Dose Of Epigallocatechin-3-gallate Research Articles

Stromal Rescue of Drug Exposed CLL Cells Can Be Overcome by the Green Tea Extract Epigallocatechin 3 Gallate

AbstractAbstract 3610 PurposeChronic Lymphocytic Leukemia (CLL) is incurable with conventional chemotherapy treatments. Nurturing of leukemic CLL B cells by marrow microenvironment appears to be one important method of chemoresistance (NEJM 2005).Epigallocatechin 3 gallate (EGCG), the major catechin in green tea, induces caspase-dependent death CLL B-cells and down regulates anti-apoptotic proteins (Mcl-1; XIAP) critical for enhanced apoptosis resistance of CLL B-cells (Blood 2004). In Phase I and II clinical testing, we found EGCG induced a decline in absolute lymphocyte count and/or lymphadenopathy in the majority of Rai stage 0-II CLL patients (JCO 2009; ASCO 2010).In these phase I/II studies, trough plasma EGCG levels as high as 8 mM were achieved. Since it does not cause myelosuppression, EGCG has the potential to be combined with other chemotherapeutic agents used for CLL. We previously reported that pharmacologically achievable plasma levels of EGCG have an additive or synergistic effect when combined with fludarabine (F), chlorambucil (C), and FC in the majority of patients (ASH 2009). In this study, we evaluated i) the effect of pharmacologically achievable doses of EGCG when combined with bendamustine (B) and ii) the ability of pharmacologically achievable doses of EGCG to overcome in vitro stromal rescue of CLL B-cells exposed to B or FC. MethodsWe first evaluated the effects of EGCG combined with B to establish if this partnership would be additive or synergistic. Primary CLL cells were treated with B (12.5-100 mM) with or without 4 uM EGCG. After 48 hours cells were stained with annexin/PI, and viability analyzed by flow cytometry. Data were analyzed using the CalcuSyn program (Chou & Talay method) to determine the combination index (indicating antagonistic, additive or synergistic effects). Next, we evaluated the ability of 4 mM EGCG to overcome stromal rescue of CLL cells treated with B or FC. To eliminate the cytotoxic effects of FC and B on stromal cells, we treated CLL cells with FC or B for 24 hours, washed with PBS, and then cultured these drug exposed cells directly on the HS-5 human stromal cell line with or without 4 mM EGCG for 48 hours (FC) or 24 hours (B). Cells were harvested and viability of CD19+ cells was assessed by annexin/PI staining. To specifically evaluate the ability of 4 mM EGCG to overcome stromal protection, co-culture samples in which no stromal protection was observed were excluded. Similar experiments were conducted using primary bone marrow mesenchymal stromal cells (MSC) from CLL patients (Leuk Res, 2007). In these experiments the fludarabine/chlorambucil combination was used as an approximation of fludarabine/cyclophosphamide since the active form of cyclophosphamide (4-hydroxycyclophosphamide) rapidly breaks down in aqueous buffer and is not readily available for in vitro use and. ResultsWe previously observed additive or synergistic FC and EGCG interactions in a majority of patient samples (ASH 2009). 4 mM EGCG was less consistently beneficial when combined with increasing doses of B in the absence of stroma. Synergistic or mixed synergistic/additive effect was seen in 5 patients, mixed additive/antagonistic effects in 3, and antagonistic effects in 2. Given previously observed synergy with FC and additive/synergistic interactions with at least some B treated samples, we next explored the effects of EGCG on stromal cell rescue. Co-culture with HS-5 stromal cells significantly reduced both B and FC mediated cell death. However the addition of 4 μM EGCG significantly attenuated stromal cell rescue of CLL B-cells exposed to B and FC therapy (Fig. 1A and 1B respectively). Remarkably in the case of FC, 4 mM EGCG almost completely abrogated stromal rescue. Experiments using primary bone marrow MSC cultures from CLL patients yields similar results (n=3, data not shown). [Display omitted] [Display omitted] ConclusionsPharmacologically achievable doses of EGCG appear to reduce stromal rescue of CLL cells treated with B or combinations of purine nucleoside analog/alkylating agents. This occurs in the absence of detectable stromal cell kill by EGCG. These findings, coupled with the favorable toxicity profile and lack of myelosuppression in phase I/II trials of EGCG make it an attractive agent to test in combination with bendamustine or purine analogue/alkylating agent chemo-immunotherapy for patients with CLL. Disclosures:Shanafelt:Polyphenon Pharma: Research Funding.

Role of (−)-epigallocatechin-3-gallate in cell viability, lipogenesis, and retinol-binding protein 4 expression in adipocytes

(-)-Epigallocatechin-3-gallate (EGCG), a bioactive compound of green tea, is known to combat obesity by reducing the viability and lipid accumulation of adipocytes. In this study, we evaluated the mechanism and clinical relevance on those actions of EGCG. We measured the viability of 3T3-L1 preadipocytes and adipocytes by the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide assay. Lipid accumulation was measured by Oil Red O staining. Intracellular accumulation of reactive oxygen species (ROS) was determined using a flow cytometer. Cellular glucose uptake was determined with 2-deoxy-[(3)H]-glucose. The protein levels of peroxisome proliferator-activated receptor (PPAR)-γ and adiponectin in 3T3-L1 adipocytes, as well as the protein level and secretion of plasma retinol-binding protein (RBP4) in human adipocytes, were measured by western blot. EGCG at concentrations higher than 10μM induced ROS generation and decreased the viability and lipid accumulation of adipocytes. It also decreased the expression of PPAR-γ and adiponectin. At concentrations readily achievable in human plasma via green tea intake (≤10μM), EGCG inhibited cellular glucose uptake and enhanced the expression and secretion of RBP4 in adipocytes. Pharmacological doses of EGCG showed cytotoxic effects in preadipocytes and adipocytes. EGCG-mediated glucose uptake inhibition in adipocytes may be clinically relevant and is probably linked to the increase in the expression and secretion of RBP4. Because secreted RBP4 from adipocytes inhibits muscular glucose uptake and enhance hepatic glucose output, the systemic effect of EGCG associated with its effect on RBP4 secretion should be further determined, as it may negatively regulate whole-body insulin sensitivity, contrary to general belief.

Inhibition of inducible nitric oxide synthase expression and cell death by (−)-epigallocatechin-3-gallate, a green tea catechin, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease

The aim of this study was to investigate the involvement of inducible nitric oxide synthase (iNOS) in the action of (−)-epigallocatechin-3-gallate (EGCG), a potential neuroprotective agent against Parkinson’s disease (PD), and to test for toxicity resulting from high doses of EGCG. EGCG was administered to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice at two different doses (10 mg/kg and 50 mg/kg). EGCG treatment reduced the neuronal death rate to less than 50%. The level of iNOS expression in the MPTP group was 20% higher than that seen in the control group, but in the EGCG groups, iNOS expression was reduced to the level observed in the negative control group. The two doses of EGCG were equally beneficial for cell rescue, and no toxicity was observed with the higher dose. Inhibition of iNOS may be an important mechanism underlying the prevention of MPTP toxicity, and EGCG may potentially be a neuroprotective agent against PD.

Green tea polyphenol epigallocatechin 3-gallate in arthritis: progress and promise

Green tea's active ingredient, epigallocatechin 3-gallate (EGCG), has gained significant attention among scientists and has been one of the leading plant-derived molecules studied for its potential health benefits. In the present review I summarize the findings from some of the most significant preclinical studies with EGCG in arthritic diseases. The review also addresses the limitations of the dose, pharmacokinetics, and bioavailability of EGCG in experimental animals and findings related to the EGCG-drug interaction. Although these findings provide scientific evidence of the anti-rheumatic activity of EGCG, further preclinical studies are warranted before phase clinical trials could be initiated with confidence for patients with joint diseases.

The Green Tea Extract EGCG Demonstrates Synergist Activity against CLL B-Cells When Combined with Fludarabine and Chlorambucil.

Abstract 3452Poster Board III-340 PURPOSEChronic Lymphocytic Leukemia (CLL) is incurable with current chemotherapy treatments. Epigallocatechin 3 gallate (EGCG), the major catechin in green tea, has been shown to induced caspase-dependent death CLL B-cells and down regulate anti-apoptotic proteins (Mcl-1; XIAP) known to increase the resistance of CLL B-cells to apoptosis (Blood 104:788-94). In Phase I testing for patients with early stage CLL (Rai 0-II), EGCG treatment was well tolerated and induced a decline in absolute lymphocyte count (ALC) and/or lymphadenopathy in the majority of treated patients without myelosuppression (JCO 27:3808-14). Although peak plasma EGCG levels were not measured in this study, trough plasma EGCG levels as high as 4 μg/ML were achieved. To further our understanding of the potential clinical applications of EGCG for patients with CLL, we evaluated the effects of EGCG on the viability of CLL B-cells when combined with fludarabine (F), chlorambucil (C), or fludarabine and chlorambucil in combination (FC). METHODSPrimary CLL B-cells were treated with various doses of F (0.25-1 μM), C (10-30 μM), or FC either alone or in combination EGCG (50 to 100 μM). In other experiments, CLL B-cells were treated with various doses of F (0.25-1 μM), C (10-30 μM), or FC with or without a fixed dose of EGCG known to be physiologically achievable in vivo (4 μM). After 48 hours (experiments with C +/− EGCG) or 72 hours (experiments with F +/− EGCG or FC +/− EGCG), cells were harvested, stained with annexin/PI and viability analyzed by flow cytometry. After concentration-effect curves were generated for each agent, data were also analyzed using the CalcuSyn software program (Biosoft, Cambridge, UK) which uses the method of Chou and Talalay to determine whether combination treatment yielded greater effects than expected from summation alone. A combination index (CI) of 0.8 – 1.2 indicates an additive effect, a CI >1.2 indicates an antagonistic effect and a CI <0.8 indicates a synergistic effect. RESULTSPrimary leukemic B-cells from 56 CLL patients were cultured in vitro with various doses of EGCG alone or in combination with F, C, or FC. The median LD50 was approximately 100 μM. Although % apoptotic cells at the 100 μM EGCG dose did not vary based on Rai stage, ZAP-70 status, IGHV gene mutation status, or cytogenetic abnormalities by FISH, CD38 negative patients had greater cell death than CD38 positive patients (67% vs. 49%; p=0.05). In co-titration experiments, EGCG had an additive (CI 0.8 – 1.2) or synergistic (CI <0.8) effect on apoptosis when combined with C in the majority of patients (13/15; additive 11, synergism 2) with rare individuals demonstrating antagonism (CI >1.2; 2/15). The effects of EGCG when combined with F in co-titration experiments were more variable with a relatively even distribution between antagonism (8/18), additive effects (4/18), and synergy (6/18). However, co-titration experiments of FC with or without EGCG demonstrated additive or synergistic effect in most patients (8/10; additive 7, synergism 1). Next we evaluated the effect of F (0.25 - 1 μM), C (10 - 30 μM), or FC with or without a fixed dose of EGCG known to be physiologically achievable in vivo (4 μM). This dose of EGCG had an additive or synergistic effect in the majority of samples across the spectrum of dose levels under all 3 conditions (F, C, and FC). For example, at the 10 μM dose of C the addition of 4 μM EGCG had an additive or synergistic effect in 11/14 patients (synergistic 6; additive 5). With F at the 2.5 μM dose level the addition of 4 μM EGCG had an additive or synergistic effect in 13/16 patients (synergistic 7, additive 6). The addition of 4 μM EGCG appeared particularly beneficial when given in combination with FC (Fig.). For example, at F 2.5 μM in combination with C 5 μM the addition of 4 μM EGCG had an additive or synergistic effect in 10/10 patients (synergism 7; additive 3). CONCLUSIONSPhysiologically achievable doses of EGCG appear to enhance the efficacy of alkylating agents, purine nucleoside analogues, and alkylating agent/purine analogue combination therapy for the majority of CLL patients on in vitro testing. The favorable toxicity profile of EGCG and lack of myelosuppression with this agent in the phase I trial (JCO 27:3808-14) make it an attractive agent to test in combination with purine analogue and alkylator based chemo-immunotherapy for patients with CLL. [Display omitted] DisclosuresKay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon Pharma : Patents & Royalties, Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding.

Hepatotoxicity of high oral dose (−)-epigallocatechin-3-gallate in mice

The tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) has been studied for chronic disease preventive effects, and is marketed as part of many dietary supplements. However, case-reports have associated the use of green tea-based supplements with liver toxicity. We studied the hepatotoxic effects of high dose EGCG in male CF-1 mice. A single dose of EGCG (1500 mg/kg, i.g.) increased plasma alanine aminotransferase (ALT) by 138-fold and reduced survival by 85%. Once-daily dosing with EGCG increased hepatotoxic response. Plasma ALT levels were increased 184-fold following two once-daily doses of 750 mg/kg, i.g. EGCG. Moderate to severe hepatic necrosis was observed following treatment with EGCG. EGCG hepatotoxicity was associated with oxidative stress including increased hepatic lipid peroxidation (5-fold increase), plasma 8-isoprostane (9.5-fold increase) and increased hepatic metallothionein and γ-histone 2AX protein expression. EGCG also increased plasma interleukin-6 and monocyte chemoattractant protein-1. Our results indicate that higher bolus doses of EGCG are hepatotoxic to mice. Further studies on the dose-dependent hepatotoxic effects of EGCG and the underlying mechanisms are important given the increasing use of green tea dietary supplements, which may deliver much higher plasma and tissue concentrations of EGCG than tea beverages.

Protective effects of epigallocatechin gallate following 3-nitropropionic acid-induced brain damage: possible nitric oxide mechanisms

The role of oxidative stress has been well known in neurodegenerative disorders. 3-Nitropropionic acid (3-NP) is a plant-based mycotoxin that produces HD like symptoms in animals. Oxidative stress and nitric oxide mechanisms have been recently proposed in the 3-NP-induced neurotoxicity. Epigallocatechin gallate (EGCG) is one of the major components of green tea, known for its potent antioxidant activity. Besides, neuroprotective effect of EGCG has also been suggested in different experimental models. The present study has been designed to examine possible effect of EGCG against 3-NP induced behavioral, oxidative stress, mitochondrial dysfunction, and striatal damage in rats and its possible interaction with nitric oxide modulators. Systemic 3-NP (10 mg/kg) administration for 14 days significantly reduced locomotor activity, body weight, grip strength, oxidative defense (raised levels of lipid peroxidation, nitrite concentration, depletion of antioxidant enzyme), and mitochondrial enzymes activity in striatum, cortex, and hippocampal regions of the brain. Fourteen days of EGCG pretreatment (10, 20, and 40 mg/kg) significantly attenuated behavioral alterations, oxidative damage, mitochondrial complex enzymes dysfunction, and striatal damage in 3-NP-treated animals. L-arginine (50 mg/kg) pretreatment with sub-effective dose of EGCG (20 mg/kg) significantly reversed the protective behavioral, biochemical, cellular, and histological effects of EGCG. However, L-NAME (10 mg/kg) pretreatment with EGCG (20 mg/kg) significantly potentiated the protective effect of EGCG which was significant as compared to their effect per se. The present study shows that EGCG attenuate 3-NP-induced neurotoxicity, and nitric oxide modulation might be involved in its protective action.

Effect of lycopene and epigallocatechin-3-gallate against 3-nitropropionic acid induced cognitive dysfunction and glutathione depletion in rat: A novel nitric oxide mechanism

Huntington’s disease is a neurodegenerative disorder characterized by symptoms like chorea and dementia. There is no exact therapeutic agent available to manage and cure this disease. 3-Nitropropionic acid, a neurotoxin causes gait and memory impairment which leads to oxidative damage and upsets glutathione defense in animals. 3-NP model is a useful tool to develop suitable therapeutic agent in the treatment of Huntington’s disease. Present study compares the effects of lycopene and epigallocatechin-3-gallate (EGCG) on memory impairment and disturbs glutathione system against 3-NP treatment. 3-NP treatment significantly impaired memory as assessed in Morris water maze and elevated plus maze tasks. On the 15 day, the levels of reduced glutathione, total glutathione and glutathione-S-transferase were also significantly decreased in the striatum, hippocampus and cortex areas of the brain. The treatment with lycopene (2.5, 5 and 10 mg/kg) and EGCG (10, 20 and 40 mg/kg) significantly improved memory and restored glutathione system functioning. Further, L-arginine and L-NAME pretreatment with the sub effective dose of lycopene (5 mg/kg) and EGCG (20 mg/kg) reversed and potentiate their protective effects respectively. In conclusion, lycopene and EGCG could be used to mange 3-NP induced behavioral and biochemical alterations by involving nitric oxide pathways.

(-)-Epigallocatechin gallate enhances the expression of genes related to insulin sensitivity and adipocyte differentiation in 3T3-L1 adipocytes at an early stage of differentiation

Objective (-)-Epigallocatechin gallate (EGCG) is thought to enhance insulin sensitivity in adipocytes, although doses used in in vitro experiments have been shown to promote apoptosis. To explore the effects of EGCG on insulin sensitivity in adipocytes, the expression of genes related to insulin sensitivity and adipocyte differentiation in 3T3-L1 adipocytes were measured in response to low doses of EGCG. Methods Increasing concentrations of low-dose EGCG were administered for 8 d to differentiating 3T3 adipocytes, either at days 0–8 (early stage) or at days 8–16 (late stage). Fat accumulation and cell activity were measured by Oil Red O staining and 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay, respectively. The expression of genes related to insulin sensitivity and adipocyte differentiation was measured by real-time reverse transcriptase–polymerase chain reaction. Results Fat accumulation and cell activity in 3T3-L1 cells at the early and late stages were reduced at EGCG concentrations ≥50 μM. However, EGCG doses of 5–10 μM reduced fat accumulation and induced the expression of genes related to insulin sensitivity (including Fabp4, Cd36, Lpl, Pck1, Acox1, Lypla3, and Ucp2) and adipocyte differentiation ( Pparg1, Pparg2, Cebps, and Ppargc1a). These increases were only seen at the early, and not late, stages of differentiation. Conclusion These data indicate that low doses of EGCG, despite reducing triacylglycerol accumulation, induce the expression of genes related to insulin sensitivity in the early stage of differentiation.

Dihydrotestosterone sensitises LNCaP cells to death induced by epigallocatechin‐3‐gallate (EGCG) or an IGF‐i receptor inhibitor

Compelling evidence has accumulated for chemopreventive effects for the active component of green tea Epigallocatechin-3-Gallate (EGCG) particularly for prostate cancer (CaP). We have assessed interactions between the effects of EGCG and two main regulators of prostate cell function, dihydrotestosterone (DHT) and insulin-like growth factor-1 (IGF-I). Using LNCaP (androgen-sensitive), PC3 and DU145 (androgen-resistant) CaP cell lines, we assessed the effect of EGCG alone on growth (0-200 microM) and on cell death (0-50 microM). EGCG decreased the proliferation of all the CaP cancer cells in a dose-dependent manner with an increase in apoptosis from 30 to 50 microM. With DU145 cells, a sub-apoptotic dose of EGCG (10-20 microM) reduced IGF-induced growth. With LNCaP cells, a sub-apoptotic dose of EGCG (8 microM) switched DHT from a growth promoter to a growth inhibitor. A similar reversal of DHT effect was seen in the presence of an IGF-I receptor inhibitor, AG1024 (1 microM). These responses appeared to be due to DHT sensitizing the cells to apoptosis by EGCG and AG1024 (P < 0.01 and P < 0.001 respectively). Our data suggests that both green tea and AG1024 are effective in inhibiting cell growth and inducing death in CaP cells but the effects of both are more effective in the presence of androgen.

Reversal of hypermethylation and reactivation of genes by dietary polyphenolic compounds

Reversal of hypermethylation and reactivation of genes by dietary polyphenolic compounds

T1781 N-Acetyl-L-Cysteine (NAC) Abrogates Changes in Cell Viability, Mitochondrial Potential and Gene Expression Caused By (-)-Epigallocatechin Gallate (EGCG) in a Human Liver (Huh7) Cell Line

Background: Green tea, a widely consumed beverage, contains polyphenolic compounds which are antioxidants with reported anti-inflammatory and anti-carcinogenic properties. Studies have shown that EGCG is one of the more abundant and active polyphenols that are responsible for these properties. Extracts based on EGCG, usually ingested as dietary supplements, are widely used as a form of complimentary and alternative medicine. However, use of these extracts is associated with severe hepatotoxicity and even acute liver failure. Aim: To determine whether treatment with NAC in HuH7 cells reverses EGCG-induced changes in cell viability, mitochondrial potential and gene expression. Design: HuH7 cells were treated with increasing doses of EGCG in the presence or absence of NAC. Cell viability was assessed by the CellTiter-Glo® assay (Promega), which measures luminescence based on an ATP-driven luciferase reaction from freshly lysed cells, in a 96 well-format. Mitochondrial membrane potential changes were measured by flow cytometry using JC-1 (Invitrogen/ Molecular Probes) fluorescent dye. Microarray analysis (using Illumina RefSeq8 Human gene array) was performed on RNA extracted (RNeasy Plus Mini Kit, Qiagen) six hours after treatment to identify genes in which expression was altered by EGCG and partially restored by NAC. Results: EGCG reduced cell viability in HuH7 cells with an IC(50) = 58μM at 48 hours. The addition of 10 mM NAC to cells treated with EGCG increased the IC(50) value to 138 μM. Treatment with EGCG showed a dose-dependent decrease in mitochondrial potential, which was significantly reversed by the addition of NAC. Microarray analysis revealed that EGCG downregulated, and NAC partially restored, the expression of genes involved with mitochondrial electron transport, energy generation, ATP synthesis and oxidative phosphorylation. Conclusion: N-acetyl-L-cysteine can reverse EGCG-induced changes in cell viability, mitochondrial potential and gene expression in HuH7 cells. NAC may prove to be a useful clinical intervention for the potential hepatotoxicity caused by EGCG.

