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1.
Sci Rep ; 12(1): 395, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013445

RESUMO

The aetiology of Amyotrophic Lateral Sclerosis (ALS) is still poorly understood. The discovery of genetic forms of ALS pointed out the mechanisms underlying this pathology, but also showed how complex these mechanisms are. Excitotoxicity is strongly suspected to play a role in ALS pathogenesis. Excitotoxicity is defined as neuron damage due to excessive intake of calcium ions (Ca2+) by the cell. This study aims to find a relationship between the proteins coded by the most relevant genes associated with ALS and intracellular Ca2+ accumulation. In detail, the profile of eight proteins (TDP-43, C9orf72, p62/sequestosome-1, matrin-3, VCP, FUS, SOD1 and profilin-1), was analysed in three different cell types induced to raise their cytoplasmic amount of Ca2+. Intracellular Ca2+ accumulation causes a decrease in the levels of TDP-43, C9orf72, matrin3, VCP, FUS, SOD1 and profilin-1 and an increase in those of p62/sequestosome-1. These events are associated with the proteolytic action of two proteases, calpains and caspases, as well as with the activation of autophagy. Interestingly, Ca2+ appears to both favour and hinder autophagy. Understanding how and why calpain-mediated proteolysis and autophagy, which are physiological processes, become pathological may elucidate the mechanisms responsible for ALS and help discover new therapeutic targets.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Autofagia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Calpaína/metabolismo , Caspases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Profilinas/genética , Profilinas/metabolismo , Proteólise , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Fatores de Tempo , Transcriptoma , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
2.
J Pathol ; 257(1): 82-95, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35064579

RESUMO

Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH). The role of OSM was investigated in (1) selected cohorts of NAFLD/NASH HCC patients, (2) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM, (3) murine HCC xenografts, and (4) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM upregulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Oncostatina M , Animais , Carcinogênese , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia
3.
Cancers (Basel) ; 11(12)2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31817100

RESUMO

BACKGROUND: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2α-dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1α and HIF-2α. SB3 acts by (i) up-regulating HIF-1α transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2α stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2α transcripts; (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2α transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2α transcripts. Conclusions: These data outline either HIF-2α and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.

4.
Environ Health Perspect ; 124(6): 776-84, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26685284

RESUMO

BACKGROUND: Chrysotile asbestos accounts for > 90% of the asbestos used worldwide, and exposure is associated with asbestosis (asbestos-related fibrosis) and other malignancies; however, the molecular mechanisms involved are not fully understood. A common pathogenic mechanism for these malignancies is represented by epithelial-mesenchymal transition (EMT), through which epithelial cells undergo a morphological transformation to assume a mesenchymal phenotype. In the present work, we propose that chrysotile asbestos induces EMT through a mechanism involving a signaling pathway mediated by tranforming growth factor beta (TGF-ß). OBJECTIVES: We investigated the role of chrysotile asbestos in inducing EMT in order to elucidate the molecular mechanisms involved in this event. METHODS: Human bronchial epithelial cells (BEAS-2B) were incubated with 1 µg/cm2 chrysotile asbestos for ≤ 72 hr, and several markers of EMT were investigated. Experiments with specific inhibitors for TGF-ß, glycogen synthase kinase-3ß (GSK-3ß), and Akt were performed to confirm their involvement in asbestos-induced EMT. Real-time polymerase chain reaction (PCR), Western blotting, and gelatin zymography were performed to detect mRNA and protein level changes for these markers. RESULTS: Chrysotile asbestos activated a TGF-ß-mediated signaling pathway, implicating the contributions of Akt, GSK-3ß, and SNAIL-1. The activation of this pathway in BEAS-2B cells was associated with a decrease in epithelial markers (E-cadherin and ß-catenin) and an increase in mesenchymal markers (α-smooth muscle actin, vimentin, metalloproteinases, and fibronectin). CONCLUSIONS: Our findings suggest that chrysotile asbestos induces EMT, a common event in asbestos-related diseases, at least in part by eliciting the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 pathway. CITATION: Gulino GR, Polimeni M, Prato M, Gazzano E, Kopecka J, Colombatto S, Ghigo D, Aldieri E. 2016. Effects of chrysotile exposure in human bronchial epithelial cells: insights into the pathogenic mechanisms of asbestos-related diseases. Environ Health Perspect 124:776-784; http://dx.doi.org/10.1289/ehp.1409627.


