to the irreversible inhibition of GAPDH activity. Mutation experiments revealed that EGCG is primarily bound to the cysteinyl thiol group of the active center, indicating that the irreversible inhibition of GAPDH is due to the covalent attachment of EGCG to the active-center cysteine. Moreover, using EGCG-treated cancer cells, we identified GAPDH as a target of EGCG covalent binding through specific interactions between catechols and aminophenyl boronate agarose resin. Based on these findings, we propose that the covalent modification of proteins by EGCG may be a novel pathway related to the biological activity of EGCG.     

Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent ([Formula: See Text] and [Formula: See Text] respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.     

Proposed mechanisms of (-)-epigallocatechin-3-gallate for anti-obesity

Green tea catechins (GTCs) are polyphenolic flavonoids formerly called vitamin P. GTCs, especially (-)-epigallocatechin-3-gallate (EGCG), lower the incidence of cancers, collagen-induced arthritis, oxidative stress-induced neurodegenerative diseases, and streptozotocin-induced diabetes. Also, inhibition of adipogenesis by green tea and green tea extract has been demonstrated in cell lines, animal models, and humans. The obesity-preventive effects of green tea and its main constituent EGCG are widely supported by results from epidemiological, cell culture, animal, and clinical studies in the last decade. Studies with adipocyte cell lines and animal models have demonstrated that EGCG inhibits extracellular signal-related kinases (ERK), activates AMP-activated protein kinase (AMPK), modulates adipocyte marker proteins, and down-regulates lipogenic enzymes as well as other potential targets. Also, the catechin components of green tea have been shown to possess anti-carcinogenic properties possibly related to their anti-oxidant activity. In addition, it was shown that dietary supplementation with EGCG could potentially contribute to nutritional strategies for the prevention and treatment of type 2 diabetes mellitus. In this review, the biological activities and multiple mechanisms of EGCG in cell lines, animal models, and clinical observations are explained.     

Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration

In the present study we demonstrate neuroprotective property of green tea extract and (-)-epigallocatechin-3-gallate in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson's disease. N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxin caused dopamine neuron loss in substantia nigra concomitant with a depletion in striatal dopamine and tyrosine hydroxylase protein levels. Pretreatment of mice with either green tea extract (0.5 and 1 mg/kg) or (-)-epigallocatechin-3-gallate (2 and 10 mg/kg) prevented these effects. In addition, the neurotoxin caused an elevation in striatal antioxidant enzymes superoxide dismutase (240%) and catalase (165%) activities, both effects being prevented by (-)-epigallocatechin-3-gallate. (-)-Epigallocatechin-3-gallate itself also increased the activities of both enzymes in the brain. The neuroprotective effects are not likely to be caused by inhibition of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine conversion to its active metabolite 1-methyl-4-phenylpyridinium by monoamine oxidase-B, as both green tea and (-)-epigallocatechin-3-gallate are very poor inhibitors of this enzyme in vitro (770 microg/mL and 660 microM, respectively). Brain penetrating property of polyphenols, as well as their antioxidant and iron-chelating properties may make such compounds an important class of drugs to be developed for treatment of neurodegenerative diseases where oxidative stress has been implicated.     

Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (-)-epigallocatechin-3-gallate: implications for neurodegenerative diseases

Accumulating evidence supports the hypothesis that brain iron misregulation and oxidative stress (OS), resulting in reactive oxygen species (ROS) generation from H2O2 and inflammatory processes, trigger a cascade of events leading to apoptotic/necrotic cell death in neurodegenerative disorders, such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases, and amyotrophic lateral sclerosis (ALS). Thus, novel therapeutic approaches aimed at neutralization of OS-induced neurotoxicity, support the application of ROS scavengers, transition metals (e.g. iron and copper) chelators and non-vitamin natural antioxidant polyphenols, in monotherapy, or as part of antioxidant cocktail formulation for these diseases. Both experimental and epidemiological evidence demonstrate that flavonoid polyphenols, particularly from green tea and blueberries, improve age-related cognitive decline and are neuroprotective in models of PD, AD and cerebral ischemia/reperfusion injuries. However, recent studies indicate that the radical scavenger property of green tea polyphenols is unlikely to be the sole explanation for their neuroprotective capacity and in fact, a wide spectrum of cellular signaling events may well account for their biological actions. In this article, the currently established mechanisms involved in the beneficial health action and emerging studies concerning the putative novel molecular neuroprotective activity of green tea and its major polyphenol (-)-epigallocatechin-3-gallate (EGCG), will be reviewed and discussed.     

