RESUMEN

INTRODUCTION: GEO- and TCGA-based data analysis suggested the differential expression of miR-29c in pancreatic cancer. However, limited data are available on the downstream mechanistic actions of miR-29c, which may fuel the in vitro and in vivo studies of pancreatic cancer. METHODS: The downstream target gene of miR-29c and the downstream ERK/MAPK pathway involved in pancreatic cancer were predicted by bioinformatics tools. Next, the expression of miR-29c and MAPK1 was determined in pancreatic cancer tissues and cells. After ectopic expression and depletion experiments in pancreatic cancer cells, oncogenic phenotypes of pancreatic cancer cells were tested by MTS assay, Transwell assay, and flow cytometry. Effects of miR-29c/MAPK1 on tumorigenic ability in vivo were evaluated in pancreatic cancer xenografts in nude mice. RESULTS: Through differential analysis, five pancreatic cancer-related miRNAs (hsa-miR-29c, hsa-miR-107, hsa-miR-324-3p, hsa-miR-375, and hsa-miR-210) were screened out, among which miR-29c was selected as the key miRNA related to prognosis of pancreatic cancer patients. miR-29c could target and inhibit MAPK1 to suppress the activation of ERK/MAPK pathway. miR-29c was downregulated in pancreatic cancer, and its high expression was related to the good prognosis of pancreatic cancer patients. Both in vitro and in vivo experiments demonstrated that restoration of miR-29c inhibited oncogenic phenotypes of pancreatic cancer cells, as well as repressed tumorigenic ability of pancreatic cancer cells in nude mice. CONCLUSIONS: Taken together, we unveil a novel miR-29c/MAPK1/ERK/MAPK axis that suppresses pancreatic cancer both in vitro and in vivo.

Asunto(s)

MicroARNs , Neoplasias Pancreáticas , Ratones , Animales , Humanos , Ratones Desnudos , Línea Celular Tumoral , MicroARNs/metabolismo , Neoplasias Pancreáticas/genética , Regulación Neoplásica de la Expresión Génica , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Neoplasias Pancreáticas

RESUMEN

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Glicocálix/metabolismo , Ácido Hialurónico/metabolismo , Sindecano-1/genética , Neoplasias de la Mama Triple Negativas/genética , Vía de Señalización Wnt/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Antígeno CD24/genética , Antígeno CD24/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Bases de Datos Factuales , Femenino , Glicocálix/química , Glicocálix/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Hialuronano Sintasas/genética , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/farmacología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Células MCF-7 , MicroARNs/genética , MicroARNs/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis de Supervivencia , Sindecano-1/antagonistas & inhibidores , Sindecano-1/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología

RESUMEN

The superiority of spaced over massed learning is an established fact in the formation of long-term memories (LTM). Here we addressed the cellular processes and the temporal demands of this phenomenon using a weak spatial object recognition (wSOR) training, which induces short-term memories (STM) but not LTM. We observed SOR-LTM promotion when two identical wSOR training sessions were spaced by an inter-trial interval (ITI) ranging from 15 min to 7 h, consistently with spaced training. The promoting effect was dependent on neural activity, protein synthesis and ERKs1/2 activity in the hippocampus. Based on the "behavioral tagging" hypothesis, which postulates that learning induces a neural tag that requires proteins to induce LTM formation, we propose that retraining will mainly retag the sites initially labeled by the prior training. Thus, when weak, consecutive training sessions are experienced within an appropriate spacing, the intracellular mechanisms triggered by each session would add, thereby reaching the threshold for protein synthesis required for memory consolidation. Our results suggest in addition that ERKs1/2 kinases play a dual role in SOR-LTM formation after spaced learning, both inducing protein synthesis and setting the SOR learning-tag. Overall, our findings bring new light to the mechanisms underlying the promoting effect of spaced trials on LTM formation.

Asunto(s)

Conducta Animal , Hipocampo/fisiología , Memoria a Largo Plazo/fisiología , Memoria a Corto Plazo/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Memoria Espacial/fisiología , Animales , Condicionamiento Psicológico , Activación Enzimática , Masculino , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Biosíntesis de Proteínas , Ratas , Ratas Wistar

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.

