Research Database

Author: Yumei Chen, Bo Zhu, Hongping Zhang, Ding Ding, Xishi Liu, Sun-Wei Guo

We have previously reported that induction of adenomyosis in mice results in progressive hyperalgesia, uterine hyperactivity, and elevated plasma corticosterone levels and that epigallocatechin-3-gallate (EGCG) treatment dose dependently suppressed myometrial infiltration and improved generalized hyperalgesia. In this study, we examined whether adenomyosis induced in mice results in the loss of GABAergic inhibition as manifested by the diminished glutamate decarboxylase (GAD) 65-expressing neurons in the brainstem nucleus raphe magnus (NRM) that could correlate with heightened hyperalgesia. We also evaluated whether EGCG treatment would reverse these changes and also improve the expression of some proteins known to be involved in adenomyosis. Adenomyosis was induced in 28 female ICR mice and additional 12 were used as blank controls, as reported previously. At the 16th week, all mice with induced adenomyosis received low- or high-dose EGCG treatment or untreated. Mice without adenomyosis received no treatment. After 3 weeks of treatment, their uterine horns and brains were harvested. The right uterine horn was used for immunohistochemistry analysis and for counting the number of macrophages infiltrating into the ectopic endometrium. The brainstem NRM sections were subjected to immunofluorescence staining for GAD65. We found that mice with induced adenomyosis had significantly diminished GAD65-expressing neurons, concomitant with heightened hyperalgesia. Treatment with EGCG increased these neurons in conjunction with improved hyperalgesia, reduced the expression of p-p65, cycloxygenase 2, oxytocin receptor, collagen I and IV, and transient receptor potential vanilloid type 1 in ectopic endometrium or myometrium, reduced the number of macrophages infiltrating into the ectopic endometrium while elevated the expression of progesterone receptor isoform B. Thus, adenomyosis-induced pain resembles neuropathic pain in that there is a remarkable central plasticity.

 

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Author: Leonarda D’Angelo, Giulia Piazzi, Annalisa Pacilli, Anna Prossomariti, Chiara Fazio, Lorenzo Montanaro, Giulia Graziani, Vincenzo Fogliano, Alessandra Munarini, Francesca Bianchi, Andrea Belluzzi, Franco Bazzoli and Luigi Ricciardiello

Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA), epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape seed [GS] extract) at low cytotoxic concentrations on CRC cells and test their activity on mTOR and translational regulation. The CRC cell lines HCT116 and SW480 were treated for 24h with combinations of EPA-FFA (0-150 µM), EGCG (0-175 µM) and GS extract (0-15 µM) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 µM, EGCG 175 µM and GS extract 15 µM completely inhibited the mTOR signaling in HCT116 and SW480 cells, reaching an effect stronger than or comparable to that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells, respectively. Moreover, the treatment led to changes of protein translation of ribosomal proteins, c-Myc and cyclin D1. In addition, we found a reduction of clonal capability in both cell lines, with block of cell cycle in G0G1 and induction of apoptosis. Our data suggest that the low cytotoxic combination of EPA-FFA, EGCG and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment.

 

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Author: Zhang D, Rajaratnam V, Al-Hendy O, Halder S, Al-Hendy A

Background/Aims: To investigate the inhibitory effect of green tea extract, epigallocatechin gallate (EGCG), on wild-type human leiomyoma (WT-HuLM) cells and its potential action via catechol-o-methyltransferase (COMT) activity. Methods: Cell proliferation of WT-HuLM and COMT gene-silenced HuLM (COMT-shRNA-HuLM) cells treated with 0 or 100 µM EGCG for 7 days was measured using the MTT method. Total RNA and protein were extracted from cells treated with 0 or 100 µM of EGCG for 48 h. Gene expression profiling was performed using Human Signal Transduction PathwayFinder. Proliferation cell nuclear antigen (PCNA), cyclin-dependent kinase 4 (Cdk4) and COMT protein levels were detected by Western blot analyses. COMT enzyme activity was evaluated by HPLC. Results: EGCG-treated WT-HuLM cells showed significantly decreased COMT expression (p < 0.001) and enzyme activity (p < 0.05) compared to untreated WT-HuLM cells, while COMT-shRNA-HuLM cells showed no significant change. At 100 μM of EGCG, survival of WT-HuLM cells was significantly lower (p < 0.05) compared to COMT-shRNA-HuLM cells. EGCG treatment modulated multiple signaling pathways in WT-HuLM compared to untreated control, while changes were minimal or reversed in COMT-shRNA-HuLM cells. EGCG significantly decreased PCNA, Cdk4 and soluble COMT protein levels (p < 0.001) in WT-HuLM, but not in COMT-shRNA-HuLM cells. Conclusions: The antiproliferative and gene-modulating effects of EGCG on HuLM cells are mediated, at least partially, via its effect on COMT expression and enzyme activity.

 

 

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Author: Mi Ran Byun, Mi Kyung Sung, A Rum Kim, Cham Han Lee, Eun Jung Jang, Mi Gyeong Jeong, Minsoo Noh, Eun Sook Hwang and Jeong-Ho Hong

Osteoporosis is a degenerative bone disease characterized by low bone mass and is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. It is known that the bioactive compounds present in green tea increase osteogenic activity and decrease the risk of fracture by improving bone mineral density. However, the detailed mechanism underlying these beneficial effects has yet to be elucidated. In this study, we investigated the osteogenic effect of (−)-epicatechin gallate (ECG), a major bioactive compound found in green tea. We found that ECG effectively stimulates osteoblast differentiation, indicated by the increased expression of osteoblastic marker genes. Up-regulation of osteoblast marker genes is mediated by increased expression and interaction of the transcriptional coactivator with PDZ-binding motif (TAZ) and Runt-related transcription factor 2 (RUNX2). ECG facilitates nuclear localization of TAZ through PP1A. PP1A is essential for osteoblast differentiation because inhibition of PP1A activity was shown to suppress ECG-mediated osteogenic differentiation. Taken together, the results showed that ECG stimulates osteoblast differentiation through the activation of TAZ and RUNX2, revealing a novel mechanism for green tea-stimulated osteoblast differentiation.

 

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Author: Federica Rizzi, Valeria Naponelli, Alessandro Silva, Alice Modernelli, Ileana Ramazzina, Martina Bonacini, Saverio Tardito, Rita Gatti, Jacopo Uggeri and Saverio Bettuzzi

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 μg/ml] than PC3 (IC50 = 145 μg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

 

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Synergistic inhibition of lung cancer cell lines by (−)-epigallocatechin-3-gallate in combination with clinically used nitrocatechol inhibitors of catechol- O -methyltransferase

Author: Sarah C. Forester and Joshua D. Lambert

(−)-Epigallocatechin-3-gallate (EGCG) has exhibited been studied for lung cancer inhibitory activity in vitroand in animal models, but it is rapidly methylated and inactivated by catechol- O -methyltransferase (COMT). Entacapone and tolcapone, COMT inhibitors, are used to mitigate the symptoms of Parkinson’s disease. We investigated the synergistic effects of entacapone/tolcapone and EGCG against lung cancer cell lines in culture. EGCG, entacapone and tolcapone inhibited the growth of H1299 human lung cancer cells (IC ₅₀ = 174.9, 76.8 and 29.3 µM, respectively) and CL-13 murine lung cancer cells (IC ₅₀ = 181.5, 50.7 and 19.7 µM, respectively) as single agents following treatment for 72h. Treatment with 1:10, 1:5, 1:2.5 and 1:1 combinations of EGCG and tolcapone or entacapone resulted in synergistically enhanced growth inhibition. The growth inhibitory effect of the combinations was mediated by induction of intracellular oxidative stress, cell cycle arrest and decreased nuclear translocation of nuclear factor-κΒ. Methylation of EGCG was dose dependently inhibited by entacapone and tolcapone (IC ₅₀ = 10 and 20 µM, respectively) in a cell-free system, and both compounds increased the intracellular levels of unmethylated EGCG. Treatment of mice with EGCG in combination with tolcapone increased the bioavailability of EGCG and decreased the methylation of plasma norepinephrine: no apparent liver or behavioral toxicity was observed. In conclusion, the combination of EGCG and entacapone/tolcapone synergistically inhibited the growth of lung cancer cells in culture, and the mechanistic basis for this synergy is likely due in part to inhibition of COMT with resultant increase in the levels of unmetabolized EGCG.

 

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Author: David Heber, Yanjun Zhang, Jieping Yang, Janice E. Ma, Susanne M. Henning, and Zhaoping Li

Green tea (GT) and caffeine in combination were shown to increase energy expenditure and fat oxidation, but less is known about the effects of black tea (BT) and oolong tea (OT). This study investigated whether decaffeinated polyphenol extracts from GT, BT, and OT decrease body fat and inflammation in male C57BL/6J mice fed high-fat/high-sucrose [HF/HS (32% energy from fat, 25% energy from sucrose)] diets. Mice were fed either an HF/HS diet with 0.25% of polyphenol from GT, OT, or BT or a low-fat/high-sucrose [LF/HS (10.6% energy from fat, 25% energy from sucrose)] diet for 20 wk. Monomeric tea polyphenols were found in the liver and adipose tissue of mice fed the HF/HS diet with GT polyphenols (GTPs) and OT polyphenols (OTPs) but not BT polyphenols (BTPs). Treatment with GTPs, OTPs, BTPs, and an LF/HS diet led to significantly lower body weight, total visceral fat volume by MRI, and liver lipid weight compared with mice in the HF/HS control group. Only GTPs reduced food intake significantly by ∼10%. GTP, BTP, and LF/HS-diet treatments significantly reduced serum monocyte chemotactic protein-1 (MCP-1) compared with HF/HS controls. In mesenteric fat, monocyte chemotactic protein-1 (Mcp1) gene expression was significantly decreased by treatment with GTPs, BTPs, OTPs, and an LF/HS diet and in liver tissue by GTP and BTP treatments. Mcp1 gene expression in epididymal fat was significantly decreased by the BTP and LF/HS diet interventions. In epididymal fat, consistent with an anti-inflammatory effect, adiponectin gene expression was significantly increased by GTPs and OTPs. Angiogenesis during adipose tissue expansion is anti-inflammatory by maintaining adipocyte perfusion. We observed significantly increased gene expression of vascular endothelial growth factor A by GTPs and vascular endothelial growth factor receptor 2 by BTPs and the LF/HS diet and a decrease in pigment epithelium-derived factor gene expression by OTPs and BTPs. In summary, all 3 tea polyphenol extracts induced weight loss and anti-inflammatory and angiogenic effects, although the tissue content of polyphenols differed significantly.

 

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Author: Sachiko Matsuzaki and Claude Darcha

STUDY QUESTION: Is epigallocatechin-3-gallate (EGCG) treatment effective in the treatment of fibrosis in endometriosis? SUMMARY ANSWER: EGCG appears to have antifibrotic properties in endometriosis. WHAT IS KNOWN ALREADY: Histologically, endometriosis is characterized by dense fibrous tissue surrounding the endometrial glands and stroma. However, only a few studies to date have evaluated candidate new therapies for endometriosis-associated fibrosis. STUDY DESIGN, SIZE, DURATION: For this laboratory study, samples from 55 patients (45 with and 10 without endometriosis) of reproductive age with normal menstrual cycles were analyzed. A total of 40 nude mice received single injection proliferative endometrial fragments from a total of 10 samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: The in vitro effects of EGCG and N-acetyl-l-cysteine on fibrotic markers (alpha-smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin) with and without transforming growth factor (TGF)-β1 stimulation, as well as on cell proliferation, migration and invasion and collagen gel contraction of endometrial and endometriotic stromal cells were evaluated by real-time PCR, immunocytochemistry, cell proliferation assays, in vitro migration and invasion assays and/or collagen gel contraction assays. The in vitro effects of EGCG on mitogen-activated protein kinase (MAPK) and Smad signaling pathways in endometrial and endometriotic stromal cells were evaluated by western blotting. Additionally, the effects of EGCG treatment on endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with EGCG significantly inhibited cell proliferation, migration and invasion of endometrial and endometriotic stromal cells from patients with endometriosis. In addition, EGCG treatment significantly decreased the TGF-β1-dependent increase in the mRNA expression of fibrotic markers in both endometriotic and endometrial stromal cells. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels were significantly attenuated at 8, 12 and 24 h after treatment with EGCG. Epigallocatechin-3-gallate also significantly inhibited TGF-β1-stimulated activation of MAPK and Smad signaling pathways in endometrial and endometriotic stromal cells. Animal experiments showed that EGCG prevented the progression of fibrosis in endometriosis. LIMITATIONS, REASONS FOR CAUTION: The attractiveness of epigallocatechin-3-gallate as a drug candidate has been diminished by its relatively low bioavailability. However, numerous alterations to the EGCG molecule have been patented, either to improve the integrity of the native compound or to generate a more stable yet similarly efficacious molecule. Therefore, EGCG and its derivatives, analogs and prodrugs could potentially be developed into agents for the future treatment and/or prevention of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Epigallocatechin-3-gallate is a potential drug candidate for the treatment and/or prevention of endometriosis. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

