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1.
J. appl. oral sci ; 27: e20180150, 2019. graf
Article in English | LILACS, BBO | ID: biblio-975883

ABSTRACT

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.


Subject(s)
Animals , Mice , Osteoclasts/drug effects , Root Canal Filling Materials/pharmacology , Bone Marrow Cells/drug effects , Cell Differentiation/drug effects , Silicates/pharmacology , Calcium Compounds/pharmacology , Alendronate/pharmacology , Bone Density Conservation Agents/pharmacology , Osteoclasts/physiology , Osteogenesis/drug effects , Phosphorylation/drug effects , Root Resorption/prevention & control , Time Factors , Bone Marrow Cells/cytology , Cell Survival/drug effects , Cells, Cultured , Blotting, Western , Reproducibility of Results , MAP Kinase Signaling System/drug effects , I-kappa B Proteins/drug effects , RANK Ligand/analysis , RANK Ligand/drug effects , Real-Time Polymerase Chain Reaction , Tartrate-Resistant Acid Phosphatase
2.
Arq. bras. cardiol ; 110(1): 44-51, Jan. 2018. graf
Article in English | LILACS | ID: biblio-887998

ABSTRACT

Resumo Background: Melatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis. Objective: Demonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes. Methods: In vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats. Results: In vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059. Conclusion: melatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1.


Resumo Fundamento: A melatonina é um hormônio neuroendócrino sintetizado principalmente pela glândula pineal que é indicado para prevenir efetivamente a lesão de reperfusão miocárdica. Não está claro se a melatonina protege a função cardíaca da lesão de reperfusão através da modulação da homeostase do cálcio intracelular. Objetivo: Demonstrar que a melatonina protege contra a lesão de reperfusão miocárdica através da modulação de IP3R e SERCA para manter a homeostase de cálcio por meio da ativação de ERK1 em cardiomiócitos. Métodos: Foram realizados experimentos in vitro usando células H9C2 submetidas a indução de hipoxia / reoxigenação simulada (H/R). O nível de expressão de ERK1, IP3R e SERCA foi avaliado por Western Blots. A apoptose de cardiomiócitos foi detectada por TUNEL. A coloração de faloidina foi utilizada para avaliar a alteração da organização de filamentos de actina dos cardiomiócitos. Fura-2 / AM foi utilizado para medir a concentração intracelular de Ca2+. Realizando experiências in vivo, a expressão miocárdica de IP3R e SERCA foi detectada por coloração com imunofluorescência usando modelo de isquemia miocárdica / reperfusão (I/R) em ratos. Resultados: resultados in vitro mostraram que a melatonina induz a ativação de ERK1 em cardiomiócitos contra H/R que foi inibida por PD98059 (inibidor de ERK1). Os resultados mostraram que a melatonina inibe a apoptose dos cardiomiócitos e melhora a organização do filamento de actina em cardiomiócitos contra H/R, pois ambas poderiam ser revertidas pela PD98059. A melatonina mostrou reduzir a sobrecarga de cálcio, além de inibir a expressão de IP3R e promover a expressão de SERCA através da via ERK1 em cardiomiócitos contra H/R. A melatonina induziu menor IP3R e maior expressão de SERCA no miocárdio que foram revertidas pela PD98059. Conclusão: a cardioproteção induzida pela melatonina contra lesão de reperfusão é pelo menos parcialmente através da modulação de IP3R e SERCA para manter a homeostase de cálcio intracelular via ativação de ERK1.


Subject(s)
Animals , Male , Rats , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/prevention & control , MAP Kinase Signaling System/drug effects , Myocytes, Cardiac/drug effects , Sarcoplasmic Reticulum Calcium-Transporting ATPases/drug effects , Inositol 1,4,5-Trisphosphate Receptors/drug effects , Melatonin/pharmacology , Myocardial Reperfusion Injury/pathology , Rats, Sprague-Dawley , Myocytes, Cardiac/pathology , Disease Models, Animal , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism
3.
Biol. Res ; 51: 17, 2018. graf
Article in English | LILACS | ID: biblio-950903

ABSTRACT

BACKGROUND: Improper control on reactive oxygen species (ROS) elimination process and formation of free radicals causes tissue dysfunction. Pineal hormone melatonin is considered a potent regulator of such oxidative damage in different vertebrates. Aim of the current communication is to evaluate the levels of oxidative stress and ROS induced damage, and amelioration of oxidative status through melatonin induced activation of signaling pathways. Hepatocytes were isolated from adult Labeo rohita and exposed to H2O2 at three different doses (12.5, 25 and 50 µM) to observe peroxide induced damage in fish hepatocytes. Melatonin (25, 50 and 100 µg/ml) was administered against the highest dose of H2O2. Enzymatic and non-enzymatic antioxidants such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) was measured spectrophotometrically. Expression level of heat shock proteins (HSP70 and HSP90), HSPs-associated signaling molecules (Akt, ERK, cytosolic and nuclear NFkB), and melatonin receptor was also measured by western blotting analysis. RESULTS: H2O2 induced oxidative stress significantly altered (P < 0.05) MDA and GSH level, SOD and CAT activity, and up regulated HSP70 and HSP90 expression in carp hepatocytes. Signaling proteins exhibited differential modulation as revealed from their expression patterns in H2O2-exposed fish hepatocytes, in comparison with control hepatocytes. Melatonin treatment of H2O2-stressed fish hepatocytes restored basal cellular oxidative status in a dose dependent manner. Melatonin was observed to be inducer of signaling process by modulation of signaling molecules and melatonin receptor. CONCLUSIONS: The results suggest that exogenous melatonin at the concentration of 100 µg/ml is required to improve oxidative status of the H2O2-stressed fish hepatocytes. In H2O2 exposed hepatocytes, melatonin modulates expression of HSP70 and HSP90 that enable the hepatocytes to become stress tolerant and survive by altering the actions of ERK, Akt, cytosolic and nuclear NFkB in the signal transduction pathways. Study also confirms that melatonin could act through melatonin receptor coupled to ERK/Akt signaling pathways. This understanding of the mechanism by which melatonin regulates oxidative status in the stressed hepatocytes may initiate the development of novel strategies for hepatic disease therapy in future.


Subject(s)
Animals , Signal Transduction/drug effects , Oxidative Stress/drug effects , Hepatocytes/drug effects , Hydrogen Peroxide/pharmacology , Melatonin/pharmacology , Spectrophotometry , Superoxide Dismutase/drug effects , Catalase/drug effects , Catalase/metabolism , Blotting, Western , NF-kappa B/drug effects , NF-kappa B/metabolism , Reactive Oxygen Species/metabolism , MAP Kinase Signaling System/drug effects , Hepatocytes/metabolism , Proto-Oncogene Proteins c-akt/drug effects , Fishes , Glutathione/drug effects , Glutathione/metabolism , Malondialdehyde/metabolism
4.
Braz. j. med. biol. res ; 51(10): e7476, 2018. graf
Article in English | LILACS | ID: biblio-951713

ABSTRACT

Tubular-interstitial nephritis (TIN) is characterized by tubular cell damage and inflammatory lesions of kidneys. Baicalein (BAI) is a flavonoid compound found in the roots of Scutellaria baicalensis Georgi. The present study was undertaken to explore the anti-inflammatory and anti-oxidative effects of BAI on TIN patients and a lipopolysaccharide (LPS)-induced TIN cell model. The expression levels of interleukin-6 (IL-6), IL-10, and tumor necrosis factor α in serum samples of TIN patients and culture supernatants of renal proximal tubular epithelial cells (RPTECs) were evaluated using enzyme-linked immunosorbent assay. Creatinine clearance was calculated using the Cockcroft-Gault equation. Activities of malondialdehyde, superoxide dismutase, and glutathione peroxidase were also determined. Viability and apoptosis of RPTECs were measured using MTT assay and Guava Nexin assay, respectively. qRT-PCR was performed to determine the expressions of Bax, Bcl-2, nuclear factor kappa B (IκBα), and p65. Protein levels of Bax, Bcl-2, IκBα, p65, c-Jun N-terminal kinase, extracellular regulated protein kinases, and p38 were analyzed using western blotting. We found that BAI reduced inflammation and oxidative stress in vivo and in vitro. Moreover, BAI alleviated the LPS-induced RPTECs viability inhibition and apoptosis enhancement, as well as nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) activation. Phorbol ester, an activator of NF-κB, attenuated the effects of BAI on LPS-induced inflammatory cytokine expressions in RPTECs. In conclusion, BAI had anti-inflammatory and anti-oxidative effects on TIN patients and LPS-induced RPTECs by down-regulating NF-κB and MAPK pathways.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Young Adult , NF-kappa B/metabolism , Flavanones/administration & dosage , Anti-Inflammatory Agents/administration & dosage , Nephritis, Interstitial/drug therapy , Antioxidants/administration & dosage , Enzyme-Linked Immunosorbent Assay , Signal Transduction/drug effects , Down-Regulation , Lipopolysaccharides , NF-kappa B/drug effects , MAP Kinase Signaling System/drug effects
5.
J. appl. oral sci ; 26: e20170231, 2018. graf
Article in English | LILACS, BBO | ID: biblio-893679

ABSTRACT

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.


Subject(s)
Animals , Mice , Gene Expression/drug effects , Calcium/pharmacology , Fibroblast Growth Factor 2/drug effects , Cyclic AMP-Dependent Protein Kinases/drug effects , Mitogen-Activated Protein Kinase 1/drug effects , Dental Papilla/drug effects , Mitogen-Activated Protein Kinase 3/drug effects , Time Factors , Calcium Chloride/pharmacology , Enzyme-Linked Immunosorbent Assay , Cells, Cultured , Blotting, Western , Reproducibility of Results , Fibroblast Growth Factor 2/analysis , Fibroblast Growth Factor 2/genetics , Cyclic AMP-Dependent Protein Kinases/analysis , Mitogen-Activated Protein Kinase 1/analysis , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 3/analysis , Real-Time Polymerase Chain Reaction
6.
Acta cir. bras ; 32(5): 350-358, May 2017. tab, graf
Article in English | LILACS | ID: biblio-837705

ABSTRACT

Abstract Purpose: To investigate the mechanisms by which PD98059 and LY294002 interfere with the abnormal deposition of extracellular matrix regulated by connective tissue growth factor (CTGF) of rat pulmonary artery smooth muscle cells (PASMCs). Methods: Rat PASMCs were cultured and separated into a control group. Real-time fluorescence quantitative PCR was performed to detect the expression of collagen III and fibronectin mRNA. Immunohistochemistry and western blot analyses were performed to detect the expression of collagen III protein. Results: The expression of collagen III and fibronectin mRNA was greater in PASMCs stimulated with CTGF for 48 h, than in the control group. After 72h of stimulation, the expression of collagen III protein in the PASMCs was greater than in the control. The equivalent gene and protein expression of the CPL group were much more significant. Conclusions: CTGF can stimulate the gene expression of collagen III and fibronectin in PASMCs, which may be one of the factors that promote pulmonary vascular remodeling (PVR) under the conditions of pulmonary arterial hypertension (PAH). PD98059 and LY294002 can inhibit the ERK1/2 and PI3K/PKB signaling pathways, respectively, thus interfering with the biological effects of CTGF. This may be a new way to reduce PAH-PVR.


Subject(s)
Animals , Male , Flavonoids/pharmacology , Chromones/pharmacology , Fibronectins/metabolism , MAP Kinase Signaling System/drug effects , Collagen Type III/metabolism , Connective Tissue Growth Factor/pharmacology , Pulmonary Artery/cytology , Gene Expression/drug effects , Cells, Cultured , Gene Expression Regulation , Fibronectins/genetics , Rats, Sprague-Dawley , Phosphatidylinositol 3-Kinases/metabolism , Models, Animal , Collagen Type III/genetics , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Connective Tissue Growth Factor/metabolism
7.
Braz. j. med. biol. res ; 50(7): e5920, 2017. graf
Article in English | LILACS | ID: biblio-951704

ABSTRACT

As an active constituent of the beetle Mylabris used in traditional Chinese medicine, cantharidin is a potent and selective inhibitor of protein phosphatase 2A (PP2A) that plays a crucial role in cell cycle progression, apoptosis, and cell fate. The role and possible mechanisms exerted by cantharidin in cell growth and metastasis of breast cancer were investigated in this study. Cantharidin was found to inhibit cell viability and clonogenic potential in a time- and dose-dependent manner. Cell cycle analysis revealed that cell percentage in G2/M phase decreased, whereas cells in S and G1 phases progressively accumulated with the increasing doses of cantharidin treatment. In a xenograft model of breast cancer, cantharidin inhibited tumor growth in a dose-dependent manner. Moreover, high doses of cantharidin treatment inhibited cell migration in wound and healing assay and downregulated protein levels of major matrix metalloproteinases (MMP)-2 and MMP-9. MDA-MB-231 cell migration and invasion were dose-dependently inhibited by cantharidin treatment. Interestingly, the members of the mitogen-activated protein kinase (MAPK) signaling family were less phosphorylated as the cantharidin dose increased. Cantharidin was hypothesized to exert its anticancer effect through the MAPK signaling pathway. The data of this study also highlighted the possibility of using PP2A as a therapeutic target for breast cancer treatment.


Subject(s)
Humans , Animals , Female , Rabbits , Breast Neoplasms/drug therapy , Cantharidin/pharmacology , Signal Transduction/drug effects , MAP Kinase Signaling System/drug effects , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Drug Screening Assays, Antitumor , Cell Movement/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects
8.
Braz. j. med. biol. res ; 49(3): e5043, Mar. 2016. graf
Article in English | LILACS | ID: lil-771931

ABSTRACT

Ovarian cancer is one of the most common causes of death from gynecologic tumors and is an important public health issue. Ghrelin is a recently discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR). Several studies have identified the protective effects of ghrelin on the mammalian reproductive system. However, little research has been done on the effects of ghrelin on ovarian cancer cells, and the underlying mechanisms of these effects. We sought to understand the potential involvement of mitogen-activated protein kinases (MAPKs) in ghrelin-mediated inhibition of growth of the ovarian line HO-8910. We applied different concentrations of ghrelin and an inhibitor of the ghrelin receptor (D-Lys3-GHRP-6) to HO-8910 cells and observed the growth rate of cells and changes in phosphorylation of the MAPKs ERK1/2, JNK and p38. We discovered that ghrelin-induced apoptosis of HO-8910 cells was though phosphorylated ERK1/2, and that this phosphorylation (as well as p90rsk phosphorylation) was mediated by the GHSR. The ERK1/2 pathway is known to play an essential part in the ghrelin-mediated apoptosis of HO-8910 cells. Hence, our study suggests that ghrelin inhibits the growth of HO-8910 cells primarily through the GHSR/ERK pathway.


Subject(s)
Humans , Female , Middle Aged , Gene Expression Regulation, Neoplastic/genetics , Ghrelin/pharmacology , MAP Kinase Signaling System/physiology , Ovarian Neoplasms/genetics , Apoptosis/drug effects , MAP Kinase Signaling System/drug effects , Oligopeptides/metabolism , Ovarian Neoplasms/metabolism , Phosphorylation/drug effects , Receptors, Ghrelin/antagonists & inhibitors , Receptors, Ghrelin/metabolism , Tumor Cells, Cultured
9.
Acta cir. bras ; 31(2): 143-149, Feb. 2016. graf
Article in English | LILACS | ID: lil-775565

ABSTRACT

PURPOSE: To investigate the anticancer activity of ellagic acid (EA) in U251 human glioblastoma cells and its possible molecular mechanism. METHODS: The cells were treated with EA at various concentrations for different time periods. Cell viability and cell proliferation were detected by cell counting kit-8(CCK-8) assay and live/dead assay respectively. Cell apoptosis were measured with Annexin V-FITC/PI double staining method by flow cytometry and Mitochondrial membrane potential assay separately. Cell cycle was measured with PI staining method by flow cytometry. The expressions of Bcl-2, Survivin, XIAP, Caspase-3, Bax, JNK, p-JNK, ERK1/2, p-ERK1/2, p38, p-p38, DR4, DR5, CHOP and GRP78-related proteins were detected by western blot after EA treatment. RESULTS: Cell viability and proliferation of glioblastoma cells treated with EA were significantly lower than the control group. EA caused robust apoptosis of the glioblastoma cells compared to the control group. EA significantly decreased the proportion at G0/G1 phases of cell cycling accompanied by increased populations at S phase in U251 cell lines. And the expressions of anti-apoptotic proteins were dramatically down-regulated. CONCLUSION: Ellagic acid potentially up-regulated DR4, DR5 and MAP kinases (JNK, ERK1/2 and p38). EA also caused significant increase in the expressions of CHOP and GRP78. Our findings suggest that EA would be beneficial for the treatment of glioblastoma.


Subject(s)
Humans , Apoptosis/drug effects , Glioblastoma/metabolism , Cell Proliferation/drug effects , Ellagic Acid/pharmacology , Cell Survival/drug effects , Apoptosis/physiology , MAP Kinase Signaling System/drug effects , Ellagic Acid/metabolism , Transcription Factor CHOP/drug effects , Transcription Factor CHOP/metabolism , Caspase 3/metabolism , Heat-Shock Proteins/drug effects , Heat-Shock Proteins/metabolism
10.
Yonsei Medical Journal ; : 588-598, 2016.
Article in English | WPRIM | ID: wpr-52542

ABSTRACT

PURPOSE: Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to exhibit antitumor effects in various cancers apart from nasopharyngeal cancer (NPC). NPC exhibits high invasiveness, as well as metastatic potential, and patients continue to suffer from residual, recurrent, or metastatic disease even after chemoradiation therapy. Therefore, new treatment strategies are needed for NPC. In this study, we investigated the efficacy and molecular mechanisms of TA in NPC treatment. MATERIALS AND METHODS: TA-induced cell death was detected by cell viability assay in the NPC cell lines, HNE1 and HONE1. Wound healing assay, invasion assay, and Western blot analysis were used to evaluate the antitumor effects of TA in NPC cell lines. RESULTS: Treatment with TA suppressed the migration and invasion of HNE1 and HONE1 cells. Hepatocyte growth factor enhanced the proliferation, migration, and invasion abilities of NPC cells. This enhancement was successfully inhibited by TA treatment. Treatment with TA increased phosphorylation of p38, and the inhibition of p38 with SB203580 reversed the cytotoxic, anti-invasive, and anti-migratory effects of TA treatment in NPC cell lines. Moreover, inhibition of p38 also reversed the decrease in expression of Slug that was induced by TA treatment. CONCLUSION: In conclusion, the activation of p38 plays a role in mediating TA-induced cytotoxicity and inhibition of invasion and migration via down-regulation of Slug.


Subject(s)
Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Down-Regulation , Gastropoda , Gene Expression Regulation, Neoplastic/drug effects , Hepatocyte Growth Factor/metabolism , Humans , Imidazoles , MAP Kinase Signaling System/drug effects , Nasopharyngeal Neoplasms/drug therapy , Neoplasm Invasiveness/prevention & control , Phosphorylation/drug effects , Pyridines , ortho-Aminobenzoates/pharmacology
11.
Salud colect ; 11(1): 99-114, ene.-mar. 2015. ilus, tab
Article in Spanish | LILACS | ID: lil-746687

ABSTRACT

El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.


In 2008, Brazil's Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMF's ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.


Subject(s)
Animals , Rats , Apoptosis/genetics , Gene Expression Regulation, Enzymologic/genetics , Heme/deficiency , Nerve Degeneration/genetics , Neurons/metabolism , Porphyrias/complications , Apoptosis/drug effects , Caspases/drug effects , Caspases/metabolism , Cell Survival/drug effects , Cell Survival/genetics , Collagen Type XI/drug effects , Collagen Type XI/metabolism , Cyclic AMP Response Element-Binding Protein/drug effects , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Down-Regulation/drug effects , Down-Regulation/physiology , Enzyme Inhibitors , Gene Expression Regulation, Enzymologic/drug effects , Heptanoates , Heme/biosynthesis , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Membrane Proteins/drug effects , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Cell Adhesion Molecules/drug effects , Neural Cell Adhesion Molecules/genetics , Neural Cell Adhesion Molecules/metabolism , Neurons/drug effects , Neurons/pathology , Poly(ADP-ribose) Polymerases , Porphyrias/metabolism , Porphyrias/physiopathology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , RNA-Binding Proteins/drug effects , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , SMN Complex Proteins , Up-Regulation/drug effects , Up-Regulation/physiology , Vesicular Transport Proteins/drug effects , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism
12.
Biol. Res ; 48: 1-8, 2015. ilus, graf
Article in English | LILACS | ID: biblio-950833

ABSTRACT

BACKGROUND: Human amnion mesenchymal cells (hAMCs), isolated from the amniotic membrane of human placenta, are a unique population of mesenchymal stem cells. Recent studies demonstrated that hAMCs could inhibit the activities and functions of several immune cells. However, their effect on inflammatory macrophages is largely unknown. This study investigated the effect of hAMCs on expression of inflammatory cytokines and mitogen-activated protein kinases (MAPKs)/NF-kB pathway in human THP-1 macrophages induced by lipopolysaccharide (LPS). RESULTS: The levels of TNF-α and IL-1ß secreted by LPS- stimulated THP-1 cells were increased significantly compared with those in the control group. After co-culture with different numbers of hAMCs, the levels of TNF-α and IL-1ß in LPS-stimulated THP-1 cells were significantly reduced compared with the LPS group. The mRNA expression of TNF-α and IL-1ß were also markedly inhibited. Moreover, treating LPS-stimulated THP-1 cells with hAMCs supernatants could also suppress TNF-α and IL-1ß production in THP-1 cells. Important signaling pathways involved in the production of TNF-α and IL-1ß were affected by hAMCs co-culture: hAMCs remarkably suppressed NF-kB activation and down-regulated the phosphorylation of ERK and JNK in LPS- stimulated THP-1 cells. CONCLUSIONS: Human amnion mesenchymal cells inhibited the production of TNF-α and IL-1ß secreted by LPS-stimulated THP-1 cells, partly through the suppression of NF-kB activation and ERK and JNK phosphorylation.


Subject(s)
Humans , Lipopolysaccharides/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Interleukin-1beta/biosynthesis , Mesenchymal Stem Cells/physiology , Amnion/cytology , Macrophages/metabolism , Tumor Necrosis Factor-alpha/drug effects , MAP Kinase Signaling System/drug effects , Interleukin-1beta/drug effects
13.
Article in English | WPRIM | ID: wpr-70183

ABSTRACT

Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.


Subject(s)
Acer/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival , Extracellular Signal-Regulated MAP Kinases/metabolism , Hep G2 Cells , Human Umbilical Vein Endothelial Cells/drug effects , Humans , MAP Kinase Signaling System/drug effects , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/metabolism , Neoplasm Invasiveness/pathology , Neovascularization, Pathologic/drug therapy , Nitric Oxide Synthase Type III/metabolism , Phosphorylation/drug effects , Plant Extracts/pharmacology , Rats , Rats, Sprague-Dawley , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors
14.
Braz. j. med. biol. res ; 47(9): 773-779, 09/2014. graf
Article in English | LILACS | ID: lil-719311

ABSTRACT

The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.


Subject(s)
Animals , Rats , Anti-Inflammatory Agents/therapeutic use , Glutamic Acid/toxicity , Glycyrrhizic Acid/therapeutic use , Neuroprotective Agents/therapeutic use , /drug effects , Signal Transduction/drug effects , Apoptosis/drug effects , /isolation & purification , Cell Differentiation/drug effects , Cell Survival/drug effects , Chromones/pharmacology , Cytochromes c/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , MAP Kinase Signaling System/drug effects , Mitochondria/drug effects , Morpholines/pharmacology , /classification , /cytology , Proto-Oncogene Proteins c-akt/drug effects , /isolation & purification , /isolation & purification
15.
Article in English | WPRIM | ID: wpr-191059

ABSTRACT

Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-kappaB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-kappaB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.


Subject(s)
Active Transport, Cell Nucleus/drug effects , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Hyaluronic Acid/pharmacology , Interleukin-6/pharmacology , Keratinocytes/metabolism , MAP Kinase Signaling System/drug effects , NF-kappa B/metabolism , Phosphorylation/drug effects , Protein Transport/drug effects , Wound Healing , p38 Mitogen-Activated Protein Kinases/metabolism
16.
Korean Journal of Urology ; : 542-550, 2014.
Article in English | WPRIM | ID: wpr-156581

ABSTRACT

PURPOSE: Transforming growth factor beta1 (TGF-beta1) inhibits the growth of bladder cancer cells and this effect is prominent and constant in 253J bladder cancer cells. We performed a microarray analysis to search for genes that were altered after TGF-beta1 treatment to understand the growth inhibitory action of TGF-beta1. MATERIALS AND METHODS: 253J bladder cancer cells were exposed to TGF-beta1 and total RNA was extracted at 6, 24, and 48 hours after exposure. The RNA was hybridized onto a human 22K oligonucleotide microarray and the data were analyzed by using GeneSpring 7.1. RESULTS: In the microarray analysis, a total of 1,974 genes showing changes of more than 2.0 fold were selected. The selected genes were further subdivided into five highly cohesive clusters with high probability according to the time-dependent expression pattern. A total of 310 genes showing changes of more than 2.0 fold in repeated arrays were identified by use of simple t-tests. Of these genes, those having a known function were listed according to clusters. Microarray analysis showed increased expression of molecules known to be related to Smad-dependent signal transduction, such as SARA and Smad4, and also those known to be related to the mitogen-activated protein kinase (MAPK) pathway, such as MAPKK1 and MAPKK4. CONCLUSIONS: A list of genes showing significantly altered expression profiles after TGF-beta1 treatment was made according to five highly cohesive clusters. The data suggest that the growth inhibitory effect of TGF-beta1 in bladder cancer may occur through the Smad-dependent pathway, possibly via activation of the extracellular signal-related kinase 1 and Jun amino-terminal kinases Mitogen-activated protein kinase pathway.


