RESUMO
BACKGROUND: Outcomes for organ transplantation are constantly improving because of advances in organ preservation, surgical techniques, immune clinical monitoring, and immunosuppressive treatment preventing acute transplant rejection. However, chronic rejection including transplant vasculopathy still limits long-term patient survival. Transplant vasculopathy is characterized by progressive neointimal hyperplasia leading to arterial stenosis and ischemic failure of the allograft. This work sought to decipher the manner in which the humoral immune response, mimicked by W6/32 anti-HLA antibody, contributes to transplant vasculopathy. METHODS AND RESULTS: Studies were performed in vitro on cultured human smooth muscle cells, ex vivo on human arterial segments, and in vivo in a model consisting of human arterial segments grafted into severe combined immunodeficiency/beige mice injected weekly with anti-HLA antibodies. We report that anti-HLA antibodies are mitogenic for smooth muscle cells through a signaling mechanism implicating matrix metalloproteinases (MMPs) (membrane type 1 MMP and MMP2) and neutral sphingomyelinase-2. This mitogenic signaling and subsequent DNA synthesis are blocked in smooth muscle cells silenced for MMP2 or for neutral sphingomyelinase-2 by small interfering RNAs, in smooth muscle cells transfected with a vector coding for a dominant-negative form of membrane type 1 MMP, and after treatment by pharmacological inhibitors of MMPs (Ro28-2653) or neutral sphingomyelinase-2 (GW4869). In vivo, Ro28-2653 and GW4869 reduced the intimal thickening induced by anti-HLA antibodies in human mesenteric arteries grafted into severe combined immunodeficiency/beige mice. CONCLUSIONS: These data highlight a crucial role for MMP2 and neutral sphingomyelinase-2 in vasculopathy triggered by a humoral immune response and open new perspectives for preventing transplant vasculopathy with the use of MMP and neutral sphingomyelinase inhibitors, in addition to conventional immunosuppression.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Artérias/transplante , Antígenos HLA/imunologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Doenças Vasculares/fisiopatologia , Compostos de Anilina/farmacologia , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Artérias/patologia , Artérias/fisiopatologia , Compostos de Benzilideno/farmacologia , Células Cultivadas , Constrição Patológica/etiologia , Constrição Patológica/fisiopatologia , Modelos Animais de Doenças , Humanos , Hiperplasia/etiologia , Hiperplasia/fisiopatologia , Técnicas In Vitro , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos SCID , Modelos Animais , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Neointima/patologia , Neointima/fisiopatologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Doenças Vasculares/etiologia , Enxerto VascularRESUMO
Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23-mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii-induced immunopathology. Moreover, IL-23-dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4(+) T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.
Assuntos
Interleucina-17/fisiologia , Interleucina-23/fisiologia , Interleucinas/fisiologia , Intestino Delgado/patologia , Metaloproteinase 2 da Matriz/fisiologia , Toxoplasma/patogenicidade , Animais , Feminino , Intestino Delgado/imunologia , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Piperazinas/farmacologia , Pirimidinas/farmacologia , Toxoplasmose Animal/imunologia , Interleucina 22RESUMO
Connecting fluorescence signals with anatomic structures enhances our ability to monitor biologic processes in mice. Here, we present a semiautomated approach to correlate two-dimensional (2D) noninvasive near-infrared fluorescence (NIRF) imaging with three-dimensional (3D), high-resolution, flat-panel volume computed tomography (fpVCT). We developed an algorithm to colocalize fluorescence signals of NIRF-labeled antibodies directed against matriptase and urokinase plasminogen activator receptor (uPAR) to orthotopic carcinomas in mice visualized by fpVCT. For this purpose, mice were anesthetized and fixed on a multimodality animal bed containing fiducial markers filled with iodine-containing contrast agent and fluorescent dye. After intravenous administration of contrast agent and Cy5.5-labeled antibodies, NIRF and fpVCT images were obtained, without repositioning the mice. Binding of Cy5.5-labeled matriptase-specific antibody to pancreatic tumors and Cy5.5-labeled uPAR-specific antibody to mammary carcinomas was assessed by time-domain NIRF imaging measuring the location of fluorescence intensity and its lifetime. In summary, we developed a novel 2D-3D registration technique for image fusion with NIRF imaging and fpVCT to provide complementary information in tumor models on the in vivo association of functional information with anatomic structures. The combination of fpVCT with NIRF imaging will now allow targeted and effective monitoring of preclinical tumor therapies.
