RESUMO
Glioblastoma (GBM) is the most aggressive and lethal among the primary brain tumors, with a low survival rate and resistance to radio and chemotherapy. The P2Y12 is an adenosine diphosphate (ADP) purinergic chemoreceptor, found mainly in platelets. In cancer cells, its activation has been described to induce proliferation and metastasis. Bearing in mind the need to find new treatments for GBM, this study aimed to investigate the role of the P2Y12R in the proliferation and migration of GBM cells, as well as to evaluate the expression of this receptor in patients' data obtained from the TCGA data bank. Here, we used the P2Y12R antagonist, ticagrelor, which belongs to the antiplatelet agent's class. The different GBM cells (cell line and patient-derived cells) were treated with ticagrelor, with the agonist, ADP, or both, and the effects on cell proliferation, colony formation, ADP hydrolysis, cell cycle and death, migration, and cell adhesion were analyzed. The results showed that ticagrelor decreased the viability and the proliferation of GBM cells. P2Y12R antagonism also reduced colony formation and migration potentials, with alterations on the expression of metalloproteinases, and induced autophagy in GBM cells. Changes were observed at the cell cycle level, and only the U251 cell line showed a significant reduction in the ADP hydrolysis profile. TCGA data analysis showed a higher expression of P2Y12R in gliomas samples when compared to the other tumors. These data demonstrate the importance of the P2Y12 receptor in gliomas development and reinforce its potential as a pharmacological target for glioma treatment.
Assuntos
Glioblastoma , Humanos , Ticagrelor/metabolismo , Ticagrelor/farmacologia , Difosfato de Adenosina/metabolismo , Glioblastoma/tratamento farmacológico , Plaquetas , Autofagia , Proliferação de Células , Receptores Purinérgicos P2Y12/metabolismo , Antagonistas do Receptor Purinérgico P2Y/metabolismoRESUMO
Gliomas are the most common malignant brain tumors in adults, characterized by a high proliferation and invasion. The tumor microenvironment is rich in growth-promoting signals and immunomodulatory pathways, which increase the tumor's aggressiveness. In response to hypoxia and glioma therapy, the amounts of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) strongly increase in the extracellular space, and the purinergic signaling is triggered by nucleotides' interaction in P2 receptors. Several cell types are present in the tumor microenvironment and can facilitate tumor growth. In fact, tumor cells can activate platelets by the ADP-P2Y12 engagement, which plays an essential role in the cancer context, protecting tumors from the immune attack and providing molecules that contribute to the growth and maintenance of a rich environment to sustain the protumor cycle. Besides platelets, the P2Y12 receptor is expressed by some tumors, such as renal carcinoma, colon carcinoma, and gliomas, being related to tumor progression. In this context, this review aims to depict the glioma microenvironment, focusing on the relationship between platelets and tumor malignancy.
Assuntos
Glioma/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y12/fisiologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/fisiopatologia , Humanos , Receptores Purinérgicos/metabolismo , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologiaRESUMO
Chronic wounds are a public health problem worldwide, especially those related to diabetes. Besides being an enormous burden to patients, it challenges wound care professionals and causes a great financial cost to health system. Considering the absence of effective treatments for chronic wounds, our aim was to better understand the pathophysiology of tissue repair in diabetes in order to find alternative strategies to accelerate wound healing. Nucleotides have been described as extracellular signaling molecules in different inflammatory processes, including tissue repair. Adenosine-5'-diphosphate (ADP) plays important roles in vascular and cellular response and is immediately released after tissue injury, mainly from platelets. However, despite the well described effect on platelet aggregation during inflammation and injury, little is known about the role of ADP on the multiple steps of tissue repair, particularly in skin wounds. Therefore, we used the full-thickness excisional wound model to evaluate the effect of local ADP application in wounds of diabetic mice. ADP accelerated cutaneous wound healing, improved new tissue formation, and increased both collagen deposition and transforming growth factor-ß (TGF-ß) production in the wound. These effects were mediated by P2Y12 receptor activation since they were inhibited by Clopidogrel (Clop) treatment, a P2Y12 receptor antagonist. Furthermore, P2Y1 receptor antagonist also blocked ADP-induced wound closure until day 7, suggesting its involvement early in repair process. Interestingly, ADP treatment increased the expression of P2Y12 and P2Y1 receptors in the wound. In parallel, ADP reduced reactive oxygen species (ROS) formation and tumor necrosis factor-α (TNF-α) levels, while increased IL-13 levels in the skin. Also, ADP increased the counts of neutrophils, eosinophils, mast cells, and gamma delta (γδ) T cells (Vγ4+ and Vγ5+ cells subtypes of γδ+ T cells), although reduced regulatory T (Tregs) cells in the lesion. In accordance, ADP increased fibroblast proliferation and migration, myofibroblast differentiation, and keratinocyte proliferation. In conclusion, we provide strong evidence that ADP acts as a pro-resolution mediator in diabetes-associated skin wounds and is a promising intervention target for this worldwide problem.
