RESUMO
Regulatory T (Treg) cells are essential for immune tolerance of embryo implantation, and insufficient Treg cells are implicated in early pregnancy loss. An abortion-prone mouse model was used to evaluate the utility of IL-2 complexed with JES6-1 anti-IL-2 antibody (IL-2/JES6-1) to boost uterine Treg cells and improve reproductive success. IL-2/JES6-1, but not IL-2/IgG control, administered in the periconception phase to CBA/J females mated with DBA/2 males elicited a greater than twofold increase in the proportion of CD4+ T cells expressing forkhead box P3 (FOXP3), and an increase in the ratio of FOXP3+ Treg cells/FOXP3- T conventional cells, in the uterus and its draining lymph nodes at embryo implantation that was sustained into midgestation. An attenuated phenotype was evident in both thymic-derived and peripheral Treg cells with elevated cytotoxic T-lymphocyte antigen-4, CD25, and FOXP3, indicating improved suppressive function, as well as increased proliferative marker Ki-67. IL-2/JES6-1 treatment reduced fetal loss from 31% to 10%, but this was accompanied by a 6% reduction in late gestation fetal weight, despite comparable placental size and architecture. Similar effects of IL-2/JES6-1 on Treg cells and fetal growth were seen in CBA/J females with healthy pregnancies sired by BALB/c males. These findings show that expanding the uterine Treg cell pool through targeting IL-2 signaling is a strategy worthy of further investigation for mitigating immune-mediated fetal loss.
RESUMO
Our understanding of the aetiology and pathophysiology of endometriosis remains limited. Disease modelling in the field is problematic as many versions of induced mouse models of endometriosis exist. We integrated bioluminescent imaging of 'lesions' generated using luciferase-expressing donor mice. We compared longitudinal bioluminescence and histology of lesions, sensory behaviour of mice with induced endometriosis and the impact of the gonadotropin-releasing hormone antagonist Cetrorelix on lesion regression and sensory behaviour. Four models of endometriosis were tested. We found that the nature of the donor uterine material was a key determinant of how chronic the lesions were, as well as their cellular composition. The severity of pain-like behaviour also varied across models. Although Cetrorelix significantly reduced lesion bioluminescence in all models, it had varying impacts on pain-like behaviour. Collectively, our results demonstrate key differences in the progression of the 'disease' across different mouse models of endometriosis. We propose that validation and testing in multiple models, each of which may be representative of the different subtypes/heterogeneity observed in women, should become a standard approach to discovery science in the field of endometriosis.
Assuntos
Endometriose , Animais , Modelos Animais de Doenças , Endometriose/diagnóstico por imagem , Endometriose/patologia , Feminino , Antagonistas de Hormônios/farmacologia , Humanos , CamundongosRESUMO
BACKGROUND: Allergic asthma is a prevalent inflammatory disease of the airways caused by dysregulated immune balance in the lungs with incompletely understood pathogenesis. The recently identified type 2 innate lymphoid cells (ILC2s) play significant roles in the pathogenesis of asthma. Although ILC2-activating factors have been identified, the mechanisms that suppress ILC2s remain largely unknown. Plasmacytoid dendritic cells (pDCs) are important in antiviral immunity and in maintaining tolerance to inert antigens. OBJECTIVE: We sought to address the role of pDCs in regulating ILC2 function and ILC2-mediated airway hyperreactivity (AHR) and lung inflammation. METHODS: We used several murine models, including BDCA-2-diphtheria toxin receptor (DTR) transgenic and IFN-α receptor 1-deficient mice, as well as purified primary ILC2s, to reach our objective. We extended and validated our findings to human ILC2s. RESULTS: We show that activation of pDCs through Toll-like receptor 7/8 suppresses ILC2-mediated AHR and airway inflammation and that depletion of pDCs reverses this suppression. We further show that pDCs suppress cytokine production and the proliferation rate while increasing the apoptosis rate of ILC2s through IFN-α production. Transcriptomic analysis of both human and murine ILC2s confirms the activation of regulatory pathways in ILC2s by IFN-α. CONCLUSION: Activation of pDCs alleviates AHR and airway inflammation by suppressing ILC2 function and survival. Our findings reveal a novel regulatory pathway in ILC2-mediated pulmonary inflammation with important clinical implications.