Green tea constituent (--)-epigallocatechin-3-gallate inhibits topoisomerase I activity in human colon carcinoma cells

DNA topoisomerases I and II are essential for cell survival and play critical roles in DNA metabolism and structure. Inhibitors of topoisomerase constitute a novel family of antitumor agents with demonstrated clinical activity in human malignancies. The clinical use of these agents is limited due to severe toxic effects on normal cells. Therefore, there is a need to develop novel, nontoxic topoisomerase inhibitors that have the ability to spare normal cells. Recent studies have shown that green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG), impart growth inhibitory responses to cancer cells but not to normal cells. Based on the knowledge that EGCG induces DNA damage, cell cycle arrest, and apoptosis, we considered the possibility of the involvement of topoisomerase in the antiproliferative response of EGCG. Here, for the first time, we show that EGCG inhibits topoisomerase I, but not topoisomerase II in several human colon carcinoma cell lines. Based on this study it is tempting to suggest that combination of EGCG with other conventional topoisomerase inhibitors could be an improved strategy for treatment of colon cancer. The possible role of EGCG as a chemotherapeutic agent needs to be investigated.     

Study of the green tea polyphenols catechin-3-gallate (CG) and epicatechin-3-gallate (ECG) as proteasome inhibitors

The green tea polyphenol catechin-3-gallate (CG) and epicatechin-3-gallate (ECG) were synthesized enantioselectively via a Sharpless hydroxylation reaction followed by a diastereoselective cyclization. Their potencies to inhibit the proteasome activity were measured. The unnatural enantiomers were found to be equally potent to the natural compounds.     

Green tea polyphenol (-) -epigallocatechin-3-gallate promotes the rapid protein kinase C- and proteasome-mediated degradation of Bad: implications for neuroprotection

The aim of the present study was to gain a deeper insight into the cell signaling pathways involved in the neuroprotection/neurorescue activity of the major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG). EGCG (1 micro m) caused an immediate (30 min) down-regulation (approximately 40%) of Bad protein levels, and a more pronounced reduction after 24 h (55%) in the human neuroblastoma cell line SH-SY5Y. Co-treatment with EGCG and the protein synthesis inhibitor cycloheximide prominently shortened Bad half-life, with as little as 30% of the Bad protein content remaining after 2 h, suggesting an effect of EGCG on Bad protein degradation. Accordingly, the proteasome inhibitors MG-132 and lactacystin damped Bad down-regulation by EGCG. The general protein kinase C (PKC) inhibitor GF109203X, or the down-regulation of conventional and novel PKC isoforms, abolished EGCG-induced Bad decline. However, no inhibition was seen with the cell-permeable myristoylated pseudosubstrate inhibitor of the atypical PKCzeta isoform. The enforced expression of Bad for up to 72 h rendered the cells more susceptible to serum deprivation-induced cell death, whereas EGCG treatment significantly improved cell viability (up to 1.6-fold). The present study reveals a novel pathway in the neuroprotective mechanism of the action of EGCG, which involves a rapid PKC-mediated degradation of Bad by the proteasome.     

Glucuronidation of the Green Tea Catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate,by Rat Hepatic and Intestinal Microsomes

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent ([Formula: See Text] and [Formula: See Text] respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

In vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate on the scuticociliate fish pathogen Philasterides dicentrarchi

This study investigated the in vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate (EGCG) on the histiophagous ciliate Philasterides dicentrarchi, which causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus L. Of the 3 polyphenols, resveratrol showed strongest antiprotozoal activity, reducing ciliate density after 1 wk culture by, on average, 91% at 50 microM, and 96% at 500 microM. EGCG reduced ciliate density by, on average, 93% at 500 microM, with no significant effect at 50 microM. Mangiferin reduced ciliate density by, on average, 56% at 500 microM, again with no significant effect at 50 microM. In view of these findings, we discuss the potential utility of chemotherapy with polyphenols as a strategy for the control of scuticociliatosis in farmed turbot.     

