RESUMEN
In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 µs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.
Asunto(s)
Mitocondrias , Neoplasias , Mitocondrias/metabolismo , Membrana Celular/metabolismo , Electricidad , Citoesqueleto/metabolismo , Encéfalo/metabolismo , Neoplasias/metabolismoRESUMEN
Extracellular High-mobility group box 1 (HMGB1) contributes to the pathogenesis of inflammatory disorders, including inflammatory bowel diseases (IBD). Poly (ADP-ribose) polymerase 1 (PARP1) has been recently reported to promote HMGB1 acetylation and its secretion outside cells. In this study, the relationship between HMGB1 and PARP1 in controlling intestinal inflammation was explored. C57BL6/J wild type (WT) and PARP1-/- mice were treated with DSS to induce acute colitis, or with the DSS and PARP1 inhibitor, PJ34. Human intestinal organoids, which are originated from ulcerative colitis (UC) patients, were exposed to pro-inflammatory cytokines (INFγ + TNFα) to induce intestinal inflammation, or coexposed to cytokines and PJ34. Results show that PARP1-/- mice develop less severe colitis than WT mice, evidenced by a significant decrease in fecal and serum HMGB1, and, similarly, treating WT mice with PJ34 reduces the secreted HMGB1. The exposure of intestinal organoids to pro-inflammatory cytokines results in PARP1 activation and HMGB1 secretion; nevertheless, the co-exposure to PJ34, significantly reduces the release of HMGB1, improving inflammation and oxidative stress. Finally, HMGB1 release during inflammation is associated with its PARP1-induced PARylation in RAW264.7 cells. These findings offer novel evidence that PARP1 favors HMGB1 secretion in intestinal inflammation and suggest that impairing PARP1 might be a novel approach to manage IBD.
Asunto(s)
Colitis , Proteína HMGB1 , Enfermedades Inflamatorias del Intestino , Poli(ADP-Ribosa) Polimerasa-1 , Animales , Humanos , Ratones , Colitis/inducido químicamente , Citocinas , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamación , Organoides , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
OBJECTIVE: The importance of autophagy in mechanisms underlying inflammation has been highlighted. Downstream effects of the bacterial sensor NOD2 include autophagy induction. Recently, a relationship between defects in autophagy and adherent/invasive Escherichia coli (AIEC) persistence has emerged. The present study aims at investigating the interplay between autophagy, NOD2 and AIEC bacteria and assessing the expression level of autophagic proteins in intestinal biopsies of pediatric patients with inflammatory bowel disease (IBD). METHODS: A human epithelial colorectal adenocarcinoma (Caco2) cell line stably over-expressing NOD2 was produced (Caco2NOD2). ATG16L1, LC3 and NOD2 levels were analysed in the Caco2 cell line and Caco2NOD2 after exposure to AIEC strains, by western blot and immunofluorescence. AIEC survival inside cells and TNFα, IL-8 and IL-1ßmRNA expression were analysed by gentamicin protection assay and real time PCR. ATG16L1 and LC3 expression was analyzed in the inflamed ileum and colon of 28 patients with Crohn's disease (CD), 14 with ulcerative colitis (UC) and 23 controls by western blot. RESULTS: AIEC infection increased ATG16L1 and LC3 in Caco2 cells. Exposure to AIEC strains increased LC3 and ATG16L1 in Caco2 overexpressing NOD2, more than in Caco2 wild type, while a decrease of AIEC survival rate and cytokine expression was observed in the same cell line. LC3 expression was increased in the inflamed colon of CD and UC children. CONCLUSIONS: The NOD2-mediated autophagy induction is crucial to hold the intramucosal bacterial burden, especially towards AIEC, and to limit the resulting inflammatory response. Autophagy is active in inflamed colonic tissues of IBD pediatric patients.
Asunto(s)
Autofagia , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Infecciones por Escherichia coli/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Adolescente , Proteínas Relacionadas con la Autofagia/inmunología , Células CACO-2 , Niño , Preescolar , Citocinas/genética , Células Epiteliales/microbiología , Femenino , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Intestinos/citología , Masculino , Proteínas Asociadas a Microtúbulos/inmunologíaRESUMEN
The arrest of differentiation is a feature of both chronic myelogenous leukemia cells in myeloid blast crisis and myeloid precursors that ectopically express the p210BCR-ABL oncoprotein; however, its underlying mechanisms remain poorly understood. Here we show that expression of BCR-ABL in myeloid precursor cells leads to transcriptional suppression of the granulocyte colony-stimulating factor receptor G-CSF-R (encoded by CSF3R), possibly through down-modulation of C/EBPalpha-the principal regulator of granulocytic differentiation. Expression of C/EBPalpha protein is barely detectable in primary marrow cells taken from individuals affected with chronic myeloid leukemia in blast crisis. In contrast, CEBPA RNA is clearly present. Ectopic expression of C/EBPalpha induces granulocytic differentiation of myeloid precursor cells expressing BCR-ABL. Expression of C/EBPalpha is suppressed at the translational level by interaction of the poly(rC)-binding protein hnRNP E2 with CEBPA mRNA, and ectopic expression of hnRNP E2 in myeloid precursor cells down-regulates both C/EBPalpha and G-CSF-R and leads to rapid cell death on treatment with G-CSF (encoded by CSF3). Our results indicate that BCR-ABL regulates the expression of C/EBPalpha by inducing hnRNP E2-which inhibits the translation of CEBPA mRNA.
Asunto(s)
Apoptosis/fisiología , Proteína alfa Potenciadora de Unión a CCAAT/biosíntesis , Proteínas de Unión al ADN , Proteínas de Fusión bcr-abl/fisiología , Regulación de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas , Proteínas de Unión al ARN/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/biosíntesis , Factores de Transcripción , Animales , Apoptosis/genética , Benzamidas , Crisis Blástica/metabolismo , Crisis Blástica/patología , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT , Proteínas Portadoras/metabolismo , Células Cultivadas/metabolismo , Regulación hacia Abajo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Células Mieloides/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , Piperazinas/farmacología , Biosíntesis de Proteínas , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Pirimidinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , TransfecciónRESUMEN
Microalgae are natural sources of valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs), that show antioxidant, anti-inflammatory, anticancer and antimicrobial activities. The marine microalga Isochrysis galbana (I. galbana) is extremely rich in ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Probiotics are currently suggested as adjuvant therapy in the management of diseases associated with gut dysbiosis. The Lactobacillus reuteri (L. reuteri), one of the most widely used probiotics, has been shown to produce multiple beneficial effects on host health. The present study aimed to present an innovative method for growing the probiotic L. reuteri in the raw seaweed extracts from I. galbana as an alternative to the conventional medium, under conditions of oxygen deprivation (anaerobiosis). As a result, the microalga I. galbana was shown for the first time to be an excellent culture medium for growing L. reuteri. Furthermore, the gas-chromatography mass-spectrometry analysis showed that the microalga-derived ω3 PUFAs were still available after the fermentation by L. reuteri. Accordingly, the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis, was able to significantly reduce the adhesiveness and invasiveness of the harmful adherent-invasive Escherichia coli to intestinal epithelial cells, due to a cooperative effect between L. reuteri and microalgae-released ω3 PUFAs. These findings open new perspectives in the use of unicellular microalgae as growth medium for probiotics and in the production of biofunctional compounds.
Asunto(s)
Técnicas de Cultivo Celular por Lotes/métodos , Haptophyta/microbiología , Limosilactobacillus reuteri/crecimiento & desarrollo , Medios de Cultivo/química , Ácidos Docosahexaenoicos/química , Ácido Eicosapentaenoico/química , Ácidos Grasos Omega-3 , Ácidos Grasos Insaturados/química , Fermentación , Haptophyta/metabolismo , Microalgas/química , Probióticos/metabolismoRESUMEN
In metastatic cancer cells, the process of invasion is regulated by several transcription factors that induce changes required for migration and resistance to apoptosis. Slug (SNAI2, Snail2) is involved in epithelial mesenchymal transition in physiological and in pathological contexts. We show here that in embryonic kidney, colon carcinoma, chronic myeloid leukemia-blast crisis, and in neuroblastoma cells, expression of Slug is transcriptionally regulated by c-Myb via Myb binding sites in the 5'-flanking region and in the first intron of the slug gene. In embryonic kidney and neuroblastoma cells, c-Myb induced vimentin, fibronectin, and N-cadherin expression and membrane ruffling via actin polymerization consistent with the acquisition of a mesenchymal-like phenotype. Furthermore, down-regulation of endogenous c-Myb levels in colon carcinoma cells led to increased expression of E-cadherin and reduced levels of vimentin. Some of these changes are predominantly Slug-dependent as Slug silencing via RNA interference (RNAi) reverts the cells to a quasi-parental condition. Changes in gene expression and morphology induced by c-Myb-activated Slug correlated with increased ability to migrate (embryonic kidney) and to invade through a Matrigel membrane (embryonic kidney, colon carcinoma, neuroblastoma). c-Myb-dependent Slug expression was also essential for the homing of chronic myeloid leukemia K562 cells to the bone marrow. In summary, we show here that the proto-oncogene c-Myb controls Slug transcription in tumor cells of different origin. Such a regulatory pathway contributes to the acquisition of invasive properties that are important for the metastatic process.
Asunto(s)
Médula Ósea/patología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Etopósido/farmacología , Citometría de Flujo , Humanos , Intrones/genética , Ratones , Ratones SCID , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/fisiopatología , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myb/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genéticaRESUMEN
Stage 4 neuroblastoma (NB) is a devastating childhood cancer whose poor outcome has remained essentially unchanged in the last 20 years. Receptor tyrosine kinases have important roles in the control of proliferation, differentiation and apoptosis of NB cells. Thus, we tested the activity of second-generation tyrosine kinase inhibitor Dasatinib in human NB cell lines in vitro and in an orthotopic mouse model. Dasatinib inhibited cell viability with an IC(50) in the submicromolar range in 7 of 10 tested cell lines. In sensitive cells, Dasatinib reduced anchorage-independent growth and, in some instances, induced senescence and apoptosis. In HTLA-230 cells, Dasatinib treatment caused down-regulation of c-Kit and c-Src phosphorylation in conjunction with strong inhibition of Erk1/2 and Akt activity. To test the efficacy of Dasatinib in vivo, HTLA-230 and SY5Y cells were orthotopically injected in the adrenal gland of nude mice and drug treatments carried out until day 40. In mice injected with HTLA-230 cells, tumour growth was significantly inhibited at the dose of 30 mg/(kg day) when treatment was started 7 days after injection. In animals injected with SY5Y cells that were exquisitely sensitive in vitro (IC(50)= 92 nM), the antitumour effect of Dasatinib was observed at the dose of 60 mg/(kg day) but only when treatment was started 1 day after injection. However, the anti-tumour effect of Dasatinib in vivo was partial in both orthotopic models, emphasizing the importance of testing candidate new drugs in animal environments closely mimicking the human tumour.
Asunto(s)
Modelos Animales de Enfermedad , Neuroblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Dasatinib , Femenino , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neuroblastoma/metabolismo , Neuroblastoma/patología , Fosforilación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: We assessed the relevance of Slug (SNAI2) for apoptosis resistance and invasion potential of neuroblastoma cells in vitro and in vivo. EXPERIMENTAL DESIGN: We evaluated the effect of imatinib mesylate on invasion and analyzed the genes modulated by imatinib mesylate treatment in neuroblastoma cells. Slug expression, inhibited by imatinib mesylate treatment, was knocked down in neuroblastoma cells by RNA interference, and the effects on invasion and apoptosis were evaluated in vitro. A pseudometastatic model of neuroblastoma in severe combined immunodeficient mice was used to assess the effects of Slug silencing alone or in combination with imatinib mesylate treatment on metastasis development. RESULTS: Microarray analysis revealed that several genes, including Slug, were down-regulated by imatinib mesylate. Slug expression was detectable in 8 of 10 human neuroblastoma cell lines. Two Slug-expressing cell lines were infected with a vector encoding a microRNA to Slug mRNA. Infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (p53, Bax, and Bcl-2) identified previously as Slug targets. Bcl-2 was down-regulated in Slug-interfered cells. Slug down-regulation increased sensitivity to apoptosis induced by imatinib mesylate, etoposide, or doxorubicin. Invasion of Slug-silenced cells was reduced in vitro. Animals injected with Slug-silenced cells had fewer tumors than controls and the inhibition of tumor growth was even higher in animals treated with imatinib mesylate. CONCLUSIONS: Slug down-regulation facilitates apoptosis induced by proapoptotic drugs in neuroblastoma cells and decreases their invasion capability in vitro and in vivo. Slug inhibition, possibly combined with imatinib mesylate, may represent a novel strategy for treatment of metastatic neuroblastoma.
Asunto(s)
Apoptosis/fisiología , Invasividad Neoplásica , Neuroblastoma/metabolismo , Factores de Transcripción/metabolismo , Animales , Antineoplásicos/farmacología , Benzamidas , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib , Ratones , Neuroblastoma/tratamiento farmacológico , Análisis de Secuencia por Matrices de Oligonucleótidos , Piperazinas/farmacología , Pirimidinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia SnailRESUMEN
Gut mucosal healing (MH) is considered a key therapeutic target and prognostic parameter in the management of inflammatory bowel disease (IBD). The dipotassium glycyrrhizate (DPG), a salt of the glycoconjugated triterpene glycyrrhizin, has been shown to inhibit the High Mobility Group Box 1 (HMGB1) protein, an allarmin strongly implicated in the pathogenesis of most inflammatory and auto-immune disorders. Here we discuss new insights on how DPG acts on MH comparing the acute phase and the recovery phase from experimental colitis in mice. We found that DPG strongly accelerates MH by differently regulating pro-inflammatory (CXCL1, CXCL3, CXCL5, PTGS2, IL-1ß, IL-6, CCL12, CCL7) and wound healing (COL3A1, MMP9, VTN, PLAUR, SERPINE, CSF3, FGF2, FGF7, PLAT, TIMP1) genes as observed only during the recovery phase of colitis. Relevant issue is the identification of extracellular matrix (ECM) remodeling genes, VTN, and PLAUR, as crucial genes to achieve MH during DPG treatment. Furthermore, a noticeable recovery of intestinal epithelial barrier structural organization, wound repair ability, and functionality is observed in two human colorectal adenocarcinoma cell lines exposed to DPG during inflammation. Thus, our study identifies DPG as a potent tool for controlling intestinal inflammation and improving MH.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Ácido Glicirrínico/farmacología , Mucosa Intestinal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Células CACO-2 , Línea Celular , Línea Celular Tumoral , Colitis/tratamiento farmacológico , Colitis/metabolismo , Citocinas/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Femenino , Proteína HMGB1/metabolismo , Células HT29 , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND AIMS: Recent evidence implicates gut microbiota (GM) and immune alterations in autism spectrum disorders (ASD). We assess GM profile and peripheral levels of immunological, neuronal and bacterial molecules in ASD children and controls. Alarmin HMGB1 was explored as a non-invasive biomarker to monitor gastrointestinal (GI) symptoms. METHODS: Thirty ASD children and 14 controls entered into the study. GM metagenomic analysis was performed for 16 ASD patients and 7 controls. GM functional profile was assessed by GO term analysis. Blood levels of IL-1ß, TNFα, TGFß, IL-10, INFγ, IL-8, lipopolysaccharide, Neurotensin, Sortilin1 and GSSG/GSH ratio were analyzed in all subjects by ELISA. Fecal HMGB1 was analyzed by Western blot. RESULTS: We observed a significant decrease in bacterial diversity. Furthermore, 82 GO terms underrepresented in ASD. Four of them pointed at 3,3 phenylpropionate catabolism and were imputable to Escherichia coli (E. coli) group. Serum levels of TNFα, TGFß, NT, and SORT-1 increased in ASD patients. Fecal levels of HMGB1 correlated with GI sign severity in ASD children. CONCLUSIONS: We suggest that a decrease of E. coli might affect the propionate catabolism in ASD. We report occurrence of peripheral inflammation in ASD children. We propose fecal HMGB1 as a non-invasive biomarker to detect GI symptoms.
Asunto(s)
Trastorno del Espectro Autista/microbiología , Enfermedades Gastrointestinales/inmunología , Microbioma Gastrointestinal , Inflamación , Trastorno del Espectro Autista/inmunología , Trastorno del Espectro Autista/fisiopatología , Estudios de Casos y Controles , Niño , Desarrollo Infantil , Preescolar , Comorbilidad , Citocinas/sangre , Escherichia coli/aislamiento & purificación , Heces/microbiología , Femenino , Humanos , Masculino , Fenilpropionatos/metabolismoRESUMEN
We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.
Asunto(s)
Apoptosis/efectos de los fármacos , Topoisomerasa de ADN IV/administración & dosificación , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
Background and aims: Recent evidences reveal the occurrence of a close relationship among epithelial to mesenchymal transition (EMT), chronic inflammation and fibrosis. ZNF281 is an EMT-inducing transcription factor (EMT-TF) involved in the regulation of pluripotency, stemness, and cancer. The aim of this study was to investigate in vitro, in vivo, and ex vivo a possible role of ZNF281 in the onset and progression of intestinal inflammation. A conceivable contribution of the protein to the development of intestinal fibrosis was also explored. Methods: Human colorectal adenocarcinoma cell line, HT29, and C57BL/6 mice were used for in vitro and in vivo studies. Mucosal biopsy specimens were taken during endoscopy from 29 pediatric patients with Crohn's disease (CD), 24 with ulcerative colitis (UC) and 16 controls. ZNF281 was knocked down by transfecting HT29 cells with 20 nM small interference RNA (siRNA) targeting ZNF281 (siZNF281). Results: We show for the first time that ZNF281 is induced upon treatment with inflammatory agents in HT29 cells, in cultured uninflamed colonic samples from CD patients and in DSS-treated mice. ZNF281 expression correlates with the disease severity degree of CD and UC patients. Silencing of ZNF281 strongly reduces both inflammatory (IL-8, IL-1beta, IL-17, IL-23) and EMT/fibrotic (SNAIL, Slug, TIMP-1, vimentin, fibronectin, and α-SMA) gene expression; besides, it abolishes the increase of extracellular-collagen level as well as the morphological modifications induced by inflammation. Conclusions: The identification of transcription factor ZNF281 as a novel player of intestinal inflammation and fibrosis allows a deeper comprehension of the pathogenetic mechanisms underlying inflammatory bowel disease (IBD) and provide a new target for their cure.
Asunto(s)
Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , Enterocolitis/genética , Mucosa Intestinal/metabolismo , Transactivadores/genética , Adolescente , Animales , Niño , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Sulfato de Dextran , Enterocolitis/metabolismo , Fibrosis , Regulación de la Expresión Génica , Células HT29 , Humanos , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Proteínas Represoras , Transactivadores/metabolismoRESUMEN
hnRNP A1 is a nucleocytoplasmic shuttling heterogeneous nuclear ribonucleoprotein that accompanies eukaryotic mRNAs from the active site of transcription to that of translation. Although the importance of hnRNP A1 as a regulator of nuclear pre-mRNA and mRNA processing and export is well established, it is unknown whether this is relevant for the control of proliferation, survival, and differentiation of normal and transformed cells. We show here that hnRNP A1 levels are increased in myeloid progenitor cells expressing the p210(BCR/ABL) oncoprotein, in mononuclear cells from chronic myelogenous leukemia (CML) blast crisis patients, and during disease progression. In addition, in myeloid progenitor 32Dcl3 cells, BCR/ABL stabilizes hnRNP A1 by preventing its ubiquitin/proteasome-dependent degradation. To assess the potential role of hnRNP A1 nucleocytoplasmic shuttling activity in normal and leukemic myelopoiesis, a mutant defective in nuclear export was ectopically expressed in parental and BCR/ABL-transformed myeloid precursor 32Dcl3 cells, in normal murine marrow cells, and in mononuclear cells from a CML patient in accelerated phase. In normal cells, expression of this mutant enhanced the susceptibility to apoptosis induced by interleukin-3 deprivation, suppressed granulocytic differentiation, and induced massive cell death of granulocyte colony-stimulating factor-treated cultures. In BCR/ABL-transformed cells, its expression was associated with suppression of colony formation and reduced tumorigenic potential in vivo. Moreover, interference with hnRNP A1 shuttling activity resulted in downmodulation of C/EBPalpha, the major regulator of granulocytic differentiation, and Bcl-X(L), an important survival factor for hematopoietic cells. Together, these results suggest that the shuttling activity of hnRNP A1 is important for the nucleocytoplasmic trafficking of mRNAs that encode proteins influencing the phenotype of normal and BCR/ABL-transformed myeloid progenitors.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Fusión bcr-abl/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucopoyesis , Ribonucleoproteínas/metabolismo , Animales , Transporte Biológico , Diferenciación Celular , Línea Celular , Supervivencia Celular , Cisteína Endopeptidasas/metabolismo , Regulación Leucémica de la Expresión Génica , Granulocitos/citología , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Ratones , Complejos Multienzimáticos/metabolismo , Mutación , Células Progenitoras Mieloides/citología , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Ribonucleoproteínas/genética , Células Tumorales Cultivadas , Ubiquitina/metabolismoRESUMEN
BACKGROUND: Large evidence supports the role of microRNAs as new important inflammatory mediators by regulating both the adaptive and innate immunity. In the present study, we speculated that miR-320 controls NOD2 (nucleotide-binding oligomerization domain) expression, because it contains multiple binding sites in the 3'-untranslated region of the gene. NOD2, the first gene associated to increased susceptibility to Crohn's disease, is a cytosolic receptor that senses wall peptides of bacteria and promotes their clearance through initiation of a proinflammatory transcriptional program. This study aims at demonstrating that NOD2 is a target of miR-320 as well as investigating the role of inflammation in modulating the miR-320 control on NOD2 expression and analyzing miR-320 expression in intestinal biopsies of children with inflammatory bowel disease. METHODS: The colonic adenocarcinoma cell line HT29 was used to assess the miR-320-mediated regulation of NOD2 expression. MiR-320 and NOD2 expression were analyzed in mucosal samples of 40 children with inflammatory bowel disease. RESULTS: During inflammation, NOD2 expression is inversely correlated with miR-320 expression in vitro and ex vivo. Exogenous miR-320 transfection in HT29 cells leads to a significant decrease of NOD2 expression, whereas the miR-320 inhibitor transfection leads to increase of NOD2 expression, nuclear translocation of nuclear factor κB, and activation of downstream cytokines. CONCLUSIONS: We show for the first time that NOD2 expression is under the control of miR-320. We also show in vitro and ex vivo that inflammation induces a decrease of miR-320 and the latter correlates negatively with NOD2 expression.
Asunto(s)
Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Regulación de la Expresión Génica , Inflamación/patología , MicroARNs/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Adolescente , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Colitis Ulcerosa/genética , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Citocinas/genética , Citocinas/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Estudios de Seguimiento , Células HT29 , Humanos , Inmunidad Innata , Técnicas para Inmunoenzimas , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Proteína Adaptadora de Señalización NOD2/genética , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Krill oil is a marine derived oil rich in phospholipids, astaxanthin and omega-3 fatty acids. Several studies have found benefits of krill oil against oxidative and inflammatory damage. AIMS: We aimed at assessing the ability of krill oil to reduce intestinal inflammation by improving epithelial barrier integrity, increasing cell survival and reducing pathogenicity of adherent-invasive Escherichia coli. METHODS: CACO2 and HT29 cells were exposed to cytomix (TNFα and IFNγ) to induce inflammation and co-exposed to cytomix and krill oil. E-cadherin, ZO-1 and F-actin levels were analyzed by immunofluorescence to assess barrier integrity. Scratch test was performed to measure wound healing. Cell survival was analyzed by flow cytometry. Adherent-invasive Escherichia coli LF82 was used for adhesion/invasion assay. RESULTS: In inflamed cells E-cadherin and ZO-1 decreased, with loss of cell-cell adhesion, and F-actin polymerization increased stress fibres; krill oil restored initial conditions and improved wound healing, reduced bacterial adhesion/invasion in epithelial cells and survival within macrophages; krill oil reduced LF82-induced mRNA expression of pro-inflammatory cytokines. CONCLUSIONS: Krill oil improves intestinal barrier integrity and epithelial restitution during inflammation and controls bacterial adhesion and invasion to epithelial cells. Thus, krill oil may represent an innovative tool to reduce intestinal inflammation.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Euphausiacea , Ácidos Grasos Omega-3/farmacología , Viabilidad Microbiana/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Actinas/metabolismo , Animales , Células CACO-2 , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Escherichia coli/patogenicidad , Escherichia coli/fisiología , Infecciones por Escherichia coli/inmunología , Células HT29 , Humanos , Interferón gamma/farmacología , Interleucina-8/genética , Interleucina-8/metabolismo , Ratones , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Cicatrización de Heridas/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismoAsunto(s)
Antibacterianos/uso terapéutico , Bacteriocinas/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Metagenómica/tendencias , Farmacorresistencia Bacteriana Múltiple/genética , Europa (Continente) , Humanos , Microbiota/genética , Probióticos/administración & dosificaciónRESUMEN
Neuroblastoma (NB) is a childhood tumor that depends on insulin-like growth factors (IGFs) for its growth and metastatic spread. Some metastatic NBs acquire independence from the paracrine support of IGF by activating autocrine production of IGF-2. Insulin-like growth factor binding protein-5 (IGFBP-5), a member of the IGF binding protein family, is able to optimize binding between IGF itself and its receptor. NB cell lines retain the ability to differentiate in vitro toward neuronal, schwann-like or melanocytic phenotypes upon treatment with retinoic acid (RA). Retinoids are currently used in NB therapy to achieve a mature postmitotic phenotype. Here, we present evidence that the expression of IGFBP-5 is a common feature of neuroblastoma cell lines and that IGFBP-5 acts in concert with IGF-2 in inducing cell proliferation. RA-induced differentiation causes a sharp increase of IGFBP-5. Functional assays carried out in differentiating conditions demonstrate that IGFBP-5 transcription is sensitive to RA treatment. We show that the effect of RA on the IGFBP-5 promoter is exerted, at least in part, through a proximal 5'-CACCC-3' tandem repeat (-147 bp to -137 bp from the transcription start site) that has previously been described as a cis-acting element involved in the progesterone-mediated response in osteoblasts. Given the relevance of IGF-2 in determining the proliferative and metastatic behavior of NB, the role of IGFBP-5 as a modulator of the IGF signal transduction pathway should be studied further for potential therapeutic applications.
Asunto(s)
Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neuroblastoma/metabolismo , Western Blotting , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Cartilla de ADN/química , Relación Dosis-Respuesta a Droga , Humanos , Factor II del Crecimiento Similar a la Insulina/metabolismo , Luciferasas/metabolismo , Mitosis , Modelos Genéticos , Mutación , Fenotipo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
BACKGROUND: Endoplasmic reticulum stress and unfolded protein response have been recently associated with the development of inflammatory bowel diseases in adults. We aimed at assessing the involvement of these pathways also in paediatric inflammatory bowel disease by analysing the expression of the main genes involved in endoplasmic reticulum stress and correlating them with the degree of intestinal inflammation. METHODS: Real-time PCR and Western blot analysis of the expression of the endoplasmic reticulum stress marker HSPA5 and of selected genes representing the three pathways of unfolded protein response (IRE-XBP1, PERK-ATF4, ATF6p90-p50) in inflamed and uninflamed biopsies from 28 inflammatory bowel disease paediatric patients and 10 controls. RESULTS: HSPA5, PDIA4, as well as unspliced and spliced XBP1 mRNAs were significantly increased in patients' inflamed colonic mucosa compared to uninflamed mucosa and controls. HSPA5, PDIA4, ATF6, and phospho-IRE proteins were also upregulated, indicating the activation of the IRE-XBP1 and ATF6p90-p50 branches of unfolded protein response. A positive significant correlation between interleukin-8 levels, as a marker of inflammation, and upregulated genes was found in the inflamed colonic mucosa. CONCLUSION: A deregulation of the genes involved in the endoplasmic reticulum stress and unfolded protein response pathways may be a key component of the inflammatory response in paediatric patients with inflammatory bowel disease.
Asunto(s)
Colon/metabolismo , Colonoscopía/métodos , Estrés del Retículo Endoplásmico , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Respuesta de Proteína Desplegada/fisiología , Colon/patología , Chaperón BiP del Retículo Endoplásmico , Femenino , Estudios de Seguimiento , Humanos , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
Advanced breast cancer cells acquire metastatic properties in response to TGFß. We show here that the expression of c-Myb increases in TGFß-treated ER (+) breast cancer cells by protein stabilization, transcription activation and release from miR200-dependent down-regulation. In particular, we mapped 2 sites for miR200b, miR200c and miR429 binding in the 3' UTR of the human c-myb gene. These microRNAs decreased the expression of c-Myb when transfected in MCF-7 cells. In addition, luciferase activity from a vector containing the 3' UTR of the c-myb gene was inhibited by miR200s through a binding-dependent mechanism. siRNA- and shRNA-mediated down-regulation was used to investigate the role of c-Myb for the effects induced by TGFß in ER(+) breast cancer MCF-7 and ZR-75.1 cells. Transfection with c-Myb siRNAs blocked the increase of Slug (SNAI2) and Bcl-2 expression and reversed the decrease in E-cadherin expression induced by TGF-ß treatment. Conversely, c-Myb down-regulation decreased invasion and anchorage-independent growth of breast cancer cells expressing a constitutively active TGFß receptor I. Finally, apoptosis induced by etoposide increased in c-Myb-silenced TGFß-treated ER(+) cell lines. In summary, exposure of ER(+) breast cancer cells to TGFß induces an increase of c-Myb expression which is required for expression of EMT-associated markers, in vitro invasion and anchorage-independent growth. Furthermore, our findings suggest a potentially detrimental effect of TGFß and c-Myb co-expression in breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-myb/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Clonación Molecular , Transición Epitelial-Mesenquimal , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Etopósido/farmacología , Femenino , Citometría de Flujo , Genes myb , Humanos , Lentivirus/genética , Lentivirus/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Mutagénesis Sitio-Dirigida , Invasividad Neoplásica , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-myb/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
Signal transduction mediated by insulin-like growth factors is implicated in the aggressive behavior of neuroblastoma (NB), a childhood tumor originating from the neural crest. IGFBP-5, a protein that binds IGFs with high affinity, is expressed in many NB cell lines exerting opposite effects, depending on its concentration. We found that IGFBP-5 expression increased during retinoic acid (RA)-mediated differentiation of NB cells. This was due to transcriptional activation as demonstrated by reporter assays carried out in basal and differentiating conditions. We defined the shortest region of the human IGFBP-5 promoter (from nucleotide -83 to +53) which is sensitive to RA. Mutation of a CCAAT enhancer binding protein (C/EBP) element inside this region increased transcription, suggesting a repressive role of this sequence. DNA Affinity Precipitation Assays (DAPA) and chromatin immunoprecipitation demonstrated that the binding of C/EBPalpha and beta to the C/EBP site decreased upon treatment with RA. C/EBPalpha and beta induced an increase in IGFBP-5 transcription in human and murine NB cells similar to that obtained upon RA treatment. Activation by C/EBP alpha and beta did not depend on their binding to the C/EBP site, since they still activated IGFBP-5 promoter carrying a mutation in the C/EBP site. Of interest, we found that both transcription factors were able to interact with the TATA box, but only C/EBPalpha interaction increased during RA-induced differentiation.