Assuntos
Asma/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Plasmócitos/imunologia , Animais , Asma/genética , Asma/patologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Plasmócitos/patologiaRESUMO
BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is characterized by type 2 inflammation with high levels of Th2 cytokines. Although T helper cytokines are released from T cells, innate lymphoid cells (ILC) are also known to produce high levels of the same cytokines. However, the presence of various types of ILC in CRS is poorly understood. OBJECTIVE: The objective of this study was to fully characterize the presence of all ILC subsets in CRS and to identify phenotypical differences of group 2 ILC (ILC2) in CRSwNP compared to ILC2 from non-type 2 inflamed areas. METHODS: We investigated the presence of ILC subsets in peripheral blood mononuclear cells (PBMC) from healthy subjects, tonsil tissue, ethmoid tissue from control subjects and patients with non-polypoid CRS (CRSsNP) and CRSwNP, as well as nasal polyp (NP) tissue from CRSwNP by flow cytometry. Sorted ILC2 were cultured in the presence and absence of IL-33 and production of IL-5 and IL-13 was assessed by Luminex. RESULTS: We found that all ILC subsets were present in NP but ILC2 were dominant and significantly elevated compared to PBMC, tonsil, CRSsNP, and normal sinus tissue. We also found that inducible T-cell co-stimulator (ICOS) and side scatter were increased and CD127 was down-regulated in ILC2 from NP compared to blood or tonsil ILC2. Thymic stromal lymphopoietin, IL-7, and IL-33 were able to down-regulate expression of CD127 and increase side scatter in blood ILC2. Furthermore, sorted NP ILC2 but not blood ILC2 spontaneously released type 2 cytokines including IL-5 and IL-13. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that ILC2 are not only elevated but also activated in CRSwNP in vivo and that ILC2 may play important roles in the type 2 inflammation in CRSwNP.
Assuntos
Imunidade Inata , Linfócitos , Pólipos Nasais , Rinite , Sinusite , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Citocinas/imunologia , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-7/imunologia , Linfócitos/imunologia , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/imunologia , Pólipos Nasais/patologia , Rinite/imunologia , Rinite/patologia , Sinusite/imunologia , Sinusite/patologiaRESUMO
Chronic respiratory diseases are a significant health problem requiring novel approaches to both complement existing therapies and provide breakthrough medicines. Recent clinical advances in understanding the behavior of inhaled oligonucleotides provide the impetus for application of this technology to microRNA therapeutics. MicroRNAs are evolutionarily conserved small regulatory RNA molecules involved in tuning gene networks controlling biological and pathological processes. Deletion or overexpression of microRNAs results in phenotypic changes in animal models of disease such as cancer, fibrosis, diabetes, and inflammation. Inhibition of microRNAs in preclinical models of asthma, cystic fibrosis, and idiopathic pulmonary fibrosis has shown therapeutic promise. In animals, inhibitors of microRNAs directly delivered to the airway at doses suitable for nebulizers or hand-held inhalers up-regulate expression of cohorts of genes containing complementary "seed" sequences for specific and directed microRNA binding within their mRNA untranslated regions. These observations suggest the opportunity to exploit intervention in microRNA biology to create new therapies for chronic pulmonary disorders.
