Inhibitory effect of (-)-epigallocatechin 3-gallate, a polyphenol of green tea, on neutrophil chemotaxis in vitro and in vivo

The effect of (-)-epigallocatechin 3-gallate (EGCG), a major polyphenol of green tea, on neutrophil migration has been studied using multiwell-type Boyden chambers in vitro and a fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA)-induced rat allergic inflammation model in vivo. EGCG inhibited rat neutrophil chemotaxis toward cytokine-induced neutrophil chemoattractant-1 (CINC-1) in a concentration-dependent manner. In addition, CINC-1-induced neutrophil chemotaxis was suppressed by the pretreatment of rat neutrophils with EGCG at the concentration over 15 microg/mL. EGCG caused concentration-dependent suppression of the transient increase in CINC-1-induced intracellular free calcium level in both rat neutrophils and rat CXC chemokine receptor 2 (CXCR2)-transfected HEK 293 cells. EGCG inhibited CINC-1 production by IL-1beta-stimulated rat fibroblasts (NRK-49F cells) and lipopolysaccharide-stimulated rat macrophages at the concentration over 50 microg/mL, a comparatively high concentration. Oral administration of EGCG (1.0 mg or 1.5 mg/rat) at 1 h before the challenge with FITC-OVA suppressed neutrophil infiltration into the air pouch (inflammatory site) in the air-pouch type FITC-OVA-induced allergic inflammation in rats. Chemokine levels in the pouch fluids, however, were not influenced by EGCG administration. The results suggest that EGCG suppressed neutrophil infiltration by a direct action on neutrophils, but not by indirect actions, including the suppression of chemokine production at the inflammatory site.     

Inhibition of gap junctional intercellular communication by the green tea polyphenol (-)-epigallocatechin gallate in normal rat liver epithelial cells

(-)-Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, is a promising chemopreventive agent against cancer due to its strong antiproliferative effects on cancer cells; however, its possible toxicity and carcinogenicity must be investigated before EGCG can be used as a dietary supplement for chemoprevention. The inhibition of gap junctional intercellular communication (GJIC) is strongly associated with carcinogenesis, particularly the tumor promotion process; thus, we investigated the effects of EGCG on GJIC in WB-F344 normal rat liver epithelial (RLE) cells. EGCG, but not (-)-epicatechin (EC), another polyphenol found in green tea, inhibited GJIC in a dose-dependent and reversible manner in RLE cells. EGCG also induced the phosphorylation of connexin 43 (Cx43), a major regulator of GJIC. The phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) was also observed in EGCG-treated RLE cells. The inhibition of GJIC and phosphorylation of Cx43 and ERK1/2 by EGCG were completely blocked by U0126, a pharmacological inhibitor of mitogen-activated protein kinase/ERK kinase. EGCG generated a larger amount of hydrogen peroxide than EC in a dose-dependent manner. Furthermore, catalase partially inhibited the EGCG-induced inhibition of GJIC and the phosphorylation of Cx43 and ERK1/2. These results indicated that EGCG inhibited GJIC mainly due to its prooxidant activity.     

Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice

The aim of this study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on newly developed high-fat/Western-style diet-induced obesity and symptoms of metabolic syndrome. Male C57BL/6J mice were fed a high fat/Western-style (HFW; 60% energy as fat and lower levels of calcium, vitamin D(3), folic acid, choline bitartrate, and fiber) or HFW with EGCG (HFWE; HFW with 0.32% EGCG) diet for 17 wks. As a comparison, two other groups of mice fed a low-fat diet (LF; 10% energy as fat) and high-fat diet (HF; 60% energy as fat) were also included. The HFW group developed more body weight gain and severe symptoms of metabolic syndrome than the HF group. The EGCG treatment significantly reduced body weight gain associated with increased fecal lipids and decreased blood glucose and alanine aminotransferase (ALT) levels compared to those of the HFW group. Fatty liver incidence, liver damage, and liver triglyceride levels were also decreased by the EGCG treatment. Moreover, the EGCG treatment attenuated insulin resistance and levels of plasma cholesterol, monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interlukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Our results demonstrate that the HFW diet produces more severe symptoms of metabolic syndrome than the HF diet and that the EGCG treatment can alleviate these symptoms and body fat accumulation. The beneficial effects of EGCG are associated with decreased lipid absorption and reduced levels of inflammatory cytokines.     

Tea polyphenol (-)-epigallocatechin 3-gallate suppresses heregulin-beta1-induced fatty acid synthase expression in human breast cancer cells by inhibiting phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase cascade signaling

Tumor-associated fatty acid synthase (FAS) is implicated in tumorigenesis and connected to HER2 (human epidermal growth factor receptor 2) by systemic analyses. Suppression of FAS in cancer cells may lead to growth inhibition and cell apoptosis. Our previous study demonstrated that (-)-epigallocatechin 3-gallate (EGCG), the green tea catechin, could down-regulate FAS expression by suppressing EGFR (epidermal growth factor receptor) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/Akt activation in the MCF-7 breast cancer cell line. Herein, we examined the effects of EGCG on FAS expression modulated by another member of the erbB family, that is, HER2 or HER3. We identified that heregulin-beta1 (HRG-beta1), a HER3 ligand, stimulated dose-dependent FAS expression in breast cancer cell lines MCF-7 and AU565, but not MDA-MB-453. The time-dependent increase in FAS expression after HRG-beta1 stimulation was also observed in MCF-7 cells, and this up-regulation was de novo RNA synthesis dependent. Treatment of MCF-7 cells with EGCG markedly inhibited HRG-beta1-dependent induction of mRNA and protein of FAS. EGCG also decreased the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 that were demonstrated as selected downstream HRG-beta1-responsive kinases required for FAS expression using dominant-negative Akt, PI3K inhibitors (LY294002 and wortmannin), or MEK inhibitor (PD98059). FAS induction by HRG-beta1 was also blocked by AG825, a selective HER2 inhibitor, and by genistein, a selective tyrosine kinase inhibitor, indicating the formation of a heterodimer between HER2 and HER3, and their tyrosine kinase activities are essential for HRG-beta1-mediated elevation of FAS. Additionally, growth inhibition of HRG-beta1-treated cells was parallel to suppression of FAS by EGCG. Taken together, these findings extend our previous study to indicate that EGCG may be useful in the chemoprevention of breast carcinoma in which FAS overexpression results from HER2 or/and HER3 signaling.     

Relationship between the biological activities of methylated derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) and their cell surface binding activities

It was previously reported that (-)-epigallocatechin-3-O-gallate (EGCG) suppresses the expression of the high-affinity IgE receptor FcepsilonRI in human basophilic cells and that this suppressive effect is associated with EGCG binding to the cell surface. This study examined the effects of five methylated derivatives of EGCG, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG 3' 'Me), (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4' 'Me), (-)-4'-O-methyl-epigallocatechin-3-O-gallate (EGCG 4'Me), (-)-epigallocatechin-3-O-(3,4-O-methyl)gallate (EGCG 3' '4' 'diMe), and (-)-4'-O-methyl-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4'4' 'diMe) on FcepsilonRI expression and ERK1/2 phosphorylation, and each of their cell surface binding activities was measured. Of these five methylated derivatives, three that are methylated at the 3' '- and/or 4' '-position, EGCG 3' 'Me, EGCG 4' 'Me, and EGCG 3' '4' 'diMe, suppressed FcepsilonRI expression and ERK1/2 phosphorylation, although the suppressive effects were lower than that of EGCG. EGCG 4'Me and EGCG 4'4' 'diMe, both of which are methylated at the 4'-position, did not demonstrate a suppressive effect. Furthermore, it was found that EGCG 3' 'Me, EGCG 4' 'Me, EGCG 3' '4' 'diMe, and EGCG 4'Me, which are methylated at the 3' '- and/or 4' '-positions or the 4'-position, could bind to the cell surface even though their binding activities were lower than that of EGCG. Only EGCG 4'4' 'diMe, which is methylated at both the 4'- and 4' '-positions, could not bind. These results suggest that the trihydroxyl structure of the B ring is essential for EGCG to exert the suppressive effects and that the hydroxyl groups on both the 4'-position in the B ring and the 4' '-position in the gallate are crucial for the cell surface binding activity of EGCG.     

