Inhibition of NADPH oxidase subunits translocation by tea catechin EGCG in mast cell

The inhibitory mechanism of tea catechins for allergy remains undefined. We studied the effect of catechins, mainly EGCG, on the activation of mast cell line canine cutaneous mastocytoma cells (CM-MC). Compound 48/80 induced the degranulation in CM-MC dose dependently, whereas its release of beta-hexosaminidase was inhibited by EGCG and O-methylated EGCG (EGCG-Me). Both catechins were found to inhibit intracellular ROS generation dose dependently together with DPI. Intracellular ROS generation in human polymorphonuclear leukocytes was also inhibited by EGCG. Neither L-NAME, ebeselen nor NAC inhibited ROS generation. From the Western blot analysis of the subunits components of NADPH oxidase, we detected cytosolic subunits; p47(phox), p67(phox), p40(phox), rac2 and membrane subunits; gp91(phox), p22(phox) in CM-MC. Cytosolic subunits were translocated from cytosol to membrane time dependently after stimulation with compound 48/80. EGCG and DPI inhibited cytosolic subunits from translocating into membrane. These data suggest that EGCG inhibits the activation of NADPH oxidase in CM-MC.     

Prevention of quinone-mediated DNA arylation by antioxidants.

High performance liquid chromatographic (HPLC) analysis showed that the prototype antioxidant ascorbate (vitamin C) inhibits the DNA adducts induced by synthetic estrogen diethylstilbestrol (DES) and the antiestrogen metabolite 4-hydroxytamoxifen (4-OHTam). Treatment of salmon testes DNA with 4-OHTam quinone or 4-OHTam in the presence of horseradish peroxidase and hydrogen peroxide (H(2)O(2)) generated the same DNA adduct profile. Vitamin C and N-acetylcysteine (NAC) inhibited the formation of 4-OHTam-dG adducts in a dose-dependent manner. To determine whether the same antioxidants also protect cellular DNA, HL-60 cells were used as cell culture model. Cells treated with 10 microM 4-OHTam in the presence of 1 microM H(2)O(2 )for 24 h gave 4-OHTam-dG adducts approximately 4 x 10(-7), n = 3. Treatment of the cells with 100 microM 4-OHTam, without H(2)O(2), produced the same level of adducts. Supplementation of the incubation media with vitamin C (2.5 mM) or NAC (5 mM) inhibited the formation of DNA adducts. Thus, antioxidants may protect susceptible cells from genotoxicity associated with 4-OHTam activation.     

Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

A major constituent of green tea,EGCG, inhibits the growth of a human cervical cancer cell line,CaSki cells,through apoptosis,G(1) arrest,and regulation of gene expression

A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG) has been known to possess antiproliferative properties. In this study, we investigated the anticancer effects of EGCG in human papillomavirus (HPV)-16 associated cervical cancer cell line, CaSki cells. The growth inhibitory mechanism(s) and regulation of gene expression by EGCG were also evaluated. EGCG showed growth inhibitory effects in CaSki cells in a dose-dependent fashion, with an inhibitory dose (ID)(50) of approximately 35 microM. When CaSki cells were further tested for EGCG-induced apoptosis, apoptotic cells were significantly observed after 24 h at 100 microM EGCG. In contrast, an insignificant induction of apoptotic cells was observed at 35 microM EGCG. However, cell cycles at the G1 phase were arrested at 35 microM EGCG, suggesting that cell cycle arrests might precede apoptosis. When CaSki cells were tested for their gene expression using 384 cDNA microarray, an alteration in the gene expression was observed by EGCG treatment. EGCG downregulated the expression of 16 genes over time more than twofold. In contrast, EGCG upregulated the expression of four genes more than twofold, suggesting a possible gene regulatory role of EGCG. This data supports that EGCG can inhibit cervical cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene expression in vitro. Furthermore, in vivo antitumor effects of EGCG were also observed. Thus, EGCG likely provides an additional option for a new and potential drug approach for cervical cancer patients.     

Acute lung inflammation and ventilator-induced lung injury caused by ATP via the P2Y receptors: an experimental study

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

Limitations of MTT and MTS-Based Assays for Measurement of Antiproliferative Activity of Green Tea Polyphenols

Background

The chemopreventive effect of green tea polyphenols, such as (-)-epigallocatechin-3-gallate (EGCG), has been well demonstrated in cell culture studies. However, a wide range of IC₅₀ concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature.

Methodology/Principal Findings

We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP) and deoxynucleic acid (DNA) showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95). However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC₅₀ concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays.

Conclusions/Significance

These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.     

Epigallocatechin-3-gallate (EGCG) addition as an antioxidant in a cryo-diluent media improves microscopic parameters,and fertility potential,and alleviates oxidative stress parameters of buffalo spermatozoa

The disparity between the endogenous antioxidants concentration and free radicals in spermatozoa results in reactive oxygen species (ROS) generation. In this prospect, epigallocatechin-3-gallate (EGCG) preserves vigorous antioxidant features. Current study explored the influence of EGCG in a cryo-diluent media on microscopic parameters, oxidative stress parameters, and fertility potential of buffalo spermatozoa during cryopreservation. Concisely, collected semen from three donor bulls for four times were then evaluated for volume, motility, concentrations and then dilution in a cryo-diluent media with different concentrations of EGCG (EGCG-0 = control; EGCG-50 = 50 μM, EGCG-100 = 100 μM, EGCG-200 = 200 μM, and EGCG-300 = 300 μM) at 37 °C, cooled to 4 °C in 2 h, equilibrated for 4 h at 4 °C, and cryopreserved. At post-thawing, Computer-Assisted Sperm motion Analysis motilities (total and progressive, %) and rapid velocity (%), plasma membrane functionality, supravital plasma membrane integrity, and mitochondrial potential (%) were found higher (P < 0.05) in EGCG-200, and EGCG-300 than control, whereas average-path, straight-line, and curved-linear velocities (μm/sec), and acrosome integrity (%) were recorded higher in EGCG-300 than control. Further, comet length (μm), and tail length (μm), LPO (lipid peroxidation, μM/mL), and apoptosis-like changes (%) in spermatozoa were significantly decreased in EGCG-300 than control. Seminal plasma antioxidant enzymes activities (glutathione peroxidase, U/mL, and superoxide dismutase, U/mL) were increased with EGCG-300 than control. Moreover, EGCG-300 addition in a cryo-diluent media improves the fertility potential (%) of buffalo spermatozoa. In a nutshell, the inclusion of EGCG-300 in a cryo-diluent media enhances post-thaw microscopic parameters, and fertility potential, whereas decreases oxidative stress parameters in buffalo spermatozoa.     

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.     

Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kappaB in cancer cells versus normal cells

Green tea has shown remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays, cell culture systems, and epidemiological studies. Many of these biological effects of green tea are mediated by epigallocatechin 3-gallate (EGCG), the major polyphenol present therein. We have earlier shown that EGCG treatment results in apoptosis of several cancer cells, but not of normal cells (J. Natl. Cancer Inst. 89, 1881-1886 (1997)). The mechanism of this differential response of EGCG is not known. In this study, we investigated the involvement of NF-kappaB during these differential responses of EGCG. EGCG treatment resulted in a dose-dependent (i) inhibition of cell growth, (ii) G0/G1-phase arrest of the cell cycle, and (iii) induction of apoptosis in human epidermoid carcinoma (A431) cells, but not in normal human epidermal keratinocytes (NHEK). Electromobility shift assay revealed that EGCG (10-80 microM) treatment results in lowering of NF-kappaB levels in both the cytoplasm and nucleus in a dose-dependent manner in both A431 cells and NHEK, albeit at different concentrations. EGCG treatment was found to result in a dose-based differential inhibition of TNF-alpha- and LPS-mediated activation of NF-kappaB in these cells. The inhibition of NF-kappaB constitutive expression and activation in NHEK was observed only at high concentrations. The immunoblot analysis also demonstrated a similar pattern of inhibition of the constitutive expression as well as activation of NF-kappaB/p65 nuclear protein. This inhibition of TNF-alpha-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha. Taken together, EGCG was found to impart differential dose-based NF-kappaB inhibitory response in cancer cells vs normal cells; i.e., EGCG-mediated inhibition of NF-kappaB constitutive expression and activation was found to occur at much higher dose of EGCG in NHEK as compared to A431 cells. This study suggests that EGCG-caused cell cycle deregulation and apoptosis of cancer cells may be mediated through NF-kappaB inhibition.     

Involvement of multidrug resistance-associated proteins in regulating cellular levels of (-)-epigallocatechin-3-gallate and its methyl metabolites

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol of green tea, has many interesting biological activities. The uptake of EGCG and involvement of specific efflux pumps were studied in MDCKII cells transfected with hPgp, hMRP1, and hMRP2 genes. Total cell associated [3H]EGCG increased 7-fold in the presence of the MRP inhibitors, indomethacin and probenecid, in MDCKII/MRP1 cells, compared to a 2-fold increase in wild-type cells. Intracellular levels of EGCG, 4(")-O-methyl EGCG, and 4('),4(")-di-O-methyl EGCG were increased by 13-, 11-, and 3-fold, respectively, by indomethacin in MDCKII/MRP1 cells. Accumulation of EGCG and its methyl metabolites was also increased approximately 10-fold in the presence of MK-571 in MDCKII/MRP2 cells. Co-treatment with isoflavones, curcumin and tetrahydrocurcumin, increased [3H]EGCG accumulation significantly in MDCKII/MRP1 and HT-29 cells. The results indicate that EGCG and its methyl metabolites are substrates for MRP1 and MRP2, but not for Pgp. MRP type efflux pumps may limit the bioavailability of EGCG.     

MicroRNA-449a Enhances Radiosensitivity in CL1-0 Lung Adenocarcinoma Cells

Lung cancer is the leading cause of cancer-related mortality worldwide. Radiotherapy is often applied for treating lung cancer, but it often fails because of the relative non-susceptibility of lung cancer cells to radiation. MicroRNAs (miRNAs) have been reported to modulate the radiosensitivity of lung cancer cells and have the potential to improve the efficacy of radiotherapy. The purpose of this study was to identify a miRNA that can adjust radiosensitivity in lung adenocarcinoma cells. Two lung adenocarcinoma cell lines (CL1-0 and CL1-5) with different metastatic ability and radiosensitivity were used. In order to understand the regulatory mechanisms of differential radiosensitivity in these isogenic tumor cells, both CL1-0 and CL1-5 were treated with 10 Gy radiation, and were harvested respectively at 0, 1, 4, and 24 h after radiation exposure. The changes in expression of miRNA upon irradiation were examined using Illumina Human microRNA BeadChips. Twenty-six miRNAs were identified as having differential expression post-irradiation in CL1-0 or CL1-5 cells. Among these miRNAs, miR-449a, which was down-regulated in CL1-0 cells at 24 h after irradiation, was chosen for further investigation. Overexpression of miR-449a in CL1-0 cells effectively increased irradiation-induced DNA damage and apoptosis, altered the cell cycle distribution and eventually led to sensitization of CL1-0 to irradiation.     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.     

Human thioredoxin attenuates hypoxia-reoxygenation injury of murine endothelial cells in a thiol-free condition

The adult T cell leukemia-derived factor (ADF), or human thioredoxin (hTRX), has a radical scavenging effect similar to that of N-acetyl cysteine (NAC). We have recently shown that ADF/hTRX protects the lung and the heart from ischemia-reperfusion induced injury. To elucidate mechanisms of the protective effect, a hypoxia-reoxygenation (H-R) injury model was developed using a murine endothelial cell line, cultured in a thiol-free medium. In this condition, cells became much more vulnerable to H-R injury. The viability of cells decreased significantly after 1 h of hypoxic incubation followed by 1 h of reoxygenation. The injury was reduced by ADF/hTRX (100 microM) or NAC (10 mM). These two agents also demonstrated an additive protective effect. When cells were cultured in thiol-free medium for 2 h in a normoxic condition, intracellular hydrogen peroxide production was increased, which was associated with a decrease in glutathione level. NAC (10 mM) attenuated these changes whereas ADF/hTRX (100 microM) did not. These results suggest that although both ADF/hTRX and NAC protected cells from H-R injury, the underlying mechanisms are different. Because the cytoprotective effect of ADF/hTRX occurs in the thiol-free condition, it must be mediated via a novel mechanism other than enhancing thiol uptake. The additive cytoprotective effect between ADF/hTRX and NAC suggests that we should combine these two agents clinically.     

Synergistic effect of curcumin on epigallocatechin gallate-induced anticancer action in PC3 prostate cancer cells

Epigallocatechin gallate (EGCG) and curcumin are well known to naturally-occurring anticancer agents. The aim of this study was to verify the combined beneficial anticancer effects of curcumin and EGCG on PC3 prostate cancer cells, which are resistant to chemotherapy drugs and apoptosis inducers. EGCG showed weaker inhibitory effect on PC3 cell proliferation than two other prostate cancer cell lines, LNCaP and DU145. Co-treatment of curcumin improved antiproliferative effect of EGCG on PC3 cells. The protein expressions of p21 were significantly increased by the co-treatment of EGCG and curcumin, whereas it was not changed by the treatment with each individual compound. Moreover, treatments of EGCG and curcumin arrested both S and G2/M phases of PC3 cells. These results suggest that the enhanced inhibitory effect of EGCG on PC3 cell proliferation by curcumin was mediated by the synergic up-regulation of p21-induced growth arrest and followed cell growth arrest. [BMB Reports 2015; 48(8): 461-466]     

Biosynthesis of d-Erythroascorbic Acid by Candida

Epigallocatechin 3-gallate (EGCG) has cytotoxic effects in many cancer cells. It has been reported that A549 lung cancer cells are markedly resistant to cell death induced by EGCG. In the present study, the effects of EGCG on A549 lung cancer cell growth and angiogenesis were studied. We found that EGCG dose-dependently suppressed A549 cell growth, while A549 cells were markedly resistant to cell death in vitro. Next we found that EGCG increased endostatin expression and suppressed vascular endothelial growth factor (VEGF) expression. We further studied to determine whether EGCG would suppress A549 tumor growth in nude mouse and angiogenesis. EGCG in drinking water significantly suppressed A549 tumor growth in nude mice. Histological analysis revealed that the number of CD34 positive vessels had a tendency to decrease in the tumor. In sum, EGCG had anti-proliferative effects of A549 on tumor growth and showed a tendency to suppress angiogenesis.     

Activity of matrix metalloproteinases in normal and transformed mouse fibroblasts exposed to antioxidants

The effects of two antioxidants on the activity of matrix metalloproteinases (MMP) secreted by normal (3T3) and transformed (3T3-SV40) mouse fibroblasts were examined. We compared the action of N-acetylcysteine (NAC) and alpha-lipoic acid (ALA) on two gelatinases, MMP-2 and MMP-9. Gel zymography demonstrated that activity of MMP-2 was higher in normal 3T3 cells, whereas, in transformed 3T3-SV40 cells, the MMP-9 activity was higher. NAC treatment for 2–6 h completely suppressed MMP-2 and MMP-9 activity in both cell lines. The inhibitory effect did not depend on NAC concentration within the range of 1–10 mM. ALA (1.2 mM) did not affect the cells very dramatically; it decreased the MMP-2 activity in both types of cells. MMP-9 activity in the presence of ALA was decreased in 3T3 cells and slightly increased in 3T3-SV40 cells. The activity of the membrane bound and intracellular MMP was not changed under the same conditions. In conclusion, the altered activity of MMP in the presence of antioxidant may influence the intracellular signaling and cell functions.     

Changes in ceramide levels upon catechins-induced apoptosis in LoVo cells

It has been demonstrated that there is difference in the induction of apoptosis in LoVo cells by (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin (EC). In this study, we explored changes in ceramide levels upon the three catechins-induced apoptosis in LoVo cells. Addition of C2- and C6-ceramide to LoVo cells mimicked EGCG or EGC in leading to apoptotic death. Further measurement of intracellular ceramide content showed that the treatment of LoVo cells with EGCG or EGC resulted in a rapidly transient increase in ceramide content, and then back gradually to base line level, whereas the action of EC was just opposite to that of EGCG or EGC. These results suggest there is difference in the generation of intracellular ceramide by the three catechins and ceramide may take part in the regulation of EGCG- or EGC-induced apoptosis in LoVo cells.     

Mechanism-based inhibition of cancer metastasis with (−)-epigallocatechin gallate

Cell motility and cell stiffness are closely related to metastatic activity of cancer cells. (−)-Epigallocatechin gallate (EGCG) has been shown to inhibit spontaneous metastasis of melanoma cell line into the lungs of mice, so we studied the effects of EGCG on cell motility, cell stiffness, and expression of vimentin and Slug, which are molecular phenotypes of epithelial–mesenchymal transition (EMT). Treatments of human non-small cell lung cancer cell lines H1299 and Lu99 with 50 and 100 μM EGCG reduced cell motility to 67.5% and 43.7% in H1299, and 71.7% and 31.5% in Lu99, respectively in in vitro wound healing assay. Studies on cell stiffness using atomic force microscope (AFM) revealed that treatment with 50 μM EGCG increased Young’s modulus of H1299 from 1.24 to 2.25 kPa and that of Lu99 from 1.29 to 2.28 kPa, showing a 2-fold increase in cell stiffness, i.e. rigid elasticity of cell membrane. Furthermore, treatment with 50 μM EGCG inhibited high expression of vimentin and Slug in the cells at a leading edge of scratch. Methyl-β-cyclodextrin, a reagent to deplete cholesterol in plasma membrane, showed inhibition of EMT phenotypes similar that by EGCG, suggesting that EGCG induces inhibition of EMT phenotypes by alteration of membrane organization.