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1.
Curr Probl Cardiol ; 46(3): 100418, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31030854

RESUMO

Non-ST-segment elevation acute coronary syndromes (NSTE-ACSs) are a group of clinical conditions characterized by acute myocardial ischemia. Conventional echocardiography is generally used to evaluate cardiac function using wall motion analysis and left ventricular ejection fraction but may be insufficient to explore all the complex features of NSTE-ACSs, which may vary substantially from patient to patient in terms of severity of ischemia and extent of involved myocardium. In the last years, speckle tracking echocardiography (STE) has become a widely available technique for the non-invasive assessment of cardiac function and has been repeatedly applied in the setting of NSTE-ACSs. In this review we summarize current evidence about the use of STE in patients with NSTE-ACSs, trying to underline advantages and limitations in comparison with conventional echocardiography for: diagnosis of NSTE-ACS, differential diagnosis, identification of high-risk patients, and prediction of outcome.


Assuntos
Síndrome Coronariana Aguda , Síndrome Coronariana Aguda/diagnóstico por imagem , Ecocardiografia , Humanos , Volume Sistólico , Função Ventricular Esquerda
2.
Osteoarthritis Cartilage ; 25(4): 600-610, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27914878

RESUMO

OBJECTIVE: Nutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1). METHODS: Human primary and C-28/I2 chondrocytes were pre-treated with 100 µM HT 30 min before 100 µM H2O2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes. RESULTS: MiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H2O2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H2O2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2. CONCLUSIONS: These results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.


Assuntos
Morte Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , MicroRNAs/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Álcool Feniletílico/análogos & derivados , Sirtuína 1/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/genética , MicroRNAs/genética , Oxidantes/farmacologia , Álcool Feniletílico/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Sirtuína 1/genética , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética
3.
Osteoarthritis Cartilage ; 24(6): 1082-91, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26805019

RESUMO

OBJECTIVE: Autophagy dysfunction has been reported in osteoarthritis (OA) cartilage. The objective of this study was to investigate the role of microRNA-155 (miR-155), which is overexpressed in OA, in the regulation of autophagy in human chondrocytes. DESIGN: Rapamycin (50 nM) and 2-deoxyglucose (2-DG) (5 mM) were used to stimulate autophagy in primary human articular chondrocytes and in the T/C28a2 human chondrocyte cell line. Cells were transfected with LNA GapmeR or mimic specific for miR-155 and autophagy flux was assessed by LC3 western blotting and by Cyto-ID(®) dye quantification in autophagic vacuoles. Expression of predicted miR-155 targets in the autophagy pathway were analyzed by real-time PCR and western blotting. RESULTS: Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy regulators including Ulk1, FoxO3, Atg14, Atg5, Atg3, Gabarapl1, and Map1lc3. CONCLUSION: MiR-155 is an inhibitor of autophagy in chondrocytes and contributes to the autophagy defects in OA.


Assuntos
Autofagia , Cartilagem Articular , Células Cultivadas , Condrócitos , Humanos , MicroRNAs , Osteoartrite
4.
Apoptosis ; 19(10): 1471-83, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25015553

RESUMO

Chondrocyte death and loss of extracellular matrix are the central features in articular cartilage degeneration during osteoarthritis pathogenesis. Cartilage diseases and, in particular, osteoarthritis are widely correlated to apoptosis but, chondrocytes undergoing apoptosis "in vivo" more often display peculiar features that correspond to a distinct process of programmed cell death termed "chondroptosis". Programmed cell death of primary human chondrocyte has been here investigated in micromasses, a tridimensional culture model, that represents a convenient means for studying chondrocyte biology. Cell death has been induced by different physical or chemical apoptotic agents, such as UVB radiation, hyperthermia and staurosporine delivered at both 1 and 3 weeks maturation. Conventional electron microscopy was used to analyse morphological changes. Occurrence of DNA fragmentation and caspase involvement were also investigated. At Transmission Electron Microscopy, control cells appear rounding or slightly elongated with plurilobated nucleus and diffusely dispersed chromatin. Typically UVB radiation and staurosporine induce chromatin apoptotic features, while hyperthermia triggers the "chondroptotic" phenotype. A weak TUNEL positivity appears in control, correlated to the well known cell death patterns occurring along cartilage differentiation. UVB radiation produces a strong positivity, mostly localized at the micromass periphery. After hyperthermia a higher number of fluorescent nuclei appears, in particular at 3 weeks. Staurosporine evidences a diffuse, but reduced, positivity. Therefore, DNA fragmentation is a common pattern in dying chondrocytes, both in apoptotic and "chondroptotic" cells. Moreover, all triggers induce caspase pathway activation, even if to a different extent, suggesting a fundamental role of apoptotic features, in chondrocyte cell death.


