Abstract

Kombucha, a fermented tea (KT) is claimed to possess many beneficial properties. Recent studies have suggested that KT prevents paracetamol and carbon tetrachloride-induced hepatotoxicity. We investigated the beneficial role of KT was against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity and cell death in murine hepatocytes. TBHP is a well known reactive oxygen species (ROS) inducer, and it induces oxidative stress in organ pathophysiology. In our experiments, TBHP caused a reduction in cell viability, enhanced the membrane leakage and disturbed the intra-cellular antioxidant machineries while simultaneous treatment of the cells with KT and this ROS inducer maintained membrane integrity and prevented the alterations in the cellular antioxidant status. These findings led us to explore the detailed molecular mechanisms involved in the protective effect of KT. TBHP introduced apoptosis as the primary phenomena of cell death as evidenced by flow cytometric analyses. In addition, ROS generation, changes in the mitochondrial membrane potential, cytochrome c release, activation of caspases (3 and 9) and Apaf-1 were detected confirming involvement of mitochondrial pathway in this pathophysiology. Simultaneous treatment of KT with TBHP, on the other hand, protected the cells against oxidative injury and maintained their normal physiology. In conclusion, KT was found to modulate the oxidative stress induced apoptosis in murine hepatocytes probably due to its antioxidant activity and functioning via mitochondria dependent pathways and could be beneficial against liver diseases, where oxidative stress is known to play a crucial role.

Pathophysiology. 2011 Jun;18(3):221-34. Epub 2011 Mar 8.

Observational studies on tea consumption and prostate cancer (PCa) risk
are still inconsistent. The authors conducted a meta-analysis to
investigate the association between green tea and black tea consumption
with PCa risk. Thirteen studies providing data on green tea or black tea
consumption were identified by searching PubMed and ISI Web of Science
databases and secondary referencing qualified for inclusion. A
random-effects model was used to calculate the summary odds ratios (OR)
and their corresponding 95% confidence intervals (CIs). For green tea,
the summary OR of PCa indicated a borderline significant association in
Asian populations for highest green tea consumption vs. non/lowest (OR =
0.62; 95% CI: 0.38-1.01); and the pooled estimate reached statistically
significant level for case-control studies (OR = 0.43; 95% CI:
0.25-0.73), but not for prospective cohort studies (OR = 1.00; 95% CI:
0.66-1.53). For black tea, no statistically significant association was
observed for the highest vs. non/lowest black tea consumption (OR =
0.99; 95% CI: 0.82-1.20). In conclusion, this meta-analysis supported
that green tea but not black tea may have a protective effect on PCa,
especially in Asian populations. Further research regarding green tea
consumption across different regions apart from Asia is needed.

http://www.ncbi.nlm.nih.gov/pubmed/21667398

Extracts and fluid extracts from the aerial parts from Passiflora incarnata L. are widely used as components of herbal sedatives. Many pharmacological investigations confirm the sedative effects of Passiflorae herba. From some of the studies also anxiolytic effects can be deduced. As Passionflower is mainly used in combinations, clinical studies of the single drug are not available. Based on pharmacological data, the experiences of traditional use and the use in combinations Passiflora extracts are an important factor in the phytotherapy of tenseness, restlessness and irritability with difficulty in falling asleep.

Wien Med Wochenschr. 2002;152(15-16):404-6.

A relaxed, awake state is associated with Alpha waves, and an awake and excited brain will emit high frequency Beta waves. Caffeine can be shown to suppress theta and alpha waves, while promoting the beta waves that are linked with stress and anxiety. So, what does theanine do?

A number of studies have confirmed that within 30 minutes of ingesting theanine, there is a measurable enhancement of alpha wave activity, implying an alert but relaxed state.

And while clinical studies to date don’t show an improvement in memory or mental function in humans, rodent studies show some promise. The dose of theanine in the Gatorade product, 25 mg per serving, is below the levels used in studies, but tea itself does fall within the range of use that affects alpha brain waves.

For all the latest news on tea, click Tea under Categories to the right.

Tea Antiinflammatory
Green tea polyphenols such as EGCG have potent anti-inflammatory properties. Prior research had shown that EGCG inhibits tumor necrosis factor through a mechanism that was thought to have implications for inflammation generally. Epidemiological studies link regular consumption of tea with decreased cancer risk and a reduction in mortality during the 12 month period following a heart attack. “Considerably less is known regarding the mechanisms by which tea confers these health benefits.” The present research demonstrates one important mechanism in the inhibition of interleukin-1 mediated signal transduction. “Given the long safety record of tea consumption, the use of EGCG and related compounds may represent a novel pharmacological strategy for the modulation of inflammation. EGCG and related compounds could potentially be used as a nutritional supplement in patients with inflammatory disease processes. The next steps to further substantiate these assertions are to test the efficacy of green tea derived polyphenols such as EGCG in animal models of inflammation associated organ injury and to further elucidate the mechanisms by which these compounds modulate no inflammatory signal transduction pathways.” FULL ARTICLE –>

Epigallocatechin-3-gallate, a Green Tea-Derived Polyphenol, Inhibits IL-1[beta]-Dependent Proinflammatory Signal Transduction in Cultured Respiratory Epithelial Cells1,2
The Journal of Nutrition . May 1, 2004 . Catravas, John D; Denenberg, Alvin; Et al; Odoms, Kelli; Wheeler, Derek S
ABSTRACT Polyphenolic components of green tea, such as epigallocatechin-3-gallate (EGCG), have potent anti-inflammatory properties. We previously showed that EGCG inhibits tumor necrosis factor-[alpha] (TNF-[alpha])-mediated activation of the nuclear factor-[kappa]B (NF-[kappa]B) pathway, partly through inhibition of I[kappa]B kinase (IKK). The NF-[kappa]B pathway may also be activated in response to interleukin-1[beta] (IL-1[beta]) stimulation through a distinct signal transduction pathway. We therefore hypothesized that EGCG inhibits IL-1 [beta]-mediated activation of the NF-[kappa]?B pathway. Because the gene expression of interleukin-8 (IL-8), the major human neutrophil chemoattractant, is dependent on activation of NF-[kappa]B, IL-8 gene expression in human lung epithelial (A549) cells treated with human IL-1[beta] was used as a model of IL-1[beta] signal transduction. The EGCG markedly inhibited IL-1[beta]-mediated IL-1[beta] receptor-associated kinase (IRAK) degradation and the signaling events downstream from IRAK degradation: IKK activation, I[kappa]B[alpha] degradation, and NF-[kappa]B activation. In addition, EGCG inhibited phosphorylation of the p65 subunit of NF-[kappa]B. The functional consequence of this inhibition was evident by inhibition of IL-8 gene expression. Therefore, the green tea polyphenol EGCG is a potent inhibitor of IL-1[beta] signal transduction in vitro. The proximal mechanisms of this effect involve inhibition of IRAK-dependent signaling and phosphorylation of p65. J. Nutr. 134: 1039-1044, 2004.

KEY WORDS: * transcription factors * inflammation * signal transduction * chemokines * polyphenols

During the initial host inflammatory response to an infection or other inciting event, several pro inflammatory cytokines are released into the systemic circulation, which, if left unchecked, can ultimately cause a dysregulated inflammatory cascade that results in significant autoinjury to the host (1). The systemic administration of either recombinant interleukin-1[beta] (IL-1[beta])4 or tumor necrosis factor-[alpha] (TNF-[alpha]) rapidly induces a shocklike state in experimental animals (2,3) and causes fever and hypotension in healthy human volunteers (4-6). These two important proinflammatory cytokines appear to orchestrate the inflammatory response through the activation of transcription factors, such as nuclear factor-[kappa]B (NF-[kappa]B) and activated protein-1, with the subsequent induetion of proinflammatory gene expression. Although these two cytokines share many biologic and physiologic properties, the signaling mechanisms that lead to IL-1[beta]-dependent signal transduction are distinct from that of TNF-[alpha]-dependent signal transduction.