Enhancement of (−)-epigallocatechin-3-gallate and theaflavin-3-3′-digallate induced apoptosis by ascorbic acid in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells via MAPK pathways

Tea polyphenols (−)-epigallocatechin-3-gallate (EGCG) and theaflavin-3-3′-digallate (TF₃) are two prospective compounds in cancer prevention and treatment. Ascorbic acid (Vc) is essential to a healthy diet as well as being a highly effective antioxidant. In this work, the effects of the combination of EGCG or TF₃ with Vc on the apoptosis and caspases-3/9 activities in human lung adenocarcinoma SPC-A-1 cells and esophageal carcinoma Eca-109 cells were determined. Furthermore, the role of mitogen-activated protein kinases (MAPK) pathways in the apoptosis induced by TF₃ or EGCG together with Vc were studied using three MAPK inhibitors (ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580). Our results showed that Vc could enhance the EGCG and TF₃ induced apoptosis in SPC-A-1 and Eca-109 cells, and this effect involved the activation of caspase-3 and 9. EGCG, TF₃ and Vc could activate MAPK pathways respectively, and each compound activated different MAPK subfamilies in different cells. This may explain the enhancement of EGCG and TF₃ induced apoptosis by Vc in SPC-A-1 and Eca-109 cells, and will ultimately aid the design of more effective anti-cancer treatments.     

Molecular bases of thioredoxin and thioredoxin reductase-mediated prooxidant actions of (-)-epigallocatechin-3-gallate

Thioredoxin (Trx) and thioredoxin reductase (TrxR) function as antioxidant and anti-apoptotic proteins, which are often up-regulated in drug-resistant cancer cells. (-)-epigallocatechin-3-gallate (EGCG) is a naturally occurring antioxidant in green tea, but also exhibits prooxidant and apoptosis-inducing properties. We have previously showed a linkage between EGCG-induced inactivation of TrxR and decreased cell survival, revealing TrxR as a new target of EGCG. However, the molecular events underlying the importance of Trx/TrxR in EGCG-induced cytotoxicity remain unclear. Here, we show that the crosstalk between EGCG and Trx/TrxR occurred in a redox-dependent manner, and EGCG induced inactivation of Trx/TrxR in parallel with increased ROS levels in HeLa cells. Moreover, EGCG displayed great reactivity with Cys/Sec residues that have low pK(a) values. The structure of EGCG suggests that its quinone form would readily react with thiolate and selenolate nucleophiles. Using mass spectrometry, we have demonstrated the formation of EGCG-Trx1 (Cys(32)) and EGCG-TrxR (Cys/Sec) conjugates, confirming that EGCG quinone specifically conjugates with active-site Cys(32) in Trx or C-terminal Cys/Selenocysteine (Sec) couple in TrxR under conditions where Trx/TrxR are reduced. Non-reduced form of Trx/TrxR could escape from EGCG inhibition. These data reveal a potential mechanism for enhancing EGCG-induced cancer cell death by the NADPH-dependent reduction of Trx/TrxR.     

A novel approach of proteomics and transcriptomics to study the mechanism of action of the antioxidant-iron chelator green tea polyphenol (-)-epigallocatechin-3-gallate

Previous findings suggest that the antioxidant-iron chelator green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) may have a neurorescue impact in aging and neurodegenerative diseases to retard or even reverse the accelerated rate of neuronal degeneration. The present study sought a deeper elucidation of the molecular neurorescue activity of EGCG in a progressive neurotoxic model of long-term serum deprivation of human SH-SY5Y neuroblastoma cells. In this model, proteomic analysis revealed that EGCG (0.1-1 microM) affected the expression levels of diverse proteins, including proteins related to cytoskeletal components, metabolism, heat shock, and binding. EGCG induced the levels of cytoskeletal proteins, such as beta tubulin IV and tropomyosin 3, playing a role in facilitating cell assembly. In accordance, EGCG increased the levels of the binding protein 14-3-3 gamma, involved in cytoskeletal regulation and signal transduction pathways in neurons. Additionally, EGCG decreased protein levels and mRNA expression of the beta subunit of the enzyme prolyl 4-hydroxylase, which belongs to a family of iron-oxygen sensors of hypoxia-inducible factor (HIF) prolyl hydroxylases that negatively regulate the stability and degradation of several proteins involved in cell survival and differentiation. Accordingly, EGCG decreased protein levels of two molecular chaperones that were associated with HIF regulation, the immunoglobulin-heavy-chain binding protein and the heat shock protein 90 beta. Thus, the present study sheds some light on the antioxidative-iron chelating activities of EGCG underlying its neuroprotective/neurorescue mechanism of action, further suggesting a potential neurodegenerative-modifying effect for EGCG.     