Assuntos
Apoptose , Cartilagem Articular/citologia , Condrócitos/citologia , Osteoartrite/fisiopatologia , Cartilagem Articular/metabolismo , Cartilagem Articular/efeitos da radiação , Cartilagem Articular/ultraestrutura , Caspases/metabolismo , Morte Celular , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/efeitos da radiação , Condrócitos/ultraestrutura , Fragmentação do DNA , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Osteoartrite/enzimologia , Raios Ultravioleta
5.
Amino Acids ; 42(2-3): 667-78, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21814786

RESUMO

Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.


Assuntos
Poliaminas Biogênicas/metabolismo , Diferenciação Celular , Condrócitos/patologia , Osteoartrite/patologia , Condrócitos/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Confocal , Osteoartrite/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
6.
Amino Acids ; 38(2): 525-31, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19960215

RESUMO

Recent studies have shown that aldosterone may play a critical role in the transition to heart failure and that heart is a direct target of the action of aldosterone, which can provoke hypertrophy and apoptosis of isolated cardiomyocytes and also increase the expression of genes that favor tissue fibrosis. Early work from this and other laboratories has established a link between the aliphatic polyamines and cardiac hypertrophy, while more recently an involvement of polyamines even in cell death and survival has emerged. In the present study we have treated cardiac cells, i.e. rat H9c2 cardiomyoblasts and neonatal cardiomyocytes, with (D, L)-2-(difluoromethyl)ornithine, a specific inhibitor of polyamine biosynthesis, to investigate the effects of polyamines in relation to the hypertrophic, pro-fibrotic and pro-apoptotic actions of aldosterone. The results indicate that inhibition of polyamine biosynthesis may prevent or attenuate the adverse actions of aldosterone, by modulating the expression of genes related to cardiac hypertrophy and fibrosis, as well as the levels of proteins and the activities of enzymes that control apoptosis.


Assuntos
Aldosterona/farmacologia , Eflornitina/farmacologia , Cardiopatias/patologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Poliaminas Biogênicas/biossíntese , Células Cultivadas , Eflornitina/química , Fibrose/metabolismo , Expressão Gênica/efeitos dos fármacos , Cardiopatias/tratamento farmacológico , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Hipertrofia/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Wistar
7.
Amino Acids ; 33(2): 197-202, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17578652

RESUMO

Growing evidence suggests a role for polyamines in apoptosis, although the relationship appears to be complex. alpha-Difluoromethylornithine (DFMO), a largely used ornithine decarboxylase inhibitor, is cytostatic, hardly cytotoxic and may even increase the resistance of tumour cells to some apoptotic stimuli. This may represent a problem in cancer therapy, where the killing of tumoral cells would be a desired effect, but could be an advantage in other pathological contexts related to an excess of apoptosis, such as cardiovascular diseases, stem cell transplantation, arthritis and infections. In different cellular models, polyamine depletion following treatment with polyamine biosynthesis inhibitors appears to inhibit mitochondrial and death receptor pathways of apoptosis by affecting key proteins. These studies indicate that inhibition of polyamine biosynthesis may prevent or reduce the apoptotic response triggered by a variety of stimuli in non-tumoral cells, such as cardiac cells, stem cells, chondrocytes, macrophages and intestinal epithelial cells.


Assuntos
Apoptose/efeitos dos fármacos , Poliaminas Biogênicas/biossíntese , Amidinas/farmacologia , Animais , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Eflornitina/farmacologia , Humanos , Indanos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Inibidores da Ornitina Descarboxilase , Peptídeo Hidrolases/metabolismo , Putrescina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermidina/metabolismo
8.
Amino Acids ; 27(2): 207-14, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15378410

RESUMO

In a previous research, we have shown that adequate levels of polyamines are required in transformed mouse fibroblasts for the correlated activations of MAPK subtypes (ERK and JNK) and caspases induced by etoposide and leading to apoptosis. We report now that the treatment of fibroblasts with etoposide also elicited a progressive and sustained increase of NF-kappaB activation. The DNA binding activity of p65 NF-kappaB subunit was increased up to approximately 4-fold and was accompanied by enhancement of p65 phosphorylation. A two days pre-treatment of fibroblasts with alpha-difluoromethylornithine (DFMO), which caused polyamine depletion, provoked a slight activating effect when given alone, but markedly inhibited the etoposide-induced increases in p65 DNA binding and phosphorylation. The NF-kappaB inhibiting effect of DFMO was prevented by the addition of exogenous putrescine, which restored the intracellular content of polyamines. Selective inhibitors of the etoposide-stimulated MAPK subtypes also reduced NF-kappaB activation. Moreover, pharmacological NF-kappaB inhibition reduced the increase in caspase activity and cell death elicited by etoposide, suggesting that NF-kappaB is involved in signaling to apoptosis. The results of the present study, together with our previous findings, suggest that polyamines play a permissive role in the pathways triggered by etoposide and leading to cell death of fibroblasts, by supporting the activation of MAPKs, NF-kappaB and caspases.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Etoposídeo/farmacologia , Fibroblastos/metabolismo , NF-kappa B/metabolismo , Poliaminas/química , Animais , Apoptose , Western Blotting , Caspases/metabolismo , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Cumarínicos/química , DNA/metabolismo , Eflornitina/química , Inibidores Enzimáticos/farmacologia , Etoposídeo/química , Flavonoides/farmacologia , Marcação In Situ das Extremidades Cortadas , Sistema de Sinalização das MAP Quinases , Camundongos , Fosforilação , Ligação Proteica , Fatores de Tempo
9.
Amino Acids ; 27(3-4): 359-65, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15452702

RESUMO

Polyamines are important multifunctional cellular components and are classically considered as mediators of cell growth and division. Recently polyamines have been also implicated in cell death. Now it appears that polyamines are bivalent regulators of cellular functions, promoting proliferation or cell death depending on the cell type and on environmental signals. This review draws a picture about the role of polyamines in signalling pathways related to apoptotic cell death and the proposed molecular targets of these polycations at the level of the apoptotic cascade. Solid evidence indicates that polyamines may affect the mitochondrial and postmitochondrial phases of apoptosis, by modulating cytochrome c release from mitochondria and activation of caspases. Recently, polyamines have been also implicated in the regulation of the premitochondrial phase of apoptosis, during which upstream apoptotic signal transduction pathways are activated. The studies reviewed here suggest that polyamines may participate in loops involving interaction with signal transduction pathways and activation/expression of proteins that may control cell death or cell growth.


Assuntos
Apoptose/fisiologia , Poliaminas/metabolismo , Transdução de Sinais , Animais , Ciclo Celular/fisiologia , Humanos , Mitocôndrias/metabolismo
10.
Biochem Pharmacol ; 62(3): 319-28, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11434904

RESUMO

Intracellular signaling pathways involved in the survival of proliferating L1210 leukemia cells were investigated by using specific modulators. Among the various inhibitors tested, only 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase (sGC) inhibitor, was found to induce a marked increase in caspase activity, which was associated with a loss of cell viability and a reduction in cGMP content. ODQ also provoked the processing of caspases-3 and -9, release of cytochrome c and, as early events, reduction of Bcl-2 content and dephosphorylation of Bad at Ser 112. Furthermore, YC-1, an sGC activator, and 8-Br-cGMP, a cell-permeant analogue of cGMP, exerted some protection against various apoptotic stimuli, such as serum deprivation or spermine accumulation. Although PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of the p44/42 mitogen-activated protein kinase (MAPK) pathway, did not increase basal caspase activity, and ODQ did not affect p44/42 MAPK phosphorylation significantly, phorbol myristate acetate stimulated p44/42 MAPK and reduced caspase activation induced by ODQ, serum deprivation, and spermine in a p44/42-dependent manner. SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)1H-imidazole), a p38 MAPK inhibitor, also partially protected against ODQ-induced apoptosis by increasing p44/42 MAPK phosphorylation. In conclusion, these results suggest that sGC may be relevant both for survival of L1210 cells under basal growing conditions and for protection against various apoptotic stimuli. p44/42 MAPK activation may also confer some protection from apoptosis, but apparently through a pathway largely independent of cGMP.


Assuntos
Guanilato Ciclase/fisiologia , Leucemia L1210/patologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Animais , Apoptose , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , GMP Cíclico/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Imidazóis/farmacologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Oxidiazóis/farmacologia , Oxirredução , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Piridinas/farmacologia , Quinoxalinas/farmacologia , Transdução de Sinais , Células Tumorais Cultivadas
11.
Biochem J ; 355(Pt 1): 199-206, 2001 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11256964

RESUMO

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.