Nuclear factor-[kappa]B belongs to the Rel family of transcription factors, which share common structural motifs for dimerization and DNA binding. Five known subunits belong to the mammalian NF-[kappa]B/Rel family: c-Rel, NF-[kappa]B1 (p50/p105), NF-[kappa]B2 (p52/p100), Rel A (p65), and Rel B. Nuclear factor-[kappa]B consists of 2 such subunits arranged as either homodimers (e.g., p50/p50) or heterodimers (e.g., p65/p50), although the most common form of activated NF-[kappa]B consists of a p65 (Rel A) and p50 heterodimer. Nuclear factor-[kappa]B activation appears to be a master switch, or control point, for the expression of a large number of proinflammatory genes, including several cytokines, chemokines, and adhesion molecules (7). Nuclear factor-[kappa]B is usually present in the cytoplasm of cells in an inactive state bound to a related inhibitory protein known as 1[kappa]B[alpha], an association that physically masks the nuclear translocation sequence of NF-[kappa]B, thereby retaining it in the cytoplasm.

The regulation of NF-[kappa]B activation following stimulation with IL-1[beta] appears to involve at least 2 independent signal transduction pathways. The best-characterized mechanism for the activation of NF-[kappa]B involves the phosphorylation of the inhibitory protein, I[kappa]B[alpha]. Interleukin-1[beta] binds to its receptor, the IL-1 receptor type 1 (IL-1R1), which forms a complex with a related accessory protein, IL-1 receptor accessory proteins (IL-1RAcP). This interaction between IL-1R1 and IL-1RAcP is possible due to the presence of a shared region of homology in the cytoplasmic domain of each protein called the Toll/ IL-1R domain (8). The cytosolic adaptor protein MyD88 (9) and the Toll-interacting protein (10) interact with this receptor complex, which is a necessary step for association with a serine-threonine kinase, IL-1 receptor-associated kinase (IRAK) (11-13). The IRAK then recruits several additional adaptor proteins, including TNF receptor-associated factor 6 (14), transforming growth factor-[beta]-activated kinase-1 (TAK1), and the TAK1 binding proteins 1 and 2 (14,15). Autophosphorylation of IRAK promotes its dissociation from this complex, which is followed by its polyubiquitination, and subsequent degradation by the 26S proteosome system (16). Interestingly, the kinase activity of IRAK may not be an essential step in the IL-1[beta] signal transduction pathway and may function to terminate signal transduction instead (1719). Nevertheless, this sequence of events is temporally and functionally associated with the downstream activation of I[kappa]B kinase (IKK), which phosphorylates the serine-32 and -36 residues of I[kappa]B[alpha] (20). Phosphorylated I[kappa]B[alpha] is targeted for rapid ubiquitination and degradation by the 26S proteosome system, which unmasks the nuclear translocation sequence of NF-[kappa]B and allows it to enter the nucleus and bind to the NF-[kappa]B consensus sequence to direct the transcription of target proinflammatory genes (20).

An alternative mechanism for the activation of NF-[kappa]B is I[kappa]B[alpha]-independent and involves direct phosphorylation of the p65 subunit of NF-[kappa]B at multiple sites by several candidate kinases (21). In addition, phosphorylation of specific tyrosine residues on I[kappa]B[alpha] causes activation of NF-[kappa]B without the proteolytic degradation of I[kappa]B[alpha] (22). It is likely that further study in this area will yield additional mechanisms of ????independent NF-[kappa]B activation.

Given the important role that NF-kB plays in the regulation of a large number of proinflammatory genes, there is growing interest in targeting NF-kB directly in order to affect the inherent redundancy of the inflammatory cascade. A potential novel, safe, and nontoxic strategy for inhibiting NF-[kappa]B activation involves the polyphenolic compounds found in green tea, especially epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea (23). Apart from their antioxidant properties, the catechins, especially EGCG, inhibit several proteins involved in inflammation, including NF-[kappa]B (23-25). We previously showed that EGCG inhibits the TNF[alpha]-mediated activation of NF-[kappa]B in cultured respiratory epithelial cells, partly through the inhibition of IKK (26). Accordingly, we hypothesized that EGCG would inhibit IL1[beta]-mediated activation of NF-[kappa]B.

MATERIALS AND METHODS

Cell culture. Epithelium A549 cells (American Type Culture Collection), a human lung adenocarcinoma cell line representative of the distal respiratory epithelium, were maintained in an incubator with room air:CO^sub 2^ (95:5, v:v) at 37°C, using DMEM containing 8% FBS and 1% penicillin/streptomycin (Gibco BRL).

Experimental conditions. Cells were treated with either 1 µg/L of human IL-1[beta] (Boehringer Mannheim) or vehicle. Epigallocatechin gallate (EGCG; Sigma Chemical) was diluted in filtered PBS to a stock concentration of 10 mmol/L. We noticed an oxidative color change and deterioration in the anti-inflammatory effects noted below when the EGCG stock was used after 24 h (data not shown), and for this reason, EGCG stock was prepared immediately before each use. The EGCG stock was further diluted to experimental concentrations ranging from 3 to 100 µmol/L in DMEM. Cells were treated with EGCG for l h before incubation with IL-1[beta]. Cells not treated with EGCG were preincubated in DMEM alone. The concentration of EGCG used and the duration of treatment did not affect the viability of these cells, as previously reported (26).

Western blot analysis for IRAK and I[kappa]B[alpha] degradation. Whole cell lysates of treated cells were prepared and electrophoretically separated as previously described (26) on 8 to 16% Tris-glycine gradient gels (Novex) and subsequently transferred to nitrocellulose membranes using the Novex Xcell Mini-Gel system (Novex).

For IRAK immunoblotting, membranes were blocked with nonfat dried milk:PBS (3:97, v:v) for 30 min. Primary antibody against IRAK (Upstate Biotechnology) was applied at a concentration of 2 mg/L in milk:PBS (3:97) overnight at 4°C. After washing twice with distilled H2O, the secondary antibody (peroxidase-conjugated antirabbit IgG; Stressgen) was applied at a 1:5000 dilution in milk:PBS (3:97) for 1 h.

I[kappa]B[alpha] immunoblotting was performed as previously described (26), using a primary antibody directed against human I[kappa]B[alpha] (Santa Cruz Biotechnology). Blots were incubated in commercial enhanced chemiluminescence reagents (ECL; Amersham), and exposed to photographic film (26).

Western blot analysis for phospho-NF-[kappa]B (p65). Treated cells were washed twice in ice-cold PBS. Cells were then lysed in ice-cold lysis buffer containing 50 mmol/L Tris (pH 8.0), 110 mmol/L NaCL, 5 mmol/L EDTA, and 1% Triton X-100, to which 100 mmol/L Na^sub 3^VO^sub 4^, 2 g/L leupeptin, 2 mol/L [beta]-glycerol phosphate, and 100 g/L phenylmethysulfonyl fluoride were added. Electrophoresis and protein transfer were carried out as described above. For immunoblotting, membranes were blocked in nonfat milk:TBS:Tween (5.0:94.9:0.1, by vol) for 1 h. A primary antibody against phospho-p65 (Cell Signaling Technology) was applied at a 1:1000 dilution in milk:TBS:Tween (5:95, v:v) overnight at 4°C. After washing 3 times with TBS:Tween (99.9:0.1, v:v), secondary antibody (peroxidase-conjugated antirabbit IgG; Stressgen) was applied at 1:2000 dilution for 1 h. Blots were washed in TBS:Tween twice for 10 min, incubated in commercial enhanced chemiluminescence reagents (ECL; Amersham), and exposed to photographic film.