A synthetic naringenin derivative, 5-hydroxy-7,4'-diacetyloxyflavanone-N-phenyl hydrazone (N101-43), induces apoptosis through up-regulation of Fas/FasL expression and inhibition of PI3K/Akt signaling pathways in non-small-cell lung cancer cells

Naringenin, a well-known naturally occurring flavonone, demonstrates cytotoxicity in a variety of human cancer cell lines; its inhibitory effects on tumor growth have spurred interest in its therapeutic application. In this study, naringenin was derivatized to produce more effective small-molecule inhibitors of cancer cell proliferation, and the anticancer effects of its derivative, 5-hydroxy-7,4'-diacetyloxyflavanone-N-phenyl hydrazone (N101-43), in non-small-cell lung cancer (NSCLC) cell lines NCI-H460, A549, and NCI-H1299 were investigated. Naringenin itself possesses no cytotoxicity against lung cancer cells. In contrast, N101-43 inhibits proliferation of both NCI-H460 and A549 cell lines; this capacity is lost in p53-lacking NCI-H1299 cells. N101-43 induces apoptosis via sub-G1 cell-cycle arrest in NCI-H460 and via G0/G1 arrest in A549 cells. Expression of apoptosis and cell-cycle regulatory factors is altered: Cyclins A and D1 and phospho-pRb are down-regulated, but expression of CDK inhibitors such as p21, p27, and p53 is enhanced by N101-43 treatment; N101-43 also increases expression levels of the extrinsic death receptor Fas and its binding partner FasL. Furthermore, N101-43 treatment diminishes levels of cell survival factors such as PI3K and p-Akt dose-dependently, and N101-43 additionally induces cleavage of the pro-apoptotic factors caspase-3, caspase-8, and poly ADP-ribose polymerase (PARP). Cumulatively, these investigations show that the naringenin derivative N101-43 induces apoptosis via up-regulation of Fas/FasL expression, activation of caspase cascades, and inhibition of PI3K/Akt survival signaling pathways in NCI-H460 and A549 cells. In conclusion, these data indicate that N101-43 may have potential as an anticancer agent in NSCLC.     

EGCG与Cd2+对前列腺癌细胞PC-3生长的抑制作用研究

采用MTT法检测了EGCG、Cd2+及二者相互作用对PC-3细胞生长的抑制作用;通过倒置显微镜观察了前列腺癌细胞PC-3形态的变化;采用细胞凋亡罗丹明123染色试剂盒检测了PC-3细胞凋亡;利用ESR法检测了PC-3细胞膜流动性的改变。结果表明,EGCG与Cd2+都可抑制前列腺癌细胞的生长;二者都可改变细胞的外观形态;80 µmol/L的EGCG处理并未观察到凋亡的PC-3细胞,20 µmol/L的Cd2+处理主要导致PC-3细胞坏死,而80 µmol/L EGCG与20 µmol/L Cd2+共存可诱导细胞凋亡;同时,EGCG参与的处理都降低了PC-3细胞膜的流动性。     

Different effects of five catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Five catechins [(-)-epigallocatechins gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), and (+)-catechin (C)] were compared with regard to their effects on 6-hydroxydopamine (OHDA)-induced apoptosis in PC12 cells--the vitro model of Parkinson's disease. Measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 6-OHDA inhibited cell viability in a time- and concentration-dependent manner. When PC12 cells were pretreated with the five catechins for 30 min before exposure to 250 microM 6-OHDA, MTT results showed that the five catechins had different effects: EGCG and ECG had obvious concentration-dependent protective effects at 50-400 microM; EC and (+)-C had almost no effects; and EGC especially decreased cell viability. Catechins also had different effects on apoptotic morphology. Only 200-400 microM EGCG and ECG kept cells adhering well. When pretreated with other catechins at any concentration, PC12 cells became round and some of them were detached as when treated with 6-OHDA. In addition, typical apoptotic characteristics of PC12 cells were determined by fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis after the cells were treated with 250 microM 6-OHDA for 24 h or pretreated with catechins before it. Preincubation with 200-400 microM EGCG and ECG led to significant inhibitory effects against PC12 cell apoptosis, as shown by flow cytometry. The other catechins have little protective effect. Therefore, at 200-400 microM, the classified protective effects of the five catechins were in the order ECG > EGCG > EC > (+)-C > EGC. The data also indicated that EGCG and ECG might be potent neuroprotective agents for Parkinson's disease. The results of fluorescence microscopy and DNA fragment analysis supported the conclusion.     

