RESUMO
Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, we identified grindelwald (grnd), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. Here we show that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of Ras(V12)/scrib(-/-) tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptores do Fator de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/genética , Moléculas de Adesão Celular/metabolismo , Divisão Celular/genética , Polaridade Celular/genética , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Dados de Sequência Molecular , Invasividade Neoplásica/genética , Neoplasias/enzimologia , Neoplasias/genética , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive "Fas apoptosis" to "Fas multisignals" paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.
Assuntos
Evolução Molecular , Neoplasias/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptor fas/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Apoptose , Endocitose , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Estrutura Terciária de ProteínaRESUMO
The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.
Assuntos
Morte Celular/efeitos dos fármacos , Proteína Ligante Fas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Tirosina Quinases/genética , Receptor fas/genética , Tirosina Quinase da Agamaglobulinemia , Morte Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Regulação da Expressão Gênica , Células HT29 , Humanos , Células Matadoras Naturais , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Fosfatidilinositol 4,5-Difosfato/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1α (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome and suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3 and caspase 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress.
Assuntos
Apoptose , Regulação para Baixo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Defeitos do Tubo Neural/etiologia , Neurogênese , Gravidez em Diabéticas/fisiopatologia , Resposta a Proteínas não Dobradas , Animais , Biomarcadores/metabolismo , Linhagem Celular , Diabetes Mellitus Experimental/fisiopatologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Feminino , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multienzimáticos/metabolismo , Proteínas Mutantes/metabolismo , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Gravidez , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismoRESUMO
Activated T cells secrete Fas ligand (FasL)-containing vesicles (secreted vesicles) that induce death of target cells. We provide evidence that secreted vesicles from culture supernatants (Csup) of various origins are able to generate both Fas-dependent apoptotic and Fas-independent, nonapoptotic cell death. In the absence of Fas, the nonapoptotic, Fas-independent pathway could still induce cell death. In contrast to RIP-independent classical Fas-induced cell death triggered by cross-linked or membrane-bound FasL, CSup-derived stimuli-induced apoptosis exhibited unique molecular and enzymatic characteristics. It could be partially inhibited by blocking cathepsin D enzyme activity and required the presence of RIP. Whereas stimulation with CSup, derived from both FasL-overexpressing Jurkat cells and PBMC, could induce cell death, the requirements for Fas-associated death domain protein and caspase-9 were different between the two systems. Our study highlights an important distinction between cell contact-mediated and secreted vesicle-generated activation-induced cell death and also demonstrates that the type of the secreted vesicles can also modify the cell death route. We propose that besides cell-to-cell interaction-mediated Fas triggering, stimuli induced by secreted vesicles can mediate important additional cell death signals regulating activation-induced cell death under physiological conditions.
Assuntos
Apoptose/imunologia , Vesículas Citoplasmáticas/imunologia , Vesículas Citoplasmáticas/metabolismo , Ativação Linfocitária/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor fas/fisiologia , Comunicação Celular/imunologia , Morte Celular/imunologia , Transformação Celular Neoplásica/imunologia , Técnicas de Cocultura , Meios de Cultivo Condicionados , Citidina Desaminase/fisiologia , Vesículas Citoplasmáticas/enzimologia , Testes Imunológicos de Citotoxicidade , Humanos , Células Jurkat , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Subpopulações de Linfócitos T/enzimologiaRESUMO
Fas ligand (FasL) not only induces apoptosis in Fas receptor-bearing target cells, it is also able to transmit signals into the FasL-expressing cell via its intracellular domain (ICD). Recently, we described a Notch-like proteolytic processing of FasL that leads to the release of the FasL ICD into the cytoplasm and subsequent translocation into the nucleus where it may influence gene transcription. To study the molecular mechanism underlying such reverse FasL signaling in detail and to analyze its physiological importance in vivo, we established a knockout/knockin mouse model, in which wild-type FasL was replaced with a deletion mutant lacking the ICD. Our results demonstrate that FasL ICD signaling impairs activation-induced proliferation in B and T cells by diminishing phosphorylation of phospholipase C γ, protein kinase C, and extracellular signal-regulated kinase 1/2. We also demonstrate that the FasL ICD interacts with the transcription factor lymphoid-enhancer binding factor-1 and inhibits lymphoid-enhancer binding factor-1-dependent transcription. In vivo, plasma cell numbers, generation of germinal center B cells, and, consequently, production of antigen-specific immunoglobulin M antibodies in response to immunization with T cell-dependent or T cell-independent antigen are negatively affected in presence of the FasL ICD, suggesting that FasL reverse signaling participates in negative fine-tuning of certain immune responses.
