RESUMO
BACKGROUND: It has been suggested that diabetes medications, such as metformin, may have effects that inhibit abdominal aortic aneurysm (AAA) growth. The aim of this study was to examine the association of diabetes treatments with AAA growth in three patient cohorts. METHODS: AAA growth was studied using ultrasound surveillance in cohort 1, repeated CT in cohort 2 and more detailed repeat CT in cohort 3. Growth was estimated by the mean annual increase in maximum AAA diameter. RESULTS: A total of 1697 patients with an AAA were studied, of whom 118, 39 and 16 patients were prescribed metformin for the treatment of diabetes in cohorts 1, 2 and 3 respectively. Prescription of metformin was associated with a reduced likelihood of median or greater AAA growth in all three cohorts (cohort 1: adjusted odds ratio (OR) 0·59, 95 per cent c.i. 0·39 to 0·87, P = 0·008; cohort 2: adjusted OR 0·38, 0·18 to 0·80, P = 0·011; cohort 3: adjusted OR 0·13, 0·03 to 0·61, P = 0·010). No other diabetes treatment was significantly associated with AAA growth in any cohort. CONCLUSION: These findings suggest a potential role for metformin in limiting AAA growth.
Assuntos
Aneurisma da Aorta Abdominal/diagnóstico por imagem , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Idoso , Estudos de Coortes , Diabetes Mellitus/tratamento farmacológico , Feminino , Humanos , Modelos Logísticos , MasculinoRESUMO
PURPOSE: Neointimal hyperplasia (NIH), a pathophysiological event identified in bypass graft and stent re-stenosis, is characterised by aberrant vascular smooth muscle cell (VSMC) migration and proliferation. Recent evidence identifies histone deacetylase modulation as a regulator of VSMC proliferation and migration and a potential therapeutic target in the treatment of NIH. The purpose of our study was to determine the in vitro and in vivo potential of a novel agent, MCT-3, to modulate VSMC migration, proliferation and NIH. METHODS: In vitro VSMC studies utilized reverse transcriptase and real time Q-PCR gene expression analysis, western blot, elisa assay and cellular proliferation and migration scratch assay's. In vivo studies utilized the partial carotid artery ligation model of NIH together with immunohistochemistry in FVB/N mice. RESULTS: MCT-3 treatment induced histone H3 and H4 acetylation and inhibited VSMC migration and proliferation in vitro and significantly attenuated NIH in vivo. MCT-3-mediated regulation of orphan nuclear receptor NUR77, Plasminogen Activator Inhibitor Type-1 (PAI-1) and cyclin dependent kinase inhibitors (CDKI) p21(CIP1/WAF1) and p27(KIP1) expression was also identified. CONCLUSIONS: Together these observations identify a novel agent, MCT-3, with histone deacetylase inhibitory activity, able to inhibit NIH and identify a potential molecular mechanism responsible for these effects. Additional pre-clinical studies may be warranted to determine the potential clinical utility of this compound.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Hiperplasia/tratamento farmacológico , Neointima/tratamento farmacológico , Acetilação , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Histonas/metabolismo , Humanos , Hiperplasia/metabolismo , Masculino , Camundongos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , RatosRESUMO
A combination of demethylating agents and histone deacetylase inhibitors (HDACi) has been proposed as a novel therapy in leukemia and myelodysplasia. In HL-60 cells azacytidine (AZA) and Metacept-1 (MCT-1), a novel HDACi augmented inhibition of cell growth and increased apoptosis. In identifying a molecular mechanism responsible for these effects MCT-1 alone and in combination with AZA induced p15INK4b, p21WAF1/CIP1 and Caspase-3 whilst attenuating Bcl-XL expression. Interestingly, MCT-1 in combination with AZA significantly induced the recently identified suppressor of leukemogenesis Nur77 and attenuated AZA-induced MMP-9 expression. The combination of MCT-1 and AZA is more effective in inhibiting leukemic cell growth and induction of apoptosis. Regulation of a recently identified tumour suppressor gene together with cell cycle, apoptosis and matrix degrading proteases may underpin the molecular mechanism responsible for these effects.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Proteínas de Ligação a DNA/genética , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/uso terapêutico , Leucemia/tratamento farmacológico , Metaloproteinase 9 da Matriz/genética , Receptores de Esteroides/genética , Sulfonamidas/uso terapêutico , Apoptose/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p15/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HL-60/efeitos dos fármacos , Humanos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Localized deformation patterns are a common motif in morphogenesis and are increasingly finding applications in materials science and engineering, in such instances as mechanical memories. Here, we describe the emergence of spatially localized deformations in a minimal mechanical system by exploring the impact of growth and shear on the conformation of a semi-flexible filament connected to a pliable shearable substrate. We combine numerical simulations of a discrete rod model with theoretical analysis of the differential equations recovered in the continuum limit to quantify (in the form of scaling laws) how geometry, mechanics and growth act together to give rise to such localized structures in this system. We find that spatially localized deformations along the filament emerge for intermediate shear modulus and increasing growth. Finally, we use experiments on a 3D-printed multi-material model system to demonstrate that external control of the amount of shear and growth may be used to regulate the spatial extent of the localized strain texture.
