RESUMO
SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.
Assuntos
Aldeído Redutase/metabolismo , Células HeLa/fisiologia , Pressão Osmótica/fisiologia , Sirtuínas/fisiologia , Células U937/fisiologia , Simulação por Computador , Regulação Enzimológica da Expressão Gênica/fisiologia , Células HeLa/enzimologia , Células HeLa/metabolismo , Humanos , Immunoblotting , Técnicas In Vitro , Simulação de Acoplamento Molecular , Células U937/enzimologia , Células U937/metabolismo , Regulação para CimaRESUMO
Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/enzimologia , Membrana Celular/metabolismo , Isoantígenos/fisiologia , Glicoproteínas de Membrana/fisiologia , Mieloblastina/biossíntese , Neutrófilos/enzimologia , Receptores de Superfície Celular/fisiologia , Adulto , Idoso , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/sangue , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Doadores de Sangue , Indução Enzimática , Feminino , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/enzimologia , Hemoglobinúria Paroxística/imunologia , Humanos , Isoantígenos/biossíntese , Isoantígenos/genética , Transplante de Rim , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Mieloblastina/genética , Mieloblastina/farmacologia , Neutrófilos/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , Células U937/efeitos dos fármacos , Células U937/enzimologiaRESUMO
PURPOSE: The mechanisms of thymidine phosphorylase (TP) regulation induced by radiation therapy (XRT) in various tumors are poorly understood. We investigated the effect and mechanisms of preoperative XRT on TP expression in rectal cancer tissues. METHODS AND MATERIALS: TP expression and CD68 and monocyte chemoattractant protein-1 (MCP-1) levels in rectal cancer tissues and cancer cell lines were evaluated before and after XRT in Western blotting, immunohistochemistry, enzyme-linked immunoassay, and reverse transcription-polymerase chain reaction studies. Isolated peripheral blood monocytes were used in the study of chemotaxis under the influence of MCP-1 released by irradiated colon cancer cells. RESULTS: Expression of TP was significantly elevated by 9 Gy of XRT in most rectal cancer tissues but not by higher doses of XRT. In keeping with the close correlation of the increase in both TP expression and the number of tumor-associated macrophages (TAMs), anti-TP immunoreactivity was found in the CD68-positive TAMs and not the neoplastic cells. Expression of MCP-1 was increased in most cases after XRT, and this increase was strongly correlated with TP expression. However, this increase in MCP-1 expression occurred in tumor cells and not stromal cells. The XRT upregulated MCP-1 mRNA and also triggered the release of MCP-1 protein from cultured colon cancer cells. The supernatant of irradiated colon cancer cells showed strong chemotactic activity for monocyte migration, but this activity was completely abolished by neutralizing antibody. CONCLUSIONS: Use of XRT induces MCP-1 expression in cancer cells, which causes circulating monocytes to be recruited into TAMs, which then upregulate TP expression in rectal cancer tissues.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Quimiocina CCL2/metabolismo , Monócitos/fisiologia , Neoplasias Retais/metabolismo , Timidina Fosforilase/metabolismo , Regulação para Cima/efeitos da radiação , Linhagem Celular Tumoral/enzimologia , Linhagem Celular Tumoral/efeitos da radiação , Ensaios de Migração de Leucócitos/métodos , Ensaios de Migração de Macrófagos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HT29/enzimologia , Células HT29/efeitos da radiação , Humanos , Macrófagos/fisiologia , RNA Mensageiro/metabolismo , Dosagem Radioterapêutica , Neoplasias Retais/radioterapia , Células U937/enzimologia , Células U937/efeitos da radiação , Regulação para Cima/fisiologiaAssuntos
Antineoplásicos/farmacologia , Clioquinol/farmacologia , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Mieloma Múltiplo/enzimologia , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Proteínas Arqueais/antagonistas & inibidores , Povo Asiático , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Clioquinol/efeitos adversos , Clioquinol/uso terapêutico , Cobre/antagonistas & inibidores , Cobre/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Endopeptidases , Humanos , Japão , Células K562/efeitos dos fármacos , Células K562/enzimologia , Células K562/transplante , Leucemia Experimental/enzimologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/patologia , Doenças do Nervo Óptico/induzido quimicamente , Doenças do Nervo Óptico/etnologia , Inibidores de Proteases/efeitos adversos , Inibidores de Proteases/uso terapêutico , Complexo de Endopeptidases do Proteassoma , Doenças da Medula Espinal/induzido quimicamente , Doenças da Medula Espinal/etnologia , Células U937/efeitos dos fármacos , Células U937/enzimologia , Células U937/transplanteRESUMO
Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Inibidores Enzimáticos/farmacologia , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Consumo de Oxigênio/efeitos dos fármacos , Células U937/enzimologia , Células U937/patologiaRESUMO
OBJECTIVE: The roles of phosphatidylinositol 3 (PI3K) and mitogen-activated protein kinases (MAPK) have been widely studied in terms of the differentiation process induced by several drugs (phorbol ester, vitamin D-3, retinoic acid, etc.), but their exact functions in leukemic cells' phenotype and their potential therapeutic role remain incompletely clarified. MATERIALS AND METHODS: In order to investigate this query, leukemia cells were cultured in presence of kinase inhibitors (KIs). Proliferation, apoptosis, and differentiation were analyzed at the cellular and molecular levels, using flow cytometry and reverse transcriptase quantitative polymerase chain reaction. RESULTS: SB203580, a P38 MAPK inhibitor, had no effect on cell proliferation, whereas LY294002, a PI3K inhibitor, and PD098059, a selective inhibitor of mitogen-activated extracellular regulated kinase (MEK) phosphorylation, arrested cells in G(0)/G(1). However, LY294002 and PD098059 acted using different mechanisms: LY294002 decreased the expression of phosphorylated S6RP, whereas PD098059 increased P21/waf1 antigen expression. SP600125, an inhibitor of N-terminal c-jun kinases, arrested cells in G(2) and induced an endoreplicative process. SP600125 increased p21 at both the mRNA and protein levels. G(2) blockage is dependent on the PI3K pathway and the endoreplicative process is dependent on the PI3K and extracellular regulated kinase (ERK) pathways and mRNA synthesis. On the other hand, PD098059 potentiated the apoptotic process induced by either SP600125 or LY294002. Modulation of the expression of CD11, CD15, CD18, and CD54 was cell-dependent. CONCLUSION: Our results suggest that KIs modulate proliferation of leukemia cells and that the MEK/ERK inhibitor, PD098059, in combination with either SP600125 or LY294002, could have clinical value.
Assuntos
Leucemia/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Antracenos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Cromonas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Flavonoides/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Células HL-60/enzimologia , Humanos , Imidazóis/farmacologia , Leucemia/tratamento farmacológico , Leucemia/patologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Morfolinas/farmacologia , Piridinas/farmacologia , Células U937/efeitos dos fármacos , Células U937/enzimologiaRESUMO
BACKGROUND AND OBJECTIVE: Periodontal diseases are a group of inflammatory disorders that are initiated by specific gram-negative bacteria and lead to connective tissue destruction. Proteolytic enzymes, including matrix metalloproteinases (MMPs) and elastase, produced by resident and inflammatory cells in response to periodontopathogens and their products, play a major role in gingival tissue destruction. The aim of this study was to investigate the effect of a high-molecular-weight fraction prepared from cranberry juice concentrate on MMP-3, MMP-9 and elastase activities, as well as on MMP production by human cells stimulated with lipopolysaccharide of Actinobacillus actinomycetemcomitans. MATERIAL AND METHODS: MMP-3 and MMP-9 production by gingival fibroblasts and macrophages treated with the cranberry fraction and then stimulated with lipopolysaccharide was measured by enzyme-linked immunosorbent assay. MMP-3, MMP-9 and elastase activities in the presence of the cranberry fraction were evaluated using colorimetric or fluorogenic substrates. The changes in expression and phosphorylation state of fibroblast intracellular signaling proteins induced by A. actinomycetemcomitans lipopolysaccharide and the cranberry fraction were characterized by antibody microarrays. RESULTS: The lipopolysaccharide-induced MMP-3 and MMP-9 responses of fibroblasts and macrophages were inhibited in a dose-dependent manner by the cranberry fraction. This fraction was found to inhibit fibroblast intracellular signaling proteins, a phenomenon that may lead to a down-regulation of activating protein-1 activity. MMP-3, MMP-9 and elastase activities were also efficiently inhibited by the cranberry fraction, even when it was used at low concentrations. CONCLUSION: These results suggest that cranberry compounds offer promising perspectives for the development of novel host-modulating strategies for an adjunctive treatment of periodontitis.
Assuntos
Matriz Extracelular/enzimologia , Elastase de Leucócito/antagonistas & inibidores , Inibidores de Metaloproteinases de Matriz , Extratos Vegetais/farmacologia , Vaccinium macrocarpon , Aggregatibacter actinomycetemcomitans , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Peso Molecular , Proantocianidinas/farmacologia , Análise Serial de Proteínas , Células U937/efeitos dos fármacos , Células U937/enzimologiaRESUMO
The role of topoisomerase (topo) II in DNA repair has yet to be fully elucidated. Current evidence suggesting a role for topo II in the repair of DNA damage has been obtained by using in vitro model systems or inferred from correlative data in drug resistant cell lines. In this study we directly examined the role of topo IIalpha and beta in mediating the repair of melphalan-induced crosslinks in cellular DNA. To accomplish this, we used siRNA technology to knock down either topo IIalpha or beta in human chronic myelogenous leukemia K562 and histiocytic lymphoma U937 cell line. Our data demonstrate that topo IIbeta levels, (but not alpha), are a determinant of melphalan-induced crosslinks and sensitivity to melphalan. Furthermore, we show that knocking down topo IIbeta inhibits the repair of melphalan-induced crosslinks in K562 cells. These studies represent the first direct evidence that topo IIbeta participates in the repair of DNA damage induced by an alkylating agent in cellular DNA. Finally, these results suggest non-redundant roles for these two isoforms in mediating repair of DNA crosslinks.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/efeitos dos fármacos , Melfalan/farmacologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Células K562/efeitos dos fármacos , Células K562/enzimologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Inibidores da Topoisomerase II , Transfecção , Células U937/efeitos dos fármacos , Células U937/enzimologiaRESUMO
The chimaeric protein Bcr/Abl, the hallmark of chronic myeloid leukaemia, has been connected with several signalling pathways, such as those involving protein kinase B/Akt, JNK (c-Jun N-terminal kinase) or ERKs (extracellular-signal-regulated kinases) 1 and 2. However, no data about the p38 MAPK (mitogen-activated protein kinase) have been reported. Here, we present evidence showing that Bcr/Abl is able to modulate this signalling pathway. Transient transfection experiments indicated that overexpression of Bcr/Abl in 293T cells is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl, as the use of STI571 demonstrated. In fact, Bcr alone was able to induce p38 MAPK activation specifically through MKK3 (MAP kinase kinase 3). Supporting these observations, chronic myeloid leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared with Bcr/Abl-negative cells. While Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C (1-beta-D-arabinofuranosylcytosine), Bcr/Abl-positive cells were unable to activate p38 MAPK, suggesting that the p38 MAPK pathway is not sensitive to Abl-dependent stimuli in Bcr/Abl-positive cells. Our results demonstrate that the involvement of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C, and could provide a clue for new therapeutic approaches based on the use of specific Abl inhibitors.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Benzamidas , Linhagem Celular , Linhagem Celular Tumoral , Citarabina/antagonistas & inibidores , Citarabina/metabolismo , Citarabina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Genes Supressores de Tumor , Humanos , Mesilato de Imatinib , Rim/química , Rim/citologia , Rim/embriologia , Rim/metabolismo , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/farmacologia , Proteína Tumoral p73 , Proteínas Supressoras de Tumor , Células U937/enzimologia , Células U937/metabolismo , Células U937/patologiaRESUMO
Plaque destabilization leading to myocardial infarction is observed after surgery even if the intervention is of noncardiovascular nature. Mediators of peri- or postoperative stress responsible for such events could include catecholamines and lipopolysaccharide (LPS). Monocytes may be involved in destabilization of atherosclerotic plaques by production of matrix metalloproteinases (MMP). We examined whether catecholamines could affect the expression of MMPs in human monocytes/macrophages and whether catecholamines could modulate LPS-stimulated expression of particular MMPs in these cells. Epinephrine and norepinephrine up-regulated MMP-1 and potentiated LPS-induced expression of MMP-1 in peripheral blood monocytes and monocyte-derived macrophages. We further characterized this effect employing the monocytic cell line U937 and showed that catecholamines potentiate LPS-induced effects on MMP-1 and MMP-9 antigen and activity. mRNA levels of the respective MMPs also increased. These effects did not result from higher mRNA stability but rather from increased transcription possibly induced by enhanced DNA binding of AP-1 and were mediated by either beta1- or beta 2-receptors. If this mechanism is also effective in vivo, our findings might, at least in part, help to explain the observation that cardiac events are important causes of morbidity and mortality after noncardiac surgery and support the findings that peri-operative beta-blockade has been shown to reduce postoperative mortality from cardiac events.
Assuntos
Epinefrina/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Monócitos/efeitos dos fármacos , Norepinefrina/farmacologia , Células U937/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Arteriosclerose/complicações , Arteriosclerose/enzimologia , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Humanos , Macrófagos/enzimologia , Macrófagos/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Monócitos/enzimologia , Monócitos/metabolismo , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/prevenção & controle , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , RNA Mensageiro/biossíntese , Receptores Adrenérgicos beta/fisiologia , Ruptura Espontânea , Estresse Fisiológico/complicações , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células U937/enzimologia , Células U937/metabolismoRESUMO
Phospholipase A(2) type IVA (IVAPLA(2)) is a cytosolic enzyme that on activation selectively releases arachidonic acid (AA) from cell membrane phospholipids. Both AA and lysophospholipid, products of the enzymic reaction, can function as signal transducers in cellular interactions. The enzyme is present in most cells, including polymorphs, eosinophils, and platelets. This study used affinity purification to extract IVAPLA(2) from red cell lysate prepared from leukocyte- and platelet-depleted human blood to overcome the masking effect of hemoglobin on Western blot detection. We show that IVAPLA(2) is present in red cells as a 90-kDa protein.
Assuntos
Citosol/enzimologia , Eritrócitos/enzimologia , Fosfolipases A/isolamento & purificação , Western Blotting , Cromatografia de Afinidade , Fosfolipases A2 do Grupo IV , Humanos , Peso Molecular , Fosfolipases A/análise , Fosfolipases A2 , Células U937/enzimologiaRESUMO
OBJECTIVE: It has been shown that plasma level of C-reactive protein (CRP) is an independent predictor for acute coronary syndromes and is associated with plaque weakening. However, the underlying mechanisms are not well understood. In this study, we investigated the effect of CRP on the expression of matrix metalloproteinase-1 (MMP-1) that has been implicated in plaque vulnerability by human U937 histiocytes and monocyte-derived macrophages. METHODS AND RESULTS: Enzyme-linked immunosorbent assay of MMP-1 in conditioned medium showed that treatment of U937 cells with 100 microg/mL of CRP for 24 hour led to a 3- to 5-fold increase in MMP-1 secretion. CRP also markedly stimulated MMP-1 release from human monocyte-derived macrophages. In contrast, CRP had no effect on tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion. Northern blot showed that CRP upregulated MMP-1 mRNA expression. Collagenase activity assay showed that CRP increased collagen-degrading activity in cell-conditioned medium. Furthermore, results showed that the stimulation of MMP-1 secretion by CRP was inhibited by anti-CD32, but not by anti-CD64 antibody, and by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor PD98059. Finally, Western blot showed that CRP stimulated phosphorylation of extracellular signal-regulated kinase. CONCLUSIONS: This study demonstrates that CRP stimulates MMP-1 expression by U937 cells through FcgammaRII and extracellular signal-regulated kinase pathway. These findings suggest that CRP may promote matrix degradation and thus contribute to plaque vulnerability.
Assuntos
Proteína C-Reativa/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 1 da Matriz/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Receptores de IgG/efeitos dos fármacos , Células U937/efeitos dos fármacos , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Indução Enzimática/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Metaloproteinase 1 da Matriz/genética , Proteína Quinase 3 Ativada por Mitógeno , Monócitos/citologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , Receptores de IgG/fisiologia , Células U937/enzimologia , Células U937/metabolismoRESUMO
Human leukocyte elastase (HLE) interacts with HIV-1 glycoprotein (gp)41, suggesting a nonenzymatic receptor function for HLE in the context of HIV-1. HLE is found localized to the cell surface, but not granules in HIV permissive clones, and to granules, but not the cell surface of HIV nonpermissive clones. Inducing cell-surface HLE expression on HLE null, HIV nonpermissive clones permits HIV infectivity. HIV binding and infectivity diminish in proportion to HLE RNA subtraction. HIV binding and infectivity show dose dependence for the natural HLE ligand alpha1 proteinase inhibitor (alpha1antitrypsin, alpha1PI). Chemokines prevent, whereas alpha1PI promotes, copatching of HLE with the canonical HIV receptors. Recent demonstration that decreased viral RNA is significantly correlated with decreased circulating alpha1PI in HIV seropositive individuals is consistent with a model in which HLE and alpha1PI can serve as HIV coreceptor and cofactor, respectively, and potentially participate in the pathophysiology of HIV disease progression.
Assuntos
Membrana Celular/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Elastase de Leucócito/metabolismo , Proteínas de Membrana/metabolismo , Células U937/metabolismo , alfa 1-Antitripsina/fisiologia , Antígenos CD4/metabolismo , Membrana Celular/virologia , Quimiocinas/farmacologia , Células Clonais/efeitos dos fármacos , Células Clonais/enzimologia , Células Clonais/metabolismo , Células Clonais/virologia , Grânulos Citoplasmáticos/enzimologia , Progressão da Doença , Humanos , Lipopolissacarídeos/farmacologia , Substâncias Macromoleculares , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Agregação de Receptores , Receptores CXCR4/metabolismo , Células U937/efeitos dos fármacos , Células U937/enzimologia , Células U937/virologia , alfa 1-Antitripsina/farmacologiaRESUMO
Mylabris phalerata (MP) is an insect that has been used for the treatment of cancer in oriental medicine. In the present study, the butanol (BuOH) fraction of MP (BFMP) was examined to determine whether it can exert anti-cancer activity through an apoptotic pathway with little toxicity. BFMP was found to have a specific cytotoxic effect on human monocytic leukemic U937 cells (IC(50) = 140 microg/ml) rather than on peripheral blood mononuclear lymphocytes (PBML, IC(50) = over 500 microg/ml). BFMP also induced the morphological changes of apoptosis, such as chromatin condensation, cell shrinking and DNA fragmentation at a concentration of 31.25 microg/ml. In addition, BFMP significantly increased the portion of apoptotic annexin-V positive cells in a dose-dependent manner, and effectively activated caspases (cysteine aspartase) cascade involving caspases 8, 9 and 3. BFMP also effectively cleaved Bid, a death agonist member of the Bcl-2 family and (poly(ADP-ribose)polymerase) (PARP) and induced the subsequent release of cytochrome c from mitochondria into the cytosol. However, it did not affect Bcl-2 and Bax expression. Taken together, these data suggest that the BuOH extract of Mylabris phalerata can induce apoptosis in U937 cells by caspase cascade activation in conjunction with cytochrome c release, induced by a product of Bid. Therefore, we conclude that BFMP has anti-cancer activity, which is achieved through apoptosis and is associated with little toxicity.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Caspases/biossíntese , Besouros , Grupo dos Citocromos c/metabolismo , Medicina Tradicional Chinesa , Extratos de Tecidos/farmacologia , Animais , Anexina A5/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Divisão Celular/efeitos dos fármacos , Besouros/química , Fragmentação do DNA , Relação Dose-Resposta a Droga , Humanos , Marcação In Situ das Extremidades Cortadas , Concentração Inibidora 50 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Células U937/efeitos dos fármacos , Células U937/enzimologia , Células U937/patologiaRESUMO
beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermum radix, induces apoptosis in various lines of human tumor cells. To identify genes involved in beta-HIVS-induced apoptotic process, we performed cDNA array analysis and found that beta-HIVS suppresses the expression of the gene for a polo-like kinase 1 (PLK1) that is involved in control of the cell cycle. When U937 and HL60 cells were treated with 10(-6) M beta-HIVS for 0.5 h, both the amount of PLK1 itself and the kinase activity of this enzyme were decreased. By contrast, Bcr-Abl-positive K562 cells were resistant to the induction of apoptosis by beta-HIVS and this compound did not suppress the kinase activity of PLK1 in these cells. However, simultaneous treatment of K562 cells with both beta-HIVS and STI571, which selectively inhibits the protein tyrosine kinase (PTK) activity of Bcr-Abl, strongly induced apoptosis. Moreover, beta-HIVS increased the inhibitory effect of STI571 on PTK activity. Treatment of K562 cells with antisense oligodeoxynucleotides (ODNs) specific for PLK1 sensitized these cells to the beta-HIVS-induced fragmentation of DNA. These results suggest that suppression of the activity of PLK1 via inhibition of tyrosine kinase activity by beta-HIVS might play a critical role in the induction of apoptosis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide/patologia , Naftoquinonas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Benzamidas , Proteínas de Ciclo Celular , Linhagem Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , DNA Complementar/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Células HL-60/efeitos dos fármacos , Células HL-60/enzimologia , Humanos , Mesilato de Imatinib , Células K562/efeitos dos fármacos , Células K562/enzimologia , Rim , Leucemia Mieloide/enzimologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Pirimidinas/farmacologia , Células U937/efeitos dos fármacos , Células U937/enzimologia , Quinase 1 Polo-LikeRESUMO
Overexpression of manganese superoxide dismutase (MnSOD) has been postulated as one possible mechanism of protection from oxidative damage and free radicals. Doxorubicin treatment induces oxygen free radicals, leading to cytotoxicity and myelosuppression. The present study was performed to determine whether over-expression of MnSOD may play a role in resistance to doxorubicin. Retroviral constructs having the human MNSOD gene in the sense orientation and the neomycin phosphotransferase gene (NEOR) as a selectable marker were transduced into the human melanoma cell line A375 and the human histiocytic lymphoma cell line U937. Stably transduced A375 and U937 cells were subjected to 10-100 ng/ml doxorubicin for 24 h and compared with doxorubicin-treated A375 and U937 cells transduced with vector only. A colony forming assay was used to determine cell viability in semi-solid medium. Results demonstrated that wild-type A375 and U937 cells display low levels of endogenous MnSOD mRNA and protein, and are sensitive to doxorubicin treatment. In contrast, A375 and U937 cells transduced with the MNSOD gene consistently demonstrate increased colony formation in the presence of increasing concentrations of doxorubicin. MnSOD-transduced A375 and U937 cells also demonstrate increased MnSOD mRNA and protein levels when compared with wild type or those cells transduced with vector only. These results indicate that overexpression of MnSOD can enhance resistance to doxorubicin treatment.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Melanoma/enzimologia , Superóxido Dismutase/metabolismo , Células U937/enzimologia , Northern Blotting , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Melanoma/patologia , Retroviridae/genética , Superóxido Dismutase/genética , Células Tumorais Cultivadas , Células U937/patologiaRESUMO
Caspases are cysteine proteases involved in apoptosis and cytokine maturation. In erythroblasts, keratinocytes, and lens epithelial cells undergoing differentiation, enucleation has been regarded as a caspase-mediated incomplete apoptotic process. Here, we show that several caspases are activated in human peripheral blood monocytes whose differentiation into macrophages is induced by macrophage colony-stimulating factor (M-CSF). This activation is not associated with cell death and cannot be detected in monocytes undergoing dendritic cell differentiation in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanisms and consequences of caspase activation were further studied in U937 human monocytic cells undergoing phorbol ester-induced differentiation into macrophages. Differentiation-associated caspase activation involves the release of cytochrome c from the mitochondria and leads to the cleavage of the protein acinus while the poly(ADP-ribose)polymerase remains uncleaved. Inhibition of caspases by either exposure to the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-(DL)-Asp-fluoromethylketone (z-VAD-fmk) or expression of the p35 baculovirus inhibitory protein or overexpression of Bcl-2 inhibits the differentiation process. In addition, z-VAD-fmk amplifies the differentiation-associated production of radical oxygen species in both phorbol ester-differentiated U937 cells and M-CSF-treated monocytes, shifting the differentiation process to nonapoptotic cell death. Altogether, these results indicate that caspase activation specifically contributes to the differentiation of monocytes into macrophages, in the absence of cell death.