I[kappa]B kinase assay. The I[kappa]B kinase assay was performed as previously prescribed (26). Briefly, cell extracts were immunoprecipitated using anti-IKK[gamma] antibody (Santa Cruz Biotechnology). The kinase reaction was performed using ATP, GST-I[kappa]B[alpha], and [gamma]-[^sup 32^P]ATP as substrate, and the resulting proteins were separated electrophoretically using a Novex Mini-Cell System. Gels were dried, exposed overnight, and analyzed using a Phosphorlmager screen and Image-Quant software (Molecular Dynamics).

Electromobility gel shift assay. All nuclear extraction procedures were performed on ice with ice-cold reagents as previously described (26). Nuclear proteins were stored at – 70°C until used for electromobility gel shift assays (EMSAs). The NF-[kappa]B oligonucleotide probe used for EMSA (5′-GTGGAATTTCCTCTGA-3′) corresponds to the NF-[kappa]B site in the interleukin-8 (IL-8) promoter and was synthesized at the University of Cincinnati DNA Core Facility (26). The probe was labeled with [gamma]-[^sup 32^P]adenosine triphosphate using T4 polynucleotide kinase (Gibco BRL) and purified in Bio-Spin chromatography columns (BioRad). The EMSA procedure was as previously described (26).

Transient transfection and luciferase assay. Interleukin-8 promoter activity was measured using a plasmid containing the fulllength promoter region of the IL-8 gene cloned into a luciferase reporter plasmid (pGL2; Promega). Cells were transiently transfected with the IL-8 promoter-luciferase reporter plasmid as previously described (26). After transfection, cells were washed once with PBS, pretreated with EGCG for 1 h, and subsequently treated with human IL-1[beta] for 6 h. Cellular proteins were extracted and analyzed for luciferase activity according to the manufacturer’s instructions (Promega), using a Berthold AutoLumat LB953 luminometer. Luciferase activity was corrected for total cellular protein and reported as fold induction over control cells (cells that were transfected and treated with medium alone).

Northern blot analysis. Total cellular RNA was electrophoretically separated and subsequently transferred to nylon membranes (MicroSeparations) and UV autocrosslinked (UV Stratalinker 1800; Stratagene) as previously described (26). After 4 h of prehybridization at 42°C, membranes were hybridized overnight with a radiolabeled human IL-8 cDNA probe. The cDNA was labeled with a y-[^sup 32^P]deoxycytidine triphosphate (specific activity = 3000 Ci/mmol; New England Nuclear Research Products) by random priming (Pharmacia). After washing, the hybridized filters were exposed overnight and analyzed using a Phosphorlmager screen and ImageQuant software (Molecular Dynamics).

Enzyme-linked immunosorbent assay. Immunoreactive IL-8 concentrations in the media of treated cells were measured using a commercially available sandwich ELISA (Biosource). All procedures were performed as recommended by the manufacturer.

Statistical analysis. Differences in immunoreactive IL-8 level, luciferase activity, and cell viability among the experimental groups were evaluated by one-way ANOVA and Student-Newman-Keuls test. Values of P < 0.05 were considered significant.

RESULTS

Interleukin-1[beta]-mediated degradation of IRAK. Treatment with IL-1[beta] caused nearly complete degradation of IRAK compared to control cells, whereas preincubation with 30 and 100 µmol/L EGCG inhibited this degradation (Fig. 1).

Interleukin-1[beta]-induced IKK activation. Treatment with IL-1[beta] increased IKK activity compared to untreated control cells. Consistent with the IRAK degradation data above, 30 and 100 µmol/L EGCG almost completely suppressed IL-1[beta]induced activation of IKK (Fig. 2).

Interleukin-1 [beta]-mediated degradation of I[kappa]B[alpha]. Treatment with IL-1[beta] caused marked degradation of I[kappa]B[alpha] compared to control cells. Consistent with the previous data involving IRAK degradation and IKK activation, 30 and 100 µmol/L EGCG inhibited IL-1[beta]-mediated I[kappa]B[alpha] degradation (Fig. 3).

Interleukin-1[beta]-mediated activation of NF-[kappa]B. Treatment with IL-1[beta] increased the activation of NF-[kappa]B compared with control cells, as determined by EMSA. Consistent with the effects of EGCG on IL-1[beta]-induced degradation of IRAK and activation of IKK, pretreatment with 30 and 100 µmol/L EGCG inhibited activation of NF-[kappa]B (Fig. 4). However, lower concentrations of EGCG (3 and 10 /xmol/L) also moderately inhibited NF-[kappa]B activation, suggesting that additional, I[kappa]B[alpha]-independent mechanisms of NF-[kappa]B inhibition may play a role.

Phosphorylation of p65. Phosphorylated p65 was detected within 30 min after stimulation with IL-1[beta], wheras preincubation with EGCG caused a dose-dependent decrease in phosphorylatcd p65 concentration (Fig. 5).

Interleuicin-1[beta]-mediated expression of the IL-8 gene. Treatment with IL-1[beta] induced nearly 5-fold the luciferase activity in cells transfected with an IL-8 promoter-luciferase reporter plasmid, compared to control cells that were transfected and treated with media alone. Pretreatment with EGCG inhibited luciferase activity in a dose-dependent manner, with significant inhibition at 30 and 100 µmol/L EGCG (Fig. 6). Furthermore, IL-1[beta] treatment alone increased IL-8 mRNA expression (measured by Northern blot analysis) compared to control cells treated with media alone, whereas pretreatment with EGCG inhibited the expression of IL-8 mRNA in a dose-dependent manner (Fig. 7). The effects noted for IL-8 mRNA were corroborated by measurement of IL-8 peptide levels by ELISA. Treatment with IL-1[beta] alone markedly increased the production of immunoreactive IL-8 compared to control cells treated with media alone, whereas pretreatment with EGCG decreased the production of immunoreactive IL-8 in a dose-dependent manner (Fig. . Collectively, these data demonstrate that the inhibitory effects of EGCG on IL-1[beta]mediated NF-[kappa]B activation are associated with the inhibition of IL-8 gene expression.

DISCUSSION

A large body of indirect and direct evidence links the NF-[kappa]B pathway to the dysregulated inflammation that is characteristic of diseases such as sepsis and acute respiratory distress syndrome. Several of the genes that comprise the complex network contributing to this dysregulated inflammation are regulated at the transcriptional level by NF-[kappa]B, including the cytokines IL-1[beta] and TNF-[alpha] chemokines such as IL-6, IL-8, and macrophage chemotactic protein-1; cell adhesion molecules such as vascular cell adhesion molecule 1 and intercellular adhesion molecule 1; growth factors such as granulocytemacrophage colony-stimulating factor and granulocyte colonystimulating factor; as well as additional proinflammatory genes such as inducible nitric oxide synthase. There appears to be a correlation between increased NF-[kappa]B activity and the severity of illness and mortality in critically ill patients (27-29). In addition, studies with in vivo animal models of lethal septic shock demonstrate that inhibition of NF-[kappa]B activation reduces mortality (30,31). These data support the general hypothesis that increased NF-[kappa]B-dependent inflammation directly contributes to the outcome of inflammation-mediated organ injury and strongly support the concept of therapeutic strategies targeting the NF-[kappa]B pathway. An attractive feature of this strategy is the fact that NF-[kappa]B activation appears to be a master switch, or control point, for the expression of a large number of proinflammatory genes. Thus, targeting NF-[kappa]B may potentially affect the inherent redundancy of the inflammatory cascade.

Recent epidemiological studies link the regular consumption of tea with a decreased risk of cancer (32). In addition, a recent study indicates that consumption of as little as 2 cups (473 mL) of tea per day is associated with a reduction in mortality during the 12-mo period following an acute myocardial infarction (33). Considerably less is known regarding the mechanisms by which tea confers these health benefits. We previously showed that one of the active ingredients in green tea, EGCG, is a potent inhibitor of TNF-[alpha]-induced IL-8 gene expression in A549 cells, at least partially through a mechanism involving the inhibition of NF-[kappa]B signaling (26). The present study shows that EGCG inhibits IL-1[beta]-mediated signal transduction in A549 cells as well. First, our study shows that EGCG inhibits the degradation of IRAK, which appears to be a crucial event in the IL-1 signal transduction pathway, in that cells derived from IRAK-knockout mice do not respond to IL-1 stimulation (34-36). The mechanism by which EGCG affects IRAK degradation may involve the direct inhibition of the proteolytic activity of the 26S proteosome by EGCG itself (37). Similarly, EGCG may inhibit I[kappa]B[alpha] degradation via a similar mechanism (38). However, based on the current experiments, it is also possible that EGCG affects events proximal to IRAK degradation. For example, EGCG appears to disrupt the binding of epidermal growth factor to its receptor in A431 epidermoid carcinoma cells (39,40). Similarly, EGCG may modulate IL-1[beta] signaling at the receptor level, by interfering with the binding of IL-1[beta] to the IL-1R, although further experiments are needed to investigate this hypothesis. In combination, inhibition of the IRAK to I[kappa]B[alpha] pathway accounts in part for the mechanism by which EGCG inhibits NF-[kappa]B activation and subsequent NF-[kappa]B-dependent gene expression. The experimental data also demonstrate that EGCG inhibits phosphorylation of p65, thus providing an additional mechanism for the inhibition of NF-[kappa]B activation. This effect on p65 phosphorylation could be the result of IKK inhibition, because a recent report indicates that IKK can phosphorylate the p65 subunit in vitro (41).

The pharmacokinetics of the green tea polyphenols in humans are well-described (42-44), and the maximum achievable EGCG concentration in vivo is significantly less than the concentrations reported in the current in vitro study. For example, 1 cup (240 mL) of green tea contains 200 mg of EGCG (45), and a single, 200-mg dose of EGCG produces a plasma EGCG concentration of ~0.1 µmol/L (46). However, the consumption of pharmaceutically prepared formulations of green tea polyphenols produces plasma EGCG concentrations approaching 2 µmol/L (44,46,47). Moreover, little is known regarding the effective EGCG concentration required to modulate these proinflammatory signaling pathways in vivo.

In summary, the green tea-derived polyphenol EGCG appears to be a potent inhibitor of IL-1 [beta]-mediated signal transduction in A549 cells. The mechanism of this effect involves in part inhibition of IRAK degradation and the subsequent activation of the well-characterized I [kappa]B[alpha]-dependent pathway of NF-[kappa]B activation. An additional mechanism appears to involve the inhibition of p65 phosphorylation. Given the long safety record of tea consumption, the use of EGCG and related compounds may represent a novel pharmacological strategy for the modulation of inflammation dependent on the NF-[kappa]B pathway. Alternatively, EGCG and related compounds could potentially be used as a nutritional supplement in patients with inflammatory disease processes. The next steps to further substantiate these assertions are to test the efficacy of green tea-derived polyphenols such as EGCG in animal models of inflammation-associated organ injury and to further elucidate the mechanisms by which these compounds modulate proinflammatory signal transduction pathways.

[Sidebar]

0022-3166/04 $8.00 © 2004 American Society for Nutritional Sciences.

Manuscript received 3 December 2003. Initial review completed 5 January 2004. Revision accepted 3 February 2004.

[Reference]

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[Author Affiliation]

Derek S. Wheeler,3 John D. Catravas,* Kelli Odoms,[dagger] Alvin Denenberg,[dagger] Vivek Malhotra,[dagger] and Hector R. Wong[dagger]

Section of Critical Care Medicine, Children’s Medical Center, and * Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912; and [dagger] Division of Critical Care Medicine, Cincinnati Children’s Hospital Medical Center, and Children’s Hospital Research Foundation, Cincinnati, OH 45229

[Author Affiliation]

3 To whom correspondence should be addressed. E-mail: dewheeler@mcq.edu.

Alternative Medicine Review. October 1, 2000

Green tea and thermogenesis: interactions between catechin-polyphenols, caffeine and sympathetic activity.

Green tea and thermogenesis: interactions between catechin-polyphenols, caffeine and sympathetic activity. Dulloo AG, Seydoux J, Girardier L, et al. Int J Obes Relat Metab Disord 2000;24:252-258.

The thermogenic effect of tea is generally attributed to its caffeine content. We report here that a green tea extract stimulates brown adipose tissue thermogenesis to an extent which is much greater than can be attributed to its caffeine content per se, and that its thermogenic properties could reside primarily in an interaction between its high content in catechin-polyphenols and caffeine with sympathetically released noradrenaline (NA). Since catechin-polyphenols are known to be capable of inhibiting catechol-O-methyl-transferase (the enzyme that degrades NA), and caffeine to inhibit trancellular phosphodiesterases (enzymes that break down NA-induced cAMP), it is proposed that the green tea extract, via its catechin-polyphenols and caffeine, is effective in stimulating thermogenesis by relieving inhibition at different control points along the NA-cAMP axis. Such a synergistic interaction between catechin-polyphenols and caffeine to augment and prolong sympathetic stimulation of thermogenesis could be of value in assisting the management of obesity.

Green Tea Polyphenol Epigallocatechin-3 Gallate (EGCG) Affects Gene Expression of Breast Cancer Cells Transformed by the Carcinogen 7,12-Dimethylbenz[a]Anthracene1-3
The Journal of Nutrition
. December 1, 2005 . Sonenshein, Gail E; Taylor, Chad; Yang, Sanghwa; Guo, Shangqin

ABSTRACT

Since the 1980s, the incidence of late-onset breast cancer has been increasing in the United States. Known risk factors, such as genetic modifications, have been estimated to account for ~5 to 10% of breast cancer cases, and these tend to be early onset. Thus, exposure to and bioaccumulation of ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons (PAHs), have been proposed to play a role in this increased incidence. Treatment of female Sprague-Dawley rats with a single dose of the PAH 7,12-dimethylbenz[a]anthracene (DMBA) induces mammary tumors in ~90 to 95% of test animals. We showed previously that female rats treated with DMBA and given green tea as drinking fluid displayed significantly decreased mammary tumor burden and invasiveness and a significantly increased latency to first tumor. Here we used cDNA microarray analysis to elucidate the effects of the green tea polyphenol epigallocatechin-3 gallate (EGCG) on the gene expression profile in a DMBA-transformed breast cancer cell line. RNA was isolated, in quadruplicate, from D3-1 cells treated with 60 µg/mL EGCG for 2, 7, or 24 h and subjected to analysis. Semiquantitative RT-PCR and Northern blot analyses confirmed the changes in the expression of 12 representative genes seen in the microarray experiments. Overall, our results documented EGCG-altered expression of genes involved in nuclear and cytoplasmic transport, transformation, redox signaling, response to hypoxia, and PAHs.

J. Nutr. 135: 2978S-2986S, 2005.

KEY WORDS: * EGCG * DMBA * microarray * breast cancer

The rise in breast cancer incidence has been suggested to result in part from increased exposure to and bioaccumulation of lipophilic environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs)5 (1). This hypothesis is based on epidemiological studies relating increased breast cancer to carcinogen exposure (2,3) and from studies showing increased levels of aromatic hydrocarbons and their receptors in breast carcinomas (4,5) and sera from breast cancer patients (2). Furthermore, many studies have shown that PAHs can cause malignant transformation in rodent models in vivo and human mammary cells in vitro. For example, treatment with the PAH 7,12-dimethylbenz[a]anthracene (DMBA) induces mammary tumors in female Sprague-Dawley (S-D) rats (6) and transforms the human mammary epithelial cell line MCF-10F in culture, yielding the D3-1 transformed line (7).

Epidemiological studies indicated that green tea consumption protects against breast cancer (8). Green tea is rich in polyphenols, such as epigallocatechin-3 gallate (EGCG), which possess antioxidant qualities, and were shown to have anticarcinogenic activity against breast and other cancers in animal models. For example, we showed that female S-D rats given green tea as their drinking fluid display a significant decrease in DMBA-induced mammary tumor burden and invasiveness and significantly increased latency to first tumor (9). Similarly, oral consumption of green tea polyphenols was reported to inhibit prostate cancer development and improve survival in the transgenic adenocarcinoma of the mouse prostate TRAMP model (10). To begin to elucidate the exact molecular targets and mechanism for such protection, we turned to breast cancer cell lines as models. We found that EGCG inhibits Her-2/neu receptor tyrosine autophosphorylation in these cancer cells (11). EGCG was also reported to directly inhibit telomerase activity (12,13) and the chymotrypsin-like activity of the proteasome (14). In various models, EGCG was reported to interfere with multiple aspects of control of tumor cell proliferation, apoptosis, angiogenesis, invasion, and metastasis (15-21).

In the present study, we sought to identify the changes in gene expression profile induced by EGCG to probe for the targets mediating the chemopreventive action in DMBA-transformed breast cancer cells using microarray analysis. D3-1 cells were selected because growth of these cells in culture was shown to be potently inhibited by EGCG (9). More recently, EGCG was found to greatly reduce the ability of these cells to grow in soft agar, a hallmark of transformation (data not shown). Our results indicate that genes involved in nuclear and cytoplasmic transport, transformation, redox signaling and hypoxia, and PAH responses were modulated by EGCG.

Materials and methods

Cell culture and mRNA preparation. D3-1 cells were maintained as described previously (9) and grown to 60% confluence for RNA preparation. EGCG (E6234; LKT Laboratory) was dissolved in DMSO. Total RNA was extracted using the UltraspecII RNA isolation kit (Biotex), following the manufacturer’s instructions. The quality of RNA was verified by analyzing RNA samples in a 1% formaldehyde-agarose gel with visualization by ethidium bromide staining.

Reverse transcription and semiquantitative PCR. RNA was digested for 30 min at 37°C with RQ1 RNase-Free DNase (Promega), according to the manufacturer’s directions. Briefly, reverse transcription was performed using 5 µg total RNA, 1 µL random primers (200 ng), and 1 µL 10 mmol/L deoxyribonucleotide triphosphate (dNTP) mixed in 12 µL, heated to 65°C for 5 min, and quick-chilled on ice. All reagents were from InVitrogen unless otherwise specified. Subsequently, 4 µL 5X First-Strand Buffer, 2 µL 0.1 mol/L dithiothreitol, and 1 µL RNasin (Promega) RNAse inhibitor were added. After a 2-min incubation at 42°C, 1 µL (200 U) of Superscript reverse transcriptase was added, and the mixture was incubated at 37°C for 50 mm. To inactivate the reaction, the samples were heated to 70°C for 15 min. Samples (1 µL cDNA) were PCR amplified in a 15 µL reaction volume with 1X reaction buffer (InVitrogen), 2 mmol/L MgCl^sub 2^, 0.2 mmol/L dNTP, 1 µmol/L each of primers, and 0.2 µL Taq DNA polymerase. Reactions were performed in a Robocycler PCR machine (Stratagene) or PTC-100 Thermocontroller (MJ Research). The machines were programmed with a 2-min initial denaturing phase at 95°C; a cycling phase of 30 s denaturing at 95°C, 50 s annealing, and 50 s elongation at 72°C; and an extended elongation of 2 min at 72°C. Annealing temperature was set at 55°C, unless otherwise specified. PCR products from most experiments were resolved on a 1% agarose gel prepared in 40 mmol/L Tris-HCl (pH 8.0), 40 mmol/L acetic acid, and 1 mmol/L EDTA (pH 8.0) containing 0.5 µg/mL ethidium bromide or on a 5% polyacrylamide gel using 0.5X TBE running buffer and stained with GelStar nucleic acid stain (Cambrex) for 30 min (22). All gels were visualized with a UV transilluminator and photographed and quantitated with the Kodak DC210 scientific imaging system. The sequences for the primers used for the PCR reactions are listed in Table 1, along with the GenBank accession numbers. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for RNA integrity and equal loading.

Northern blot analysis. RNA samples (15-20 µg) were subjected to Northern blot analysis following published protocols (23). RNA was transferred to GeneScreen Plus (DuPont NEN) by overnight capillary transfer and UV crosslinked. The Xba I restriction fragment DNA from the pcDNAAhR vector (obtained from D. H. Sherr, Boston University School of Medicine), was used as probe for the aryl hydrocarbon receptor (AhR). Ethidium bromide staining of the 18S and 28S rRNA was used to control for equal loading.

cDNA microarray fabrication and hybridisation. A set of 7500 sequence-verified human cDNA clones was purchased from Research Genetics. Bacterial clones were amplified in 96-well culture plates. Plasmid DNA was isolated using a plasmid kit (Millipore) and open reading frames were PCR-amplified using a pair of universal primers, 5′-CTGCAAGGCGATTAAGTTGGGTAAC-3′ and 5′-GTGA-GCGGATAACAATTTC-ACACAGGAAACAGC-3′, under the following conditions: initial denaturation at 94°C for 2 min; followed by 30 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 2 min; and a final extension step at 72°C for 10 min. The PCR amplification products were examined by 1% agarose gel electrophoresis, purified using a Sephadex G-50 column, dried, and then resuspended in a 50% DMSO solution. PCR products were spotted by an OmniGrid(TM) Microarrayer (GeneMachines) onto a silanized glass slide surface (CMT-GAPS; Corning). Each slide was crosslinked with 300 mJ short-wave UV irradiation (Stratalinker) and stored in a desiccator until use.

Target preparation and hybridisation. Control (DMSO) and EGCG-treated D3-1 cells were harvested and total RNA was isolated, as above. RNA from the control DMSO-treated cells was labeled with fluorescent dye cyanine 3-dUTP (Cy3-dUTP; NEN Life Science Products) and used as the control target. RNA from EGCG-treated cells was labeled separately with cyanine 5-dUTP. The labeled cDNAs were purified using a QIAquick PCR Purification Kit (Qiagen) and concentrated through a Microcon-30 column (Millipore) and resuspended in 80 µL hybridization solution (3X SSC and 0.3% SDS). The mixture was denatured at 100°C for 2 min and applied to the DNA chip, then incubated at 65°C for 16 h in a humidified chamber. The hybridized slide was washed once each in 2X SSC for 2 min; 0.1X SSC, 0.1% SDS for 5 min; and 0.1X SSC for 5 min; then dried by spinning before scanning at room temperature with a GenePix 4000B scanner (Axon Instruments).

Data acquisition, analysis, and statistics. The fluorescence signal was calculated by subtracting the background intensity from the total intensity of a spot using GenePix Pro 4.1 software. Spots with poor signals (F532 – 1.5 × B532 < 0 or F635 – 1.5 × B635 < 0) were removed from further analysis. Normalization for the expression ratios (median Cy5:median Cy3) was achieved by dividing each ratio by a single normalization factor obtained from the GenePix Pro 4.1 scanning process. Expression ratios for each gene were collected over the time points of each treatment, clustered via the hierarchical clustering method using CLUSTER (24), and visualized using TREEVIEW (24). P-values were calculated assuming that EGCG did not affect gene expression, and the ratio of EGCG/DMSO = 1 using a 2-tailed unequal-variance t-test.

