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1.
Biol. Res ; 52: 41, 2019. tab, graf
Article in English | LILACS | ID: biblio-1019505

ABSTRACT

BACKGROUND: Di-N-butyl-phthalate (DBP) is an endocrine disrupting substance. We investigated the adverse effect of DBP on testis of male rat and reveal its potential mechanism of MAPK signaling pathway involved this effect in vivo and in vitro. Gonadal hormone, sperm quality, morphological change and the activation status of JNK, ERK1/2 and p38 was determined in vivo. Primary Sertoli cell was established and cultivated with JNK, ERK1/2 inhibitors, then determine the cell viability, apoptosis and the expression of p-JNK, p-ERK1/2. Data in this study were presented as mean ± SD and determined by one-way analysis of variance (ANOVA) followed by Bonferroni's test. Difference was considered statistically significant at P < 0.05. RESULTS: In vivo experiment, DBP impaired the normal structure of testicular tissue, reduced testosterone levels in blood serum, decreased sperm count and increased sperm abnormality, p-ERK1/2 and p-JNK in rat testicular tissue increased in a dose-dependent manner. In vitro studies, DBP could decrease the viability of Sertoli cells and increase p-ERK1/2 and p-JNK. Cell apoptosis in SP600125 + DBP group was significantly lower than in DBP group (P < 0.05). p-JNK was not significantly decreased in SP600125 + DBP group, while p-ERK1/2 was significantly decreased in U0126 + DBP group. CONCLUSIONS: These results suggest that DBP can lead to testicular damage and the activation of ERK1/2 and JNK pathways, the JNK signaling pathway may be primarily associated with its effect.


Subject(s)
Animals , Male , Rats , Testis/injuries , Testis/metabolism , Signal Transduction/physiology , Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System/physiology , JNK Mitogen-Activated Protein Kinases/metabolism , Dibutyl Phthalate/pharmacology , Testis/drug effects , Rats, Sprague-Dawley , Mitogen-Activated Protein Kinases/physiology , JNK Mitogen-Activated Protein Kinases/physiology
2.
Braz. j. med. biol. res ; 51(2): e6520, 2018. tab, graf
Article in English | LILACS | ID: biblio-889032

ABSTRACT

Multiple growth factors can be administered to mimic the natural process of bone healing in bone tissue engineering. We investigated the effects of sequential release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone regeneration. To improve the double emulsion/solvent evaporation technique, VEGF was encapsulated in PELA microcapsules, to which BMP-2 was attached. The scaffold (BMP-2/PELA/VEGF) was then fused to these microcapsules using the dichloromethane vapor method. The bioactivity of the released BMP-2 and VEGF was then quantified in rat mesenchymal stem cells (rMSCs). Immunoblotting analysis showed that BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblasts via the MAPK and Wnt pathways. Osteoblast differentiation was assessed through alkaline phosphatase expression. When compared with simple BMP-2 plus VEGF group and pure PELA group, osteoblast differentiation in BMP-2/PELA/VEGF group significantly increased. An MTT assay indicated that BMP-2-loaded PELA scaffolds had no adverse effects on cell activity. BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblast via the ERK1/2 and Wnt pathways. Our findings indicate that the sequential release of BMP-2 and VEGF from PELA microcapsule-based scaffolds is a promising approach for the treatment of bone defects.


Subject(s)
Animals , Rabbits , Rats , Polyesters/pharmacology , Polyethylene Glycols/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Vascular Endothelial Growth Factors/metabolism , Tissue Scaffolds , Bone Morphogenetic Protein 2/metabolism , Mesenchymal Stem Cells/cytology , Time Factors , Bone Regeneration , Signal Transduction/physiology , Cells, Cultured , Models, Animal , Cell Proliferation , beta Catenin/physiology , Nanoparticles , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Wnt Signaling Pathway/physiology
3.
Clinics ; 73(supl.1): e466s, 2018. graf
Article in English | LILACS | ID: biblio-952823