 

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Author: Wei Zhu, Jing Xu, Yangyang Ge, Han Cao, Xin Ge, Judong Luo, Jiao Xue, Hongying Yang, Shuyu Zhang and Jianping Cao

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, is a potent antioxidant and free radical scavenger that may have therapeutic applications for the treatment of many disorders. Radiation therapy is widely used for the treatment of various types of cancers; however, radiation-induced skin injury remains a serious concern. EGCG has not yet been reported as protecting skin cells against ionizing radiation. In the present study, we investigated whether EGCG confers cytoprotection against ionizing radiation. We found that, compared with the control, pretreatment with EGCG significantly enhanced the viability of human skin cells that were irradiated with X-rays, and decreased apoptosis induced by X-ray irradiation. Mito-Tracker assay showed that EGCG suppressed the damage to mitochondria induced by ionizing radiation via upregulation of SOD2. Reactive oxygen species (ROS) in HaCaT cells were significantly reduced when pretreated with EGCG before irradiation. Radiation-induced γH2AX foci, which are representative of DNA double-strand breaks, were decreased by pretreatment with EGCG. Furthermore, EGCG induced the expression of the cytoprotective molecule heme oxygenase-1 (HO-1) in a dose-dependent manner via transcriptional activation. HO-1 knockdown or treatment with the HO-1 inhibitor tin protoporphyrin (SnPPIX) reversed the protective role of EGCG, indicating an important role for HO-1. These results suggest that EGCG offers a new strategy for protecting skin against ionizing radiation.

 

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Author: Aaron Katz, Mitchell Efros, Jed Kaminetsky, Kelli Herrlinger, Diana Chirouzes, Michael Ceddia

Objectives: The objective of this study was to examine the effects of a green and black tea extract blend [AssuriTEA Men’s Health (AMH)] in men with lower urinary tract symptoms (LUTS). Methods: In this randomized, double-blind, placebo-controlled study, 46 men aged 30–70 with an American Urologic Association symptom score (AUAss) of at least 8 and up to 24 were randomized to 500 mg AMH, 1000 mg AMH, or placebo daily for 12 weeks. Measurements were taken at baseline (BL), week 6 and week 12 for AUAss, simple uroflowmetry, postvoid residual volume (PVR), C-reactive protein (CRP), Short-Form 36 Health Survey (SF-36), and International Index of Erectile Function (IIEF). Results: A total of 40 subjects completed the study. AUAss decreased 34.5% from BL to week 12 in the 1000 mg AMH group (p = 0.008). At week 12, CRP increased in the 500 mg AMH (p = 0.003) and placebo (p = 0.012) groups from their BL levels but not in the 1000 mg group. Average urine flow (Qmean) increased in the 500 mg (p = 0.033) and 1000 mg AMH (p = 0.002) groups versus placebo. PVR decreased in the 1000 mg AMH group (p = 0.034) from BL at week 6. Treatment group effects were observed for the physical functioning and sexual desire domains of the SF-36 and IIEF (p = 0.051 and p = 0.005 respectively). AMH was well tolerated. Conclusions: Oral administration of AMH improved LUTS and quality of life in as little as 6 weeks.

 

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