RESUMO
Patients with advanced stage ovarian clear cell carcinoma (OCCC) have a poor prognosis due to resistance to conventional platinum chemotherapy. Recent studies have demonstrated that PI3K/AKT/mTOR and ERK1/2 signaling pathways are involved in this chemoresistance. Progranulin (PGRN) overexpression contributes to cisplatin resistance of epithelial ovarian cancer cell lines. Also, PGRN expression is regulated by AKT/mTOR and ERK1/2 signaling pathways in different cell types. Thus, the present study was designed to identify if PGRN expression is regulated by AKT, mTOR, and ERK1/2 signaling pathways in the OCCC cell line TOV-21G. Cultured TOV-21G cells were incubated with different concentrations of pharmacological cell signaling inhibitors. PGRN expression and phosphorylation of ERK1/2, AKT, and mTOR were assessed by Western blotting. Inhibition of AKT, mTOR, and ERK1/2 significantly reduced PGRN expression. Cell viability was not affected, while cell proliferation significantly decreased with all inhibitors used in this study. These observations demonstrated that inhibition of PI3K/AKT/mTOR and ERK1/2 signaling pathways reduces PGRN expression in TOV-21G cells. Thus, PGRN could be considered as a candidate for explaining the high resistance to platinum-based treatment and a potential biomarker for therapy response to cell signaling inhibitors in patients with OCCC.
Assuntos
Antineoplásicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Ovarianas/metabolismo , Progranulinas , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Progranulinas/análise , Progranulinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The Mitogen-Activated Protein Kinase (MAPK) signaling pathways constitute one of the most important and evolutionarily conserved mechanisms for the perception of extracellular information in all the eukaryotic organisms. The MAPK pathways are involved in the transfer to the cell of the information perceived from extracellular stimuli, with the final outcome of activation of different transcription factors that regulate gene expression in response to them. In all species of fungi, the MAPK pathways have important roles in their physiology and development; e.g. cell cycle control, mating, morphogenesis, response to different stresses, resistance to UV radiation and to temperature changes, cell wall assembly and integrity, degradation of cellular organelles, virulence, cell-cell signaling, fungus-plant interaction, and response to damage-associated molecular patterns (DAMPs). Considering the importance of the phylogenetically conserved MAPK pathways in fungi, an updated review of the knowledge on them is discussed in this article. This information reveals their importance, their distribution in fungal species evolutionarily distant and with different lifestyles, their organization and function, and the interactions occurring between different MAPK pathways, and with other signaling pathways, for the regulation of the most complex cellular processes.
Assuntos
Proteínas Fúngicas/fisiologia , Fungos/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Sequência de Aminoácidos , Sequência Conservada , Fungos/genética , Fungos/crescimento & desenvolvimento , Fungos/patogenicidade , Proteínas de Ligação ao GTP/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Biológicos , Morfogênese/fisiologia , Filogenia , Receptores Acoplados a Proteínas G/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Virulência/fisiologiaRESUMO
Extracellular heat shock protein 70 (HSP70) is recognized by receptors on the plasma membrane, such as Toll-like receptor 4 (TLR4), TLR2, CD14, and CD40. This leads to activation of nuclear factor-kappa B (NF-κB), release of pro-inflammatory cytokines, enhancement of the phagocytic activity of innate immune cells, and stimulation of antigen-specific responses. However, the specific characteristics of HSP70 binding are still unknown, and all HSP70 receptors have not yet been described. Putative models for HSP70 complexation to the receptor for advanced glycation endproducts (RAGEs), considering both ADP- and ATP-bound states of HSP70, were obtained through molecular docking and interaction energy calculations. This interaction was detected and visualized by a proximity fluorescence-based assay in A549 cells and further analyzed by normal mode analyses of the docking complexes. The interacting energy of the complexes showed that the most favored docking situation occurs between HSP70 ATP-bound and RAGE in its monomeric state. The fluorescence proximity assay presented a higher number of detected spots in the HSP70 ATP treatment, corroborating with the computational result. Normal-mode analyses showed no conformational deformability in the interacting interface of the complexes. Results were compared with previous findings in which oxidized HSP70 was shown to be responsible for the differential modulation of macrophage activation, which could result from a signaling pathway triggered by RAGE binding. Our data provide important insights into the characteristics of HSP70 binding and receptor interactions, as well as putative models with conserved residues on the interface area, which could be useful for future site-directed mutagenesis studies.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Simulação de Acoplamento Molecular , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Células A549 , Sítios de Ligação , Corantes Fluorescentes/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptor para Produtos Finais de Glicação Avançada/química , Receptor para Produtos Finais de Glicação Avançada/genética , TermodinâmicaRESUMO
Leptin, a 16-kDa polypeptide hormone, is produced by the adipocyte and can also be synthesized by placenta. We previously demonstrated that leptin promotes proliferation and survival in placenta, in part mediated by the p53 pathway. In this work, we investigated the mechanisms involved in leptin down-regulation of p53 level. The human first trimester cytotrophoblastic Swan-71 cell line and human placental explants at term were used. In order to study the late phase of apoptosis, triggered by serum deprivation, experiments of DNA fragmentation were carried out. Exogenous leptin added to human placental explants, showed a decrease on DNA ladder formation and MAPK pathway is involved in this leptin effect. We also found that under serum deprivation condition, leptin decreases p53 levels and the inhibitory leptin effect is lost when cells were pretreated with 50 µM PD98059 or 10 µM LY29004; or were transfected with dominant negative mutants of intermediates of these pathways, suggesting that MAPK and PI3K signaling pathways are necessaries for leptin action. Additionally, leptin diminished Ser-46 p53 phosphorylation and this effect in placental explants was mediated by the activation of MAPK and PI3K pathways. Finally, in order to assess leptin effect on p53 half-life experiments with cycloheximide were performed and MDM-2 expression was analyzed. Leptin diminished p53 half-life and up-regulated MDM-2 expression. In summary, we provided evidence suggesting that leptin anti-apoptotic effect is mediated by MAPK and PI3K pathways.
Assuntos
Leptina/metabolismo , Trofoblastos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linhagem Celular , Proliferação de Células , Humanos , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
Acute kidney injury (AKI) is one of the more frequent and lethal pathological conditions seen in intensive care units. Currently available treatments are not totally effective but stem cell-based therapies are emerging as promising alternatives, especially the use of mesenchymal stromal cells (MSC), although the signaling pathways involved in their beneficial actions are not fully understood. The objective of this study was to identify signaling networks and key proteins involved in the repair of ischemia by MSC. Using an in vitro model of AKI to investigate paracrine interactions and label-free high definition 2D-NanoESI-MS(E) , differentially expressed proteins were identified in a human renal proximal tubule cell lineage (HK-2) exposed to human MSC (hMSC) after an ischemic insult. In silico analysis showed that hMSC stimulated antiapoptotic activity, normal ROS handling, energy production, cytoskeleton organization, protein synthesis, and cell proliferation. The proteomic data were validated by parallel experiments demonstrating reduced apoptosis in HK-2 cells and recovery of intracellular ATP levels. qRT-PCR for proteins implicated in the above processes revealed that hMSC exerted their effects by stimulating translation, not transcription. Western blotting of proteins associated with ROS and energy metabolism confirmed their higher abundance in HK-2 cells exposed to hMSC.
Assuntos
Injúria Renal Aguda/prevenção & controle , Proliferação de Células , Isquemia/fisiopatologia , Túbulos Renais Proximais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteoma/análise , Proteômica/métodos , Injúria Renal Aguda/metabolismo , Apoptose , Biomarcadores/análise , Western Blotting , Células Cultivadas , Técnicas de Cocultura , Eletroforese em Gel Bidimensional/métodos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Túbulos Renais Proximais/citologia , Células-Tronco Mesenquimais/citologia , Nanotecnologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
BACKGROUND & AIMS: The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS: SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS: SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS: In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.
Assuntos
Dano ao DNA , Células Epiteliais/enzimologia , Receptores ErbB/metabolismo , Mucosa Gástrica/enzimologia , Infecções por Helicobacter/enzimologia , Helicobacter pylori/metabolismo , Receptor ErbB-2/metabolismo , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Colômbia , Progressão da Doença , Ativação Enzimática , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Receptores ErbB/deficiência , Receptores ErbB/genética , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/enzimologia , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Honduras , Humanos , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fosforilação , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/microbiologia , Lesões Pré-Cancerosas/patologia , Análise de Componente Principal , Multimerização Proteica , Receptor ErbB-2/genética , Transdução de Sinais , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Tennessee , Poliamina OxidaseRESUMO
It has been long thought that the brain reorganizes itself in response to environmental needs. Sensory experiences coded in action potentials are the mean by which information on the surroundings is introduced into neuronal networks. The information approaching the brain in the form of electrochemical codes must then be translated in biochemical, epigenetic and genetic ones. Only until recently we have begun understanding the underpinning of such informational transformations and how this process is expressed as neuronal plastic responses. Central for our comprehension of this matter is the finding that signals transduction cascades can modify gene expression by remodeling the chromatin through epigenetic mechanisms. Hence, chromatin remodeling seems to be the process by which experiences are imprinted.