Assuntos
Linfócitos B/metabolismo , Proteína Ligante Fas/metabolismo , Imunomodulação/imunologia , Ativação Linfocitária/imunologia , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Proliferação de Células , Proteína Ligante Fas/imunologia , Regulação da Expressão Gênica/imunologia , Técnicas de Introdução de Genes , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Myelodysplastic syndromes (MDS) are associated with increased bone marrow vascularity and increased levels of various angiogenic factors including Vascular Endothelial Growth Factor (VEGF) which is implicated in the proliferation and survival of leukemic cells. Before the approval of hypomethylating agents in this indication, the GFM conducted a multicenter phase II trial testing the efficacy and tolerance of bevacizumab, a humanized monoclonal antibody against VEGF, in MDS with excess of marrow blasts and its impact on bone marrow angiogenesis. Twenty-one patients were enrolled (16 males and five females) with a median age of 70 years and 19 were evaluable for haematological response after treatment (5 mg/kg IV every 2 weeks for 12 weeks). WHO diagnosis at baseline was RAEB-1 (38%) and RAEB-2 (62%). Treatment was well tolerated and was associated with significant decrease of VEGF plasma level [median (low quartile-high quartile)] from 65.5 pg/ml [LQ (low-quartile)-HQ (high quartile), 35.3-87.3 to 30.4 pg/ml (LQ-HQ, 22.5-34.0 pg/ml)] (p < 0.01) and reduction of bone marrow angiogenesis from a median of 20 vessels/mm(3) (LQ-HQ, 16.5-33 vessels/mm(3)) to 15.5 vessels/mm(3) (LQ-HQ, 10-23.2 vessels/mm(3)) (p = 0.03). On the other hand, only one patient had a significant haematological response with achievement of RBC transfusion independence. Thus, although bevacizumab had a significant impact on VEGF levels and angiogenesis in our patients, very few responses were seen when this drug was used as single agent. Given its good tolerability profile, however, combination of bevacizumab with other drugs, especially hypomethylating agents, could be considered in MDS.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/sangue , Idoso , Idoso de 80 Anos ou mais , Bevacizumab , Medula Óssea/irrigação sanguínea , Células da Medula Óssea/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Fas interaction at the plasma membrane with its lipid and protein environment plays a crucial role in the early steps of Fas signalling induced by Fas ligand binding. Particularly, Fas localisation in the raft nanodomains, ezrin-mediated interaction with the actin cytoskeleton and subsequent internalization are critical steps in Fas-mediated cell death. We identified a lysine-rich region (LRR) in the cytoplasmic, membrane-proximal region of Fas as a key determinant modulating these initial events. Through a genetic approach, we demonstrate that Fas LRR represents another signal additional to palmitoylation targeting Fas to the raft nanodomains, and modulates Fas interaction with the cytoskeleton.