Assuntos
Difosfato de Adenosina/farmacologia , Diabetes Mellitus Experimental/complicações , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/metabolismo , Cicatrização/efeitos dos fármacos , Difosfato de Adenosina/uso terapêutico , Administração Cutânea , Aloxano/administração & dosagem , Aloxano/toxicidade , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Humanos , Masculino , Camundongos , Agonistas do Receptor Purinérgico P2Y/uso terapêutico , Pele/efeitos dos fármacos , Pele/lesões , Pele/patologiaRESUMO
ATP is a cotransmitter released with other neurotransmitters from sympathetic nerves, where it stimulates purinergic receptors. Purinergic adenosine P1 receptors (coupled to Gi/o proteins) produce sympatho-inhibition in several autonomic effectors by prejunctional inhibition of neurotransmitter release. Similarly, signalling through P2Y12 and P2Y13 receptors coupled to Gi/o proteins is initiated by the ATP breakdown product ADP. Hence, this study has pharmacologically investigated a possible role of ADP-induced inhibition of the cardioaccelerator sympathetic drive in pithed rats, using a stable ADP analogue (ADPßS) and selective antagonists for the purinergic P2Y1, P2Y12 and P2Y13 receptors. Accordingly, male Wistar rats were pithed and: (i) pretreated i.v. with gallamine (25 mg/kg) and desipramine (50 µg/kg) for preganglionic spinal (C7-T1) stimulation of the cardioaccelerator sympathetic drive (n = 78); or (ii) prepared for receiving i.v. injections of exogenous noradrenaline (n = 12). The i.v. continuous infusions of ADPßS (10 and 30 µg/kg/min) dose-dependently inhibited the tachycardic responses to electrical sympathetic stimulation, but not those to exogenous noradrenaline. The cardiac sympatho-inhibition produced by 30 µg/kg/min ADPßS was (after i.v. administration of compounds) (i) unchanged by 1-ml/kg bidistilled water or 300-µg/kg MRS 2500 (P2Y1 receptor antagonist), (ii) abolished by 300-µg/kg PSB 0739 (P2Y12 receptor antagonist) and (iii) partially blocked by 3000-µg/kg MRS 2211 (P2Y13 receptor antagonist). Our results suggest that ADPßS induces a cardiac sympatho-inhibition that mainly involves the P2Y12 receptor subtype and, probably to a lesser extent, the P2Y13 receptor subtype. These receptors may represent therapeutic targets for treating cardiovascular pathologies, including stroke and myocardial infarctions.
Assuntos
Difosfato de Adenosina/análogos & derivados , Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2/metabolismo , Sistema Nervoso Simpático/fisiologia , Tionucleotídeos/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Masculino , Ratos , Ratos Wistar , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
Adenine nucleotides through P2Y1 receptor stimulation are known to control retinal progenitor cell (RPC) proliferation by modulating expression of the p57KIP2, a cell cycle regulator. However, the role of Gi protein-coupled P2Y12 and P2Y13 receptors also activated by adenine nucleotides in RPC proliferation is still unknown. Gene expression of the purinergic P2Y12 subtype was detected in rat retina during early postnatal days (P0 to P5), while expression levels of P2Y13 were low. Immunohistochemistry assays performed with rat retina on P3 revealed P2Y12 receptor expression in both Ki-67-positive cells in the neuroblastic layer and Ki-67-negative cells in the ganglion cell layer and inner nuclear layer. Nonetheless, P2Y13 receptor expression could not be detected in any stratum of rat retina. Intravitreal injection of PSB 0739 or clopidogrel, both selective P2Y12 receptor antagonists, increased by 20 and 15%, respectively, the number of Ki-67-positive cells following 24 h of exposure. Moreover, the P2Y12 receptor inhibition increased cyclin D1 and decreased p57KIP2 expression. However, there were no changes in the S phase of the cell cycle (BrdU-positive cells) or in mitosis (phospho-histone-H3-positive cells). Interestingly, an increase in the number of cyclin D1/TUNEL-positive cells after treatment with PSB 0739 was observed. These data suggest that activation of P2Y12 receptors is required for the successful exit of RPCs from cell cycle in the postnatal rat retina.
Assuntos
Organogênese , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2/metabolismo , Retina/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y12/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5'-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection.