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1.
J Pharmacol Exp Ther ; 374(3): 469-478, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32631869

RESUMO

The complex pathophysiology of sickle cell anemia (SCA) involves intravascular hemolytic processes and recurrent vaso-occlusion, driven by chronic vascular inflammation, which result in the disease's severe clinical complications, including recurrent painful vaso-occlusive episodes. Hydroxyurea, the only drug frequently used for SCA therapy, is a cytostatic agent, although it appears to exert nitric oxide/soluble guanylyl cyclase (sGC) modulating activity. As new drugs that can complement or replace the use of hydroxyurea are sought to further reduce vaso-occlusive episode frequency in SCA, we investigated the effects of the sGC agonists BAY 60-2770 (sGC activator) and BAY 41-2272 (sGC stimulator) in the presence or absence of hydroxyurea on SCA vaso-occlusive mechanisms and cell recruitment both ex vivo and in vivo. These agents significantly reduced stimulated human SCA neutrophil adhesive properties ex vivo in association with the inhibition of surface ß2-integrin activation. A single administration of BAY 60-2770 or BAY 41-2272 decreased tumor necrosis factor cytokine-induced leukocyte recruitment in a mouse model of SCA vaso-occlusion. Importantly, the in vivo actions of both agonists were significantly potentiated by the coadministration of hydroxyurea. Erythroid cell fetal hemoglobin (HbF) elevation is also a major goal for SCA therapy. BAY 41-2272 but not BAY 60-2770 at the concentrations employed significantly induced γ-globin gene transcription in association with HbF production in cultured erythroleukemic cells. In conclusion, sGC agonist drugs could represent a promising approach as therapy for SCA, for use either as stand-alone treatments or in combination with hydroxyurea. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that stimulators and activators of sGC are potent inhibitors of the adhesion and recruitment of leukocytes from humans and in mice with sickle cell anemia (SCA) and may represent a promising approach for diminishing vaso-occlusive episode frequency in SCA. Hydroxyurea, a drug already frequently used for treating SCA, was found to potentiate the beneficial effects of sGC agonists in in vivo studies, implying that these classes of compounds could be used alone or in combination therapy.


Assuntos
Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Hidroxiureia/farmacocinética , Guanilil Ciclase Solúvel/metabolismo , Animais , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Hemoglobina Fetal/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Células K562 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Piridinas/farmacologia , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/metabolismo , Vasodilatadores/farmacologia
2.
Chem Biol Interact ; 328: 109190, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32652078

RESUMO

BACKGROUND: Doxorubicin (DOX) administration decreases cardiac soluble guanylate cyclase (sGC) activity. We hypothesized that bypassing impaired NO-sGC-cGMP pathway resulting from the activation of oxidized and heme-free soluble guanylate cyclase (sGC) could be a therapeutic target for DOX-mediated cardiomyopathy (DOX-CM). The present study investigated the therapeutic roles and mechanism of BAY60-2770, an activator of oxidized sGC, in alleviating DOX-CM. METHODS: H9c2 cardiomyocytes were pretreated with BAY60-2770 followed by DOX. Cell viability and intracellular reactive oxygen species (ROS) were subsequently measured. To determine the role BAY60-2770 in mitochondrial ROS generation and mitochondrial membrane potential, we examined mitoSOX RED and TMRE fluorescence under DOX exposure. As animal experiments, rats were orally administered with 5 mg/kg of BAY60-2770 at 1 h prior to every DOX treatment and then assessed by echocardiography and apoptotic marker and autophagy. RESULTS: BAY60-2770 ameliorated cell viability and DOX-induced oxidative stress in H9c2 cells, which was mediated by PKG activation. Mitochondrial ROS and TMRE fluorescence were attenuated by BAY60-2770 in DOX-treated H9c2 cells. DOX-induced caspase-3 activation decreased after pretreatment with BAY60-2770 in vivo and in vitro. Echocardiography showed that BAY60-2770 significantly improved DOX-induced myocardial dysfunction. Autophagosome was increased by BAY60-2770 in vivo. CONCLUSIONS: BAY60-2770 appears to mitigate DOX-induced mitochondrial ROS, membrane potential loss, autophagy, and subsequent apoptosis, leading to protection of myocardial injury and dysfunction. These novel results highlighted the therapeutic potential of BAY60-2770 in preventing DOX-CM.