Asunto(s)

Muerte Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/virología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , eIF-2 Quinasa/metabolismo , Apoptosis , Astrocitos/metabolismo , Astrocitos/patología , Astrocitos/virología , Supervivencia Celular , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/patología , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Transducción de Señal , Replicación Viral , eIF-2 Quinasa/genética

RESUMEN

Objective: Cocaine use disorders (CUDs) represent a major public health problem in many countries. To better understand the interaction between the environmental modulations and phenotype, the aim of the present study was to investigate the DNA methylation pattern of CUD patients, who had concomitant cocaine and crack dependence, and healthy controls. Methods: We studied DNA methylation profiles in the peripheral blood of 23 CUD patients and 24 healthy control subjects using the Illumina Infinium HumanMethylation450 BeadChip arrays. Results: Comparison between CUD patients and controls revealed 186 differentially methylated positions (DMPs; adjusted p-value [adjP] < 10-5) related to 152 genes, with a subset of CpGs confirmed by pyrosequencing. DNA methylation patterns discriminated CUD patients and control groups. A gene network approach showed that the EHMT1, EHMT2, MAPK1, MAPK3, MAP2K1, and HDAC5 genes, which are involved in transcription and chromatin regulation cellular signaling pathways, were also associated with cocaine dependence. Conclusion: The investigation of DNA methylation patterns may contribute to a better understanding of the biological mechanisms involved in CUD.

Asunto(s)

Humanos , Masculino , Adulto , Adulto Joven , Cocaína Crack , Metilación de ADN , Trastornos Relacionados con Cocaína/genética , Trastornos Relacionados con Cocaína/sangre , Estudio de Asociación del Genoma Completo/métodos , Estudios de Casos y Controles , Modelos Lineales , N-Metiltransferasa de Histona-Lisina/genética , Estadísticas no Paramétricas , Proteína Quinasa 1 Activada por Mitógenos/genética , MAP Quinasa Quinasa 1/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos de Histocompatibilidad/genética , Histona Desacetilasas/genética

RESUMEN

OBJECTIVE: Cocaine use disorders (CUDs) represent a major public health problem in many countries. To better understand the interaction between the environmental modulations and phenotype, the aim of the present study was to investigate the DNA methylation pattern of CUD patients, who had concomitant cocaine and crack dependence, and healthy controls. METHODS: We studied DNA methylation profiles in the peripheral blood of 23 CUD patients and 24 healthy control subjects using the Illumina Infinium HumanMethylation450 BeadChip arrays. RESULTS: Comparison between CUD patients and controls revealed 186 differentially methylated positions (DMPs; adjusted p-value [adjP] < 10-5) related to 152 genes, with a subset of CpGs confirmed by pyrosequencing. DNA methylation patterns discriminated CUD patients and control groups. A gene network approach showed that the EHMT1, EHMT2, MAPK1, MAPK3, MAP2K1, and HDAC5 genes, which are involved in transcription and chromatin regulation cellular signaling pathways, were also associated with cocaine dependence. CONCLUSION: The investigation of DNA methylation patterns may contribute to a better understanding of the biological mechanisms involved in CUD.

Asunto(s)

Trastornos Relacionados con Cocaína/sangre , Trastornos Relacionados con Cocaína/genética , Cocaína Crack , Metilación de ADN , Estudio de Asociación del Genoma Completo/métodos , Adulto , Estudios de Casos y Controles , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos de Histocompatibilidad/genética , Histona Desacetilasas/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Modelos Lineales , MAP Quinasa Quinasa 1/genética , Masculino , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Estadísticas no Paramétricas , Adulto Joven

RESUMEN

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.

