RESUMEN
Abrin is a toxin from the seeds of Abrus precatorius. Abrin is considerably more toxic than ricin and a potent bio-warfare agent. The mechanism of abrin induced hepatotoxicity remains unclear. Silibinin has antioxidant, anti-inflammatory and hepatoprotective activities. But, its therapeutic potential in abrin toxicity is unknown. In view of these facts, the purpose of this study was to delineate the mechanisms and ameliorative role of silibinin against abrin induced hepatotoxicity. Parameters related to liver functions, oxidative stress, inflammation, Fas pathway and histopathology were evaluated in the liver of BALB/c mice after abrin exposure. Abrin intoxication resulted in hepatotoxicity, oxidative stress, inflammation, altered histopathology and increased Fas pathway signaling. Silibinin improves survival of abrin-exposed mice by decreasing serum liver enzymes and reinstating the antioxidant capacity. Silibinin also inhibits abrin-induced inflammation and Fas pathway. Present study for the first time demonstrates the hepatoprotective potential of silibinin against abrin toxicity.
Asunto(s)
Abrina , Enfermedad Hepática Inducida por Sustancias y Drogas , Silibina , Receptor fas , Abrina/toxicidad , Animales , Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Interacciones Farmacológicas , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Ratones , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Silibina/farmacología , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
We planned to appraise the effects of ß-caryophyllene on Fas- receptor and caspase-mediated apoptosis signaling pathway and endothelial dysfunction in rats infarcted with isoproterenol. Rats were induced myocardial infarction by using isoproterenol (100 mg/kg body weight [b.w]). Serum creatine kinase-MB, serum cardiac troponin-T, heart weight, heart rate, and heart lipid peroxidation were greatly (p < 0.05) augmented, while heart enzymatic antioxidants and plasma nonenzymatic antioxidants were greatly (p < 0.05) lessened in isoproterenol-treated rats. Reverse transcription-polymerase chain reaction study revealed augmented expressions of Fas-receptor and caspases 8, 9, and 3 genes in myocardial infarcted rats. Furthermore, iNOS protein expression was amplified and eNOS protein was lessened in the myocardial infarcted heart. Three weeks pre- and cotreatment with ß-caryophyllene (20 mg/kg b.w) greatly (p < 0.05) protected isoproterenol-treated rats against these altered structural, biochemical, molecular, and immunohistochemical parameters, by its anti-cardiac hypertrophic, anti-tachycardial, antioxidant, anti-apoptotic, and anti-endothelial dysfunction effects. In conclusion, these findings projected the use of ß-caryophyllene for the therapy of human myocardial infarction after clinical trials.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Endotelio Vascular/efectos de los fármacos , Infarto del Miocardio/metabolismo , Sesquiterpenos Policíclicos/farmacología , Transducción de Señal/efectos de los fármacos , Receptor fas/antagonistas & inhibidores , Animales , Antioxidantes/metabolismo , Biomarcadores/sangre , Forma MB de la Creatina-Quinasa/sangre , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Miocardio/enzimología , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sesquiterpenos Policíclicos/administración & dosificación , Ratas , Ratas Wistar , Receptor fas/metabolismoRESUMEN
Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 is a virus-induced inflammatory disease of the airways and lungs that leads to severe multi-organ damage and death. Here we show that cellular lipid synthesis is required for SARS-CoV-2 replication and offers an opportunity for pharmacological intervention. Screening a short-hairpin RNA sublibrary that targets metabolic genes, we identified genes that either inhibit or promote SARS-CoV-2 viral infection, including two key candidate genes, ACACA and FASN, which operate in the same lipid synthesis pathway. We further screened and identified several potent inhibitors of fatty acid synthase (encoded by FASN), including the US Food and Drug Administration-approved anti-obesity drug orlistat, and found that it inhibits in vitro replication of SARS-CoV-2 variants, including more contagious new variants, such as Delta. In a mouse model of SARS-CoV-2 infection (K18-hACE2 transgenic mice), injections of orlistat resulted in lower SARS-CoV-2 viral levels in the lung, reduced lung pathology and increased mouse survival. Our findings identify fatty acid synthase inhibitors as drug candidates for the prevention and treatment of COVID-19 by inhibiting SARS-CoV-2 replication. Clinical trials are needed to evaluate the efficacy of repurposing fatty acid synthase inhibitors for severe COVID-19 in humans.
Asunto(s)
Antivirales/farmacología , COVID-19/metabolismo , COVID-19/virología , Ácidos Grasos/biosíntesis , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/uso terapéutico , COVID-19/mortalidad , Línea Celular , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta a Droga , Desarrollo de Medicamentos , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno/genética , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , Receptor fas/antagonistas & inhibidores , Receptor fas/deficiencia , Receptor fas/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
Although many studies have reported toxic effects of cadmium (Cd) and lead (Pb) in the central nervous system, few studies have investigated the combined toxicity of Cd and Pb. The mechanisms by which these combined heavy metals induce toxicity, as well as effective means to exert neuroprotection from these agents, remain poorly understood. To investigate the protective effects of alpha-lipoic acid (α-LA) on Cd- and/or Pb-induced cortical damage in rats, 48 Sprague-Dawley rats were exposed to drinking water containing 50 mg/L of Cd and/or 300 mg/L of Pb for 12 weeks, in the presence or absence of α-LA co-treatment (50 mg/kg) via gavage. We observed that exposure to Cd and/or Pb decreased the brain weight/body weight ratio and increased Cd and/or Pb contents as well as ultrastructural damage to the cerebral cortex. Cd and/or Pb also induced endoplasmic-reticulum (ER) stress and activated Fas (CD95/APO-1)/Fas ligand (FasL) and mitochondrial apoptotic pathways. Furthermore, co-treatment of Cd and Pb further exacerbated part of these phenotypes than treatment of Cd or Pb alone. However, simultaneous supplementation with α-LA attenuated Cd and/or Pb-induced neurotoxicity by increasing the brain weight/body weight ratio, reducing Cd and/or Pb contents, ameliorating both nuclear/mitochondrial damage and ER stress, and attenuating activation of Fas/FasL and mitochondrial apoptotic pathways. Collectively, our results indicate that the accumulation of Cd and/or Pb causes cortical damage and that α-LA exerts protection against Cd- and/or Pb-induced neurotoxicity. These findings highlight that α-LA may be exploited for the treatment and prevention of Cd- and/or Pb-induced neurotoxicity.
Asunto(s)
Cadmio/toxicidad , Corteza Cerebral/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteína Ligando Fas/antagonistas & inhibidores , Plomo/toxicidad , Ácido Tióctico/farmacología , Receptor fas/antagonistas & inhibidores , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Corteza Cerebral/metabolismo , Corteza Cerebral/ultraestructura , Estrés del Retículo Endoplásmico/fisiología , Proteína Ligando Fas/metabolismo , Femenino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptor fas/metabolismoRESUMEN
BACKGROUND: Glaucoma is a complex, multifactorial disease where apoptosis, microglia activation, and inflammation have been linked to the death of retinal ganglion cells (RGCs) and axon degeneration. We demonstrated previously that FasL-Fas signaling was required for axon degeneration and death of RGCs in chronic and inducible mouse models of glaucoma and that Fas activation triggered RGC apoptosis, glial activation, and inflammation. Here, we investigated whether targeting the Fas receptor with a small peptide antagonist, ONL1204, has anti-inflammatory and neuroprotective effects in a microbead-induced mouse model of glaucoma. METHODS: Intracameral injection of microbeads was used to elevate intraocular pressure (IOP) in Fas-deficient (Faslpr) mice and WT C57BL/6J mice that received an intravitreal injection of the Fas inhibitor, ONL1204 (2 µg/1 µl) (or vehicle only), on day 0 or day 7 after microbead injection. The IOP was monitored by rebound tonometry, and at 28 days post-microbead injection, Brn3a-stained RGCs and paraphenylenediamine (PPD)-stained axons were analyzed. The effects of ONL1204 on retinal microglia activation and the expression of inflammatory genes were analyzed by immunostaining of retinal flatmounts and quantitative PCR (qPCR). RESULTS: Rebound tonometry showed equivalent elevation of IOP in all groups of microbead-injected mice. At 28 days post-microbead injection, the RGC and axon counts from microbead-injected Faslpr mice were equivalent to saline-injected (no IOP elevation) controls. Treatment with ONL1204 also significantly reduced RGC death and loss of axons in microbead-injected WT mice when compared to vehicle-treated controls, even when administered after IOP elevation. Confocal analysis of Iba1-stained retinal flatmounts and qPCR demonstrated that ONL1204 also abrogated microglia activation and inhibited the induction of multiple genes implicated in glaucoma, including cytokines and chemokines (GFAP, Caspase-8, TNFα, IL-1ß, IL-6, IL-18, MIP-1α, MIP-1ß, MIP-2, MCPI, and IP10), components of the complement cascade (C3, C1Q), Toll-like receptor pathway (TLR4), and inflammasome pathway (NLRP3). CONCLUSIONS: These results serve as proof-of-principal that the small peptide inhibitor of the Fas receptor, ONL1204, can provide robust neuroprotection in an inducible mouse model of glaucoma, even when administered after IOP elevation. Moreover, Fas signaling contributes to the pathogenesis of glaucoma through activation of both apoptotic and inflammatory pathways.
Asunto(s)
Glaucoma/patología , Degeneración Nerviosa/patología , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Receptor fas/antagonistas & inhibidores , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Glaucoma/metabolismo , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/metabolismo , Péptidos/química , Células Ganglionares de la Retina/patologíaRESUMEN
A novel series of fatty acid synthase (FAS) inhibitors with D-(-)-pantolactone moiety and potential utility for the treatment of obesity were designed, synthesized and characterized, in which the structure of compound 3k was further confirmed by single X-ray diffraction. The mouse FAS inhibitory activity of synthesized compounds was evaluated. Major synthesized compounds (except 3g, 3i, 3k, 3l, and 3n) exhibited moderate FAS inhibitory properties with IC50 values in the range of 13.68⯱â¯1.52-33.19⯱â¯1.39⯵M, reference inhibitor C75 has IC50 value of 13.86⯱â¯2.79⯵M. Eight compounds (3c, 3d, 3e, 3f, 3j, 3m, 3q and 3r) also displayed inhibitory effect on lipid accumulation in human HepG2 cells. Additionally, the molecular docking study revealed that compound 3m having good inhibition activity against FAS and lipid accumulation also showed promising binding affinities with hFAS, while its binding model with hFAS (PDB ID: 4PIV) was different from that of reference compound C75.
Asunto(s)
4-Butirolactona/análogos & derivados , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Receptor fas/antagonistas & inhibidores , 4-Butirolactona/síntesis química , 4-Butirolactona/química , 4-Butirolactona/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células Hep G2 , Humanos , Lípidos/antagonistas & inhibidores , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Células Tumorales Cultivadas , Receptor fas/metabolismoRESUMEN
Cell-free DNA (cfDNA) (e.g. fetal- or tumor-derived DNA) is DNA found in the blood circulation. It is now widely investigated as a biomarker for prenatal screening, tumor diagnosis, and tumor monitoring as "liquid biopsies". However, the biological and biochemical aspects of cfDNA remain unclear. Although cfDNA is considered to be mainly derived from dead cells, information is scarce as to whether it is apoptotic or necrotic and what kinds of endonucleases or DNases are involved. We induced in vivo hepatocyte necrosis and apoptosis in mice deficient in DNase1L3 (also named DNase γ) and/or caspase-activated DNase (CAD) genes with acetaminophen overdose and anti-Fas antibody treatments. We found that (i) DNase1L3 was the endonuclease responsible for generating cfDNA in acetaminophen-induced hepatocyte necrosis and (ii) CAD and DNase1L3 cooperated in producing cfDNA for anti-Fas mediated hepatocyte apoptosis.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Desoxirribonucleasas/genética , Endodesoxirribonucleasas/genética , Necrosis/genética , Receptor fas/genética , Acetaminofén/administración & dosificación , Animales , Anticuerpos Neutralizantes/farmacología , Ácidos Nucleicos Libres de Células/sangre , Desoxirribonucleasas/sangre , Endodesoxirribonucleasas/sangre , Trampas Extracelulares/metabolismo , Regulación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis/sangre , Necrosis/inducido químicamente , Necrosis/patología , Transducción de Señal , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
Aim: Stimulation of Fas death receptor is introduced as a major cause of non-alcoholic steatohepatitis (NASH) progression through suppression of cell viability. Therefore, the blocking of death pathways is hypothesised to be express new approaches to NASH therapy. For this purpose, current experiment applied synthetic small interference RNA (SiRNA) to trigger Fas death receptor and to show its potential therapeutic role in designed NASH model. Methods: Male mice were placed on a western diet (WD) for 8 weeks and exposed to cigarette smoke during the last 4 weeks of feeding to induce NASH model. In the next step, Fas SiRNA was injected to mice aiming to examine specific Fas gene silencing, after 8 weeks. As a control, mice received scrambled SiRNA. Reversible possibility of disease was examined by 3 weeks of recovery. Results: Analysis of data is accompanied with the significant histopathological changes (steatosis, ballooning and inflammation), increased lipid profile and hepatic enzyme activities (AST, ALT, ALP) plus TBARS as well as decreased antioxidants levels in NASH model. Upon Fas-SiRNA injection, almost all measured parameters of NASH such as overexpression of Fas receptor, caspase3, NF-kB genes and marked increase of hepatic TNF-α were significantly restored and were remained nearly unchanged following recovery liking as scrambled groups. Conclusions: The suppression of Fas receptor signalling subsequent RNAi therapy may represent an applicable strategy to decline hepatocyte damages and so NASH progression in mice.
Asunto(s)
Fumar Cigarrillos/efectos adversos , Dieta Occidental/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/terapia , ARN Interferente Pequeño/genética , Receptor fas/genética , Alanina Transaminasa/genética , Alanina Transaminasa/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Aspartato Aminotransferasas/genética , Aspartato Aminotransferasas/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos , FN-kappa B/genética , FN-kappa B/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-κB responses, causing increased production of TNFα, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFNß) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNß production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNß luciferase but not LPS-induced NF-κB luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-κB luciferase activation but was seen to augment LPS-induced IFNß luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKε-, TBK-1- and TRAF3-induced IFNß luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFNß signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.
Asunto(s)
Proteína de Dominio de Muerte Asociada a Fas/genética , Interferón beta/genética , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Receptor Toll-Like 4/genética , Receptor fas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Animales , Anticuerpos/farmacología , Proteína de Dominio de Muerte Asociada a Fas/inmunología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Interferón beta/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-8/genética , Interleucina-8/inmunología , Células Jurkat , Macrófagos/citología , Macrófagos/inmunología , Ratones , FN-kappa B/genética , FN-kappa B/inmunología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Células RAW 264.7 , Transducción de Señal , Células THP-1 , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/inmunología , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Receptor fas/antagonistas & inhibidores , Receptor fas/inmunologíaRESUMEN
Ischemic stroke-induced neuronal cell death results in the permanent disabling of brain function. Apoptotic mechanisms are thought to play a prominent role in neuronal injury and ample evidence implicates Fas signaling in mediating cell death. In this study, we describe the neuroprotective effects of a Fas-blocking peptide (FBP) that by obstructing Fas signaling in cerebral ischemia inhibits apoptosis. Using an intranasal administration route in a rat model of focal cerebral ischemia, we demonstrate that nose-to-brain delivery of FBP after middle cerebral artery occlusion (MCAO) surgery results in the delivery and retention of FBP in Fas-expressing ischemic areas of the brain. A single intranasal administration of 2 mg/kg FBP resulted in significantly reduced neuronal cell death by inhibiting Fas-mediated apoptosis leading to decreased infarct volumes, reduced neurologic deficit scores and recovery from cerebral ischemia. Intranasally delivered FBP might be a promising strategy for the treatment of cerebral ischemic stroke.
Asunto(s)
Apoptosis/efectos de los fármacos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Péptidos/farmacología , Receptor fas/antagonistas & inhibidores , Animales , Biomarcadores , Encéfalo/irrigación sanguínea , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/etiología , Línea Celular , Humanos , Inmunohistoquímica , Ratones , Péptidos/administración & dosificación , RatasRESUMEN
We investigated the effect of apigenin, a dietary flavonoid, on isoproterenol hydrochloride (ISO)-induced apoptotic signaling in cardiomyoblast H9C2 cells. The results showed that apigenin treatment (10 µM) prevented ISO (31.25 µM)-induced lipid peroxidative levels and antioxidants status in H9C2 cells. Furthermore, apigenin inhibited expression of inflammatory markers in ISO-treated cells. In addition, apigenin prevented ISO-induced DNA damage and apoptotic signaling through modulating the expression of Bax, caspase-3, -8 and -9, cytochrome c, and Fas proteins in H9C2 cells. It is concluded that apigenin prevents ISO-induced antioxidants depletion, oxidative DNA damage, inflammatory, and apoptotic signaling in H9C2 cells. Thus, the present results demonstrated that apigenin has a cardioprotective effect on cardiomyoblasts cells.
Asunto(s)
Antioxidantes/farmacología , Apigenina/farmacología , Apoptosis/efectos de los fármacos , Cardiotónicos/efectos adversos , Isoproterenol/efectos adversos , Mioblastos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/farmacología , Biomarcadores/metabolismo , Cardiotónicos/antagonistas & inhibidores , Línea Celular , Daño del ADN/efectos de los fármacos , Mediadores de Inflamación/agonistas , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Isoproterenol/antagonistas & inhibidores , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/inmunología , Mitocondrias Cardíacas/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/inmunología , Membranas Mitocondriales/metabolismo , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/inmunología , Mioblastos Cardíacos/metabolismo , Ratas , Proteína X Asociada a bcl-2/agonistas , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/metabolismo , Receptor fas/agonistas , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.
Asunto(s)
Apoptosis , Respuesta al Choque Térmico/genética , Glándulas Mamarias Animales/metabolismo , MicroARNs/metabolismo , Receptor fas/genética , Animales , Bovinos , Supervivencia Celular , Células Cultivadas , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Células HEK293 , Humanos , Glándulas Mamarias Animales/citología , MicroARNs/antagonistas & inhibidores , MicroARNs/fisiología , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
BACKGROUND: Apigetrin, a flavonoid found in many plant leaves and seeds, has been known to possess antimutagenic, anti-cancer, antioxidant and anti-inflammatory properties. Here, we are investigating the effect of the apigetrin on adipocytes differentiation in 3T3-L1 adipocytes, and elucidating the mechanism of its action. METHODS: Lipids accumulation was measured by Oil Red O staining and cell cycle was analyzed by flow cytometry. The antioxidant effect of apigetrin was evaluated against hydrogen peroxide. The expression of various genes, involved in adipogenesis and inflammation, was studied by real-time PCR. RESULTS: Our results showed that apigterin treatment inhibited significantly lipid accumulation without effect on cell viability at 100 µM, and it exerted the anti-adipogenic effect during the early stages of differentiation. Flow cytometry analysis showed that apigenin-7-O-glucoside (Ap7G) inhibited cell proliferation during mitotic clonal expansion and caused cell cycle delay. Quantitative PCR analysis revealed that the mRNA levels of C/EBP-α, PPAR-γ, SREBP-1c and FAS were suppressed after apigetrin treatment at 100 µM. Moreover, the mRNA level of pro-inflammatory genes (TNF-α and IL-6) were suppressed after apigterin treatment, at high concentration preadipocyte cells. CONCLUSION: Taken together, these results indicated that apigenin-7-O-glucoside inhibits adipogenesis of 3T3-L1 preadipocytes at early stage of adipogenesis.
Asunto(s)
Adipogénesis/efectos de los fármacos , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Apigenina/farmacología , Proteínas Potenciadoras de Unión a CCAAT/genética , PPAR gamma/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , PPAR gamma/antagonistas & inhibidores , PPAR gamma/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/antagonistas & inhibidores , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
A series of caspase inhibitors containing γ-amino acid moiety have been synthesized. A systemic study on their structure-activity relationship of anti-apoptotic cellular activity is presented. These efforts led to the discovery of compound 20o as a potent caspase inhibitor, which demonstrated preclinical ameliorating total bilirubin efficacy with a significantly improved pharmacokinetic profile.
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Aminoácidos/química , Inhibidores de Caspasas/química , Animales , Bilirrubina/sangre , Sitios de Unión , Caspasa 1/química , Caspasa 1/metabolismo , Inhibidores de Caspasas/farmacocinética , Inhibidores de Caspasas/uso terapéutico , Modelos Animales de Enfermedad , Semivida , Humanos , Células Jurkat , Hepatopatías/tratamiento farmacológico , Hepatopatías/patología , Ratones , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain.
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Apoptosis , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Receptor fas/metabolismo , Proteína ADAM17/antagonistas & inhibidores , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasas/metabolismo , Células Cultivadas , Desintegrinas/antagonistas & inhibidores , Desintegrinas/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Confocal , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Estaurosporina/farmacología , Inhibidor Tisular de Metaloproteinasa-3/genética , Receptor fas/antagonistas & inhibidores , Receptor fas/genéticaRESUMEN
Off-target effects (OTE) are an undesired side effect of RNA interference (RNAi) caused by partial complementarity between the targeting siRNA and mRNAs other than the gene to be silenced. The death receptor CD95 and its ligand CD95L contain multiple sequences that when expressed as either si- or shRNAs kill cancer cells through a defined OTE that targets critical survival genes. Death induced by survival gene elimination (DISE) is characterized by specific morphological changes such as elongated cell shapes, senescence-like enlarged cells, appearance of large intracellular vesicles, release of mitochondrial ROS followed by activation of caspase-2, and induction of a necrotic form of mitotic catastrophe. Using genome-wide shRNA lethality screens with eight different cancer cell lines, we recently identified 651 genes as critical for the survival of cancer cells. To determine whether the toxic shRNAs targeting these 651 genes contained shRNAs that kill cancer cell through DISE rather than by silencing their respective target genes, we tested all shRNAs in the TRC library derived from a subset of these genes targeting tumor suppressors (TS). We now report that only by monitoring the responses of cancer cells following expression of shRNAs derived from these putative TS it was possible to identify DISE-inducing shRNAs in five of the genes. These data indicate that DISE in general is not an undefined toxic response of cells caused by a random OTE but rather a specific cellular response with shared features that points at a specific biological function involving multiple genes in the genome.
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Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteína Ligando Fas/antagonistas & inhibidores , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Receptor fas/antagonistas & inhibidores , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Purpose: Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT1 and MT2) in 661W cells, then whether MEL can prevent H2O2-induced cell death, and last, through which pathway MEL confers protection. Methods: The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H2O2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H2O2-mediated cell death, a Fas/FasL antagonist was used before the exposure to H2O2. Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H2O2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Results: Both MEL receptors (MT1 and MT2) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H2O2-mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H2O2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H2O2, and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H2O2. Conclusions: The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H2O2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.
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Antioxidantes/farmacología , Caspasa 3/metabolismo , Proteína Ligando Fas/antagonistas & inhibidores , Peróxido de Hidrógeno/toxicidad , Melatonina/farmacología , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Receptor fas/antagonistas & inhibidores , Animales , Caspasa 3/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Inmunohistoquímica , Ratones , Microscopía Confocal , Oxidantes/toxicidad , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/genética , Receptor de Melatonina MT2/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Despite impressive clinical success, cancer immunotherapy based on immune checkpoint blockade remains ineffective in many patients due to tumoral resistance. Here we use the autochthonous TiRP melanoma model, which recapitulates the tumoral resistance signature observed in human melanomas. TiRP tumors resist immunotherapy based on checkpoint blockade, cancer vaccines or adoptive T-cell therapy. TiRP tumors recruit and activate tumor-specific CD8+ T cells, but these cells then undergo apoptosis. This does not occur with isogenic transplanted tumors, which are rejected after adoptive T-cell therapy. Apoptosis of tumor-infiltrating lymphocytes can be prevented by interrupting the Fas/Fas-ligand axis, and is triggered by polymorphonuclear-myeloid-derived suppressor cells, which express high levels of Fas-ligand and are enriched in TiRP tumors. Blocking Fas-ligand increases the anti-tumor efficacy of adoptive T-cell therapy in TiRP tumors, and increases the efficacy of checkpoint blockade in transplanted tumors. Therefore, tumor-infiltrating lymphocytes apoptosis is a relevant mechanism of immunotherapy resistance, which could be blocked by interfering with the Fas/Fas-ligand pathway.
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Inmunoterapia , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Animales , Apoptosis/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Proteína Ligando Fas/antagonistas & inhibidores , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Femenino , Humanos , Inmunoterapia/métodos , Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor/patología , Masculino , Melanoma Experimental/patología , Ratones , Ratones Transgénicos , Microambiente Tumoral/inmunología , Receptor fas/antagonistas & inhibidores , Receptor fas/inmunologíaRESUMEN
Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3'UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells.
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Antineoplásicos/metabolismo , Muerte Celular , Proteína Ligando Fas/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Receptor fas/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular , Humanos , Interferencia de ARNRESUMEN
Our previous study indicated that anti-Fas antibody/actinomycin D (AF/AD) induced apoptosis of human hepatocellular carcinoma Bel-7402 cells; however, crosstalk influence between P38MAPK and autophagy on mitochondria-mediated apoptosis induced by AF/AD in Bel-7402 cells remains unclear. Therefore, effect of AF/AD on apoptosis, autophagy, phosphorylated-P38MAPK (p-P38MAPK), and membrane potential (ΔΨm) with or without the P38MAPK inhibitor SB203580 or the autophagy inhibitor 3-methyladenine (3-MA) in Bel-7402 cells was investigated in the present study. The results showed that AF/AD resulted in induction of apoptosis concomitant with autophagy, upregulation of p-P38MAPK and autophagy-associated gene proteins (Atg5-Atg12 protein complex, Atg7, Atg10, Beclin-1, LC3 I, and LC3 II), and downregulation of ΔΨm in Bel-7402 cells. In contrast, SB203580 attenuated the effects of AF/AD in Bel-7402 cells. Furthermore, the findings also demonstrated that 3-MA inhibited the impact of AF/AD on autophagy, Atg5-Atg12 protein complex, Atg7, Atg10, Beclin-1, LC3 I, LC3 II, and ΔΨm, and promoted the influence of AF/AD on apoptosis and p-P38MAPK in Bel-7402 cells. Taken together, we conclude that crosstalk between P38MAPK and autophagy regulates mitochondria-mediated apoptosis induced by AF/AD in Bel-7402 cells.