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The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ubiquitin ligase which catalyses the generation of linear ubiquitin linkages de novo LUBAC is a crucial component of various immune receptor signalling pathways. Here, we show that LUBAC forms part of the TRAIL-R-associated complex I as well as of the cytoplasmic TRAIL-induced complex II In both of these complexes, HOIP limits caspase-8 activity and, consequently, apoptosis whilst being itself cleaved in a caspase-8-dependent manner. Yet, by limiting the formation of a RIPK1/RIPK3/MLKL-containing complex, LUBAC also restricts TRAIL-induced necroptosis. We identify RIPK1 and caspase-8 as linearly ubiquitinated targets of LUBAC following TRAIL stimulation. Contrary to its role in preventing TRAIL-induced RIPK1-independent apoptosis, HOIP presence, but not its activity, is required for preventing necroptosis. By promoting recruitment of the IKK complex to complex I, LUBAC also promotes TRAIL-induced activation of NF-κB and, consequently, the production of cytokines, downstream of FADD, caspase-8 and cIAP1/2. Hence, LUBAC controls the TRAIL signalling outcome from complex I and II, two platforms which both trigger cell death and gene activation.
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Muerte Celular , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Activación Transcripcional , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , HumanosRESUMEN
Sarcomas are rare and heterogeneous cancers classically associated with a poor outcome. Sarcomas are 1% of the cancer but recent estimations indicate that sarcomas account for 2% of the estimated cancer-related deaths. Traditional treatment with surgery, radiotherapy, and chemotherapy has improved the outcome for some types of sarcomas. However, novel therapeutic strategies to treat sarcomas are necessary. TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand initially described as capable of inducing apoptosis on tumor cell while sparing normal cells. Only few clinical trials have used TRAIL-based treatments in sarcoma, but they show only low or moderate efficacy of TRAIL. Consequently, novel TRAIL formulations with an improved TRAIL bioactivity are necessary. Our group has developed a novel TRAIL formulation based on tethering this death ligand on a lipid nanoparticle surface (LUV-TRAIL) resembling the physiological secretion of TRAIL as a trasmembrane protein inserted into the membrane of exosomes. We have already demonstrated that LUV-TRAIL shows an improved cytotoxic activity when compared to soluble recombinant TRAIL both in hematological malignancies and epithelial-derived cancers. In the present study, we have tested LUV-TRAIL in several human sarcoma tumor cell lines with different sensitivity to soluble recombinant TRAIL, finding that LUV-TRAIL was more efficient than soluble recombinant TRAIL. Moreover, combined treatment of LUV-TRAIL with distinct drugs proved to be especially effective, sensitizing even more resistant cell lines to TRAIL.
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Apoptosis , Lípidos , Nanopartículas , Sarcoma/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Recombinantes , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
PURPOSE: Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. METHODS/PATIENTS: LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. RESULTS: LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. CONCLUSION: The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.
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Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Nanopartículas/química , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Liposomas , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/químicaRESUMEN
The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.
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Virus ARN , Ubiquitina-Proteína Ligasas , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Transducción de Señal , Proteína 58 DEAD Box/genética , Virus ARN/metabolismoRESUMEN
The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.
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COVID-19 , Modelos Animales de Enfermedad , Proteína Ligando Fas , SARS-CoV-2 , Animales , Ratones , Líquido del Lavado Bronquioalveolar , COVID-19/patología , COVID-19/inmunología , COVID-19/metabolismo , COVID-19/virología , COVID-19/mortalidad , Proteína Ligando Fas/metabolismo , Inflamación/patología , Inflamación/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Pulmón/patología , Pulmón/virología , Pulmón/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones Endogámicos C57BLRESUMEN
Human Apo2-ligand/TRAIL is a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells. Besides its antitumor activity, Apo2L/TRAIL is also implicated in immune regulation. Apo2L/TRAIL is stored inside activated T cells in cytoplasmic multivesicular bodies and is physiologically released to the extracellular medium inserted in the internal membrane vesicles, known as exosomes. In this study we have generated artificial lipid vesicles coated with bioactive Apo2L/TRAIL, which resemble natural exosomes, to analyze their apoptosis-inducing ability on cell lines from hematological tumors. We have tethered Apo2L/TRAIL to lipid vesicles by using a novel Ni(2+)-(N-5-amino-1-carboxylpentyl)-iminodiacetic acid, NTA)-containing liposomal system. This lipidic framework (LUVs-Apo2L/TRAIL) greatly improves Apo2L/TRAIL activity, decreasing by around 14-fold the LC50 on the T-cell leukemia Jurkat. This increase in bioactivity correlated with the greater ability of LUVs-Apo2L/TRAIL to induce caspase-3 activation and is probably due to the increase in local concentration of Apo2L/TRAIL, improving its receptor cross-linking efficiency. More important, liposome-bound Apo2L/TRAIL overcame the resistance to soluble recombinant Apo2L/TRAIL exhibited by tumor cell mutants overexpressing Bcl-xL or by a Bax and Bak-defective Jurkat cell mutant (Jurkat-shBak) and are also effective against other hematologic tumor cells. Jurkat-Bcl-xL and Jurkat-shBak cells are resistant to most chemotherapeutic drugs currently used in cancer treatment, and their sensitivity to LUVs-Apo2L/TRAIL could have potential clinical applications.
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Neoplasias Hematológicas/tratamiento farmacológico , Liposomas/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Resistencia a Antineoplásicos , Citometría de Flujo , Humanos , Leucocitos Mononucleares , Liposomas/administración & dosificación , Ligando Inductor de Apoptosis Relacionado con TNF/química , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
Cancer resistance to treatments is a challenge that researchers constantly seek to overcome. For instance, TNF-related apoptosis-inducing ligand (TRAIL) is a potential good prospect as an anti-cancer therapy, as it attacks tumor cells but not normal cells. However, treatments based in soluble TRAIL provided incomplete clinical results and diverse formulations have been developed to improve its bioactivity. In previous works, we generated a new TRAIL formulation based in its attachment to the surface of unilamellar nanoliposomes (LUV-TRAIL). This formulation greatly increased apoptosis in a wide selection of tumor cell types, albeit a few of them remained resistant. On the other hand, it has been described that a metabolic shift in cancer cells can also alter its sensitivity to other treatments. In this work, we sought to increase the sensitivity of several tumor cell types resistant to LUV-TRAIL by previous exposure to the metabolic drug dichloroacetate (DCA), which forces oxidative phosphorylation. Results showed that DCA + LUV-TRAIL had a synergistic effect on both lung adenocarcinoma A549, colorectal HT29, and breast cancer MCF7 cells. Despite DCA inducing intracellular changes in a cell-type specific way, the increase in cell death by apoptosis was clearly correlated with an increase in death receptor 5 (DR5) surface expression in all cell lines. Therefore, DCA-induced metabolic shift emerges as a suitable option to overcome TRAIL resistance in cancer cells.
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Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.
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Apoptosis , Virosis , Animales , Ratones , Caspasa 8/genética , Piel/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The use of face masks and air purification systems has been key to curbing the transmission of SARS-CoV-2 aerosols in the context of the current COVID-19 pandemic. However, some masks or air conditioning filtration systems are designed to remove large airborne particles or bacteria from the air, being limited their effectiveness against SARS-CoV-2. Continuous research has been aimed at improving the performance of filter materials through nanotechnology. This article presents a new low-cost method based on electrostatic forces and coordination complex formation to generate antiviral coatings on filter materials using silver nanoparticles and polyethyleneimine. Initially, the AgNPs synthesis procedure was optimized until reaching a particle size of 6.2 ± 2.6 nm, promoting a fast ionic silver release due to its reduced size, obtaining a stable colloid over time and having reduced size polydispersity. The stability of the binding of the AgNPs to the fibers was corroborated using polypropylene, polyester-viscose, and polypropylene-glass spunbond mats as substrates, obtaining very low amounts of detached AgNPs in all cases. Under simulated operational conditions, a material loss less than 1% of nanostructured silver was measured. SEM micrographs demonstrated high silver distribution homogeneity on the polymer fibers. The antiviral coatings were tested against SARS-CoV-2, obtaining inactivation yields greater than 99.9%. We believe our results will be beneficial in the fight against the current COVID-19 pandemic and in controlling other infectious airborne pathogens.
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Cytotoxic lymphocytes (CLs), and more specifically Tc and NK cells, are the main executors of cell death in the immune system, playing a key role during both immunosurveillance and immunotherapy. These cells induce regulated cell death (RCD) by different mechanisms, being granular exocytosis and expression of death ligands the most prominent and best characterized ones. Apoptosis, a traditionally considered low-inflammatory type of cell death, has been accepted for years as the paradigm of RCD induced by CLs. However, several recent studies have demonstrated that NK cells and Tc cells can also induce more inflammatory forms of cell death, namely, necroptosis, pyroptosis, and ferroptosis. Activation of these highly inflammatory types of cell death appears to critically contribute to the activation of a successful antitumour immune response. Additionally, the role of specific cell death pathways in immunogenic cell death is still under intense debate, especially considering the interconnections with other inflammatory forms of cell death. These evidences, together with the advent of new cancer immunotherapies, highlight the necessity to deepen our understanding of the link between the cell death triggered by CLs and inflammation. This knowledge will be instrumental to maximize the antitumour potential of immunotherapies, minimizing deleterious effects associated with these treatments. In this review, we will briefly summarize the main features of apoptosis, necroptosis, pyroptosis and ferroptosis, to subsequently discuss the most recent evidences about the role of these RCD pathways during the elimination of cancer cells mediated by CLs and its modulation to increase the efficacy of cancer immunotherapy.
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Antineoplásicos , Piroptosis , Apoptosis , Muerte Celular , Células Asesinas Naturales , NecroptosisRESUMEN
Coronavirus disease 2019 (COVID19), caused by SARS-CoV-2, is a complex disease, with a variety of clinical manifestations ranging from asymptomatic infection or mild cold-like symptoms to more severe cases requiring hospitalization and critical care. The most severe presentations seem to be related with a delayed, deregulated immune response leading to exacerbated inflammation and organ damage with close similarities to sepsis. Methods: In order to improve the understanding on the relation between host immune response and disease course, we have studied the differences in the cellular (monocytes, CD8+ T and NK cells) and soluble (cytokines, chemokines and immunoregulatory ligands) immune response in blood between Healthy Donors (HD), COVID19 and a group of patients with non-COVID19 respiratory tract infections (NON-COV-RTI). In addition, the immune response profile has been analyzed in COVID19 patients according to disease severity. Results: In comparison to HDs and patients with NON-COV-RTI, COVID19 patients show a heterogeneous immune response with the presence of both activated and exhausted CD8+ T and NK cells characterised by the expression of the immune checkpoint LAG3 and the presence of the adaptive NK cell subset. An increased frequency of adaptive NK cells and a reduction of NK cells expressing the activating receptors NKp30 and NKp46 correlated with disease severity. Although both activated and exhausted NK cells expressing LAG3 were increased in moderate/severe cases, unsupervised cell clustering analyses revealed a more complex scenario with single NK cells expressing more than one immune checkpoint (PD1, TIM3 and/or LAG3). A general increased level of inflammatory cytokines and chemokines was found in COVID19 patients, some of which like IL18, IL1RA, IL36B and IL31, IL2, IFNα and TNFα, CXCL10, CCL2 and CCL8 were able to differentiate between COVID19 and NON-COV-RTI and correlated with bad prognosis (IL2, TNFα, IL1RA, CCL2, CXCL10 and CXCL9). Notably, we found that soluble NKG2D ligands from the MIC and ULBPs families were increased in COVID19 compared to NON-COV-RTI and correlated with disease severity. Conclusions: Our results provide a detailed comprehensive analysis of the presence of activated and exhausted CD8+T, NK and monocyte cell subsets as well as extracellular inflammatory factors beyond cytokines/chemokines, specifically associated to COVID19. Importantly, multivariate analysis including clinical, demographical and immunological experimental variables have allowed us to reveal specific immune signatures to i) differentiate COVID19 from other infections and ii) predict disease severity and the risk of death.
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COVID-19/sangre , COVID-19/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Linfocitos T CD8-positivos/virología , COVID-19/mortalidad , Estudios de Casos y Controles , Quimiocinas/sangre , Citocinas/sangre , Femenino , Hospitalización , Humanos , Células Asesinas Naturales/virología , Modelos Logísticos , Masculino , Persona de Mediana Edad , Monocitos/virología , Estudios Prospectivos , Infecciones del Sistema Respiratorio/sangre , Infecciones del Sistema Respiratorio/inmunología , Índice de Severidad de la EnfermedadRESUMEN
Several hundred millions of people have been diagnosed of coronavirus disease 2019 (COVID-19), causing millions of deaths and a high socioeconomic burden. SARS-CoV-2, the causative agent of COVID-19, induces both specific T- and B-cell responses, being antibodies against the virus detected a few days after infection. Passive immunization with hyperimmune plasma from convalescent patients has been proposed as a potentially useful treatment for COVID-19. Using an in-house quantitative ELISA test, we found that plasma from 177 convalescent donors contained IgG antibodies specific to the spike receptor-binding domain (RBD) of SARS-CoV-2, although at very different concentrations which correlated with previous disease severity and gender. Anti-RBD IgG plasma concentrations significantly correlated with the plasma viral neutralizing activity (VN) against SARS-CoV-2 in vitro. Similar results were found using an independent cohort of serum from 168 convalescent health workers. These results validate an in-house RBD IgG ELISA test in a large cohort of COVID-19 convalescent patients and indicate that plasma from all convalescent donors does not contain a high enough amount of anti-SARS-CoV-2-RBD neutralizing IgG to prevent SARS-CoV-2 infection in vitro. The use of quantitative anti-RBD IgG detection systems might help to predict the efficacy of the passive immunization using plasma from patients recovered from SARS-CoV-2.
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Recent work shows that TRAILshort, a membrane-bound short form of TRAIL, is expressed by human cancer cells and protects them from TRAIL-induced cell death. A mAb that selectively targets TRAILshort enhances cancer susceptibility to TRAIL and increases the efficacy of autologous CD8+ T cells in ex vivo primary tumors.See related article by Aboulnasr et al., p. 5759.
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Neoplasias , Ligando Inductor de Apoptosis Relacionado con TNF , Apoptosis , Linfocitos T CD8-positivos , Homicidio , Humanos , Inmunoterapia , Neoplasias/terapiaRESUMEN
(TNF)-related apoptosis-inducing ligand (TRAIL) is able to activate the extrinsic apoptotic pathway upon binding to DR4/TRAIL-R1 and/or DR5/TRAIL-R2 receptors. Structural data indicate that TRAIL functions as a trimer that can engage three receptor molecules simultaneously, resulting in receptor trimerization and leading to conformational changes in TRAIL receptors. However, receptor conformational changes induced by the binding of TRAIL depend on the molecular form of this death ligand, and not always properly trigger the apoptotic cascade. In fact, TRAIL exhibits a much stronger pro-apoptotic activity when is found as a transmembrane protein than when it occurs as a soluble form and this enhanced biological activity is directly linked to its ability to cluster TRAIL receptors in supra-molecular structures. In this regard, cells involved in tumor immunosurveillance, such as activated human T cells, secrete endogenous TRAIL as a transmembrane protein associated with lipid microvesicles called exosomes upon T-cell reactivation. Consequently, it seems clear that a proper oligomerization of TRAIL receptors, which leads to a strong apoptotic signaling, is crucial for inducing apoptosis in cancer cells upon TRAIL treatment. In this review, the current knowledge of oligomerization status of TRAIL receptors is discussed as well as the implications for cancer treatment when using TRAIL-based therapies.
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: T-cell mediated immune responses should be regulated to avoid the development of autoimmune or chronic inflammatory diseases. Several mechanisms have been described to regulate this process, namely death of overactivated T cells by cytokine deprivation, suppression by T regulatory cells (Treg), induction of expression of immune checkpoint molecules such as CTLA-4 and PD-1, or activation-induced cell death (AICD). In addition, activated T cells release membrane microvesicles called exosomes during these regulatory processes. In this review, we revise the role of exosome secretion in the different pathways of immune regulation described to date and its importance in the prevention or development of autoimmune disease. The expression of membrane-bound death ligands on the surface of exosomes during AICD or the more recently described transfer of miRNA or even DNA inside T-cell exosomes is a molecular mechanism that will be analyzed.
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Enfermedades Autoinmunes/inmunología , Exosomas/metabolismo , Linfocitos T/inmunología , Animales , Humanos , Tolerancia Inmunológica , Inflamación/patología , Modelos BiológicosRESUMEN
Although TRAIL (TNF-related apoptosis-inducing ligand, also known as Apo2L) was described as capable of inducing apoptosis in transformed cells while sparing normal cells, limited results obtained in clinical trials has limited its use as an anti-tumor agent. Consequently, novel TRAIL formulations with enhanced bioactivity are necessary for overcoming resistance to conventional soluble TRAIL (sTRAIL) exhibited by many primary tumors. Our group has generated artificial liposomes with sTRAIL anchored on their surface (large unilamellar vesicle (LUV)-TRAIL), which have shown a greater cytotoxic activity both in vitro and in vivo when compared to sTRAIL against distinct hematologic and epithelial carcinoma cells. In this study, we have improved LUV-TRAIL by loading doxorubicin (DOX) in its liposomal lumen (LUVDOX-TRAIL) in order to improve their cytotoxic potential. LUVDOX-TRAIL killed not only to a higher extent, but also with a much faster kinetic than LUV-TRAIL. In addition, the concerted action of the liposomal DOX and TRAIL was specific of the liposomal DOX and was not observed when with soluble DOX. The cytotoxicity induced by LUVDOX-TRAIL was proven to rely on two processes due to different molecular mechanisms: a dynamin-mediated internalization of the doxorubicin-loaded particle, and the strong activation of caspase-8 exerted by the liposomal TRAIL. Finally, greater cytotoxic activity of LUVDOX-TRAIL was also observed in vivo in a tumor xenograft model. Therefore, we developed a novel double-edged nanoparticle combining the cytotoxic potential of DOX and TRAIL, showing an exceptional and remarkable synergistic effect between both agents.
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Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of intratumoral injection of recombinant granulysin using in vivo models of breast cancer and multiple myeloma. In the present work we have developed a granulysin gene fusion to the anti-carcinoembryonic antigen (CEA/CEACAM5) single chain Fv antibody fragment MFE23. Both granulysin and the granulysin-based immunotoxin were expressed in Pichia pastoris. The immunotoxin specifically recognized CEA, purified or expressed on the cell surface. Moreover, the bioactivity of the immunotoxin against several CEA+ cell lines was higher than that of granulysin alone. Granulysin and the immunotoxin were tested as a treatment in in vivo xenograft models in athymic mice. When injected intratumorally, both granulysin and the immunotoxin were able to inhibit tumor growth. Furthermore, systemic administration of the immunotoxin demonstrated a decrease in tumor growth in a CEA+ tumor-bearing mouse model, whereas granulysin did not exhibit a therapeutic effect. This is the first granulysin-based immunotoxin and the present work constitutes the proof of concept of its therapeutic potential.
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Cytotoxic CD8+ T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1-4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-XL or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc-based immunotherapy.
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Antígenos Virales/inmunología , Resistencia a Antineoplásicos/genética , Glicoproteínas/inmunología , Neoplasias/terapia , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/trasplante , Proteínas Virales/inmunología , Animales , Apoptosis , Caspasa 3/metabolismo , Línea Celular Tumoral , Granzimas/deficiencia , Granzimas/genética , Granzimas/metabolismo , Humanos , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Neoplasias/mortalidad , Perforina/deficiencia , Perforina/genética , Perforina/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Tasa de Supervivencia , Linfocitos T Citotóxicos/inmunología , Trasplante Heterólogo , Proteína bcl-X/metabolismoRESUMEN
The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.
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Quinasa I-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células A549 , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Células HeLa , Humanos , Ratones Noqueados , Fosforilación/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
BACKGROUND/AIM: Targeting the extrinsic apoptotic pathway is an interesting option for anticancer therapy. A protein which such ability is Apo2 ligand, also known as TNF-related apoptosis-inducing ligand (TRAIL). The aim of this study was to examine the possibility of sensitizing resistant CLBL-1 canine lymphoma cells to TRAIL-induced apoptosis by using flavopiridol (FVP) a cyclin-dependent kinase inhibitor (CDKs). MATERIALS AND METHODS: The CLBL-1 (canine B-cell lymphoma cell line) was used in the study. The effect of FVP and TRAIL treatment on apoptosis induction was assessed by flow cytometry and western blot. RESULTS: Although canine lymphoma cells were resistant to TRAIL-induced apoptosis, combination of this death ligand with FVP was able to overcome TRAIL resistance of CLBL-1 lymphoma cells. CONCLUSION: Our results demonstrated that although canine lymphoma cells were resistant to TRAIL-induced apoptosis, combination of this death ligand with FVP was able to overcome TRAIL resistance of CLBL-1 lymphoma cell line. Although further investigation is required to deepen the knowledge of TRAIL as an antitumor agent in canine cancers, our results open the door to future use of TRAIL-based treatment strategies in veterinary oncology.