Assuntos
Apoptose , Lisina/metabolismo , Microdomínios da Membrana/metabolismo , Transdução de Sinais , Receptor fas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteína Ligante Fas , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Lipoilação , Lisina/química , Lisina/genética , Camundongos , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/genéticaRESUMO
Recombinant MrNV capsid protein has been shown to effectively deliver plasmid DNA and dsRNA into Sf9 insect cells and shrimp tissues. To extend its application to cancer cell-targeting drug delivery, we created three different types of chimeric MrNV virus-like particles (VLPs) (R-MrNV, I-MrNV, and E-MrNV) that have specificity toward the epidermal growth factor receptor (EGFR), a cancer cell biomarker, by incorporating the EGFR-specific GE11 peptide at 3 different locations within the host cell recognition site of the capsid. All three chimeric MrNV-VLPs preserved the ability to form a mulberry-like VLP structure and to encapsulate EGFP DNA plasmid with an efficiency comparable to that previously reported for normal MrNV (N-MrNV). Compared to N-MrNV, the chimeric R-MrNV and E-MrNV carrying the exposed GE-11 peptide showed a significantly enhanced binding and internalization abilities that were specific towards EGFR expression in colorectal cancer cells (SW480). Specific targeting of chimeric MrNV to EGFR was proven by both EGFR silencing with siRNA vector and a competition with excess GE-11 peptide as well as the use of EGFR-negative colorectal cells (SW620) and breast cancer cells (MCF7). We demonstrated here that both chimeric R-MrNV and E-MrNV could be used to encapsulate cargo such as exogenous DNA and deliver it specifically to EGFR-positive cells. Our study presents the potential use of surface-modified VLPs of shrimp virus origin as nanocontainers for targeted cancer drug delivery.
Assuntos
Adenocarcinoma/tratamento farmacológico , Proteínas do Capsídeo/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Nodaviridae/química , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/genética , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
S-palmitoylation is a lipid modification that regulates membrane-protein association and influences protein trafficking, stability or aggregation, thus playing an important role in protein signalling. We previously demonstrated that the palmitoylation of Fas, one of the DD (death domain)-containing members of the TNFR [TNF (tumour necrosis factor) receptor] superfamily, is essential for the redistribution of this receptor into lipid rafts, an obligatory step for the death signal transmission. Here we investigate the requirement of protein palmitoylation in the activities of other DD-containing death receptors. We show that DR4 is palmitoylated, whereas DR5 and TNFR1 are not. Furthermore, DR4 palmitoylation is required for its raft localization and its ability to oligomerize, two essential features in TRAIL (TNF-related apoptosis-inducing ligand)-induced death signal transmission.
Assuntos
Morte Celular/fisiologia , Lipoilação/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Multimerização Proteica/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Camundongos , Camundongos Nus , Nitrosação , Compostos de Fenilureia , Sorafenibe/farmacologia , Tiorredoxinas/genéticaRESUMO
UNC5H receptors (UNC5H1, UNC5H2, UNC5H3) are putative tumor suppressors whose expression is lost in numerous cancers. These receptors have been shown to belong to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand netrin-1 is absent, thus conferring a state of cellular dependence towards ligand presence. Along this line, these receptors may limit tumor progression because they induce the death of tumor cells that grow in settings of ligand unavailability. We show here that UNC5H receptors are localized to cholesterol-and sphingolipid-enriched membrane domains called lipid rafts. We then demonstrate that the lipid raft localization of UNC5H2 is required for the pro-apoptotic activity of unbound UNC5H2. We also propose that this lipid raft localization is probably mediated via the recruitment of adaptor protein(s) within the death domain of UNC5H2 but is not dependent on the post-translational modification by palmitoylation of UNC5H2 even though this palmitoylation is required for UNC5H2 pro-apoptotic activity. Moreover we show that the interaction of UNC5H2 with the downstream pro-apoptotic serine threonine kinase DAPk is dependent on both UNC5H2 lipid raft localization and palmitoylation. Thus, we propose that the UNC5H dependence receptors require lipid raft localization and palmitoylation to trigger apoptosis.