Assuntos
Caspases/fisiologia , Macrófagos/citologia , Monócitos/citologia , Adulto , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Células Dendríticas/citologia , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Espécies Reativas de Oxigênio , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células U937/efeitos dos fármacos , Células U937/enzimologia , Proteínas Virais/farmacologiaRESUMO
Etoposide (VP-16) is an anticancer agent that induces apoptosis in human leukemic cell lines such as U937 and HL60. We performed RNase protection assays, with two distinct cRNA panels covering most of caspase and BCL-2-related genes, using total RNA from cell lines exposed to various concentrations of the drug. Our results show that VP-16 down-regulates expression of most surveyed genes with the noticeable exception of casp-2S mRNA that is up regulated whereas casp-2L mRNA is decreased. Since these mRNAs are produced by the alternative splicing of exon 9, we devised a reverse transcriptase-polymerase chain reaction method using primers from exons 8 and 10 to demonstrate that VP-16 stimulates the production of exon 9-containing sequences, irrespective of active transcription. However, this effect is specific of the 3'-end of the CASP-2 gene since no difference in the relative amounts of the 5'-end of the mRNA species is detected. Nevertheless, the level of full-length casp-2L mRNA together with that of procaspase-2L protein, which is pro-apoptotic, are decreased under VP-16 treatment, suggesting that an early cell response to treatment by cytotoxic agents is to down-regulate expression of selected pro-apoptotic proteins.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Caspases/genética , Etoposídeo/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Monocítica Aguda/patologia , Leucemia Promielocítica Aguda/patologia , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Caspase 2 , Caspases/biossíntese , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Dactinomicina/farmacologia , Indução Enzimática/efeitos dos fármacos , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/genética , Éxons/genética , Genes bcl-2 , Células HL-60/efeitos dos fármacos , Células HL-60/enzimologia , Humanos , Leucemia Monocítica Aguda/enzimologia , Leucemia Promielocítica Aguda/enzimologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/biossíntese , Biossíntese de Proteínas , Proteínas/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proto-Oncogenes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937/efeitos dos fármacos , Células U937/enzimologiaAssuntos
Caspases/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , Células U937/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Citosol/enzimologia , Humanos , Microssomos/enzimologia , Mitocôndrias/enzimologia , Células U937/citologia , Células U937/enzimologiaRESUMO
We have identified a novel germline mutation in the PTEN tumour suppressor gene. The mutation was identified in a patient with a glioma, and turned out to be a heterozygous germline mutation of PTEN (Arg234Gln), without loss of heterozygosity in tumour DNA. The biological consequences of this germline mutation were investigated by means of transfection studies of the mutant PTEN molecule compared to wild-type PTEN. In contrast to the wild-type molecule, the mutant PTEN protein is not capable of inducing apoptosis, induces increased cell proliferation and leads to high constitutive PKB/Akt activation, which cannot be increased anymore by stimulation with insulin. The reported patient, in addition to glioma, had suffered from benign meningioma in the past but did not show any clinical signs of Cowden disease or other hereditary diseases typically associated with PTEN germline mutations. The functional consequences of the mutation in transfection studies are consistent with high proliferative activity. Together, these findings suggest that the Arg234Gln missense mutation in PTEN has oncogenic properties and predisposes to brain tumours of multiple lineages.