RESUMO
BACKGROUND: Glioblastoma multiforme (GBM), due to its location, aggressiveness, heterogeneity and infiltrative growth, is characterized by an exceptionally dismal clinical outcome. The small molecule SI113, recently identified as a SGK1 inhibitor, has proven to be effective in restraining GBM growth in vitro and in vivo, showing also encouraging results when employed in combination with other antineoplastic drugs or radiotherapy. Our aim was to explore the pharmacological features of SI113 in GBM cells in order to elucidate the pivotal molecular pathways affected by the drug. Such knowledge would be of invaluable help in conceiving a rational offensive toward GBM. METHODS: We employed GBM cell lines, either established or primary (neurospheres), and used a Reverse-Phase Protein Arrays (RPPA) platform to assess the effect of SI113 upon 114 protein factors whose post-translational modifications are associated with activation or repression of specific signal transduction cascades. RESULTS: SI113 strongly affected the PI3K/mTOR pathway, evoking a pro-survival autophagic response in neurospheres. These results suggested the use of SI113 coupled, for maximum efficiency, with autophagy inhibitors. Indeed, the association of SI113 with an autophagy inhibitor, the antimalarial drug quinacrine, induced a strong synergistic effect in inhibiting GBM growth properties in all the cells tested, including neurospheres. CONCLUSIONS: RPPA clearly identified the molecular pathways influenced by SI113 in GBM cells, highlighting their vulnerability when the drug was administered in association with autophagy inhibitors, providing a strong molecular rationale for testing SI113 in clinical trials in associative GBM therapy.
Assuntos
Autofagia/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinacrina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteínas de Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein-protein complex from Burkitt lymphoma cells and from primary human CD4+ T cells. In addition, this unit describes an application of TIP for the isolation of transcription-factor-bound chromatin. © 2018 by John Wiley & Sons, Inc.
Assuntos
Separação Celular/métodos , Imunoprecipitação/métodos , Linhagem Celular , Células/química , Células/metabolismo , Humanos , Mapeamento de Interação de ProteínasRESUMO
The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone.
Assuntos
Proteínas de Transporte/metabolismo , Dermatite/metabolismo , Proteína Ligante Fas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Dermatite/genética , Peptídeos e Proteínas de Sinalização Intracelular , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.
Assuntos
Proteínas de Transporte/metabolismo , Morte Celular , Desenvolvimento Embrionário , Hematopoese , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular/genética , Perda do Embrião/genética , Desenvolvimento Embrionário/genética , Células Endoteliais/citologia , Feminino , Hematopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.
Assuntos
Imunoprecipitação/métodos , Complexos Multiproteicos/isolamento & purificação , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Apoptose , Biotinilação , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Humanos , Carioferinas/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteína Fosfatase 2C/metabolismo , Proteômica , Receptores Citoplasmáticos e Nucleares/metabolismo , Reprodutibilidade dos Testes , Receptor fas/metabolismoRESUMO
Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Cadeias alfa de Integrinas/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Sistemas de Liberação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Células HeLa , Humanos , Cadeias alfa de Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A meningioma is the most common primary intracranial tumor in adults. Here, we investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in 37 meningiomas. Freshly isolated primary meningioma cells were treated with TRAIL with or without different sensitizing protocols, and apoptotic cell death was then quantified. Mechanisms of TRAIL sensitization were determined by a combination of Western blotting, flow cytometry, receptor complex immunoprecipitation, and siRNA-mediated knockdown experiments. Tumor necrosis factor-related apoptosis-inducing ligand receptor expression was analyzed using immunohistochemistry and quantified by an automated software-based algorithm. Primary tumor cells from 11 (29.7%) tumor samples were sensitive to TRAIL-induced apoptosis, 12 (32.4%) were intermediate TRAIL resistant, and 14 (37.8%) were completely TRAIL resistant. We tested synergistic apoptosis-inducing cotreatment strategies and determined that only the proteasome inhibitor bortezomib potently enhanced expression of the TRAIL receptors TRAIL-R1 and/or TRAIL-R2, the formation of the TRAIL death-inducing signaling complex, and activation of caspases; this treatment resulted in sensitization of all TRAIL-resistant meningioma samples to TRAIL-induced apoptosis. Bortezomib pretreatment induced NOXA expression and downregulated c-FLIP, neither of which caused the TRAIL-sensitizing effect. Native TRAIL receptor expression could not predict primary TRAIL sensitivity. This first report on TRAIL sensitivity of primary meningioma cells demonstrates that TRAIL/bortezomib cotreatment may represent a novel therapeutic option for meningiomas.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Meningioma/metabolismo , Proteínas Mitocondriais/biossíntese , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/toxicidade , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Ácidos Borônicos/uso terapêutico , Bortezomib , Células HEK293 , Humanos , Meningioma/tratamento farmacológico , Pirazinas/uso terapêutico , Células Tumorais CultivadasRESUMO
Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1ß was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.
Assuntos
Imunidade/imunologia , Inflamação/metabolismo , Transdução de Sinais , Ubiquitinação , Animais , Ligante de CD40/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Quinase I-kappa B/metabolismo , Inflamação/patologia , Inflamação/prevenção & controle , Interleucina-1beta/metabolismo , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Pele/citologia , Pele/imunologia , Pele/metabolismo , Pele/patologia , Fatores de Transcrição , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Ubiquitina/química , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoAssuntos
Apoptose/genética , Complexos Multiproteicos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Camundongos , Modelos Biológicos , Poliubiquitina/metabolismo , Ligação ProteicaRESUMO
Despite tremendous progress in recent years, nanopatterning of hydrated polymeric systems such as hydrogels still represents a major challenge. Here, we employ block copolymer nanolithography to arrange gold nanoparticles on a solid template, followed by the transfer of the pattern to a polymeric hydrogel. In the next step, these nanoparticles serve as specific anchor points for active biomolecules. We demonstrate the engineering of poly(ethylene glycol) hydrogel surfaces with respect to elasticity, nanopatterning, and functionalization with biomolecules. For the first time, biomolecule arrangement on the nanometer scale and substrate stiffness can be varied independently from each other. Young's moduli, a measure of the compliance of the substrates, can be tuned over 4 orders of magnitude, including the values for all of the different tissues found in the human body. Structured hydrogels can be used to pattern any histidine-tagged protein as exemplified for his-protein A as an acceptor for immunoglobulin. When cell-adhesion-promoting peptide cRGDfK is selectively coupled to gold nanoparticles, the surfaces provide cues for cell-surface interaction and allow for the study of the modulation of cellular adhesion by the mechanical properties of the environment. Therefore, these substrates represent a unique multipurpose platform for studying receptor/ligand interactions with adhering cells, mechanotransduction, and cell-adhesion-dependent signaling.
Assuntos
Polímeros/química , Adesão Celular , Células Cultivadas , Elasticidade , Humanos , Hidrogéis/química , Microscopia Confocal , Microscopia de Fluorescência , Tamanho da PartículaRESUMO
TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.
Assuntos
Regulação da Expressão Gênica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Ubiquitina/metabolismo , Animais , Apoptose , Linhagem Celular , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/fisiologia , Células U937 , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of zeta-chain-associated protein of 70 kD, phospholipase-gamma, and protein kinase C- into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca(2+) mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-kappaB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion.
Assuntos
Proteína Ligante Fas/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Receptor fas/imunologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD28/genética , Antígenos CD28/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Caspases/metabolismo , Proliferação de Células , Células Cultivadas , Citocinas/imunologia , Ativação Enzimática , Proteína Ligante Fas/genética , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Microdomínios da Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linfócitos T/citologia , Receptor fas/genéticaRESUMO
Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Caspase 3/metabolismo , Sinergismo Farmacológico , Ativação Enzimática , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Cristalização/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/instrumentação , Análise Serial de Proteínas/instrumentação , Proteínas/química , Proteínas/ultraestrutura , Desenho de Equipamento , Análise de Falha de Equipamento , Ouro/química , Teste de Materiais , Miniaturização , Conformação Molecular , Nanotecnologia/métodos , Tamanho da Partícula , Análise Serial de Proteínas/métodos , Ligação Proteica , Propriedades de SuperfícieRESUMO
Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.
Assuntos
Adenocarcinoma/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Genes bcl-2/fisiologia , Neoplasias Pancreáticas/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Adenocarcinoma/patologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Embrião de Galinha , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno/farmacologia , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The extrinsic apoptosis pathway is activated when certain members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are oligomerized by their cognate ligands that are members of the TNF superfamily (TNFSF). The apoptosis-inducing capacity of a member of the TNFRSF relies on the presence of a death domain (DD) in the intracellular portion of the receptor protein. Such receptors are also referred to as death receptors. Binding of a TNFSF ligand to a TNFRSF receptor that is expressed on the surface of a cell results in the formation of a receptor proximal protein complex. This protein complex is the platform for further signaling events within the cell. In case of death receptors like TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1/DR4), TRAIL-R2 (KILLER/APO-2/DR5/TRICK), CD95 (Fas, APO-1), or TNF receptor 1 (TNF-R1), this complex is termed death-inducing signaling complex (DISC). The compositions of the various DISCs have been intensively studied in the last 12 years. For the CD95 and the TRAIL-R1/R2 DISCs, it is now clear that the adaptor protein Fas-associated DD protein (FADD) forms part of these complexes and is necessary for recruitment of the proapoptotic signaling molecules caspase-8 and caspase-10. Recruitment of these proteases allows for their activation at the DISC and subsequent induction of apoptosis. The caspase-8 homologous cellular FLICE-like inhibitory protein (cFLIP) can also be recruited to the DISC. cFLIP acts as an anti-apoptotic regulator by interfering with activation of caspases 8 and 10 at the DISC. Interestingly, treatment of TRAIL-resistant tumor cells with conventional chemotherapeutic drugs or with proteasome inhibitors renders these cells sensitive for TRAIL-induced apoptosis. By applying the methodology of the biochemical analysis of the TRAIL DISC described here, we were able to show that this sensitization is mainly due to changes in the biochemical composition of the DISC as the apoptosis-initiating protein complex of the extrinsic pathway.
Assuntos
Apoptose , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/análise , Ligante Indutor de Apoptose Relacionado a TNF/análise , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Western Blotting , Células Cultivadas , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/fisiologia , Citometria de Fluxo/métodos , Humanos , Imunoprecipitação , Modelos Biológicos , Ligação Proteica , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Death ligands not only activate a death program but also regulate inflammatory signalling pathways, for example, through NF-kappaB induction. Although tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF both activate NF-kappaB in human keratinocytes, only TRAIL potently induces apoptosis. However, when induction of NF-kappaB was inhibited with a kinase dead IKK2 mutant (IKK2-KD), TNF- but not TRAIL-induced apoptosis was dramatically enhanced. Acquired susceptibility to TNF-induced apoptosis was due to increased caspase-8 activation. To investigate the mechanism of resistance of HaCaT keratinocytes to TNF-induced apoptosis, we analyzed a panel of NF-kappaB-regulated effector molecules. Interestingly, the inhibitor of apoptosis protein (IAP) family member cIAP2, but not cIAP1, X-linked inhibitor of apoptosis, TNF receptor-associated factor (TRAF)-1, or TRAF2, was downregulated in sensitive but not in resistant HaCaT keratinocytes. Surprisingly, however, stable inducible expression of cIAP2 was not sufficient to render IKK2-KD-sensitized keratinocytes resistant to TNF, and reduction of cIAP2 alone did not increase the sensitivity of HaCaT keratinocytes to TNF. In conclusion, we demonstrate that inhibition of NF-kappaB dramatically sensitizes human keratinocytes to TNF- but not to TRAIL-induced apoptosis and that this sensitization for TNF was largely independent of cIAP2. Our data thus clearly exclude the candidates proposed to date to confer TNF apoptosis resistance and suggest the function of an unanticipated effector of NF-kappaB critical for the survival of HaCaT keratinocytes upstream or at the level of caspase-8 activation.
Assuntos
Caspase 8/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Apoptose/fisiologia , Proteína 3 com Repetições IAP de Baculovírus , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Queratinócitos/citologia , Mutagênese , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/fisiologia , Fator 1 Associado a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND AND AIMS: The constant renewal of enterocytes along the crypt-villus axis (CVA) of human small intestine is due to cell-inherent changes resulting in the apoptotic cell death of senescent enterocytes. The aim of the present study was to examine underlying molecular mechanisms of the cell death at the villus tip. METHODS: Characterization of human acyl-coenzyme A (CoA) synthetase 5 (ACSL5) was performed by cloning, recombinant protein expression, biochemical approaches, and several functional and in situ analyses. RESULTS: Our data show that different amounts of acyl-CoA synthetase 5-full length (ACSL5-fl) and a so far unknown splice variant lacking exon 20 (ACSL5-Delta 20) are found in human enterocytes. In contrast with the splice variant ACSL5-Delta 20, recombinant and purified ACSL5-fl protein is active at a highly alkaline pH. Over expression of ACSL5-fl protein is associated with a decrease of the anti-apoptotic FLIP protein in a ceramide-dependent manner and an increased cell-surface expression of the death receptor TRAIL-R1. Expression analyses revealed that the ACSL5-fl/ACSL5-Delta 20 ratio increases along the CVA, thereby sensitizing ACSL5-fl-dominated cells at the villus tip to the death ligand TRAIL, which is corroborated by functional studies with human small intestinal mucosal samples and an immortalized human small intestinal cell line. CONCLUSIONS: Our results suggest an ACSL5-dependent regulatory mechanism that contributes to the cellular renewal along the CVA in human small intestine. Deregulation of the ACSL5-fl/ACSL5-Delta 20 homeostasis in the maturation and shedding of cells along the CVA might also be of relevance for the development of intestinal neoplasia.
Assuntos
Processamento Alternativo , Apoptose/genética , Coenzima A Ligases/metabolismo , Duodeno/enzimologia , Enterócitos/enzimologia , Regulação Enzimológica da Expressão Gênica , Íleo/enzimologia , RNA Mensageiro/metabolismo , Idoso , Sequência de Aminoácidos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Células CACO-2 , Caspase 3/metabolismo , Doença Celíaca/enzimologia , Doença Celíaca/genética , Doença Celíaca/patologia , Ceramidas/biossíntese , Coenzima A Ligases/genética , Duodeno/patologia , Retículo Endoplasmático/enzimologia , Enterócitos/patologia , Ativação Enzimática , Humanos , Concentração de Íons de Hidrogênio , Íleo/patologia , Microvilosidades/enzimologia , Microvilosidades/patologia , Pessoa de Meia-Idade , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , TransfecçãoRESUMO
PURPOSE: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. The antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in gliomas has thus far only been thoroughly established in tumor cell lines. In the present study, we investigated the therapeutic potential of TRAIL in primary human glioma cells. EXPERIMENTAL DESIGN: We isolated primary tumor cells from 13 astrocytoma and oligoastrocytoma patients of all four WHO grades of malignancy and compared the levels of TRAIL-induced apoptosis induction, long-term tumor cell survival, caspase, and caspase target cleavage. RESULTS: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Regardless of the tumor heterogeneity, cotreatment with the proteasome inhibitor bortezomib efficiently sensitized all primary glioma samples for TRAIL-induced apoptosis and tremendously reduced their clonogenic survival. Due to the pleiotropic effect of bortezomib-enhanced TRAIL DISC formation upon TRAIL triggering, down-regulation of cFLIP(L) and activation of the intrinsic apoptosis pathway seem to cooperatively contribute to the antitumor effect of bortezomib/TRAIL cotreatment. CONCLUSION: TRAIL sensitivity of tumor cell lines is not a reliable predictor for the behavior of primary tumor cells. The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by bortezomib cotreatment might, however, provide a powerful therapeutic option for glioma patients.
Assuntos
Apoptose , Astrocitoma/tratamento farmacológico , Ácidos Borônicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Imunoterapia/métodos , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Adolescente , Adulto , Idoso , Astrocitoma/metabolismo , Bortezomib , Neoplasias Encefálicas/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
UNLABELLED: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents a novel promising anticancer biotherapeutic. However, TRAIL-resistant tumor cells require combinatorial regimens to sensitize tumor but not normal cells for TRAIL-induced apoptosis. Here, we investigated the mechanism of the synergistic antitumor effect of bortezomib in combination with TRAIL in hepatoma, colon, and pancreatic cancer cells in comparison to the toxicity in primary human hepatocytes (PHH). TRAIL cotreatment at high but clinically relevant concentrations of bortezomib caused toxicity in PHH which potentially limits the clinical applicability of bortezomib/TRAIL cotreatment. However, at low concentrations of bortezomib TRAIL-resistant hepatoma, colon and pancreatic cancer cell lines but not PHH were efficiently sensitized for TRAIL-induced apoptosis. RNA interference and TRAIL receptor blockage experiments revealed that in bortezomib-treated hepatoma cells TRAIL-R1/TRAIL-R2 up-regulation, enhanced TRAIL DISC formation and cFLIPL down-regulation in addition to accumulation of Bak cooperatively sensitized for TRAIL. Bim, although accumulated upon bortezomib treatment, did not play a causal role for TRAIL sensitization in Hep3b cells. Combined treatment with bortezomib and TRAIL massively reduced the clonogenic capacity of hepatoma cells in vitro. Surviving clones could be resensitized for repeated TRAIL treatment. CONCLUSION: Bortezomib/TRAIL cotreatment bears the risk of severe hepatotoxicity at high but clinically relevant concentrations of bortezomib. However, within a wide therapeutic window bortezomib sensitized different cancer cells but not PHH for TRAIL-induced apoptosis.