Assuntos
Pneumopatias/genética , Pneumopatias/terapia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Animais , Doença Crônica , Humanos , Pneumopatias/metabolismo , MicroRNAs/metabolismoRESUMO
OBJECTIVE: Mediation of RNA interference by oligonucleotides constitutes a powerful approach for the silencing of genes involved in the pathogenesis of inflammatory disease, but in vivo application of this technique requires effective delivery to immune cells and/or sites of inflammation. The aim of the present study was to develop a new carrier system to mediate systemic administration of oligonucleotides to rheumatoid arthritis (RA) joints, and to develop an antisense oligonucleotide (ASO)-based approach to interfere with CD40-CD154 interactions in an experimental model of RA. METHODS: A novel liposomal carrier with amphoteric properties, termed Nov038, was developed and assessed for its ability to systemically deliver an ASO directed against CD40 (CD40-ASO). Male DBA/1 mice with collagen-induced arthritis were treated with Nov038-encapsulated CD40-ASO, and the effects of treatment on various parameters of disease activity, including clinical score, paw swelling, lymph node responses, and inflammatory cytokine production in the joints, were assessed. RESULTS: Nov038 was well tolerated, devoid of immune-stimulatory effects, and efficacious in mediating systemic oligonucleotide delivery to sites of inflammation. In mice with collagen-induced arthritis, Nov038 enabled the therapeutic administration of CD40-ASO and improved established disease, while unassisted CD40-ASO was ineffective, and anti-tumor necrosis factor alpha (anti-TNFalpha) treatment was less effective in this model. Nov038/CD40-ASO efficacy was attributed to its tropism for monocyte/macrophages and myeloid dendritic cells (DCs), resulting in rapid down-regulation of CD40, inhibition of DC antigen presentation, and reduction in collagen-specific T cell responses, as well as decreased levels of TNFalpha, interleukin-6 (IL-6), and IL-17 in arthritic joints. CONCLUSION: Amphoteric liposomes represent a novel carrier concept for systemic and antigen-presenting cell-targeted oligonucleotide delivery with clinical applicability and numerous potential applications, including target validation in vivo and inflammatory disease therapeutics. Moreover, Nov038/CD40-ASO constitutes a potent alternative to monoclonal antibody-based approaches for interfering with CD40-CD40L interactions.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Artrite Experimental/terapia , Artrite Reumatoide/terapia , Antígenos CD40/genética , Terapia Genética/métodos , Lipossomos/farmacocinética , Animais , Células Apresentadoras de Antígenos/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , DNA Antissenso/farmacocinética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação para Baixo/imunologia , Articulações/imunologia , Articulações/metabolismo , Articulações/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Solubilidade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130(Y757F/Y757F) mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130(Y757F/Y757F) mice, when compared with unaffected gastric tissue in wild-type mice, while gp130(Y757F/Y757F) mice lacking the IL-11 ligand-binding receptor subunit (IL-11Ralpha) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130(Y757F/Y757F) mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130(Y757F/Y757F) mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1.
Assuntos
Receptor gp130 de Citocina/imunologia , Inflamação/metabolismo , Interleucina-11/imunologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/imunologia , Neoplasias Gástricas/metabolismo , Animais , Receptor gp130 de Citocina/genética , Mucosa Gástrica/metabolismo , Humanos , Interleucina-11/genética , Interleucina-6/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologia , Estômago/anatomia & histologia , Estômago/patologia , Neoplasias Gástricas/patologiaRESUMO
PURPOSE: Signal transducer and activator of transcription 5a/b (Stat5a/b) is the key mediator of prolactin effects in prostate cancer cells via activation of Janus-activated kinase 2. Prolactin is a locally produced growth factor in human prostate cancer. Prolactin protein expression and constitutive activation of Stat5a/b are associated with high histologic grade of clinical prostate cancer. Moreover, activation of Stat5a/b in primary prostate cancer predicts early disease recurrence. Here, we inhibited Stat5a/b by several different methodologic approaches. Our goal was to establish a proof of principle that Stat5a/b is critical for prostate cancer cell viability in vitro and for prostate tumor growth in vivo. EXPERIMENTAL DESIGN: We inhibited Stat5a/b protein expression by antisense oligonucleotides or RNA interference and transcriptional activity of Stat5a/b by adenoviral expression of a dominant-negative mutant of Stat5a/b in prostate cancer cells in culture. Moreover, Stat5a/b activity was suppressed in human prostate cancer xenograft tumors in nude mice. Stat5a/b regulation of Bcl-X(L) and cyclin D1 protein levels was shown by antisense suppression of Stat5a/b protein expression followed by Western blotting. RESULTS AND CONCLUSIONS: We show here that inhibition of Stat5a/b by antisense oligonucleotides, RNA interference, or adenoviral expression of dominant-negative Stat5a/b effectively kills prostate cancer cells. Moreover, we show that Stat5a/b is critical for human prostate cancer xenograft growth in nude mice. The effects of Stat5a/b on the viability of prostate cancer cells involve Stat5a/b regulation of Bcl-X(L) and cyclin D1 protein levels but not the expression or activation of Stat3. This work establishes Stat5a/b as a therapeutic target protein for prostate cancer. Pharmacologic inhibition of Stat5a/b in prostate cancer can be achieved by small-molecule inhibitors of transactivation, dimerization, or DNA binding of Stat5a/b.
Assuntos
Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Fator de Transcrição STAT5/genética , Transcrição Gênica , Adenoviridae/genética , Animais , Western Blotting , Sobrevivência Celular , Ciclina D , Ciclinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Genes Dominantes , Humanos , Masculino , Camundongos , Camundongos Nus , Oligonucleotídeos Antissenso/farmacologia , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/farmacologia , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/metabolismoRESUMO
The molecular mechanisms underlying progression of prostate cancer to the hormone-independent state are poorly understood. Signal transducer and activator of transcription 5a and 5b (Stat5a/b) is critical for the viability of human prostate cancer cells. We have previously shown that Stat5a/b is constitutively active in high-grade human prostate cancer, but not in normal prostate epithelium. Furthermore, activation of Stat5a/b in primary human prostate cancer predicted early disease recurrence. We show here that transcription factor Stat5a/b is active in 95% of clinical hormone-refractory human prostate cancers. We show for the first time that Stat5a/b synergizes with androgen receptor (AR) in prostate cancer cells. Specifically, active Stat5a/b increases transcriptional activity of AR, and AR, in turn, increases transcriptional activity of Stat5a/b. Liganded AR and active Stat5a/b physically interact in prostate cancer cells and, importantly, enhance nuclear localization of each other. The work presented here provides the first evidence of synergy between AR and the prolactin signaling protein Stat5a/b in human prostate cancer cells.
Assuntos
Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Fator de Transcrição STAT5/metabolismo , Transporte Ativo do Núcleo Celular , Androgênios/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoprecipitação , Masculino , Neoplasias Hormônio-Dependentes/genética , Neoplasias da Próstata/genética , Mapeamento de Interação de Proteínas , Receptores Androgênicos/análise , Receptores Androgênicos/genética , Fator de Transcrição STAT5/análise , Fator de Transcrição STAT5/genética , Transdução de Sinais , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Interferon (IFN)-gamma actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-gamma signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-gamma on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-gamma consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs. STAT3 activation resulted in nuclear translocation of this transcription factor. By selectively inhibiting STAT3 and not STAT1 signaling, we identified a number of candidate IFN-gamma-inducible, STAT3-dependent gene products by microarray analysis. Results for selected genes, including the pro-apoptotic molecules X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa, were verified by real time quantitative reverse transcription-PCR and immunoblot analyses. IFN-gamma-induced STAT3 and STAT1 signaling in VSMCs demonstrated reciprocal inhibition. STAT3 activation by IFN-gamma sensitized VSMCs to apoptosis triggered by both death receptor- and mitochondrial-mediated pathways. Knock down of XAF1 and Noxa expression inhibited the priming of VSMCs to apoptotic stimuli by IFN-gamma. Finally, we confirmed the in vivo relevance of our observations using a chimeric animal model of immunodeficient mice bearing human coronary artery grafts in which the expression of XAF1 and Noxa as well as the pro-apoptotic effects induced by IFN-gamma were dependent on STAT3. The data suggest STAT1-independent signaling by IFN-gamma via STAT3 that promotes the death of human VSMCs.
Assuntos
Proteínas F-Box/metabolismo , Regulação da Expressão Gênica , Interferon gama/fisiologia , Músculo Liso Vascular/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Biológicos , Transdução de Sinais , Transplante de TecidosRESUMO
The molecular mechanisms that promote progression of localized prostate cancer to hormone-refractory and disseminated disease are poorly understood. Prolactin (Prl) is a local growth factor produced in high-grade prostate cancer, and exogenously added Prl in tissue or explant cultures of normal and malignant prostate is a strong mitogen and survival factor for prostate epithelium. The key signaling proteins that mediate the biological effects of Prl in prostate cancer are Signal Transducer and Activator of Transcription (Stat)-5a/5b via activation of Janus kinase-2. Importantly, inhibition of Stat5a/b in prostate cancer cells induces apoptotic death. Using a specific Prl receptor antagonist (Delta1-9G129R-hPRL), we demonstrate here for the first time that autocrine Prl in androgen-independent human prostate cancer cells promotes cell viability via Stat5 signaling pathway. Furthermore, we examined a unique clinical material of human hormone refractory prostate cancers and metastases and show that autocrine Prl is expressed in 54% of hormone-refractory clinical human prostate cancers and 62% prostate cancer metastases. Finally, we demonstrate that autocrine Prl is expressed from both the proximal and distal promoters of the Prl gene in clinical human prostate cancers and in vivo and in vitro human prostate cancer models, independently of pituitary transcription factor-1 (Pit-1). Collectively, the data provide novel evidence for the concept that autocrine Prl signaling pathway is involved in growth of hormone-refractory and metastatic prostate cancer. The study also provides support for the use of Prl receptor antagonists or other therapeutic strategies to block the Prl-Janus kinase-2-Stat5 signaling pathway in advanced prostate cancer.
Assuntos
Janus Quinase 2/metabolismo , Prolactina/metabolismo , Neoplasias da Próstata/patologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Oligodesoxirribonucleotídeos Antissenso/genética , Fosforilação/efeitos dos fármacos , Prolactina/antagonistas & inibidores , Prolactina/genética , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/genética , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Transplante Heterólogo , Proteínas Supressoras de Tumor , Tirfostinas/farmacologiaRESUMO
The B7-family molecule CD86, expressed on the surface of pulmonary and thoracic lymph node antigen-presenting cells, delivers essential costimulatory signals for T-cell activation in response to inhaled allergens. CD86-CD28 signaling is involved in priming allergen-specific T cells, but it is unclear whether these interactions play a role in coordinating memory T-helper 2 cell responses. In the ovalbumin (OVA)-induced mouse model of asthma, administration of CD86-specific antibody before systemic sensitization suppresses inhaled OVA-induced pulmonary inflammation and airway hyper-responsiveness (AHR). In previously OVA-sensitized mice, systemic and intranasal coadministration of CD86 antibody is required to produce these effects. To directly assess the importance of pulmonary CD86 expression in secondary immune responses to inhaled allergens, mice were sensitized and locally challenged with nebulized OVA before treatment with an inhaled aerosolized CD86 antisense oligonucleotide (ASO). CD86 ASO treatment suppressed OVA-induced up-regulation of CD86 protein expression on pulmonary dendritic cells and macrophages as well as on recruited eosinophils. Suppression of CD86 protein expression correlated with decreased methacholine-induced AHR, airway inflammation, and mucus production following rechallenge with inhaled OVA. CD86 ASO treatment reduced BAL eotaxin levels, but it did not reduce CD86 protein on cells in the draining lymph nodes of the lung, and it had no effect on serum IgE levels, suggesting a local and not a systemic effect. These results demonstrate that CD86 expression on pulmonary antigen-presenting cells plays a vital role in regulating pulmonary secondary immune responses and suggest that treatment with an inhaled CD86 ASO may have utility in asthma and other chronic inflammatory lung conditions.
Assuntos
Asma/terapia , Antígeno B7-2/genética , Terapia Genética/métodos , Oligonucleotídeos Antissenso/uso terapêutico , Pneumonia/prevenção & controle , Hipersensibilidade Respiratória/prevenção & controle , Animais , Asma/induzido quimicamente , Asma/fisiopatologia , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Granulócitos/metabolismo , Interleucina-13/metabolismo , Interleucina-2/metabolismo , Interleucina-5/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Muco/metabolismo , Oligonucleotídeos Antissenso/análise , Oligonucleotídeos Antissenso/farmacocinética , Pneumonia/metabolismo , Pneumonia/patologia , Ventilação Pulmonar , Hipersensibilidade Respiratória/fisiopatologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , TransfecçãoRESUMO
The Th2 cytokines IL-4 and IL-13 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor-alpha chain (IL-4Ralpha). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4Ralpha-deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4Ralpha. We designed a chemically modified IL-4Ralpha antisense oligonucleotide (IL-4Ralpha ASO) that specifically inhibits IL-4Ralpha protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4Ralpha ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4Ralpha ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4Ralpha in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.
Assuntos
Anti-Inflamatórios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Administração por Inalação , Aerossóis , Animais , Asma/fisiopatologia , Hiper-Reatividade Brônquica/terapia , Testes de Provocação Brônquica , Quimiocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Inflamação , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Metaplasia , Camundongos , Mucinas/genética , Mucinas/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Ovalbumina , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Resultado do TratamentoRESUMO
PURPOSE: Hepatocellular carcinoma (HCC) is an aggressive malignancy and is a devastating clinical complication of chronic liver disease. Therapeutic options are limited mainly because the genetic and biochemical understanding of this disease remains fragmented. We intended to study the role of signal transducer and activator of transcription 3 (STAT3) aberrant signaling in HCC malignancy, and the therapeutic potential of inhibition of STAT3 expression for HCC. EXPERIMENTAL DESIGN: A 2'-O-methoxyethylribose-modified phosphorothioate antisense oligonucleotide (ASO) was used to knock down STAT3 expression in different human HCC cell lines, including the highly metastatic HCCLM3 derived from orthotopic implantation and subsequent lung metastasis in athymic mice. The effects of STAT3 ASO treatment on HCC cells, metastasis, and animal survival following HCCLM3 orthotopic implantation were evaluated. RESULTS: Specific suppression of phosphorylated STAT3 reduced its DNA-binding activity, inhibited the expression of vascular endothelial growth factor, survivin, matrix metalloproteinases 2 and 9, reduced cell proliferation and migratory potential, induced apoptosis in vitro, and inhibited intradermal angiogenesis and s.c. tumorigenesis upon injection in mice. In mice bearing orthotopically implanted HCCLM3, STAT3 inhibition following therapeutic treatment with STAT3 ASO reduced circulating vascular endothelial growth factor and basic fibroblast growth factor, decreased intratumor CD34-positive microvessel density, intrahepatic and intraperitoneal transmission, and lung metastasis. HCC tumor volume and weight were reduced and the survival time of mice bearing orthotopically xenografted HCC was approximately doubled in STAT3 ASO-treated mice (P < 0.05). CONCLUSIONS: Constitutively activated STAT3 is essential for the growth, survival, and metastasis of HCC, suggesting that STAT3-targeted therapy may have utility for HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/secundário , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Oligonucleotídeos Antissenso/farmacologia , Fator de Transcrição STAT3/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Fosforilação , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Taxa de Sobrevida , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
STATs are believed to play key roles in normal and abnormal cell function. In the present work, we investigated the role of STATs in an IL-2-responsive human lymphoblastic lymphoma-derived cell line, YT. Only STAT3 was found constitutively tyrosine phosphorylated, but not other STATs. Hyperactive STAT3 was not attributable to a pre-existing intermediate affinity IL-2R complex and/or hyperactive Jak activity. Depletion of STAT3 protein expression reduced tumor cell viability with protracted kinetics (72-96 h), while TUNEL assays demonstrated cell death occurred via apoptosis. Interestingly, depletion of STAT5 in this same tumor induced more pronounced cell death compared with STAT3 depletion (24 h). Although IL-2 was able to rescue STAT3-depleted cells from death, it could not compensate for the loss of STAT5. To determine the prosurvival function of STAT3 vs STAT5 within the same tumor model, genes were profiled in STAT3- or STAT5-depleted YT cells by apoptosis-specific microarrays. Several differentially expressed genes were identified. Interestingly, those genes involved in NF-kappaB regulation, such as TNFR-associated factors 2 and 5 and B cell leukemia/lymphoma 10, were readily decreased upon STAT5, but not STAT3, depletion as validated by quantitative RT-PCR. These results suggest that STAT5 and, to a lesser extent, hyperactive STAT3 provide preferential and critical cell survival signals for certain human lymphoid tumors, indicating that nonhyperactive STATs should be considered as therapeutic targets for abrogating tumorigenesis.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/fisiologia , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-2 , NF-kappa B/metabolismo , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiologia , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT5/deficiênciaRESUMO
Loss of p53 function by mutation is common in cancer. However, most natural p53 mutations occur at a late stage in tumor development, and many clinically detectable cancers have reduced p53 expression but no p53 mutations. It remains to be fully determined what mechanisms disable p53 during malignant initiation and in cancers without mutations that directly affect p53. We show here that oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Site-specific mutation of a Stat3 DNA-binding site in the p53 promoter partially abrogates Stat3-induced inhibition. Stat3 activity also influences p53 response genes and affects UV-induced cell growth arrest in normal cells. Furthermore, blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. As a point of convergence for many oncogenic signaling pathways, Stat3 is constitutively activated at high frequency in a wide diversity of cancers and is a promising molecular target for cancer therapy. Thus, repression of p53 expression by Stat3 is likely to have an important role in development of tumors, and targeting Stat3 represents a novel therapeutic approach for p53 reactivation in many cancers lacking p53 mutations.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Fator de Transcrição STAT3 , Transdução de Sinais , Transativadores/antagonistas & inibidores , Transativadores/deficiência , Transativadores/genética , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Anaplastic large cell lymphomas (ALCLs) are caused by chromosomal translocations that juxtapose the anaplastic lymphoma kinase (ALK) proto-oncogene to a dimerization partner, resulting in constitutive expression of ALK and ALK tyrosine kinase activity. One substrate of activated ALK in human ALCLs is the transcription factor Stat3, and its phosphorylation is accurately recapitulated in a new nucleophosmin (NPM)-ALK transgenic mouse model of lymphomagenesis. Here we show by gene targeting that Stat3 is required for the transformation of mouse embryonic fibroblasts in vitro, for the development of B-cell lymphoma in transgenic mice and for the growth and survival of both human and mouse NPM-ALK-transformed B and T cells. Ablation of Stat3 expression by antisense oligonucleotides significantly (P < 0.0001) impaired the growth of human and mouse NPM-ALK tumors in vivo. Pharmacological ablation of Stat3 represents a new candidate approach for the treatment of human lymphoma
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/fisiologia , Linfoma Difuso de Grandes Células B/fisiopatologia , Proteínas Tirosina Quinases/fisiologia , Transativadores/fisiologia , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular , Fibroblastos/fisiologia , Humanos , Linfoma de Células T/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Dados de Sequência Molecular , Mieloma Múltiplo/fisiopatologia , Oligonucleotídeos Antissenso/farmacologia , Proto-Oncogene Mas , Receptores Proteína Tirosina Quinases , Fator de Transcrição STAT3RESUMO
Antisense oligodeoxynucleotides formulated in cream preparations are being examined in the clinic as topical therapy for psoriasis. To produce their intended anti-inflammatory effects, these large anionic molecules must penetrate the stratum corneum and reach the living epidermis and dermis. A topically applied phosphorothioate antisense oligonucleotide targeted to intercellular adhesion molecule-1 recently was shown to modulate cytokine-inducible target gene expression in engrafted human skin. In this study, we examined the route of entry into mouse skin of fluorochrome-tagged or naked second-generation 2'-O-methoxyethyl-modified oligonucleotides that react specifically with an antibody, using topical cream-based formulations. In hairless mouse skin, immunohistochemical analysis and fluorescence microscopy were unable to detect the presence of oligonucleotide in the epidermis or dermis following topical application although immunostaining was associated with the stratum corneum and fluorescein isothiocyanate-labeled oligonucleotide was observed in hair follicles. Kinetic analysis of oligonucleotide topically applied to hair-clipped BALB/c mouse skin showed early follicular localization, diffusion of oligonucleotide from the mid-follicle, and subsequent dermal accumulation. Saline formulation resulted in oligonucleotide remaining within the hair follicle. These results suggest that oligonucleotide penetration in skin involves a follicular route and further, that topical oligonucleotide therapy may be particularly well suited for altering physiology within the hair follicle and related structures.
Assuntos
Folículo Piloso/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Pele/metabolismo , Administração Tópica , Animais , Transporte Biológico , Camundongos , Camundongos Pelados , Camundongos Endogâmicos BALB C , Oligonucleotídeos Antissenso/farmacocinética , Fatores de TempoRESUMO
The p38 mitogen-activated protein kinase (MAPK) plays a critical role in the activation of inflammatory cells. Therefore, we investigated the antiinflammatory effects of a respirable p38alpha MAPK antisense oligonucleotide (p38alpha-ASO) in a mouse asthma model. A potent and selective p38alpha-ASO was characterized in vitro. Inhalation of aerosolized p38alpha-ASO using an aerosol chamber dosing system produced measurable lung deposition of ASO and significant reduction of ovalbumin (OVA-)-induced increases in total cells, eosinophils, and interleukin 4 (IL-4), IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and dose-dependent inhibition of airway hyperresponsiveness in allergen-challenged mice. Furthermore, inhaled p38alpha-ASO markedly inhibited OVA-induced lung tissue eosinophilia and airway mucus hypersecretion. Quantitative polymerase chain reaction analysis of bronchoalveolar lavage fluid cells and peribronchial lymph node cells showed that p38alpha-ASO significantly reduced p38alpha MAPK mRNA expression. Nose-only aerosol exposure of mice verified the p38alpha-ASO-induced inhibition of OVA-induced pulmonary eosinophilia, mucus hypersecretion, and airway hyperresponsiveness. None of the effects of the p38alpha-ASO were produced by a six-base mismatched control oligonucleotide. These findings demonstrate antisense pharmacodynamic activity in the airways after aerosol delivery and suggest that a p38alpha MAPK ASO approach may have therapeutic potential for asthma and other inflammatory lung diseases.
Assuntos
Asma/prevenção & controle , Proteína Quinase 14 Ativada por Mitógeno/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Administração por Inalação , Aerossóis , Animais , Asma/fisiopatologia , Hiper-Reatividade Brônquica , Líquido da Lavagem Broncoalveolar , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Muco/metabolismo , Ovalbumina/imunologia , Reação em Cadeia da Polimerase , Eosinofilia Pulmonar/prevenção & controleRESUMO
Signal transducers and activators of transcription (STAT) were originally discovered as components of cytokine signal transduction pathways. Persistent activation of one STAT, STAT3, is a common feature of prostate cancer. Activated STAT3 was found in pathology specimens obtained from prostatectomy in the cancerous areas but not in the normal margins. Because the activation of STAT3 is mediated by the action of an upstream Janus kinase (JAK) kinase, usually JAK1 or JAK2, the activation step for STAT3 might itself be a target for therapy in prostate cancer. However, the redundancy of upstream kinases may make this strategy unreliable for therapy. To develop molecular targets for prostate cancer treatment, JAK kinase and STAT3 inhibition of two prostate cancer lines were compared. DU145 and NRP-154 cells were treated with JAK kinase inhibitors, analyzed for onset of apoptosis, and measured by annexin V binding and propidium iodide uptake. Activation of caspases in the cells was determined by measuring cleaved caspase-3 following treatment. For determining the effect on mitochondrial membrane depolarization that accompanies apoptosis, the fluorescent dye JC-1 was used. STAT3 was specifically inhibited by transfecting either a dominant-negative (DN) STAT3 plasmid or antisense STAT3 oligonucleotides into the cells. To look for reduction in STAT3 levels within cells, fixed and permeabilized prostate cancer cells were stained with antibody to STAT3. We found that more than one JAK kinase is involved in STAT3 activation in prostate cancer lines. AG490 (JAK2 specific) induced apoptosis in DU145 but not in NRP-154 prostate cancer lines, whereas piceatannol (JAK1 specific) induced apoptosis in NRP-154 but not in DU145 cells. Next, we demonstrated efficacy of specific STAT3 inhibitors in prostate cancer lines. Both induction of apoptosis and reduction in intracellular STAT3 protein were observed following treatment with antisense STAT3 oligonucleotides, while transfection of a DN-STAT3 plasmid into both prostate cancer cell lines resulted in loss of viability and onset of apoptosis. We conclude that STAT3-specific inhibitors, rather than JAK kinase-specific inhibitors, should be more useful therapeutically in treating androgen-resistant prostate cancer and that STAT3 is an appropriate target in the treatment of prostate cancer.