RESUMEN
OBJECTIVE: To compare effects of latanoprost, a topical prostaglandin analogue (PGA) commonly used to treat glaucoma and lens instability in dogs, and latanoprostene bunod, a novel PGA with a nitric oxide-donating moiety, on intraocular pressure (IOP) and pupil diameter (PD). ANIMALS STUDIED: Ten ophthalmologically normal Beagle dogs. PROCEDURES: Dogs were treated twice a day for 5 days in a randomly selected eye with either latanoprost or latanoprostene bunod. After a 6-week washout period, dogs were treated with the opposite drug. IOP and PD were measured at treatment times, at midday on days 1 and 5, and for 6 days post-treatment. RESULTS: Both drugs significantly decreased IOP and PD. At midday on day 5 of treatment, mean IOP in eyes treated with latanoprost was 4.5 mmHg lower than the fellow eye and 3.0 mmHg lower than the same eye at baseline, while mean IOP in eyes treated with latanoprostene bunod was 5.5 mmHg lower than the fellow eye and 3.6 mmHg lower than baseline. Mean PD was 0.94 mm in eyes treated with latanoprost and 0.76 mmHg in eyes treated with latanoprostene bunod. There was no significant difference between the two drugs for either parameter at that time point (p = .372 and .619, respectively, for IOP relative to control and to baseline; p = .076 for PD) or when analyzed longitudinally. Significant diurnal variation in PD was noted and may have implications for treatment of lens' instability. CONCLUSIONS: Latanoprost and latanoprostene bunod produce similar IOP reduction and miosis in normal canine eyes.
Asunto(s)
Enfermedades de los Perros , Glaucoma de Ángulo Abierto , Prostaglandinas F Sintéticas , Animales , Antihipertensivos/farmacología , Antihipertensivos/uso terapéutico , Enfermedades de los Perros/tratamiento farmacológico , Perros , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Glaucoma de Ángulo Abierto/veterinaria , Presión Intraocular , Latanoprost/farmacología , Latanoprost/uso terapéutico , Soluciones Oftálmicas/uso terapéutico , Prostaglandinas A/farmacología , Prostaglandinas A/uso terapéutico , Prostaglandinas F Sintéticas/farmacología , Prostaglandinas F Sintéticas/uso terapéutico , PupilaRESUMEN
Animals maintain homeostasis of cell numbers, constantly creating new cells and eliminating others. Programmed cell death, apoptosis, is a mechanism of cell elimination and it acts in many aspects of animal biology. Drawing on the biomedical background, several signals launch the apoptosis mechanisms, including prostaglandins (PGs). Based on this information, we posed the hypothesis that PGs similarly induce apoptosis in insect cell lines. We used three Spodoptera frugiperda cell lines, including two newly established, BCIRL-SfNS-0518B-YL derived from the central nervous system and BCIRL-Sf4FB-0614-SGS derived from fat body, and the commercially available Sf9 cells. Using a kinetic apoptosis kit, we found treating SfNS cells for 18 h with 15 or 20 µM PGA2 led to decreases in cell numbers, coupled with increased numbers of apoptotic and dead cells. Similar exposures to 10 µM PGA2 (24 h) led to substantial increases in apoptotic cells, confirmed by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay on a flow cytometer. The influence of PGA2 treatments increased with dosage, as we recorded about 20% apoptosis at 24 h post-PGA2 treatments (10 µM) and about 34% apoptosis at 24 h post-30 µM treatments. PGA2 treatments led to 10- to 30-fold increases in messenger RNAs (mRNAs) encoding apoptosis-specific caspases-1, -2, -3, and -5 at 12 h and 40- to 60-fold increases in mRNAs encoding caspases-1 and -2, 10-fold increases for caspases-3 and -5 at 24 h. These findings strongly support our hypothesis that PGs induce apoptosis in an insect cell line and confirm an additional PG action in insect biology.
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Caspasas , Prostaglandinas A/farmacología , Células Sf9/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Spodoptera/metabolismoRESUMEN
Disruption of the intracellular lipid balance leading to cholesterol accumulation is one of the features of cells that participate in the development of atherosclerotic lesions. Evidence form our laboratory indicates that anti-inflammatory cyclopentenone prostaglandins (cyPGs) of A- and J-family deviate lipid metabolism from the synthesis of cholesterol and cholesteryl esters to the synthesis of phospholipids in foam-cell macrophages. cyPGs possessing an α,ß-unsaturated cyclopentane ring are highly electrophilic substances able to promptly react with reactive cysteines of intracellular molecules through Michael addition. On the other hand, HMG-CoA reductase (HMGCR), the enzyme responsible for the rate-limiting step in cholesterol biosynthesis, presents critically reactive cysteines at the entry of catalytic domain, particularly Cys561, that could be target of cyPG inhibition. In the present study, we showed that cyPGs (but not other non-α,ß-unsaturated PGs) physically interact with HMGCR, in a dithiothreitol- and ß-mercaptoethanol-sensitive way, and block the activity of the catalytic subunit of the enzyme (IC50 for PGA2 = 0.17 µM). PGA2 inhibits HMGCR activity in cultured rat and human macrophages/macrophage-foam cells and leads to enhanced expression of HMGCR protein, as observed with statins. In cell culture models, PGA2 effectively inhibits the reductase at non-toxic doses (e.g., 1 µM) that block cell proliferation thus suggesting that part of the well-known antiproliferative effect of PGA2 may be due to its ability of blocking HMGCR activity, as cells cannot proliferate without a robust cholesterogenesis. Therefore, besides the powerfully anti-inflammatory and antiproliferative effects, the anticholesterogenic effects of PGA2 should be exploited in atherosclerosis therapeutics.
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Antiinflamatorios , Células Espumosas/enzimología , Hidroximetilglutaril-CoA Reductasas , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Prostaglandinas A , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Humanos , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Masculino , Prostaglandinas A/química , Prostaglandinas A/farmacología , Ratas , Ratas WistarRESUMEN
Prostaglandin (PG) A1 is a metabolic product of cyclooxygenase 2 (COX-2) that is potentially involved in regulating the development and progression of Alzheimer's disease (AD). PGA1 is a cyclopentenone (cy) PG characterized by the presence of a chemically reactive α,ß-unsaturated carbonyl. PGA1 is potentially involved in the regulation of multiple biological processes via Michael addition; however, the specific roles of PGA1 in AD remain unclear. TauP301S transgenic (Tg) mice were used as in vivo AD models, and neuroblastoma (N) 2a cells were used as an in vitro neuronal model. The PGA1-binding proteins were identified by HPLC-MS-MS after intracerebroventricular injection (i.c.v) of PGA1. Western blotting was used to determine tau phosphorylation in PGA1-treated Tg mice in the absence or in the presence of okadaic acid (OA), an inhibitor of protein phosphatase (PP) 2A. A combination of pull-down assay, immunoprecipitation, western blotting, and HPLC-MS-MS was used to determine that the PP2A scaffold subunit A alpha (PPP2R1A) is activated by the direct binding of PGA1 to cysteine 377. The effect of inhibiting tau hyperphosphorylation was tested in the Morris maze to determine the inhibitory effects of PGA1 on cognitive decline in tauP301S Tg mice. Incubation with N2a cells, pull-down assay, and mass spectrometry (MS) analysis revealed and indicated that PGA1 binds to more than 1000 proteins; some of these proteins are associated with AD and especially with tauopathies. Moreover, short-term administration of PGA1 in tauP301S Tg mice significantly decreased tau phosphorylation at Thr181, Ser202, and Ser404 in a dose-dependent manner. This effect was caused by the activation of PPP2R1A in tauP301S Tg mice. Importantly, PGA1 can form a Michael adduct with cysteine 377 of PPP2R1A, which is critical for the enzymatic activity of PP2A. Long-term treatment of tauP301S Tg mice with PGA1 activated PP2A and significantly reduced tau phosphorylation resulting in improvements in cognitive decline in tauP301S Tg mice. Our data provided new insight into the mechanisms of the ameliorating effects of PGA1 on cognitive decline in tauP301S Tg mice by activating PP2A via a mechanism involving the formation of a Michael adduct with cysteine 377 of PPP2R1A.
