RESUMO
The Notch signalling pathway mediates cell fate decisions and is tumour suppressive or oncogenic depending on the context. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine fate. In small-cell lung cancer, an aggressive neuroendocrine lung cancer, loss-of-function mutations in NOTCH genes and the inhibitory effects of ectopic Notch activation indicate that Notch signalling is tumour suppressive. Here we show that Notch signalling can be both tumour suppressive and pro-tumorigenic in small-cell lung cancer. Endogenous activation of the Notch pathway results in a neuroendocrine to non-neuroendocrine fate switch in 10-50% of tumour cells in a mouse model of small-cell lung cancer and in human tumours. This switch is mediated in part by Rest (also known as Nrsf), a transcriptional repressor that inhibits neuroendocrine gene expression. Non-neuroendocrine Notch-active small-cell lung cancer cells are slow growing, consistent with a tumour-suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to neuroendocrine tumour cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumour growth and delays relapse in pre-clinical models. Thus, small-cell lung cancer tumours generate their own microenvironment via activation of Notch signalling in a subset of tumour cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select patients with small-cell lung cancer.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Receptores Notch/metabolismo , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Microambiente Tumoral , Animais , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Recidiva Local de Neoplasia/prevenção & controle , Receptores Notch/agonistas , Receptores Notch/antagonistas & inibidores , Receptores Notch/deficiência , Proteínas Repressoras/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológicoRESUMO
Pancreatic cancer is one of the deadliest forms of cancer and effective treatment remains a clinical challenge. Transforming growth factor-beta (TGF-beta) has important roles in primary tumor progression and in promoting metastasis, and has become an attractive target for therapy. Previously, we reported that treatment of pancreatic cancer cells in vitro with SD-208, a small molecule inhibitor of the TGF-beta receptor I kinase (TGF-betaRI), inhibited expression of genes associated with tumor progression and inhibited invasiveness in a cell-based assay. In a demonstration of efficacy of TGF-beta signaling inhibition in an in vivo model of pancreatic cancer, we showed significantly reduced primary tumor weight and decreased incidence of metastasis in the Panc-1 orthotopic xenograft model of established pancreatic cancer. In this report, we extend these in vivo findings to examine the mechanistic consequences of TGF-betaRI inhibition on Panc-1 primary tumors and their microenvironment in situ. In a longitudinal study of TGF-betaRI inhibition in the Panc-1 orthotopic model, we show that SD-208 treatment significantly reduced tumor growth measured as bioluminescence intensity throughout the study. Histological evaluation revealed that SD-208 treatment reduced proliferation and induced apoptosis in the primary tumors, and reduced fibrosis in the tumor microenvironment. An immune contribution (greater B-cell infiltration in SD-208-treated tumors) was also suggested by the histological analyses. SD-208 not only blocked direct TGF-beta signaling in Panc-1 primary tumors (reduced phospho SMAD2/3), but also down-regulated the expression of TGF-beta-regulated genes (PAI-1 and COL7A1). Taken together, our results indicate that a TGF-betaRI kinase inhibitor has a potential therapeutic benefit for pancreatic cancer patients.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Pteridinas/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linfócitos B/imunologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, ß-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Neoplasias Colorretais , Mutação , Proteínas de Neoplasias , Paclitaxel/farmacologia , Taxoides/farmacologia , Trombospondinas , Microambiente Tumoral/efeitos dos fármacos , Via de Sinalização Wnt , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trombospondinas/antagonistas & inibidores , Trombospondinas/genética , Trombospondinas/metabolismo , Microambiente Tumoral/genética , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/ß-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.
Assuntos
Antineoplásicos/farmacologia , Mitose/efeitos dos fármacos , Taxoides/farmacologia , Proteínas Wnt/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Paclitaxel/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidoresRESUMO
The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate-dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-beta-induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.
Assuntos
Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeo Natriurético Encefálico/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Remodelação Ventricular , Adolescente , Western Blotting , Butadienos/farmacologia , Divisão Celular , Células Cultivadas/efeitos dos fármacos , GMP Cíclico/biossíntese , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Fibrose , Flavonoides/farmacologia , Perfilação da Expressão Gênica , Humanos , Inflamação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Peptídeo Natriurético Encefálico/fisiologia , Nitrilas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Deregulation of the ß-catenin signaling has long been associated with cancer. Intracellular components of this pathway, including axin, APC, and ß-catenin, are frequently mutated in a range of human tumors, but the contribution of specific extracellular ligands that promote cancer development through this signaling axis remains unclear. We conducted a reporter-based screen in a panel of human tumors to identify secreted factors that stimulate ß-catenin signaling. Through this screen and further molecular characterization, we found that R-spondin (RSPO) proteins collaborate with Wnt proteins to activate ß-catenin. RSPO family members were expressed in several human tumors representing multiple malignancies, including ovarian, pancreatic, colon, breast, and lung cancer. We generated specific monoclonal antibody antagonists of RSPO family members and found that anti-RSPO treatment markedly inhibited tumor growth in human patient-derived tumor xenograft models, either as single agents or in combination with chemotherapy. Furthermore, blocking RSPO signaling reduced the tumorigenicity of cancer cells based on serial transplantation studies. Moreover, gene-expression analyses revealed that anti-RSPO treatment in responsive tumors strongly inhibited ß-catenin target genes known to be associated with cancer and normal stem cells. Collectively, our results suggest that the RSPO family is an important stimulator of ß-catenin activity in many human tumors and highlight a new effective approach for therapeutically modulating this fundamental signaling axis.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Trombospondinas/metabolismo , beta Catenina/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais , Trombospondinas/biossíntese , Trombospondinas/genética , Trombospondinas/imunologia , Proteínas Wnt/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS mutations are frequent in colorectal cancer (CRC) and are associated with clinical resistance to treatment with the epidermal growth factor receptor (EGFR)-targeted monoclonal antibodies. Delta-like 4 ligand (DLL4) is an important component of the Notch signaling pathway and mediates stem cell self-renewal and vascular development. DLL4 inhibition in colon tumor cells reduces tumor growth and stem cell frequency. Considering the need for new drugs to treat colon cancers with oncogenic KRAS mutations, we examined in this study the efficacy of anti-DLL4 antibodies in KRAS mutant tumors in a panel of early passage colon tumor xenograft models derived from patients. Consistent with clinical findings, mutant KRAS colorectal xenograft tumors were insensitive to the EGFR therapeutic antibody cetuximab, whereas KRAS wild-type tumors responded to cetuximab. In contrast, anti-DLL4 was efficacious against both wild-type and mutant KRAS colon tumors as a single agent and in combination with irinotecan. Further analysis of mutant KRAS tumors indicated that the anti-DLL4/irinotecan combination produced a significant decrease in colon cancer stem cell frequency while promoting apoptosis in tumor cells. Our findings provide a rationale for targeting DLL4-Notch signaling for improved treatment of CRC patients with activating KRAS mutations.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Irinotecano , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras) , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transforming growth factor beta (TGFbeta) is a pleiotropic factor that regulates cell proliferation, angiogenesis, metastasis, and immune suppression. Dysregulation of the TGFbeta pathway in tumor cells often leads to resistance to the antiproliferative effects of TGFbeta while supporting other cellular processes that promote tumor invasiveness and growth. In the present study, SD-208, a 2,4-disubstituted pteridine, ATP-competitive inhibitor of the TGFbeta receptor I kinase (TGFbetaRI), was used to inhibit cellular activities and tumor progression of PANC-1, a human pancreatic tumor line. SD-208 blocked TGFbeta-dependent Smad2 phosphorylation and expression of TGFbeta-inducible proteins in cell culture. cDNA microarray analysis and functional gene clustering identified groups of TGFbeta-regulated genes involved in metastasis, angiogenesis, cell proliferation, survival, and apoptosis. These gene responses were inhibited by SD-208. Using a Boyden chamber motility assay, we demonstrated that SD-208 inhibited TGFbeta-stimulated invasion in vitro. An orthotopic xenograft mouse model revealed that SD-208 reduced primary tumor growth and decreased the incidence of metastasis in vivo. Our findings suggest mechanisms through which TGFbeta signaling may promote tumor progression in pancreatic adenocarcinoma. Moreover, they suggest that inhibition of TGFbetaRI with a small-molecule inhibitor may be effective as a therapeutic approach to treat human pancreatic cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Pteridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Receptores de Ativinas Tipo I/antagonistas & inibidores , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Genes myc , Humanos , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Serina-Treonina Quinases , Pteridinas/uso terapêutico , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteína Smad2/antagonistas & inibidores , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
Inhibitors of angiogenic factors are known to be upregulated, and their levels increase in the maternal circulation before the onset of preeclampsia. We reproduced a previously characterized model of preeclampsia by adenoviral overexpression of the soluble vascular endothelial growth factor (VEGF) receptor sFlt-1 (also referred to as sVEGFR-1) in pregnant and nonpregnant Sprague-Dawley rats. Animals were treated with VEGF121 at 0, 100, 200, or 400 microg/kg once or twice daily (n=8 per group; 64 total) and compared with normal control animals (n=4 per group) by examination of systolic blood pressure, urinary albumin and creatinine, renal histopathology, and glomerular gene expression profiling. sFlt-1 expression induced hypertension with proteinuria and glomerular endotheliosis and significant changes in gene expression. VEGF121 treatment alleviated these symptoms and reversed 125 of 268 sFlt-1-induced changes in gene expression. VEGF121 had beneficial effects in this rat model of preeclampsia without apparent harm to the fetus. Further study of VEGF121 as a potential therapeutic agent for preeclampsia is warranted.
Assuntos
Hipertensão/tratamento farmacológico , Rim/efeitos dos fármacos , Rim/patologia , Pré-Eclâmpsia/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão/fisiopatologia , Rim/metabolismo , Pré-Eclâmpsia/fisiopatologia , Gravidez , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Destruction of cartilage and bone is a poorly managed hallmark of human rheumatoid arthritis (RA). p38 Mitogen-activated protein kinase (MAPK) has been shown to regulate key proinflammatory pathways in RA, including tumor necrosis factor alpha, interleukin (IL)-1beta, and cyclooxygenase-2, as well as the process of osteoclast differentiation. Therefore, we evaluated whether a p38alpha MAPK inhibitor, indole-5-carboxamide (SD-282), could modulate cartilage and bone destruction in a mouse model of RA induced with bovine type II collagen [collagen-induced arthritis (CIA)]. In mice with early disease, SD-282 treatment significantly improved clinical severity scores, reduced bone and cartilage loss, and reduced mRNA levels of proinflammatory genes in paw tissue, including IL-1beta, IL-6, and cyclooxygenase-2. Notably, SD-282 treatment of mice with advanced disease resulted in significant improvement in clinical severity scoring and paw swelling, a reversal in bone and cartilage destruction as assessed by histology, bone volume fraction and thickness, and three-dimensional image analysis. These changes were accompanied by reduced osteoclast number and lowered levels of serum cartilage oligomeric matrix protein, a marker of cartilage breakdown. Thus, in a model of experimental arthritis associated with significant osteolysis, p38alpha MAPK inhibition not only attenuates disease progression but also reverses cartilage and bone destruction in mice with advanced CIA disease.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Experimental/enzimologia , Doenças das Cartilagens/enzimologia , Ossos do Pé/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Doenças das Cartilagens/tratamento farmacológico , Doenças das Cartilagens/patologia , Ossos do Pé/efeitos dos fármacos , Ossos do Pé/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.
Assuntos
Medula Óssea , Adesão Celular/fisiologia , Proliferação de Células , Indóis/metabolismo , Mieloma Múltiplo , Osteoclastos/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Medula Óssea/química , Medula Óssea/metabolismo , Proteínas de Transporte/metabolismo , Quimiocinas/metabolismo , Técnicas de Cocultura , Meios de Cultivo Condicionados , Humanos , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Osteoclastos/citologia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Células Estromais/citologia , Células Estromais/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Transforming growth factor-beta (TGFbeta) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGFbeta also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFbetaRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFbetaRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGFbeta-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGFbeta-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.
Assuntos
Receptores de Ativinas Tipo I/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Fibrose Pulmonar/etiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Diferenciação Celular , Células Cultivadas , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo , Citoesqueleto/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Sistema de Sinalização das MAP Quinases , Análise de Sequência com Séries de Oligonucleotídeos , Pteridinas/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/antagonistas & inibidores , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta/farmacologia , CicatrizaçãoRESUMO
p38 mitogen-activated protein (MAP) kinase is activated during ischemic/hypoxic myocardial injury. However, the role of activated p38 MAP kinase on cardiac function after myocardial injury is not well understood. In the present study, we investigated the cardioprotective effects of p38 MAP kinase inhibition in a rat model of acute myocardial injury, induced by subcutaneous injection of isoproterenol (ISO, 20 mg/kg/d for 3 days). A synthetic p38 alpha MAP kinase inhibitor, SD-282 (40 mg/kg) or vehicle (0.25% Tween 80 in saline) was given intraperitoneally twice a day for 3 days, concomitant with ISO treatment. Cardiac function, systolic blood pressure, gene expression including collagen I and III, fibronectin and COX-2, and the myocardial injury were analyzed. Results showed that administration of SD-282 remarkably improved ISO-induced reduction of cardiac function with increases in ejection fraction (P < 0.001), cardiac output (P < 0.05), stroke volume (P < 0.001), and cardiac index (P < 0.01). SD-282 abolished ISO-induced reduction of systolic blood pressure (106.7 +/- 2.2 versus 123.1 +/- 5.3 mm Hg, P < 0.05). The ISO-induced expression of COX-2, collagen I and III, and fibronectin genes was reduced significantly (P < 0.05 in all cases) by administration of SD-282. The myocardial injury induced by ISO was significantly reduced by the treatment of SD-282 as judged by the reduction of myocardial necrosis. Data suggest that p38 alpha MAP kinase may be involved in the pathogenesis of cardiac dysfunction in ischemic myocardial injury. Inhibition of this enzyme may improve cardiac function and protect myocardium from ischemic/hypoxic injury that occurs during ischemic heart disease.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Isquemia Miocárdica/prevenção & controle , Doença Aguda , Animais , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cardiotônicos/farmacologia , Expressão Gênica/efeitos dos fármacos , Injeções Intraperitoneais , Isoproterenol , Masculino , Isquemia Miocárdica/etiologia , Isquemia Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Necrose , Tamanho do Órgão/efeitos dos fármacos , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
An active role for C-reactive protein (CRP) in inflammatory vascular diseases has been recently suggested. Monocytes play an important role in vascular pathology and are activated by p38 mitogen activated protein kinase (MAPK) dependent mechanisms in many inflammatory settings. Therefore, we investigated whether CRP directly promotes a pro-inflammatory phenotype in human peripheral blood mononuclear cells (HPBMC) via p38 MAPK signaling. CRP exposure leads to a rapid phosphorylation of p38 MAPK in HPBMC. CRP-induced p38 kinase activity in HPBMC was blocked by treatment with an inhibitor of p38 kinase, SD-282. CRP-induced the expression of tissue factor protein and the secretion of IL-6, IL-8, IL-1beta, TNFalpha and PGE(2). Co-exposure to CRP and SD-282 blocked the secretion of these pro-inflammatory and pro-thrombotic mediators. CRP treatment elevated IL-6, IL-8, IL-1beta, TNFalpha, COX-2 and TF mRNA expression. These effects of CRP also required p38 activity, since SD-282 blocked mRNA induction of each. Taken together these data suggest a mechanistic relationship between p38 MAPK signaling and CRP-induced pro-inflammatory and pro-thrombotic activities in HPBMC. Thus, p38 inhibition may represent a novel approach to attenuate inflammation and its consequences in cardiovascular disease.