Assuntos
Anticorpos , Carbocianinas , Tomografia Computadorizada de Feixe Cônico/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Automação/métodos , Carcinoma/diagnóstico , Carcinoma/patologia , Tomografia Computadorizada de Feixe Cônico/instrumentação , Estudos de Viabilidade , Feminino , Imunofluorescência/métodos , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Masculino , Neoplasias Mamárias Experimentais/diagnóstico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Camundongos SCID , Espectrometria de Fluorescência/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC50 < 100 nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in near stasis (92% TGI) in an HCT116 mouse xenograft model.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Administração Oral , Animais , Aurora Quinase A , Aurora Quinase B , Aurora Quinases , Benzimidazóis/síntese química , Benzimidazóis/química , Disponibilidade Biológica , Linhagem Celular Tumoral , Cristalografia por Raios X , Cães , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Humanos , Lactamas/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/metabolismo , RatosRESUMO
Balanced expression of proteases and their inhibitors is one prerequisite of tissue homeostasis. Metastatic spread of tumor cells through the organism depends on proteolytic activity and is the death determinant for cancer patients. Paradoxically, increased expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of several endometalloproteinases, including matrix metalloproteinases and a disintegrin and metalloproteinase-10 (ADAM-10), in cancer patients is negatively correlated with their survival, although TIMP-1 itself inhibits invasion of some tumor cells. Here, we show that elevated stromal expression of TIMP-1 promotes liver metastasis in two independent tumor models by inducing the hepatocyte growth factor (HGF) signaling pathway and expression of several metastasis-associated genes, including HGF and HGF-activating proteases, in the liver. We also found in an in vitro assay that suppression of ADAM-10 is in principle able to prevent shedding of cMet, which may be one explanation for the increase of cell-associated HGF receptor cMet in livers with elevated TIMP-1. Similar TIMP-1-associated changes in gene expression were detected in livers of patients with metastatic colorectal cancer. The newly identified role of TIMP-1 to create a prometastatic niche may also explain the TIMP-1 paradoxon.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
PURPOSE: Therapeutic efficacy of the novel matrix metalloproteinase (MMP) inhibitor Ro 28-2653 has been shown in various models of different tumor entities. We hypothesized that the inhibitor effect of Ro 28-2653 on the tumor growth could be improved by combination with chemotherapeutic drugs and examined therefore the effect of Ro 28-2653 alone and in combination with etoposide or estramustine in the MatLyLu Dunning R-3327 rat tumor model characteristic for the androgen-independent prostate cancer (PCa). METHODS: In vitro effects were estimated measuring the proliferation of MatLyLu cells incubated with the three agents alone or in combination using the XTT test. The in vivo effects of the agents alone or in combination were examined by measuring the tumor weight 18 days after tumor cell injection. RESULTS: The proliferation rate was only inhibited by etoposide while that effect was increased in combination with Ro 28-2653 and estramustine. Ro 28-2653 reduced the tumor weight by 86%. That effect was significantly increased in combination with etoposide to 92%. CONCLUSIONS: The inhibitory effect of the MMP inhibitor Ro 28-2653 on the tumor growth in the Dunning PCa model is enhanced by the standard chemotherapeutic drug etoposide. A combined application of both agents could be considered as potential tool to improve the therapy of patients with advanced PCa after failure of hormonal treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Animais , Peso Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Estramustina/administração & dosagem , Etoposídeo/administração & dosagem , Humanos , Injeções Intraperitoneais , Masculino , Invasividade Neoplásica/prevenção & controle , Transplante de Neoplasias , Piperazinas/administração & dosagem , Neoplasias da Próstata/patologia , Pirimidinas/administração & dosagem , Ratos , Fatores de Tempo , Inibidores Teciduais de Metaloproteinases/administração & dosagemRESUMO
OBJECTIVE: The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ETB receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs expressing either the full-length ETB receptor or an N-terminally truncated ETB receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ETB receptor, IRL1620 (an ETB-selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22alpha, and alpha-actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of Gi proteins. In contrast, in VSMCs expressing N-terminally truncated ETB receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression. CONCLUSIONS: This study is the first to show that stimulation of full-length ETB receptors promotes expression of contractile proteins and may thus participate in the differentiation of VSMCs.