Results

Microarray analysis of EGCG-treated D3-1 cells. DMBA-transformed human D3-1 breast cancer cells were treated with 60 µg/mL (130 µmol/L) of EGCG or an equivalent amount of carrier solution DMSO for 2, 7, or 24 h, and total RNA was isolated and subjected to microarray analysis using a cDNA array with 7500 human genes. The experiment was performed in duplicate twice, resulting in quadruplicate replication of each time point. A heat map of one experiment is presented in Figure 1. The same genes that appeared to change in all experiments are summarized in Figure 2. Columns 1 to 4 designate each of the quadruplicate readings in the corresponding experiment. The numbers in the shaded area show the gene expression changes in samples treated with EGCG versus DMSO. Values > 1, shaded in dark grey, indicate genes that were upregulated by EGCG treatment, compared with the control DMSO treatment. Values < 1, shaded in light grey, indicate genes that were downregulated by EGCG treatment, versus the control treatment. Values = 1 indicate that the treatment and control samples did not differ. The gene ontology information was obtained from the Genetics Department at Stanford University (25), and gene information for Homo sapiens was obtained from the National Library of Medicine (26).

Genes that increased or decreased separated very well by 7 h; at 2 h there was considerable variability between the quadruplicate samples, perhaps because of an initial early change that was reversed later. Overall, the direction of change was maintained to 24 h of treatment. Gene changes at 24 h were chosen for further study. Several housekeeping genes were analyzed similarly to controls [including ring finger protein 5 (AA402960), inosine monophosphate dehydrogenase 2 (N73268), soluble acid phosphatase 1 (W45148), and cyclophillin (AA418410)]; these showed no variation with EGCG, as expected (data not shown). Overall, significant changes were detected in 21 genes.

LIM and SH3 protein 1 (AI003699), hypoxia upregulated 1 (AA099134), AhR (AA181307), rab3 GTPase-activating protein (AA520985), myeloid cell leukemia sequence 1 (Mcl1; AA488674), tight junction protein 1 (H50344), thrombospondin 1 (AA464532), sterol response element binding protein 2 (SREBP2; AA053886), metallothionein 1E (AA872383), human clone 23721 mRNA sequence (R45056), Ras-GTPase activating protein SH3 domain-binding protein 2 (AA151214), chromosome segregation 1-like (CSE-1; N69204), karyopherin a 6 (AI865149), LanC-like 1 (R59621), nucleosome assembly protein 1-like 4 (H92201), chord domain-containing protein 1 (AA773461), and solute family carrier protein 20 member 1 (W46972) were all downregulated by EGCG at 24 h. In contrast, aldo-keto reductase (AKR) family 1, C3 (AA916325), AKR family 1, C2 (AI924357), AKR family 1, C1 (R93124), carbonic anhydrase IX (AI023541) and peroxisome proliferator activated receptor (PPAR)γ angiopoietin-related protein (T54298) were all upregulated by EGCG at 24 h. Although changes in other genes were seen, they did not appear to reach statistical significance; these included protein tyrosine phosphatase, nonreceptor type 11 (PTPN11; AA995560), epithelial cell transforming sequence (ECTS) 2 oncogene (AI031571), H1 histone family, member 0 (W69399), and connective tissue growth factor (CTGF; AA598794).

Confirmation of gene expression changes induced by EGCG. RT-PCR analyses were performed to validate the changes in gene expression seen in the microarray analysis. RNA was freshly isolated from D3-1 cells treated with 60 µg/mL EGCG for 24 h and processed for RT-PCR using primer sequences specified in Table 1. A panel of 13 genes was selected for confirmation, in addition to ring finger protein 5 and GAPDH as controls (Fig. 3). Seven of the genes changed in both replicate experiments. The expression of the other 6 genes changed in only 2 of the 4 experiments, and these were selected for their potential relevance to breast cancer. As shown in Figure 3, RT-PCR confirmed most of the changes in gene expression observed with the microarray analysis. In particular, CSE-1, CTGF, AhR, LIM and SH3 protein 1, hypoxia upregulated 1, rab3 GTPase-activating protein, myeloid cell leukemia sequence, tight junction protein 1, SREBP2, PTPN11, metallothionein 1E, epithelial cell transforming sequence 2 oncogene, thrombospondin 1, human clone 23721 mRNA sequence, Ras-GTPase activating protein SH3 domain-binding protein 2, H1 histone family member 0, karyopherin α 6, LanC-like 1, nucleosome assembly protein 1-like 4, and chord domain-containing protein 1 were all downregulated by EGCG at 24 h. In contrast, AKR family 1 C3, C2, and C1; carbonic anhydrase IX; and PPARγ angiopoietin-related protein were all upregulated by EGCG at 24 h. The housekeeping gene ring finger protein 5 (AA402960), which did not change in the microarray, showed no change in expression by RT-PCR assay (Fig. 3). Analysis of GAPDH, which was included as an additional control, confirmed equal RNA loading. Lastly, Northern blot analysis, which was performed to assess AhR mRNA levels (Fig. 4), confirmed significant downregulation of AhR gene expression upon EGCG treatment.

Some genes seen to change in only 1 of the 2 replicate experiments were tested by RT-PCR for confirmation. These included bone morphogenic protein 6 (BMP6; AA424833), glutathione S-transferase (GST) A4 (AA152347), transforming growth factor-β1 (TGF-β1; R36467), and Wnt signaling inducible secreted protein 1 (WISP-1; (AI473336), which were all upregulated, and heat shock protein 10 kDa (HSP10; AA448396) and PTPN11 (AA995560), which were downregulated by EGCG treatment at 24 h. RNA expression of BMP6, GST A4, TGF-β1, and WISP-1 were all shown to increase by RT-PCR, whereas HSP10 was shown to decrease. In contrast, PTPN11 did not show any substantial change when assayed by RT-PCR, consistent with the original statistical analysis. Thus, overall, the RNA analysis largely confirmed the changes in gene expression identified by the microarray analysis.

Discussion

In the present study, we demonstrated that EGCG treatment of D3-1 breast cancer cells mediated changes in gene expression that promote a more normal phenotype. In particular, microarray analyses demonstrated that genes involved in nuclear and cytoplasmic transport, transformation, redox signaling, and hypoxia and PAH signaling responses were modulated by EGCG, indicating an overall chemopreventive role of EGCG, although a few minor exceptions were noted. Below, we discuss the potential physiological significance of these changes.

Downregulated genes. Two of the genes downregulated by EGCG encode proteins involved in nucleocytoplasmic transport: CSE-1 and karyopherin α 6 (Table 2). The nuclear localization signal (NLS) functions via interaction with the NLS import receptor, a heterodimer of importin α and β subunits, also known as karyopherins. Importin α binds the NLS-containing cargo in the cytoplasm, and importin β docks the complex at the cytoplasmic side of the nuclear pore complex. In the presence of nucleoside triphosphates and the small GTP-binding protein Ran, the complex moves into the nuclear pore complex, and the importin subunits dissociate. Importin α enters the nucleoplasm with its passenger protein, and importin β remains at the pore. CSE-1 is an export receptor for importin a, mediating importin a reexport from the nucleus to the cytoplasm after it has released its load into the nucleoplasm (27). CSE-1 was isolated as cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by pseudomonas exotoxin, diphtheria toxin, and tumor necrosis factor (28). Its expression is low in quiescence or on growth arrest and is highly expressed in actively dividing cells, including tumor cell lines (28), consistent with its reduced expression in the presence of EGCG. Karyopherin a 6 (importin α 7) encodes a member of the importin a family. The decreases in CSE-1 and karyopherin α 6 gene expression were significant, and the change in CSE-1 was confirmed by RT-PCR analysis.

A decrease in AhR expression was confirmed by RT-PCR and Northern blot analysis. The AhR is a cytosolic, ligand-activated receptor and transcription factor involved in the regulation of biological responses to several classes of carcinogenic environmental chemicals (e.g., DMBA and other PAHs, dioxin, and planar polychlorinated biphenyls). On activation, the receptor moves to the nucleus in a complex and induces gene transcription mediated by xenobiotic response elements, including those encoding the cytochrome P450 (CYP) enzymes CYP1A1, CYP1A2, and CYP1B1. High levels of constitutively active AhR were found in human breast cancer specimens and in DMBA-induced rat mammary tumors, and its induction occurred early in the DMBA-induced carcinogenesis (4). If EGCG similarly decreases AhR levels in the mammary glands of S-D rats, this could contribute to the observed decrease in tumor burden resulting from DMBA treatment in the rats given green tea as their drinking fluid (9). Work from our laboratory has shown a functional interaction between AhR and classical nuclear factor-κB (NF-κB), which cooperatively transactivate the c-myc oncogene (29). The reduction in the expression of AhR thus might compromise the NF-κB activity observed in these cells (29), decreasing its full oncogenic potential. Interestingly, the downregulation of AhR by EGCG was not seen in the Her-2/neu-overexpressing NF639 cells (data not shown), suggesting that the primary targets of GTPs are different depending on cell types, are related to the etiology of transformation, or both.

Although the decrease in mRNA levels of the 2 growth-promoting factors, CTGF and ECTS, was not significant when measured by microarray analysis, a clear reduction was measured by RT-PCR. CTGF is the major connective tissue mitoattractant secreted by vascular endothelial cells. Advanced breast cancers were found to overexpress CTGF by Xie et al. (30) whereas Jiang et al. (31) detected a reduced level. CTGF is induced by hypoxia, and recent evidence implicates HIF1α in direct regulation of CTGF promoter activity (32). Interestingly, hypoxia upregulated 1 gene product, a member of the heat shock protein 70 (HSP70) family, has an important cytoprotective role in hypoxia-induced cellular perturbations (33). The hypoxia upregulated 1 gene product plays an important role in protein folding and secretion in the endoplasmic reticulum, is upregulated in breast tumors, and is associated with tumor invasiveness. Expression of this gene was also significantly reduced by EGCG in the microarray analysis (~3-fold, P = 0.003). ECTS is a transforming protein related to Rho-specific exchange factors and yeast cell cycle regulators. It is expressed in a cell cycle-dependent manner during liver regeneration and plays an important role in the regulation of cytokinesis (34,35).

Expression of several additional genes displayed significant decreases in the microarray analyses as yet not confirmed by RT-PCR. For example, SREBP2 was downregulated ~2-fold by EGCG (P = 0.003). SREBPs are master transcription regulators for many important genes involved in metabolism. SREBP expression increases during malignant transformation, leading to increased expression of genes involved in lipid metabolism to sustain accelerated tumor cell growth (36). Fatty acid synthase is overexpressed in several human cancers, and inhibition of fatty acid synthase suppresses Her-2/neu overexpression in cancer cells (37). SREBP and its downstream effecter genes are upregulated during progression to androgen independence in prostate cancer models (38). Mcl-1, which is in the Bcl-2 family, is involved in the programming of differentiation and concomitant maintenance of viability. In breast cancer cells and myeloma cells, Mcl-1 possesses strong antiapoptotic function (39,40). Thus, inhibition of antiapoptotic signals might be another mechanism for EGCG to inhibit tumor formation. LIM and SH3 protein 1 mRNA encodes a member of a LIM protein subfamily, which is characterized by a LIM motif and a SH3 domain. It is overexpressed in breast cancers (41). Thrombospondin 1, HSP10, tight junction protein 1, H1 histone family member 0, nucleosome assembly protein 1-like 4, and prefoldin are other gene products that were downregulated by EGCG. The primary known cellular functions of these genes are briefly discussed in Table 2. Metallothionein 1E, LanC-like 1 (bacterial), Ras-GTPase activating protein SH3 domain-binding protein 2, Rab3 GTPase-activating protein, cysteine and histidine-rich domain-containing protein 1, prefoldin, and solute carrier family 20 (phosphate transporter) member 1 are other genes that appeared downregulated by EGCG. Although the functions and regulation of these proteins are less well understood, their collective modulation by EGCG may represent pathways for EGCG to exert its anticarcinogenic function in DMBA-induced transformation.

Upregulated genes. A brief summary of the primary functions of genes that were upregulated by EGCG is given in Table 3. EGCG induced expression of 3 of the 4 isoforms of 3α-hydroxysteroid dehydrogenases or AKRs across the time course: AKR C1, AKR C2, and AKR C3. These enzymes catalyze the conversion of aldehydes and ketones to their corresponding alcohols, using NADH, NADPH, or both as cofactors (42). They inactivate steroid hormones in the liver, regulate 5α-dihydrotestosterone levels in the prostate, and form the neurosteroid allopregnanolone in the central nervous system. These enzymes have also been implicated in the metabolic activation of PAH trans-dihydrodiols, which cause cytotoxicity. Overexpression of this class of enzyme in MCF-7 cells led to cell death (43). AKR1C4 oxidized DMBA-3,4-diol to the highly reactive DMBA-3,4-dione (44). The collective upregulation in the AKRs by EGCG may be reflective of the D3-1 cell etiology, because these cells were transformed by DMBA in vitro. The changes identified by microarray in all 3 AKRs were significant (AKR C3, P = 0.007; AKR C2, P = 0.008; and AKR C1, P = 0.001).

The increase in PPARγ angiopoietin-related protein and carbonic anhydrase (CA)IX genes may reflect changes in hypoxia-induced pathways. PPARγ angiopoietin-related protein shows hypoxia-induced expression in endothelial cells and plays important roles in angiogenesis (45). CAIX, which is membrane associated, is strongly induced by hypoxia. CAIX is overexpressed in a variety of tumor types and associated with increased metastasis and poor prognosis (46). The regulation of most proteins required for hypoxic adaptation occurs at the gene level, which involves transcriptional induction via the binding of a transcription factor HIF-1 to the hypoxia-response element on the regulated genes (47). However, the upregulation of HIF-1 itself was not seen in the microarray analysis. Additional analysis will be required to elucidate the mechanism of CAIX mRNA induction.

At present, 8 distinct classes of the soluble cytoplasmic mammalian GSTs have been identified: α, κ, μ, Ω, τ, σ, θ, and ζ. These enzymes are involved in cellular defense against toxic, carcinogenic, and pharmacologically active electrophilic compounds. GST A4 encodes a member belonging to the a class. It is distinguished by high catalytic efficiency toward the substrate 4-hydroxynon-2-enal, a cytotoxic and mutagenic lipid peroxidation product of oxidative stress (48). The upregulation of GST A4 induced by EGCG might be related to balancing the cellular redox status perturbed by EGCG, which is known to possess strong antioxidative capacity (49). Interestingly, members of the μ and θ classes GST µl (GSTM1) and GST θ1 (GSTT1) have been implicated in the sensitivity to green tea as an agent to prevent oxidative damage (50).

The increase in TGF-β1 mRNA is particularly interesting. TGF-β1 is synthesized as a precursor, which requires processing to control proliferation, and epithelial-to-mesenchymal transition (EMT). Although TGF-β1 inhibits NF-κB activity and slows growth or induces apoptosis in less transformed cells (51,52), it promotes EMT of Ras-transformed cells (53). Interestingly, we showed that NF-κB activity in Ras-transformed liver epithelial cells is resistant to inhibition by TGF-β1 (54). The primary known cellular functions of the above-discussed proteins, as well as those of WISP-1 and BMP6, are briefly given in Table 3.