Assuntos
Caspases/metabolismo , Poliaminas/metabolismo , Espermina Sintase/metabolismo , Animais , Western Blotting , Células Cultivadas , Cicloeximida/farmacologia , Eflornitina/farmacologia , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Inibidores da Síntese de Proteínas/farmacologia , Espermina Sintase/genética
12.
Cardiovasc Res ; 49(2): 408-16, 2001 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11164851

RESUMO

OBJECTIVE: We have recently shown that tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response. METHODS: CMs were isolated from 10-day-old chick embryos and grown to confluence. After 20 h of serum starvation the cells were treated with TNFalpha and LPS, and/or specific agonists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities and cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of [3H]-thymidine. RESULTS: Treatment of CMs with TNFalpha and LPS increased cell number and [3H]-thymidine incorporation. Addition of TNFalpha and LPS provoked an induction of ODC, with consequent polyamine accumulation, and a more delayed enhancement of NOS activity, which appeared to be independent of the activation of the ODC-polyamine system. TNFalpha and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments with specific inhibitors of ODC and NOS, as well as with inhibitors of sGC and cGMP-dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP-dependent pathways are required for the mitogenic action of TNFalpha and LPS. Moreover, addition of exogenous polyamines to untreated cells raised the cGMP level in a NO-dependent fashion, and enhanced [3H]-thymidine incorporation. The latter effect was inhibited by sGC or PKG inhibitors. Treatment of quiescent cells with NO donors, 8-bromo-cGMP or YC-1, an sGC activator, also promoted DNA synthesis. Furthermore, putrescine and NO donor can additively activate sGC in cell-free extracts. CONCLUSION: TNFalpha and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP.


Assuntos
Carbazóis , GMP Cíclico/metabolismo , DNA/biossíntese , Indóis , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Poliaminas/metabolismo , Alcaloides/farmacologia , Aminoquinolinas/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Eflornitina/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Lipopolissacarídeos/farmacologia , Azul de Metileno/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Inibidores de Proteínas Quinases , Estimulação Química , Fator de Necrose Tumoral alfa/farmacologia , ômega-N-Metilarginina/farmacologia
13.
Biochem Pharmacol ; 61(1): 25-32, 2001 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-11137705

RESUMO

Treatment of serum-starved, human ECV304 cells with histamine or ATP elicited a transient induction of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis, to an extent similar to that provoked by phorbol myristate acetate or serum re-addition. All these agents also provoked an increase in active phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The involvement of p44/42 MAPK and p38 MAPK in the induction of ODC was investigated by using selective inhibitors. U0126 and PD98059, two specific p44/42 MAPK kinase inhibitors, prevented the induction of ODC elicited by any stimulus employed, whereas SB203580 and SB202190, which are widely used as p38 MAPK inhibitors, enhanced ODC induction in a way that appeared dependent on p44/42 MAPK activation. By using inhibitors of other key signaling proteins that may lead to activation of p44/42 MAPK, we provide evidence that protein kinase C, but not phosphoinositide 3-kinase, is involved in histamine-stimulated ODC induction. These results show that the p44/42 MAPK pathway, but not p38 MAPK, is essential for ODC induction stimulated either by agonists of G-protein-coupled receptors, phorbol esters, or serum, and suggest that the inhibition of ODC induction may be an important event in the antiproliferative response to p44/42 MAPK pathway inhibitors.


Assuntos
Inibidores Enzimáticos/farmacologia , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Ornitina Descarboxilase/biossíntese , Trifosfato de Adenosina/farmacologia , Butadienos/farmacologia , Células Cultivadas , Meios de Cultura Livres de Soro , Ativação Enzimática , Indução Enzimática , Histamina/farmacologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Proteína Quinase C/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno
14.
Biochem J ; 347 Pt 3: 875-80, 2000 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-10769194

RESUMO

Cytochrome c release from mitochondria to the cytosol represents a critical step in apoptosis, correlated to the activation of the caspase cascade. In this report, we show that addition of micromolar concentrations of polyamines to isolated rat heart mitochondria induces the release of cytochrome c. Spermine, which is effective at concentrations of 10-100 microM, is more potent than spermidine, whereas putrescine has no effect up to 1 mM. The release of cytochrome c caused by spermine is a rapid, saturable and selective process that is independent of mitochondria damage. Spermine, unlike polylysine, is able to release a discrete amount of cytochrome c from intact, functional mitochondria. The cytochrome c-releasing power of spermine is not affected by cyclosporin A, differently from the effect of permeability transition inducers. In a cardiac cell-free model of apoptosis, the latent caspase activity of cytosolic extracts from cardiomyocytes could be activated by cytochrome c released from spermine-treated heart mitochondria. These data indicate a novel mechanism of cytochrome c release from the mitochondrion, and suggest that prolonged and sustained elevation of polyamines, characteristic of some pathologies such as heart hypertrophy, could be involved in the development of apoptosis.


Assuntos
Grupo dos Citocromos c/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Poliaminas/farmacologia , Animais , Apoptose , Caspases/metabolismo , Extratos Celulares , Embrião de Galinha , Ciclosporina/farmacologia , Citosol/efeitos dos fármacos , Citosol/enzimologia , Citosol/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Cinética , Miocárdio/citologia , Permeabilidade/efeitos dos fármacos , Polilisina/farmacologia , Putrescina/farmacologia , Ratos , Ratos Wistar , Espermidina/farmacologia , Espermina/farmacologia
15.
Biochem J ; 341 ( Pt 2): 363-9, 1999 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-10393094

RESUMO

The involvement of p44/42 mitogen-activated protein kinase (MAPK) in the induction of ornithine decarboxylase (ODC) was investigated by using PD98059, a specific MAPK-kinase (MEK1/2) inhibitor, and other signal-transduction inhibitors. In d,l-alpha-difluoromethylornithine (DFMO)-resistant L1210 cells stimulated to grow from quiescence, treatment with PD98059 inhibited p44/42 MAPK phosphorylation and the induction of ODC activity and protein. A marked reduction of the accumulation of mature ODC mRNA and its intron-containing precursor was observed, whereas ODC turnover was hardly affected. PD98059 also reduced the content of antizyme, but not that of antizyme mRNA. U0126, a novel and more potent inhibitor of MEK1/2, provoked a dose-dependent inhibition of ODC induction at lower concentrations with respect to PD98059. Other effective inhibitors of ODC induction proved to be genistein, manumycin A, herbimycin A, LY294002, wortmannin and KT5823, suggesting the involvement of other key proteins of signal-transduction pathways, i.e. Ras, Src, phosphatidylinositol 3-kinase and cGMP-dependent protein kinase, which may have a positive impact on MAPK. Cells kept in a DFMO-free medium, and thus containing high levels of putrescine and spermidine, showed enhanced MAPK phosphorylation and lower sensitivity to PD98059, compared with cells maintained in the presence of DFMO. In conclusion, these results indicate that the activation of p44/42 MAPK may favour the expression of ODC, and that polyamines, in turn, may affect the phosphorylation state of MAPK.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Leucemia Experimental/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Ornitina Descarboxilase/biossíntese , Transdução de Sinais , Animais , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Células Tumorais Cultivadas
16.
FEBS Lett ; 451(2): 95-8, 1999 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-10371145

RESUMO

Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post-nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP-dependent processing of pro-caspase-3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro-apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.


Assuntos
Apoptose , Caspases/fisiologia , Espermina/farmacologia , Espermina/fisiologia , Animais , Caspase 3 , Sistema Livre de Células , Relação Dose-Resposta a Droga , Humanos , Mitocôndrias/enzimologia , Miocárdio/enzimologia , Poliaminas/metabolismo , Ratos , Fatores de Tempo , Células U937
17.
FEBS Lett ; 437(3): 233-6, 1998 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-9824297

RESUMO

Exposure of several leukaemia cell types to the polyamine spermine triggered caspase activation. In HL60 cells, the onset of caspase activity correlated with the accumulation of spermine, and was accompanied by the processing of the caspase-3 precursor and the digestion of the substrate proteins PARP and gelsolin. Spermine also induced the accumulation of cytochrome c in the cytosol. Caspase activation triggered by spermine was not blocked by antioxidants or inhibition of polyamine oxidase. The deregulation of polyamine uptake strongly sensitised the cells to spermine-induced caspase activation. These data show that an excessive intracellular level of spermine triggers caspase activation that is not mediated by oxidative mechanisms, and suggest a model where elevated free cytosolic polyamines may act as transducers of a death message.


Assuntos
Caspases/metabolismo , Leucemia/enzimologia , Espermina/farmacologia , Animais , Caspase 3 , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Células HL-60 , Humanos , Células Jurkat , Leucemia L1210 , Poliaminas/metabolismo , Células U937
18.
Mol Cell Biochem ; 185(1-2): 47-53, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9746211

RESUMO

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for delta-receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic kappa-selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by kappa or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific kappa-antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, kappa-opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.


Assuntos
Ornitina Descarboxilase/biossíntese , Receptores Opioides/agonistas , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Analgésicos não Narcóticos/farmacologia , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Células Cultivadas , Encefalina Leucina/farmacologia , Indução Enzimática/efeitos dos fármacos , Sistema Imunitário/citologia , Sistema Imunitário/efeitos dos fármacos , Ligantes , Masculino , Miocárdio/citologia , Miocárdio/metabolismo , Especificidade de Órgãos , Ornitina Descarboxilase/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Wistar , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos
19.
Biochem Biophys Res Commun ; 243(3): 821-6, 1998 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-9500985

RESUMO

The AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases that are believed to protect cells against environmental and nutritional stress. In the present study the hypothesis of a protective role for AMPK against thymocyte apoptosis has been tested. It is shown that AMPK is expressed in rat thymocytes that contain the transcript for the a1 isoform of the AMPK catalytic subunit and can be activated by treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a well-established activator of AMPK. AICAR is not toxic and prevents glucocorticoid-induced apoptosis in the same concentration range used to activate AMPK. At concentrations higher than 1 mM, AICAR fully restores cell viability and inhibits DNA laddering in dexamethasone-treated thymocytes. Furthermore, AICAR blocks the dexamethasone-induced activation of caspase 3-like enzymes, which are believed to play a pivotal role in apoptotic cell death. Activation of AMPK by oligomycin, which depletes thymocytes of ATP, is also correlated to inhibition of caspase 3-like activity in dexamethasone-treated cells. However, AICAR and oligomycin do not exert any protective action when apoptosis is induced by staurosporine. These results indicate that AICAR is a powerful inhibitor of glucocorticoid-induced apoptosis and suggest that AMPK activation may interfere with a step in the apoptotic cascade triggered by dexamethasone.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Apoptose/efeitos dos fármacos , Caspases , Glucocorticoides/farmacologia , Complexos Multienzimáticos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Ribonucleotídeos/farmacologia , Timo/citologia , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/farmacologia , Animais , Caspase 3 , Sobrevivência Celular , Cisteína Endopeptidases/metabolismo , DNA/metabolismo , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Complexos Multienzimáticos/genética , Reação em Cadeia da Polimerase , Proteínas Quinases/genética , RNA/análise , Ratos , Ratos Wistar , Timo/enzimologia
20.
Biochem Biophys Res Commun ; 239(3): 729-33, 1997 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-9367837

RESUMO

The involvement of phosphatidylinositol 3-kinase (PI3K) in the induction of ornithine decarboxylase (ODC) was investigated by using specific PI3K inhibitors. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, treatment with LY294002 inhibited cell growth and provoked a complete block of the induction of ODC activity (IC50 approximately 2 microM) and ODC protein. Some reduction in the accumulation of ODC mRNA was also observed, whereas ODC turnover was not affected significantly. Wortmannin, another specific inhibitor of PI3K, structurally unrelated to LY294002, also inhibited ODC induction with an IC50 of about 10 nM. These results indicate that PI3K activity is required for the induction of ODC, possibly affecting both ODC mRNA level and translation. Since p70 S6 kinase (p70S6K) is considered an important mediator of PI3K action in several experimental systems, the effect of rapamycin, which can lead to selective inhibition of p70S6K, was also investigated. Rapamycin inhibited p70S6K activity and produced ODC inhibiting effects similar to those elicited by LY294002. However, LY294002 and wortmannin at concentrations which inhibited almost completely PI3K activity did not decrease p70S6K activity, suggesting that p70S6K does not mediate the PI3K effects on ODC, but may lie on a separate pathway in this experimental model.


Assuntos
Leucemia L1210/enzimologia , Ornitina Descarboxilase/biossíntese , Fosfatidilinositol 3-Quinases/fisiologia , Androstadienos/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Cromonas/farmacologia , Eflornitina/farmacologia , Indução Enzimática/efeitos dos fármacos , Leucemia L1210/genética , Leucemia L1210/patologia , Camundongos , Morfolinas/farmacologia , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Polienos/farmacologia , Sirolimo , Wortmanina
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