Assuntos
Asbestos Serpentinas/toxicidade , Linhagem Celular , Transição Epitelial-Mesenquimal/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Fatores de Transcrição da Família Snail/metabolismo
5.
Oncotarget ; 6(4): 2206-21, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25544768

RESUMO

SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells. Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively. Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.


Assuntos
Antígenos de Neoplasias/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Serpinas/genética , Regulação para Cima , Antígenos de Neoplasias/metabolismo , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células HT29 , Células Hep G2 , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/metabolismo
6.
Histol Histopathol ; 29(1): 33-44, 2014 01.
Artigo em Inglês | MEDLINE | ID: mdl-23996844

RESUMO

Fibrogenic progression of chronic liver diseases (CLDs) towards the end-point of cirrhosis is currently regarded, whatever the aetiology, as a dynamic and highly integrated cellular response to chronic liver injury. Liver fibrogenesis (i.e., the process) is sustained by hepatic populations of highly proliferative, pro-fibrogenic and contractile myofibroblast-like cells (MFs) that mainly originate from hepatic stellate cells (HSC) or, to a less extent, from portal fibroblasts or bone marrow-derived cells. As is well known, liver fibrosis (i.e., the result) is accompanied by perpetuation of liver injury, chronic hepatitis and persisting activation of tissue repair mechanisms, leading eventually to excess deposition of extracellular matrix (ECM) components. In this scenario, hypoxic areas represent a very common and major feature of fibrotic and cirrhotic liver during the progression of CLDs. Cells exposed to hypoxia respond by means of heterodimeric hypoxia-inducible factors (HIFs) that translocate into the nucleus and binds to a specific core sequence defined hypoxia-responsive element (HRE), present in the promoter on several genes which are considered as hypoxia-regulated target genes. HIFs transcription factors can activate a complex genetic program designed to sustain several changes necessary to efficiently counteract the decrease in oxygen tension. Accordingly, hypoxia, through up-regulation of angiogenesis, is currently believed to significantly contribute to fibrogenic progression of CLDs, mostly by affecting the pro-fibrogenic and pro-angiogenic behaviour of hepatic MFs. In addition, experimental and clinical evidence generated in the last decade also indicates that angiogenesis and fibrogenesis in CLDs may also be sustained by HIF-dependent but hypoxia-independent mediators.


Assuntos
Hipóxia Celular/fisiologia , Hepatopatias/metabolismo , Hepatopatias/patologia , Neovascularização Patológica/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doença Crônica , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Miofibroblastos/metabolismo , Neovascularização Patológica/patologia , Proteínas Repressoras
7.
Expert Opin Ther Targets ; 18(2): 121-35, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24325753

RESUMO

INTRODUCTION: Cyclooxygenase-2 (COX-2) is overexpressed in several malignancies and is implicated in breast cancer progression. OBJECTIVES: We investigated whether changes in COX-2 expression may affect epithelial-to-mesenchymal transition (EMT) and then invasive potential of human breast cancer cells, in relationship with hypoxia. COX-2-null MCF-7 human breast cancer cells, MCF-7 cells transiently expressing COX-2 and COX-2-expressing MDA-MB-231 cells were employed. RESULTS: COX-2 overexpression resulted in downregulation of E-cadherin and ß-catenin, upregulation of vimentin, N-cadherin and SNAI1, suggesting EMT occurrence. COX-2-overexpressing MCF-7 cells were also characterized by increased invasiveness and release of matrix-metalloproteinase-9. The above-mentioned characteristics, homologous to those detected in highly invasive MDA-MB-231 cells, were reverted by treatment of COX-2-overexpressing MCF-7 cells with celecoxib, a COX-2-specific inhibitor, partly through the inhibition of COX-2-related intracellular generation of reactive oxygen species. Hypoxia further exacerbated COX-2 expression, EMT changes and invasive ability in both COX-2-overexpressing MCF-7 cells and MDA-MB-231 cells. Finally, immunohistochemistry performed on samples from normal and neoplastic human breast tissues revealed that COX-2-positive malignant cells were also positive for EMT-related antigens, hypoxia-inducible factor (HIF)-2α and the oxidative stress marker heme oxygenase. CONCLUSIONS: These findings support the existence of a direct link between COX-2 overexpression, EMT and invasiveness in human breast cancer cells, emphasizing the role of hypoxic microenvironment.


Assuntos
Neoplasias da Mama/metabolismo , Ciclo-Oxigenase 2/metabolismo , Transição Epitelial-Mesenquimal , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , DNA Complementar/genética , Dinoprostona/metabolismo , Feminino , Heme Oxigenase-1/metabolismo , Humanos , Hipóxia/metabolismo , Células MCF-7 , Invasividade Neoplásica , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo
8.
J Pathol ; 226(4): 588-97, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21959987

RESUMO

Liver fibrogenesis is sustained by pro-fibrogenic myofibroblast-like cells (MFs), mainly originating from activated hepatic stellate cells (HSC/MFs) or portal (myo)fibroblasts, and is favoured by hypoxia-dependent angiogenesis. Human HSC/MFs were reported to express vascular-endothelial growth factor (VEGF) and VEGF-receptor type 2 and to migrate under hypoxic conditions. This study was designed to investigate early and delayed signalling mechanisms involved in hypoxia-induced migration of human HSC/MFs. Signal transduction pathways and intracellular generation of reactive oxygen species (ROS) were evaluated by integrating morphological, cell, and molecular biology techniques. Non-oriented and oriented migration were evaluated by using wound healing assay and the modified Boyden's chamber assay, respectively. The data indicate that hypoxia-induced migration of HSC/MFs is a biphasic process characterized by the following sequence of events: (a) an early (15 min) and mitochondria-related increased generation of intracellular ROS which (b) was sufficient to switch on activation of ERK1/2 and JNK1/2 that were responsible for the early phase of oriented migration; (c) a delayed and HIF-1α-dependent increase in VEGF expression (facilitated by ROS) and its progressive, time-dependent release in the extracellular medium that (d) was mainly responsible for sustained migration of HSC/MFs. Finally, immunohistochemistry performed on HCV-related fibrotic/cirrhotic livers revealed HIF-2α and haem-oxygenase-1 positivity in hepatocytes and α-SMA-positive MFs, indicating that MFs were likely to be exposed in vivo to both hypoxia and oxidative stress. In conclusion, hypoxia-induced migration of HSC/MFs involves an early, mitochondrial-dependent ROS-mediated activation of ERK and JNK, followed by a delayed- and HIF-1α-dependent up-regulation and release of VEGF.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Movimento Celular/fisiologia , Células Estreladas do Fígado/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Células Cultivadas , Cultura em Câmaras de Difusão , Inativação Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Estreladas do Fígado/citologia , Hepatite C/metabolismo , Hepatite C/patologia , Hepatite C/virologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Mitocôndrias/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologia
9.
Amino Acids ; 42(2-3): 783-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21901470

RESUMO

Epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition are biologic processes responsible for conversion of epithelial cells into a mesenchymal phenotype or viceversa, respectively. They occur during embryo- and foetal-development and, in adult organisms, are involved in wound healing, in the genesis and progression of organ fibrosis as well as in the invasiveness of epithelial cancer cells. The key event of epithelial-to-mesenchymal transition is the loss of E-cadherin expression due to repressor activity of the transcriptional factor Snai1. Intracellular Snai1 levels are controlled through translational and post-translational events such as phosphorylation and de-phosphorylation, potentially modulated by polyamine content. Epithelial MDCK cells exposed to TGF-ß(1) acquired a fibroblastoid phenotype and expressed mesenchymal markers. These changes were emphasized in cells that were also exposed to DFMO in order to decrease the intracellular levels of polyamines. Addition of exogenous polyamines almost completely abolished the combined action of DFMO and TGF-ß(1) and rapidly reverted to epithelial phenotype MDCK cells previously undergone to mesenchymal phenotype. Nuclear extracts of cells treated with DFMO + TGF-ß(1) revealed the presence of Snai1 immunopositive bands in a range of molecular weight between 55 and 72 kDa, with additional positive bands detected at MW greater than 170 kDa. Same bands resulted positive to anti-Sumo 2/3 antibody, suggesting that an intracellular low level of polyamines favours Snai1 nuclear accumulation under the form of polysumoylated proteins.


Assuntos
Poliaminas Biogênicas/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Animais , Western Blotting , Linhagem Celular , Cães , Humanos , Fosforilação
10.
Amino Acids ; 42(2-3): 769-74, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21901471

RESUMO

During our study of the cytostatic effect of agmatine, we were able to isolate an agmatine resistant clone from a parental hepatoma cell line, HTC. These cells, called Agres, had slower growth rate than the parental cells when cultured in normal medium. The modification in polyamine content induced by agmatine was much lower in these cells and ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine/spermine acetyltransferase activities were much less affected. By investigating the mechanism responsible for these modifications, it was shown that agmatine and polyamines were not taken up by Agres cells. Their resistance to the antiproliferative effects of agmatine may thus arise from a lack of the polyamine transport system. Moreover, Agres cells were able to take up both glutamic acid and arginine at a rate significantly higher than that detected for HTC cells, most likely to provide components for compensatory increase of PA synthesis. These results emphasize the importance of polyamine transport for cell growth.


Assuntos
Agmatina/farmacologia , Neoplasias Hepáticas Experimentais/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Ratos
11.
Stem Cells ; 29(6): 952-63, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21563276

RESUMO

Hypoxic conditions have been reported to facilitate preservation of undifferentiated mesenchymal stem cell (MSC) phenotype and positively affect their colony-forming potential, proliferation, and migration/mobilization. In this study, designed to dissect mechanisms underlying hypoxia-dependent migration of bone marrow-derived human MSC (hMSC), signal transduction, and molecular mechanisms were evaluated by integrating morphological, molecular, and cell biology techniques, including the wound healing assay (WHA) and modified Boyden's chamber assay (BCA) to monitor migration. Exposure of hMSCs to moderate hypoxia resulted in a significant increase of migration of hMSCs in both WHA (from 6 to 20 hours) and BCA (within 6 hours). Mechanistic experiments outlined the following sequence of hypoxia-dependent events: (a) very early (15 minutes) increased generation of intracellular reactive oxygen species (ROS), which (b) was sufficient to switch on activation of extracellular regulated kinase 1/2 and c-Jun N-terminal protein kinase 1/2, found to be relevant for the early phase of hMSC migration; (c) hypoxia inducible factor-1 (HIF-1)-dependent increased expression of vascular endothelial growth factor (VEGF) (facilitated by ROS) and its progressive release that was responsible for (d) a delayed and sustained migration of hMSCs. These results suggest that hypoxia-dependent migration relies on a previously unrecognized biphasic scenario involving an early phase, requiring generation of ROS, and a delayed phase sustained by HIF-1-dependent expression and release of VEGF.


Assuntos
Células da Medula Óssea/fisiologia , Movimento Celular , Células-Tronco Mesenquimais/fisiologia , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Hipóxia Celular , Células Cultivadas , Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estabilidade Proteica , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
J Hepatol ; 54(5): 964-74, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21145826

RESUMO

BACKGROUND & AIMS: Liver fibrogenesis is sustained by myofibroblast-like cells originating from hepatic stellate cells (HSC/MFs), portal fibroblasts or bone marrow-derived cells, including mesenchymal stem cells (MSCs). Herein, we investigated the mechanistic role of intracellular generation of reactive oxygen species (ROS) and redox-sensitive signal transduction pathways in mediating chemotaxis, a critical profibrogenic response for human HSC/MFs and for MSC potentially engrafting chronically injured liver. METHODS: Intracellular generation of ROS and signal transduction pathways were evaluated by integrating morphological and molecular biology techniques. Chemokinesis and chemotaxis were evaluated by wound healing assay and modified Boyden's chamber assay, respectively. Additional in vivo evidence was obtained in human specimens from HCV-related cirrhosis. RESULTS: Human MSCs and HSC/MFs migrate in response to a panel of polypeptide chemoattractants and extracellularly generated superoxide anion. All polypeptides induced a NADPH-oxidase-dependent intracellular rise in ROS, resulting in activation of ERK1/2 and JNK1/2. Moreover, menadione or 2,3-dimethoxy-1,4-naphthoquinone, which generate intracellular superoxide anion or hydrogen peroxide, respectively, induced ERK1/2 and JNK1/2 activation and migration. JNK1 activation was predominant for migration as shown by specific silencing. Finally, activation of ERK1/2 and JNK1/2 was found in extracts obtained from HSC/MFs during the course of an oxidative stress-mediated model of liver injury and phosphorylated JNK1/2 isoforms were detected in α-smooth muscle actin-positive myofibroblasts lining fibrotic septa in human cirrhotic livers. CONCLUSIONS: Intracellular generation of ROS, through activation of specific signaling pathways, is a critical event for directional migration of HSC/MFs and MSCs.


Assuntos
Células da Medula Óssea/citologia , Células Estreladas do Fígado/citologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Células-Tronco Mesenquimais/citologia , Espécies Reativas de Oxigênio/metabolismo , Células da Medula Óssea/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Fatores Quimiotáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Estreladas do Fígado/metabolismo , Hepatite C Crônica/metabolismo , Hepatite C Crônica/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Mesenquimais/metabolismo
13.
Lab Invest ; 90(6): 929-39, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20212449

RESUMO

Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis.


Assuntos
Rim/citologia , Rim/fisiologia , Mesoderma/fisiologia , Animais , Comunicação Celular/fisiologia , Cães , Regulação para Baixo , Desenvolvimento Embrionário , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Metaloproteinases da Matriz/metabolismo , Mesoderma/efeitos dos fármacos , Poliaminas/metabolismo , Desnaturação Proteica , RNA Mensageiro/genética , Espermidina Sintase/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia
14.
Antioxid Redox Signal ; 12(12): 1383-430, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19903090

RESUMO

Epithelial to mesenchymal transition (EMT) is a fundamental process, paradigmatic of the concept of cell plasticity, that leads epithelial cells to lose their polarization and specialized junctional structures, to undergo cytoskeleton reorganization, and to acquire morphological and functional features of mesenchymal-like cells. Although EMT has been originally described in embryonic development, where cell migration and tissue remodeling have a primary role in regulating morphogenesis in multicellular organisms, recent literature has provided evidence suggesting that the EMT process is a more general biological process that is also involved in several pathophysiological conditions, including cancer progression and organ fibrosis. This review offers first a comprehensive introduction to describe major relevant features of EMT, followed by sections dedicated on those signaling mechanisms that are known to regulate or affect the process, including the recently proposed role for oxidative stress and reactive oxygen species (ROS). Current literature data involving EMT in both physiological conditions (i.e., embryogenesis) and major human diseases are then critically analyzed, with a special final focus on the emerging role of hypoxia as a relevant independent condition able to trigger EMT.


Assuntos
Desdiferenciação Celular/fisiologia , Células Epiteliais/citologia , Mesoderma/citologia , Hipóxia Celular , Polaridade Celular , Citoesqueleto/ultraestrutura , Desenvolvimento Embrionário/fisiologia , Células Endoteliais/citologia , Proteínas da Matriz Extracelular/fisiologia , Fibrose/patologia , Regulação da Expressão Gênica , Humanos , MicroRNAs/fisiologia , Neoplasias/patologia , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Cicatrização/fisiologia
15.
Int J Biochem Cell Biol ; 41(11): 2089-93, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19782946

RESUMO

Hepatic myofibroblasts constitute a heterogeneous population of highly proliferative, pro-fibrogenic, pro-inflammatory, pro-angiogenic and contractile cells that sustain liver fibrogenesis and then fibrotic progression of chronic liver diseases of different aetiology to the common advanced-stage of cirrhosis. These alpha-smooth muscle actin-positive myofibroblast-like cells, according to current literature, mainly originate by a process of activation and trans-differentiation that involves either hepatic stellate cells or fibroblasts of portal areas. Hepatic myofibroblasts can also originate from bone marrow-derived cells, including mesenchymal stem cells or circulating fibrocytes able to engraft chronically injured liver, as well as, in certain conditions, by a process of epithelial to mesenchymal transition involving hepatocytes and cholangiocytes. Hepatic myofibroblasts may have also additional crucial roles in modulating immune response and in the cross talk with hepatic progenitor (stem) cells as well as with malignant cells of either primary hepatocellular carcinomas or of metastatic cancers.


Assuntos
Fibroblastos/patologia , Cirrose Hepática/patologia , Fígado/patologia , Animais , Células Estreladas do Fígado/patologia , Humanos
16.
Am J Physiol Cell Physiol ; 296(6): C1411-9, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19321739

RESUMO

Agmatine, an endogenous metabolite of arginine, selectively suppresses growth in cells with high proliferative kinetics, such as transformed cells, through depletion of intracellular polyamine levels. In the present study, we depleted intracellular polyamine content with agmatine to determine if attrition by cell death contributes to the growth-suppressive effects. We did not observe an increase in necrosis, DNA fragmentation, or chromatin condensation in Ha-Ras-transformed NIH-3T3 cells administered agmatine. In response to Ca(2+)-induced oxidative stress in kidney mitochondrial preparations, agmatine demonstrated attributes of a free radical scavenger by protecting against the oxidation of sulfhydryl groups and decreasing hydrogen peroxide content. The functional outcome was a protective effect against Ca(2+)-induced mitochondrial swelling and mitochondrial membrane potential collapse. We also observed decreased expression of proapoptotic Bcl-2 family members and of execution caspase-3, implying antiapoptotic potential. Indeed, we found that apoptosis induced by camptothecin or 5-fluorourocil was attenuated in cells administered agmatine. Agmatine may offer an alternative to the ornithine decarboxylase inhibitor difluoromethyl ornithine for depletion of intracellular polyamine content while avoiding the complications of increasing polyamine import and reducing the intracellular free radical scavenger capacity of polyamines. Depletion of intracellular polyamine content with agmatine suppressed cell growth, yet its antioxidant capacity afforded protection from mitochondrial insult and resistance to cellular apoptosis. These results could explain the beneficial outcomes observed with agmatine in models of injury and disease.


Assuntos
Agmatina/metabolismo , Antioxidantes/metabolismo , Apoptose , Arginina/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Camptotecina/toxicidade , Caspase 3/metabolismo , Proliferação de Células , Citoproteção , Fragmentação do DNA , Fluoruracila/toxicidade , Rim/efeitos dos fármacos , Rim/patologia , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial , Células NIH 3T3 , Poliaminas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos
17.
J Neurochem ; 108(4): 1045-56, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19196431

RESUMO

While it is well established that stroke and cerebral hypoperfusion are both significant risk factors for Alzheimer's disease, the molecular link between ischemia and amyloid precursor protein processing has only been recently established. Specifically, hypoxia significantly increases beta-site APP cleaving enzyme (BACE1) gene transcription through the over-expression of hypoxia inducible factor 1alpha, resulting in increased BACE1 secretase activity and amyloid-beta production. In this study, we significantly extend these findings both in vitro, in differentiated SK-N-BE neuroblastoma cells, and in vivo, in rats subjected to cerebral ischemia, showing that hypoxia up-regulates BACE1 expression through a biphasic mechanism. The early post-hypoxic up-regulation of BACE1 depends on the production of reactive oxygen species mediated by the sudden interruption of the mitochondrial electron transport chain, while the later expression of BACE1 is caused by hypoxia inducible factor 1alpha activation. The involvement of reactive oxygen species released by mitochondria in the BACE1 up-regulation was confirmed by the complete protection exerted by complex I inhibitors such as rotenone and diphenyl-phenylen iodonium. Moreover, the oxidative stress-mediated up-regulation of BACE1 is mediated by c-jun N terminal kinase pathway as demonstrated by the protection exerted by the silencing of c-jun N-terminal kinase isoforms 1 and 2. Our study strengthens the hypothesis that oxidative stress is a basic common mechanism of amyloid-beta accumulation.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Estresse Oxidativo/fisiologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/fisiopatologia , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Desacopladores/farmacologia , Regulação para Cima/fisiologia
18.
Int J Biochem Cell Biol ; 41(3): 586-94, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18708157

RESUMO

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.


Assuntos
Cobalto/toxicidade , Hepatócitos/fisiologia , Mitocôndrias Hepáticas/fisiologia , Estresse Oxidativo/fisiologia , Animais , Apoptose/fisiologia , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Fracionamento Celular , Células Cultivadas , Cobalto/análise , Citocromos c/análise , Hepatócitos/ultraestrutura , Peróxido de Hidrogênio/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Membranas Intracelulares , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias Hepáticas/química , Permeabilidade , Ratos , Ratos Wistar
19.
Carcinogenesis ; 29(12): 2267-78, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18791199

RESUMO

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.


Assuntos
Hipóxia Celular/fisiologia , Transformação Celular Neoplásica/metabolismo , Transdução de Sinais/fisiologia , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Epitélio/metabolismo , Epitélio/patologia , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mesoderma/metabolismo , Mesoderma/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Oxirredução , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
20.
Int J Biochem Cell Biol ; 40(9): 1861-71, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18343708

RESUMO

Disruption of cell-to-cell contacts, as observed in many pathophysiological conditions, prime hepatocytes for compensatory hyperplastic response that involves induction of several genes, including proto-oncogenes and other gene targets of beta-catenin signaling pathway. By using cultured hepatocytes and experimental models of adherens junction disruption we have investigated changes in beta-catenin subcellular localization and their relationships with inducible nitric oxide synthase (iNOS) expression. Two experimental models were employed: (a) rat hepatocytes obtained by collagenase liver perfusion within the first 48 h of culture; (b) 48-h old cultured hepatocytes, transiently transfected or not with a plasmid encoding for dominant/negative inhibitory kappa B-alpha, exposed to ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid/LiCl treatment. beta-Catenin signaling and cellular localization, iNOS expression and nuclear factor kappaB involvement, were investigated using morphological, cell and molecular biology techniques. E-cadherin-mediated disruption of cell-to-cell contacts induces early beta-catenin translocation from membrane to cytoplasm and nuclear compartments, events that are followed by up-regulation of c-myc, cyclin D1 and beta-transducin repeat-containing protein expression. This, in turn, resulted eventually in iNOS induction that was mechanistically related to nuclear factor kappaB activation, as unequivocally shown in cells expressing dominant negative inhibitory kappa B-alpha. Our data indicate that E-cadherin disassembly and concomitant inactivation of glycogen synthase kinase-3beta result in nuclear factor kappaB-dependent induction of iNOS in hepatocytes.


Assuntos
Hepatócitos/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Regulação para Cima , beta Catenina/metabolismo , Animais , Caderinas/metabolismo , Bovinos , Comunicação Celular , Células Cultivadas , Ácido Egtázico/farmacologia , Indução Enzimática/efeitos dos fármacos , Hepatócitos/enzimologia , Cloreto de Lítio/farmacologia , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Transporte Proteico , Ratos , Reprodutibilidade dos Testes , Regulação para Cima/efeitos dos fármacos , Proteínas Contendo Repetições de beta-Transducina/metabolismo
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