Asunto(s)

Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Enfermedad de Parkinson/metabolismo , Fosfolipasa D/biosíntesis , alfa-Sinucleína/metabolismo , Línea Celular Tumoral , Mutación con Ganancia de Función , Humanos , Filamentos Intermedios/genética , Filamentos Intermedios/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fosfolipasa D/genética , alfa-Sinucleína/genética

RESUMEN

Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a ß-1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the "caspofungin paradoxical effect" (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase-encoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies.IMPORTANCE Systemic Aspergillus fumigatus infections are often accompanied by high mortality rates. The fungal cell wall is important for infection as it has immunomodulatory and immunoevasive properties. Paradoxical growth of A. fumigatus in the presence of high concentrations of the cell wall-disturbing agent caspofungin has been observed for more than a decade, although the mechanistic nature of this phenomenon remains largely uncharacterized. Here, we show that the CWI pathway components MpkA and RlmA as well as the calcium/calcineurin-responsive transcription factor CrzA regulate the expression of cell wall biosynthetic genes during the caspofungin paradoxical effect (CPE). Furthermore, an additional, novel calcium/calcineurin-responsive transcription factor was identified to play a role in cell wall biosynthesis gene expression during the CPE. This work paints a crucial role for calcium metabolism in the CPE and provides further insight into the complex regulation of cell wall biosynthesis, which could ultimately lead to the development of more efficient antifungal therapies.

Asunto(s)

Aspergillus fumigatus/genética , Pared Celular/metabolismo , Quitina Sintasa/genética , Equinocandinas/farmacología , Proteínas Fúngicas/metabolismo , Lipopéptidos/farmacología , Factores de Transcripción/metabolismo , Aspergilosis/microbiología , Aspergillus fumigatus/efectos de los fármacos , Caspofungina , Pared Celular/efectos de los fármacos , Pared Celular/genética , Quitina/metabolismo , Equinocandinas/genética , Equinocandinas/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Lipopéptidos/genética , Lipopéptidos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética

RESUMEN

Parathyroid hormone-related peptide (PTHrP) is associated with several human cancers such as colon carcinoma. This disease is a complex multistep process that involves enhanced cell cycle progression and migration. Recently we obtained evidence that in the human colorectal adenocarcinoma Caco2 cells, exogenous PTHrP increases the proliferation and positively modulates cell cycle progression via ERK1/2, p38 MAPK and PI3K. The purpose of this study was to explore if the serine/threonine kinase RSK, which is involved in the progress of many cancers and it is emerging as a potential therapeutic target, mediates PTHrP effects on cancer colon cells. Western blot analysis revealed that PTHrP increases RSK phosphorylation via ERK1/2 signaling pathway but not through p38 MAPK. By performing subcellular fractionation, we found that the peptide also induces the nuclear localization of activated RSK, where many of its substrates are located. RSK participates in cell proliferation, in the upregulation of cyclin D1 and CDK6 and in the downregulation of p53 induced by PTHrP. Wound healing and transwell filter assays revealed that cell migration increased after PTHrP treatment. In addition, the hormone increases the protein expression of the focal adhesion kinase FAK, a regulator of cell motility. We observed that PTHrP induces cell migration and modulates FAK protein expression through ERK/RSK signaling pathway but not via p38 MAPK pathway. Finally, in vivo studies revealed that the hormone activates RSK in xenografts tumor. Taken together, our findings provide new insights into the deregulated cell cycle and migration that is characteristic of tumor intestinal cells.

Asunto(s)

Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas/genética , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Proteínas Serina-Treonina Quinasas/genética , Animales , Células CACO-2 , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Células HCT116 , Humanos , Inyecciones Intralesiones , Masculino , Ratones , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Trasplante de Neoplasias , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

STUDY QUESTION: How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? SUMMARY ANSWER: hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. WHAT IS KNOWN ALREADY: hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. STUDY DESIGN, SIZE, DURATION: This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such induction could not be blocked by inhibitors for PKA, PKC and PI3K. Epac inhibition and knockdown with siRNA prevented pErk1/2 induction by hCG. ESCs stimulated with hCG for up to 72 h showed a significant increase in PR mRNA and immunofluorescent label at 48 h only; an effect that was abrogated with the mitogen-activated protein kinase kinase inhibitor UO126. In addition, the hCG-activated Erk1/2 pathway significantly decreased the mRNA levels for secreted frizzled-related protein 4 (SFRP4) at 24 h, whereas it increased those for homeobox A10 (HOXA10) at 48 h (P = 0.041 and P = 0.022 versus control, respectively). Prolactin mRNA levels were not significantly modified. HOXA10 mRNA up-regulation by hCG was not enhanced by co-stimulation with progesterone; however, it was completely abolished in the presence of RU486 (P = 0.036 hCG versus hCG + RU486). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study utilizing stromal cell cultures from human endometrial tissues. Furthermore, results obtained should also be confirmed in vivo in the context of the whole human endometrial tissue and hormonal milieu. The in vitro experiments using hCG have been conducted without other hormones/factors that may also modulate the ESCs response to hCG. WIDER IMPLICATIONS OF THE FINDINGS: We have determined that hCG induces the PR through the Erk1/2 pathway in ESCs which may render them more sensitive to progesterone, increasing our understanding about the effects of hCG at the embryo-maternal interface. The activation of such a pathway in the context of the hormonal milieu during the window of implantation might contribute to a successful dialog between the embryo and the uterus, leading to appropriate endometrial function. Defective hCG signaling in the endometrial stromal tissue may lead to an incomplete uterine response, compromising embryo implantation and early pregnancy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Fund for Scientific and Technological Development, Government of Chile (FONDECYT) grants 11100443 and 1140614 (A.T.-P.). The authors have no conflicts of interest to declare.

Asunto(s)

Gonadotropina Coriónica/farmacología , Factores de Intercambio de Guanina Nucleótido/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Receptores de Progesterona/genética , Células del Estroma/efectos de los fármacos , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Adulto , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endometrio/citología , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/agonistas , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Homeobox A10 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Embarazo , Cultivo Primario de Células , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Progesterona/metabolismo , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismo , Transcripción Genética

RESUMEN

Radiotherapy is widely used for advanced rectal tumors. However, refractory metastasis has become the major cause of therapy failure in rectal cancer patients. Understanding the molecular mechanism that controls the aggressive cellular response to this treatment is essential for developing new therapeutic applications and improving radiotherapy response in colorectal cancer patients. Using the progeny of cells that were submitted to irradiation, we have demonstrated that the PI3K/AKT, Wnt/ß-catenin signaling pathways as well as ERK1/2 downstream of EPHA4 receptor activation, play an important role in the regulation of events related with the EMT development, which may be associated with the therapeutic failure in rectal cancer after radiotherapy. Here, we further discuss about EphA4 receptor as a potential therapeutic target for the treatment of this cancer type. J. Cell. Biochem. 118: 442-445, 2017. © 2016 Wiley Periodicals, Inc.

Asunto(s)

Transición Epitelial-Mesenquimal/efectos de la radiación , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Receptor EphA4/metabolismo , Neoplasias del Recto/metabolismo , Neoplasias del Recto/radioterapia , Vía de Señalización Wnt/efectos de la radiación , Transición Epitelial-Mesenquimal/genética , Humanos , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor EphA4/genética , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Vía de Señalización Wnt/genética

RESUMEN

Transcriptomic analyses were performed in the green macroalga Ulva compressa cultivated with 10µM copper for 24h. Nucleotide sequences encoding antioxidant enzymes, ascorbate peroxidase (ap), dehydroascorbate reductase (dhar) and glutathione reductase (gr), enzymes involved in ascorbate (ASC) synthesis l-galactose dehydrogenase (l-gdh) and l-galactono lactone dehydrogenase (l-gldh), in glutathione (GSH) synthesis, γ-glutamate-cysteine ligase (γ-gcl) and glutathione synthase (gs), and metal-chelating proteins metallothioneins (mt) were identified. Amino acid sequences encoded by transcripts identified in U. compressa corresponding to antioxidant system enzymes showed homology mainly to plant and green alga enzymes but those corresponding to MTs displayed homology to animal and plant MTs. Level of transcripts encoding the latter proteins were quantified in the alga cultivated with 10µM copper for 0-12 days. Transcripts encoding enzymes of the antioxidant system increased with maximal levels at day 7, 9 or 12, and for MTs at day 3, 7 or 12. In addition, the involvement of calmodulins (CaMs), calcium-dependent protein kinases (CDPKs), and the mitogen-activated protein kinase kinase (MEK1/2) in the increase of the level of the latter transcripts was analyzed using inhibitors. Transcript levels decreased with inhibitors of CaMs, CDPKs and MEK1/2. Thus, copper induces overexpression of genes encoding antioxidant enzymes, enzymes involved in ASC and GSH syntheses and MTs. The increase in transcript levels may involve the activation of CaMs, CDPKs and MEK1/2 in U. compressa.