A component of green tea, (-)-epigallocatechin-3-gallate, promotes apoptosis in T24 human bladder cancer cells via modulation of the PI3K/Akt pathway and Bcl-2 family proteins

Bladder cancer is the fourth most common cancer in men and ninth most common in women. It has a protracted course of progression and is thus an ideal candidate for chemoprevention strategies and trials. This study was conducted to evaluate the chemopreventive/antiproliferative potential of (-)-epigallocatechin gallate (EGCG, the major phytochemical in green tea) against bladder cancer and its mechanism of action. Using the T24 human bladder cancer cell line, we found that EGCG treatment caused dose- and time-dependent inhibition of cellular proliferation and cell viability, and induced apoptosis. Mechanistically, EGCG inhibits phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells. These findings suggest that EGCG may be an important chemoprevention agent for the management of bladder cancer.     

Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

Coxsackievirus B3 (CVB3) is the most common human pathogen for viral myocarditis. We have previously shown that the signaling protein p21(ras) GTPase-activating protein (RasGAP) is cleaved and that mitogen-activated protein kinases (MAPKs) ERK1/2 are activated in the late phase of CVB3 infection. However, the role of intracellular signaling pathways in CVB3-mediated myocarditis and the relative advantages of such pathways to host or virus remain largely unclear. In this study we extended our prior studies by examining the interaction between CVB3 replication and intracellular signaling pathways in HeLa cells. We observed that CVB3 infection induced a biphasic activation of ERK1/2, early transient activation versus late sustained activation, which were regulated by different mechanisms. Infection by UV-irradiated, inactivated virus capable of receptor binding and endocytosis triggered early ERK1/2 activation, but was insufficient to trigger late ERK1/2 activation. By using a general caspase inhibitor (zVAD.fmk) we further demonstrated that late ERK1/2 activation was not a result of CVB3-mediated caspase cleavage. Treatment of cells with U0126, a selective inhibitor of MAPK kinase (MEK), significantly inhibited CVB3 progeny release and decreased virus protein production. Furthermore, inhibition of ERK1/2 activation circumvented CVB3-induced apoptosis and viral protease-mediated RasGAP cleavage. Taken together, these data suggest that ERK1/2 activation is important for CVB3 replication and contributes to virus-mediated changes in host cells. Our findings demonstrate coxsackievirus takeover of a particular host signaling mechanism and uncover a prospective approach to stymie virus spread and preserve myocardial integrity.     

Attenuation of intestinal ischemia/reperfusion induced liver and lung injury by intraperitoneal administration of (-)-epigallocatechin-3-gallate

The aim of this study was to evaluate the effect of ( - )-epigallocatechin-3-gallate (EGCG), a natural antioxidant, on liver and lungs after warm intestinal ischemia/reperfusion (I/R). Thirty male Wistar rats were equally divided into a sham-operation group, an intestinal I/R group and an intestinal I/R group pretreated with EGCG intraperitoneally. Intestinal ischemia was induced by occlusion of the superior mesenteric artery for 60 min followed by reperfusion for 120 min. Immediately after reperfusion, liver, lung and blood samples were collected and analyzed. Results showed that intestinal I/R increased the levels of aspartate (AST) and alanine (ALT) transaminase in serum to 987 and 752 IU/l, respectively. Malondialdehyde (MDA) increased in liver to 1.524 nmol/g in the group subjected to intestinal I/R compared to 0.995 nmol/g in the sham operation group. MDA was also increased in lungs to 1.581 nmol/g compared to 0.896 nmol/g in the sham operation group. Myeloperoxidase (MPO) increased in liver, after intestinal I/R, to 5.16 U/g compared to 1.59 U/g in the sham operation group. MPO was also increased in lungs to 3.89 U/g compared to 1.65 U/g in the sham operation group. Pretreatment with EGCG decreased serum levels of AST and ALT to 236 and 178 IU/l, respectively. It also decreased mean MDA levels in liver and lungs to 1.061 and 1.008 nmol/g, respectively, and mean MPO levels in liver and lungs to 1.88 and 1.71 U/g, respectively. Light microscopy and transmission electron microscopy examinations showed significant alteration in liver and lungs and protection of liver and lung parenchyma in the animals treated with EGCG.