Subject(s)
Antineoplastic Agents/pharmacology , Cluster Analysis , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Genes, Neoplasm , Humans , MAP Kinase Signaling System/drug effects , Neoplasm Proteins/genetics , Oligonucleotide Array Sequence Analysis/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Signal Transduction/drug effects , Smad Proteins/genetics , Transforming Growth Factor beta1/pharmacology , Tumor Cells, Cultured/drug effects , Urinary Bladder Neoplasms/genetics
17.
Article in English | WPRIM | ID: wpr-73119

ABSTRACT

Cyclooxygenase-2 (COX-2) is an important enzyme in inflammation. In this study, we investigated the underlying molecular mechanism of the synergistic effect of rottlerin on interleukin1beta (IL-1beta)-induced COX-2 expression in MDA-MB-231 human breast cancer cell line. Treatment with rottlerin enhanced IL-1beta-induced COX-2 expression at both the protein and mRNA levels. Combined treatment with rottlerin and IL-1beta significantly induced COX-2 expression, at least in part, through the enhancement of COX-2 mRNA stability. In addition, rottlerin and IL-1beta treatment drove sustained activation of p38 Mitogen-activated protein kinase (MAPK), which is involved in induced COX-2 expression. Also, a pharmacological inhibitor of p38 MAPK (SB 203580) and transient transfection with inactive p38 MAPK inhibited rottlerin and IL-1beta-induced COX-2 upregulation. However, suppression of protein kinase C delta (PKC delta) expression by siRNA or overexpression of dominant-negative PKC delta (DN-PKC-delta) did not abrogate the rottlerin plus IL-1beta-induced COX-2 expression. Furthermore, rottlerin also enhanced tumor necrosis factor-alpha (TNF-alpha), phorbol myristate acetate (PMA), and lipopolysaccharide (LPS)-induced COX-2 expression. Taken together, our results suggest that rottlerin causes IL-1beta-induced COX-2 upregulation through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells.


Subject(s)
Acetophenones/pharmacology , Benzopyrans/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cyclooxygenase 2/genetics , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Interleukin-1beta/immunology , MAP Kinase Signaling System/drug effects , Mallotus Plant/chemistry , NF-kappa B/immunology , Protein Kinase C-delta/antagonists & inhibitors , Reactive Oxygen Species/immunology , p38 Mitogen-Activated Protein Kinases/immunology
18.
Article in English | WPRIM | ID: wpr-131300

ABSTRACT

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.


Subject(s)
Animals , Arginine , Cell Dedifferentiation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Elongation Factor 2 Kinase/metabolism , Fibroblast Growth Factor 2/metabolism , Fibroblasts/metabolism , Flavonoids/pharmacology , MAP Kinase Signaling System/drug effects , Methylation , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Myofibroblasts/pathology , NIH 3T3 Cells , Protein Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/metabolism , RNA, Small Interfering/genetics
19.
Article in English | WPRIM | ID: wpr-131297

ABSTRACT

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.


Subject(s)
Animals , Arginine , Cell Dedifferentiation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Elongation Factor 2 Kinase/metabolism , Fibroblast Growth Factor 2/metabolism , Fibroblasts/metabolism , Flavonoids/pharmacology , MAP Kinase Signaling System/drug effects , Methylation , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Myofibroblasts/pathology , NIH 3T3 Cells , Protein Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/metabolism , RNA, Small Interfering/genetics
20.
Braz. j. med. biol. res ; 43(9): 828-836, Sept. 2010. ilus
Article in English | LILACS | ID: lil-556864

ABSTRACT

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.


Subject(s)
Animals , Mice , Cell Proliferation/drug effects , Endothelins/pharmacology , Rhodopsin/drug effects , Vasoconstrictor Agents/pharmacology , Viper Venoms/pharmacology , Cell Line , Gene Expression Regulation , Goldfish , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Polymerase Chain Reaction , Protein Kinase C/drug effects , Protein Kinase C/genetics , RNA, Messenger/drug effects , RNA, Messenger/genetics , Rhodopsin/genetics , Rhodopsin/metabolism
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