Inhibitory effects of (-)-epigallocatechin gallate on the mutation,DNA strand cleavage,and DNA adduct formation by heterocyclic amines

Green tea is known to be a potential chemopreventive agent against cancer. In this study, we investigated the inhibitory activities of tea extracts, and in particular the polyphenolic component (-)-epigallocatechin gallate (EGCG), against heterocyclic amine-induced genotoxicity. The tea extracts displayed inhibition of 2-hydroxyamino-6-methyldipyrido[1,2-a,3',2'-d]imidazole (Glu-P-1(NHOH))-induced mutagenicity. This inhibition can be accounted for by the presence of EGCG in the extracts. The mutagenic effect of Glu-P-1(NHOH), which induces single-strand cleavage in supercoiled circular DNA under neutral conditions, was inhibited by EGCG. Using the Drosophila repair test, a test for gross DNA damage, and DNA adduct detection by (32)P-postlabeling, we showed that EGCG prevented 2-amino-3,8-dimethylimidazo[4,5-f]quinoline-induced DNA damage and adduct formation in insect DNA. EGCG was found to accelerate the degradation of Glu-P-1(NHOH) in vitro. This observation suggested that the inhibition by EGCG is associated with an accelerated degradation of metabolically activated heterocyclic amines.     

Dietary docosahexaenoic acid can alter the surface expression of CD4 and CD8 on T cells in peripheral blood

The effect of dietary docosahexaenoic acid (DHA) on T cell states in peripheral blood was investigated. Weanling male C57Bl/6N mice were kept on one of three 10% fat diets containing various amounts of DHA and linoleic acid for 4 weeks. Changing the concentration of dietary DHA did not alter the proportion of T cells expressing CD4 or CD8. However, increasing the concentration of dietary DHA lowered the expression of CD4 and CD8 on the cell surface. The decreased expression of these surface molecules involved in T cell proliferation has serious implications in the role of DHA as an immunosuppressant.     

Oligosaccharide and peptidoglycan of Ganoderma lucidum activate the immune response in human mononuclear cells

The acid-hydrolyzed fragments of Ganoderma lucidum polysaccharides (GLPS) obtained by Smith degradation were separated by size-exclusion chromatography into two major water-soluble fractions: peptidoglycans (GLPS-SF1) and oligosaccharides (GLPS-SF2). Both fractions induced CD69 in human peripheral blood mononuclear cells (hPB-MNCs), and they displayed distinct immunomodulating properties. GLPS-SF1, with a molecular weight of around 20 kDa, were heterogeneous peptidoglycans composed of glucose/mannose (4:1) that exhibited biological activities with Th1 cytokines IL-12, IL-2, TNF-α, and IFN-γ in hPB-MNCs and stimulated macrophage cytokine expression via Toll-like receptor 4 (TLR4) signaling. For GLPS-SF2, with a molecular weight of around several kilodaltons, its sugar sequence was elucidated by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as [-α-1,4-Glc-(β-1,4-GlcA)(3)-](n). This oligosaccharide displayed specific immune property with low monocyte induction, greatly stimulated cell activation and proliferation of NK and T cells. This oligosaccharide isolated from G. lucidum polysaccharides with internal glucuronic acids/glucose repeat unit in a 3:1 ratio may be responsible for the active stimulation of NK and T cells.