Assuntos
Microdomínios da Membrana/metabolismo , Ácido Palmítico/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Morte Celular , Linhagem Celular , Proteínas Quinases Associadas com Morte Celular , Humanos , Receptores de Netrina , Estrutura Terciária de Proteína , Receptores de Superfície Celular/químicaRESUMO
Gangliosides have been involved in multiple cellular processes such as growth, differentiation and adhesion, and more recently as regulators of cell death signaling pathways. Some of these molecules can be considered as tumor-associated antigens, in particular, N-glycolyl sialic acid-containing gangliosides, which are promising candidates for cancer-targeted therapy because of their low expression in normal human tissues. In this study, we provided the molecular and cellular characterization of a novel cell death mechanism induced by the anti-NGcGM3 14F7 monoclonal antibody (mAb) in L1210 murine tumor cell line but not in mouse normal cells (B and CD4(+) T lymphocytes) that expressed the antigen. Impairment of ganglioside synthesis in tumor cells abrogated the 14F7 mAb cytotoxic effect; however, exogenous reincorporation of the ganglioside did not restore tumor cell sensitivity to 14F7 mAb-induced cytotoxicity. 14F7 F(ab')(2) but not Fab fragments retained the cytotoxic capacity of the whole mAb. By contrary, other mAb, which recognizes N-glycolylated gangliosides, did not show any cytotoxic effect. These mAbs showed quite different capacities to bind NGcGM3-positive cell lines measured by binding inhibition experiments. Interestingly, this complement-independent cell death mechanism did not resemble apoptosis, because no DNA fragmentation, caspase activation, or Fas mediation were observed. However, NGcGM3 ganglioside-mediated 14F7 mAb-induced cell death was accompanied by cellular swelling, membrane lesion formation, and cytoskeleton activation, suggesting an oncosis-like phenomenon. This novel mechanism of cell death lets us to support further therapeutic approaches using NGcGM3 as a molecular target for antibody-based cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Gangliosídeo G(M3)/imunologia , Neoplasias/patologia , Animais , Afinidade de Anticorpos/efeitos dos fármacos , Especificidade de Anticorpos/efeitos dos fármacos , Sítios de Ligação , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL/TNFSF10) and Fas Ligand (FasL/TNFSF6), two major cytokines of the TNF (Tumor Necrosis Factor) superfamily, exert their main functions from the immune system compartment. Mice model studies revealed that TRAIL and FasL-mediated signalling both control the homeostasis of the immune cells, mainly from the lymphoid lineage, and function on cytotoxic cells as effector proteins to eliminate the compromised cells. The first clues in the physiological functions of TRAIL arose from the analysis of TRAIL deficient mice, which, even though they are viable and fertile, are prone to cancer and autoimmune diseases development, revealing TRAIL as an important safeguard against autoimmunity and cancer. The naturally occurring gld (generalized lymphoproliferative disease) and lpr (lymphoproliferation) mutant mice develop lymphadenopathy and lupus-like autoimmune disease. The discovery that they are mutated in the fasl and the fas receptor gene, respectively, demonstrates the critical role of the FasL/Fas system in lymphocyte homeostasis and autoimmunity. This review summarizes the state of current knowledge regarding the key death and non-death immune functions that TRAIL and FasL play in the initiation and progression of cancer and autoimmune diseases.
RESUMO
BACKGROUND: Apoptosis has been described extensively in acute myocardial infarction and chronic heart failure. Because Daxx (death-associated protein) appears to be essential for stress-induced cell death and acts as an antisurvival molecule, we tested the hypothesis that Daxx is involved in myocardial ischemia/reperfusion-induced cell death in vivo. METHODS AND RESULTS: Transgenic mice overexpressing a dominant-negative form of Daxx (Daxx-DN) under the control of the beta-actin promoter and control wild-type mice underwent an ischemia/reperfusion protocol: 40 minutes of left coronary artery occlusion and 60 minutes of reperfusion. Area at risk and infarct size were measured after dual staining by triphenyltetrazolium chloride and phthalocyanine blue dye. Apoptosis was measured in the ischemic versus the nonischemic part of the left ventricle by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling staining, enzyme-linked immunosorbent assay, and Western blotting of caspase-3, caspase-8, and poly(ADP-ribose) polymerase. The mitogen-activated protein kinase status was investigated by Western blot analysis. Comparison between groups was assessed by ANOVA or Student t test (statistical significance: P<0.05). Left ventricle tissues from transgenic mice expressed Daxx-DN at the protein level. Area at risk/left ventricle values were comparable among groups. Infarct size/area at risk was 45% reduced in Daxx-DN versus wild-type mice (P<0.001). This cardioprotection was maintained for a 4-hour reperfusion. Ischemia/reperfusion-induced apoptosis was significantly decreased and ERK1/2 prosurvival pathway was activated in ischemic Daxx-DN hearts. CONCLUSIONS: Our study clearly indicates that Daxx participates in myocardial ischemia/reperfusion proapoptotic signaling in vivo.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Genes Dominantes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Doença Aguda , Animais , Proteínas de Transporte/genética , Caspase 3/metabolismo , Caspase 8/metabolismo , Doença Crônica , Proteínas Correpressoras , Modelos Animais de Doenças , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Chaperonas Moleculares , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Proteínas Nucleares/genéticaRESUMO
Tyrosine phosphorylation of Fas (TNFRSF6/CD95) in its death domain turns off Fas-mediated apoptosis, turns on the pro-survival signal, and has implications in different cancers types. We show here that Fas in its pro-survival state, phosphorylated at Y291 (pY291-Fas), functionally interacts with the epidermal growth factor receptor (EGFR), a key cancer-driving protein and major therapeutic target. Using an evolution-guided pY291-Fas proxy, RNA interference, and site-specific phospho-protein detection, we show that pY291-Fas significantly intensifies EGFR signaling in anti-EGFR-resistant colorectal cancer cells via the Yes-1/STAT3-mediated pathway. The pY291-Fas is essential for the EGF-induced formation of the Fas-mediated nuclear EGFR/STAT3 signaling complex consisting of Fas, EGFR, Yes-1, Src, and STAT3. The pY291-Fas accumulates in the nucleus upon EGF treatment and promotes the nuclear localization of phospho-EGFR and phospho-STAT3, the expression of cyclin D1, the activation of STAT3-mediated Akt and MAPK pathways, and cell proliferation and migration. This novel cancer-promoting function of phosphorylated Fas in the nuclear EGFR signaling constitutes the foundation for developing pro-survival-Fas targeted anti-cancer therapies to overcome disease recurrence in patients with anti-EGFR resistant cancer.
Assuntos
Neoplasias Colorretais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fosforilação/fisiologia , Fator de Transcrição STAT3/metabolismo , Tirosina/metabolismo , Receptor fas/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Ciclina D1/metabolismo , Receptores ErbB/metabolismo , Células HCT116 , Humanos , Recidiva Local de Neoplasia/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Efficient adjuvants have the potential to trigger both innate and adaptive immune responses simultaneously. Flagellin is a unique pathogen-derived protein, which is recognized by pattern recognition receptors (PRRs) as well as by B-cell and T cell receptors thus providing an important link between innate and adaptive immunity. The aforementioned properties define flagellin as an optimal adjuvant. The induction of immunogenic cell death could be an additional expectation for adjuvants in the context of cancer immunotherapy due to their ability to activate dendritic cells (DC) to present tumor antigens through the engulfment of dying cells. The immunostimulatory potential of flagellin in the course of DC and lymphocyte activation is well documented, however the exact mechanism is not fully explored. Based on this limitation we sought to investigate the potential modulatory effects of flagellin on various cell death processes knowing that it plays detrimental roles in regulating the final outcome of various types of immune responses. Here we provide evidence that the pre-treatment of Jurkat T-cells with recombinant flagellin is able to increase the degree of cell death provoked by FasL or TNF-α, and concomitantly increases the cytotoxic potential of phytohemagglutinin activated T-lymphocytes in a TLR5 dependent way. In contrast to these flagellin-mediated effects on the death receptor-induced signaling events, the mitochondrial apoptotic pathway remained unaffected. Furthermore, the cell culture supernatant of wild type Salmonella enteritidis bacteria, but not their flagellin deficient variant, was able to enhance the Fas-induced cell death process. To define the molecular mechanisms of flagellin-mediated elevated levels of cell death we were able to detect the upregulation of RIP1-dependent signaling events. These findings demonstrate that the cooperative actions of pattern recognition and different death receptors are able to initiate the cell death process with the mobilization of RIP-dependent cell death modalities. This finding highlights the capability of flagellin to act as a potential adjuvant which is relevant for tumor immunotherapy.
Assuntos
Adjuvantes Imunológicos , Flagelina/metabolismo , Receptores de Morte Celular/metabolismo , Salmonella enteritidis/genética , Linfócitos T/imunologia , Imunidade Adaptativa , Apoptose , Células Dendríticas/fisiologia , Proteína Ligante Fas/metabolismo , Flagelina/genética , Humanos , Imunidade Inata , Células Jurkat , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de SinaisRESUMO
Finely tuned regulation of epithelial cell death maintains tissue integrity and homeostasis. At the cellular level, life and death decisions are controlled by environmental stimuli such as the activation of death receptors. We show that cell polarity and adherens junction formation prevent proapoptotic signals emanating from the Fas death receptor. Fas is sequestered in E-cadherin actin-based adhesion structures that are less able to induce downstream apoptosis signaling. Using a proteomic-based approach, we find that the polarity molecule Dlg1 interacts with the C-terminal PDZ-binding site in Fas and that this interaction decreases formation of the death-inducing complex upon engagement with Fas ligand (FasL), thus acting as an additional cell death protection mechanism. We propose that E-cadherin and Dlg1 inhibit FasL-induced cell death by two complementary but partially independent mechanisms that help to maintain epithelial homeostasis by protecting normal polarized epithelia from apoptosis. When polarity is lost, the Fas-cadherin-Dlg1 antiapoptotic complex is disrupted, and FasL can promote the elimination of compromised nonpolarized cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Células Epiteliais/metabolismo , Proteína Ligante Fas/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Receptor fas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Junções Aderentes/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Polaridade Celular , Proteína 1 Homóloga a Discs-Large , Células Epiteliais/citologia , Proteína Ligante Fas/genética , Humanos , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Domínios Proteicos , Proteômica , Receptor fas/genéticaRESUMO
Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).
Assuntos
Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Fosfoproteínas/isolamento & purificação , Fosfotirosina/metabolismo , Receptor fas/isolamento & purificação , Receptor fas/metabolismo , Linhagem Celular , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Immunoblotting , Fosfoproteínas/química , Fosforilação , Fosfotirosina/química , Receptor fas/químicaRESUMO
S-acylation is the covalent addition of a fatty acid, most generally palmitate onto cysteine residues of proteins through a labile thioester linkage. The death receptor CD95 is S-palmitoylated and this post-translational modification plays a crucial role on CD95 organization in cellular membranes and thus on CD95-mediated signaling. Here, we describe the nonradioactive detection of CD95 S-acylation by acyl-biotin exchange chemistry in which a biotin is substituted for the CD95-linked fatty acid. This sensitive technique, which depends on the ability of hydroxylamine to specifically cleave the thioester linkage between fatty acids and proteins, relies on three chemical steps: (1) blockage of free thiols of non-modified cysteine residues, (2) hydroxylamine-mediated cleavage of thioester-linked fatty acids to restore free thiols and (3) biotinylation of free thiols with a thiol reactive biotinylation agent. Resulting biotinylated proteins can be easily purified by an avidin capture and analyzed by SDS-PAGE and immunoblotting.