Asunto(s)

Proteínas Algáceas/metabolismo , Antioxidantes/metabolismo , Cobre/toxicidad , Expresión Génica/efectos de los fármacos , Ulva/metabolismo , Contaminantes Químicos del Agua/toxicidad , Calmodulina/genética , Calmodulina/metabolismo , Perfilación de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN de Planta/química , ARN de Planta/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ulva/enzimología

RESUMEN

The present study screened potential genes related to lung adenocarcinoma, with the aim of further understanding disease pathogenesis. The GSE2514 dataset including 20 lung adenocarcinoma and 19 adjacent normal tissue samples from 10 patients with lung adenocarcinoma aged 45-73 years was downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) between the two groups were screened using the t-test. Potential gene functions were predicted using functional and pathway enrichment analysis, and protein-protein interaction (PPI) networks obtained from the STRING database were constructed with Cytoscape. Module analysis of PPI networks was performed through MCODE in Cytoscape. In total, 535 upregulated and 465 downregulated DEGs were identified. These included ATP5D, UQCRC2, UQCR11 and genes encoding nicotinamide adenine dinucleotide (NADH), which are mainly associated with mitochondrial ATP synthesis coupled electron transport, and which were enriched in the oxidative phosphorylation pathway. Other DEGs were associated with DNA replication (PRIM1, MCM3, and RNASEH2A), cell surface receptor-linked signal transduction and the enzyme-linked receptor protein signaling pathway (MAPK1, STAT3, RAF1, and JAK1), and regulation of the cytoskeleton and phosphatidylinositol signaling system (PIP5K1B, PIP5K1C, and PIP4K2B). Our findings suggest that DEGs encoding subunits of NADH, PRIM1, MCM3, MAPK1, STAT3, RAF1, and JAK1 might be associated with the development of lung adenocarcinoma.

Asunto(s)

Humanos , Masculino , Femenino , Persona de Mediana Edad , Anciano , Adenocarcinoma/genética , Perfilación de la Expresión Génica/métodos , Neoplasias Pulmonares/genética , Proteínas de Microfilamentos/genética , Regulación hacia Abajo/genética , Redes Reguladoras de Genes , Proteína Quinasa 1 Activada por Mitógenos/genética , NAD/genética , Mapas de Interacción de Proteínas/genética , Proteínas Proto-Oncogénicas c-raf/genética , Regulación hacia Arriba/genética

RESUMEN

The present study screened potential genes related to lung adenocarcinoma, with the aim of further understanding disease pathogenesis. The GSE2514 dataset including 20 lung adenocarcinoma and 19 adjacent normal tissue samples from 10 patients with lung adenocarcinoma aged 45-73 years was downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) between the two groups were screened using the t-test. Potential gene functions were predicted using functional and pathway enrichment analysis, and protein-protein interaction (PPI) networks obtained from the STRING database were constructed with Cytoscape. Module analysis of PPI networks was performed through MCODE in Cytoscape. In total, 535 upregulated and 465 downregulated DEGs were identified. These included ATP5D, UQCRC2, UQCR11 and genes encoding nicotinamide adenine dinucleotide (NADH), which are mainly associated with mitochondrial ATP synthesis coupled electron transport, and which were enriched in the oxidative phosphorylation pathway. Other DEGs were associated with DNA replication (PRIM1, MCM3, and RNASEH2A), cell surface receptor-linked signal transduction and the enzyme-linked receptor protein signaling pathway (MAPK1, STAT3, RAF1, and JAK1), and regulation of the cytoskeleton and phosphatidylinositol signaling system (PIP5K1B, PIP5K1C, and PIP4K2B). Our findings suggest that DEGs encoding subunits of NADH, PRIM1, MCM3, MAPK1, STAT3, RAF1, and JAK1 might be associated with the development of lung adenocarcinoma.

