RESUMO
BACKGROUND: Oolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (-)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PURPOSE: OHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. METHODS: The effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. RESULTS: Cell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. CONCLUSIONS: Taken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.
Assuntos
Catequina , Melanoma Experimental , Animais , Humanos , Proteína Fosfatase 2/metabolismo , Polifenóis/farmacologia , Catequina/farmacologia , Ciclo Celular , Melanoma Experimental/tratamento farmacológico , Receptores de Laminina/química , Receptores de Laminina/metabolismoRESUMO
The body is equipped with a "food factor-sensing system" that senses food factors, such as polyphenols, sulfur-containing compounds, and vitamins, taken into the body, and plays an essential role in manifesting their physiological effects. For example, (-)-epigallocatechin-3-O-gallate (EGCG), the representative catechin in green tea (Camellia sinensi L.), exerts various effects, including anti-cancer, anti-inflammatory, and anti-allergic effects, when sensed by the cell surficial protein 67-kDa laminin receptor (67LR). Here, we focus on three representative effects of EGCG and provide their specific signaling mechanisms, the 67LR-mediated EGCG-sensing systems. Various components present in foods, such as eriodictyol, hesperetin, sulfide, vitamin A, and fatty acids, have been found to act on the food factor-sensing system and affect the functionality of other foods/food factors, such as green tea extract, EGCG, or its O-methylated derivative at different experimental levels, i.e., in vitro, animal models, and/or clinical trials. These phenomena are observed by increasing or decreasing the activity or expression of EGCG-sensing-related molecules. Such functional interaction between food factors is called "functional food pairing". In this review, we introduce examples of functional food pairings using EGCG.
Assuntos
Catequina , Animais , Catequina/análogos & derivados , Alimento Funcional , Polifenóis/farmacologia , Receptores de Laminina/metabolismo , Proteínas Ribossômicas , CháRESUMO
(-)-Epigallocatechin-3-O-(3-O-methyl) gallate (1, EGCG3â³Me), an antiallergic O-methylated catechin, is present in high quantities in the green tea cultivar "Benifuuki" (Camellia sinensis L.). Previous studies have shown that EGCG3â³Me inhibited basophil degranulation mediated through the cell-surface 67-kDa laminin receptor (67LR), but the mechanisms are not fully elucidated. This study aimed to investigate the mechanisms underlying the inhibitory effect of EGCG3â³Me on IgE/antigen (Ag)-mediated degranulation and the combined effect of EGCG3â³Me with eriodictyol (2), a bioactive flavanone. EGCG3â³Me inhibited ß-hexosaminidase release from the rat basophilic/mast cell line RBL-2H3 stimulated by IgE/Ag and induced acid sphingomyelinase (ASM) activity. This induction was inhibited by anti-67LR antibody treatment. The ASM-specific inhibitor desipramine inhibited EGCG3â³Me-induced suppression of degranulation. The soluble guanylate cyclase (sGC) inhibitor NS2028 weakened the potency of EGCG3â³Me, and the sGC activator BAY41-2272 suppressed degranulation. The ability of EGCG3â³Me to induce ASM activity and inhibit degranulation was amplified by eriodictyol. Furthermore, oral administration of the lemon-peel-derived eriodyctiol-7-O-glucoside (3) potentiated the suppressive effect of EGCG3â³Me-rich "Benifuuki" green tea on the IgE/Ag-induced passive cutaneous anaphylaxis (PCA) reaction in BALB/c mice. These results suggest that EGCG3â³Me inhibits IgE/Ag-mediated degranulation by inducing the 67LR/sGC/ASM signaling pathway, and eriodictyol amplifies this signaling.