Assuntos
Proteínas Contráteis/metabolismo , Receptores ErbB/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Peptídeo Hidrolases/metabolismo , Receptor de Endotelina B/química , Receptor de Endotelina B/metabolismo , Ativação Transcricional , Animais , Células Cultivadas , Endotelinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Fluorescência Verde/genética , Humanos , Músculo Liso Vascular/citologia , Mutação , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ratos , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/genética , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
In amyotrophic lateral sclerosis (ALS), there is increased expression of matrix metalloproteinases (MMPs) and degradation of the extracellular matrix in postmortem spinal cord tissue. We used zymography and in situ zymography to analyze the expression of MMP-2 and MMP-9 in spinal cord tissue from the G93A transgenic mouse model of ALS. Expression of MMP-9 was increased in the spinal cord of G93A mice. For functional analysis of the role of MMPs, we investigated the effects of oral administration of the MMP inhibitor Ro 28-2653 (100 mg/kg), starting at the age of 30 days (n = 19) and on disease onset (starting at the age of 90 days (n = 10)). Treatment with the MMP inhibitor Ro 28-2653 starting at 30 days of age improved motor performance and significantly (P < 0.05) prolonged the survival time of the animals (136 +/- 12 versus 123 +/- 12 days, mean +/- SD), however, administration at disease onset did not significantly improve survival time. Our experiments show that MMPs are expressed in an animal model of ALS and may play a role in the complex pathophysiologic changes. Early pharmacologic inhibition with a synthetic MMP inhibitor extends survival of the animals which suggest a role of MMPs in the early phase of the disease.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/enzimologia , Inibidores de Metaloproteinases de Matriz , Piperazinas/uso terapêutico , Inibidores de Proteases/uso terapêutico , Pirimidinas/uso terapêutico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/mortalidade , Animais , Feminino , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Inibidores de Proteases/farmacologia , Taxa de Sobrevida , Fatores de TempoRESUMO
Matrix metalloproteinase 9 (MMP-9) is implicated in various physiological processes by its ability to degrade the extracellular matrix (ECM) and process multiple regulatory proteins. Normally, MMP-9 expression is tightly controlled in cells. Sustained or enhanced MMP-9 secretion, however, has been demonstrated to contribute to the pathophysiology of numerous diseases, including arthritis and tumor progression, rendering this enzyme a major target for clinical interventions. Here we show that constitutive MMP-9 secretion was abrogated in THP-1 monocytic leukemia cells by addition of neutralizing antibodies against tumor necrosis factor alpha (TNF-alpha) or TNF receptor type 1 (TNF-R1), as well as by inhibition of TNF-alpha converting enzyme (TACE). This indicates that MMP-9 production in these cells is maintained by autocrine stimulation, with TNF-alpha acting via TNF-R1. To investigate the intracellular signaling routes involved in MMP-9 gene transcription, cells were treated with different inhibitors of major mitogen-activated protein kinase (MAPK) pathways. Interruption of the extracellular signal-regulated kinase pathway 1/2 (ERK1/2) using PD98059 significantly downregulated constitutive MMP-9 release. In contrast, blockage of p38 kinase activity by addition of SB203580 or SB202190, as well as inhibition of c-Jun N-terminal kinase (JNK) using L-JNK-I1, clearly augmented MMP-9 expression and secretion by an upregulation of ERK1/2 phosphorylation. Moreover, exogenously added TNF-alpha augmented MMP-9 synthesis and secretion in THP-1 cells via enhancement of ERK1/2 activity. Taken together, our results indicate that ERK1/2 activity plays a pivotal role in TNF-alpha-induced MMP-9 production and demonstrate its negative modulation by p38 and JNK activity. These findings suggest ERK1/2 rather than p38 and JNK as a reasonable target to specifically block MMP-9 expression using MAPK inhibitors in therapeutic applications.