Taken together, EGCG treatment induced changes in expression of a large number of genes that have potential relevance to tumor biology. Other groups have used microarray analysis to study the action of EGCG in other cellular systems. Human lung cancer cell line PC-9 cells were treated with 200 µmol/L (92 µg/mL) of EGCG for 7 h, and gene expression was profiled using an Atlas Human Cancer cDNA Expression Array containing 588 genes (55). Human prostate cancer cell line LNCaP cells treated with 12 µmol/L (5.5 µg/mL) EGCG for 12 h were analyzed using a Micromax Direct System (56,57). In another study, human papillomavirus-16-associated cervical cancer cell line CaSki cells were treated with 35 µmol/L (16 µg/mL) of EGCG for 12, 24, and 48 h, and gene expression was profiled using a Macrogen 384-cDNA chip (16). Human vascular endothelial cells were exposed to green tea extracts for 6 and 48 h, and gene expression was profiled using an Affymetrix chip containing 12,625 genes (58). These studies reported genes up- or downregulated by >2-fold. These included gene categories involved in proliferation control, cell cycle control, and apoptosis, confirming the findings with conventional molecular and cellular biology studies. However, the differences between the cell types, the duration and dose of treatment, and the array chips used for these experiments make a direct comparison almost impossible. Our current data provide a catalogue of genes involved in breast cancer, with particular emphasis on the DMBA-transformed etiology.

In addition to EGCG, the effects of other green tea polyphenols including epicatechin, epicatechin-3-gallate, and epigallocatechin are also of interest for examination, because they have been reported to have anticarcinogenic activity as well. The ability of EGCG and other tea polyphenols to inhibit carcinogenesis make EGCG a good template for deriving small-molecule drugs. Modifications in structure may improve the pharmacokinetics and effectiveness. As a readily available dietary substance, it holds promise for prevention of early-stage cancer. Our very recent studies with the DMBA-induced mammary tumorigenesis model demonstrated that in situ tumors in rats drinking green tea versus water have a less-invasive phenotype (unpublished observation). New target identification with gene expression profiling may help in designing new effective adjuvant therapy treatments. The present study was designed to evaluate the protective effect of EGCG on a specific environmental carcinogen (DMBA). It would be important to also evaluate the protective effect in other oncogenic settings. For example, we previously showed that EGCG inhibits Her-2/neu receptor tyrosine phosphorylation and downstream signaling. These findings suggest that EGCG may also be effective in the treatment of breast cancer overexpressing this oncogene, especially when combined with other chemotherapeutic agents. It would be of great interest to compare the data from the present study with similar high-throughput analysis of Her-2/neu tumors with regard to the different and common target genes for EGCG. These studies would allow for identification of key molecules and pathways and provide a list of candidate genes whose functional role might be critical for the chemopreventive and antiinvasive role of green tea polyphenols.

ACKNOWLEDGMENTS

We thank Zidong Zhang for technical assistance in performing the RT-PCR analysis, and D. H. Sherr, Boston University School of Medicine, for generously providing cloned AhR DNA.

[Reference]

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[Author Affiliation]

Shangqin Quo, Sanghwa Yang,* Chad Taylor, and Gail E. Sonenshein4

Department of Biochemistry and Women’s Health Interdisciplinary Research Center, Boston University School of Medicine, Boston, MA 02118-2394; and * Cancer Metastasis Research Center, Yonsei University College of Medicine, Seodaemun-Gu, Seoul 120-752, Korea

[Author Affiliation]

4 To whom correspondence should be addressed. E-mail: gsonensh@bu.edu.

Am J Physiol Endocrinol Metab, 2007 Jan 16; [Epub ahead of print]Potenza MA, Marasciulo FL, Tarquinio M, et al.

Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, may augment metabolic and vascular actions of insulin. We investigated effects of EGCG treatment to simultaneously improve cardiovascular and metabolic function in SHR rats (model of metabolic syndrome with hypertension, insulin resistance, and overweight). In acute studies, EGCG (1-100 microM) elicited dose-dependent vasodilation in mesenteric vascular beds (MVB) from SHR ex vivo, inhibitable by L-NAME (NOS antagonist) or wortmannin (PI 3-kinase inhibitor). In chronic studies, 9-wk old SHR were treated by gavage for 3 weeks with EGCG (200 mg/kg/d), enalapril (30 mg/kg/d), or vehicle. A separate group of SHR receiving L-NAME (80 mg/L in drinking water) was treated for 3 weeks with either EGCG or vehicle. Vasodilator actions of insulin were significantly improved in MVB from EGCG- or enalapril-treated SHR (compared with vehicle-SHR). Both EGCG and enalapril therapy significantly lowered systolic blood pressure (SBP) in SHR. EGCG therapy of SHR significantly reduced infarct size and improved cardiac function in Langendorff-perfused hearts exposed to ischemia/reperfusion injury (I/R). In SHR given L-NAME, effects of EGCG on SBP and I/R were not observed. Both enalapril and EGCG treatment of SHR improved insulin sensitivity and raised plasma adiponectin. We conclude that acute actions of EGCG to stimulate production of NO from endothelium using PI 3-kinase dependent pathways may explain, in part, beneficial effects of EGCG therapy to simultaneously improve metabolic and cardiovascular pathophysiology in SHR. These findings may be relevant to understanding potential benefits of green tea consumption in patients with metabolic syndrome.

J Allergy Cli, Immunol Williamson ME McCormick TG, Nance CL, Shearer WT. 2006;118:1369-1374.
Epigallocatechin gallate, the main polyphenol in green tea, binds to
the T-cell receptor, CD4: potential for HIV-1 therapy.(Recent
Abstracts)(Brief article)
BACKGROUND: 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.
OBJECTIVE: To demonstrate that EGCG binds to the CD4 molecule at the
gpl20 attachment site and inhibits gp120 binding at physiologically
relevant levels, thus establishing EGCG as a potential therapeutic
treatment for HIV-1 infection. METHODS: 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. RESULTS: 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
gpl20. Physiologically relevant concentrations of EGCG (0.2 micromol/L)
inhibited binding of gp120 to isolated human CD4+ T cells. CONCLUSION:
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
ofgpl20 binding to human CD4+ T cells. CLINICAL IMPLICATIONS:
Epigallocatechin gallate has potential use as adjunctive therapy in
HIV-1 infection.

Safety of Green Tea Extracts : A Systematic Review by the US Pharmacopeia.
Drug Saf. 2008;31(6):469-84.. Sarma DN, Barrett ML, Chavez ML, Gardiner
P, Ko R, Mahady GB, Marles RJ, Pellicore LS, Giancaspro GI, Low Dog T.

Green tea [Camellia sinensis (L.) Kuntze] is the fourth most
commonly used dietary supplement in the US. Recently, regulatory
agencies in France and Spain suspended market authorization of a
weight-loss product containing green tea extract because of
hepatotoxicity concerns. This was followed by publication of adverse
event case reports involving green tea products. In response, the US
Pharmacopeia (USP) Dietary Supplement Information Expert Committee (DSI
EC) systematically reviewed the safety information for green tea
products in order to re-evaluate the current safety class to which
these products are assigned. DSI EC searched PubMed (January 1966-June
2007) and EMBASE (January 1988-June 2007) for clinical case reports and
animal pharmacological or toxicological information. Reports were also
obtained from a diverse range of other sources, including published
reviews, the US FDA MedWatch programme, USP’s MEDMARX((R))
adverse event reporting system, the Australian Therapeutic Goods
Administration, the UK Medicines and Healthcare products Regulatory
Agency, and Health Canada’s Canadian Adverse Drug Reaction
Monitoring Program. Case reports pertaining to liver damage were
evaluated according to the Naranjo causality algorithm scale. In
addition, the Committee analysed information concerning historical use,
regulatory status, and current extent of use of green tea products. A
total of 216 case reports on green tea products were analysed,
including 34 reports concerning liver damage. Twenty-seven reports
pertaining to liver damage were categorized as possible causality and
seven as probable causality. Clinical pharmacokinetic and animal
toxicological information indicated that consumption of green tea
concentrated extracts on an empty stomach is more likely to lead to
adverse effects than consumption in the fed state. Based on this safety
review, the DSI EC determined that when dietary supplement products
containing green tea extracts are used and formulated appropriately the
Committee is unaware of significant safety issues that would prohibit
monograph development, provided a caution statement is included in the
labelling section. Following this decision, USP’s DSI ECs may
develop monographs for green tea extracts, and USP may offer its
verification programmes related to that dietary ingredient.