ABSTRACT

Protein tyrosine phosphatases have long been considered key regulators of biological processes and are therefore implicated in the origins of various human diseases. Heterozygosity, mutations, deletions, and the complete loss of some of these enzymes have been reported to cause neurodegenerative diseases, autoimmune syndromes, genetic disorders, metabolic diseases, cancers, and many other physiological imbalances. Vaccinia H1-related phosphatase, also known as dual-specificity phosphatase 3, is a protein tyrosine phosphatase enzyme that regulates the phosphorylation of the mitogen-activated protein kinase signaling pathway, a central mediator of a diversity of biological responses. It has been suggested that vaccinia H1-related phosphatase can act as a tumor suppressor or tumor-promoting phosphatase in different cancers. Furthermore, emerging evidence suggests that this enzyme has many other biological functions, such as roles in immune responses, thrombosis, hemostasis, angiogenesis, and genomic stability, and this broad spectrum of vaccinia H1-related phosphatase activity is likely the result of its diversity of substrates. Hence, fully identifying and characterizing these substrate-phosphatase interactions will facilitate the identification of pharmacological inhibitors of vaccinia H1-related phosphatase that can be evaluated in clinical trials. In this review, we describe the biological processes mediated by vaccinia H1-related phosphatase, especially those related to genomic stability. We also focus on validated substrates and signaling circuitry with clinical relevance in human diseases, particularly oncogenesis.


Subject(s)
Humans , Dual Specificity Phosphatase 3/physiology , Neoplasms/enzymology , Signal Transduction , Survival Analysis , Mitogen-Activated Protein Kinases/metabolism , Neoplasms/mortality
4.
Article in English | WPRIM | ID: wpr-30206

ABSTRACT

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-theta (PKCtheta) and protein kinase C-beta (PKCbeta) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCtheta and PKCbeta stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox-/- mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.


Subject(s)
Animals , Aorta/cytology , Cell Line , Cell Proliferation , Cells, Cultured , Humans , Lipoproteins, LDL/metabolism , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , NADPH Oxidases/metabolism , Phosphorylation , Protein Kinase C/metabolism , Signal Transduction , Superoxides/metabolism
5.
Article in English | WPRIM | ID: wpr-160903

ABSTRACT

Trichomonas vaginalis is a flagellated protozoan parasite that causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. Mast cells have been reported to be predominant in vaginal smears and vaginal walls of patients infected with T. vaginalis. Mitogen-activated protein kinase (MAPK), activated by various stimuli, have been shown to regulate the transcriptional activity of various cytokine genes in mast cells. In this study, we investigated whether MAPK is involved in ROS generation and exocytotic degranulation in HMC-1 cells induced by T. vaginalis-derived secretory products (TvSP). We found that TvSP induces the activation of MAPK and NADPH oxidase in HMC-1 cells. Stimulation with TvSP induced phosphorylation of MAPK and p47phox in HMC-1 cells. Stimulation with TvSP also induced up-regulation of CD63, a marker for exocytosis, along the surfaces of human mast cells. Pretreatment with MAPK inhibitors strongly inhibited TvSP-induced ROS generation and exocytotic degranulation. Finally, our results suggest that TvSP induces intracellular ROS generation and exocytotic degranulation in HMC-1 via MAPK signaling.


Subject(s)
Cell Degranulation , Cell Line , Exocytosis , Humans , Mast Cells/drug effects , Mitogen-Activated Protein Kinases/metabolism , Reactive Oxygen Species/metabolism , Trichomonas vaginalis/metabolism , Virulence Factors/metabolism
6.
Article in English | WPRIM | ID: wpr-210689

ABSTRACT

Traumatic brain injury (TBI) is associated with poor neurological outcome, including necrosis and brain edema. In this study, we investigated whether agmatine treatment reduces edema and apoptotic cell death after TBI. TBI was produced by cold injury to the cerebral primary motor cortex of rats. Agmatine was administered 30 min after injury and once daily until the end of the experiment. Animals were sacrificed for analysis at 1, 2, or 7 days after the injury. Various neurological analyses were performed to investigate disruption of the blood-brain barrier (BBB) and neurological dysfunction after TBI. To examine the extent of brain edema after TBI, the expression of aquaporins (AQPs), phosphorylation of mitogen-activated protein kinases (MAPKs), and nuclear translocation of nuclear factor-kappaB (NF-kappaB) were investigated. Our findings demonstrated that agmatine treatment significantly reduces brain edema after TBI by suppressing the expression of AQP1, 4, and 9. In addition, agmatine treatment significantly reduced apoptotic cell death by suppressing the phosphorylation of MAPKs and by increasing the nuclear translocation of NF-kappaB after TBI. These results suggest that agmatine treatment may have therapeutic potential for brain edema and neural cell death in various central nervous system diseases.