Assuntos
Autofagia/efeitos dos fármacos , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Cardiotoxicidade/patologia , Doxorrubicina/efeitos adversos , Hidrocarbonetos Fluorados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
3.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
4.
Int J Hematol ; 112(5): 725-727, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32557126

RESUMO

Chemotherapy is the mainstay of treatment for advanced pancreatic cancer however, due to possible myelotoxicity, it is used with caution in patients with thrombocytopenia, especially when severe. TPO-receptor agonists have been employed for chemotherapy-induced thrombocytopenia, however treatment with TPO-receptor agonists to allow chemotherapy in patients with inherited thrombocytopenia has not been reported so far. We report the first successful use of eltrombopag to prevent chemotherapy-induced thrombocytopenia in a patient with MHY9-related disorder and pancreatic cancer. Treatment with eltrombopag allowed to attain a safe and stable platelet count for several months sufficient to permit chemotherapy and to allow the patient to undergo endoscopic placement of a biliary stent with no bleeding complications.


Assuntos
Benzoatos/uso terapêutico , Hidrazinas/uso terapêutico , Cadeias Pesadas de Miosina , Neoplasias Pancreáticas/tratamento farmacológico , Pirazóis/uso terapêutico , Trombocitopenia/tratamento farmacológico , Trombocitopenia/genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Benzoatos/farmacologia , Perda Sanguínea Cirúrgica/prevenção & controle , Endoscopia do Sistema Digestório , Feminino , Humanos , Hidrazinas/farmacologia , Neoplasias Pancreáticas/cirurgia , Contagem de Plaquetas , Pirazóis/farmacologia , Receptores de Trombopoetina/agonistas , Trombocitopenia/sangue , Trombocitopenia/congênito
5.
Chem Biol Interact ; 325: 109088, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32360554

RESUMO

Osteoarthritis (OA) is one of the most common degenerative joint diseases in aging people. The activation of chondrocytes and their dysregulation are closely related to the pathogenesis of OA. GPR55 is an unique orphan G-receptor which binds to cannabinoids. In this study, we explored the role of GPR55 in advanced glycation end productions (AGEs)- induced chondrocytes activation in cultured cells. We showed that AGEs dose dependently induced GPR55 expression in ATDC5 chondrocytes. The blockage of GPR55 by its newly discovered antagonist-CID16020046 mitigated AGEs- induced increase in cellular ROS and decrease in antioxidant NRF2. Moreover, CID16020046 showed a dose-response suppressive effect on AGEs- induced expression of the major inflammatory mediators, including COX-2 and iNOS, and the production of NO and PGE2. CID16020046 also dose responsively inhibited AGEs- induced key effectors of cartilage degradation such as MMP-3 and MMP-13. In consequence, CID16020046 showed robust inhibition on AGEs- induced type II collagen degradation. Mechanistically, our data demonstrated that CID16020046 mediated GPR55 blockage ameliorated AGEs- induced NF-κB activation as revealed by its inhibition on IκBα, nuclear p65 translocation and NF-κB promoter activity. Collectively, our study demonstrates that GPR55 signaling mediates AGEs- induced chondrocyte activation, and the targeted blockage of GPR55 pathway could be therapeutic choice in the treatment of osteoarthritis.


Assuntos
Compostos Azabicíclicos/farmacologia , Benzoatos/farmacologia , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Receptores de Canabinoides/metabolismo , Linhagem Celular , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Proteólise/efeitos dos fármacos
6.
Invest Ophthalmol Vis Sci ; 61(5): 33, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32428234

RESUMO

Purpose: This study aimed to investigate the role and pathophysiological mechanism of ATP binding cassette transporter A1 (ABCA1) in regulating the IOP and aqueous humor outflow. Methods: ABCA1 expression was measured in trabecular meshwork samples obtained from patients with POAG and human donor eyes by Western blot. To further evaluate the functional significance of ABCA1, porcine angular aqueous plexus (AAP) cells, which are equivalent to human Schlemm's canal endothelial cells, were either treated with ABCA1 agonist GW3965 or transduced with lentivirus expressing ABCA1-shRNA. Transendothelial electrical resistance, protein expression, and nitric oxide (NO) concentration were measured. GW3965 was administered by intracameral injection. IOP and aqueous humor outflow facility were also measured. Results: ABCA1 expression was significantly higher in the trabecular meshwork tissue of patients with POAG compared with controls. ABCA1 upregulation in angular aqueous plexus cells decreased the transendothelial electrical resistance in the angular aqueous plexus monolayers accompanied by a 0.56-fold decrease in caveolin-1 expression and a 2.85-fold and 1.17-fold increase in endothelial NO synthase expression and NO concentration, respectively (n = 3, P < 0.05). Conversely, ABCA1 downregulation increased transendothelial electrical resistance and caveolin-1 expression and decreased endothelial NO synthase expression and NO production (n = 3, P < 0.05). GW3965 decreased IOP and significantly increased conventional outflow facility (P < 0.05). Conclusions: Regulation of aqueous humor outflow via the caveolin-1/endothelial NO synthase/NO pathway is a newly defined function of ABCA1 that is different from its traditional role in mediating cholesterol efflux. ABCA1 is a compelling, novel therapeutic candidate for the treatment of glaucoma and ocular hypertension.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/fisiologia , Caveolina 1/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Pressão Intraocular/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Animais , Humor Aquoso/fisiologia , Benzoatos/farmacologia , Benzilaminas/farmacologia , Western Blotting , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Glaucoma de Ângulo Aberto/cirurgia , Humanos , Lentivirus/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Malha Trabecular/metabolismo , Trabeculectomia , Transfecção
7.
Int J Mol Sci ; 21(4)2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32093330

RESUMO

In vitro chondrogenically differentiated mesenchymal stem cells (MSCs) have a tendency to undergo hypertrophy, mirroring the fate of transient "chondrocytes" in the growth plate. As hypertrophy would result in ossification, this fact limits their use in cartilage tissue engineering applications. During limb development, retinoic acid receptor (RAR) signaling exerts an important influence on cell fate of mesenchymal progenitors. While retinoids foster hypertrophy, suppression of RAR signaling seems to be required for chondrogenic differentiation. Therefore, we hypothesized that treatment of chondrogenically differentiating hMSCs with the RAR inverse agonist, BMS204,493 (further named BMS), would attenuate hypertrophy. We induced hypertrophy in chondrogenic precultured MSC pellets by the addition of bone morphogenetic protein 4. Direct activation of the RAR pathway by application of the physiological RAR agonist retinoic acid (RA) further enhanced the hypertrophic phenotype. However, BMS treatment reduced hypertrophic conversion in hMSCs, shown by decreased cell size, number of hypertrophic cells, and collagen type X deposition in histological analyses. BMS effects were dependent on the time point of application and strongest after early treatment during chondrogenic precultivation. The possibility of modifing hypertrophic cartilage via attenuation of RAR signaling by BMS could be helpful in producing stable engineered tissue for cartilage regeneration.


Assuntos
Benzoatos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/metabolismo , Estilbenos/farmacologia , Proteína Morfogenética Óssea 4/metabolismo , Condrogênese/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/patologia
8.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936485