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Cisteína/metabolismo , Prostaglandinas A/farmacología , Proteína Fosfatasa 2/metabolismo , Proteínas tau/metabolismo , Animales , Línea Celular Tumoral , Disfunción Cognitiva/patología , Células HEK293 , Humanos , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Prostaglandinas A/administración & dosificación , Subunidades de Proteína/metabolismoRESUMEN
Prostaglandin (PG) A2, one of cyclopentenone PGs, is known to induce activation of apoptosis in various cancer cells. Although PGA2 has been reported to cause activation of apoptosis by altering the expression of apoptosis-related genes, the role of p53, one of the most critical pro-apoptotic genes, on PGA2-induced apoptosis has not been clarified yet. To address this issue, we compared the apoptosis in HCT116 p53 null cells (HCT116 p53-/-) to that in HCT116 cells containing the wild type p53 gene. Cell death induced by PGA2 was associated with phosphorylation of histone H2A variant H2AX (H2AX), activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase 1 in HCT116 cells. Induction of apoptosis in PGA2-treated cells was almost completely prevented by pretreatment with a pan-caspase inhibitor, z-VAD-Fmk, or an inhibitor of protein synthesis, cycloheximide. While PGA2 induced apoptosis in HCT116 cells, phosphorylation of p53 and transcriptional induction of p53-target genes such as p21WAF1, PUMA, BAX, NOXA, and DR5 occurred. Besides, pretreatment of pifithrin-α (PFT-α), a chemical inhibitor of p53's transcriptional activity, interfered with the induction of apoptosis in PGA2-treated HCT116 cells. Pretreatment of NU7441, a small molecule inhibitor of DNA-activated protein kinase (DNA-PK) suppressed PGA2-induced phosphorylation of p53 and apoptosis as well. Moreover, among target genes of p53, knockdown of DR5 expression by RNA interference, suppressed PGA2-induced apoptosis. In the meanwhile, in HCT116 p53-/- cells, PGA2 induced apoptosis in delayed time points and with less potency. Delayed apoptosis by PGA2 in HCT116 p53-/- cells was also associated with phosphorylation of H2AX but was not inhibited by either PFT-ï¡ or NU7441. Collectively, these results suggest the following. PGA2 may induce p53-dependent apoptosis in which DNA-PK activates p53, and DR5, a transcriptional target of p53, plays a pivotal role in HCT116 cells. In contrast to apoptosis in HCT116 cells, PGA2 may induce apoptosis in a fashion of less potency, which is independent of p53 and DNA-PK in HCT116 p53-/- cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Prostaglandinas A/farmacología , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Fosforilación/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Prostaglandins (PGs) are early and key contributors to chronic neurodegenerative diseases. As one important member of classical PGs, PGA1 has been reported to exert potential neuroprotective effects. However, the mechanisms remain unknown. To this end, we are prompted to investigate whether PGA1 is a useful neurological treatment for Alzheimer's disease (AD) or not. Using high-throughput sequencing, we found that PGA1 potentially regulates cholesterol metabolism and lipid transport. Interestingly, we further found that short-term administration of PGA1 decreased the levels of the monomeric and oligomeric ß-amyloid protein (oAß) in a cholesterol-dependent manner. In detail, PGA1 activated the peroxisome proliferator-activated receptor-gamma (PPARγ) and ATP-binding cassette subfamily A member 1 (ABCA1) signalling pathways, promoting the efflux of cholesterol and decreasing the intracellular cholesterol levels. Through PPARγ/ABCA1/cholesterol-dependent pathway, PGA1 decreased the expression of presenilin enhancer protein 2 (PEN-2), which is responsible for the production of Aß. More importantly, long-term administration of PGA1 remarkably decreased the formation of Aß monomers, oligomers, and fibrils. The actions of PGA1 on the production and deposition of Aß ultimately improved the cognitive decline of the amyloid precursor protein/presenilin1 (APP/PS1) transgenic mice.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Colesterol/metabolismo , Cognición/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , PPAR gamma/metabolismo , Prostaglandinas A/farmacología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Disfunción Cognitiva/metabolismo , Ratones , Ratones Transgénicos , Presenilina-1/genética , Presenilina-1/metabolismo , Prostaglandinas A/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).
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Animales , Bovinos , Antivirales/farmacología , Prostaglandinas A/farmacología , Diarrea Mucosa Bovina Viral/virología , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Antivirales/análisis , Prostaglandinas A/análisis , Replicación Viral/efectos de los fármacos , Diarrea Mucosa Bovina Viral/tratamiento farmacológico , Línea Celular , Virus de la Diarrea Viral Bovina/fisiología , Virus de la Diarrea Viral Bovina/genética , DiarreaRESUMEN
INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6µg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.
Asunto(s)
Alphavirus/efectos de los fármacos , Células Epiteliales/virología , Proteínas HSP70 de Choque Térmico/farmacología , Prostaglandinas A/farmacología , Replicación Viral/efectos de los fármacos , Alphavirus/ultraestructura , Animales , Antivirales/farmacología , Western Blotting , Bovinos , Línea Celular , Electroforesis en Gel de Poliacrilamida , Células Epiteliales/ultraestructura , Humanos , Microscopía Electrónica de TransmisiónRESUMEN
Abstract INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.
Asunto(s)
Humanos , Animales , Bovinos , Prostaglandinas A/farmacología , Replicación Viral/efectos de los fármacos , Alphavirus/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/farmacología , Células Epiteliales/virología , Antivirales/farmacología , Línea Celular , Western Blotting , Alphavirus/ultraestructura , Microscopía Electrónica de Transmisión , Electroforesis en Gel de Poliacrilamida , Células Epiteliales/ultraestructuraRESUMEN
Reactive electrophile species (RES), including prostaglandins, phytoprostanes and 12-oxo phytodienoic acid (OPDA), activate detoxification responses in plants and animals. However, the pathways leading to the activation of defense reactions related to abiotic or biotic stress as a function of RES formation, accumulation or treatment are poorly understood in plants. Here, the thiol-modification of proteins, including the RES-activated basic region/leucine zipper transcription factor TGA2, was studied. TGA2 contains a single cysteine residue (Cys186) that was covalently modified by reactive cyclopentenones but not required for induction of detoxification genes in response to OPDA or prostaglandin A1. Activation of the glutathione-S-transferase 6 (GST6) promoter was responsive to cyclopentenones but not to unreactive cyclopentanones, including jasmonic acid suggesting that thiol reactivity of RES is important to activate the TGA2-dependent signaling pathway resulting in GST6 activation We show that RES modify thiols in numerous proteins in vivo, however, thiol reactivity alone appears not to be sufficient for biological activity as demonstrated by the failure of several membrane permeable thiol reactive reagents to activate the GST6 promoter.
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Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Cisteína/metabolismo , Proteínas Nucleares/metabolismo , Aminoácidos/farmacología , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Ciclopentanos/farmacología , Escherichia coli , Ácidos Grasos Insaturados/farmacología , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas Nucleares/química , Oxilipinas/farmacología , Ácidos Pipecólicos/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Prostaglandinas A/farmacología , Proteínas Recombinantes/metabolismo , Plantones/efectos de los fármacos , Plantones/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10µg/mL).
Asunto(s)
Antivirales/farmacología , Diarrea Mucosa Bovina Viral/virología , Virus de la Diarrea Viral Bovina/efectos de los fármacos , Prostaglandinas A/farmacología , Animales , Antivirales/análisis , Diarrea Mucosa Bovina Viral/tratamiento farmacológico , Bovinos , Línea Celular , Diarrea , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina/fisiología , Prostaglandinas A/análisis , Replicación Viral/efectos de los fármacosRESUMEN
The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.
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Apoptosis/efectos de los fármacos , Membranas Intracelulares/metabolismo , Prostaglandinas A/farmacología , Proteínas ras/metabolismo , Animales , Calpaína/metabolismo , Línea Celular Tumoral , Cisteína/metabolismo , Embrión de Mamíferos/citología , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Membranas Intracelulares/efectos de los fármacos , Ratones , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.
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Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/metabolismo , Prostaglandinas A/metabolismo , Prostaglandinas A/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Prostaglandinas/metabolismo , Prostaglandinas/farmacología , Unión Proteica/fisiología , Ratas , Ratas WistarRESUMEN
Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit the replication of a wide variety of DNA and RNA viruses in different mammalian cell types. We investigated a new role for prostaglandin A1 (PGA1) in the inhibition of hepatitis C virus (HCV)-IRES-mediated translation. PGA1 exhibited dose-dependent inhibitory effects on HCV translation in HCV replicon cells. Furthermore, repetitive PGA1 treatment demonstrated the potential to safely induce the suppression of HCV translation. We also validated a new role for PGA1 in the inhibition of HCV-IRES-mediated translation by targeting cellular translation factors, including the small ribosomal subunit (40S) and eukaryotic initiation factors (eIFs). In pull-down assays, biotinylated PGA1 co-precipitated with the entire HCV IRES RNA/eIF3-40S subunit complex. Moreover, the interactions between PGA1 and the elongation factors and ribosomal subunit were dependent upon HCV IRES RNA binding, and the PGA1/HCV IRES RNA/eIF3-40S subunit complex inhibited HCV-IRES-mediated translation. The novel mechanism revealed in this study may aid in the search for more effective anti-HCV drugs.
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Hepacivirus/crecimiento & desarrollo , Hepacivirus/genética , Hepacivirus/metabolismo , Prostaglandinas A/metabolismo , Prostaglandinas A/farmacología , Replicón/efectos de los fármacos , Subunidades Ribosómicas Pequeñas/efectos de los fármacos , Línea Celular Tumoral , Factor 3 de Iniciación Eucariótica/metabolismo , Humanos , Sitios Internos de Entrada al Ribosoma , Biosíntesis de Proteínas/efectos de los fármacos , ARN Viral/genética , Replicón/fisiología , Subunidades Ribosómicas Pequeñas/metabolismoRESUMEN
Influenza A viruses (IAV) have the potential to cause devastating pandemics. In recent years, the emergence of new avian strains able to infect humans represents a serious threat to global human health. The increase in drug-resistant IAV strains underscores the need for novel approaches to anti-influenza chemotherapy. Herein we show that prostaglandin-A1 (PGA1) possesses antiviral activity against avian IAV, including H5N9, H7N1 and H1N1 strains, acting at a level different from the currently available anti-influenza drugs. PGA1 acts at postentry level, causing dysregulation of viral protein synthesis and preventing virus-induced disassembly of host microtubular network and activation of pro-inflammatory factor NF-κB. The antiviral activity is dependent on the presence of a cyclopentenone ring structure and is associated with activation of a cytoprotective heat shock response in infected cells. The results suggest that cyclopentenone prostanoids or prostanoids-derived molecules may represent a new tool to combat avian influenza virus infection.
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Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Prostaglandinas A/farmacología , Proteínas Virales/biosíntesis , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Pollos , Perros , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H7N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N1 del Virus de la Influenza A/fisiología , Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , FN-kappa B/metabolismo , Alveolos PulmonaresRESUMEN
Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A1 (PGA1) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin-proteasome pathway, we examined the effects of PGA1 on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA1 blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA1-treated 293T cells. The induction of HSP70 synthesis by PGA1 significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA1. Thus, we showed that PGA1 inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.
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Fármacos Anti-VIH/farmacología , Citidina Desaminasa/metabolismo , Infecciones por VIH/enzimología , VIH-1/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Prostaglandinas A/farmacología , Regulación hacia Arriba/efectos de los fármacos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Línea Celular , Citidina Desaminasa/genética , Regulación hacia Abajo/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteolisis/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Jasmonates and phytoprostanes are oxylipins that regulate stress responses and diverse physiological and developmental processes. 12-Oxo-phytodienoic acid (OPDA) and phytoprostanes are structurally related electrophilic cyclopentenones, which activate similar gene expression profiles that are for the most part different from the action of the cyclopentanone jasmonic acid (JA) and its biologically active amino acid conjugates. Whereas JA-isoleucine signals through binding to COI1, the bZIP transcription factors TGA2, TGA5, and TGA6 are involved in regulation of gene expression in response to phytoprostanes. Here root growth inhibition and target gene expression were compared after treatment with JA, OPDA, or phytoprostanes in mutants of the COI1/MYC2 pathway and in different TGA factor mutants. Inhibition of root growth by phytoprostanes was dependent on COI1 but independent of jasmonate biosynthesis. In contrast, phytoprostane-responsive gene expression was strongly dependent on TGA2, TGA5, and TGA6, but not dependent on COI1, MYC2, TGA1, and TGA4. Different mutant and overexpressing lines were used to determine individual contributions of TGA factors to cyclopentenone-responsive gene expression. Whereas OPDA-induced expression of the cytochrome P450 gene CYP81D11 was primarily regulated by TGA2 and TGA5, the glutathione S-transferase gene GST25 and the OPDA reductase gene OPR1 were regulated by TGA5 and TGA6, but less so by TGA2. These results support the model that phytoprostanes and OPDA regulate differently (i) growth responses, which are COI1 dependent but jasmonate independent; and (ii) lipid stress responses, which are strongly dependent on TGA2, TGA5, and TGA6. Identification of molecular components in cyclopentenone signalling provides an insight into novel oxylipin signal transduction pathways.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Nucleares/metabolismo , Oxilipinas/farmacología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Ciclopentanos/metabolismo , Ciclopentanos/farmacología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos Insaturados/farmacología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Isoleucina/metabolismo , Proteínas Nucleares/genética , Oxilipinas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Prostaglandinas A/farmacología , Transducción de Señal , Estrés Fisiológico , Transcripción Genética , TranscriptomaRESUMEN
We have previously reported that members of the NR4A family of orphan nuclear receptors can augment insulin's ability to stimulate glucose transport in adipocytes. In the current study, we endeavored to test for an insulin-sensitizing effect in muscle cells and to identify a potential transactivator. Lentiviral constructs were used to engineer both hyperexpression and shRNA silencing of NR4A3 in C2C12 myocytes. The NR4A3 hyper-expression construct led to a significant increase in glucose transport rates in the presence of maximal insulin while the NR4A3 knock-down exhibited a significant reduction in insulin-stimulated glucose transport rates. Consistently, insulin-mediated AKT phosphorylation was increased by NR4A3 hyperexpression and decreased following shRNA NR4A3 suppression. Then, we examined effects of prostaglandin A2 (PGA2) on insulin action and NR4A3 transactivation. PGA2 augmented insulin-stimulated glucose uptake in C2C12 myocytes and AKT phosphorylation after 12-h treatment, without significant effects on basal transport or basal AKT phosphorylation. More importantly, we demonstrated that PGA2 led to a greater improvement in insulin-stimulated glucose rates in NR4A3 overexpressing C2C12 myocytes, when compared with Lac-Z controls stimulated with insulin and PGA2. Moreover, the sensitizing effect of PGA2 was significantly diminished in NR4A3 knockdown myocytes compared to scramble controls. These results show for the first time that: (i) PGA2 augments insulin action in myocytes as manifested by enhanced stimulation of glucose transport and AKT phosphorylation; and (ii) the insulin sensitizing effect is dependent upon the orphan nuclear receptor NR4A3.
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Resistencia a la Insulina , Insulina/farmacología , Miembro 3 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Prostaglandinas A/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Células HEK293 , Humanos , Lentivirus/efectos de los fármacos , Lentivirus/metabolismo , Ratones , Células Musculares/citología , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Factores de Tiempo , Transducción GenéticaRESUMEN
The antiproliferative properties of cyclopentenone prostaglandins of the A-class have long been known. Considerable research has led to the elucidation of some of the mechanisms of action of these pleiotropic compounds. A-class prostaglandins or derived molecules (A-PG) may block the cell cycle, inhibit anti-apoptotic transcription factors, activate apoptotic cascades, induce a stress response and inhibit protein synthesis in a cell type-dependent manner. In addition, recent reports indicate that A-class PG may interact with various cellular detoxification systems and drug metabolizing enzymes used by cancer cells as mechanisms of chemoresistance. Some of these findings may open new perspectives for the development of strategies aimed at overcoming cancer resistance to widely used antitumor drugs. Here we outline the mechanisms of action for the antitumoral effects of PGA and related compounds, emphasizing those with impact on cellular defence systems which may contribute to cancer chemoresistance. The ability of A-PG to form covalent adducts with thiol groups in proteins and in glutathione is essential for their biological actions. Therefore, identification of the protein targets and elucidation of the interactions of A-PG with the glutathione biotransformation system will be critical for understanding the antitumoral effects of these compounds per se or through their ability to sensitize cancer cells towards other drugs.
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Resistencia a Antineoplásicos , Prostaglandinas A/farmacología , Glutatión/metabolismo , Humanos , Prostaglandinas A/química , Relación Estructura-ActividadRESUMEN
Prostaglandins (PGs) and other eicosanoids are oxygenated metabolites of arachidonic acid and two other C(20) polyunsaturated fatty acids. While most well studied in mammals, PGs exert important actions in insects and virtually all other invertebrates. We have been researching the mechanisms of PG actions in established insect cell lines and reported earlier that two PGs, PGA(1) and PGE(1), influence gene and protein expression in HzAM1 cells. Here we report on further experiments with three 2-series PGs, PGA(2), PGE(2) and PGF(2α). In separate experiments we treated cells with each of the three PGs for 12 and 24h and then analyzed cell lysates by 2-D electrophoresis. Analysis of the gels by Delta2D software showed that PGA(2) influenced expression of 60 proteins while PGE(2) and PGF(2α) treatments led to expression changes for only a few proteins. All spots representing changes in protein expression were processed for analysis by MALDI TOF/TOF mass spectrometry. Bioinformatic analysis of the resulting sequences yielded in silico identifications of all proteins. The apparent changes in some proteins were confirmed by quantitative PCR, which demonstrated that changes in protein expression were parallel to changes in mRNA expression. We assorted the proteins into functional categories, including 1/cell structure and function; 2/cell protection and immunity; 3/energetics and metabolism; 4/nucleotide processing; 5/protein action and processing and 6/signal transduction. These findings substantially extend our idea that one mechanism of PG actions in insect cells is the modulation of gene and protein expression.