RESUMO
Disorders of fibrinogen are usually caused by genetic mutations that result in low protein levels (hypofibrinogenemia) or an abnormal molecule (dysfibrinogenemia). However, environmental and plasma factors can have an acquired effect on its expression or function. For example, antibodies can bind fibrinogen and/or fibrin to interfere with polymerization and inhibit coagulation. The objective here was to determine the cause of dysfibrinogenemia in a 63-year-old man. Despite a low functional fibrinogen concentration and prolonged thrombin time, no inherited fibrinogen abnormality could be detected after extensive protein analysis and gene sequencing. Thus, electrophoresis methods and fibrinogen binding studies were used to establish the cause of the acquired dysfibrinogenemia. An immunoglobulin lambda light chain was found to bind fibrinogen as a monomer. It had no significant effect on fibrinopeptide release, but caused substantial defects in all other stages of thrombin-catalyzed fibrin polymerization. Binding to fibrinogen also seemed to prevent the light chain from being filtered through the kidneys, causing only low levels of it in the urine. Once in the urine, the lambda chain lost its anti-fibrinogen activity, apparently due to dimerization. The 63-year-old patient acquired dysfibrinogenemia from a monoclonal production of lambda light chain that bound and inhibited the function of fibrinogen. At age 64.5 he was diagnosed with monoclonal gammopathy of undetermined significance, explaining the abnormal immunoglobulin chain production. This case was particularly unusual in that the inhibition of fibrin polymerization was caused by a single immunoglobulin light chain, rather than by a whole antibody molecule.
Assuntos
Afibrinogenemia/etiologia , Fibrinogênio/metabolismo , Humanos , Cadeias lambda de Imunoglobulina/metabolismo , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/complicações , Gamopatia Monoclonal de Significância Indeterminada/diagnósticoAssuntos
Hidrocarboneto de Aril Hidroxilases/genética , Doenças Cardiovasculares/genética , Genótipo , Ticlopidina/análogos & derivados , Hidrocarboneto de Aril Hidroxilases/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/enzimologia , Clopidogrel , Citocromo P-450 CYP2C19 , Humanos , Inibidores da Agregação Plaquetária/uso terapêutico , Ticlopidina/uso terapêuticoRESUMO
Cell surface, urokinase (u-PA)-mediated, plasminogen activation has recently been recognised as a process integral to extracellular matrix degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor. The malignant metastatic phenotype is associated with excessive and uncontrolled, tumour cell-associated, u-PA-mediated, extracellular matrix degradation. Inhibition of the malignant metastatic phenotype via induction of PAI-2 expression and/or inhibition of u-PA expression may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI-2 and/or inhibiting u-PA activity may restrict u-PA-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease. We have identified the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras-transformed NIH-3T3 cells, as capable of upregulating PAI-2 and simultaneously suppressing u-PA expression in two different cell systems. In addition, zymographic analysis indicated that oxamflatin treatment results in a significant reduction in u-PA proteolytic activity in both HT-1080 fibrosarcoma and U-937 histiocytic lymphoma cells. We postulate that oxamflatin represents a novel means by which induction of PAI-2 and concomitant inhibition of u-PA gene and protein expression can be achieved and may be of benefit in inhibiting the malignant metastatic phenotype.
Assuntos
Antineoplásicos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Inibidor 2 de Ativador de Plasminogênio/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Humanos , Proteínas Nucleares/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidor 2 de Ativador de Plasminogênio/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células U937 , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Urokinase-type plasminogen activator (u-PA) bound to its receptor, u-PAR, initiates signal transduction pathways able to induce expression of the activator protein-1 (AP-1) family member c-fos [1]. Since transcription factors bound to AP-1 recognition sequences within the PAI-2 gene promoter play a role in basal and phorbol ester-mediated induction of PAI-2 gene expression, we hypothesised that u-PA/u-PAR-mediated modulation of AP-1 activity would in turn influence constitutive and inducible PAI-2 gene expression. Treatment of HT-1080 or U-937 cells with high molecular weight u-PA (HMW u-PA) resulted in induction of nuclear proteins binding to a functional AP-1 element in the proximal PAI-2 promoter. This increase in AP-1 activity correlated with a transactivation of the PAI-2 gene promoter in transiently transfected HT-1080 cells. We also demonstrate the u-PA treatment potentiated phorbol ester (PMA)-mediated induction of PAI-2 mRNA, indicating that u-PA binding produces a bone fide response in vivo.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/biossíntese , Inibidor 2 de Ativador de Plasminogênio/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/biossíntese , Sequência Consenso , Fibrossarcoma , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B , Oligodesoxirribonucleotídeos , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: In recent years it has become clear that the molecular investigation of hypofibrinogenemia provides unique insight into regions of the fibrinogen molecule that are important in molecular assembly, secretion and stability. OBJECTIVES: To investigate a case of hypofibrinogenemia at the molecular level. PATIENTS AND METHODS: The study was conducted on a 37-year-old woman from Mannheim, Germany, who had an antigenic plasma fibrinogen concentration of 0.86 g L(-1). Mutation screening was performed by DNA sequencing and the effect of the identified mutation was investigated at the protein level. RESULTS: Analysis of exon 8 of the fibrinogen gamma gene identified a heterozygous CAT-->TAT transition at codon 307. This novel His-->Tyr substitution was not detected when plasma fibrinogen was analyzed by electrospray ionization mass spectrometry. The mutation predicts a mass increase of 26 Da in the gamma chain, but purified gamma chains had a normal mass, indicating non-expression of the gamma(Tyr307) chain in plasma fibrinogen. CONCLUSIONS: This work reports a novel gamma307 His-->Tyr mutation (fibrinogen Mannheim II) that causes hypofibrinogenemia. Crystal structures show that His307 is located immediately adjacent to three residues that have been implicated in fibrin polymerization at the D:D interface. However, the histidine residue appears critical in maintaining structure of the fibrinogen gammaD domain, rather than in determining function.
Assuntos
Fibrinogênio/genética , Mutação , Adulto , Coagulação Sanguínea , Testes de Coagulação Sanguínea , Códon , DNA/metabolismo , Análise Mutacional de DNA , Éxons , Saúde da Família , Feminino , Fibrinogênio/biossíntese , Fibrinogênio/química , Fibrinólise , Heterozigoto , Histidina/química , Humanos , Masculino , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Espectrometria de Massas por Ionização por Electrospray , Tirosina/químicaRESUMO
Although a well documented phenomenon in Hodgkin's disease, malignant pulmonary lymphoid disease in other lymphoproliferative diseases, such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), is less frequently described. We present 3 patients, 2 with NHL and one with CLL, all demonstrating pulmonary malignant lymphoid involvement. We briefly review the probable mechanism underlying the development of this disease process and identify anatomical distribution of malignant pulmonary lymphoid disease as a prognostic marker in this condition.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Pulmonares/patologia , Transtornos Linfoproliferativos/patologia , Idoso , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Reports of the impact of larynx cancer treatment modality on quality of life are conflicting, in part due to varying study methodology. The aims of this study were to (1) provide preliminary comparisons of quality of life following radiotherapy or combination therapy; (2) evaluate a number of measures of quality of life and thereby (3) inform future prospective studies. Thirty-six laryngeal cancer patients, 24 following radiotherapy, 12 following radiotherapy and laryngectomy completed the Functional Assessment of Cancer Therapy (FACT) - General/Head and Neck subscale; Nottingham Health Profile (NHP); and the Hospital Anxiety and Depression scale (HAD), three to 12 months post-treatment. Results showed trends towards a less good quality of life in the combined therapy group over a wide range of outcomes, significant for the disease specific FACT head and neck subscale, NHP emotion (p = 0.04) and isolation (p = 0.027). To the authors' knowledge, however, this is the first demonstration of greater impact of laryngeal cancer on quality of life in younger subjects, who had lower scores among others on emotional wellbeing (p = 0.015) and anxiety (p = 0.035). Younger patients thus appear more likely to need more intensive support through treatment. Many of the physical and psychosocial domains derived from the three tools used were highly correlated. In other words, given the known high morbidity of the disease and its treatment, the selection of tools for head and neck quality of life assessment may be much less important than their universal application.
Assuntos
Neoplasias Laríngeas/reabilitação , Qualidade de Vida/psicologia , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Ansiedade/etiologia , Terapia Combinada/métodos , Depressão/etiologia , Emoções , Feminino , Humanos , Neoplasias Laríngeas/radioterapia , Neoplasias Laríngeas/cirurgia , Laringectomia , Masculino , Pessoa de Meia-Idade , Medição da Dor/métodos , Estudos Retrospectivos , Isolamento Social/psicologia , Resultado do TratamentoRESUMO
Combination epigenetic treatment (EGT) utilizing DNA methyl transferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) may be more efficacious than single agent treatment in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The molecular mechanisms behind the potential clinical efficacy of combination EGT treatment are incompletely understood and the frequently lengthy EGT regimes required to determine clinical response have generated a significant demand for early molecular markers of treatment response. Our study aimed to identify the effect of combination azacitidine (AZA) and panobinostat (LBH589) on expression levels of a panel of genes implicated in the pathogenesis of high-risk MDS or AML in HL-60 cells. We also characterized gene expression profiles in peripheral blood mononuclear (PBMCs) from patients in a recently reported phase Ib/II clinical trial using the combination of AZA and LBH589 and correlated these findings with clinical response to treatment. In vitro analysis demonstrated increased expression of caspase-3, Nor-1, NUR77, p15INK4B and p21WAF1/CIP1 and decreased expression of BclxL in HL-60 cells treated with combination EGT. Analysis of patient samples prior to treatment demonstrated a significant reduction in NUR77 and p21WAF1/CIP1 expression compared to healthy controls. NUR77 and p21WAF1/CIP1 levels were similar between treatment nonresponders and responders at screening. Early post first cycle treatment (day 25) analysis demonstrated a significant increase in expression of both NUR77, and p21WAF1/CIP1. A significant increase in NUR77, and p21WAF1/CIP1 together with a trend to increase in p15INK4B first cycle expression was observed in treatment responders compared to non-responders. In summary, combination AZA and LBH589 epigenetic treatment is associated with in vitro and in vivo modulation of genes implicated in the pathogenesis of MDS/AML. Early expression of NUR77 and p21WAF1/CIP1 correlated with clinical response to combination EGT suggesting investigation for potential use as molecular markers of early treatment response may be warranted.
Assuntos
Azacitidina/administração & dosagem , Biomarcadores Tumorais/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Ácidos Hidroxâmicos/administração & dosagem , Indóis/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Epigênese Genética/efeitos dos fármacos , Feminino , Células HL-60 , Humanos , Ácidos Hidroxâmicos/uso terapêutico , Indóis/uso terapêutico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Panobinostat , Resultado do TratamentoRESUMO
Therapeutic options are limited for elderly patients with acute myeloid leukemia (AML). A phase Ib/II study was undertaken to evaluate the maximum-tolerated dose (MTD) and preliminary efficacy of the pan-histone deacetylase inhibitor panobinostat (LBH589) in combination with azacitidine in patients with AML or high-risk myelodysplastic syndrome (MDS) naïve to intensive chemotherapy. Thirty-nine patients (AML=29, MDS=10) received azacitidine 75 mg/m(2) subcutaneously (days 1-5) and oral panobinostat (starting on day 5, thrice weekly for seven doses) in 28-day cycles until toxicity or disease progression. Dose-limiting toxicities during the phase Ib stage were observed in 0/4 patients receiving 10 mg panobinostat, in 1/7 patients (fatigue) receiving 20 mg, in 1/6 patients (fatigue) receiving 30 mg and in 4/5 patients (fatigue, syncope, hyponatremia and somnolence) receiving 40 mg. In phase II, an additional 17 patients received panobinostat at a MTD of 30 mg. The overall response rate (ORR=CR+CRi+PR) in patients with AML was 31% (9/29) and that in patients with MDS was 50% (5/10). After a median follow-up of 13 months, the median overall survival was 8 and 16 months in patients with AML and MDS, respectively. Increased histone H3 and H4 acetylation was a useful early biomarker of clinical response. Combining panobinostat with azacitidine was tolerable and clinically active in high-risk MDS/AML patients, warranting further exploration.
Assuntos
Afibrinogenemia/genética , Fibrinogênio/genética , Ataque Isquêmico Transitório/genética , Mutação Puntual , Adulto , Afibrinogenemia/sangue , Criança , Feminino , Genes Recessivos , Heterozigoto , Homozigoto , Humanos , Íntrons , Ataque Isquêmico Transitório/sangue , Masculino , Splicing de RNA/genéticaRESUMO
Histone deacetylase inhibitors (HDACi) demonstrate considerable in vitro and in vivo activity and clinical efficacy in the treatment of hematological malignancies. Pre-clinical and early phase clinical trials identify therapeutic activity using a combination of HDACi and demethylating agents which may be more efficacious than single agent treatment. Our studies aimed to determine the effects and molecular mechanisms of action of novel hydroxamate (MCT-3) and benzamide [MGCD0103 (MG)] HDACi's in the HL-60 cell line alone and in combination with the demethylating agent 5-aza-cytidine (AZA). MG, MCT-3 and AZA treatment significantly inhibited HL-60 cell growth in vitro with MG being the most potent agent. MG in combination with AZA demonstrated no significant increase in inhibition of cell growth over MG treatment alone whilst MCT-3 in combination with AZA demonstrated increased inhibition of cell growth over either agent alone although no more significant than MG alone. MG alone or MCT-3 in combination with AZA significantly increased p15 and caspase-3 expression. MG and MCT-3 significantly attenuated AZA-induced MMP-9 mRNA expression and proteolytic activity. Interestingly, MCT-3, MG and AZA alone and in combination increased expression of the novel tumour suppressor gene Nur77, important in leukemogenesis, with MG a more potent inducer as a single agent. These observations suggest the enhanced anti-leukemia activity of the combination of AZA and HDACi may only reside with certain HDACi classes and may be in-part explained by regulation of genes associated with cell cycle arrest, apoptosis and tumour suppression.
Assuntos
Antineoplásicos/farmacologia , Azacitidina/farmacologia , Benzamidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Leucemia/tratamento farmacológico , Caspase 3/genética , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Células HL-60 , Humanos , Metaloproteinase 9 da Matriz/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genéticaRESUMO
Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Emu-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Emu-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Proteína 11 Semelhante a Bcl-2 , Genes myc , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Vorinostat , Proteína bcl-X/fisiologiaRESUMO
Increased plasminogen activator inhibitor type 1 (PAI-1) levels are observed in endothelial cells stimulated by tumour necrosis factor alpha (TNFalpha). Thiazolidinediones (TZDs) may inhibit elevated endothelial cell PAI-1 accounting, in part, for the putative atheroprotective effects of TZDs. In an endothelial cell line, Rosiglitazone (RG) and Pioglitazone (PG) inhibited induction of PAI-1 by TNFalpha. The specific peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor, SR-202, failed to modulate this effect. RG also inhibited the effect of TNFalpha on a reporter gene construct harbouring the proximal PAI-1 promoter and PAI-1 mRNA in cells co-transfected with a dominant-negative PPARgamma construct. RG and PG attenuated TNFalpha-mediated induction of trans-acting factor(s) Nur77/Nurr1 and binding of nuclear proteins (NP) to the cis-acting element (NBRE). SR-202 failed to modulate these effects. The observations suggest TZDs inhibit TNFalpha-mediated PAI-1 induction independent of inducible PPARgamma activation and this may involve in the modulation of Nur77/Nurr1 expression and NP binding to the PAI-1 NBRE.