Assuntos
Comunicação Autócrina/fisiologia , Metaloproteinase 9 da Matriz/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Humanos , MAP Quinase Quinase 4/metabolismo , Modelos Biológicos , Fosforilação , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Chronic exposure to the chloracetanilide herbicide alachlor has been shown to cause olfactory mucosal neoplasms. Genomic analysis of olfactory mucosa from rats given alachlor (126 mg/kg/d) for from 1 day to 18 mo suggested that matrix metalloproteinases MMP-2 and MMP-9 were upregulated in the month following initiation of treatment. The present studies were designed to confirm this latter finding and to explore the potential role of MMPs in alachlor-induced olfactory carcinogenesis. Zymographic analysis of olfactory mucosal extracts confirmed that MMP-2 activity is higher in the olfactory mucosa of alachlor-treated rats. Therefore, rats were fed alachlor (126 mg/kg/d in the diet for 1 year) either with or without the MMP-2/MMP-9 inhibitor Ro 28-2653 (100 mg/kg daily by gavage for the first 2 months of alachlor treatment). The number of olfactory mucosal neoplasms was reduced by 25% after 1 year of alachlor treatment in rats that received both alachlor and Ro 28-2653. The morphology of alachlor-induced olfactory tumors was similar whether or not Ro 28-2653 had been given; the MMP inhibitor itself had no impact on olfactory mucosal histology. These data confirm that olfactory mucosal MMP-2 activity is increased following short-term alachlor exposure and show that administration of an MMP-2/9 inhibitor reduced the incidence of olfactory neoplasms in alachlor-treated rats, thereby implicating MMP-2 activity as a mediator of alachlor-induced carcinogenicity.
Assuntos
Inibidores de Metaloproteinases de Matriz , Mucosa Nasal/patologia , Neoplasias Nasais/patologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Acetamidas , Animais , Masculino , Mucosa Nasal/efeitos dos fármacos , Neoplasias Nasais/induzido quimicamente , Ratos , Ratos Long-EvansRESUMO
Therapeutic efficacy of the novel matrix metalloproteinase (MMP) inhibitor, Ro 28-2653 (5-biphenyl-4-yl-5-[4-(-nitro-phenyl)-piperazin-1-yl]-pyrimidine-2,4,6-trione), has been shown in various models of different tumor entities. The tumor growth-reducing effect has been demonstrated in the orthotopic rat prostate Dunning model (subline MatLyLu). Based on these results we investigated Ro 28-2653 in combination with estramustine on the G subline of the Dunning tumor. This subline is characterized by a low metastatic ability and androgen sensitivity. Efficacy was determined by recording tumor growth in vivo by magnetic resonance imaging (MRI). Tumor cells were injected into the prostates of 81 Copenhagen rats. MRI was performed at day 100 and at day 126 after tumor cell injection. The duration of therapy was 17 days with daily oral application of Ro 28-2653 (100 mg/kg) and four i.p. injections of estramustine (7.5 mg/kg). Histological evaluations were conducted to provide further information about the effects on tumor morphology. Orthotopic tumor induction was successful in 100% of the animals. Tumor volume calculations with MRI showed a significant difference between the control groups, the animals treated with Ro 28-2653, and the animals treated with the combination of Ro 28-2653 and estramustine. The new MMP inhibitor Ro 28-2653 reduces tumor growth and provides a compatible therapeutic alternative for patients with prostate cancer.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Estramustina/uso terapêutico , Inibidores de Metaloproteinases de Matriz , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Piperazinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Pirimidinas/uso terapêutico , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Masculino , Metaloproteinases da Matriz/metabolismo , Neoplasias Hormônio-Dependentes/secundário , Neoplasias da Próstata/patologia , Ratos , Ratos Endogâmicos , Taxa de Sobrevida , Fatores de Tempo , Células Tumorais CultivadasRESUMO
BACKGROUND: Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions. OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs. METHODS AND RESULTS: OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2-/- cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti-MMP-2 and anti-membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2-/- cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis. CONCLUSIONS: Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.
