RESUMEN

In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The effect of lyophilized latex (LL), its hydroethanolic extract (E80), triterpene (F-TRI)- and diterpene (F-DIT)-enriched fractions, along with six isolated phorbol esters from LL and phorbol 12-myristate 13-acetate (PMA) on J774A.1, THP-1, SK-MEL-28, and B16-F10 cell line viability were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The compounds were identified by 2D-NMR and HRESIMS. The effect of the LL, extract and fractions on cell viability was also assessed through a resazurin reduction assay. At 100 µg/ml, LL, and its fractions moderately inhibited J774A.1 (37.5-59.5%) and THP-1 (12.6-43.6%) metabolism. LL (IC50 70 µg/ml) and F-TRI (IC50 68 µg/ml) were barely more effective against B16-F10 cells, and only F-TRI exerted an inhibitory effect on SK-MEL-28 cells (IC50 66-75 µg/ml). The samples did not effectively inhibit THP-1 growth (IC50 69-87 µg/ml, assessed by MTT). B16-F10 was susceptible to PMA (IC50 53 µM) and two 12-phenylacetate esters (IC50 56-60 µM), while SK-MEL-28 growth was inhibited (IC50 58 µM) by one of these kinds of esters with an additional 4ß-deoxy structure. Synagrantol A (IC50 39 µM) was as effective as PMA (IC50 47 µM) in inhibiting J774A.1 growth in a dose-dependent manner. Furthermore, an in silico study with target receptors indicated a high interaction of the compounds with the PKC proteins. These results provide useful knowledge on the effect of tigliane-type diterpenes on tumor cell from the perspective of medicinal chemistry.

Asunto(s)

Euphorbia , Látex , Ésteres del Forbol , Euphorbia/química , Látex/química , Ésteres del Forbol/farmacología , Humanos , Ratones , Animales , Línea Celular Tumoral , Estructura Molecular , Extractos Vegetales/farmacología , Extractos Vegetales/química , Brasil , Monocitos/efectos de los fármacos , Fitoquímicos/farmacología , Fitoquímicos/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Diterpenos/farmacología , Diterpenos/aislamiento & purificación , Terpenos/farmacología , Terpenos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Acetato de Tetradecanoilforbol , Melanoma/tratamiento farmacológico

RESUMEN

In this research work, a series of 16 quinazoline derivatives bearing ibuprofen and an amino acid were designed as inhibitors of epidermal growth factor receptor tyrosine kinase domain (EGFR-TKD) and cyclooxygenase-2 (COX-2) with the intention of presenting dual action in their biological behavior. The designed compounds were synthesized and assessed for cytotoxicity on epithelial cancer cells lines (AGS, A-431, MCF-7, MDA-MB-231) and epithelial non-tumorigenic cell line (HaCaT). From this evaluation, derivative 6 was observed to exhibit higher cytotoxic potency (IC50) than gefitinib (reference drug) on three cancer cell lines (0.034 µM in A-431, 2.67 µM in MCF-7, and 3.64 µM in AGS) without showing activity on the non-tumorigenic cell line (>100 µM). Furthermore, assessment of EGFR-TKD inhibition by 6 showed a discreet difference compared to gefitinib. Additionally, 6 was used to conduct an in vivo anti-inflammatory assay using the 12-O-tetradecanoylphorbol-3-acetate (TPA) method, and it was shown to be 5 times more potent than ibuprofen. Molecular dynamics studies of EGFR-TKD revealed interactions between compound 6 and M793. On the other hand, one significant interaction was observed for COX-2, involving S531. The RMSD graph indicated that the ligand remained stable in 50 ns.

