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1.
Int J Mol Sci ; 25(7)2024 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-38612585

RESUMEN

Hypercortisolism is known to affect platelet function. However, few studies have approached the effect of exogenous cortisol on human platelets, and the results obtained are conflicting and unconvincing. In this study, the effect of exogenous cortisol on several parameters indicative of oxidative status in human platelets has been analysed. We have found that cortisol stimulates ROS production, superoxide anion formation, and lipid peroxidation, with these parameters being in strict correlation. In addition, cortisol decreases GSH and membrane SH-group content, evidencing that the hormone potentiates oxidative stress, depleting platelet antioxidant defence. The involvement of src, syk, PI3K, and AKT enzymes in oxidative mechanisms induced by cortisol is shown. The main sources of ROS in cells can include uncontrolled increase of NADPH oxidase activity and uncoupled aerobic respiration during oxidative phosphorylation. Both mechanisms seem to be involved in ROS formation induced by cortisol, as the NADPH oxidase 1 inhibitor 2(trifluoromethyl)phenothiazine, and rotenone and antimycin A, complex I and III inhibitor, respectively, significantly reduce oxidative stress. On the contrary, the NADPH oxidase inhibitor gp91ds-tat, malate and NaCN, complex II and IV inhibitor, respectively, have a minor effect. It is likely that, in human platelets, oxidative stress induced by cortisol can be associated with venous and arterial thrombosis, greatly contributing to cardiovascular diseases.


Asunto(s)
Hidrocortisona , Estrés Oxidativo , Humanos , Hidrocortisona/farmacología , Especies Reactivas de Oxígeno , Plaquetas , NADPH Oxidasas
2.
Int J Mol Sci ; 25(9)2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38731825

RESUMEN

Aminopyrazoles represent interesting structures in medicinal chemistry, and several derivatives showed biological activity in different therapeutic areas. Previously reported 5-aminopyrazolyl acylhydrazones and amides showed relevant antioxidant and anti-inflammatory activities. To further extend the structure-activity relationships in this class of derivatives, a novel series of pyrazolyl acylhydrazones and amides was designed and prepared through a divergent approach. The novel compounds shared the phenylamino pyrazole nucleus that was differently decorated at positions 1, 3, and 4. The antiproliferative, antiaggregating, and antioxidant properties of the obtained derivatives 10-22 were evaluated in in vitro assays. Derivative 11a showed relevant antitumor properties against selected tumor cell lines (namely, HeLa, MCF7, SKOV3, and SKMEL28) with micromolar IC50 values. In the platelet assay, selected pyrazoles showed higher antioxidant and ROS formation inhibition activity than the reference drugs acetylsalicylic acid and N-acetylcysteine. Furthermore, in vitro radical scavenging screening confirmed the good antioxidant properties of acylhydrazone molecules. Overall, the collected data allowed us to extend the structure-activity relationships of the previously reported compounds and confirmed the pharmaceutical attractiveness of this class of aminopyrazole derivatives.


Asunto(s)
Amidas , Antineoplásicos , Antioxidantes , Proliferación Celular , Hidrazonas , Pirazoles , Humanos , Pirazoles/química , Pirazoles/farmacología , Hidrazonas/química , Hidrazonas/farmacología , Hidrazonas/síntesis química , Antioxidantes/farmacología , Antioxidantes/química , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Amidas/química , Amidas/farmacología , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Células MCF-7 , Células HeLa
3.
Molecules ; 29(10)2024 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-38792163

