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The biomedical consequences of allogeneic blood transfusions and the possible pathomechanisms of transfusion-related morbidity and mortality are still not entirely understood. In retrospective studies, allogeneic transfusion was associated with increased rates of cancer recurrence, metastasis and death in patients with colorectal cancer. However, correlation does not imply causation. The purpose of this study was to elucidate this empirical observation further in order to address insecurity among patients and clinicians. We focused on the in vitro effect of microparticles derived from red blood cell units (RMPs). We incubated different colon carcinoma cells with RMPs and analyzed their effects on growth, invasion, migration and tumor marker expression. Furthermore, effects on Wnt, Akt and ERK signaling were explored. Our results show RMPs do not seem to affect functional and phenotypic characteristics of different colon carcinoma cells and did not induce or inhibit Wnt, Akt or ERK signaling, albeit in cell culture models lacking tumor microenvironment. Allogeneic blood transfusions are associated with poor prognosis, but RMPs do not seem to convey tumor-enhancing effects. Most likely, the circumstances that necessitate the transfusion, such as preoperative anemia, tumor stage, perioperative blood loss and extension of surgery, take center stage.
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Carcinoma , Micropartículas Derivadas de Células , Neoplasias del Colon , Carcinoma/complicaciones , Micropartículas Derivadas de Células/patología , Neoplasias del Colon/patología , Humanos , Recurrencia Local de Neoplasia/etiología , Proteínas Proto-Oncogénicas c-akt , Estudios Retrospectivos , Microambiente TumoralRESUMEN
The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.
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Ácidos Grasos/metabolismo , Herpes Simple/metabolismo , Herpesvirus Humano 2/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Animales , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/patología , Herpes Simple/genética , Herpes Simple/patología , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Lipoilación , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/patología , Células RAW 264.7RESUMEN
Sphingosine-1-phosphate (S1P) is involved in the regulation of important cellular processes, including immune-cell trafficking and proliferation. Altered S1P signaling is strongly associated with inflammation, cancer progression, and atherosclerosis; however, the mechanisms underlying its pathophysiologic effects are only partially understood. This study evaluated the effects of S1P in vitro and in vivo on the biosynthesis of leukotrienes (LTs), which form a class of lipid mediators involved in the pathogenesis of inflammatory diseases. Here, we report for the first time that S1P potently suppresses LT biosynthesis in Ca2+-ionophore-stimulated intact human neutrophils. S1P treatment resulted in intracellular Ca2+ mobilization, perinuclear translocation, and finally irreversible suicide inactivation of the LT biosynthesis key enzyme 5-lipoxygenase (5-LO). Agonist studies and S1P receptor mRNA expression analysis provided evidence for a S1P receptor 4-mediated effect, which was confirmed by a functional knockout of S1P4 in HL60 cells. Systemic administration of S1P in wild-type mice decreased both macrophage and neutrophil migration in the lungs in response to LPS and significantly attenuated 5-LO product formation, whereas these effects were abrogated in 5-LO or S1P4 knockout mice. In summary, targeting the 5-LO pathway is an important mechanism to explain S1P-mediated pathophysiologic effects. Furthermore, agonism at S1P4 represents a novel effective strategy in pharmacotherapy of inflammation.-Fettel, J., Kühn, B., Guillen, N. A., Sürün, D., Peters, M., Bauer, R., Angioni, C., Geisslinger, G., Schnütgen, F., Meyer zu Heringdorf, D., Werz, O., Meybohm, P., Zacharowski, K., Steinhilber, D., Roos, J., Maier, T. J. Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.
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Antiinflamatorios/farmacología , Araquidonato 5-Lipooxigenasa/efectos de los fármacos , Lisofosfolípidos/farmacología , Receptores de Lisoesfingolípidos/fisiología , Esfingosina/análogos & derivados , Animales , Araquidonato 5-Lipooxigenasa/biosíntesis , Araquidonato 5-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Calcio/metabolismo , Línea Celular , Femenino , Humanos , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neutrófilos/enzimología , Neutrófilos/metabolismo , Neumonía/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/metabolismo , Esfingosina/farmacología , Especificidad por SustratoRESUMEN
It is well known that sepsis and inflammation reduce male fertility. Within the testis, toll-like receptor 3 (TLR3) is constitutively expressed and recognizes double-stranded RNA (dsRNA) from viruses, degraded bacteria, damaged tissues and necrotic cells. To characterize the potential role of TLR3 in response to testicular infections, its expression and downstream signaling were investigated upon challenge with lipopolysaccharides (LPS) in two mouse strains that differ in their immuno-competence regarding T cell-regulated immunity. Thereto, Balb/c and Foxn1nu mice were randomized into six interventional groups treated with either i.v. application of saline or LPS followed by 20 min, 5 h 30 min and 18 h of observation and two sham-treated control groups. LPS administration induced a significant stress response; the amplification was manifested for TLR3 and interleukin 6 (IL6) mRNA in the impaired testis 5 h 30 min after LPS injection. TLR3 immunostaining revealed that TLR3 was primarily localized in spermatocytes. The TLR3 expression displayed different temporal dynamics between both mouse strains. However, immunofluorescence staining indicated only punctual interferon regulatory factor 3 (IRF3) expression upon LPS treatment along with minor alterations in interferon ß (IFNß) mRNA expression. Induction of acute inflammation was closely followed by a significant shift of the Bax/Bcl2 ratio to pro-apoptotic signaling accompanied by augmented TUNEL-positive cells 18 h after LPS injection with again differing patterns in both mouse strains. In conclusion, this study shows the involvement of TLR3 in response to LPS-induced testicular inflammation in immuno-competent and -incompetent mice, yet lacking transmission into its signaling pathway.
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Apoptosis/inmunología , Orquitis/inmunología , Espermatocitos/inmunología , Testículo/metabolismo , Receptor Toll-Like 3/inmunología , Animales , Factor 3 Regulador del Interferón/inmunología , Interferón beta/inmunología , Lipopolisacáridos/química , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Orquitis/inducido químicamente , Espermatocitos/citología , Testículo/patologíaRESUMEN
RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of covalent 5-lipoxygenase inhibitors is challenging due to unknown amino acid specificity and low posttranslational modification (PTM)-identification rates. The analysis of the amino-acid specificity and of the characteristic fragmentation of chemically modified peptides is considered to improve knowledge for the analysis of chemically modified peptides and proteins by MALDI-MS. METHODS: Various compounds were used to investigate the modification of synthetic peptides carrying reactive amino acid residues. Mass spectra were recorded using a MALDI-LTQ Orbitrap XL for high-resolution mass spectrometry and ion trap MALDI-MS2 . UV-Vis-based reduction and radical scavenging analysis was conducted. The on-plate digestion method described by Rühl et al was utilized for modification-site analysis at 5-lipoxygenase. RESULTS: The analysis of amino-acid-specific reactivity revealed the reactivity of quinones towards cysteine residues and the potential occurrence of a subsequent oxidative process was observed by an UV-Vis-based reduction assay. MALDI collision-induced dissociation tandem mass spectrometry (CID-MS2 ) indicated a prominent fragmentation mechanism of modified cysteine and histidine residues. Fragmentation included highly abundant neutral-loss signals which could be used to identify new modifications induced by chemical modifiers at the cysteine-159 residue of 5-lipoxygenase. CONCLUSIONS: Specificity and fragmentation analysis provides crucial information for the analysis of chemically modified cysteines and histidines by MALDI-MS. Elucidation of binding sites by MALDI-MS has been significantly improved using an easy-to-run peptide assay and gives background information for the analysis in the case of chemically modified 5-lipoxygenase.
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Péptidos/química , Proteínas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Sitios de Unión , Cisteína/análisis , Cisteína/química , Cisteína/metabolismo , Histidina/análisis , Histidina/química , Histidina/metabolismo , Lipooxigenasa , Inhibidores de la Lipooxigenasa , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Péptidos/análisis , Péptidos/metabolismo , Unión Proteica , Proteínas/análisis , Proteínas/metabolismo , Quinonas/químicaRESUMEN
Alzheimer's disease (AD) is the most common form of dementia in the elderly; important risk factors are old age and inheritance of the apolipoprotein E4 (APOE4) allele. Changes in amyloid precursor protein (APP) binding, trafficking, and sorting may be important AD causative factors. Secretase-mediated APP cleavage produces neurotoxic amyloid-beta (Aß) peptides, which form lethal deposits in the brain. In vivo and in vitro studies have implicated sortilin-related receptor (SORL1) as an important factor in APP trafficking and processing. Recent in vitro evidence has associated the APOE4 allele and alterations in the SORL1 pathway with AD development and progression. Here, we analyzed SORL1 expression in neural stem cells (NSCs) from AD patients carrying null, one, or two copies of the APOE4 allele. We show reduced SORL1 expression only in NSCs of a patient carrying two copies of APOE4 allele with increased Aß/SORL1 localization along the degenerated neurites. Interestingly, SORL1 binding to APP was largely compromised; this could be almost completely reversed by γ-secretase (but not ß-secretase) inhibitor treatment. These findings may yield new insights into the complex interplay of SORL1 and AD pathology and point to NSCs as a valuable tool to address unsolved AD-related questions in vitro.
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Enfermedad de Alzheimer/metabolismo , Apolipoproteína E4/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Células Cultivadas , Femenino , Humanos , Recién Nacido , Masculino , Neuritas/metabolismo , FenotipoRESUMEN
The p53 tumor suppressor plays a critical role in cancer, and more than 50% of human tumors contain mutations or deletions of the TP53 gene. p53 can transactivate or repress target genes in response to diverse stress signals, such as transient growth arrest, DNA repair, cellular differentiation, senescence and apoptosis. Through an unbiased genome-wide ChIP-seq analysis, we have found that 5-lipoxygenase (ALOX5, 5-LO) which is a key enzyme of leukotriene (LT) biosynthesis, is a direct target gene of p53 and its expression is induced by genotoxic stress via actinomycin D (Act.D) or etoposide (Eto) treatment. 5-LO and LTs play a role in immunological diseases as well as in tumorigenesis and tumor growth. p53 binds to a specific binding site consisting of a complete p53 consensus-binding motif in ALOX5 intron G which is located about 64kbp downstream of the transcriptional start site. We confirmed the strong binding of p53 to the 5-LO target site in ChIP-qPCR experiments. Expression analyses by qRT-PCR and immunoblot further revealed that genotoxic stress induces the ALOX5 mRNA and protein expression in a p53-dependent manner. Knockdown of p53 in U2OS cells leads to a downregulation of 5-LO mRNA and protein expression. In addition, immunofluorescence and immunoprecipitation assays indicate the direct binding of 5-LO to p53 protein. Furthermore, we found that 5-LO can inhibit the transcriptional activity of p53 suggesting that 5-LO acts in a negative feedback loop to limit induction of p53 target genes.
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Araquidonato 5-Lipooxigenasa/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteína p53 Supresora de Tumor/fisiología , Sitios de Unión , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Dactinomicina/farmacología , Etopósido/farmacología , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is a cytokine-activated transcription factor controlling inflammation, cell proliferation, survival, and differentiation in normal tissue as well as in tumor growth. One of its most important negative regulators is the suppressor of cytokine signaling 3 (SOCS3). Here, we analyzed SOCS3 and other tumor-associated local immune regulators in human clear cell renal cell carcinoma (ccRCC). Analyses were performed in tumor and adjacent tumor-free healthy renal tissue from 35 patients with ccRCC. For functional analysis, ccRCC Caki-1 cell lines were stimulated with IL-6 and IFNγ in cell culture assays. We observed significantly lower SOCS3 messenger RNA (mRNA) levels in tumor tissue compared to healthy tissue. SOCS3 mRNA strongly correlated within tumor and healthy tissue. Interestingly vice versa, SOCS3 protein levels were significantly higher in tumor tissue than in healthy tissue. IL-22 and IL-22R1 mRNA displayed no differences in tumor and healthy tissue. Stimulation of Caki-1 cells with IFNγ resulted in markedly increased SOCS3 mRNA levels. We conclude that SOCS3 along with STAT3 participates in regulatory mechanisms in ccRCC, which certainly features only one of multiple factors involved but nevertheless merits further attention.
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Carcinoma de Células Renales/genética , Proteína 3 Supresora de la Señalización de Citocinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Femenino , Regulación de la Expresión Génica/genética , Humanos , Inflamación/genética , Interferón gamma/genética , Interleucina-6/genética , Interleucinas/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Factor de Transcripción STAT3/genética , Interleucina-22RESUMEN
Platelets are activated by the interaction with cancer cells and release enhanced levels of lipid mediators [such as thromboxane (TX)A2 and prostaglandin (PG)E2, generated from arachidonic acid (AA) by the activity of cyclooxygenase (COX)-1], granule content, including ADP and growth factors, chemokines, proteases and Wnt proteins. Moreover, activated platelets shed different vesicles, such as microparticles (MPs) and exosomes (rich in genetic material such as mRNAs and miRNAs). These platelet-derived products induce several phenotypic changes in cancer cells which confer high metastatic capacity. A central event involves an aberrant expression of COX-2 which influences cell-cycle progression and contribute to the acquisition of a cell migratory phenotype through the induction of epithelial mesenchymal transition genes and down-regulation of E-cadherin expression. The identification of novel molecular determinants involved in the cross-talk between platelets and cancer cells has led to identify novel targets for anti-cancer drug development.
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Plaquetas/citología , Ciclooxigenasa 2/metabolismo , Neoplasias/patología , Activación Plaquetaria , Plaquetas/metabolismo , Comunicación Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis de la Neoplasia , Neoplasias/metabolismoRESUMEN
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Cyclooxygenase (COX)-2-derived prostanoids can influence several processes that are linked to carcinogenesis. We aimed to address the hypothesis that platelets contribute to aberrant COX-2 expression in HT29 colon carcinoma cells and to reveal the role of platelet-induced COX-2 on the expression of proteins involved in malignancy and marker genes of epithelial-mesenchymal transition (EMT). Human platelets cocultured with HT29 cells rapidly adhered to cancer cells and induced COX-2 mRNA expression, but not protein synthesis, which required the late release of platelet-derived growth factor and COX-2 mRNA stabilization. Platelet-induced COX-2-dependent prostaglandin E2 (PGE2) synthesis in HT29 cells was involved in the downregulation of p21(WAF1/CIP1) and the upregulation of cyclinB1 since these effects were prevented by rofecoxib (a selective COX-2 inhibitor) and rescued by exogenous PGE2. Galectin-3, which is highly expressed in HT29 cells, is unique among galectins because it contains a collagen-like domain. Thus, we studied the role of galectin-3 and platelet collagen receptors in platelet-induced COX-2 overexpression. Inhibitors of galectin-3 function (ß-lactose, a dominant-negative form of galectin-3, Gal-3C, and anti-galectin-3 antibody M3/38) or collagen receptor-mediated platelet adhesion (revacept, a dimeric platelet collagen receptor GPVI-Fc) prevented aberrant COX-2 expression. Inhibition of platelet-cancer cell interaction by revacept was more effective than rofecoxib in preventing platelet-induced mRNA changes of EMT markers, suggesting that direct cell-cell contact and aberrant COX-2 expression synergistically induced gene expression modifications associated with EMT. In conclusion, our findings provide the rationale for testing blockers of collagen binding sites, such as revacept, and galectin-3 inhibitors in the prevention of colon cancer metastasis in animal models, followed by studies in patients.
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Plaquetas/efectos de los fármacos , Plaquetas/patología , Comunicación Celular/efectos de los fármacos , Neoplasias del Colon/sangre , Neoplasias del Colon/enzimología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Sitios de Unión , Plaquetas/enzimología , Plaquetas/metabolismo , Comunicación Celular/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Ciclina B1/genética , Ciclina B1/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/genética , Dinoprostona/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Galectina 3/antagonistas & inhibidores , Galectina 3/genética , Galectina 3/metabolismo , Expresión Génica/efectos de los fármacos , Glicoproteínas/farmacología , Células HT29 , Humanos , Fragmentos Fc de Inmunoglobulinas/farmacología , Lactonas/farmacología , Lactosa/farmacología , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Mensajero/genética , Receptores de Colágeno/genética , Receptores de Colágeno/metabolismo , Sulfonas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The SARS-CoV-2 variant Omicron has spread world-wide and is responsible for rapid increases in infections, including in populations with high vaccination rates. Here, we analysed in the sera of vaccinated individuals the antibody binding to the receptor-binding domain (RBD) of the spike protein and the neutralization of wild-type (WT), Delta (B.1.617.2), and Omicron (B.1.1.529; BA.1) pseudotyped vectors. Although sera from individuals immunized with vector vaccines (Vaxzevria; AZ and COVID-19 Janssen, Ad26.COV2.S; J&J) were able to bind and neutralize WT and Delta, they showed only background levels towards Omicron. In contrast, mRNA (Comirnaty; BNT) or heterologous (AZ/BNT) vaccines induced weak, but detectable responses against Omicron. While RBD-binding antibody levels decreased significantly six months after full vaccination, the SARS-CoV-2 RBD-directed avidity remained constant. However, this still coincided with a significant decrease in neutralization activity against all variants. A third booster vaccination with BNT significantly increased the humoral immune responses against all tested variants, including Omicron. In conclusion, only vaccination schedules that included at least one dose of mRNA vaccine and especially an mRNA booster vaccination induced sufficient antibody levels with neutralization capacity against multiple variants, including Omicron.
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Waning immunity against SARS-CoV-2 and the emergence of variants, especially of the most distant variant, Omicron, affect titers of neutralizing antibodies in the sera of vaccinated individuals. Thus, two vaccinations with the mRNA vaccine BNT162b fail to induce neutralizing antibodies against the Omicron variant. A first booster vaccination increases Omicron-RBD-binding IgG and IgA and neutralizing capacity. In comparison, the Wuhan isolate titers of the Omicron variant binding antibodies are 8.5 lower. After a third vaccination, induction of Omicron-RBD- and Wuhan-RBD-binding antibodies follows the same kinetic. Five to six months after the third vaccination, there are still Omicron-RBD-binding antibodies detectable, but 35.9 percent of the analyzed sera fail to neutralize the Omicron variant, while all sera efficiently neutralize the Delta isolate. In the case of the Wuhan-RBD, a significantly larger number of stable antigen-antibody complexes is formed than in Omicron-RBD. A fourth vaccination with mRNA-1273 temporarily restores levels of Omicron-, Delta- and Wuhan-specific antibodies. Comparing different booster strategies revealed that the breadth of the immune response is not affected by the vaccination regimen. Taken together, these data indicate that booster vaccinations (third and fourth dose) increase the breadth of the immune response, but there is a qualitative difference of antibodies with respect to the stability of antigen-antibody complexes and persistence of antibody titers.
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Sulindac is a non-selective inhibitor of cyclooxygenases (COX) used to treat inflammation and pain. Additionally, non-COX targets may account for the drug's chemo-preventive efficacy against colorectal cancer and reduced gastrointestinal toxicity. Here, we demonstrate that the pharmacologically active metabolite of sulindac, sulindac sulfide (SSi), targets 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs). SSi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human polymorphonuclear leukocytes (IC(50) approximately 8-10 microM). Importantly, SSi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC(50) = 18.7 microM). SSi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone (SSo), failed to inhibit 5-LO. Mechanistic analysis demonstrated that SSi directly suppresses 5-LO with an IC(50) of 20 muM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy.
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Antiinflamatorios/farmacología , Inhibidores de la Lipooxigenasa , Sulindac/análogos & derivados , Proteínas Activadoras de la 5-Lipooxigenasa , Antiinflamatorios/uso terapéutico , Araquidonato 5-Lipooxigenasa/metabolismo , Sangre/efectos de los fármacos , Sangre/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Sistema Libre de Células/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/metabolismo , Concentración Osmolar , Transporte de Proteínas/efectos de los fármacos , Sulindac/farmacología , Sulindac/uso terapéuticoRESUMEN
Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here, we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion but also fusion from without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies, as well as sera from convalescent patients, inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during coronavirus disease 2019 (COVID-19) can proceed at low levels of S protein and may not be effectively prevented by antibodies.
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5-Lipoxygenase (5-LO) is the key enzyme in the formation of pro-inflammatory leukotrienes (LT) which play an important role in a number of inflammatory diseases. Accordingly, 5-LO inhibitors are frequently used to study the role of 5-LO and LT in models of inflammation and cancer. Interestingly, the therapeutic efficacy of these inhibitors is highly variable. Here we show that the frequently used 5-LO inhibitors AA-861, BWA4C, C06, CJ-13,610 and the FDA approved compound zileuton as well as the pan-LO inhibitor nordihydroguaiaretic acid interfere with prostaglandin E2 (PGE2) release into the supernatants of cytokine-stimulated (TNFα/IL-1ß) HeLa cervix carcinoma, A549 lung cancer as well as HCA-7 colon carcinoma cells with similar potencies compared to their LT inhibitory activities (IC50 values ranging from 0.1-9.1 µM). In addition, AA-861, BWA4C, CJ-13,610 and zileuton concentration-dependently inhibited bacterial lipopolysaccharide triggered prostaglandin (PG) release into human whole blood. Western Blot analysis revealed that inhibition of expression of enzymes involved in PG synthesis was not part of the underlying mechanism. Also, liberation of arachidonic acid which is the substrate for PG synthesis as well as PGH2 and PGE2 formation were not impaired by the compounds. However, accumulation of intracellular PGE2 was found in the inhibitor treated HeLa cells suggesting inhibition of PG export as major mechanism. Further, experiments showed that the PG exporter ATP-binding cassette transporter multidrug resistance protein 4 (MRP-4) is targeted by the inhibitors and may be involved in the 5-LO inhibitor-mediated PGE2 inhibition. In conclusion, the pharmacological effects of a number of 5-LO inhibitors are compound-specific and involve the potent inhibition of PGE2 export. Results from experimental models on the role of 5-LO in inflammation and pain using 5-LO inhibitors may be misleading and their use as pharmacological tools in experimental models has to be revisited. In addition, 5-LO inhibitors may serve as new scaffolds for the development of potent prostaglandin export inhibitors.
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Endogenous nitro-fatty acids (NFA) are potent electrophilic lipid mediators that exert biological effects in vitro and in vivo via selective covalent modification of thiol-containing target proteins. The cytoprotective, anti-inflammatory, and anti-tumorigenic effects of NFA in animal models of disease caused by targeted protein nitroalkylation are a valuable basis for the development of future anti-phlogistic and anti-neoplastic drugs. Considering the complexity of diseases and accompanying comorbidities there is an urgent need for clinically effective multifunctional drugs. NFA are composed of a fatty acid backbone containing a nitroalkene moiety triggering Michael addition reactions. However, less is known about the target-specific structure-activity relationships and selectivities comparing different NFA targets. Therefore, we analyzed 15 NFA derivatives and compared them with the lead structure 9-nitro-oleic acid (9NOA) in terms of their effect on NF-κB (nuclear factor kappa B) signaling inhibition, induction of Nrf-2 (nuclear factor erythroid 2-related factor 2) gene expression, sEH (soluble epoxide hydrolase), LO (lipoxygenase), and COX-2 (cyclooxygenase-2) inhibition, and their cytotoxic effects on colorectal cancer cells. Minor modifications of the Michael acceptor position and variation of the chain length led to drugs showing increased target preference or enhanced multi-targeting, partly with higher potency than 9NOA. This study is a significant step forward to better understanding the biology of NFA and their enormous potential as scaffolds for designing future anti-inflammatory drugs.
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Alzheimer's disease (AD) is an incurable neurodegenerative disorder with a few early detection strategies. We previously proposed the amyloid precursor protein (APP) tyrosine 682 (Tyr682) residue as a valuable target for the development of new innovative pharmacologic or diagnostic interventions in AD. Indeed, when APP is phosphorylated at Tyr682, it is forced into acidic neuronal compartments where it is processed to generate neurotoxic amyloid ß peptides. Of interest, Fyn tyrosine kinase (TK) interaction with APP Tyr682 residue increases in AD neurons. Here we proved that when Fyn TK was overexpressed it elicited APP Tyr682 phosphorylation in neurons from healthy donors and promoted the amyloidogenic APP processing with Aß peptides accumulation and neuronal death. Phosphorylation of APP at Tyr (pAPP-Tyr) increased in neurons of AD patients and AD neurons that exhibited high pAPP-Tyr also had higher Fyn TK activity. Fyn TK inhibition abolished the pAPP-Tyr and reduced Aß42 secretion in AD neurons. In addition, the multidomain adaptor protein Fe65 controlled the Fyn-mediated pAPP-Tyr, warranting the possibility of targeting the Fe65-APP-Fyn pathway to develop innovative strategies in AD. Altogether, these results strongly emphasize the relevance of focusing on pAPP Tyr682 either for diagnostic purposes, as an early biomarker of the disease, or for pharmacological targeting, using Fyn TKI.
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Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Tirosina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Persona de Mediana Edad , Células-Madre Neurales/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-fyn/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fyn/genética , TransfecciónRESUMEN
Nitro fatty acids (NFAs) are endogenously generated lipid mediators deriving from reactions of unsaturated electrophilic fatty acids with reactive nitrogen species. Furthermore, Mediterranean diets can be a source of NFA. These highly electrophilic fatty acids can undergo Michael addition reaction with cysteine residues, leading to post-translational modifications (PTM) of selected regulatory proteins. Such modifications are capable of changing target protein function during cell signaling or in biosynthetic pathways. NFA target proteins include the peroxisome proliferator-activated receptor γ (PPAR-γ), the pro-inflammatory and tumorigenic nuclear factor-κB (NF-κB) signaling pathway, the pro-inflammatory 5-lipoxygenases (5-LO) biosynthesis pathway as well as soluble epoxide hydrolase (sEH), which is essentially involved in the regulation of vascular tone. In several animal models of inflammation and cancer, the therapeutic efficacy of well-tolerated NFA has been demonstrated. This has already led to clinical phase II studies investigating possible therapeutic effects of NFA in subjects with pulmonary arterial hypertension. Albeit Michael acceptors feature a broad spectrum of bioactivity, they have for a rather long time been avoided as drug candidates owing to their presumed unselective reactivity and toxicity. However, targeted covalent modification of regulatory proteins by Michael acceptors became recognized as a promising approach to drug discovery with the recent FDA approvals of the cancer therapeutics, afatanib (2013), ibrutinib (2013), and osimertinib (2015). Furthermore, the Michael acceptor, neratinib, a dual inhibitor of the human epidermal growth factor receptor 2 and epidermal growth factor receptor, was recently approved by the FDA (2017) and by the EMA (2018) for the treatment of breast cancer. Finally, a number of further Michael acceptor drug candidates are currently under clinical investigation for pharmacotherapy of inflammation and cancer. In this review, we focus on the pharmacology of NFA and other Michael acceptor drugs, summarizing their potential as an emerging class of future antiphlogistics and adjuvant in tumor therapeutics.
RESUMEN
Cysteinyl leukotriene receptor 1 antagonists (CysLT1RA) are frequently used as add-on medication for the treatment of asthma. Recently, these compounds have shown protective effects in cardiovascular diseases. This prompted us to investigate their influence on soluble epoxide hydrolase (sEH) and peroxisome proliferator activated receptor (PPAR) activities, two targets known to play an important role in CVD and the metabolic syndrome. Montelukast, pranlukast and zafirlukast inhibited human sEH with IC50 values of 1.9, 14.1, and 0.8 µM, respectively. In contrast, only montelukast and zafirlukast activated PPARγ in the reporter gene assay with EC50 values of 1.17 µM (21.9% max. activation) and 2.49 µM (148% max. activation), respectively. PPARα and δ were not affected by any of the compounds. The activation of PPARγ was further investigated in 3T3-L1 adipocytes. Analysis of lipid accumulation, mRNA and protein expression of target genes as well as PPARγ phosphorylation revealed that montelukast was not able to induce adipocyte differentiation. In contrast, zafirlukast triggered moderate lipid accumulation compared to rosiglitazone and upregulated PPARγ target genes. In addition, we found that montelukast and zafirlukast display antagonistic activities concerning recruitment of the PPARγ cofactor CBP upon ligand binding suggesting that both compounds act as PPARγ modulators. In addition, zafirlukast impaired the TNFα triggered phosphorylation of PPARγ2 on serine 273. Thus, zafirlukast is a novel dual sEH/PPARγ modulator representing an excellent starting point for the further development of this compound class.