Asunto(s)

Aminoácidos , Antineoplásicos , Ciclooxigenasa 2 , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB , Ibuprofeno , Quinazolinas , Ibuprofeno/farmacología , Ibuprofeno/química , Ibuprofeno/síntesis química , Humanos , Quinazolinas/farmacología , Quinazolinas/química , Quinazolinas/síntesis química , Ciclooxigenasa 2/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Estructura-Actividad , Aminoácidos/química , Aminoácidos/farmacología , Aminoácidos/síntesis química , Estructura Molecular , Línea Celular Tumoral , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/síntesis química , Acetato de Tetradecanoilforbol/farmacología , Proliferación Celular/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Simulación del Acoplamiento Molecular , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antiinflamatorios/síntesis química , Supervivencia Celular/efectos de los fármacos

RESUMEN

Dermatitis is defined as a set of inflammatory diseases that affect the skin, with varied causes. Among the different types of dermatitis, contact dermatitis is the most prevalent. Although the current therapy is often effective, it is associated with adverse effects and the possibility of drug tolerance. N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline is a L-proline amino acid derivative found in the leaves of Sideroxylon obtusifolium, a species traditionally used to treat inflammatory diseases. The aim of this study was to investigate the topical anti-inflammatory effect of N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline (NMP) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritant contact dermatitis in mice. Topically administered NMP, at doses of 0.03 - 0.50 mg/ear, reduced TPA-induced ear edema and neutrophil migration, as evidenced by low tissue myeloperoxidase activity and verified by histological examination. In addition, NMP (0.06 mg/ear) reduced tissue levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, INF-γ and MCP-1) and of the anti-inflammatory cytokine IL-10, and reduced gene expression of TNF-α, IL-6 and IL-1ß increased by TPA. The data suggest that N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline acts as a topical anti-inflammatory agent that decreases the expression of inflammatory cytokines, making it useful for the treatment of skin inflammation. Further investigations are necessary for its development as a therapeutic agent.

Asunto(s)

Dermatitis por Contacto , Dermatitis , Sapotaceae , Ratones , Animales , Acetato de Tetradecanoilforbol/farmacología , Acetato de Tetradecanoilforbol/uso terapéutico , Irritantes/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6 , Dermatitis por Contacto/tratamiento farmacológico , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Dermatitis/tratamiento farmacológico , Citocinas

RESUMEN

Previous studies have demonstrated the role of γ-aminobutyric acid type B (GABAB) receptors in skin-related conditions and pain. However, most studies have focused on the main effects of GABAB on the central nervous system. Therefore, this study has aimed to determine the potential topical anti-inflammatory and anti-proliferative effects of baclofen cream in an inflammatory skin disease model. The effects of the baclofen cream were evaluated using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears. Histological and immunohistochemical evaluations were performed using an ear oedema assay. The effect of baclofen on keratinocyte proliferation was assessed in PAM212, the murine keratinocyte cell line. The results demonstrate that a single topical application of 5% baclofen, 7.5% baclofen, and 1% dexamethasone each inhibited acute TPA-induced ear oedema (58.94 ± 6.14%, 47.73 ± 11.26%, and 87.33 ± 4.59%, respectively). These results were confirmed by histological analysis. In the chronic model, baclofen (5%) and dexamethasone (1%) each inhibited ear oedema and the maximum inhibitory effect was reached at the end of the experiment (9th day of TPA application) with a percentage inhibition of 54.60 ± 6.15% for baclofen and 71.68 ± 3.45% for dexamethasone, when compared to the vehicle. These results were confirmed by histological analysis. Baclofen and dexamethasone also reduced proliferating cell nuclear antigen expression by 62.01 ± 6.65% and 70.42 ± 6.11%, respectively. However, baclofen did not inhibit keratinocyte proliferation in PAM212 cells. In conclusion, these results demonstrate that baclofen exhibits notable topical antiproliferative and anti-inflammatory properties and could be a potential therapeutic alternative for treating inflammatory and proliferative skin diseases.

Asunto(s)

Dermatitis , Enfermedades de la Piel , Animales , Ratones , Baclofeno/farmacología , Baclofeno/uso terapéutico , Agonistas de Receptores GABA-B/farmacología , Agonistas de Receptores GABA-B/uso terapéutico , Enfermedades de la Piel/tratamiento farmacológico , Antiinflamatorios/efectos adversos , Inflamación/tratamiento farmacológico , Dexametasona/uso terapéutico , Edema/inducido químicamente , Edema/tratamiento farmacológico , Edema/metabolismo , Acetato de Tetradecanoilforbol/uso terapéutico

RESUMEN

LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA1-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA1-Rab5 interaction and receptor internalization. LPA-induced LPA1-Rab9 interaction was only observed at 60 min, and LPA1-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA1-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA1-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA1 receptor internalization varies with the stimuli.

Asunto(s)

Transferencia Resonante de Energía de Fluorescencia , Receptores del Ácido Lisofosfatídico , Receptores del Ácido Lisofosfatídico/metabolismo , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Endosomas/metabolismo , Lisofosfolípidos/farmacología , Lisofosfolípidos/metabolismo

RESUMEN

Squamous cell carcinoma (SCC) is a non-melanoma skin cancer, with chronic sun exposure as the main risk factor. Excisional surgery is the most indicated treatment; however, patients can suffer functional, aesthetic, and psychological damage depending on the lesion site. Topical administration of 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-Tetradecanoylphorbol-13- acetate (TPA) induce to the appearance of benign skin tumors in mice, some of which develop into SCC. This protocol has been used to analyze the effects of many chemopreventive agents that may block or inhibit the mechanisms of action of chemical carcinogenesis. We compared the effects of chemopreventive agents in an induced skin carcinogenesis animal model. In the Scopus, PubMed, and EMBASE databases, we searched for manuscripts published between June 16, 2011, and June 16, 2021. We excluded studies conducted in vitro or on transgenic mice; in addition, studies without drug dosage, route of administration, or tumor incidence were excluded. We selected 26 studies and analyzed their main characteristics and the outcomes of tumorigenesis analysis. Most chemopreventive agents have shown excellent potential to inhibit the development of skin tumors. This review also discusses the standardization of studies in animal models to ensure better responses and future randomized clinical trials for cancer treatment and prevention.

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O carcinoma espinocelular cutâneo (CEC) é um câncer de pele não melanoma, com a exposição solar crônica como o principal fator de risco. A cirurgia excisional é o tratamento mais indicado; entretanto, os pacientes podem sofrer danos funcionais, estéticos e psicológicos dependendo do local da lesão. A administração tópica de 7,12-dimetilbenz[a]antraceno (DMBA) e 12-O- Tetradecanoilforbol-13-acetato (TPA) induz ao aparecimento de tumores cutâneos benignos em camundongos, alguns dos quais evoluíram para CEC. Este protocolo tem sido utilizado para analisar os efeitos de muitos agentes quimiopreventivos que podem bloquear ou inibir os mecanismos de ação da carcinogênese química. Comparamos os efeitos de agentes quimiopreventivos em um modelo animal que foi induzido à carcinogênese de pele. Nas bases de dados Scopus, PubMed e EMBASE, buscamos manuscritos publicados entre 16 de junho de 2011 e 16 de junho de 2021. Excluímos estudos realizados in vitro ou em camundongos transgênicos; além disso, estudos sem dosagem de drogas, via de administração ou incidência de tumores foram excluídos. Selecionamos 26 estudos e analisamos suas principais características e os resultados da análise da tumorigênese. A maioria dos agentes quimiopreventivos tem demonstrado excelente potencial para inibir o desenvolvimento de tumores cutâneos. Esta revisão também discute a padronização de estudos em modelos animais para garantir melhores respostas e futuros ensaios clínicos randomizados para tratamento e prevenção do câncer.

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El carcinoma de células escamosas (CCE) es un cáncer de piel no melanoma, cuyo principal factor de riesgo es la exposición crónica al sol. La cirugía de escisión es el tratamiento más indicado; sin embargo, los pacientes pueden sufrir daños funcionales, estéticos y psicológicos dependiendo de la localización de la lesión. La administración tópica de 7,12-dimetilbenz[a]antraceno (DMBA) y 12-O-Tetradecanoilforbol-13-acetato (TPA) inducen a la aparición de tumores cutáneos benignos en ratones, algunos de los cuales se convierten en CCE. Este protocolo se ha utilizado para analizar los efectos de muchos agentes quimiopreventivos que pueden bloquear o inhibir los mecanismos de acción de la carcinogénesis química. Comparamos los efectos de los agentes quimiopreventivos en un modelo animal de carcinogénesis cutánea inducida. En las bases de datos Scopus, PubMed y EMBASE, se buscaron los manuscritos publicados entre el 16 de junio de 2011 y el 16 de junio de 2021. Se excluyeron los estudios realizados in vitro o en ratones transgénicos; además, se excluyeron los estudios sin dosis de fármacos, vía de administración o incidencia tumoral. Se seleccionaron 26 estudios y se analizaron sus características principales y los resultados del análisis de la tumorigénesis. La mayoría de los agentes quimiopreventivos han mostrado un excelente potencial para inhibir el desarrollo de tumores cutáneos. Esta revisión también analiza la estandarización de los estudios en modelos animales para garantizar mejores respuestas y futuros ensayos clínicos aleatorios para el tratamiento y la prevención del cáncer.

