Binding Of Epigallocatechin-3-gallate Research Articles

Epigallocatechin-gallate Suppresses Tumorigenesis by Directly Targeting Pin1

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). The human peptidyl prolyl cis/trans isomerase (Pin1) plays a critical role in oncogenic signaling. Herein, we report the X-ray crystal structure of the Pin1/EGCG complex resolved at 1.9 Å resolution. Notably, the structure revealed the presence of EGCG in both the WW and PPIase domains of Pin1. The direct binding of EGCG with Pin1 was confirmed and the interaction inhibited Pin1 PPIase activity. In addition, proliferation of cells expressing Pin1 was inhibited and tumor growth in a xenograft mouse model was suppressed. The binding of EGCG with Arg17 in the WW domain prevented the binding of c-Jun, a well-known Pin1 substrate. EGCG treatment corresponded with a decreased abundance of cyclin D1 and diminution of 12-O-tetradecanoylphorbol-l3-acetate-induced AP-1 or NF-κB promoter activity in cells expressing Pin1. Overall, these results showed that EGCG directly suppresses the tumor-promoting effect of Pin1.

Green Tea Gets Molecular

Green tea and its major polyphenolic flavonoid, epigallocatechin gallate (EGCG), have been credited with cancer chemopreventive activity for many years; the mechanism for this activity, however, has remained obscure. Now, as reported in this issue of the journal (beginning on page 1366), Urusova and colleagues showed direct binding of EGCG to the peptidyl prolyl cis/trans isomerase Pin1, which inhibited Pin1 enzymatic activity. They showed that Pin1 expression is required for EGCG effects on cell growth, c-Jun activation, and transcription regulation mediated by NF-κB and activator protein-1. The data provide a glimpse of the mechanism of action of EGCG and set a new bar for the future study of natural products with chemopreventive activity.

(−)-Epigallocatechin-3-gallate suppresses growth of AZ521 human gastric cancer cells by targeting the DEAD-box RNA helicase p68

(−)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active polyphenol in green tea, induces apoptosis and suppresses proliferation of cancer cells by modulating multiple signal transduction pathways. However, the fundamental mechanisms responsible for these cancer-preventive effects have not been clearly elucidated. Recently, we found that EGCG can covalently bind to cysteine residues in proteins through autoxidation and subsequently modulate protein function. In this study, we demonstrate the direct binding of EGCG to cellular proteins in AZ521 human gastric cancer cells by redox-cycle staining. We comprehensively explored the binding targets of EGCG from EGCG-treated AZ521 cells by proteomics techniques combined with the boronate-affinity pull-down method. The DEAD-box RNA helicase p68, which is overexpressed in a variety of tumor cells and plays an important role in cancer development and progression, was identified as a novel EGCG-binding target. Exposure of AZ521 cells to EGCG lowered the p68 level dose dependently. The present findings show that EGCG inhibits AZ521 cell proliferation by preventing β-catenin oncogenic signaling through proteasomal degradation of p68 and provide a new perspective on the molecular mechanism of EGCG action.

Catechin inhibits adhesion and migration of peripheral blood B cells by blocking CD11b

Context: Previously, we demonstrated that CD11b is expressed on peripheral blood memory B cells, and it plays an important role in the migration of B cells. And epigallocatechin gallate (EGCG), a bioactive component of green tea, by binding to CD11b, expressed on CD8(+) cytotoxic T cells, inhibited their migratory ability, one possible mechanism of the antiallergic activity of EGCG.Objective: Here, we investigated whether EGCG also affected CD11b expressed on B cells, similar to cytotoxic T cells.Materials and methods: Isolated peripheral blood CD19(+) B cells were treated with EGCG and the change in the expression of CD11b was analyzed using flow cytometry. The effects of EGCG on the ability of B cells to adhere to and to transmigrate through the endothelial cell layer were evaluated using the transwell assay.Results: EGCG significantly suppressed the apparent expression of CD11b on B cells, in the flow-cytometric analysis, and this apparent suppression was speculated to be dependent on the competitive binding of EGCG to CD11b. EGCG also significantly suppressed CD11b-mediated migration and adhesion of B cells to endothelial cells.Discussion and conclusion: EGCG has a strong suppressive activity on the adhesive and migratory abilities of peripheral blood B cells. This suppressive activity was mediated by the binding of EGCG to CD11b on B cells, and the consequent suppression of B-cell extravasation to the extravascular space. Because B cell plays an important role in the humoral immunity, EGCG could be a promising drug for the prevention and/or treatment of allergic and/or autoimmune diseases.

Thermodynamic Analysis of the Molecular Interactions between Amyloid β-Peptide 42 and (−)-Epigallocatechin-3-gallate

One of the key factors of Alzheimer's disease (AD) is the conversion of amyloid beta-peptide (Abeta) from its soluble random coil form into various aggregated forms. (-)-Epigallocatechin-3-gallate (EGCG) has been proved effective in preventing the aggregation of Abeta, but the thermodynamic mechanisms are still unclear. In this work, isothermal titration calorimetry (ITC) was utilized to study the interactions between Abeta42 and EGCG at different temperatures, salt concentrations, pH values, and EGCG and Abeta42 concentrations. Molecular dynamics (MD) simulations were performed to study the hydrogen bonding between Abeta42 and EGCG. The results indicate that the binding stoichiometry N is linearly related to the EGCG/Abeta42 ratio. Hydrophobic interaction and hydrogen bonding are both substantial in the binding process, but the extent of their contributions changes with experimental conditions. Namely, the predominant interaction gradually shifts from a hydrogen bonding to a hydrophobic interaction with the increase of the EGCG/Abeta42 ratio, resulting in a transition of the binding from enthalpy-driven to entropy-driven. This experimental observation is validated by the MD simulations. The binding of EGCG to Abeta42 can be promoted by increasing temperature and salt concentration and changing pH away from Abeta42's pI. The findings have provided new insight into the molecular interactions between Abeta42 and EGCG from a thermodynamic perspective and are expected to facilitate the research on the inhibition of Abeta42 aggregation.

Commentary

Age-related neurodegenerative diseases represent a growing socio-economic burden to developed societies due to an increased life-expectancy and the lack of effective treatments. The pathological hallmarks of the majority of these diseases, including Alzheimers (AD) and Parkinsons disease (PD), are proteinaceous inclusions and the progressive loss of neurons. These inclusions are characterised by β-sheet-rich fibrillar aggregates commonly termed amyloids that accumulate in the extracellular milieu or intracellular compartments of affected neurons. Protein aggregates are commonly derived from misfolded proteins that are prone to amyloidogenesis due to structural modifications, such as mutant forms of α-synuclein (in PD) or aberrantly cleaved forms of amyloid precursor protein (Aβ in AD). Amyloidogenesis occurs when the normally reversible balance between monomers and oligomers destabilises towards the predominant formation of oligomers, which subsequently undergo a conformational conversion into amyloid fibrils. Although still debated, the prevalent hypothesis is that amyloid fibrils represent a cellular protection mechanism, whereas soluble intermediate oligomeric and pre-fibrillar protein species are the major cause of cellular toxicity. However, the precise mechanism(s) leading to cell death are not understood. Despite this, there is an urgent need for new therapeutic strategies aimed at slowing or halting the progression of neurodegenerative diseases. A significant contribution in this direction has been the discovery that catechins derived from green tea are potent inhibitors of protein fibrillarization. Namely, epi-gallocatechin-3-gallate (EGCG) has been shown to antagonise both the formation of aggregates and the cellular toxicity of several aggregation-prone proteins including α-synuclein and Aβ. These studies already showed that EGCG can prevent amyloidogenesis, at least in cell-based model systems. The potential clinical application of such a drug could be to halt, or at least slow disease progression. However, because the majority of proteinopathies are sporadic, they are usually diagnosed once clinical symptoms have become apparent due to the loss of large populations of nerve cells. The evident question then arises whether EGCG might also be effective in disassembling toxic aggregation intermediates. To address this question, Erich Wanker and colleagues used in vitro aggregation assays and cell-based models of disease to demonstrate that EGCG remodels mature α-synuclein and Aβ fibrils and reduces cellular toxicity. Following on their previous work on EGCG, Bieschke et al. now show that EGCG affects the ordered fibrillar structure of α-synuclein and Aβ aggregates, leading to amyloid remodelling. They show that EGCG binding to aggregates precedes, and is necessary and sufficient for structural change. Most importantly, this study provides evidence that EGCG treatment remodels toxic β-sheet-rich amyloid protofibrils into smaller amorphous protein aggregates, thereby reducing cellular toxicity. The authors then tested several other EGCG-related polyphenols and found that the gallate moiety present in a variety of green tea catechins harbours the critical structural module required for efficient amyloid conversion from toxic proto-fibrils into amorphous, benign protein aggregates. The work by Bieschke et al. increases our understanding of the causes underlying amyloid-driven neurodegeneration, and demonstrates a potential therapeutic benefit of gallate in neurodegenerative diseases characterised by the formation of amyloid lesions. The fact that gallate can change the properties of amyloids from being neurotoxic to benign should make it a vital tool that allows further dissection of the structural and molecular basis of amyloid toxicity. In addition, this report represents a major step towards the identification of drugs with the potential to target proteinopathies. So far, the efficacy of EGCG has been shown in vitro and in cell-based model systems, but also in fly models of Huntington disease. The next step, of course, will be to show whether this holds true also for mammalian in vivo models, with the ultimate goal (and hope) that EGCG will be successful in clinical trials.

The Crystal Structure of the Green Tea Polyphenol (−)-Epigallocatechin Gallate−Transthyretin Complex Reveals a Novel Binding Site Distinct from the Thyroxine Binding Site,

Amyloid fibril formation is associated with protein misfolding disorders, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. Familial amyloid polyneuropathy (FAP) is a hereditary disease caused by a point mutation of the human plasma protein, transthyretin (TTR), which binds and transports thyroxine (T(4)). TTR variants contribute to the pathogenesis of amyloidosis by forming amyloid fibrils in the extracellular environment. A recent report showed that epigallocatechin 3-gallate (EGCG), the major polyphenol component of green tea, binds to TTR and suppresses TTR amyloid fibril formation. However, structural analysis of EGCG binding to TTR has not yet been conducted. Here we first investigated the crystal structure of the EGCG-V30M TTR complex and found novel binding sites distinct from the thyroxine binding site, suggesting that EGCG has a mode of action different from those of previous chemical compounds that were shown to bind and stabilize the TTR tetramer structure. Furthermore, EGCG induced the oligomerization and monomer suppression in the cellular system of clinically reported TTR variants. Taken together, these findings suggest the possibility that EGCG may be a candidate compound for FAP therapy.

The interactions of epigallocatechin-3-gallate with human whole saliva and parotid saliva

Objective The aim of the present study was to assess the null hypothesis that the astringency and loss of lubrication in the oral cavity are not related to the properties of the epigallocatechin-3-gallate (EGCG) adlayer, the affinity and the entropy-drive of EGCG binding to saliva. Methods The mass, thickness, and viscoelasticity of the EGCG adlayer and the temperature-dependence of EGCG adsorption onto saliva surfaces were determined by quartz crystal microbalance with dissipation (QCM-D). The affinities of EGCG to human whole saliva (WS) and to parotid saliva (PS) were carried out by QCM-D monitoring and fluorescence quenching. Results The stiffer and more compact EGCG adlayers were formed on saliva surfaces at various concentrations of EGCG. The affinity for EGCG binding to WS was higher than that to PS. The precipitation of EGCG/saliva was temperature-dependent. The driving force of EGCG binding to saliva is dominated by the hydrogen bond, the hydrophobic reaction, and the entropy-drive, which were confirmed by the FTIR spectra and the measurement of temperature-dependence, respectively. Conclusion The viscoelasticity of the EGCG adlayer, the affinity of EGCG to saliva, and the priority of EGCG binding to hydrophobic proteins on the mucosa may account for the oral astringency and loss of lubrication.

Biochemical Characterization of Epigallocatechin-3-gallate as an Effective Stimulator for the Phosphorylation of Its Binding Proteins by Glycogen Synthase Kinase-3β <i>in Vitro</i>

The stimulatory and inhibitory effects of epigallocatechin-3-gallate (EGCG) and its related two compounds (luteolin and quercetin) on the phosphorylation of four proteins [bovine myelin basic protein (bMBP), human recombinant tau protein (hrTP), human recombinant vimentin (hrVM) and rat collapsin response mediator protein-2 (rCRMP-2)] by glycogen synthase kinase-3β (GSK-3β) were comparatively determined in vitro. We found that (i) EGCG, not quercetin and luteolin, highly stimulated the GSK-3β-mediated phosphorylation of hrTP and significantly stimulated the phosphorylation of bMBP and hrVM by the kinase; (ii) these three polyphenols inhibited dose-dependently the phosphorylation of rCRMP-2 by GSK-3β; (iii) only EGCG significantly enhanced autophosphorylation of GSK-3β; and (iv) EGCG had a binding-affinity with two basic proteins (bMBP and hrTP) and a low affinity with rCRMP-2 rather than hrVM in vitro. In addition, the binding of EGCG to these two basic proteins induced to highly stimulate their phosphorylation, including novel potent sites for GSK-3β, and to significantly reduce the K(m) value and increase the V(max) value of these two substrate proteins for the kinase in vitro. These results provided here suggest that EGCG acts as an effective stimulator for the GSK-3β-mediated phosphorylation of its binding proteins containing EGCG-inducible phosphorylation sites for the kinase in vitro.

Solution Structure of Human Cardiac Troponin C in Complex with the Green Tea Polyphenol, (−)-Epigallocatechin 3-Gallate

Heart muscle contraction is regulated by Ca(2+) binding to the thin filament protein troponin C. In cardiovascular disease, the myofilament response to Ca(2+) is often altered. Compounds that rectify this perturbation are of considerable interest as therapeutics. Plant flavonoids have been found to provide protection against a variety of human illnesses such as cancer, infection, and heart disease. (-)-Epigallocatechin gallate (EGCg), the prevalent flavonoid in green tea, modulates force generation in isolated guinea pig hearts (Hotta, Y., Huang, L., Muto, T., Yajima, M., Miyazeki, K., Ishikawa, N., Fukuzawa, Y., Wakida, Y., Tushima, H., Ando, H., and Nonogaki, T. (2006) Eur. J. Pharmacol. 552, 123-130) and in skinned cardiac muscle fibers (Liou, Y. M., Kuo, S. C., and Hsieh, S. R. (2008) Pflugers Arch. 456, 787-800; and Tadano, N., Yumoto, F., Tanokura, M., Ohtsuki, I., and Morimoto, S. (2005) Biophys. J. 88, 314a). In this study we describe the solution structure of the Ca(2+)-saturated C-terminal domain of troponin C in complex with EGCg. Moreover, we show that EGCg forms a ternary complex with the C-terminal domain of troponin C and the anchoring region of troponin I. The structural evidence indicates that the binding site of EGCg on the C-terminal domain of troponin C is in the hydrophobic pocket in the absence of troponin I, akin to EMD 57033. Based on chemical shift mapping, the binding of EGCg to the C-terminal domain of troponin C in the presence of troponin I may be to a new site formed by the troponin C.troponin I complex. This interaction of EGCg with the C-terminal domain of troponin C.troponin I complex has not been shown with other cardiotonic molecules and illustrates the potential mechanism by which EGCg modulates heart contraction.

Interaction of Polyphenols with Proteins: Binding of (-)-Epigallocatechin Gallate to Serum Albumin, Estimated by Induced Circular Dichroism

The binding of (-)-epigallocatechin gallate (EGCG), a representative natural polyphenol, to human serum albumin (HSA) and bovine serum albumin (BSA) was investigated using induced circular dichroism (CD). The site of the binding EGCG-HSA was analyzed based on the competition with drugs with known binding sites on HSA, such as phenylbutazone (PB) and diazepam (DP). Double-reciprocal plot analyses showed the competitive relations with the site-I- (PB and tolbutamide, TB) and site-II-binding drugs (DP and ibuprofen, IP) indicating the binding of EGCG to sites I and II on HSA, while digitoxin (DG), a site-III-binding drug, did not affect the binding of EGCG. In an analogous way, the competitive relations were observed between EGCG and the site-I- (PB and TB) and site-II-binding (ethacrynic acid, EA) drugs for the binding of EGCG and BSA. The site-III drug DG also showed competitive binding with EGCG to BSA. The binding of EGCG to the albumins indicated its affinity to sites I and II on HSA, while competitive binding for all three sites was observed on BSA.

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.

Binding of (-)-epigallocatechin-3-gallate to the Hsp70 ATPase domain may promote apoptosis in colorectal cancer

Colorectal cancer (CRC) patients frequently have a poor prognosis because of metastases and drug resistance. Heat-shock protein 70 (Hsp70) over-expression in cancer may exacerbate these factors by inhibiting apoptosis, thus providing a potential therapeutic target. The green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG) induces apoptosis in numerous cancer cell lines by unclear mechanisms. This study proposed and investigated a novel mechanism by which EGCG might inhibit the anti-apoptotic activity of Hsp70, namely, by competing with adenosine triphosphate (ATP) for binding to the Hsp70 ATPase domain. It also examined the impact of EGCG on the CRC cell line HT-29, which is known to over-express Hsp70. HT-29 cells were treated with 10–300 mM EGCG for 8, 24 and/or 48 h, then analysed using an MTS proliferation-assay or flow cytometry with Annexin V-FITC and propidium iodide. The competitive binding of EGCG to the Hsp70 ATPase domain was assessed using ATP-agarose, a dot blot and chemiluminescence techniques. EGCG significantly (P, 0.001) and dose-dependently inhibited HT-29 cell viability. Viability was inhibited by 50% (IC50) at 89 mM and 74 mM after 24and 48-h treatments, respectively, also suggesting a time-dependent effect. Apoptosis was induced both doseand timedependently, commencing at 50 mM after 8and 24-h treatments. Further, apoptosis correlated significantly (P, 0.01) with reduced viability as measured by MTS at 24 h, indicating 98% causality. The dot blot suggested 200 mM EGCG competed with ATP for binding to Hsp70, presumably by binding its ATPase domain, theoretically implying this flavonoid could inhibit the anti-apoptotic effect of Hsp70.

Differential effects of a green tea-derived polyphenol (−)-epigallocatechin-3-gallate on the acidosis-induced decrease in the Ca2+ sensitivity of cardiac and skeletal muscle

(-)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined its effects on the acidosis-induced change in the Ca(2+) sensitivity of myofilaments in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. EGCg (0.1 mM) significantly inhibited the decrease caused by lowering the pH from 7.0 to 6.0 in the Ca(2+) sensitivity of myofibrillar ATPase activity in cardiac muscle, but not in skeletal muscle. Studies on recombinant mouse cardiac troponin C (cTnC) and chicken fast skeletal troponin C (sTnC) using circular dichroism and intrinsic and extrinsic fluorescence spectroscopy showed that EGCg bound to cTnC with a dissociation constant of approximately 3-4 muM, but did not bind to sTnC. By presumably binding to the cTnC C-lobe, EGCg decreased Ca(2+) binding to cTnC and overcame the depressant effect of protons on the Ca(2+) sensitivity of the cardiac contractile response. To demonstrate isoform-specific effects of the action of EGCg, the pH sensitivity of the Ca(2+) response was examined in cardiac myofibrils in which endogenous cTnC was replaced with exogenous sTnC or cTnC and in skeletal myofibrils in which the endogenous sTn complex was replaced with whole cardiac Tn complex (cTn). The results suggest that the binding of EGCg to the cardiac isoform-specific TnC or Tn complex alters the effect of pH on myofilament Ca(2+) sensitivity in striated muscle.

Fyn is a novel target of (−)‐epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation

The cancer preventive action of (-)-epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)-induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF-induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF-induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)-Fyn-JB6 cells. With an in vitro protein-binding assay, we found that EGCG directly bound with the GST-Fyn-SH2 domain but not the GST-Fyn-SH3 domain. The K(d) value for EGCG binding to the Fyn SH2 domain was 0.367 +/- 0.122 microM and B(max) was 1.35 +/- 0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF-induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF-2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA-Fyn-JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF-2, and STAT1. The DNA binding ability of AP-1, STAT1, and ATF-2 was also decreased in siRNA-Fyn-JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF-induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG-induced inhibition of cell transformation.

Single GUV Method Reveals Interaction of Tea Catechin (−)-Epigallocatechin Gallate with Lipid Membranes

Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (−)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration (∼150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 μM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.

Pharmacokinetics of (−)-Epigallocatechin-3-gallate in Conscious and Freely Moving Rats and Its Brain Regional Distribution

A liquid chromatography technique coupled with tandem mass spectrometry (LC-MS/MS) electrospray ionization was used to measure (-)-epigallocatechin-3-gallate (EGCG) in rat plasma. This method was applied to investigate the pharmacokinetics of EGCG in a conscious and freely moving rat by an automated blood sampling device. Multiple reaction monitoring (MRM) was used to monitor the transition of the deprotonated molecule m/z of 457 [M - H]- to the product ion 169 for EGCG and the m/z of 187 to 164 for the internal standard. The limit of quantification (LOQ) of EGCG in rat plasma was determined to be 5 ng/mL, and the linear range was 5-5000 ng/mL. The protein binding of EGCG in rat plasma was 92.4 +/- 2.5%. The brain distribution result indicated that EGCG may potentially penetrate through the blood-brain barrier at a lower rate. The disposition of EGCG in the rat blood was fitted well by the two-compartmental model after intravenous administration (10 mg/kg, iv). The elimination half-life of EGCG was 62 +/- 11 and 48 +/- 13 min for intravenous (10 mg/kg) and oral (100 mg/kg) administration, respectively. The pharmacokinetic data indicate that the oral bioavailability of EGCG in a conscious and freely moving rat was about 4.95%.

Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy

The green tea flavonoid, epigallocatechin gallate (EGCG), has been proposed to have an anti-HIV-1 effect by preventing the binding of HIV-1 glycoprotein (gp) 120 to the CD4 molecule on T cells. To demonstrate that EGCG binds to the CD4 molecule at the gp120 attachment site and inhibits gp120 binding at physiologically relevant levels, thus establishing EGCG as a potential therapeutic treatment for HIV-1 infection. Nuclear magnetic resonance spectroscopy was used to examine the binding of EGCG and control, (-)-catechin, to CD4-IgG2 (PRO 542). Gp120 binding to human CD4+ T cells was analyzed by flow cytometry. Addition of CD4 to EGCG produced a linear decrease in nuclear magnetic resonance signal intensity from EGCG but not from the control, (-)-catechin. In saturation transfer difference experiments, addition of 5.8 micromol/L CD4 to 310 micromol/L EGCG produced strong saturation at the aromatic rings of EGCG, but identical concentrations of (-)-catechin produced much smaller effects, implying EGCG/CD4 binding strong enough to reduce gp120/CD4 binding substantially. Molecular modeling studies suggested a binding site for EGCG in the D1 domain of CD4, the pocket that binds gp120. Physiologically relevant concentrations of EGCG (0.2 micromol/L) inhibited binding of gp120 to isolated human CD4+ T cells. We have demonstrated clear evidence of high-affinity binding of EGCG to the CD4 molecule with a Kd of approximately 10 nmol/L and inhibition of gp120 binding to human CD4+ T cells. Epigallocatechin gallate has potential use as adjunctive therapy in HIV-1 infection.

(−)−Epigallocatechin Gallate Overcomes Resistance to Etoposide-Induced Cell Death by Targeting the Molecular Chaperone Glucose-Regulated Protein 78

Many beneficial properties have been attributed to (-)-epigallocatechin gallate (EGCG), including chemopreventive, anticarcinogenic, and antioxidant actions. In this study, we investigated the effects of EGCG on the function of glucose-regulated protein 78 (GRP78), which is associated with the multidrug resistance phenotype of many types of cancer cells. Our investigation was directed at elucidating the mechanism of the EGCG and GRP78 interaction and providing evidence about whether EGCG modulates the activity of anticancer drugs through the inhibition of GRP78 function. We found that EGCG directly interacted with GRP78 at the ATP-binding site of protein and regulated its function by competing with ATP binding, resulting in the inhibition of ATPase activity. EGCG binding caused the conversion of GRP78 from its active monomer to the inactive dimer and oligomer forms. Further, we showed that EGCG interfered with the formation of the antiapoptotic GRP78-caspase-7 complex, which resulted in an increased etoposide-induced apoptosis in cancer cells. We also showed that EGCG significantly suppressed the transformed phenotype of breast cancer cells treated with etoposide. Overall, these results strongly suggested that EGCG could prevent the antiapoptotic effect of GRP78, which usually suppresses the caspase-mediated cell death pathways in drug-treated cancer cells, contributing to the development of drug resistance.

Interaction of (−)‐epigallocatechin‐3‐gallate with human serum albumin: Fluorescence, fourier transform infrared, circular dichroism, and docking studies

(-)-Epigallocatechin-3-gallate (EGCG), the major constituent of green tea has been reported to prevent many diseases by virtue of its antioxidant properties. The binding of EGCG with human serum albumin (HSA) has been investigated for the first time by using fluorescence, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and protein-ligand docking. We observed a quenching of fluorescence of HSA in the presence of EGCG. The binding parameters were determined by a Scatchard plot and the results were found to be consistent with those obtained from a modified Stern-Volmer equation. From the thermodynamic parameters calculated according to the van't Hoff equation, the enthalpy change deltaH degrees and entropy change deltaS degrees were found to be -22.59 and 16.23 J/mol K, respectively. These values suggest that apart from an initial hydrophobic association, the complex is held together by van der Waals interactions and hydrogen bonding. Data obtained by fluorescence spectroscopy, CD, and FTIR experiments along with the docking studies suggest that EGCG binds to residues located in subdomains IIa and IIIa of HSA. Specific interactions are observed with residues Trp 214, Arg 218, Gln 221, Asn 295 and Asp 451. We have also looked at changes in the accessible surface area of the interacting residues on binding EGCG for a better understanding of the interaction.