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.     

Inhibition of dopamine uptake by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a cause of parkinsonism

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine has been reported to cause parkinsonism in man and monkeys, producing behavioral effects within 5 min of administration. The compound reversibly and competively inhibited (IC50 = 2 microM) dopamine uptake into PC12, a clonal line of rat pheochromocytoma cells that store and secrete dopamine and acetylcholine. Uptake of choline and 2-deoxyglucose was not affected. Prolonged exposure to the compound was lethal to PC12; survivors of this treatment lost the ability to store dopamine and acetylcholine and to extend neurites upon incubation with nerve growth factor.     

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.     

Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease

Elucidation of the biochemical steps leading to the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of the nigrostriatal dopamine (DA) pathway has provided new clues to the pathophysiology of Parkinson's disease. In line with the enhancement of MPTP toxicity by diethyldithiocarbamate (DDC), here we demonstrate how other cytochrome P450 (CYP) 2E1 inhibitors, such as diallyl sulphide (DAS) and phenylethylisothiocyanate (PIC), also potentiate the selective DA neurone degeneration in C57/bl mice. In addition, we show that CYP 2E1 is present in the brain and in the basal ganglia of this mouse strain, as measured by RT-PCR, western blot analysis and immunohistochemistry. A kinetic analysis of MPTP and its metabolites, by means of the microdialysis technique in the striatum, indicates that no detoxification metabolic pathway is affected by any of these inhibitors. This does not rule out, however, that an undetected detoxification pathway involving CYP 2E1 is operating. In order to provide direct evidence for this isozyme involvement, CYP 2E1 knockout mice were challenged with MPTP or the combined treatment. Here we show that these transgenic mice have a low sensitivity to MPTP alone, similar to their wild-type counterparts, suggesting that it is likely that transgenic mice compensate for the missing enzyme. However, DDC pretreatment completely fails to enhance MPTP toxicity in CYP 2E1 knockout mice, whereas this enhancement is regularly present in wild-type animals. This study indicates that the occurrence of CYP 2E1 in C57/bl mouse brain is relevant to MPTP toxicity, and suggests that this isozyme may have a detoxificant role related to the efflux transporter of the toxin.     

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.     

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.     

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.     

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.     

Formation of stacking complexes between caffeine (1,2,3-trimethylxanthine) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine may attenuate biological effects of this neurotoxin

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin causing symptoms that may resemble those observed in patients suffering from Parkinson's disease. Therefore, MPTP-treated laboratory animals are currently the most favored models to study therapeutic intervention strategies in this disease. It was demonstrated recently that caffeine (1,2,3-trimethylxanthine) intake decreases the risk of Parkinson's disease in various human populations and attenuates MPTP-induced neurological effects in animal models. Since the effects of caffeine on MPTP-treated animals were mimicked by several antagonists of the adenosine A(2A) receptor, it was suggested that caffeine attenuates MPTP toxicity by blocking this receptor. Here, using microcalorimetry and molecular modeling, we demonstrate that caffeine can form stacking (pi-pi) complexes with MPTP. We found that a biological activity of MPTP (induction of mutations in a microbiological mutagenicity assay), which is completely independent on the A(2A) receptor blockade, is significantly reduced by caffeine. Therefore, we suggest that caffeine may attenuate neurotoxicity of MPTP (and possibly other polycyclic aromatic toxins) and reveal its protective effects on the risk of Parkinson's disease not only by blocking the A(2A) receptor but also by sequestering neurotoxin molecules in mixed complexes, especially in stomach.     

Interactions of the neurotoxin MPTP and its demethylated derivative (PTP) with monoamine oxidase-B

The kinetics of the interactions of MPTP and its N-des-methyl-derivative (PTP) have been studied. Both were mechanism-based inhibitors as well as substrates for the enzyme. Analysis of the reaction progress-curves for the formation of the corresponding dihydropyridine derivatives allowed the kinetic parameters for the process and the partition ratio, which corresponds to the number of mol. of product formed per mol. of enzyme inactivated, to be determined for both compounds. The conversion of MPTP to its corresponding pyridinium-ion derivative through the action of MAO-B is known to be essential for its neurotoxicity. PTP has been reported not to be neurotoxic, although it appears to be a relatively good substrate for MAO-B as well as acting a mechanism-based inhibitor. Studies of the changes in absorbance spectra during the MAO-B catalysed oxidation were consistent with the formation of the corresponding pyridinium-ion derivative (MPP+), which is known to be the effective neurotoxin, as the end-product when MPTP was oxidized. In contrast the oxidation of PTP appeared to stop at the dihydropyridine stage with no significant further oxidation to the corresponding pyridine-derivative.     