Subject(s)
Active Transport, Cell Nucleus/drug effects , Agmatine/therapeutic use , Animals , Apoptosis/drug effects , Aquaporins/metabolism , Blood-Brain Barrier/physiopathology , Brain Edema/drug therapy , Brain Injuries/pathology , Male , Mitogen-Activated Protein Kinases/metabolism , Motor Cortex/pathology , NF-kappa B/metabolism , Phosphorylation/drug effects , Rats , Rats, Sprague-Dawley
7.
Article in English | WPRIM | ID: wpr-188235

ABSTRACT

OBJECTIVES: With recent advances in nanoparticle manufacturing and applications, potential exposure to nanoparticles in various settings is becoming increasing likely. No investigation has yet been performed to assess whether respiratory tract exposure to cerium oxide (CeO2) nanoparticles is associated with alterations in protein signaling, inflammation, and apoptosis in rat lungs. METHODS: Specific-pathogen-free male Sprague-Dawley rats were instilled with either vehicle (saline) or CeO2 nanoparticles at a dosage of 7.0 mg/kg and euthanized 1, 3, 14, 28, 56, or 90 days after exposure. Lung tissues were collected and evaluated for the expression of proteins associated with inflammation and cellular apoptosis. RESULTS: No change in lung weight was detected over the course of the study; however, cerium accumulation in the lungs, gross histological changes, an increased Bax to Bcl-2 ratio, elevated cleaved caspase-3 protein levels, increased phosphorylation of p38 MAPK, and diminished phosphorylation of ERK-1/2-MAPK were detected after CeO2 instillation (p<0.05). CONCLUSIONS: Taken together, these data suggest that high-dose respiratory exposure to CeO2 nanoparticles is associated with lung inflammation, the activation of signaling protein kinases, and cellular apoptosis, which may be indicative of a long-term localized inflammatory response.


Subject(s)
Animals , Apoptosis/drug effects , Caspase 3/metabolism , Cerium/chemistry , Inflammation , Lung/drug effects , Male , Metal Nanoparticles/chemistry , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , bcl-2-Associated X Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
8.
Braz. j. microbiol ; 44(4): 1241-1250, Oct.-Dec. 2013. ilus, graf, tab
Article in English | LILACS | ID: lil-705264

ABSTRACT

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.


Subject(s)
Colletotrichum/growth & development , Colletotrichum/pathogenicity , Fungal Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Spores, Fungal/growth & development , Cloning, Molecular , DNA, Fungal/chemistry , DNA, Fungal/genetics , Fungal Proteins/genetics , Gene Deletion , Hyphae/growth & development , Molecular Sequence Data , Mutagenesis, Insertional , Mangifera/microbiology , Mitogen-Activated Protein Kinases/genetics , Open Reading Frames , Promoter Regions, Genetic , Plant Diseases/microbiology , Sequence Analysis, DNA , Virulence
9.
Article in English | WPRIM | ID: wpr-199828

ABSTRACT

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.


Subject(s)
Acute-Phase Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Lipopolysaccharide Receptors/metabolism , Bone Marrow Cells/cytology , CD8-Positive T-Lymphocytes/immunology , Carrier Proteins/metabolism , Cell Differentiation/drug effects , Cell Nucleus/drug effects , Cell Proliferation/drug effects , Cytokines/biosynthesis , Dendritic Cells/cytology , Enzyme Activation/drug effects , Lymphocyte Activation/drug effects , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/metabolism , Neoplasms/immunology , Pectins/pharmacology , Phenotype , Protein Transport/drug effects , Receptors, Chemokine/metabolism , Signal Transduction/drug effects , T-Lymphocytes, Cytotoxic/cytology , Toll-Like Receptor 4/agonists
10.
Article in English | WPRIM | ID: wpr-152458

ABSTRACT

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO2) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/alpha-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proteomics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-met/metabolism , Quinazolines/pharmacology
11.
Article in English | WPRIM | ID: wpr-35840

ABSTRACT

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.


Subject(s)
Animals , Apoptosis , Embryo, Mammalian/cytology , Fibroblasts/enzymology , Gene Expression Regulation , Intracellular Space/metabolism , Mice , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Oxidative Stress/genetics , Reactive Oxygen Species/metabolism , Signal Transduction , Transcription Factors/deficiency
12.
J. appl. oral sci ; 20(2): 128-138, Mar.-Apr. 2012. ilus, tab
Article in English | LILACS | ID: lil-626410

ABSTRACT

Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.


Subject(s)
Humans , Inflammation Mediators/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/therapeutic use , Molecular Targeted Therapy/methods , Periodontal Diseases/therapy , Signal Transduction/drug effects , Biofilms , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Intracellular Signaling Peptides and Proteins/immunology , Janus Kinases/immunology , Janus Kinases/metabolism , Mitogen-Activated Protein Kinases/immunology , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/immunology , NF-kappa B/metabolism , Periodontal Diseases/etiology , Periodontal Diseases/immunology , STAT Transcription Factors/immunology , STAT Transcription Factors/metabolism
13.
Biocell ; 35(3): 71-79, Dec. 2011. tab
Article in English | LILACS | ID: lil-653213

ABSTRACT

Apigenin, a nonmutagenic flavonoid, has been shown to possess free radical scavenging activities, anticarcinogenic properties, antioxidant and anti-inflammatory effects. Recently, apigenin was reported to cause gastric relaxation in murine. To assess possible effects of apigenin on migration of bladder smooth muscle (SM) cell, we isolated SM cells from peri-cancer tissue of human bladder and established a cell model that was capable to overexpress transiently MEKK1 (MEK kinase 1). Results showed that overexpression of active human MEKK1 by adenoviruses infection induced migration of human bladder smooth muscle (hBSM) cells and phosphorylation of MAPKs, ERK, JNK and p38, which are the downstream molecules of MEKK1. Then, hBSM cell overexpressing MEKK1 were exposed to apigenin (50 microM). Our data indicated that apigenin inhibited significantly activation/phosphorylation of MAPKs and migration of hBSM cells induced by MEKK1 overexpression. Besides, apigenin inhibited actin polymerization, which underlines muscle contraction and cell migration. The results suggest that apigenin inhibits activation of MAPKs and thereby the cell migration. The mechanism might be that apigenin blocks signal transmission from MEKK1 to MAPKs.


Subject(s)
Humans , Animals , Rats , Apigenin/pharmacology , Flavonoids , MAP Kinase Kinase Kinase 1/metabolism , Myocytes, Smooth Muscle , Myocytes, Smooth Muscle/metabolism , Cell Movement , Urinary Bladder , Urinary Bladder/metabolism , Cells, Cultured , Phosphorylation , Immunoblotting , MAP Kinase Kinase Kinase 1/genetics , Mitogen-Activated Protein Kinases/metabolism
14.
Article in English | WPRIM | ID: wpr-227749

ABSTRACT

Epidermal growth factor receptor (EGFR)-targeted therapies have been effective in some cancers, but not in hepatocellular carcinoma (HCC). The aim of this study was to investigate the drug potential to overcome multi-drug resistance in HCC cells. Thirteen drug-sensitive HCC cells were assessed using the CCK-8 assay. G0-G1 arrest was measured by FACS. Western blot analysis was used to detect the key enzymes in both the Ras/Raf and PI3K pathways. When establishing the IC50 of HCC to several drugs, including EKB-569, sorafenib, erlotinib, gefitinib, pazopanib, and brivanib, SK-Hep1 cells treated with EKB-569 have shown the highest (72.8%-86.4%) G0-G1 arrest and decreased the phosphorylation of AKT and ERK at the protein level. We found that EKB-569 had higher efficacy in HCC, compared to first generation, reversible EGFR-TK inhibitors. Furthermore, the combination of sorafenib and EKB-569 showed a synergistic effect to inhibit proliferation of SNU-475, previously the most resistant cell to EGFR-TKIs. Therefore, novel EKB-569 in combination with sorafenib may be able to overcome HCC resistance to EGFR-TK inhibitors.


Subject(s)
Aminoquinolines/pharmacology , Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzenesulfonates/pharmacology , Carcinoma, Hepatocellular/drug therapy , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Drug Synergism , Humans , Liver Neoplasms/drug therapy , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Pyridines/pharmacology , ErbB Receptors/antagonists & inhibitors
15.
Mem. Inst. Oswaldo Cruz ; 105(3): 269-277, May 2010. ilus, graf
Article in English | LILACS | ID: lil-547311

ABSTRACT

In this paper, we provide evidence that both the mRNA and protein levels of the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CDK-interacting protein 1 (Cip1) increase upon infection of A431 cells with Vaccinia virus (VACV). In addition, the VACV growth factor (VGF) seems to be required for the gene expression because infection carried out with the mutant virus VACV-VGF- revealed that this strain was unable to stimulate its transcription. Our findings are also consistent with the notion that the VGF-mediated change in p21WAF1/Cip1 expression is dependent on tyrosine kinase pathway(s) and is partially dependent on mitogen-activated protein kinase/extracellular-signal regulated kinase 1/2. We believe that these pathways are biologically significant because VACV replication and dissemination was drastically affected when the infection was carried out in the presence of the relevant pharmacological inhibitors.


Subject(s)
Humans , /metabolism , Vaccinia virus/physiology , Cell Line, Tumor , /genetics , Gene Expression Regulation, Viral/genetics , Mitogen-Activated Protein Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/genetics , Virus Replication/genetics
16.
Braz. j. med. biol. res ; 43(5): 500-505, May 2010. tab, ilus
Article in English | LILACS | ID: lil-546327

ABSTRACT

The regulatory function of á1B-adrenoceptors in mammalian heart homeostasis is controversial. The objective of the present study was to characterize the expression/activity of key proteins implicated in cardiac calcium handling (Na+/K+-ATPase and Ca2+-ATPases) and growth (ERK1/2, JNK1/2 and p38) in mice with cardiac-selective overexpression of constitutively active mutant á1B-adrenoceptor (CAMá1B-AR), which present a mild cardiac hypertrophy phenotype. Immunoblot assays showed that myocardial plasma membrane Ca2+-ATPase (PMCA) expression was increased by 30 percent in CAMá1B-AR mice (N = 6, P < 0.05), although there was no change in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) expression. Moreover, total Ca2+-ATPase activity was not modified, but a significant increase in the activity of the thapsigargin-resistant (PMCA) to thapsigargin-sensitive (SERCA) ratio was detected. Neither Na+/K+-ATPase activity nor the expression of á1 and á2 subunit isoforms was changed in CAMá1B-AR mouse hearts. Moreover, immunoblot assays did not provide evidence for an enhanced activation of the three mitogen-activated protein kinases studied in this stage of hypertrophy. Therefore, these findings indicate that chronic cardiac á1B-AR activation in vivo led to mild hypertrophy devoid of significant signs of adaptive modifications concerning primary intracellular calcium control and growth-related proteins, suggesting a minor pathophysiological role of this adrenergic receptor in mouse heart at this stage of development.


Subject(s)
Animals , Male , Mice , Adenosine Triphosphatases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Myocardium/enzymology , Receptors, Adrenergic, alpha-1/metabolism , Calcium Signaling/physiology , Mice, Transgenic , Up-Regulation
17.
Article in English | WPRIM | ID: wpr-122577

ABSTRACT

Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6alpha, and the phosphorylation of eukaryotic initiation factor-2alpha and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.


Subject(s)
Animals , Apoptosis , Brefeldin A/pharmacology , Cell Line , Cytoprotection , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/drug effects , Flavanones/pharmacology , Heat-Shock Proteins/biosynthesis , Hippocampus/cytology , Membrane Potential, Mitochondrial/drug effects , Mice , Mitogen-Activated Protein Kinases/metabolism , Neurons/drug effects , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Thapsigargin/pharmacology , Transcription Factor CHOP/biosynthesis , Transcription Factors/metabolism , Unfolded Protein Response/drug effects
18.
Article in English | WPRIM | ID: wpr-203595

ABSTRACT

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.


Subject(s)
Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Line , Cell Lineage/drug effects , Cells, Cultured , Down-Regulation/drug effects , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Endoderm/cytology , Enzyme Activation/drug effects , Free Radical Scavengers/pharmacology , Humans , Mesoderm/cytology , Mitogen-Activated Protein Kinases/metabolism , Pluripotent Stem Cells/cytology , Reactive Oxygen Species/metabolism , Up-Regulation/drug effects
19.
Article in English | WPRIM | ID: wpr-202554

ABSTRACT

Glycosphingolipids including gangliosides play important regulatory roles in cell proliferation and differentiation. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs (shRNAs) to evaluate the roles of glycosphingolipids in proliferation and neural differentiation of mouse embryonic stem cells (mESCs). HPTLC/immunofluorescence analyses of shRNA-transfected mESCs revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM3 and GD3. Furthermore, MTT and Western blot/immunofluorescence analyses demonstrated that inhibition of the Ugcg expression in mESCs resulted in decrease of cell proliferation (P < 0.05) and decrease of activation of the ERK1/2 (P < 0.05), respectively. To further investigate the role of glycosphingolipids in neural differentiation, the embryoid bodies formed from Ugcg-shRNA transfected mESCs were differentiated into neural cells by treatment with retinoic acid. We found that inhibition of Ugcg expression did not affect embryoid body (EB) differentiation, as judged by morphological comparison and expression of early neural precursor cell marker, nestin, in differentiated EBs. However, RT-PCR/immunofluorescence analyses showed that expression of microtubule- associated protein 2 (MAP-2) for neurons and glial fibrillary acidic protein (GFAP) for glial cells was decreased in neural cells differentiated from the shRNA-transfected mESCs. These results suggest that glycosphingolipids are involved in the proliferation of mESCs through ERK1/2 activation, and that glycosphingolipids play roles in differentiation of neural precursor cells derived from mESCs.


Subject(s)
Animals , Cell Proliferation , Cells, Cultured , Down-Regulation , Embryonic Stem Cells/cytology , Glucosyltransferases/genetics , Glycosphingolipids/genetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Neurogenesis , Neurons/cytology , RNA, Messenger/genetics
20.
Indian J Pathol Microbiol ; 2008 Apr-Jun; 51(2): 167-71
Article in English | IMSEAR | ID: sea-73881

ABSTRACT

Renal oncocytoma, conventional RCC (granular cell type) and chromophobe RCC have different prognosis. Sometimes differentiation between them is difficult in HandE slides. In a 5-year study of 128 renal tumors, we selected 76 cases [30 conventional RCC (CRCC), 16 papillary RCC, 21 chromophobe RCC (ChRCC), 8 oncocytoma, 1 collecting duct carcinoma (cdc)] and staining with Hale's colloidal iron, CK7, CK8, CK18, CK19, CK20, Vimentin, EMA, CD10 and RCC marker were done. No significant difference was seen between renal tumor subtypes with CK8, CK18, CK19, CK20 and EMA. The most useful markers were Vimentin, CK7, CD10, RCC marker and Hale's colloidal iron. Hale's colloidal iron staining with diffuse reticular fine cytoplasmic pattern was present in ChRCCs, but was absent in other subtypes and oncocytomas. Vimentin, CK7, CD10, RCC marker and Hale's colloidal iron can be used for the differential diagnosis of problematic epithelial tumors of kidney (CRCC, ChRCC and oncocytoma) - i.e. ChRCC: Vimentin, CD10 and RCC marker - negative, CK7 - positive and positive diffuse fine reticular cytoplasmic pattern of Hale's colloidal iron; oncocytoma: Vimentin, CK7, RCC marker and CD10 - negative and Hale's colloidal iron - negative; CRCC: CK7 - negative, Vimentin, CD10 and RCC marker - positive and Hale's colloidal iron - negative.


Subject(s)
Adenoma, Oxyphilic/diagnosis , Adult , Aged , Aged, 80 and over , Antigens, Neoplasm/metabolism , Carcinoma, Renal Cell/diagnosis , Diagnosis, Differential , Female , Humans , Keratin-7/metabolism , Kidney Neoplasms/diagnosis , Male , Middle Aged , Mitogen-Activated Protein Kinases/metabolism , Neprilysin/metabolism , Biomarkers, Tumor/metabolism , Vimentin/metabolism
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