RESUMEN

To further extend the structure-activity relationships (SARs) of 5-aminopyrazoles (5APs) and identify novel compounds able to interfere with inflammation, oxidative stress, and tumorigenesis, 5APs 1-4 have been designed and prepared. Some chemical modifications have been inserted on cathecol function or in aminopyrazole central core; in detail: (i) smaller, bigger, and more lipophilic substituents were introduced in meta and para positions of catechol portion (5APs 1); (ii) a methyl group was inserted on C3 of the pyrazole scaffold (5APs 2); (iii) a more flexible alkyl chain was inserted on N1 position (5APs 3); (iv) the acylhydrazonic linker was moved from position 4 to position 3 of the pyrazole scaffold (5APs 4). All new derivatives 1-4 have been tested for radical scavenging (DPPH assay), anti-aggregating/antioxidant (in human platelets) and cell growth inhibitory activity (MTT assay) properties. In addition, in silico pharmacokinetics, drug-likeness properties, and toxicity have been calculated. 5APs 1 emerged to be promising anti-proliferative agents, able to suppress the growth of specific cancer cell lines. Furthermore, derivatives 3 remarkably inhibited ROS production in platelets and 5APs 4 showed interesting in vitro radical scavenging properties. Overall, the collected results further confirm the pharmaceutical potentials of this class of compounds and support future studies for the development of novel anti-proliferative and antioxidant agents.


Asunto(s)
Antineoplásicos , Antioxidantes , Pirazoles , Humanos , Pirazoles/química , Pirazoles/farmacología , Pirazoles/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antioxidantes/farmacología , Antioxidantes/química , Relación Estructura-Actividad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estructura Molecular
4.
J Cell Biochem ; 124(1): 46-58, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36260649

RESUMEN

Reactive oxygen species (ROS) are known to regulate platelet activation. Since endocannabinoids behave as platelet agonists, we investigated the effect of two endocannabinoids, 2-arachidonoylglycerol (2AG) and anandamide (AEA) on the oxidative status of human platelets. We have demonstrated that 2AG and AEA stimulate ROS production, superoxide anion formation and lipid peroxidation. The effect is dose and time dependent and mainly occurs through the involvement of cannabinoid receptor 1 (CB1) since all tested parameters are greatly reduced by SR141716, the CB1 specific inhibitor. The specific inhibitor of cannabinoid receptor 2 (CB2) SR144528 produces a very small inhibition. The involvement of syk/PI3K/AKT/mTor pathway in oxidative stress induced by endocannabinoids is shown. Nicotinamide adenine dinucleotide phosphate oxidase seems to be poorly involved in the endocannabinoids effect. Concerning the aerobic metabolism, it has been demonstrated that endocannabinoids reduce the oxygen consumption and adenosine triphosphate synthesis, both in the presence of pyruvate + malate or succinate. In addition, endocannabinoids inhibit the activity of respiratory complexes II, III and IV and increase the activity of respiratory complex I. The endocannabinoids effect on aerobic metabolism seems to be also a CB1 mediated mechanism. Thus, in human platelets oxidative stress induced by endocannabinoids, mainly generated in the respiratory chain through the activation of complex I and the inhibition of complex II, III and IV, may lead to thrombotic events, contributing to cardiovascular diseases.


Asunto(s)
Plaquetas , Endocannabinoides , Humanos , Endocannabinoides/farmacología , Especies Reactivas de Oxígeno , Fosfatidilinositol 3-Quinasas , Alcamidas Poliinsaturadas/farmacología , Estrés Oxidativo , Receptores de Cannabinoides , Receptor Cannabinoide CB1
5.
Molecules ; 26(19)2021 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-34641279

RESUMEN

(1) Background: different previously synthesized pyrazoles and imidazo-pyrazoles showed interesting anti-angiogenic action, being able to interfere with ERK1/2, AKT and p38MAPK phosphorylation in different manners and with different potency; (2) Methods: here, a new small compound library, endowed with the same differently decorated chemical scaffolds, has been synthetized to obtain new agents able to inhibit different pathways involved in inflammation, cancer and human platelet aggregation. (3) Results: most of the new synthesized derivatives resulted able to block ROS production, platelet aggregation and p38MAPK phosphorylation both in platelets and Human Umbilical Vein Endothelial cells (HUVEC). This paves the way for the development of new agents with anti-angiogenic activity.


Asunto(s)
Antiinflamatorios/síntesis química , Imidazoles/química , Pirazoles/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antiinflamatorios/química , Antiinflamatorios/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fosforilación/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad
6.
Molecules ; 25(4)2020 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-32085423

RESUMEN

Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure-activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets.


Asunto(s)
Antiinflamatorios/farmacología , Pirazoles/farmacología , Especies Reactivas de Oxígeno/metabolismo , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Supervivencia Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/farmacología , Humanos , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Oxidación-Reducción , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/farmacología , Agregación Plaquetaria/efectos de los fármacos , Pirazoles/síntesis química , Pirazoles/química , Relación Estructura-Actividad
7.
Biol Cell ; 110(5): 97-108, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29537672

RESUMEN

BACKGROUND INFORMATION: Energy demand in human platelets is very high, to carry out their functions. As for most human cells, the aerobic metabolism represents the primary energy source in platelets, even though mitochondria are negligibly represented. Following the hypothesis that other structures could be involved in chemical energy production, in this work, we have investigated the functional expression of an extramitochondrial aerobic metabolism in platelets. RESULTS: Oximetric and luminometric analyses showed that platelets consume large amounts of oxygen and produce ATP in the presence of common respiring substrates, such as pyruvate + malate or succinate, although morphological electron microscopy analysis showed that these contain few mitochondria. However, evaluation of the anaerobic glycolytic metabolism showed that only 13% of consumed glucose was converted to lactate. Interestingly, the highest OXPHOS activity was observed in the presence of NADH, not a readily permeant respiring substrate for mitochondria. Also, oxygen consumption and ATP synthesis fuelled by NADH were not affected by atractyloside, an inhibitor of the adenine nucleotide translocase, suggesting that these processes may not be ascribed to mitochondria. Functional data were confirmed by immunofluorescence microscopy and Western blot analyses, showing a consistent expression of the ß subunit of F1 Fo -ATP synthase and COXII, a subunit of Complex IV, but a low signal of translocase of the inner mitochondrial membrane (a protein not involved in OXPHOS metabolism). Interestingly, the NADH-stimulated oxygen consumption and ATP synthesis increased in the presence of the physiological platelets agonists, thrombin or collagen. CONCLUSIONS: Data suggest that in platelets, aerobic energy production is mainly driven by an extramitochondrial OXPHOS machinery, originated inside the megakaryocyte, and that this metabolism plays a pivotal role in platelet activation. SIGNIFICANCE: This work represents a further example of the existence of an extramitochondrial aerobic metabolism, which can contribute to the cellular energy balance.


Asunto(s)
Plaquetas/fisiología , Metabolismo Energético , Consumo de Oxígeno , Adenosina Trifosfato/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Glucosa/metabolismo , Glucólisis , Voluntarios Sanos , Humanos , Ácido Láctico/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción
8.
J Cell Biochem ; 119(1): 876-884, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-28661046

RESUMEN

The objective of this study was to determine whether AMPK is activated by 2-arachidonoylglycerol (2-AG) and participates to the cytoskeleton control in human platelets. We found that 2-AG stimulates the AMPKα activation through a Ca2+ /Calmodulin-dependent pathway as the specific inhibition of the CaMKKß by STO-609 inhibits the AMPKα phosphorylation/activation. Moreover, the CaMKKß/AMPKα pathway activated by 2-AG is involved in the phosphorylation of cofilin, vasodilator stimulated phosphoprotein (VASP), and myosin light chain (MLCs). These proteins participate to actin cytoskeletal remodelling during aggregation. We found that the phosphorylation/activation inhibition of these proteins is associated with a significant reduction in actin polymerization, aggregation, ATP, and α-granule secretion. Finally, AMPKα activation, Cofilin, VASP, and MLCs phosphorylation are significantly reduced by SR141716, the specific inhibitor of type 1 cannabinoid (CB1) receptor, suggesting that the CB1 receptor is involved in the 2-AG effect. In conclusion, we have shown that the CaMKKß/AMPKα pathway is activated by 2-AG in human platelets and controls the phosphorylation of key proteins involved in actin polymerization and aggregation.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Ácidos Araquidónicos/farmacología , Plaquetas/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Endocannabinoides/farmacología , Glicéridos/farmacología , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Bencimidazoles/farmacología , Plaquetas/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Cicloheximida/farmacología , Humanos , Proteínas de Microfilamentos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Naftalimidas/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/metabolismo , Rimonabant , Transducción de Señal/efectos de los fármacos
9.
Biol Chem ; 398(12): 1335-1346, 2017 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-28779561

RESUMEN

We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the function of human platelets. We have found that WGA is more active than PHA in stimulating platelet activation/aggregation, while LCA has no effect. Studies on the mechanisms involved show that WGA and PHA induce phosphorylation/activation of PLCγ2 and increase [Ca2+]i. For the first time, it has been shown that Src/Syk pathway, the adapter protein SLP-76 and the exchange protein VAV, participate in the PLCγ2 activation by these lectins. Moreover WGA and PHA stimulate the PI3K/AKT pathway. PI3K, through its product phosphatidylinositol-3,4,5-trisphosphate activates Bruton's tyrosine kinase (BTK) and contributes to PLCγ2 activation. In conclusion, our findings suggest that PLCγ2 activation induced by WGA and PHA is regulated by Src/Syk and by PI3K/BTK pathways through their concerted action.


Asunto(s)
Fitohemaglutininas/farmacología , Lectinas de Plantas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Aglutininas del Germen de Trigo/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Fosfolipasa C gamma/metabolismo , Fitohemaglutininas/química , Lectinas de Plantas/química , Relación Estructura-Actividad , Aglutininas del Germen de Trigo/química
10.
J Cell Biochem ; 117(5): 1240-9, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26460717

RESUMEN

We demonstrated that in human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) decreased dose- and time-dependently cAMP intracellular levels. No effect on cAMP decrease induced by 2-AG was observed in the presence of the adenylate cyclase inhibitor SQ22536 as well in platelets pretreated with the thromboxane A2 receptor antagonist, SQ29548 or with aspirin, inhibitor of arachidonic acid metabolism through the cyclooxygenase pathway. An almost complete recovering of cAMP level was measured in platelets pretreated with the specific inhibitor of phosphodiesterase (PDE) 3A, milrinone. In platelets pretreated with LY294002 or MK2206, inhibitors of PI3K/AKT pathway, and with U73122, inhibitor of phospholipase C pathway, only a partial prevention was shown. cAMP intracellular level depends on synthesis by adenylate cyclase and hydrolysis by PDEs. In 2-AG-stimulated platelets adenylate cyclase activity seems to be unchanged. In contrast PDEs appear to be involved. In particular PDE3A was specifically activated, as milrinone reversed cAMP reduction by 2-AG. 2-AG enhanced PDE3A activity through its phosphorylation. The PI3K/AKT pathway and PKC participate to this PDE3A phosphorylation/activation mechanism as it was greatly inhibited by platelet pretreatment with LY294002, MK2206, U73122, or the PKC specific inhibitor GF109203X. Taken together these data suggest that 2-AG potentiates its power of platelet agonist reducing cAMP intracellular level.


Asunto(s)
Ácidos Araquidónicos/farmacología , Plaquetas/efectos de los fármacos , AMP Cíclico/metabolismo , Endocannabinoides/farmacología , Glicéridos/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa/farmacología , Adenilil Ciclasas/metabolismo , Plaquetas/citología , Plaquetas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes , Agonistas de Receptores de Cannabinoides/farmacología , Células Cultivadas , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Estrenos/farmacología , Ácidos Grasos Insaturados , Humanos , Hidrazinas/farmacología , Immunoblotting , Indoles/farmacología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Maleimidas/farmacología , Milrinona/farmacología , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Fosfodiesterasa 3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Agregación Plaquetaria/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Pirrolidinonas/farmacología , Receptores de Tromboxanos/antagonistas & inhibidores , Receptores de Tromboxanos/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo
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