Green tea polyphenol epigallocatechin-3-gallate (EGCG) induced intermolecular cross-linking of membrane proteins

Increasing evidence has demonstrated that EGCG possesses prooxidant potential in biological systems, including modifying proteins, breaking DNA strands and inducing the generation of reactive oxygen species. In the present study, the prooxidant effect of EGCG on erythrocyte membranes was investigated. SDS–PAGE and NBT-staining assay were utilized to detect the catechol-protein adducts that generated upon treating the membranes with EGCG. The results indicated that EGCG was able to bind covalently to sulfhydryl groups of membrane proteins, leading to the formation of protein aggregates with intermolecular cross-linking. We suggested that the catechol-quinone originated from the oxidation of EGCG acted as a cross-linker on which peptide chains were combined through thiol-S-alkylation at the C2- and C6-sites of the gallyl ring. EGC showed similar effects as EGCG on the ghost membranes, whereas ECG and EC did not, suggesting that a structure with a gallyl moiety is a prerequisite for a catechin to induce the aggregation of membrane proteins and to deplete membrane sulfhydryls. EDTA and ascorbic acid inhibited the EGCG-induced aggregation of membrane proteins by blocking the formation of catechol-quinone. The information of the present study may provide a fresh insight into the prooxidant effect and cytotoxicity of tea catechins.     

Food-drug interaction of (-)-epigallocatechin-3-gallate on the pharmacokinetics of irinotecan and the metabolite SN-38

The aim of the present study was to investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on the pharmacokinetics of irinotecan (CPT-11) and its metabolite SN-38. EGCG was potentially used to modulate the ATPase activity of P-glycoprotein (P-gp). Experimental Sprague-Dawley rats were treated with EGCG (20mg/kg, i.v.) 10min before CPT-11 (10mg/kg, i.v.) administration, whereas the control group received CPT-11 (10mg/kg, i.v.) only. The biological samples were prepared by the protein precipitation and detected by HPLC-fluorescence detection which provided a good separation of CPT-11 and SN-38 within 10min. The pharmacokinetic data indicate that the area under the plasma concentration-time curves (AUC) of CPT-11 and SN-38 were increased by 57.7 and 18.3%, and AUC in bile were decreased by 15.8 and 46.8%, respectively, for the group pretreated with EGCG. The blood to bile distribution ratio (AUC(bile)/AUC(blood)) was significantly reduced after group coadministration of EGCG, it can be seen that the bile efflux transport system of CPT-11 and SN-38 may be markedly reduced by the treatment of EGCG which plays the role of P-gp inhibitor. In conclusion, EGCG was found to inhibit the transport of CPT-11 and SN-38 into the biliary elimination and their half-lives in plasma could be substantially prolonged. Based on the food-drug interaction, persons taking daily nutritional supplements should be warned of this interaction possibility.     

Acetyl-L-carnitine prevents total body hydroxyl free radical and uric acid production induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the rat

Acetyl-L-carnitine (ALCAR) is intimately involved in the transport of long chain fatty acids across the inner mitochondrial membrane during oxidative phosphorylation. ALCAR also has been reported to attenuate the occurrence of parkinsonian symptoms associated with 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in vivo, and protects in vitro against the toxicity of the neurotoxic 1-methyl-4-phenylpyridinium (MPP+) metabolite of MPTP. The mechanism for these protective effects remains unclear. ALCAR may attenuate hydroxyl (HO*) free radical production in the MPTP/MPP+ neurotoxic pathway through several mechanisms. Most studies on MPTP/MPP+ toxicity and protection by ALCAR have focused on in vivo brain chemistry and in vitro neuronal culture studies. The present study investigates the attenuative effects of ALCAR on whole body oxidative stress markers in the urine of rats treated with MPTP. In a first study, ALCAR totally prevented the MPTP-induced formation of HO* measured by salicylate radical trapping. In a second study, the production of uric acid after MPTP administration-a measure of oxidative stress mediated through xanthine oxidase-was also prevented by ALCAR. Because ALCAR is unlikely to be a potent radical scavenger, these studies suggest that ALCAR protects against MPTP/MPP+-mediated oxidative stress through other mechanisms. We speculate that ALCAR may operate through interference with organic cation transporters such as OCTN2 and/or carnitine-acylcarnitine translocase (CACT), based partly on the above findings and on semi-empirical electronic similarity calculations on ALCAR, MPP+, and two other substrates for these transporters.     

Functional Reconstitution of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate (AMPA) Receptors from Rat Brain

Glutamate receptors belonging to the subclass specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were solubilized from rat forebrain membranes with Triton X-100 and partially purified through a series of three chromatographic steps. Specific [3H]AMPA binding increased 30-60-fold during the isolation procedure. A protein band recognized by antibodies against specific amino acid sequences of the glutamate receptor-A subunit was enriched with each purification step; the molecular mass of this band (105 kDa) corresponded to that of cloned AMPA receptor subunits. Photoaffinity labeling of forebrain membranes with 6-cyano-7-[3H]nitroquinoxaline-2,3-dione, a specific antagonist of the AMPA receptor, labeled a single band that comigrated with the immunolabeled protein. On reconstitution of the partially purified material into bilayer patches, single-channel current fluctuations were elicited by 300 nM AMPA and blocked by 1 microM 6,7-dinitroquinoxaline-2,3-dione.     

Effects of (-)-epigallocatechin-3-gallate in Ca2+ -permeable non-selective cation channels and voltage-operated Ca2+ channels in vascular smooth muscle cells

The effects of (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of tea, on Ca(2+)-permeable non-selective cation currents (NSCC) and voltage-operated Ca(2+) channels (VOCC) have been investigated in cultured rat aortic smooth muscle cells using the whole-cell voltage-clamp technique. Under the Cs(+)/tetraethylammonium (TEA)-containing internal solution, and in the presence of nifedipine (1 microM), EGCG (30 microM) activated a long-lasting inward current, with a reversal potential (E(rev)) of approximately 0 mV. This current was not significantly altered by the replacement of [Cl(-)](i) or [Cl(-)](o), implying that the inward current was not a chloride channel, but a NSCC. SKF 96365 (30 microM) and Cd(2+) (500 microM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 microM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 microM) also potentiated a nifedipine-sensitive voltage-dependent Ba(2+)-current during the first 5 min of incubation. However, after > 10 min of incubation with EGCG, this current was significantly inhibited. Our results suggest that EGCG caused a Ca(2+) influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd(2+)-sensitive Ca(2+)-permeable channels. The action described here may be responsible for the contraction induced by EGCG in rat aortic rings and for the rise of the intracellular concentration of Ca(2+) in rat aortic smooth muscle cells evoked by this catechin. On the other hand, the inhibition of VOCC after > 10 min of incubation may be, in part, responsible for the relaxation of rat aorta induced by EGCG.     

(-)-Epigallocatechin-3-gallate prevents oxidative damage in both the aqueous and lipid compartments of human plasma

When human plasma was exposed to the hydrophilic radical initiator, AAPH, (-)-epigallocatechin-(3)-gallate (EGCG) dose-dependently inhibited the aqueous compartment oxidation (IC(50)=0.72 microM) (monitored by DCFH oxidation) and spared the lipophilic antioxidants, alpha-tocopherol, and carotenoids, but not ascorbic acid. When radicals were selectively induced in the lipid compartment by the lipophilic radical initiator, MeO-AMVN, EGCG spared alpha-tocopherol, but not carotenoids and inhibited the lipid compartment oxidation (monitored by BODIPY 581/591) with a potency lower than that found in the aqueous compartment (IC(50)=4.37 microM). Our results indicate that EGCG, mainly localized in the aqueous compartment, effectively quenches aqueous radical species, thus limiting their diffusion into the lipid compartment and preventing lipid-soluble antioxidant depletion. Further, ESR experiments confirmed that EGCG recycled alpha-tocopherol through a H-transfer mechanism at the aqueous/lipid interface affording an additional protective mechanism to the lipid compartment of plasma.     

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.     

Neuroprotective effects of (-)-epigallocatechin-3-gallate (EGCG) on paraquat-induced apoptosis in PC12 cells

Green tea, owing to its beneficial effect on health, is becoming more and more popular worldwide. (-)-Epigallocatechin-3-gallate (EGCG), the main ingredient of green tea polyphenols, is a known protective effect on injured neurons in neurodegenerative disease, such as Alzheimer's disease and Parkinson's disease. Paraquat (PQ) is a widely used herbicide that possesses a similar structure to MPP(+) and is toxic to mesencephalic dopaminergic neurons. In the present study, PQ-injured PC12 cells were chosen as an in vitro cell model of Parkinson's disease and the neuroprotective effects of EGCG were investigated. The results showed that EGCG attenuated apoptosis of PC12 cells induced by PQ. The possible mechanism may be associated with maintaining mitochondrial membrane potential, inhibiting caspase-3 activity and downregulating the expression of pro-apoptotic protein Smac in cytosol. The present study supports the notion that EGCG could be used as a neuroprotective agent for treatment of neurodegenerative diseases.     

Evidence that High- and Low-Affinity DL-α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Binding Sites Reflect Membrane-Dependent States of a Single Receptor

Binding of DL-alpha-[3H]amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([3H]AMPA) to lysed rat brain membranes in the presence of potassium thiocyanate resulted in curvilinear Scatchard plots that could be resolved by regression analysis into a large low-affinity component and a small high-affinity component. Solubilization with Triton X-100 resulted in solubilized and nonsolubilized fractions that were considerably enriched in the high-affinity component and correspondingly reduced in the low-affinity component. It thus appears that solubilization converts low-affinity AMPA receptors into high-affinity receptors. Also, synaptic plasma membranes were found to be greatly enriched in the low-affinity form and deficient in the high-affinity form of the AMPA receptor. These experiments provide evidence for the hypothesis that the high- and low-affinity components of AMPA binding are interconvertible states of the same receptor rather than separate binding sites and that the conversion of these receptors from their native high-affinity state to the low-affinity state occurs on insertion of the receptors into synapses.