Asunto(s)

Adenocarcinoma/genética , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Neoplasias Pulmonares/genética , Proteínas de Microfilamentos/genética , Adenocarcinoma del Pulmón , Anciano , Regulación hacia Abajo/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/genética , NAD/genética , Mapas de Interacción de Proteínas/genética , Proteínas Proto-Oncogénicas c-raf/genética , Regulación hacia Arriba/genética

RESUMEN

Tremendous efforts have been made in renal cell carcinoma (RCC) patients' research; however, clinical findings in patients have been disappointing. The aims of our study were to identify better or alternative therapeutic methods that can reverse chemotherapy resistance and to enhance sensitivity to docetaxel (DOX)-based chemotherapy drugs. We evaluated the anti-proliferative effect of DOX against RCC cells. DOX was found to suppress proliferation of RCC cells under in vitro and in vivo settings. Flow cytometric analysis revealed that DOX suppressed cell growth by induction of both apoptosis and G2/M cell cycle arrest in a dose-dependent manner. Various patterns of gene expression were observed by cluster analysis. In addition, based on network analysis using the ingenuity pathway analysis software, DOX was found to suppress phosphorylation of extracellular signal-regulated kinase 1/2 and p38, suggesting that the mitogen-activated protein kinase signaling pathway plays a vital role in the anti-proliferative effect of DOX against RCC.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Taxoides/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Melanoma is a very aggressive tumor that arises from melanocytes. Late stage and widely spread diseases do not respond to standard therapeutic approaches. The kallikrein-kinin system (KKS) participates in biological processes such as vasodilatation, pain and inflammatory response. However, the role of KKS in tumor formation and progression is not completely understood. The role of the host kinin B1 receptor in melanoma development was evaluated using a syngeneic melanoma model. Primary tumors and metastasis were respectively induced by injecting B16F10 melanoma cells, which are derived from C57BL/6 mice, subcutaneously or in the tail vein in wild type C57BL/6 and B1 receptor knockout mice (B1(-/-)). Tumors developed in B1(-/-) mice presented unfavorable prognostic factors such as increased incidence of ulceration, higher levels of IL-10, higher activation of proliferative pathways such as ERK1/2 and Akt, and increased mitotic index. Furthermore, in the metastasis model, B1(-/-) mice developed larger metastatic colonies in the lung and lower CD8(+)immune effector cells when compared with WT animals. Altogether, our results provide evidences that B1(-/-) animals developed primary tumors with multiple features associated with poor prognosis and unfavorable metastatic onset, indicating that the B1 receptor may contribute to improve the host response against melanoma progression.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Melanoma Experimental/genética , Receptor de Bradiquinina B1/genética , Neoplasias Cutáneas/genética , Animales , Progresión de la Enfermedad , Femenino , Interleucina-10/genética , Interleucina-10/metabolismo , Sistema Calicreína-Quinina/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Melanoma Experimental/metabolismo , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Índice Mitótico , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Bradiquinina B1/deficiencia , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología

RESUMEN

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5). S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL) did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM) did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000-10000 nM) through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts.

Asunto(s)

Quimiotaxis/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Lisofosfolípidos/farmacología , ARN Mensajero/genética , Receptores de Lisoesfingolípidos/genética , Esfingosina/análogos & derivados , Linfocitos T/efectos de los fármacos , Anilidas/farmacología , Línea Celular Tumoral , Humanos , Lisofosfolípidos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Organofosfonatos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/metabolismo , Esfingosina/farmacología , Linfocitos T/metabolismo , Linfocitos T/patología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