Asunto(s)

Animales , Ratas , Neoplasias Cutáneas/tratamiento farmacológico , Quimioprevención , Antineoplásicos , Acetato de Tetradecanoilforbol , Modelos Animales , 9,10-Dimetil-1,2-benzantraceno/análisis , Carcinogénesis , Fitoquímicos

RESUMEN

This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, ß and δ contribute to ROS production while PKC ßII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1ß release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine proteases are inhibited, NETs are formed by citrullination through the PAD4 pathway. This review puts together results from the last 31 years of research on the effects of PMA on the neutrophil and proposes new insights on their interpretation.

Asunto(s)

Trampas Extracelulares , Neutrófilos , Actinas/metabolismo , Trampas Extracelulares/metabolismo , FN-kappa B/metabolismo , Neutrófilos/metabolismo , Elastasa Pancreática/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Serina Proteasas/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Neutrophil extracellular traps (NETs) were first reported as a microbicidal strategy for activated neutrophils. Through an immunologic response against several stimuli, neutrophils release their DNA together with proteins from granules, nucleus, and cytoplasm (e.g., elastase and myeloperoxidase). To date, NETs have been implicated in tissue damage during intense inflammatory processes, mainly when their release is dependent on oxygen radical generation. Flavonoids are antioxidant and anti-inflammatory agents; of these, quercetin is commonly found in our daily diet. Therefore, quercetin could exert some protective activity against tissue damage induced by NETs. In our in vitro assays, quercetin reduced NETs, myeloperoxidase (MPO), and elastase release from neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). The activity of these enzymes also decreased in the presence of quercetin. Quercetin also reduced the cytotoxic effect of NETs on alveolar cells (A549 cell line). Further, in silico assays indicated favorable interactions between quercetin and NET proteins (MPO and elastase). Overall, our results demonstrate that quercetin decreases deleterious cellular effects of NETs by reducing their release from activated neutrophils, and diminishing the enzymatic activity of MPO and elastase, possibly through direct interaction.

Asunto(s)

Trampas Extracelulares , Células A549 , Trampas Extracelulares/metabolismo , Humanos , Elastasa Pancreática/metabolismo , Peroxidasa , Quercetina/farmacología , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Reactive oxygen species (ROS) have been extensively studied in biology in the past years. This class of molecules can be derived from endogenous sources (e.g., phagocytic cells as neutrophils, eosinophils, monocytes, macrophages, and organelles as mitochondria and peroxisomes) and participate in physiological and pathological conditions. The beneficial and harmful effects of ROS depend on redox regulation, which establishes the balance between their production and the activity of antioxidant systems to prevent oxidative stress in vivo. Neutrophils are the immune effectors most well depicted with an intense oxidative burst in response to tissue inflammation. Several proteins and members of the galectin family are involved in this fine modulation of ROS production by neutrophils. Interestingly, studies have indicated that Galectin-1 (Gal-1) can up- or downregulate ROS production by neutrophils even when exposed to N-formyl-Met-Leu-Phe (fMLP) or Phorbol Myristate Acetate (PMA), both of which are potent neutrophil stimulants that trigger high levels of ROS production. Similarly, Galectin-3 (Gal-3) induces ROS in neutrophils from a sterile or nonsterile inflammatory environment, possibly creating a negative loop that could control ROS production. Besides, superoxide production is also induced by Galectin-8 (Gal-8) and Galectin-9 (Gal-9) in neutrophils but in a different manner. We describe herein the luminol and lucigenin-dependent chemiluminescence technique by using a luminometer as a method of assessment to measure ROS production by human neutrophils isolated and exposed to purified human recombinant Gal-1. The protocol described herein could be applied for the investigation of the role of other galectins in the modulation of ROS production by neutrophils.

Asunto(s)

Galectinas , Neutrófilos , Especies Reactivas de Oxígeno , Galectinas/genética , Galectinas/metabolismo , Galectinas/farmacología , Humanos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

OBJECTIVES: The objective of this work was to evaluate the anti-inflammatory activity of the aqueous extract, fractions and major compounds, which are isolated and identified from Passiflora edulis f. edulis (purple passion fruit) leaves extract. METHODS: For the isolation of the major compounds, reversed-phase chromatography and normal phase countercurrent chromatography were used. The separation was followed by thin layer chromatography and HPLC-DAD-ELSD. One-dimensional and two-dimensional NMR and ESI-TOF-MS/MS were used for structural elucidation. The anti-inflammatory activity was evaluated on a TPA multiple dose model of skin chronic inflammation in mice. Additionally, myeloperoxidase (MPO) and nitric oxide synthase (NOS) activity assays were performed as possible mechanisms of action studies. KEY FINDINGS AND CONCLUSIONS: The study of the butanolic fraction mainly showed the presence of saponins and flavonoids. Three minor flavonoids were detected; and three known saponins, cyclopassiflosides IX, XI and III were isolated and identified. This is the first unequivocal report of the presence of these compounds in P. edulis f. edulis leaves. The most favourable results of anti-inflammatory activity were obtained for the flavonoid-rich fraction. All the fractions and isolated compounds evaluated, presented high percentages of inhibition of nitric oxide synthase activity.

Asunto(s)

Antiinflamatorios/uso terapéutico , Flavonoides/uso terapéutico , Inflamación/prevención & control , Passiflora/química , Fitoterapia , Saponinas/uso terapéutico , Piel/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Cromatografía Líquida de Alta Presión , Femenino , Flavonoides/análisis , Flavonoides/farmacología , Inflamación/inducido químicamente , Inflamación/metabolismo , Ratones Endogámicos ICR , Óxido Nítrico Sintasa/antagonistas & inhibidores , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Hojas de la Planta/química , Saponinas/aislamiento & purificación , Saponinas/farmacología , Enfermedades de la Piel/metabolismo , Enfermedades de la Piel/prevención & control , Espectrometría de Masas en Tándem , Acetato de Tetradecanoilforbol/análogos & derivados

RESUMEN

An insult can trigger a protective response or even cell death depending on different factors that include the duration and magnitude of the event and the ability of the cell to activate protective intracellular signals, including inflammatory cytokines. Our previous work showed that the treatment of Lister Hooded rat retinal cell cultures with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, increases the survival of retinal ganglion cells (RGCs) kept in culture for 48 h after axotomy. Here we aim to analyze how PMA modulates the levels of TNF-α and IL-1ß (both key inflammatory mediators) and the impact of this modulation on RGCs survival. We hypothesize that the increase in RGCs survival mediated by PMA treatment depends upon modulation of the levels of IL-1ß and TNF-α. The effect of PMA treatment was assayed on cell viability, caspase 3 activation, TNF-α and IL-1ß release and TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) levels. PMA treatment increases IL-1ß and TNF-α levels in 15 min in culture and increases the release of both cytokines after 30 min and 24 h, respectively. Both IL-1ß and TNF-α levels decrease after 48 h of PMA treatment. PMA treatment also induces an increase in TNFRII levels while decreasing TNFRI after 24 h. PMA also inhibited caspase-3 activation, and decreased ROS production and EthD-1/calcein ratio in retinal cell cultures leading to an increase in cell viability. The neutralization of IL-1ß (anti-IL1ß 0,1ng/mL), the neutralization of TNF-α (anti-TNF-α 0,1ng/mL) and the TNF-α inhibition using a recombinant soluble TNFRII abolished PMA effect on RGCs survival. These data suggest that PMA treatment induces IL1ß and TNF-α release and modulation of TNFRI/TNFRII expression promoting RGCs survival after axotomy.

Asunto(s)

Proteína Quinasa C/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Animales Recién Nacidos , Axotomía/efectos adversos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Interleucina-1beta/metabolismo , Masculino , Cultivo Primario de Células , Ratas , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Células Ganglionares de la Retina/metabolismo , Inhibidores del Factor de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores

RESUMEN

Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.

Asunto(s)

Senescencia Celular , Metilación de ADN , Epigénesis Genética , Fibroblastos/enzimología , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Interleucina-1alfa/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Puntos de Control del Ciclo Celular , Proliferación Celular , Femenino , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Interleucina-1alfa/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Fluorescente , Papiloma/inducido químicamente , Papiloma/genética , Papiloma/metabolismo , Papiloma/patología , Fenotipo , Vías Secretoras , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol

RESUMEN

Neutrophil extracellular traps (NETs) are networks of decondensed chromatin loaded with antimicrobial peptides and enzymes produced against microorganisms or biochemical stimuli. Since their discovery, numerous studies made separately have revealed multiple triggers that induce similar NET morphologies allowing to classify them as lytic or non-lytic. However, the variability in NET composition depending on the inducer agent and the local milieu under similar conditions has been scarcely studied. In this work, a comparative study was conducted to evaluate structural and enzymatic divergences in NET composition induced by biochemical (phorbol myristate acetate [PMA] and hypochlorous acid [HOCl]) and microbiologic (Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa) stimuli, along with the presence of plasma from healthy donors or patients with systemic lupus erythematosus (SLE). The results showed a differential composition of DNA and the antimicrobial peptide cathelicidin (LL37) and a variable enzymatic activity (neutrophil elastase, cathepsin G, myeloperoxidase) induced by the different stimuli despite showing morphologically similar NETs. Additionally, SLE plasma´s presence increased DNA and LL37 release during NET induction independently of the trigger stimulus but with no enzymatic activity differences. This work provides new evidence about NET composition variability depending on the inducer stimulus and the local milieu.

Asunto(s)

Trampas Extracelulares/metabolismo , Lupus Eritematoso Sistémico/inmunología , Neutrófilos/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Candida albicans/inmunología , Estudios de Casos y Controles , Catelicidinas/análisis , Catelicidinas/metabolismo , Catepsina G/análisis , Catepsina G/metabolismo , Células Cultivadas , Trampas Extracelulares/inmunología , Voluntarios Sanos , Humanos , Ácido Hipocloroso/inmunología , Elastasa de Leucocito/análisis , Elastasa de Leucocito/metabolismo , Lupus Eritematoso Sistémico/sangre , Neutrófilos/inmunología , Peroxidasa/análisis , Peroxidasa/metabolismo , Cultivo Primario de Células , Pseudomonas aeruginosa/inmunología , Staphylococcus aureus/inmunología , Acetato de Tetradecanoilforbol/inmunología

RESUMEN

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

Asunto(s)

Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Lisofosfolípidos/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Receptores del Ácido Lisofosfatídico/genética , Animales , Fraccionamiento Celular , Membrana Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Estrenos/farmacología , Regulación de la Expresión Génica , Transporte Iónico/efectos de los fármacos , Isoxazoles/farmacología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Naftalenos/farmacología , Fosforilación/efectos de los fármacos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/antagonistas & inhibidores , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Cultivo Primario de Células , Propionatos/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Pirrolidinonas/farmacología , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal , Porcinos , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo

RESUMEN

Non-Steroidal Anti-inflammatory Drugs (NSAIDs) are some of the most prescribed medications for pain but the incidence of adverse effects -especially during chronic treatment- points out the requirement of new analgesics. In this study, we showed an efficient two-steps synthesis of diphenylamine-containing dipeptides consisting of a multicomponent process followed by a Buchwald-Hartwig cross-coupling reaction. We prepared 16 diphenylamine derivatives and evaluated their in vivo anti-inflammatory activity through an ear edema model using 12-O-tetradecanoylpholbol-13-acetate. Furthermore, the toxicity of the more potent compounds in the Artemia salina model and their cell viability using murine RAW 264.7 cells is reported. The fluorinated compound 10k becomes a reliable candidate since it reduced the TPA-induced edema to 92%, lacked cytotoxicity against murine macrophages, and had minimal toxicity in Artemia salina.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Artemia/efectos de los fármacos , Difenilamina/farmacología , Edema/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Supervivencia Celular/efectos de los fármacos , Difenilamina/síntesis química , Difenilamina/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Interacciones Hidrofóbicas e Hidrofílicas , Macrófagos/efectos de los fármacos , Ratones , Estructura Molecular , Células RAW 264.7 , Relación Estructura-Actividad , Acetato de Tetradecanoilforbol/análogos & derivados

RESUMEN

OBJECTIVE AND DESIGN: Biochanin A (BCA), a phytoestrogen, has various pharmacological properties. This study was conducted to compare BCA's therapeutic property against 17-ß estradiol replacement therapy in zymosan-induced arthritis (ZIA) in mice. Additionally, we further investigated in vitro the anti-inflammatory action on neutrophils. TREATMENT: Ovariectomized (OVX) and non-OVX mice were pretreated with BCA (1, 3 and 9 mg/kg) or estrogen (50 µg/kg) for 14 days prior to ZIA. Neutrophils were pretreated with BCA (1, 10 and 100 µM) for 1 h prior to phorbol 12-myristate 13-acetate. METHODS: Anti-inflammatory effects of BCA were evaluated by cellular infiltrate, paw edema and cytokine measurement. In vitro, apoptosis was assessed by morphology and flow cytometry. Neutrophil extracellular traps (NET) were determined by fluorescent microscopy and DNA release. Statistical differences were determined by one- or two-way ANOVA. RESULTS: BCA inhibited neutrophil accumulation, paw edema and proinflammatory cytokine (TNF-α and IFN-γ) and increased anti-inflammatory cytokines (IL-4 and IL-10) in OVX and non-OVX mice, similar to 17-ß estradiol replacement therapy. In vitro, BCA increased apoptosis and consequently reduced NETs. CONCLUSION: BCA has a notable anti-inflammatory effect, similar to 17-ß estradiol, and is especially effective for treatment of ZIA. These results suggest that BCA may be promising for the treatment of postmenopausal arthritis.

Asunto(s)

Antiinflamatorios no Esteroideos/uso terapéutico , Artritis/tratamiento farmacológico , Estradiol/uso terapéutico , Terapia de Reemplazo de Estrógeno , Genisteína/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Artritis/inducido químicamente , Citocinas/metabolismo , ADN/metabolismo , Edema/inducido químicamente , Edema/tratamiento farmacológico , Femenino , Ratones , Neutrófilos/efectos de los fármacos , Ovariectomía , Acetato de Tetradecanoilforbol , Zimosan

RESUMEN

Human embryonic kidney (HEK) 293 cells were co-transfected with plasmids for the expression of mCherry fluorescent protein-tagged FFA4 receptors and the enhanced green fluorescent protein-tagged Rab proteins involved in retrograde transport and recycling, to study their possible interaction through Förster Resonance Energy Transfer (FRET), under the action of agents that induce FFA4 receptor phosphorylation and internalization through different processes, i.e., the agonist, docosahexaenoic acid, the protein kinase C activator phorbol myristate acetate, and insulin. Data indicate that FFA4 receptor internalization varied depending on the agent that induced the process. Agonist activation (docosahexaenoic acid) induced an association with early endosomes (as suggested by interaction with Rab5) and rapid recycling to the plasma membrane (as indicated by receptor interaction with Rab4). More prolonged agonist stimulation also appears to allow the FFA4 receptors to interact with late endosomes (interaction with Rab9), slow recycling (interaction with Rab 11), and target to degradation (Rab7). Phorbol myristate acetate, triggered a rapid association with early endosomes (Rab5), slow recycling to the plasma membrane (Rab11), and some receptor degradation (Rab7). Insulin-induced FFA4 receptor internalization appears to be associated with interaction with early endosomes (Rab5) and late endosomes (Rab9) and fast and slow recycling to the plasma membrane (Rab4, Rab11). Additionally, we observed that agonist- and PMA-induced FFA4 internalization was markedly reduced by paroxetine, which suggests a possible role of G protein-coupled receptor kinase 2.

Asunto(s)

Ácidos Docosahexaenoicos/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Ácidos Docosahexaenoicos/farmacología , Células HEK293 , Humanos , Insulina/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Isoformas de Proteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Intense dance training leads to inflammation, which may impair the health and performance of the practitioners. Herein, we evaluate the effect of a single street dancing class on the profile of muscle enzymes, lymphocyte activation, and cell surface CD62L expression. We also investigated the correlation between muscle enzymes, adhesion molecules, and lymphocyte activation in dancers. Fifteen male participants (mean ± standard error: age 22.4 ± 1.08 years, body mass index 24.8 ± 0.69 kg/m2, body fat 12.3 ± 1.52%), who were amateur dancers, had blood samples collected previously and subsequent to a high-intensity street dance class. After the class, dancers showed an increase in total lymphocyte count (2.0-fold), creatine kinase (CK)-NAC (4.87%), and CK-MB (3.36%). We also observed a decrease (2.5-fold) in reactive oxygen species (ROS) produced by lymphocytes, under phorbol myristate acetate-stimulated environments. Following the dance class, CD62L expression in lymphocytes decreased (51.42%), while there was a negative correlation between the intensity of the exercise and CD62L expression (r = -0.73; p = 0.01). Lymphocytes were less responsive to stimuli after a single bout of street dancing, indicating transient immunosuppression.

Asunto(s)

Baile/fisiología , Selectina L/análisis , Activación de Linfocitos , Forma MB de la Creatina-Quinasa/sangre , Baile/educación , Frecuencia Cardíaca , Humanos , Inflamación , Activación de Linfocitos/efectos de los fármacos , Recuento de Linfocitos , Masculino , Especies Reactivas de Oxígeno/sangre , Acetato de Tetradecanoilforbol/farmacología , Adulto Joven

RESUMEN

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.

Asunto(s)

Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Ésteres del Forbol/farmacología , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Endosomas/efectos de los fármacos , Humanos , Proteínas Luminiscentes , Metoxamina/farmacología , Norepinefrina/farmacología , Oximetazolina/farmacología , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Proteínas de Unión al GTP rab5/efectos de los fármacos , Red trans-Golgi/efectos de los fármacos , Proteína Fluorescente Roja

RESUMEN

Sea cucumber body wall contains several naturally occurring bioactive components that possess health-promoting properties. Isostichopus badionotus from Yucatan, Mexico is heavily fished, but little is known about its bioactive constituents. We previously established that I. badionotus meal had potent anti-inflammatory properties in vivo. We have now screened some of its constituents for anti-inflammatory activity in vitro. Glycosaminoglycan and soluble protein preparations reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory responses in HaCaT cells while an ethanol extract had a limited effect. The primary glycosaminoglycan (fucosylated chondroitin sulfate; FCS) was purified and tested for anti-inflammatory activity in vivo. FCS modulated the expression of critical genes, including NF-ĸB, TNFα, iNOS, and COX-2, and attenuated inflammation and tissue damage caused by TPA in a mouse ear inflammation model. It also mitigated colonic colitis caused in mice by dextran sodium sulfate. FCS from I. badionotus of the Yucatan Peninsula thus had strong anti-inflammatory properties in vivo.

Asunto(s)

Antiinflamatorios , Sulfatos de Condroitina/aislamiento & purificación , Sulfatos de Condroitina/farmacología , Glicosaminoglicanos/aislamiento & purificación , Glicosaminoglicanos/farmacología , Otitis/tratamiento farmacológico , Pepinos de Mar/química , Extractos de Tejidos/aislamiento & purificación , Extractos de Tejidos/farmacología , Animales , Sulfatos de Condroitina/uso terapéutico , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Células HaCaT , Humanos , Técnicas In Vitro , México , Ratones , Otitis/inducido químicamente , Acetato de Tetradecanoilforbol/efectos adversos