Assuntos
Antialérgicos/farmacologia , Catequina/farmacologia , Flavanonas/farmacologia , Receptores de Laminina/metabolismo , Animais , Camellia sinensis/química , Linhagem Celular , Feminino , Mastócitos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Ratos , Transdução de Sinais/efeitos dos fármacos , CháRESUMO
(-)-epigallocatechin-3-O-gallate (EGCG) is a bioactive polyphenol in green tea. Previous studies have demonstrated the beneficial effects of EGCG on muscle mass and muscle atrophy. In the current study, we investigated the mechanisms underlying effect of EGCG on muscle atrophy. It was demonstrated that EGCG suppressed muscle-specific ubiquitin ligase, muscle RING Finger 1 (MuRF1) expression through 67-kDa laminin receptor (67LR). Previous studies have shown that eriodictyol potentiates the anti-tumor activities of EGCG by amplifying 67LR signaling. Therefore, we investigated the effects of EGCG and eriodictyol on the MuRF1 expression in C2C12 myotubes. The combined treatment of EGCG and eriodictyol significantly suppressed MuRF1 expression in dexamethasone-treated C2C12 myotubes. Tail suspension was maintained for 10 consecutive days using C57BL6/J mice, and during this time EGCG and eriodictyol were orally administered. In the gastrocnemius muscle, the muscle mass loss was inhibited by the combination of EGCG and eriodictyol. Therefore, EGCG may prevent muscle atrophy by inducing 67LR signaling and eriodictyol amplifies this pathway.
Assuntos
Catequina/análogos & derivados , Flavanonas/metabolismo , Proteínas Musculares/metabolismo , Plantas/química , Receptores de Laminina/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Catequina/química , Regulação para Baixo , Camundongos , Transdução de SinaisRESUMO
Epigallocatechin-3-gallate (EGCG), a main active catechin in green tea, was reported to attenuate renal injury and hypertension. However, its effects on salt-induced hypertension and renal injury remain unclear. In the present study, we explored its effects on hypertension and renal damage in Dahl rats with salt-sensitive hypertension. We found that EGCG could lower blood pressure after 6 weeks of oral administration, reduce 24 h urine protein levels and decrease creatinine clearance, and attenuate renal fibrosis, indicating that it could attenuate hypertension by protecting against renal damage. Furthermore, we studied the renal protective mechanisms of EGCG, revealing that it could lower malondialdehyde levels, reduce the numbers of infiltrated macrophages and T cells, and induce the apoptosis of NRK-49F cells. Considering that the 67 kD laminin receptor (67LR) binds to EGCG, its role in EGCG-induced fibroblast apoptosis was also investigated. The results showed that an anti-67LR antibody partially abrogated the apoptosis-inducing effects of EGCG on NRK-49F cells. In summary, EGCG may attenuate renal damage and salt-sensitive hypertension via exerting anti-oxidant, anti-inflammatory, and apoptosis-inducing effects on fibroblasts; the last effect is partially mediated by 67LR, suggesting that EGCG represents a potential strategy for treating salt-sensitive hypertension.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/etiologia , Catequina/análogos & derivados , Hipertensão/tratamento farmacológico , Hipertensão/etiologia , Fitoterapia , Cloreto de Sódio na Dieta/efeitos adversos , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Catequina/administração & dosagem , Catequina/metabolismo , Catequina/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/patologia , Malondialdeído/metabolismo , Ratos , Ratos Endogâmicos Dahl , Receptores de Laminina/metabolismoRESUMO
Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3â³Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3â³Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation.
Assuntos
Catequina/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Receptores de Laminina/metabolismo , Chá/química , Receptor 4 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Catequina/farmacologia , Células Cultivadas , Hiperinsulinismo/metabolismo , Hiperinsulinismo/prevenção & controle , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/prevenção & controle , Inflamação/etiologia , Inflamação/prevenção & controle , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Obesidade/etiologia , Obesidade/prevenção & controle , Receptores de Laminina/agonistas , Receptores de Laminina/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/genética , Regulação para CimaRESUMO
Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been attributed to the activation of its cell surface sensing receptor 67 kDa laminin receptor (67LR). However, the action of EGCG to activate 67LR remains unknown. Here we show that EGCG undergoes oligomer formation on its surface receptor 67LR.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Catequina/análogos & derivados , Receptores de Laminina/metabolismo , Chá/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Catequina/antagonistas & inibidores , Catequina/química , Catequina/farmacologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Estrutura Molecular , Peptídeos/química , Peptídeos/farmacologia , Receptores de Laminina/química , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
Various low-molecular-weight phytochemicals in green tea (Camellia sinensis L.), especially (-)-epigallocatechin-3-O-gallate (EGCG), are known to be involved in health promotion and disease risk reduction. However, the underlying mechanism has remained elusive because of the absence of an analytical technique that can easily detect the precise behavior of such a small molecule. Recently, we have identified a cell-surface EGCG-sensing receptor and the related signaling molecules that control the physiological functions of EGCG. We also developed a novel in situ label-free imaging technique for visualizing spatially resolved biotransformations based on simultaneous mapping of EGCG and its phase II metabolites. Furthermore, we established a chemometric method capable of evaluating the functionality of multicomponent green tea extracts by focusing on their compositional balances. This review highlights our proposed small molecule-sensing techniques for detecting the complex behavior of green tea components and linking such information to an enhanced understanding of green tea functionality.
Assuntos
Antialérgicos/farmacologia , Camellia sinensis/química , Catequina/análogos & derivados , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Catequina/análise , Catequina/farmacocinética , Catequina/farmacologia , Humanos , Receptores de Laminina/genética , Proteínas Ribossômicas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Chá/química , Distribuição TecidualRESUMO
Excessive activation of the microglia in the brain is involved in the development of several neurodegenerative diseases. Previous studies have indicated that (-)-epigallocatechin gallate (EGCG), a major active constituent of green tea, exhibits potent suppressive effects on the activation of microglia. As the 67 kDa laminin receptor (67LR) is a key element in cellular activation and migration, we investigated the effect of EGCG on cell migration and 67LR in lipopolysaccharide (LPS)-activated macrophagic RAW264.7 cells. The presence of EGCG (1-25 µM) markedly attenuated LPS-induced cell migration in a dose-dependent manner. However, the total amount of 67LR protein in the RAW264.7 cells was unaffected by EGCG, as revealed by Western blot analysis. In addition, confocal immunofluorescence microscopy indicated that EGCG caused a marked membrane translocation of 67LR from the membrane surface towards the cytoplasm. Cell-surface biotinylation analysis confirmed that EGCG induced a significant internalization of 67LR by 24-68% in a dose-dependent manner. This study helps to explain the pharmacological action of EGCG on 67LR, suggesting its potential use in the treatment of diseases associated with macrophage/microglia activation, such as neurodegenerative diseases and cancer.
Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Extratos Vegetais/farmacologia , Receptores de Laminina/metabolismo , Chá/química , Animais , Catequina/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microscopia ConfocalRESUMO
Delivery of optimal amounts of brain-derived neurotrophic factor (BDNF) to regions of the brain affected by neurodegenerative diseases is a daunting task. Using natural products with neuroprotective properties, such as green tea polyphenols, would be a highly useful complementary approach for inexpensive long-term treatment of these diseases. In this study, we used PC12(TrkB) cells which ectopically express TrkB, a high affinity receptor for BDNF. They differentiate and induce neurite outgrowth in response to BDNF. Using this model, we show for the first time that treatment with extremely low concentrations (<0.1 µg/ml) of unfractionated green tea polyphenols (GTPP) and low concentrations (<0.5 µM) of their active ingredient, epigallocatechin-3-gallate (EGCG), potentiated the neuritogenic ability of a low concentration (2 ng/ml) of BDNF. A synergistic interaction was observed between GTPP constituents, where epigallocatechin and epicatechin, both individually lacking this activity, promoted the action of EGCG. GTPP-induced potentiation of BDNF action required the cell-surface associated 67 kDa laminin receptor (67LR) to which EGCG binds with high affinity. A cell-permeable catalase abolished GTPP/EGCG-induced potentiation of BDNF action, suggesting the possible involvement of H2O2 in the potentiation. Consistently, exogenous sublethal concentrations of H2O2, added as a bolus dose (5 µM) or more effectively through a steady-state generation (1 µM), potentiated BDNF action. Collectively, these results suggest that EGCG, dependent on 67 LR and H2O2, potentiates the neuritogenic action of BDNF. Intriguingly, this effect requires only submicromolar concentrations of EGCG. This is significant as extremely low concentrations of polyphenols are believed to reach the brain after drinking green tea.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Catequina/farmacologia , Neuritos/efeitos dos fármacos , Chá/química , Animais , Antioxidantes/farmacologia , Catequina/análogos & derivados , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Peso Molecular , Neuritos/fisiologia , Oxidantes/metabolismo , Oxidantes/farmacologia , Células PC12 , Polifenóis/farmacologia , Ratos , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores de Laminina/química , Receptores de Laminina/metabolismo , Receptores de Laminina/fisiologiaRESUMO
This study investigated the pathways involved in EGCG modulation of insulin-like growth factor (IGF)-stimulated glucose uptake in 3T3-L1 adipocytes. EGCG inhibited IGF-I and IGF-II stimulation of adipocyte glucose uptake with dose and time dependencies. EGCG at 20µM for 2h decreased IGF-I- and IGF-II-stimulated glucose uptake by 59% and 64%, respectively. Pretreatment of adipocytes with antibody against the EGCG receptor (also known as the 67-kDa laminin receptor; 67LR), prevented the effects of EGCG on IGF-increased glucose uptake, but pretreatment with normal rabbit immunoglobulin did not. This suggests that the 67LR mediates the anti-IGF effect of EGCG on adipocyte glucose uptake. Further analysis indicated EGCG, IGF-I, and IGF-II did not alter total levels of GLUT1 or GLUT4 protein. However, EGCG prevented the IGF-increased GLUT4 levels in the plasma membrane and blocked the IGF-decreased GLUT4 levels in low-density microsomes. Neither EGCG nor its combination with IGF altered GLUT1 protein levels in the plasma membrane and low-density microsomes. EGCG also suppressed the IGF-stimulated phosphorylation of IGF signaling molecules, PKCζ/λ, but not AKT and ERK1/2, proteins. This study suggests that EGCG suppresses IGF stimulation of 3T3-L1 adipocyte glucose uptake through inhibition of the GLUT4 translocation, but not through alterations of the GLUT1 pathway.
Assuntos
Adipócitos/metabolismo , Catequina/análogos & derivados , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Chá/química , Células 3T3-L1 , Acetilcisteína/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Anticorpos/metabolismo , Catequina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Transporte Proteico , Receptores de Laminina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Since the administration of synthetic medicines is associated with drug resistance and undesired side effects, utilization of natural compounds could be an alternative and complementary modality to inhibit or prevent the development of tumors. Epigallocatechin 3-O-gallate (EGCG, 1), the major flavan component of green tea, and genistein (2), a soy isoflavonoid, are known to have chemopreventive and chemotherapeutic effects against cancer. This study demonstrated that both flavonoids inhibit cell proliferation, an effect enhanced under serum-free conditions. Compound 1, but not 2, induced downregulation of ErbB1 and ErbB2 in mammary and epidermoid carcinoma cells, and its inhibitory effect on cell viability was mediated by the 67 kDa laminin receptor (67LR). While 1 was superior in inducing cell death, 2 was more efficient in arresting the tumor cells in the G2/M phase. Furthermore, number and brightness analysis revealed that 1 decreased the homoclustering of a lipid raft marker, glycosylphosphatidylinositol-anchored GFP, and it also reduced the co-localization between lipid rafts and 67LR. The main conclusion made is that the primary target of 1 may be the lipid raft component of the plasma membrane followed by secondary changes in the expression of ErbB proteins. Compound 2, on the other hand, must have other unidentified targets.
Assuntos
Anticarcinógenos/química , Anticarcinógenos/farmacologia , Catequina/análogos & derivados , Flavonoides/farmacologia , Genisteína/farmacologia , Receptores de Laminina/efeitos dos fármacos , Proteínas Ribossômicas/efeitos dos fármacos , Chá/química , Algoritmos , Anticarcinógenos/isolamento & purificação , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Flavonoides/química , Flavonoides/isolamento & purificação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Genes erbB-1/genética , Genes erbB-1/fisiologia , Genes erbB-2/genética , Genes erbB-2/fisiologia , Genisteína/química , Genisteína/isolamento & purificação , Humanos , Estrutura Molecular , Polifenóis/farmacologia , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Receptores de Laminina/fisiologia , Glycine max/químicaRESUMO
PURPOSE: Neuroblastoma is a rare childhood cancer whose high risk, metastatic form has a dismal outcome in spite of aggressive therapeutic interventions. The toxicity of drug treatments is a major problem in this pediatric setting. In this study, we investigated whether Polyphenon E, a clinical grade mixture of green tea catechins under evaluation in multiple clinical cancer trials run by the National Cancer Institute (Bethesda, MD), has anticancer activity in mouse models of neuroblastoma. EXPERIMENTAL DESIGN: We used three neuroblastoma models: (i) transgenic TH-MYCN mouse developing spontaneous neuroblastomas; (ii) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenotransplanted with human SHSY5Y cells; and (iii) A/J mice transplanted with syngeneic Neuro 2A cells. Mice were randomized in control and Polyphenon E-drinking groups. Blood from patients with neuroblastoma and normal controls was used to assess the phenotype and function of myeloid cells. RESULTS: Polyphenon E reduced the number of tumor-infiltrating myeloid cells, and inhibited the development of spontaneous neuroblastomas in TH-MYCN transgenic mice. In therapeutic models of neuroblastoma in A/J, but not in immunodeficient NOD/SCID mice, Polyphenon E inhibited tumor growth by acting on myeloid-derived suppressor cells (MDSC) and CD8 T cells. In vitro, Polyphenon E impaired the development and motility of MDSCs and promoted differentiation to more neutrophilic forms through the 67 kDa laminin receptor signaling and induction of granulocyte colony-stimulating factor. The proliferation of T cells infiltrating a patient metastasis was reactivated by Polyphenon E. CONCLUSIONS: These findings suggest that the neuroblastoma-promoting activity of MDSCs can be manipulated pharmacologically in vivo and that green tea catechins operate, at least in part, through this mechanism.
Assuntos
Catequina/análogos & derivados , Células Mieloides/imunologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/imunologia , Linfócitos T/imunologia , Chá/química , Animais , Catequina/farmacologia , Células Cultivadas , Criança , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Neuroblastoma/mortalidade , Receptores de Laminina/metabolismo , Taxa de Sobrevida , Linfócitos T/efeitos dos fármacosRESUMO
The major constituent of green tea, (-)-epigallocatechin-3-O-gallate (EGCG), has been shown to have cancer-preventive and therapeutic activities. Numerous molecular targets for EGCG have been proposed, but the mechanisms of its anticancer activities are not clearly understood. In this issue of the JCI, Kumazoe et al. report that EGCG activates 67-kDa laminin receptor (67LR), elevates cGMP levels, and induces cancer cell apoptosis. Furthermore, a phosphodiesterase 5 inhibitor, vardenafil, synergizes with EGCG to induce cancer cell death. This is a provocative observation with important implications for cancer therapy. It also raises several issues for further investigation, such as the mechanism by which EGCG specifically activates 67LR.
Assuntos
Apoptose/fisiologia , GMP Cíclico/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Laminina/metabolismo , Animais , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: In traditional Chinese medicine, green tea is considered to have a life-prolonging effect, possibly as a result of its rich content of antioxidant tea polyphenols, and hence has the potential to prevent cancer. This study investigated the role of the major tea secondary plant compound epigallocatechin gallate (EGCG) for its inhibitory effects on the metastasis-associated 67 kDa laminin receptor (67LR). METHODS: To clarify the impact of EGCG on siRNA-silenced expression of 67LR, we applied an adenoviral-based intestinal in vitro knockdown model, porcine IPEC-J2 cells. Quantitative real-time polymerase chain reaction was performed to analyze 67LR gene expression following treatment with physiological and pharmacological concentrations of EGCG (1.0 g/l, 0.1 g/l, 0.02 g/l and 0.002 g/l). RESULTS: We report co-regulation of EGCG and 67LR, which is known to be an EGCG receptor. siRNA selectively and highly significantly suppressed expression of 67LR under the impact of EGCG in a synergetic manner. CONCLUSIONS: Our findings suggest that 67LR expression is regulated by EGCG via a negative feedback loop. The explicit occurrence of this effect in synergy with a small RNA pathway and a plant-derived drug reveals a new mode of action. Our findings may help to provide insights into the many unsolved health-promoting activities of other natural pharmaceuticals.
Assuntos
Camellia sinensis/química , Regulação para Baixo/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Extratos Vegetais/farmacologia , Receptores de Laminina/genética , Animais , Catequina/análogos & derivados , Catequina/metabolismo , Linhagem Celular , Humanos , Metástase Neoplásica/prevenção & controle , Neoplasias/patologia , Receptores de Laminina/metabolismoRESUMO
Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.
Assuntos
Camellia sinensis , Catequina/análogos & derivados , Células Dendríticas/efeitos dos fármacos , Receptores de Laminina/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Catequina/farmacologia , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/imunologia , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Lipopolissacarídeos/imunologia , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKCε from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKCε and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKCε, resulting in preconditioning against cell death induced by OGD/R.
Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Membrana Celular/enzimologia , Citosol/enzimologia , Glucose , Oxigênio , Polifenóis/farmacologia , Proteína Quinase C-épsilon/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Laminina/metabolismo , Chá/química , Animais , Antioxidantes/química , Catequina/química , Catequina/farmacocinética , Catequina/farmacologia , Morte Celular , Ativação Enzimática/efeitos dos fármacos , Células PC12 , Polifenóis/química , Ligação Proteica/efeitos dos fármacos , Proteína Quinase C-épsilon/genética , Transporte Proteico/efeitos dos fármacos , Ratos , Receptores de Laminina/genéticaRESUMO
BACKGROUND: We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K(166) and H(169), have a critical role in neutralizing EGCG's activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG. CONCLUSIONS/SIGNIFICANCE: Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions.
Assuntos
Catequina/análogos & derivados , Polifenóis/metabolismo , Receptores de Laminina/química , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Chá/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Catequina/metabolismo , Catequina/farmacologia , Proliferação de Células/efeitos dos fármacos , Espaço Extracelular/metabolismo , Células Hep G2 , Humanos , Dados de Sequência Molecular , Polifenóis/farmacologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
SCOPE: This study investigated the pathways involved in epigallocatechin gallate (EGCG) modulation of insulin-like growth factor (IGF)-I-stimulated and IGF-II-stimulated mitogenesis in 3T3-L1 preadipocytes. METHODS AND RESULTS: We found that this process was dose and time dependent, and caused by suppression of IGF-I-stimulated and IGF-II-stimulated phosphorylation of p66Shc and mitogen-activated protein kinase (MAPK) pathway proteins, including MEK1 kinase (RAF1), extracellular signal-regulated protein kinase (ERK) kinase (MEK1), and ERK 1 and ERK 2 (ERK1/2), but not phospho-Jun-N-terminal kinase, protein kinase B, p52Shc, or p46Shc. Furthermore, EGCG inhibited the IGF-I-stimulated phosphorylation of the IGF-I receptor-beta (IGF-IR ß), the association of IGF-IR with the p66Shc protein, and the IGF-II-stimulated associations of the IGF-II receptor with G(αi-2) and p66Shc proteins, suggesting that EGCG selectively affects particular types of Shc and MAPK family members. Pretreatment with antiserum against the EGCG receptor (also known as the 67-kDa laminin receptor; 67LR), but not with an adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, prevented the inhibitory actions of EGCG on IGF-I- and IGF-II-stimulated ERK1/2 phosphorylation and subsequent preadipocyte proliferation. CONCLUSION: The results of this study suggest that EGCG mediates anti-IGF-I and anti-IGF-II signals in preadipocyte mitogenesis via the 67LR but not the AMPK pathway.
Assuntos
Adipócitos/efeitos dos fármacos , Catequina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Receptores de Laminina/metabolismo , Células 3T3-L1 , Monofosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Catequina/farmacologia , Imunoprecipitação , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Extratos Vegetais/farmacologia , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Chá/químicaRESUMO
EGCG [(-)-epigallocatechin-3-O-gallate], the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In the present study, we show that EGCG-induced apoptotic activity is attributed to a lipid-raft clustering mediated through 67LR (67 kDa laminin receptor) that is significantly elevated in MM (multiple myeloma) cells relative to normal peripheral blood mononuclear cells, and that aSMase (acid sphingomyelinase) is critical for the lipid-raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and PKCδ (protein kinase Cδ) phosphorylation at Ser664, which was necessary for aSMase/ceramide signalling via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell-death pathway triggered by EGCG for the specific killing of MM cells.