Assuntos
Lipoproteínas LDL/farmacologia , Metaloproteinase 2 da Matriz/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fenilalanina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Ceramidas/fisiologia , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lisofosfolipídeos/fisiologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/farmacologia , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/farmacologia , Metaloendopeptidases/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenilalanina/farmacologia , Piperazinas/farmacologia , Inibidores de Proteases/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Coelhos , Receptores do Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/fisiologia , Esfingosina/fisiologia , Tiofenos/farmacologiaRESUMO
PURPOSE: The implication of matrix metalloproteinases (MMPs) in the major stages of cancer progression has fueled interest in the design of synthetic MMP inhibitors (MMPIs) as a novel anticancer therapy. Thus far, drugs used in clinical trials are broad-spectrum MMPIs the therapeutic index of which proved disappointingly low. The development of selective MMPIs for tumor progression-associated MMPs is, thus, likely to offer improved therapeutic possibilities. EXPERIMENTAL DESIGN: The anti-invasive capacity of a series of pyrimidine-trione derivatives was tested in vitro in a chemoinvasion assay, and the most potent compound was further evaluated in vivo in different human tumor xenograft models. The activity of this novel selective MMPI was compared with BB-94, a broad-spectrum inhibitor. RESULTS: Ro-28-2653, an inhibitor with high selectivity for MMP-2, MMP-9, and membrane type 1 (MT1)-MMP, showed the highest anti-invasive activity in vitro. In vivo, Ro-28-2653 reduced the growth of tumors induced by the inoculation of different cell lines producing MMPs and inhibited the tumor-promoting effect of fibroblasts on breast adenocarcinoma cells. Furthermore, Ro-28-2653 reduced tumor vascularization and blocked angiogenesis in a rat aortic ring assay. In contrast, BB-94 up-regulated MMP-9 expression in tumor cells and promoted angiogenesis in the aortic ring assay. CONCLUSION: Ro-28-2653, a selective and orally bioavailable MMPI with inhibitory activity against MMPs expressed by tumor and/or stromal cells, is a potent antitumor and antiangiogenic agent. In contrast to broad-spectrum inhibitors, the administration of Ro-28-2653 was not associated with the occurrence of adverse side effects that might hamper the therapeutic potential of these drugs.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Metaloproteinases de Matriz , Fenilalanina/análogos & derivados , Piperazinas/farmacologia , Pirimidinas/farmacologia , Adenocarcinoma/tratamento farmacológico , Administração Oral , Animais , Aorta/patologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , DNA Complementar/metabolismo , Progressão da Doença , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Humanos , Concentração Inibidora 50 , Metaloproteinase 2 da Matriz/metabolismo , Microscopia de Fluorescência , Modelos Químicos , Invasividade Neoplásica , Transplante de Neoplasias , Neovascularização Patológica , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Ratos , Tiofenos/farmacologia , Fatores de Tempo , Regulação para CimaRESUMO
Meprin is a zinc endopeptidase of the astacin family, which is expressed as a membrane-bound or secreted protein in mammalian epithelial cells, in intestinal leucocytes and in certain cancer cells. There are two types of meprin subunits, alpha and beta, which form disulphide-bonded homo- and hetero-oligomers. Here we report on the cleavage of matrix proteins by hmeprin (human meprin) alpha and beta homo-oligomers, and on the interactions of these enzymes with inhibitors. Despite their completely different cleavage specificities, both hmeprin alpha and beta are able to hydrolyse basement membrane components such as collagen IV, nidogen-1 and fibronectin. However, they are inactive against intact collagen I. Hence the matrix-cleaving activity of hmeprin resembles that of gelatinases rather than collagenases. Hmeprin is inhibited by hydroxamic acid derivatives such as batimastat, galardin and Pro-Leu-Gly-hydroxamate, by TAPI-0 (tumour necrosis factor alpha protease inhibitor-0) and TAPI-2, and by thiol-based compounds such as captopril. Therapeutic targets for these inhibitors are MMPs (matrix metalloproteases), TACE (tumour necrosis factor alpha-converting enzyme) and angiotensin-converting enzyme respectively. The most effective inhibitor of hmeprin alpha in the present study was the naturally occurring hydroxamate actinonin ( K(i)=20 nM). The marked variance in the cleavage specificities of hmeprin alpha and beta is reflected by their interaction with the TACE inhibitor Ro 32-7315, whose affinity for the beta subunit (IC50=1.6 mM) is weaker by three orders of magnitude than that for the alpha subunit ( K(i)=1.6 microM). MMP inhibitors such as the pyrimidine-2,4,6-trione derivative Ro 28-2653 that are more specific for gelatinases do not bind to hmeprin, presumably due to the subtle differences in the mode of zinc binding and active-site structure between the astacins and the MMPs.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Metaloendopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Animais , Membrana Basal/enzimologia , Humanos , Cinética , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Metaloproteases , Camundongos , Modelos Moleculares , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ratos , Especificidade da Espécie , Homologia Estrutural de ProteínaRESUMO