RESUMO

Understanding of adipogenesis is important to find remedies for obesity and related disorders. In addition, it is also critical in bone disorders because there is a reciprocal relationship between adipogenesis and osteogenesis in bone micro-environment. Oxysterols are pro-osteogenic and anti-adipogenic molecules via hedgehog activation in pluripotent bone marrow stomal cells. However, no study has evaluated the role of specific oxysterols in C3H10T1/2 cells, which are a good cell model for studying osteogenesis and adipogenesis in bone-marrows. Thus, we investigated the effects of specific oxysterols on adipogenesis and expression of adipogenic transcripts in C3H10T1/2 cells. Treatment of cells with DMITro significantly induced mRNA expression of Pparγ. This induction was significantly inhibited by 25-HC. The expression of C/cepα, Fabp4 and Lpl was also inhibited by 25-HC. To determine the mechanism by which 25-HC inhibits adipogenesis, the effects of the hedgehog signalling pathway inhibitor, cyclopamine and CUR61414, were evaluated. Treatment of C3H10T1/2 cells with DMITro + cyclopamine or DMITro + CUR61414 for 96h did not modulate adipocyte differentiation; cyclopamine and CUR61414 did not reverse the inhibitory effects of 25-HC, suggesting that the canonical hedgehog signalling may not play a role in the anti-adipogenic effects of 25-HC in C3H10T1/2 cells. In addition, LXR agonist did not inhibit adipogenesis, but 25-HC strongly inhibits adipogenesis of C3H10T1/2 cells. Our observations showed that 25-HC was the most potent oxysterol in inhibiting adipogenesis and the expression of key adipogenic transcripts in C3H10T1/2 cells among the tested oxysterols, suggesting its potential application in providing an intervention in osteoporosis and obesity. We also report that the inhibitory effects of 25-HC on adipogenic differentiation in C3H10T1/2 cells are not mediated by hedgehog signaling and LXR.


Assuntos
Adipogenia/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Células-Tronco Pluripotentes/citologia , Adipogenia/genética , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Camundongos , Oxisteróis/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Fatores de Tempo
9.
Blood ; 135(12): 948-953, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31978223

RESUMO

Mutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.


Assuntos
Benzoatos/farmacologia , Predisposição Genética para Doença , Hidrazinas/farmacologia , Mutação , Pirazóis/farmacologia , Receptores de Trombopoetina/agonistas , Receptores de Trombopoetina/genética , Trombocitemia Essencial/genética , Alelos , Substituição de Aminoácidos , Linhagem Celular , Estudos de Associação Genética , Humanos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/metabolismo
10.
Arch Biochem Biophys ; 681: 108254, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31904362

RESUMO

Atherosclerosis is a commonplace cardiovascular disease which affects most people in old age. While its causes are currently poorly understood, continuous study is being performed in order to elucidate both the pathogenesis and treatment of this insidious disease. Atherosclerosis is presently thought to be linked to several factors such as endothelial dysfunction, monocyte adhesion to the intima of the artery, and increased oxidative stress. Oxidized low-density lipoprotein (ox-LDL), colloquially known as the "bad cholesterol", is known to play a critical role in the previously mentioned atherosclerotic processes. In this study, our goal was to elucidate the role of the lysophospholipid receptor G protein-coupled receptor 55 (GPR55) and its antagonist, the cannabinoid CID16020046, in endothelial dysfunction. While their existence and especially their role in atherosclerosis has only semi-recently been elucidated, a growing body of research has begun to link their interaction to antiatherosclerosis. In our research, we found CID16020046 to have distinct atheroprotective properties such as anti-inflammation, antioxidant, and inhibition of monocyte attachment to endothelial cells. While there was previously a small body of research regarding the potential of cannabinoids to treat or prevent atherosclerosis, studies on the treatment potential of CID16020046 were even fewer. Thus, this study is one of the first to explore the effects of cannabinoids in atherosclerosis. Our findings in the present study provide a strong argument for the use of CID16020046 in the treatment of atherosclerosis as well as a basis for further experimentation using cannabinoids as therapy against atherosclerosis.


Assuntos
Compostos Azabicíclicos/farmacologia , Benzoatos/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Inflamação/prevenção & controle , Lipoproteínas LDL/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Humanos , Inflamação/metabolismo , Receptores de Canabinoides/metabolismo
11.
Nitric Oxide ; 96: 20-28, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31940502

RESUMO

BACKGROUND: We test if inhaled nitric oxide (NO) attenuates platelet functional and metabolic hyper-reactivity in subjects with submassive pulmonary embolism (PE). METHODS: Participants with PE were randomized to either 50 ppm NO + O2 or O2 only for 24 h with blood sampling at enrollment and after treatment; results were compared with healthy controls. Platelet metabolic activity was assessed by oxygen consumption (basal and uncoupled) and reactivity was assessed with agonist-stimulated thromboelastography (TEG) and fluorometric measurement of agonist-stimulated cytosolic [Ca++] without and with pharmacological soluble guanylate (sGC) modulation. RESULTS: Participants (N = 38 per group) were well-matched at enrollment for PE severity, comorbidities as well as TEG parameters and platelet O2 consumption. NO treatment doubled the mean plasma [NO3-] (P < 0.001) indicating successful delivery, but placebo treatment produced no change. After 24 h, neither TEG nor O2 consumption parameters differed significantly between treatment groups. Platelet cytosolic [Ca++] was elevated with PE versus controls, and was decreased by treatment with cinaciguat (an sGC activator), but not riociguat (an sGC stimulator). Stimulated platelet lysate sGC activity was increased with PE compared with controls. CONCLUSIONS: In patients with acute submassive PE, despite evidence of adequate drug delivery, inhaled NO had no major effect on platelet O2 consumption or agonist-stimulated parameters on TEG. Pharmacological activation, but not stimulation, of sGC effectively decreased platelet cytosolic [Ca++], and platelet sGC activity was increased with PE, confirming the viability of sGC as a therapeutic target.


Assuntos
Plaquetas/efeitos dos fármacos , Óxido Nítrico/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Embolia Pulmonar/sangue , Administração por Inalação , Adulto , Benzoatos/farmacologia , Cálcio/metabolismo , Método Duplo-Cego , Ativadores de Enzimas/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/administração & dosagem , Embolia Pulmonar/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Guanilil Ciclase Solúvel/metabolismo
12.
Insect Mol Biol ; 29(1): 104-111, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31390480

RESUMO

Acetylation is an important, reversible posttranslational modification to a protein. In a previous study, we found that there were a large number of acetylated sites in various nutrient storage proteins of the silkworm haemolymph. In this study, we confirmed that acetylation can affect the stability of nutrient storage protein Bombyx mori apolipophorin-III (BmApoLp-III). First, the expression of BmApoLp-III could be upregulated when BmN cells were treated with the deacetylase inhibitor panobinostat (LBH589); similarly, the expression was downregulated when the cells were treated with the acetylase inhibitor C646. Furthermore, the increase in acetylation by LBH589 could inhibit the degradation and improve the accumulation of BmApoLp-III in BmN cells treated with cycloheximide and MG132 respectively. Moreover, we found that an increase in acetylation could decrease the ubiquitination of BmApoLp-III and vice versa; therefore, we predicted that acetylation could improve the stability of BmApoLp-III by competing for ubiquitination and inhibiting the protein degradation pathway mediated by ubiquitin. Additionally, BmApoLp-III had an antiapoptosis function that increased after LBH589 treatment, which might have been due to the improved protein stability after acetylation. These results have laid the foundation for further study on the mechanism of acetylation in regulating the storage and utilization of silkworm nutrition.


Assuntos
Apolipoproteínas/química , Bombyx/química , Proteínas de Insetos/química , Estabilidade Proteica/efeitos dos fármacos , Acetilação , Animais , Apolipoproteínas/metabolismo , Benzoatos/farmacologia , Bombyx/efeitos dos fármacos , Linhagem Celular , Cicloeximida/farmacologia , Proteínas de Insetos/metabolismo , Leupeptinas/farmacologia , Panobinostat/farmacologia , Pirazóis/farmacologia
13.
Nat Prod Res ; 34(6): 893-897, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30445863

RESUMO

This study aims to isolate the potential antiproliferative and cytotoxic compounds from ginkgo biloba sarcotestas (GBS) and investigates the underlying mechanism in human MDA-MB-231 and mouse 4T-1 triple-negative breast cancer cells. Our results showed that 2-Hydroxy-6-tridecylbenzoic acid was isolated by cytotoxicity-guided fractionation where different fractions were assessed using MTT assay against MDA-MB-231 and 4T-1 cells. Colony formation assay showed that 2-Hydroxy-6-tridecylbenzoic acid significantly inhibited cell proliferation. The inhibition was associated with the enhancement of cytochrome P450 (CYP) 1B1 expression in a dose- and time-dependent manner and no significant change of CYP1A1 expression by qPCR and Western blot assays in MDA-MB-231 and 4T-1 cells. The mechanism was further demonstrated by the activation of aryl hydrocarbon receptor (AhR) pathway with the upregulation of AhR, AhR nuclear translocator (ARNT) and AhR-dependent xenobiotic response elements (XRE) activity. These findings may have implications for development of anticancer agents containing 2-Hydroxy-6-tridecylbenzoic acid as functional additives.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzoatos/farmacologia , Proliferação de Células/efeitos dos fármacos , Ginkgo biloba/química , Extratos Vegetais/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Benzoatos/uso terapêutico , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Feminino , Humanos , Camundongos , Extratos Vegetais/uso terapêutico , Neoplasias de Mama Triplo Negativas/patologia
14.
Nat Prod Res ; 34(12): 1663-1668, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30470138

RESUMO

A new phenolic compound (1) and together with 12 known compounds-eight flavonoids (2 ∼ 9), two phenolic compounds (10 and 11) and two benzoic acid (12 and 13)-were isolated from Phedimus middendorffianus (Maxim.). The structures of all compound were determined on the basis of spectroscopic (MS and NMR) analyses. Compounds 4, 5, 7 and 11 ∼ 13 were showed anti-proliferative activities against MCF-7 than PC-3 cell line. Also compound 12 and 13 showed the significant cytotoxic activities against two cancer cell lines, PC-3 and MCF-7.


Assuntos
Antineoplásicos/isolamento & purificação , Benzoatos/isolamento & purificação , Flavonoides/isolamento & purificação , Fenóis/isolamento & purificação , Sedum/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzoatos/química , Benzoatos/farmacologia , Linhagem Celular Tumoral , Flavonoides/química , Flavonoides/farmacologia , Humanos , Células MCF-7/efeitos dos fármacos , Estrutura Molecular , Células PC-3/efeitos dos fármacos , Fenóis/química , Fenóis/farmacologia
15.
Surg Today ; 50(9): 974-983, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31720801

RESUMO

The success of liver surgery, including resection and transplantation, is largely dependent on the ability of the liver to regenerate. Despite substantial improvement in surgical techniques and perioperative care, one of the main concerns is post-hepatectomy liver failure and early allograft dysfunction, both of which are associated with impaired liver regeneration. Recent studies have demonstrated the positive role of platelets in promoting liver regeneration and protecting hepatocytes; however, the underlying mechanisms responsible for these effects are not fully understood. In this review, we updated the accumulated evidence of the role of platelets in promoting liver regeneration, with a focus on liver resection and liver transplantation. The goal of these studies was to support the clinical implementation of platelet agents, such as thrombopoietin receptor agonists, to augment liver regeneration after liver surgery. This "platelet therapy" may become a treatment choice for post-hepatectomy liver failure and early allograft dysfunction.


Assuntos
Plaquetas/fisiologia , Hepatectomia , Hepatócitos/fisiologia , Regeneração Hepática/fisiologia , Transplante de Fígado , Receptores de Trombopoetina/agonistas , Aloenxertos , Benzoatos/administração & dosagem , Benzoatos/farmacologia , Plaquetas/metabolismo , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Hidrazinas/administração & dosagem , Hidrazinas/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Falência Hepática/tratamento farmacológico , Falência Hepática/etiologia , Regeneração Hepática/efeitos dos fármacos , Transfusão de Plaquetas , Complicações Pós-Operatórias/tratamento farmacológico , Complicações Pós-Operatórias/etiologia , Disfunção Primária do Enxerto/tratamento farmacológico , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
J Steroid Biochem Mol Biol ; 198: 105558, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31783151

RESUMO

Liver X receptor (LXR) agonists have the potential to alleviate obesity related diseases, particularly atherosclerosis. However, LXRs are transcriptional regulators that induce de novo lipogenesis and lipid accumulation in hepatocytes which represents a serious adverse effect. In this work, we sought to characterize the LXR agonist GW3965 effects on fatty acid (FA) and phospholipid (PL) remodelling and the correlation with gene expression in order to better understand the underlying effects leading to hepatic pathology upon LXR activation. Human primary hepatocytes treated for 48 h with GW3965 were analysed for changes in lipid metabolism gene expression by qPCR, variations in the FA profile was evaluated by GC-FID and in PL profiles using thin layer chromatography, ESI-MS and MS/MS analysis. Changes in cell membrane biochemical properties were studied using bilayer models generated with CHARMM-GUI. ELOLV6 and SCD1 mRNA increase was consistent with higher C16:1 and C18:1n9 at the expense of C16:0 and C18:0. The reduction of C18:2n6 and increase in C20:2n6 was in agreement with ELOVL5 upregulation. Phosphatydilethanolamine (PE) levels tended to decrease and phosphatidylinositol to increase; although differences did not reach significance, they correlated with changes in AGXT2L1, CDS1 and LPIN1 mRNA levels that were increased. The overall effect of GW3965 on PEs molecular profiles was an increase of long-chain polyunsaturated FA chains and a decrease of C16/C18 saturated and monounsaturated FAs chains. Additionally, PC (32:1) and PC (34:2) were decreased, and PC (36:1) and PC (34:1) were increased. AGXT2L1 is an enzyme with strict substrate specificity for phosphoethanolamine, which is converted into ammonia in GW3965-treated hepatocytes and could explain the PE reduction. In summary, LXR activation by GW3965 targets PE biosynthesis and FA elongation/desaturation, which tends to decrease PE in relation to total PL levels, and remodelling of PC and PE molecular species. We identified the human AGXT2L1 gene as induced by LXR activation by both synthetic and endogenous agonist treatment. The increase in acetaldehyde-induced oxidative stress, and in the lipid species identified have the potential to enhance the inflammatory process and impair membrane function. Future studies should focus on inhibition of AGXT2L1 activity with the aim of reverting the steatosis induced by LXR activation.


Assuntos
Benzoatos/farmacologia , Benzilaminas/farmacologia , Hepatócitos/metabolismo , Lipidômica , Receptores X do Fígado/metabolismo , Fosfatidiletanolaminas/metabolismo , Transaminases/metabolismo , Acetaldeído/metabolismo , Animais , Células Cultivadas , Ácidos Graxos/metabolismo , Feminino , Glutationa/metabolismo , Hepatócitos/citologia , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Estresse Oxidativo , Fosfatidilcolinas/metabolismo , Ratos , Especificidade por Substrato
17.
J Pharmacol Exp Ther ; 372(1): 107-118, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31732698

RESUMO

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e., function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC50 value <10 nM. SIGNIFICANCE STATEMENT: To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in cystic fibrosis, AbbVie-Galapagos has developed ABBV-2222/GLPG2222, a novel, potent, and orally bioavailable C1 corrector of this protein. ABBV-2222/GLPG2222, which is currently in clinical trials, exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR and substantial improvements over the existing C1 correctors.


Assuntos
Benzoatos/farmacologia , Benzopiranos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Células Cultivadas , Cloretos/metabolismo , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células HEK293 , Humanos , Moduladores de Transporte de Membrana/farmacologia , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo
18.
Expert Opin Investig Drugs ; 29(1): 1-4, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31825681

RESUMO

Introduction: Paroxysmal supraventricular tachycardia (SVT) can be very bothersome and may potentially lead to considerable health-care utilization. Non-parenteral medication is currently unavailable for the rapid termination of paroxysmal SVT. However, an intranasal spray formulation of etripamil, a short-acting calcium-channel blocker, is under investigation as a convenient, safe, and rapidly efficacious means to terminate paroxysmal SVT.Areas covered: This review summarizes the clinical rationale, potential benefit, and clinical trials safety and efficacy data for the use of etripamil nasal spray to terminate paroxysmal SVT.Expert opinion: Based on the efficacy and tolerability demonstrated in phase 1 and 2 clinical trials, etripamil nasal spray is a potential convenient, safe, and effective means for patients to terminate paroxysmal SVT. It has the potential to improve quality of life, reduce health-care burden, and alter the current management paradigm for many patients with SVT. Further ongoing evaluation in ambulatory patients will help to determine its real-life practicality, safety, and effectiveness.


Assuntos
Benzoatos/administração & dosagem , Bloqueadores dos Canais de Cálcio/administração & dosagem , Sprays Nasais , Taquicardia Paroxística/tratamento farmacológico , Taquicardia Supraventricular/tratamento farmacológico , Benzoatos/efeitos adversos , Benzoatos/farmacologia , Bloqueadores dos Canais de Cálcio/efeitos adversos , Bloqueadores dos Canais de Cálcio/farmacologia , Humanos , Qualidade de Vida , Taquicardia Paroxística/fisiopatologia , Taquicardia Supraventricular/fisiopatologia , Fatores de Tempo
19.
Biochim Biophys Acta Mol Basis Dis ; 1866(4): 165314, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30412793

RESUMO

Osteoporosis is a silent systemic disease that causes bone deterioration, and affects over 10 million people in the US alone. This study was undertaken to develop a potential stem cell therapy for osteoporosis. We have isolated and expanded human dental pulp-derived stem cells (DPSCs), characterized them, and confirmed their multipotential differentiation abilities. Stem cells often remain quiescent and require activation to differentiate and function. Herein, we show that ferutinin activates DPSCs by modulating the Wnt/ß-catenin signaling pathway and key osteoblast-secreted proteins osteocalcin and collagen 1A1 both mRNA and protein levels. To confirm that ferutinin modulates the Wnt pathway, we inhibited glycogen synthase kinase 3 (GSK3) and found that protein expression patterns were similar to those found in ferutinin-treated DPSCs. To evaluate the role of ferutinin in epigenetic regulation of canonical Wnt signaling, the pathway molecules Wnt3a and Dvl3 were analyzed using chromatin immunoprecipitation (ChIP)-quantitative PCR approaches. We confirmed that active marks of both H3K9 acetylation and H3K4 trimethylation were significantly enhanced in the promoter sites of the WNT3A and DVL3 genes in DPSCs after addition of ferutinin. These data provide evidence that ferutinin activates and promotes osteogenic differentiation of DPSCs, and could be used as an inducer as a potentially effective stem cell therapy for osteoporosis.


Assuntos
Benzoatos/farmacologia , Cicloeptanos/farmacologia , Polpa Dentária/metabolismo , Epigênese Genética/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Sesquiterpenos/farmacologia , Células-Tronco/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Compostos Bicíclicos com Pontes/farmacologia , Polpa Dentária/citologia , Humanos , Células-Tronco/citologia
20.
Cells ; 9(1)2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861948

RESUMO

The thrombopoietin receptor agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated thrombocytopenia refractory to immunomodulatory and antiviral drugs. These effects were ascribed to the effects of eltrombopag on megakaryocytes. Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interfered with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and the addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after hematopoietic stem cell transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses, and synergistically increase the effects of ganciclovir, eltrombopag is also a drug-repurposing candidate for the treatment of therapy-refractory HCMV disease.


Assuntos
Benzoatos/farmacologia , Citomegalovirus/fisiologia , Fibroblastos/virologia , Hidrazinas/farmacologia , Quelantes de Ferro/farmacologia , Células-Tronco Mesenquimais/virologia , Pirazóis/farmacologia , Animais , Células Cultivadas , Citomegalovirus/efeitos dos fármacos , Sinergismo Farmacológico , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Ganciclovir/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Replicação Viral/efeitos dos fármacos
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