Limited Sampling Strategies to Estimate Exposure to the Green Tea Polyphenol, Epigallocatechin Gallate, in Fasting and Fed Conditions

The objective of this study was to develop an efficient sampling strategy to predict epigallocatechin gallate (EGCG) pharmacokinetics after green tea administration. Ten healthy subjects received a single 800-mg oral dose of EGCG administered as Polyphenon E under both fasting and fed conditions. Plasma samples were serially collected over 24 hours and EGCG concentrations were determined. A one-compartment model with a lag time for absorption best fit the concentration-time data. Maximum A Posteriori Bayesian (MAPB) priors were developed by simultaneously fitting pharmacokinetic parameters from both study phases. The D-optimal sampling designs were determined and Monte Carlo simulations were performed. The original model with the estimators was used to fit the simulated data with the optimized sampling schemes. Two and three optimal sampling strategies (OSS-2 and OSS-3, respectively) were developed. The median two sampling times for OSS-2 were 1.3 and 6.9 hours (fasting conditions) and 3.4 and 8.7 hours (fed conditions). The median three sampling times for OSS-3 were 0.7, 1.4, and 7.0 hours (fasting conditions) and 1.4, 3.6, and 8.7 hours (fed conditions). The predictive power of OSS-3 was greater than that of OSS-2, under both fasted and fed conditions, and both strategies had greater predictive performance under fasting conditions. The sampling schemes were accurate and precise in predicting EGCG oral clearance (or area under the curve with known doses), and hence exposure, under both fasting and fed conditions. The increased predictive performance for estimating pharmacokinetic parameters under fasting conditions appeared to be the result of a decreased variability in absorption.

A Phase 1 Trial of Daily Oral Green Tea Extract in Asymptomatic, Rai Stage 0–II Patients with Chronic Lymphocytic Leukemia.

BACKGROUND: Green tea has long been touted as a health promoting substance. The active chemical compounds in green tea are called polyphenols or catechins. Epigallocatechin gallate(EGCG) is the major catechin in green tea. We previously reported the in vitro ability of EGCG to induce apoptotic cell death in chronic lymphocytic leukemia(CLL) B-cells in vitro (Blood 104:788). After publication of our findings, clinical activity in individuals using over the counter green tea extracts were reported (Leuk Res 30:707). Based on this information, we opened a phase I/II trial of green tea extracts for patients with asymptomatic, early stage CLL in fall of 2005.METHODS: The purpose of the phase I portion of this trial was to determine the optimal dose of EGCG in the Polyphenon E preparation for chronic daily administration and define tolerability in CLL patients. Previously untreated patients with asymptomatic, Rai stage 0–II CLL not currently meeting National Cancer Institute(NCI) Working Group(WG) Criteria for treatment were eligible for participation. Polyphenon E with a standardized dose of EGCG was obtained from NCI. The phase I portion of the trial was designed with 8 dose levels(range 400–2000 mg orally BID) using the standard 3 patient per dose level design. Patients remained on study up to 6 months. Grade 2 adverse events attributed to study treatment that did not respond to supportive care were considered dose limiting toxicity. The trial was designed to administer Polyphenon E in the fasting state. After accrual to dose levels 1 and 2, the U.S. FDA mandated all U.S. trials of Polyphenon E administer drug in the fed state. Accordingly, drug was administered in the fed state for dose levels 3–8. Trough plasma EGCG levels were measured 1 month after initiation of therapy. Response was classified using the NCI WG Criteria.RESULTS: As of August 2007, 33 patients have been accrued to dose levels 1–8. The maximum tolerated dose(MTD) has not been reached. Side effects have generally been mild. The most common toxicities were nausea(grade 1: 42%; grade 2: 3%), elevation in SGOT (42%; all grade 1), and abdominal pain (36%; all grade 1). To date, no patient has had a sustained 50% reduction in both absolute lymphocyte count (ALC) and lymphadenopathy that would meet the NCI WG criteria for partial response. A majority of patients have had a reduction in ALC(Table). Among the 10 patients who had palpable adenopathy at study enrollment, 7 patients experienced at least a 50% reduction in the sum of the products of all nodal areas at some point during treatment. Trough plasma EGCG levels after 1 month of treatment ranged from 2.93974 ng/mL(median 40.5 ng/mL). Plasma levels did not clearly relate to the degree of reduction in ALC suggesting sensitivity to Polyphenon E may relate more to characteristics of the leukemic clone than plasma EGCG levels.CONCLUSION: Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase I trial. The MTD has not been reached. As classified by the NCI WG criteria, no partial or complete remissions have been observed to date, however declines in ALC and lymphadenopathy have been observed in the majority of patients. The phase II portion of this trial will open at Mayo Clinic Fall 2007.Best reduction in ALCn% of patientsAt least 10% decline2576%At least 20% decline1442%At least 30% decline824%At least 40% decline412%At least 50% decline26%

Inhibition Of Tumorigenesis in ApcMin/+ Mice by a Combination of (–)-Epigallocatechin-3-gallate and Fish Oil

The effect of a combination of (-)-epigallocatechin-3-gallate (EGCG) with fish oil on intestinal tumorigenesis in Apc (Min/+) mice fed a high-fat diet was investigated in the present study. The combined treatment of EGCG and fish oil for 9 weeks reduced the tumor number by 53% as compared to controls while neither agent alone had a significant effect. Apoptosis was significantly increased in all treatment groups. beta-Catenin nuclear positivity in adenomas from the combination group was lower than control mice, implicating the modulation of Wnt signaling by the combination. Fish oil and the combination significantly reduced prostaglandin E 2 (PGE 2) levels in small intestinal tumors as compared to controls, suggesting modulation of aberrant arachidonic acid metabolism by fish oil. Akt phosphorylation in adenomas was significantly reduced in all treatment groups, which may have contributed to the observed increase in apoptosis. The results indicate that a combination of low doses of EGCG and fish oil can inhibit tumor multiplicity in Apc (Min/+) mice.

Inhibition of Epigallocatechin Gallate on Orthotopic Colon Cancer by Upregulating the Nrf2-UGT1A Signal Pathway in Nude Mice

Epigallocatechin gallate (EGCG), a key active ingredient in green tea, has many anti-carcinogenic activities. The aim of the present study was to investigate whether EGCG could prevent the occurrence or metastases of orthotopic colon cancer and probe the underlined mechanisms. We observed the inhibition of EGCG on growth and metastases of colon tumor implanted orthotopically in the cecum of nude mice. Immunohistochemistry and Western-blotting analysis were used to detect NF-E2-related factor 2 (Nrf2) protein expressions. RT-PCR was also applied to detect the mRNA levels of Nrf2, uridine 5′-diphosphate-glucuronosyltransferase (UGT) 1A, UGT1A8 and UGT1A10 in colon tumors. As a result, the inhibition rates on tumor growth in the 3 EGCG groups were significantly different (all p < 0.001) compared with the control group. In addition, different doses of EGCG were able to inhibit liver and pulmonary metastases to varying degrees. The protein level of Nrf2 and the mRNA levels of Nrf2, UGT1A, UGT1A8 and UGT1A10 significantly increased in EGCG-treated mice in comparison with the control group (all p < 0.01). The results demonstrated that EGCG has a preventive effect on the growth and liver and pulmonary metastases of orthotopic colon cancer in nude mice, and this anticancer effect could be partly caused by activating the Nrf2-UGT1A signal pathway.

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 Ca2+-permeable non-selective cation currents (NSCC) and voltage-operated Ca2+ 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 μM), EGCG (30 μM) activated a long-lasting inward current, with a reversal potential (Erev) 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 μM) and Cd2+ (500 μM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 μM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 μM) also potentiated a nifedipine-sensitive voltage-dependent Ba2+-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 Ca2+ influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd2+-sensitive Ca2+-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 Ca2+ 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.

