RESUMO
Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.
Assuntos
Imunoterapia/métodos , Melanoma/imunologia , Melanoma/patologia , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Linfócitos T Reguladores/imunologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismoRESUMO
Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.
Assuntos
Proteínas de Ciclo Celular/agonistas , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Endoderma/citologia , Endoderma/metabolismo , Genes Reporter , Proteína Goosecoid/genética , Proteína Goosecoid/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/genética , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Elementos Reguladores de Transcrição , Sítio de Iniciação de TranscriçãoRESUMO
Several lines of evidence indicate that dopamine (DA) plays a key role in the cross-talk between the nervous and immune systems. In this study, we disclose a novel immune-regulatory role for DA: inhibition of effector functions of activated NK lymphocytes via the selective upregulation of the D5 dopaminergic receptor in response to prolonged cell stimulation with rIL-2. Indeed, engagement of this D1-like inhibitory receptor following binding with DA suppresses NK cell proliferation and synthesis of IFN-γ. The inhibition of IFN-γ production occurs through blocking the repressor activity of the p50/c-REL dimer of the NF-κB complex. Indeed, the stimulation of the D5 receptor on rIL-2-activated NK cells inhibits the binding of p50 to the microRNA 29a promoter, thus inducing a de novo synthesis of this miRNA. In turn, the increased levels of microRNA 29a were inversely correlated with the ability of NK cells to produce IFN-γ. Taken together, our findings demonstrated that DA switches off activated NK cells, thus representing a checkpoint exerted by the nervous system to control the reactivity of these innate immune effectors in response to activation stimuli and to avoid the establishment of chronic and pathologic inflammatory processes.
Assuntos
Dopamina/imunologia , Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , MicroRNAs/biossíntese , Receptores de Dopamina D5/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Citotoxicidade Imunológica/imunologia , Células HEK293 , Humanos , Inflamação/imunologia , Interleucina-2/farmacologia , Ativação Linfocitária/imunologia , MicroRNAs/genética , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Regulação para Cima/imunologiaRESUMO
Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Tirosina/análogos & derivados , Células 3T3 , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Células L , Lipopolissacarídeos , Macrófagos/metabolismo , Camundongos , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Fator de Transcrição RelA/antagonistas & inibidores , Tirosina/farmacologia , Proteína bcl-X/biossínteseRESUMO
Nuclear factor κB regulates various genes involved in the immune response, inflammation, cell survival, and development. NF-κB activation is controlled by proteins possessing ankyrin repeats, such as IκBs. A precursor of the NF-κB2 (p52) subunit, p100, contains ankyrin repeats in its C-terminal portion and has been found to act as a cytoplasmic inhibitor of RelA in the canonical pathway of NF-κB activation. Here, we demonstrate that p100 also suppresses c-Rel function in dendritic cells. Expression of the p19 and p40 subunits of IL-23, a c-Rel-dependent cytokine, was enhanced in p100-deficient cells, although expression of a RelA-dependent cytokine, TNF-α, was reduced. Nuclear translocation of c-Rel was enhanced in p100-deficient cells. p100, and not the processed p52 form, associated with c-Rel in the steady state and dissociated immediately after lipopolysaccharide stimulation in wild-type dendritic cells. Four hours after the stimulation, p100 was newly synthesized and associated with c-Rel again. In cells expressing both c-Rel and RelA, c-Rel is preferentially suppressed by p100.
Assuntos
Células Dendríticas/metabolismo , Interleucina-23/metabolismo , Subunidade p52 de NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Animais , Sequência de Bases , Primers do DNA , Células HEK293 , Humanos , Camundongos , Subunidade p52 de NF-kappa B/genética , Reação em Cadeia da PolimeraseRESUMO
Despite intense efforts to develop treatments against pancreatic cancer, agents that cure this highly resistant and metastasizing disease are not available. Considerable attention has focused on broccoli compound sulforaphane (SF), which is suggested as combination therapy for targeting of pancreatic cancer stem cells (CSCs). However, there are concerns that antioxidative properties of SF may interfere with cytotoxic drugs-as suggested, e.g., for vitamins. Therefore we investigated a combination therapy using established pancreatic CSCs. Although cisplatin (CIS), gemcitabine (GEM), doxorubicin, 5-flurouracil, or SF effectively induced apoptosis and prevented viability, combination of a drug with SF increased toxicity. Similarly, SF potentiated the drug effect in established prostate CSCs revealing that SF enhances drug cytotoxicity also in other tumor entities. Most importantly, combined treatment intensified inhibition of clonogenicity and spheroid formation and aldehyde dehydrogenase 1 (ALDH1) activity along with Notch-1 and c-Rel expression indicating that CSC characteristics are targeted. In vivo, combination treatment was most effective and totally abolished growth of CSC xenografts and tumor-initiating potential. No pronounced side effects were observed in normal cells or mice. Our data suggest that SF increases the effectiveness of various cytotoxic drugs against CSCs without inducing additional toxicity in mice.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Tiocianatos/farmacologia , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Isotiocianatos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Retinal Desidrogenase , Esferoides Celulares , Sulfóxidos , Ensaio Tumoral de Célula-Tronco/métodosRESUMO
Tripartite motif-containing 9 (TRIM9) has been demonstrated to exert important roles in regulation of innate immune signaling. In this study, a novel TRIM9 homolog was identified from Penaeus monodon (named PmTRIM9). The open reading frame (ORF) of PmTRIM9 was 2064 bp, which encoding a 687-amino-acid polypeptide. Following Vibrio parahaemolyticus challenge, the expression levels of PmTRIM9 mRNA were significantly down-regulated in tested tissues. RNA interference and recombinant protein injection experiments were performed to explore the function of PmTRIM9, and the results showed it could facilitate V. parahaemolyticus replication and lead P. monodon more vulnerable to V. parahaemolyticus challenge. The dual-luciferase reporter assay showed that PmTRIM9 possessed the ability to inhibit the promoter activity in HEK293 T cells. Silencing of PmTRIM9 could increase the expression of the major NF-κB transcription factor, PmRelish. Further studies showed that knockdown of PmRelish promoted the V. parahaemolyticus infection and decreased the expression of specific antimicrobial peptides (AMPs), including PmCRU5, PmCRU7, PmALF6, PmALF3, PmLYZ and PmPEN5. However, knockdown of PmTRIM9 increased expression levels of the same AMPs, but except for PmCRU5, indicating that PmTRIM9 may negatively regulate the PmRelish-mediated expression of AMPs. All these results suggest that PmTRIM9 was involved in facilitating V. parahaemolyticus infection by inhibition of Relish pathway in P. monodon.
Assuntos
Proteínas de Peixes/genética , Penaeidae/microbiologia , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas com Motivo Tripartido/genética , Vibrio parahaemolyticus/crescimento & desenvolvimento , Animais , Linhagem Celular , Inativação Gênica , Células HEK293 , Humanos , Penaeidae/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transdução de Sinais/imunologiaRESUMO
Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.
Assuntos
Epitélio/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Macrófagos/patologia , Proteínas Proto-Oncogênicas c-rel/genética , Animais , Polaridade Celular/genética , Marcação de Genes , Hepatócitos/patologia , Hidroxiprolina/metabolismo , Cirrose Hepática/prevenção & controle , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/genética , Comunicação Parácrina/genética , Fosfofrutoquinase-2/genética , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/metabolismoRESUMO
In retroviral infections, different immunological mechanisms are involved in the development of a chronic infection. In the Friend virus (FV) model, regulatory T cells (Tregs) were found to induce CD8+ T cell dysfunction before viral clearance is achieved and thus contribute to viral chronicity. Although studied for decades, the exact suppressive mechanisms of Tregs in the FV model remain elusive and an unavailable therapeutic target. However, extracellular IL-2 and intracellular NF-κB signaling were shown to be important pathways for Treg expansion and activation. Therefore, we decided to focus on these two pathways to test therapeutic approaches inhibiting Treg activation during FV infection. In this study, we show that the inhibition of either IL-2 or the NF-κB subunit c-Rel, impaired Treg expansion and activation at 2 weeks post-FV infection. Total numbers of Tregs as well as activated Tregs were reduced in FV-infected mice after treatment with anti-IL-2 antibodies or the c-Rel blocking reagent pentoxifylline. Surprisingly, this did not affect the expansion or function of virus-specific CD8+ T cells nor viral loads in the spleen. However, our data suggest that neutralization of IL-2 as well as blocking c-Rel efficiently inhibits virus-induced Treg expansion.
Assuntos
Interleucina-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Infecções por Retroviridae/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Feminino , Vírus da Leucemia Murina de Friend , Interleucina-2/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pentoxifilina/administração & dosagem , Proteínas Proto-Oncogênicas c-rel/metabolismo , Linfócitos T Reguladores/patologia , Carga ViralRESUMO
The molecular mechanisms underlying fate decisions of human neural stem cells (hNSCs) between neurogenesis and gliogenesis are critical during neuronal development and neurodegenerative diseases. Despite its crucial role in the murine nervous system, the potential role of the transcription factor NF-κB in the neuronal development of hNSCs is poorly understood. Here, we analyzed NF-κB subunit distribution during glutamatergic differentiation of hNSCs originating from neural crest-derived stem cells. We observed several peaks of specific NF-κB subunits. The most prominent nuclear peak was shown by c-REL subunit during a period of 2-5 days after differentiation onset. Furthermore, c-REL inhibition with pentoxifylline (PTXF) resulted in a complete shift towards oligodendroglial fate, as demonstrated by the presence of OLIG2+/O4+-oligodendrocytes, which showed PDGFRα, NG2 and MBP at the transcript level. In addition c-REL impairment further produced a significant decrease in neuronal survival. Transplantation of PTXF-treated predifferentiated hNSCs into an ex vivo oxidative-stress-mediated demyelination model of mouse organotypic cerebellar slices further led to integration in the white matter and differentiation into MBP+ oligodendrocytes, validating their functionality and therapeutic potential. In summary, we present a human cellular model of neuronal differentiation exhibiting a novel essential function of NF-κB-c-REL in fate choice between neurogenesis and oligodendrogenesis which will potentially be relevant for multiple sclerosis and schizophrenia.
Assuntos
Diferenciação Celular , Células-Tronco Neurais/citologia , Neurônios/citologia , Oligodendroglia/citologia , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doenças Desmielinizantes/patologia , Glutamatos/metabolismo , Humanos , Camundongos , Modelos Biológicos , Bainha de Mielina/metabolismo , NF-kappa B/metabolismo , Crista Neural/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Pentoxifilina/farmacologia , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transplante de Células-TroncoRESUMO
The c-Rel transcription factor is a unique member of the NF-kB family that has a role in apoptosis, proliferation and cell survival. Overexpression of c-Rel is detected in many human B cell tumors, including B-cell leukemia and several cancers. The study aimed to investigate the effects of c-Rel siRNA on the proliferation and apoptosis of relapsed pre-B acute leukemia cells. The c-Rel siRNA was transfected into Leukemia cells using an Amaxa cell line Nucleofector kit L (Lonza). Quantitative real-time RT-PCR (qRT-PCR) and western blot were done to measure the expression levels of mRNA and protein, respectively. The flow cytometry was used to analyze the effect of c-Rel siRNA on the apoptosis and proliferation of Leukemia cells. Observed c-Rel expression in the 5 pre-B Acute lymphoblastic leukemia (ALL) patients were higher than the normal cells. The c-Rel siRNA transfection significantly blocked the expression of c-Rel mRNA in a time-dependent manner, leading to a strong growth inhibition and enhanced apoptosis (P<0.05). Our results demonstrated that c-Rel plays a fundamental role in the survival. Therefore, c-Rel can be considered as an attractive target for gene therapy in ALL patients. Also siRNA-mediated silencing of this gene may be a novel strategy in ALL treatment.
Assuntos
Apoptose/fisiologia , Técnicas de Silenciamento de Genes/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Adolescente , Western Blotting , Proliferação de Células/genética , Células Cultivadas , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Terapia Genética/métodos , Humanos , Masculino , Proteínas Proto-Oncogênicas c-rel/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
We demonstrate that KIOM-79, combined extracts obtained from Magnolia officinalis, Pueraria lobata, Glycyrrhiza uralensis, and Euphorbia pekinensis, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. Treatment of RAW 264.7 cells with KIOM-79 inhibited LPS-stimulated nitric oxide production in a dose-related manner. Immunohisto-chemical staining of iNOS and RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression. Immunostaining of p65, EMSA, and reporter gene assay showed that KIOM-79 inhibited NF-kappa/Rel nuclear translocation, DNA binding, and transcriptional activation, respectively. Western immunoblot analysis of p38 kinase showed KIOM-79 significantly inhibited the phosphoylation of p38 kinase which is important in the regulation of iNOS gene expression. Collectively, this series of experiments indicates that KIOM inhibits iNOS gene expression by blocking NF-kappa/Rel and p38 kinase signaling. Due to the critical role that NO release plays in mediating inflammatory responses, the inhibitory effects of KIOM-79 on iNOS suggest that KIOM-79 may represent a useful anti-inflammatory agent.
Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Euphorbia/química , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glycyrrhiza uralensis/química , Macrófagos/enzimologia , Macrófagos/metabolismo , Magnolia/química , Camundongos , NF-kappa B/metabolismo , Fitoterapia , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-rel/metabolismoRESUMO
NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB-dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease.
Assuntos
NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Animais , Feminino , Neoplasias Hematológicas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
I kappa B alpha is a dual regulator of Rel/NF-kappa B transcription factors. I kappa B alpha retains inactive NF-kappa B dimers in the cytoplasm, and inhibits their DNA-binding and transcriptional activities in the nucleus. Our previous studies identified discrete functional domains in I kappa B alpha responsible for the cytoplasmic and nuclear regulation of c-Rel. Determinants necessary for regulating c-Rel in the nucleus mapped to the central ankyrin domain of I kappa B alpha and a few negatively-charged amino acids that follow in the C-terminal PEST region. In contrast, sequences involved in the cytoplasmic regulation of c-Rel reside in the N-terminal and central ankyrin domains of I kappa B alpha. Here, we present a refined mapping of the N-terminal determinants of I kappa B alpha necessary for the cytoplasmic regulation of c-Rel homodimers. We demonstrate that amino acids 48 - 58 in p40/I kappa B alpha are essential to block the nuclear localization of c-Rel dimers. These data define a region of I kappa B alpha that may be required for optimal masking of the c-Rel NLS, or for the nuclear export of c-Rel/I kappa B alpha complexes. These findings highlight a novel function for the N-terminus of I kappa B alpha in the control of the subcellular localization of Rel/NF-kappa B dimers. Given the implication of deregulated NF-kappa B activity in hematopoietic and solid tumors, our findings predict that certain alterations in this domain of I kappa B alpha may have severe biological repercussions.
Assuntos
Citoplasma/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas I-kappa B , Fragmentos de Peptídeos/fisiologia , Proteínas Proto-Oncogênicas c-rel/metabolismo , Animais , Sítios de Ligação , Células COS , Citoplasma/metabolismo , DNA/antagonistas & inibidores , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/fisiologia , Deleção de Sequência , Transativadores/antagonistas & inibidores , Transativadores/fisiologia , TransfecçãoRESUMO
Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule-based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel-deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Ativação Linfocitária/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Feminino , Regulação da Expressão Gênica , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Tumor/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Transplante HomólogoRESUMO
The nuclear factor κB (NF-κB) family members p65 and c-Rel chiefly orchestrate lymphocytes activation following T-cell receptor (TCR) engagement. In contrast to p65, which is rapidly mobilized, c-Rel activation occurs subsequently as it involves a nuclear factor of activated T-cells (NFAT)-dependent upregulation step. However, how TCR ligation drives p65 and c-Rel activation is not fully understood. Because several ubiquitylated components of NF-κB signaling cascade accumulate in close proximity to membranes, we screened a siRNA library against E3-ligases that contain transmembrane domains on TCR-mediated NF-κB activation. Here, we report the identification of the endoplasmic reticulum resident TRIM13 protein as an enhancer of NF-κB promoter activity. We found that knocking down TRIM13 by RNA interference reduced the activation of p65, while the translocation of c-Rel into the nucleus was blunted. We further observed that c-Rel induction was diminished without TRIM13, as NFAT activation was compromised. These results unveil that TRIM13 is a selective regulator of p65 and of c-Rel activation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Fator de Transcrição RelA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Imunoprecipitação , Ativação Linfocitária , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T/metabolismo , Fator de Transcrição RelA/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin α(v)ß3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, α(v)ß3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, α(v)ß3 expression and the resulting KRAS-RalB-NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify α(v)ß3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Integrina beta3/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos Fase II como Assunto , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Feminino , Humanos , Integrina alfaVbeta3/metabolismo , Integrina beta3/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Quinazolinas/uso terapêutico , Interferência de RNA , Ensaios Clínicos Controlados Aleatórios como Assunto , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ral de Ligação ao GTP/genética , Proteínas ras/genéticaRESUMO
Rel proteins in bacteria synthesize the signal molecules (p)ppGpp that trigger the Stringent Response, responsible for bacterial survival. Inhibiting the activity of such enzymes prevents the Stringent Response, resulting in the inactivation of long-term bacterial survival strategies, leading to bacterial cell death. Herein, we describe a series of deoxyguanosine-based analogs of the Relacin molecule that inhibit in vitro the synthetic activity of Rel proteins from Gram positive and Gram negative bacteria, providing a deeper insight on the SAR for a better understanding of their potential interactions and inhibitory activity. Among the inhibitors evaluated, compound 2d was found to be more effective and potent than our previously reported Relacin.
Assuntos
Desoxiguanosina/análogos & derivados , Dipeptídeos/farmacologia , Desenho de Fármacos , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Deinococcus/química , Desoxiguanosina/síntese química , Desoxiguanosina/química , Desoxiguanosina/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/química , Relação Dose-Resposta a Droga , Escherichia coli/química , Estrutura Molecular , Proteínas Proto-Oncogênicas c-rel/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
The hepatoma upregulated protein (HURP) represents a putative oncogene that is overexpressed in many human cancers, especially hepatocellular carcinoma (HCC). HURP plays an important role during mitotic spindle formation, a process that is targeted by various anti-cancer drugs like taxol. However, the role of HURP during the establishment of taxol chemoresistance in HCC remains unclear. In this study, we observed that high HURP protein level correlates with taxol resistance in HCC cells. Following HURP knockdown, HCC cells show a more sensitive response to taxol treatment. Notably, sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, inhibits HURP expression primarily at the transcriptional level and sensitizes HCC cells to sub-lethal doses of taxol. By using real-time PCR and chromatin immunoprecipitation assays, we observed that the NF-κB family member c-Rel represents a putative transcription factor that activates HURP gene expression. In addition, the inhibitory effect of sorafenib on HURP expression was attributed to a reduced translation and nuclear translocation of c-Rel. Accordingly, downregulation of c-Rel using short-hairpin RNA was shown to reduce HURP protein level and enhance taxol-induced cell death. Taken together, our results indicate that HURP acts as a novel survival protein that protects HCC cells against taxol-induced cell death. In addition, the regulation of HURP gene expression by NF-κB signaling appears to be critical for the response of HCC cells to taxol.