Matrix metalloproteinase 2 (MMP-2) and MMP-9 are increased in human choroidal neovascularization (CNV) occurring during the exudative most aggressive form of age-related macular degeneration (AMD), but their precise role and potential interactions remain unclear. To address the question of MMP-2 and MMP-9 functions, mice deficient in the expression of MMP-2 (MMP-2 KO), MMP-9 (MMP-9 KO), and both MMP-2 and MMP-9 (MMP-2,9 KO) with their corresponding wild-type mice (WT) underwent CNV induction by laser-induced rupture of the Bruch's membrane. Both the incidence and the severity of CNV were strongly attenuated in double deficient compared with single gene deficient mice or corresponding WT controls. The reduced neovascularization was accompanied by fibrinogen/fibrin accumulation. Furthermore, overexpression of the endogenous MMP inhibitors TIMP-1 or TIMP-2 (delivered by adenoviral vectors) in WT mice or daily injection of a synthetic and gelatinase selective MMP inhibitor (Ro 26-2853) significantly decreased the pathological reaction. These findings suggest that MMP-2 and MMP-9 may cooperate in the development of AMD and that their selective inhibition represents an alternative strategy for the treatment of choroidal neovascularization.
Assuntos
Corioide/enzimologia , Neovascularização de Coroide/etiologia , Metaloproteinase 2 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/fisiologia , Animais , Corioide/química , Corioide/patologia , Neovascularização de Coroide/enzimologia , Neovascularização de Coroide/patologia , Fibrina/análise , Fibrinogênio/análise , Regulação da Expressão Gênica , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Knockout , Modelos Biológicos , Inibidores de Proteases/farmacologiaRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in invasion and metastatic spread of cancer cells. The objective of this study was to assess MMPs in plasma of Dunning tumor rats as indicators of the progression of prostate cancer and follow-up parameters after treatment. METHODS: Prostate cancer was induced in male Copenhagen rats by either subcutaneous or orthotopic implantation of R3327-MatLyLu cells. During the development of the tumor, plasma MMP-2, and MMP-9 were measured by gelatin-substrate zymography and Western blot technique with densitometry in untreated animals, rats treated with laser-induced hyperthermia, or with the new synthetic MMP inhibitor RO 28-2653. RESULTS: Normal prostatic tissue of the Copenhagen rats predominantly expressed proMMP-2 but not proMMP-9 whereas MMP-9 was only found in cancerous tissue. Elevated plasma MMP-9 values were demonstrated in rats with subcutaneous or orthotopic tumors. Animals with tumors and treated with the MMP inhibitor RO 28-2653 had both a lower tumor volume and a lower plasma MMP-9 concentration compared with controls. CONCLUSIONS: The determination of plasma MMP-9 in Dunning tumor rats is a useful tool to monitor the progression of prostate cancer and to assess the efficacy of drugs like MMP inhibitors or other treatment protocols.
Assuntos
Biomarcadores Tumorais/sangue , Metaloproteinase 9 da Matriz/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Animais , Biomarcadores , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Masculino , Metaloproteinase 2 da Matriz/sangue , Inibidores de Metaloproteinases de Matriz , Piperazinas/farmacologia , Pirimidinas/farmacologia , Ratos , Ratos EndogâmicosRESUMO
The recognition that matrix metalloproteinases (MMPs) facilitate tumor cell invasion and metastasis has led to the development of synthetic MMP inhibitors (MMPIs) as cancer therapeutic agents. Because several Phase III trials failed recently to show efficacy of broad-spectrum MMPIs in advanced cancer, the feasibility of MMPs as therapeutic targets has been challenged. However, it has not yet been determined whether MMPIs that have increased specificity may have greater benefit. We show that MMP-9 expression closely correlates with the progression of liver metastasis in a T-cell lymphoma model. MMPIs with greater selectivity/specificity for MMP-9 in vitro showed greater efficacy against liver metastasis in vivo. These data demonstrate a link between increased specificity of MMPIs and enhanced anticancer activity.
Assuntos
Inibidores Enzimáticos/farmacologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Linfoma de Células T/enzimologia , Inibidores de Metaloproteinases de Matriz , Animais , Divisão Celular/efeitos dos fármacos , Feminino , Fígado/citologia , Fígado/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/secundário , Linfoma de Células T/tratamento farmacológico , Linfoma de Células T/patologia , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinases da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos DBA , Neovascularização Patológica/tratamento farmacológico , Especificidade por SubstratoRESUMO
The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.
Assuntos
Metaloendopeptidases/metabolismo , Receptores de Endotelina/química , Receptores de Endotelina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Fosfatos de Inositol/metabolismo , Cinética , Ligantes , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Mutação , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Receptor de Endotelina B , Proteínas Recombinantes de Fusão/metabolismo , Temperatura , Fatores de Tempo , TransfecçãoRESUMO
The therapeutic efficacy of synthetic inhibitors of matrix-metalloproteinases (MMPs) in various cancers has been demonstrated. A novel inhibitor, Ro 28-2653, with high selectivity for MMP2, MMP9 and membrane type 1-MMP was evaluated in an orthotopic prostate cancer rat model. Efficacy was determined by recording tumor growth and survival endpoints. Prostate cancer was induced by inoculating R3327 Dunning tumor cells (MatLyLu) into the ventral lobe of the prostates of 148 Copenhagen rats. Daily oral treatment with Ro 28-2653 (10-300 mg/kg per day) was started on day 1 or on day 6 after tumor cell injection. Animals were sacrificed on day 20 for determination of tumor weights. For survival studies, rats received daily oral Ro 28-2653 (100 mg/kg per day) or vehicle for up to 30 days. Tumor induction was successful in 100% of the animals. Ro 28-2653 reproducibly reduced the tumor weights by up to 90% in a dose-dependent manner. In addition, an inhibitory effect in rats with established tumors (treatment start at day 6) was shown. A significantly prolonged survival of Ro 28-2653-treated rats was also demonstrated. Selective inhibition of MMP activity is a novel therapeutic approach, which bears promise for studies in patients with prostate cancer.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Inibidores de Metaloproteinases de Matriz , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Animais , Divisão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/sangue , Masculino , Metaloproteinases da Matriz/metabolismo , Transplante de Neoplasias , Ratos , Taxa de Sobrevida , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Enhanced expression and activation of matrix metalloproteinase-2 (MMP-2) and MMP-9 have been associated with tumor progression, invasion, and metastasis. The use of synthetic MMP inhibitors to block the proteolytic activity of these enzymes recently emerged as a potential therapeutic tool to treat cancer. In this study, we report that GI129471, a synthetic broad-spectrum MMP inhibitor, efficiently reduced the in vitro invasiveness of HT1080 cells through type IV collagen, a major component of basement membranes. This reduced invasion was paralleled by a complete inhibition of pro-MMP-2 activation; however, GI129471 strongly increased the amount of secreted pro-MMP-9, which could be subsequently activated through a plasminogen-dependent mechanism. Quantitative RT-PCR and northern blot analysis revealed that GI129471 specifically increased the MMP-9 mRNA steady-state level. Moreover, transient transfection of HT1080 cells with beta-galactosidase reporter vectors containing different lengths of the 5'-flanking region of the MMP-9 gene revealed an upregulation of the transcriptional activity of the corresponding promoter. Well-known modulators of MMP-9 expression such as Il-1beta and TNF-alpha were not involved in this upregulation. These findings emphasize the complexity of the regulation of MMP expression and the requirement for a detailed characterization of the potential adverse side effects associated with the use of broad-spectrum MMPIs.