Extracellular nucleotides are signaling elements present in the tumor microenvironment; however, their role in tumor growth is not completely understood. In the present study, we asked whether nucleotides regulate cell migration in ovarian carcinoma-derived cells. We observed that 100 µM UTP induced migration in SKOV-3 cells (1.57 ± 0.08 fold over basal), and RT-PCR showed expression of transcripts for the P2RY2 and P2RY4 receptors. Knockdown of P2RY2 expression in SKOV-3 cells (P2RY2-KD) abolished the UTP-induced migration. The mechanism activated by UTP to induce migration involves transactivation of the epidermal growth factor receptor (EGFR) since we observed that the EGFR kinase inhibitor AG1478 and the PI3K inhibitor Wortmannin inhibit this response (to 0.76 ± 0.23 and 0.46 ± 0.14 relative to the control, respectively). In agreement with these observations, UTP was able to modify the phosphorylation state of the EGFR; likewise, the induction of ERK1/2 phosphorylation promoted by UTP was abolished by a 30-60 min treatment with AG1478. Our data also suggested that the enhanced cell migration involves the epithelium to mesenchymal transition (EMT) process, since a 12 h stimulation of SKOV-3 cells with 100 µM UTP showed an increase in vimentin and SNAIL protein levels (459.8 ± 132.4% over basal for SNAIL). Interestingly, treatment with apyrase (10 U/mL) reduces the migration of control cells and induces a considerable enrichment of E-cadherin in the cell-cell contacts, favoring an epithelial phenotype and strongly suggesting that the nucleotides released by tumor cells and acting through the P2RY2 receptor are potential regulators of invasiveness.

Asunto(s)

Células Epiteliales/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Receptor Cross-Talk/efectos de los fármacos , Receptores Purinérgicos P2Y2/genética , Uridina Trifosfato/farmacología , Androstadienos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/metabolismo , Femenino , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Ovario/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tirfostinos/farmacología , Uridina Trifosfato/metabolismo , Vimentina/genética , Vimentina/metabolismo , Wortmanina

RESUMEN

PURPOSE: This study aimed to clarify the molecular mechanism mediating the cytotoxicity of axitinib, a selective inhibitor of the vascular endothelial growth factor receptor (VEGFR), in sunitinib-resistant renal cell carcinoma (RCC). METHODS: In our previous study (Sakai et al. in BJU Int 112:E211-E220, 2013), a human RCC cell line, ACHN, resistant to sunitinib (ACHN/R), was developed from a parental cell line (ACHN/P). Differences in molecular phenotypes following treatment with sunitinib or axitinib between these two cell lines were compared. RESULTS: ACHN/R showed an approximately fivefold higher IC50 of sunitinib than ACHN/P; however, there was no significant difference in the sensitivity to axitinib between these two cell lines. In ACHN/R, despite the lack of a difference in the phosphorylated (p)-Akt or STAT-3 expression between treatment with sunitinib and axitinib, the expression of p-p44/42 mitogen-activated protein kinase (MAPK) and p-VEGFR-2 after treatment with axitinib was markedly down-regulated compared with those after treatment with sunitinib. Furthermore, additional treatment of ACHN/R with an inhibitor of MAPK kinase significantly enhanced the cytotoxic activity of sunitinib, but not that of axitinib. In vivo growth of ACHN/R in nude mice after treatment with axitinib was significantly inhibited compared with that following treatment with sunitinib, accompanying the marked inhibition of angiogenesis. CONCLUSIONS: Antitumor activity of axitinib in RCC cells even after the acquisition of resistance to sunitinib could be explained, at least in part, by the inactivation of p44/42 MAPK and VEGFR-2, which were persistently phosphorylated in sunitinib-resistant RCC cells under treatment with sunitinib.

Asunto(s)

Carcinoma de Células Renales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Imidazoles/farmacología , Indazoles/farmacología , Neoplasias Renales/patología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/farmacología , Axitinib , Western Blotting , Línea Celular Tumoral , Proliferación Celular/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Indoles/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Pirroles/farmacología , Sunitinib , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na(+)/H(+) exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

Asunto(s)

Interleucina-8/metabolismo , Glicoproteínas de Membrana/genética , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/genética , Neutrófilos/efectos de los fármacos , Especies Reactivas de Oxígeno/inmunología , Transducción de Señal/efectos de los fármacos , Acetofenonas/farmacología , Amilorida/farmacología , Bloqueadores del Canal de Sodio Epitelial/farmacología , Regulación de la Expresión Génica , Humanos , Interleucina-8/genética , Interleucina-8/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/inmunología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Neutrófilos/citología , Neutrófilos/inmunología , Compuestos Onio/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología