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DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.
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ADN Mitocondrial , Efectores Tipo Activadores de la Transcripción , Animales , Humanos , Ratones , Adenina , Citosina , ADN Mitocondrial/genética , Edición Génica , ARN , Efectores Tipo Activadores de la Transcripción/metabolismo , Ingeniería de ProteínasRESUMEN
Inducing external strains on highly oriented thin films transferred onto mechanically deformable substrates enables a drastic enhancement of their ferroelectric, magnetic, and electronic performances, which cannot be achieved in films on rigid single crystals. Herein, the growth and diffusion behaviors of BiFeO3 thin films grown at various temperatures is reported on α-MoO3 layers of different thicknesses using sputtering. When the BiFeO3 thin films are deposited at a high temperature, significant diffusion of Fe into α-MoO3 occurs, producing the Fe1.89Mo4.11O7 phase and suppressing the maintenance of the 2D structure of the α-MoO3 layers. Although lowering the deposition temperature alleviates the diffusion yielding the survival of the α-MoO3 layer, enabling exfoliation, the BiFeO3 is amorphous and the formation of the Fe1.89Mo4.11O7 phase cannot be suppressed at the crystallization temperature. High-temperature-grown BiFeO3 thin films are successfully transferred onto flexible substrates via mechanical exfoliation by introducing a blocking layer of Au and measured the ferroelectric properties of the transferred films.
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Skin microbiota, such as acne-related Cutibacterium acnes, Staphylococcus aureus, and fungal Candida albicans, can form polymicrobial biofilms with greater antimicrobial tolerance to traditional antimicrobial agents and host immune systems. In this study, the phytopigment shikonin was investigated against single-species and multispecies biofilms under aerobic and anaerobic conditions. Minimum inhibitory concentrations of shikonin were 10 µg/mL against C. acnes, S. aureus, and C. albicans, and at 1-5 µg/mL, shikonin efficiently inhibited single biofilm formation and multispecies biofilm development by these three microbes. Shikonin increased porphyrin production in C. acnes, inhibited cell aggregation and hyphal formation by C. albicans, decreased lipase production, and increased hydrophilicity in S. aureus. In addition, shikonin at 5 or 10 µg/mL repressed the transcription of various biofilm-related genes and virulence-related genes in C. acnes and downregulated the gene expression levels of the quorum-sensing agrA and RNAIII, α-hemolysin hla, and nuclease nuc1 in S. aureus, supporting biofilm inhibition. In addition, shikonin prevented multispecies biofilm development on porcine skin, and the antimicrobial efficacy of shikonin was recapitulated in a mouse infection model, in which it promoted skin regeneration. The study shows that shikonin inhibits multispecies biofilm development by acne-related skin microbes and might be useful for controlling bacterial infections.
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Acné Vulgar , Antiinfecciosos , Naftoquinonas , Infecciones Estafilocócicas , Animales , Ratones , Candida albicans/genética , Staphylococcus aureus , Biopelículas , Antiinfecciosos/farmacologíaRESUMEN
VISTA (V domain immunoglobulin suppressor of T cell activation, also called PD-1H [programmed death-1 homolog]), a novel immune regulator expressed on myeloid and T lymphocyte lineages, is upregulated in mouse and human idiopathic pulmonary fibrosis (IPF). However, the significance of VISTA and its therapeutic potential in regulating IPF has yet to be defined. To determine the role of VISTA and its therapeutic potential in IPF, the expression profile of VISTA was evaluated from human single-cell RNA sequencing data (IPF Cell Atlas). Inflammatory response and lung fibrosis were assessed in bleomycin-induced experimental pulmonary fibrosis models in VISTA-deficient mice compared with wild-type littermates. In addition, these outcomes were evaluated after VISTA agonistic antibody treatment in the wild-type pulmonary fibrosis mice. VISTA expression was increased in lung tissue-infiltrating monocytes of patients with IPF. VISTA was induced in the myeloid population, mainly circulating monocyte-derived macrophages, during bleomycin-induced pulmonary fibrosis. Genetic ablation of VISTA drastically promoted pulmonary fibrosis, and bleomycin-induced fibroblast activation was dependent on the interaction between VISTA-expressing myeloid cells and fibroblasts. Treatment with VISTA agonistic antibody reduced fibrotic phenotypes accompanied by the suppression of lung innate immune and fibrotic mediators. In conclusion, these results suggest that VISTA upregulation in pulmonary fibrosis may be a compensatory mechanism to limit inflammation and fibrosis, and stimulation of VISTA signaling using VISTA agonists effectively limits the fibrotic innate immune landscape and consequent tissue fibrosis. Further studies are warranted to test VISTA as a novel therapeutic target for the IPF treatment.
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Fibrosis Pulmonar Idiopática , Humanos , Ratones , Animales , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/patología , Fibrosis , Bleomicina/farmacología , Inflamación/metabolismo , Fibroblastos/metabolismoRESUMEN
Coronaviruses are a major health care threat to humankind. Currently, the host factors that contribute to limit disease severity in healthy young patients are not well defined. Interferons are key antiviral molecules, especially type I and type III interferons. The role of these interferons during coronavirus disease is a subject of debate. Here, using mice that are deficient in type I (IFNAR1-/-), type III (IFNLR1-/-), or both (IFNAR1/LR1-/-) interferon signaling pathways and murine-adapted coronavirus (MHV-A59) administered through the intranasal route, we define the role of interferons in coronavirus infection. We show that type I interferons play a major role in host survival in this model, while a minimal role of type III interferons was manifested only in the absence of type I interferons or during a lethal dose of coronavirus. IFNAR1-/- and IFNAR1/LR1-/- mice had an uncontrolled viral burden in the airways and lung and increased viral dissemination to other organs. The absence of only type III interferon signaling had no measurable difference in the viral load. The increased viral load in IFNAR1-/- and IFNAR1/LR1-/- mice was associated with increased tissue injury, especially evident in the lung and liver. Type I but not type III interferon treatment was able to promote survival if treated during early disease. Further, we show that type I interferon signaling in macrophages contributes to the beneficial effects during coronavirus infection in mice. IMPORTANCE The antiviral and pathological potential of type I and type III interferons during coronavirus infection remains poorly defined, and opposite findings have been reported. We report that both type I and type III interferons have anticoronaviral activities, but their potency and organ specificity differ. Type I interferon deficiency rendered the mice susceptible to even a sublethal murine coronavirus infection, while the type III interferon deficiency impaired survival only during a lethal infection or during a sublethal infection in the absence of type I interferon signaling. While treatment with both type I and III interferons promoted viral clearance in the airways and lung, only type I interferons promoted the viral clearance in the liver and improved host survival upon early treatment (12 h postinfection). This study demonstrates distinct roles and potency of type I and type III interferons and their therapeutic potential during coronavirus lung infection.
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Infecciones por Coronavirus/inmunología , Interferón Tipo I/inmunología , Interferones/inmunología , Pulmón , Animales , Femenino , Pulmón/inmunología , Pulmón/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Interferón lambdaRESUMEN
Mandibular step osteotomy, performed for mandibular prognathism, is a difficult and time-consuming procedure. Virtual computer surgery and computer-aided design & computer-aided manufacturing have demonstrated accurate results in orthognathic surgery, though not used for mandibular step osteotomy yet. In this study, the authors report the case of a 21-year-old man with severe mandibular prognathism, with a reverse overjet of 12 mm. Step osteotomy, a modified method of body osteotomy, was planned virtually and performed using 3-dimensional (3D) printed titanium surgical guides and osteosynthesis plates, using computer-aided design & computer-aided manufacturing. At the 6-month postoperative follow-up, there were no notable complications, and normal healing was observed. Each segment was stably in place with the prefabricated plates. The proximal segments were not sagged medially or laterally. With 3D-printed surgical guides and osteosynthesis plates, intraoperative complications, such as injury to adjacent teeth and nerves, could be avoided. They also showed reasonable accuracy and helped reduce operative time and improve outcomes. Unlike surgical guides made of resin/polyamide, titanium surgical guides can be made thinner, which can reduce the extent of detachment. They also did not undergo any deterioration during the operation. Cutting guides and prefabricated plates using virtual surgical planning and 3D printing have many advantages, including reduced preoperative preparation time and operative time, reduced incidence of intraoperative complications, and improved outcomes. However, limitations still exist and further studies are required.
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Maloclusión de Angle Clase III , Procedimientos Quirúrgicos Ortognáticos , Prognatismo , Cirugía Asistida por Computador , Adulto , Diseño Asistido por Computadora , Humanos , Complicaciones Intraoperatorias , Masculino , Maloclusión de Angle Clase III/cirugía , Osteotomía Mandibular , Nylons , Procedimientos Quirúrgicos Ortognáticos/métodos , Impresión Tridimensional , Prognatismo/diagnóstico por imagen , Prognatismo/cirugía , Cirugía Asistida por Computador/métodos , Titanio , Adulto JovenRESUMEN
Staphylococcus aureus is one of the major pathogens responsible for antimicrobial resistance-associated death. S. aureus can secrete various exotoxins, and staphylococcal biofilms play critical roles in antibiotic tolerance and the persistence of chronic infections. Here, we investigated the inhibitory effects of 18 hydroquinones on biofilm formation and virulence factor production by S. aureus. It was found that 2,5-bis(1,1,3,3-tetramethylbutyl) hydroquinone (TBHQ) at 1 µg/mL efficiently inhibits biofilm formation by two methicillin-sensitive and two methicillin-resistant S. aureus strains with MICs of 5 µg/mL, whereas the backbone compound hydroquinone did not (MIC > 400 µg/mL). In addition, 2,3-dimethylhydroquinone and tert-butylhydroquinone at 50 µg/mL also exhibited antibiofilm activity. TBHQ at 1 µg/mL significantly decreased the hemolytic effect and lipase production by S. aureus, and at 5−50 µg/mL was non-toxic to the nematode Caenorhabditis elegans and did not adversely affect Brassica rapa seed germination or growth. Transcriptional analyses showed that TBHQ suppressed the expression of RNAIII (effector of quorum sensing). These results suggest that hydroquinones, particularly TBHQ, are potentially useful for inhibiting S. aureus biofilm formation and virulence.
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Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Antibacterianos/farmacología , Biopelículas , Exotoxinas/farmacología , Humanos , Hidroquinonas/farmacología , Lipasa , Meticilina/farmacología , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus , Factores de Virulencia/farmacologíaRESUMEN
Bacterial biofilm formation is a major cause of drug resistance and bacterial persistence; thus, controlling pathogenic biofilms is an important component of strategies targeting infectious bacterial diseases. Cinnamaldehyde (CNMA) has broad-spectrum antimicrobial and antibiofilm activities. In this study, we investigated the antibiofilm effects of ten CNMA derivatives and trans-CNMA against Gram-negative uropathogenic Escherichia coli (UPEC) and Gram-positive Staphylococcus aureus. Among the CNMA analogs tested, 4-nitrocinnamaldehyde (4-nitroCNMA) showed antibacterial and antibiofilm activities against UPEC and S. aureus with minimum inhibitory concentrations (MICs) for cell growth of 100 µg/mL, which were much more active than those of trans-CNMA. 4-NitroCNMA inhibited UPEC swimming motility, and both trans-CNMA and 4-nitroCNMA reduced extracellular polymeric substance production by UPEC. Furthermore, 4-nitroCNMA inhibited the formation of mixed UPEC/S. aureus biofilms. Collectively, our observations indicate that trans-CNMA and 4-nitroCNMA potently inhibit biofilm formation by UPEC and S. aureus. We suggest efforts be made to determine the therapeutic scope of CNMA analogs, as our results suggest CNMA derivatives have potential therapeutic use for biofilm-associated diseases.
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Escherichia coli Uropatógena , Acroleína/análogos & derivados , Antibacterianos/farmacología , Biopelículas , Matriz Extracelular de Sustancias Poliméricas , Pruebas de Sensibilidad Microbiana , Staphylococcus aureusRESUMEN
Mitochondria have emerged as important signaling organelles where intracellular perturbations are integrated and, consequently, intracellular signaling pathways are modulated to execute appropriate cellular functions. MAVS (mitochondrial antiviral signaling protein) represents such an example that functions as a platform molecule to mediate mitochondrial innate immune signaling. Recently, multimeric aggregation of MAVS has been identified as a key molecular process for its signaling. The underlying mechanisms to regulate this, however, are still incompletely understood. We hypothesized that PINK1 (PTEN-induced kinase 1) plays an important role in the regulation of multimeric MAVS aggregation and its consequent pathobiology. To test whether PINK1 interacts with MAVS, bimolecular fluorescence complementation analysis and IP were performed. RLH (RIG-I-like helicase) and NLRP3 inflammasome signaling were evaluated by in vitro assay. In vivo functional significance of PINK1 in the regulation of MAVS signaling was evaluated from both murine modeling of influenza viral infection and bleomycin-induced experimental pulmonary fibrosis, wherein MAVS plays important roles. Multimeric MAVS aggregation was induced by mitochondria dysfunction, and, during this event, the stabilized PINK1 interacted physically with MAVS and antagonized multimeric MAVS aggregation. Accordingly, the MAVS-mediated antiviral innate immune and NLRP3 inflammasome signaling were enhanced in PINK1 deficiency. In addition, in vivo studies revealed that MAVS-mediated pulmonary antiviral innate immune responses and fibrotic responses after bleomycin injury were enhanced in PINK1 deficiency. In conclusion, these results establish a new role of PINK1 in the regulation of MAVS signaling and the consequent pulmonary pathobiology.
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Proteínas Adaptadoras Transductoras de Señales/genética , Mitocondrias/metabolismo , Infecciones por Orthomyxoviridae/genética , Proteínas Quinasas/genética , Fibrosis Pulmonar/genética , Transducción de Señal/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Bleomicina/administración & dosificación , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inmunidad Innata , Inflamasomas/genética , Inflamasomas/inmunología , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Pulmón/inmunología , Pulmón/virología , Ratones , Ratones Noqueados , Mitocondrias/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Peroxisomas/inmunología , Peroxisomas/metabolismo , Agregado de Proteínas/genética , Unión Proteica , Proteínas Quinasas/deficiencia , Proteínas Quinasas/inmunología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Transducción de Señal/inmunologíaRESUMEN
Danger signals, or damage-associated molecular patterns (DAMPs), instigate mitochondrial innate immune responses wherein mitochondrial antiviral signaling protein (MAVS) functions as a key platform molecule to mediate them. The role of MAVS in the pathogenesis of idiopathic pulmonary fibrosis (IPF), however, has not yet been identified. Whether MAVS signalling can be modulated by currently existing drugs has also not been explored.We used an established model of pulmonary fibrosis to demonstrate that MAVS is a critical mediator of multiple DAMP signalling pathways and the consequent lung fibrosis after bleomycin-induced injury in vivoAfter bleomycin injury, MAVS expression was mainly observed in macrophages. Multimeric MAVS aggregation, a key event of MAVS signalling activation, was significantly increased and persisted in bleomycin-injured lungs. A proapoptotic BH3 mimetic, ABT-263, attenuated the expression of MAVS and its signalling and, consequently, the development of experimental pulmonary fibrosis. In contrast, the therapeutic effects of nintedanib and pirfenidone, two drugs approved for IPF treatment, were not related to the modulation of MAVS or its signalling. Multimeric MAVS aggregation was significantly increased in lungs from IPF patients as well.MAVS may play an important role in the development of pulmonary fibrosis, and targeting MAVS with BH3 mimetics may provide a novel and much needed therapeutic strategy for IPF.
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Fibrosis Pulmonar Idiopática , Antivirales/farmacología , Antivirales/uso terapéutico , Bleomicina/farmacología , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Pulmón , Transducción de SeñalRESUMEN
BACKGROUND: The in vivo evaluation of antipollution products has attracted attention due to increasing global pollution levels; however, it is expensive, time-consuming, and dangerous because of the harmful nature of fine dust. Therefore, this paper proposes an alternative in vitro assessment method and compares the fine dust blocking effectiveness of both methods for different antipollution products. MATERIALS AND METHODS: Initially, tests were conducted by spraying fine dust on human forearms and artificial leather without pretreatment for in vivo and in vitro samples, respectively. However, the same results were not obtained for both the methods. Therefore, we evaluated different leather conditions (color, drying time, and temperature) to determine the optimal artificial material for testing antipollution products before adopting beige artificial leather dried at 32°C for 30 minutes for further tests. RESULTS: The initial tests exhibited a significant difference (P < .05) between the two methods; however, the revised tests exhibited no significant difference (P > .05) between the two methods for either beige leather dried at room temperature (20°C-25°C) for 60 minutes or at 32°C for 30-60 minutes or white leather dried at 32°C for 60 min. Therefore, the in vitro method was deemed equivalent to the in vivo method. The effectiveness of fine dust blocking (P < .05) and the equivalence between the evaluation methods (P > .05) were confirmed for each antipollution product. CONCLUSION: The proposed method is economical, efficient, and safe, making it a novel and valid alternative for the evaluation of antipollution products.
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Polvo , Material Particulado , HumanosRESUMEN
Pentachlorophenol (PCP) is a widespread and persistent hydrophobic organic pollutant in the environment despite its restricted public use. Risk assessment of such hydrophobic organic compounds (HOCs) is challenging because sorption and volatilization issues during toxicity test often lead to inconsistent exposure concentration. Considering the hydrophobicity of the PCP, in this study, a passive dosing format was applied by adopting a silicone O-ring as a reservoir and evaluated its applicability on the determination of PCP on Daphnia magna. Results obtained with passive dosing method were compared with that of solvent spiking method. We hypothesized that the passive dosing method may provide more reliable and accurate toxicity results than conventional solvent spiking approach. As a result, the partition coefficient of PCP between methanol and a test medium (log KMeOH:ISO) was 2.1, which enabled the maintenance of reliable exposure concentration throughout the experiment. In the acute toxicity tests, passive dosing and solvent spiking showed similar EC50 values of 576 and 485 µg/L for 24 h, and 362 and 374 µg/L for 48 h, respectively, which overlap with EC50 values of previous studies. Altogether, both methods were suitable for the acute toxicity assessment of hydrophobic PCP.
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Daphnia/fisiología , Pentaclorofenol/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales , Interacciones Hidrofóbicas e Hidrofílicas , Medición de Riesgo , Solventes , Pruebas de Toxicidad AgudaRESUMEN
We found that the activated carbon-molecular oxygen system promotes the conversion of hydroquinones to benzoquinones, naphthoquinones, and anthraquinones, which are often found in natural products and pharmaceuticals. In particular, the one-pot synthesis of naphthoquinones and anthraquinones involving a Diels-Alder reaction is a useful protocol for this purpose.
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Antraquinonas/química , Benzoquinonas/química , Carbono/química , Naftoquinonas/química , Oxígeno/química , HidrogenaciónRESUMEN
Influenza viruses can result in significant lung injury with significant morbidity and mortality. In this study, we evaluated the impact of cigarette smoke (CS) exposure on the pulmonary fibroblastic response after influenza infection. We used a murine model in which animals were exposed to CS or room air and subsequently infected with H1N1 influenza virus. Inflammatory and fibrotic responses were measured at different time points after influenza infection. Primary fibroblasts were isolated from the lungs of mice and their characteristics were evaluated. Exposure to CS significantly increased the amount of collagen in the lungs of mice infected with influenza virus compared with the nonsmoking group at 30 days after infection. Furthermore, the presence of fibroblast-specific protein-positive cells increased in the lungs of influenza-infected mice that were exposed to CS compared with the infection-alone group. The smoking group also showed delays in weight recovery and higher cell counts in BAL fluid after infection. Active transforming growth factor ß1 levels in BAL fluid increased in both groups; however, CS-exposed mice had a later surge in active transforming growth factor ß1 (Day 24). Ex vivo cultures of lung-derived fibroblasts from CS-exposed mice with influenza infection showed rapid proliferation, increased expression of α-smooth muscle actin-stained stress fibers, and higher expression of growth factors compared with fibroblasts from room air-exposed lungs after infection. These results suggest that CS exposure changes the fibroblastic potential, leading to increased fibrosis after influenza infection.
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Fumar Cigarrillos/efectos adversos , Fibroblastos/inmunología , Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/complicaciones , Neumonía Viral/complicaciones , Fibrosis Pulmonar/etiología , Animales , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Neumonía Viral/patología , Neumonía Viral/virología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patologíaRESUMEN
Salinomycin, a monocarboxylic ionophore in Streptomyces albus, has been studied as an anti-cancer agent. However, we wondered whether salinomycin has another effect such as an anti-oxidant and hepatic protectant, because some chemical drugs treating human diseases were sometimes related with their toxic effects. Therefore, this study was conducted to examine the effects of salinomycin against oxidative stress and mitochondrial impairment in vivo and in vitro as well as the cellular mechanisms of action. In hepatocyte, salinomycin inhibited arachidonic acid (AA)â¯+â¯iron-induced apoptosis, mitochondrial dysfunction and ROS production. As a molecular mechanism, salinomycin induced autophagy through AMP-activated protein kinase (AMPK) activation, as assessed by the accumulation of acidic vesicle organelles, p62 and LC3-II. Moreover, these protective effects were blocked by AMPK inhibition, which indicates the importance of AMPK in the process of salinomycin's effects. In mice, oral administration of salinomycin protected against carbon tetrachloride (CCl4)-induced oxidative stress and liver injury, and also activated AMPK as well as autophagy-related proteins in the liver. Collectively, salinomycin had the ability to protect hepatocytes against AA+iron-induced reactive oxygen species production and mitochondrial dysfunction, as well as CCl4-induced liver injury. Although this beneficial effect was demonstrated under severe oxidative stress, this study showed that salinomycin protected the liver against the oxidative stress and liver damage through AMPK and autophagy, and suggest that salinomycin has a possibility to treat a broad range of diseases.
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Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia/efectos de los fármacos , Hepatopatías/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Piranos/farmacología , Animales , Apoptosis/efectos de los fármacos , Ácido Araquidónico/farmacología , Tetracloruro de Carbono/farmacología , Línea Celular Tumoral , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hepatopatías/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Antioxidant enzymes are related to oral diseases. We investigated the roles of heme oxygenase-1 (HO-1) and catalase in cadmium (Cd)-induced oxidative stress and the underlying molecular mechanism in oral cancer cells. Exposing YD8 cells to Cd reduced the expression levels of catalase and superoxide dismutase 1/2 and induced the expression of HO-1 as well as autophagy and apoptosis, which were reversed by N-acetyl-l-cysteine (NAC). Cd-exposed YD10B cells exhibited milder effects than YD8 cells, indicating that Cd sensitivity is associated with antioxidant enzymes and autophagy. Autophagy inhibition via pharmacologic and genetic modulations enhanced Cd-induced HO-1 expression, caspase-3 cleavage, and the production of reactive oxygen species (ROS). Ho-1 knockdown increased autophagy and apoptosis. Hemin treatment partially suppressed Cd-induced ROS production and apoptosis, but enhanced autophagy and CHOP expression, indicating that autophagy induction is associated with cellular stress. Catalase inhibition by pharmacological and genetic modulations increased Cd-induced ROS production, autophagy, and apoptosis, but suppressed HO-1, indicating that catalase is required for HO-1 induction. p38 inhibition upregulated Cd-induced phospho-JNK and catalase, but suppressed HO-1, autophagy, apoptosis. JNK suppression exhibited contrary results, enhancing the expression of phospho-p38. Co-suppression of p38 and JNK1 failed to upregulate catalase and procaspase-3, which were upregulated by JNK1 overexpression. Overall, the balance between the responses of p38 and JNK activation to Cd appears to have an important role in maintaining cellular homeostasis via the regulation of antioxidant enzymes and autophagy induction. In addition, the upregulation of catalase by JNK1 activation can play a critical role in cell protection against Cd-induced oxidative stress.
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Autofagia/efectos de los fármacos , Cadmio/toxicidad , Catalasa/metabolismo , Hemo-Oxigenasa 1/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acetilcisteína/farmacología , Antioxidantes/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Catalasa/genética , Línea Celular Tumoral , Hemo-Oxigenasa 1/genética , Humanos , Proteína Quinasa 8 Activada por Mitógenos/genética , Neoplasias de la Boca/inducido químicamente , Neoplasias de la Boca/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
In freshwater environments, aquatic organisms are generally exposed to mixtures of various chemical substances. In this study, we tested the toxicity of three organic UV-filters (ethylhexyl methoxycinnamate, octocrylene, and avobenzone) to Daphnia magna in order to evaluate the combined toxicity of these substances when in they occur in a mixture. The values of effective concentrations (ECx) for each UV-filter were calculated by concentration-response curves; concentration-combinations of three different UV-filters in a mixture were determined by the fraction of components based on EC25 values predicted by concentration addition (CA) model. The interaction between the UV-filters were also assessed by model deviation ratio (MDR) using observed and predicted toxicity values obtained from mixture-exposure tests and CA model. The results from this study indicated that observed ECxmix (e.g., EC10mix, EC25mix, or EC50mix) values obtained from mixture-exposure tests were higher than predicted ECxmix (e.g., EC10mix, EC25mix, or EC50mix) values calculated by CA model. MDR values were also less than a factor of 1.0 in a mixtures of three different UV-filters. Based on these results, we suggest for the first time a reduction of toxic effects in the mixtures of three UV-filters, caused by antagonistic action of the components. Our findings from this study will provide important information for hazard or risk assessment of organic UV-filters, when they existed together in the aquatic environment. To better understand the mixture toxicity and the interaction of components in a mixture, further studies for various combinations of mixture components are also required.
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Acrilatos/toxicidad , Cinamatos/toxicidad , Daphnia/efectos de los fármacos , Propiofenonas/toxicidad , Protectores Solares/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Organismos Acuáticos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones FarmacológicasRESUMEN
Recently, the interplay between autophagy and apoptosis has become an important factor in chemotherapy for cancer treatment. Inhibition of autophagy may be an effective strategy to improve the treatment of chemo-resistant cancer by consistent exposure to chemotherapeutic drugs. However, no reports have clearly elucidated the underlying mechanisms. Therefore, in this study, we assessed whether salinomycin, a promising anticancer drug, induces apoptosis and elucidated potential antitumor mechanisms in chemo-resistant prostate cancer cells. Cell viability assay, Western blot, annexin V/propidium iodide assay, acridine orange (AO) staining, caspase-3 activity assay, reactive oxygen species (ROS) production, and mitochondrial membrane potential were assayed. Our data showed that salinomycin alters the sensitivity of prostate cancer cells to autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the salinomycin-induced apoptosis. Notably, salinomycin decreased phosphorylated of AKT and phosphorylated mammalian target of rapamycin (mTOR) in prostate cancer cells. Pretreatment with LY294002, an autophagy and PI3K inhibitor, enhanced the salinomycin-induced apoptosis by decreasing the AKT and mTOR activities and suppressing autophagy. However, pretreatment with PD98059 and SB203580, an extracellular signal-regulated kinases (ERK), and p38 inhibitors, suppressed the salinomycin-induced autophagy by reversing the upregulation of ERK and p38. In addition, pretreatment with N-acetyl-l-cysteine (NAC), an antioxidant, inhibited salinomycin-induced autophagy by suppressing ROS production. Our results suggested that salinomycin induces apoptosis, which was related to ROS-mediated autophagy through regulation of the PI3K/AKT/mTOR and ERK/p38 MAPK signaling pathways.
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Autofagia/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Piranos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore. It was reported to anticancer activity on various cancer cell lines. In this study, salinomycin was examined on apoptosis and autophagy through generation of reactive oxygen species (ROS) in osteosarcoma U2OS cells. Apoptosis, autophagy, mitochondrial membrane potential (MMP) and ROS were analyzed using flow cytometry. Also, expressions of apoptosis- and autophagy-related proteins were determined by western blotting. As a result, salinomycin triggered apoptosis of U2OS cells, which was accompanied by change of MMP and cleavage of caspases-3 and poly (ADP-ribose) polymerase. And salinomycin increased the expression of autophagy-related protein and accumulation of acidic vesicular organelles (AVO). Salinomycin-induced ROS production promotes both apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-l-cysteine (NAC), a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of autophagy by 3-methyladenine (3 MA) enhanced the salinoymcin-induced apoptosis. Taken together, these results suggested that salinomycin-induced autophagy, as a survival mechanism, might be a potential strategy through ROS regulation in cancer therapy.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias Óseas/tratamiento farmacológico , Osteosarcoma/tratamiento farmacológico , Piranos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Humanos , Ionóforos/farmacología , Osteosarcoma/patologíaRESUMEN
BACKGROUND: Silibinin, a biologically active compound of milk thistle, has chemopreventive effects on cancer cell lines. Recently it was reported that silibinin inhibited tumor growth through activation of the apoptotic signaling pathway. Although various evidences showed multiple signaling pathways of silibinin in apoptosis, there were no reports to address the clear mechanism of ROS-mediated pathway in prostate cancer PC-3 cells. Several studies suggested that reactive oxygen species (ROS) play an important role in various signaling cascades, but the primary source of ROS was currently unclear. METHODS: The effect of silibinin was investigated on cell growth of prostate cell lines by MTT assay. We examined whether silibinin induced apoptosis through production of ROS using flow cytometry. Expression of apoptosis-, endoplasmic reticulum (ER)-related protein and gene were determined by western blotting and RT-PCR, respectively. RESULTS: Results showed that silibinin triggered mitochondrial ROS production through NOX4 expression and finally led to induce apoptosis. In addition, mitochondrial ROS caused ER stress through disruption of Ca(2+) homeostasis. Co-treatment of ROS inhibitor reduced the silibinin-induced apoptosis through the inhibition of NOX4 expression, resulting in reduction of both Ca(2+) level and ER stress response. CONCLUSIONS: Taken together, silibinin induced mitochondrial ROS-dependent apoptosis through NOX4, which is associated with disruption of Ca(2+) homeostasis and ER stress response. Therefore, the regulation of NOX4, mitochondrial ROS producer, could be a potential target for the treatment of prostate cancer.