Assuntos
Células Endoteliais/metabolismo , PPAR gama/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Tiazolidinedionas/farmacologia , Ativação Transcricional/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Humanos , Pioglitazona , Rosiglitazona , Ativação Transcricional/efeitos dos fármacosRESUMO
Since its discovery over a decade ago, evidence has accumulated implicating the cell-surface urokinase receptor (u-PAR), in numerous biological processes. Most notable has been the identification of a critical role for u-PAR in the regulation of cell-surface plasminogen activation in physiological and pathological conditions. Recent evidence suggests that u-PAR, a glycosylphosphatidylinositol-linked receptor, lacking transmembrane and cytoplasmic domains, is also involved in processes not related to plasminogen activation, including cellular adhesion and the transmission of extracellular signals across the plasma membrane. Involvement of activated u-PAR in these events identifies previously unsuspected roles for this molecule and defines a new field of research in u-PAR biology. We discuss the molecular biology of u-PAR together with the underlying mechanisms responsible for the novel functional roles recently ascribed to this pleiotropic molecule.
Assuntos
Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Adesão Celular/fisiologia , Membrana Celular/química , Membrana Celular/fisiologia , Cromossomos Humanos Par 19/genética , Ativação Enzimática/fisiologia , Regulação da Expressão Gênica/genética , Humanos , Modelos Moleculares , Receptores de Superfície Celular/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais/fisiologiaRESUMO
Plasminogen-activator inhibitor type 2 (PAI-2), a serine protease inhibitor involved in the regulation of urokinase-dependent proteolysis, is also implicated in the inhibition of tumor-necrosis-factor-(TNF)-mediated apoptosis. The PAI-2 gene is one of the most TNF-responsive genes known and is also highly induced by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the phosphatase inhibitor, okadaic acid, in both HT-1080 fibrosarcoma and U-937 histiocytic cells. We sought to identify and characterize regulatory cis-acting DNA elements and trans-acting factors which mediate basal and inducible PAI-2 gene transcription. A series of promoter deletion mutants (nucleotides -1859 to -91) fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into HT-1080 cells. Two repressor regions were identified; one distally between positions -1859 and -1100, and one proximally between positions -259 and -219. Cells transfected with constructs harboring more than 259 bp promoter sequence produced a 10-15-fold increase in CAT activity when treated with PMA or okadaic acid, but produced only a minimal (2.5-fold) increase in response to TNF. Removal of the proximal repressor by deletion to position -219, or by internal deletion from the -1100 PAI-2 CAT construct, resulted in a selective increase in TNF responsiveness, suggesting that induction of PAI-2 gene transcription by TNF is associated with derepression. Detailed analysis of the proximal repressor utilizing the electrophoretic mobility shift assay (EMSA), identified two novel and distinct protein-binding sites (A and B). Site A is located within the 40-bp proximal repressor while site B is situated immediately adjacent to the 3' boundary. Treatment of cells with PMA or okadaic acid produced no change in the binding activity of proteins recognising sites A or B. However, treatment of cells with TNF results in a profound selective reduction in site-B-binding activity, suggesting that this site plays a significant role in TNF-mediated regulation of PAI-2 gene expression. Our findings suggest that TNF-mediated induction of PAI-2 gene expression involves derepression and is associated with cis-acting and trans-acting factors located within and adjacent to the proximal repressor region.
Assuntos
Regulação da Expressão Gênica/genética , Inibidor 2 de Ativador de Plasminogênio/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Bases , Sítios de Ligação , Sondas de DNA/química , Sondas de DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/farmacologia , Análise de Sequência , Deleção de Sequência/genética , Transfecção/genética , Células Tumorais CultivadasRESUMO
A knowledge of the sugar content of molasses is of commercial importance to a number of industrial fermentations. Hence the feasibility of using a glucose oxidase biosensor to determine the glucose content of molasses samples was investigated. This method was compared with standard high-performance liquid chromatographic (HPLC) and gas-liquid chromatographic (GLC) procedures and with the use of a commercially available glucose analyser. A good correlation was obtained between the standard acetic anhydride GLC and glucose oxidase biosensor results (correlation coefficient = 0.98). Rapid and accurate measurements could be carried out using the biosensor without the need to employ the sample preparation step required in standard GLC methods. It was concluded that the use of the biosensor technique for the determination of glucose in molasses samples has distinct advantages over conventional methods.