RESUMEN
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.
RESUMEN
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.
Asunto(s)
Inmunidad Innata , Linfocitos , Pólipos Nasales , Rinitis , Sinusitis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad Crónica , Citocinas/inmunología , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-7/inmunología , Linfocitos/inmunología , Linfocitos/patología , Masculino , Persona de Mediana Edad , Pólipos Nasales/inmunología , Pólipos Nasales/patología , Rinitis/inmunología , Rinitis/patología , Sinusitis/inmunología , Sinusitis/patologíaRESUMEN
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.
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Enfermedades Pulmonares/genética , Enfermedades Pulmonares/terapia , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Animales , Enfermedad Crónica , Humanos , Enfermedades Pulmonares/metabolismo , MicroARNs/metabolismoRESUMEN
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.
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Receptor gp130 de Citocinas/inmunología , Inflamación/metabolismo , Interleucina-11/inmunología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT3/inmunología , Neoplasias Gástricas/metabolismo , Animales , Receptor gp130 de Citocinas/genética , Mucosa Gástrica/metabolismo , Humanos , Interleucina-11/genética , Interleucina-6/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/fisiología , Estómago/anatomía & histología , Estómago/patología , Neoplasias Gástricas/patologíaRESUMEN
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.
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Proteínas F-Box/metabolismo , Regulación de la Expresión Génica , Interferón gamma/fisiología , Músculo Liso Vascular/patología , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Biológicos , Transducción de Señal , Trasplante de TejidosRESUMEN
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.
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Asma/terapia , Antígeno B7-2/genética , Terapia Genética/métodos , Oligonucleótidos Antisentido/uso terapéutico , Neumonía/prevención & control , Hipersensibilidad Respiratoria/prevención & control , Animales , Asma/inducido químicamente , Asma/fisiopatología , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Líquido del Lavado Bronquioalveolar/química , Línea Celular , Quimiocina CCL11 , Quimiocina CCL5/metabolismo , Quimiocinas CC/metabolismo , Técnicas de Cocultivo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Granulocitos/metabolismo , Interleucina-13/metabolismo , Interleucina-2/metabolismo , Interleucina-5/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Moco/metabolismo , Oligonucleótidos Antisentido/análisis , Oligonucleótidos Antisentido/farmacocinética , Neumonía/metabolismo , Neumonía/patología , Ventilación Pulmonar , Hipersensibilidad Respiratoria/fisiopatología , Linfocitos T/inmunología , Linfocitos T/metabolismo , TransfecciónRESUMEN
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.
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Antiinflamatorios/metabolismo , Oligonucleótidos Antisentido/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Administración por Inhalación , Aerosoles , Animales , Asma/fisiopatología , Hiperreactividad Bronquial/terapia , Pruebas de Provocación Bronquial , Quimiocinas/biosíntesis , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Células Caliciformes/patología , Inflamación , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Masculino , Metaplasia , Ratones , Mucinas/genética , Mucinas/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Ovalbúmina , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Células Th2/efectos de los fármacos , Células Th2/inmunología , Resultado del TratamientoRESUMEN
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.
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Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/secundario , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/secundario , Oligonucleótidos Antisentido/farmacología , Factor de Transcripción STAT3/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Fosforilación , Valor Predictivo de las Pruebas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Relación Estructura-Actividad , Tasa de Supervivencia , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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
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Transformación Celular Neoplásica , Proteínas de Unión al ADN/fisiología , Linfoma de Células B Grandes Difuso/fisiopatología , Proteínas Tirosina Quinasas/fisiología , Transactivadores/fisiología , Quinasa de Linfoma Anaplásico , Animales , Línea Celular , Fibroblastos/fisiología , Humanos , Linfoma de Células T/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Datos de Secuencia Molecular , Mieloma Múltiple/fisiopatología , Oligonucleótidos Antisentido/farmacología , Proto-Oncogenes Mas , Proteínas Tirosina Quinasas Receptoras , Factor de Transcripción STAT3RESUMEN
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.
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Folículo Piloso/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Piel/metabolismo , Administración Tópica , Animales , Transporte Biológico , Ratones , Ratones Pelados , Ratones Endogámicos BALB C , Oligonucleótidos Antisentido/farmacocinética , Factores de TiempoRESUMEN
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.
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Asma/prevención & control , Proteína Quinasa 14 Activada por Mitógenos/administración & dosificación , Oligonucleótidos Antisentido/administración & dosificación , Administración por Inhalación , Aerosoles , Animales , Asma/fisiopatología , Hiperreactividad Bronquial , Líquido del Lavado Bronquioalveolar , Masculino , Ratones , Ratones Endogámicos BALB C , Moco/metabolismo , Ovalbúmina/inmunología , Reacción en Cadena de la Polimerasa , Eosinofilia Pulmonar/prevención & controlRESUMEN
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.
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Apoptosis , Proteínas de Unión al ADN/metabolismo , Neoplasias de la Próstata/metabolismo , Transactivadores/metabolismo , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Humanos , Janus Quinasa 2 , Janus Quinasa 3 , Masculino , Oligonucleótidos Antisentido/genética , Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT3 , Estilbenos/farmacología , Transactivadores/genética , Transfección , Tirfostinos/farmacologíaRESUMEN
Overexpression of receptor tyrosine kinases including the epidermal growth factor receptor (EGF-R) as well as nonreceptor tyrosine kinases, such as Src, have been implicated in the formation of human lung cancers. In addition, cytokines like interleukin-6 (IL-6) have been demonstrated to modulate lung cancer cell growth and elevated levels of IL-6 have been shown to be an adverse prognostic factor for patients with lung cancer. Despite a large body of evidence pointing to their potential importance, few direct studies into the role of signal transducers and activators of transcription (STAT) pathways in human lung cancer have been undertaken. Here we demonstrate that multiple nonsmall cell lung cancer cell lines demonstrate constitutive Stat3 DNA-binding activity. Stat3 DNA-binding activity is specifically upregulated by the addition of epidermal growth factor (EGF), IL-6, and hepatocyte-derived growth factor (HGF). Furthermore, the stimulation of Stat3 DNA-binding activity by EGF requires the activity of EGF-R tyrosine kinase as well as Src-kinase, while the upregulation of Stat3 activity by IL-6 or HGF requires only Src-kinase activity. Treatment of A549 lung cancer cells with PD180970 or SU6656, both pharmacological inhibitors of Src-kinase, resulted in reduced Src and Stat3 activity, cell cycle arrest in G2, and reduced viability of cells accompanied by induction of apoptosis. Treatment of Stat3-positive A549 and H358 cells with antisense Stat3 oligonucleotides results in complete loss of Stat3 DNA-binding activity and apoptosis, while Stat3-positive H1299 cells remained healthy. Finally, an adenoviral vector expressing a dominant-negative Stat3 isoform results in loss of Stat3 DNA-binding activity, apoptosis, and reduced cellular viability. These results demonstrate a role of Stat3 in transducing survival signals downstream of tyrosine kinases such as Src, EGF-R, and c-Met, as well as cytokines such as IL-6, in human nonsmall cell lung cancers.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transactivadores/metabolismo , Adenoviridae/genética , Apoptosis , Western Blotting , Ciclo Celular , Núcleo Celular/metabolismo , Supervivencia Celular , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fase G2 , Genes Dominantes , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Interleucina-6/metabolismo , Modelos Biológicos , Oligonucleótidos Antisentido/farmacología , Unión Proteica , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Factor de Transcripción STAT3 , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)-expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-X(L). Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.