Acute EGCG Supplementation Reverses Endothelial Dysfunction in Patients with Coronary Artery Disease

Background: Epidemiological studies demonstrate an inverse relation between dietary flavonoid intake and cardiovascular risk. Recent studies with flavonoid-containing beverages suggest that the benefits of these nutrients may relate, in part, to improved endothelial function.Objective: We hypothesized that dietary supplementation with epigallocatechin gallate (EGCG), a major catechin in tea, would improve endothelial function in humans.Design: We examined the effects of EGCG on endothelial function in a double blind, placebo-controlled, crossover design study. We measured brachial artery flow-mediated dilation by vascular ultrasound at six time points: prior to treatment with EGCG or placebo, two hours after an initial dose of EGCG (300 mg) or placebo, and after two weeks of treatment with EGCG (150 mg twice daily) or placebo. The order of treatments (EGCG or placebo) was randomized and there was a one-week washout period between treatments.Results: A total of 42 subjects completed the study, and brachial artery flow-mediated dilation improved from 7.1 ± 4.1 to 8.6 ± 4.7% two hours after the first dose of 300 mg of EGCG (P = 0.01), but was similar to baseline (7.8 ± 4.2%, P = 0.12) after two weeks of treatment with the final measurements made approximately 14 hours after the last dose. Placebo treatment had no significant effect, and there were no changes in reactive hyperemia or the response to sublingual nitroglycerin. The changes in vascular function paralleled plasma EGCG concentrations, which increased from 2.6 ± 10.9 to 92.8 ± 78.7 ng/ml after acute EGCG (P < 0.001), but were unchanged from baseline after two weeks of treatment (3.4 ± 13.1 ng/ml).Conclusion: EGCG acutely improves endothelial function in humans with coronary artery disease, and may account for a portion of the beneficial effects of flavonoid-rich food on endothelial function.

Protein kinase CK2 modulates apoptosis induced by resveratrol and epigallocatechin-3-gallate in prostate cancer cells

Resveratrol and epigallocatechin-3-gallate (EGCG) are important candidates as chemopreventive agents by virtue of their ability to induce apoptosis in cancer cells. Casein kinase 2 (CK2) is a ubiquitous protein ser/thr kinase that plays diverse roles in cell proliferation and apoptosis. We have previously shown that overexpression of CK2 suppresses apoptosis induced by a variety of agents, whereas down-regulation of CK2 sensitizes cells to induction of apoptosis. We therefore investigated whether or not CK2 played a role in resveratrol and EGCG signaling in androgen-sensitive (ALVA-41) and androgen-insensitive (PC-3) prostate cancer cells. Resveratrol- and EGCG-induced apoptosis is associated with a significant down-regulation of CK2 activity and protein expression in both the ALVA-41 and PC-3 cells. Overexpression of CK2alpha protected prostatic cancer cells against resveratrol- and EGCG-induced apoptosis. Relatively low doses (10 mumol/L) of resveratrol and EGCG induced a modest proliferative response in cancer cells that could be switched to cell death by moderate inhibition of CK2. These findings characterize, for the first time, the effects of polyphenolic compounds on CK2 signaling in androgen-sensitive and androgen-insensitive prostatic carcinoma cells and suggest that resveratrol and EGCG may mediate their cellular activity, at least in part, via their targeting of CK2. Further, the data hint at the potential of using these polyphenols alongside CK2 inhibitors in combination chemotherapy.

Effects of Epigallocatechin Gallate on Diethyldithiocarbamate-Induced Pancreatic Fibrosis in Rats

Epigallocatechin gallate (EGCG), a major component of green tea extracts, is known to have anti-fibrotic properties in many organs. The aim of present study was to investigate effects of EGCG on rat pancreatic fibrosis induced by diethyldithiocarbamate (DDC). Oral gavages of different dose of EGCG (50, 100 and 200 mg/kg daily for 8 weeks) ameliorated histological changes and significantly suppressed collagen deposition in a dose-dependent manner. Meanwhile, administration of EGCG inhibited overexpression of TGF-beta1 and alpha-smooth muscle actin (a symbol of activation of pancreatic stellate cells). Moreover, EGCG has a potent influence on expression of Smads (downstream transcription factor of TGF-beta1). EGCG suppressed the expression of Smad3 and enhanced the expression of Smad7. In conclusion, our results demonstrated that EGCG attenuated rat pancreatic fibrosis induced by DDC and therefore may be an anti-fibrogenic candidate in the pancreatic fibrosis.