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1.
Neuropharmacology ; 239: 109674, 2023 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-37541383

RESUMEN

The molecular processes that underlie long-term memory formation involve signaling pathway activation by neurotransmitter release, which induces the expression of immediate early genes, such as Zif268, having a key role in memory formation. In this work, we show that the cannabinoid CB1 receptor signaling is necessary for the effects of dexamethasone on the behavioral response in an inhibitory avoidance task, on dexamethasone-induced ERK phosphorylation, and on dexamethasone-dependent Zif268 expression. Furthermore, we provide primary evidence for the mechanism responsible for this crosstalk between cannabinoid and glucocorticoid-mediated signaling pathways, showing that dexamethasone regulates endocannabinoid metabolism by inhibiting the activity of the Fatty acid amide hydrolase (FAAH), an integral membrane enzyme that hydrolyzes endocannabinoids and related amidated signaling lipids. Our results provide novel evidence regarding the role of the endocannabinoid system, and in particular of the CB1 receptor, as a mediator of the effects of glucocorticoids on the consolidation of aversive memories.


Asunto(s)
Cannabinoides , Consolidación de la Memoria , Endocannabinoides/metabolismo , Receptor Cannabinoide CB1/genética , Cannabinoides/farmacología , Transducción de Señal , Glucocorticoides/farmacología , Dexametasona/farmacología , Amidohidrolasas , Moduladores de Receptores de Cannabinoides/farmacología
2.
Food Chem Toxicol ; 177: 113822, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37169060

RESUMEN

Breast cancer is one of the leading cancers among women worldwide. Given the evidence that pesticides play an important role in breast cancer, interest has grown in pesticide impact on disease progression. Hexachlorobenzene (HCB), an aryl hydrocarbon receptor (AhR) ligand, promotes triple-negative breast cancer cell migration and invasion. Estrogen receptor ß (ERß) inhibits cancer motility, while G protein-coupled ER (GPER) modulates the neoplastic transformation. Tryptophan is metabolized through the kynurenine pathway by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), with kynurenine signaling activation often predicting worse prognosis in cancer. In this context, we examined the HCB (0.005; 0.05; 0.5 and 5 µM) effect on LM3 cells, a human epidermal growth factor receptor 2 (HER2)-positive breast cancer model. Results show that HCB increases IDO and TDO mRNA levels and promotes cell viability, proliferation and migration through the AhR pathway. Moreover, HCB boosts mammosphere formation, vascular endothelial growth factor and cyclooxygenase-2 expression and reduces IL-10 levels. For some parameters, U-shaped or inverted U-shaped dose-response curves are shown. HCB alters ER levels, reducing ERß while increasing GPER. These results demonstrate that exposure to environmentally relevant concentrations of HCB up-regulates the kynurenine pathway and dysregulates ERß and GPER levels, collaborating in HER2-positive breast cancer progression.


Asunto(s)
Dioxigenasas , Plaguicidas , Neoplasias de la Mama Triple Negativas , Femenino , Humanos , Hexaclorobenceno/toxicidad , Quinurenina , Triptófano , Receptor beta de Estrógeno , Factor A de Crecimiento Endotelial Vascular , Línea Celular Tumoral , Neoplasias de la Mama Triple Negativas/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo
3.
Biochim Biophys Acta Gen Subj ; 1867(4): 130322, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36773726

RESUMEN

BACKGROUND: Ceefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice. METHODS: U937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice. RESULTS: Ceefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage. CONCLUSIONS: These results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine. GENERAL SIGNIFICANCE: This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.


Asunto(s)
Histamina , Leucemia Mieloide Aguda , Animales , Humanos , Ratones , Apoptosis , Transportadoras de Casetes de Unión a ATP , Histamina/farmacología , Leucemia Mieloide Aguda/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos
4.
Pharmacol Res Perspect ; 10(2): e00913, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35184416

RESUMEN

G protein-coupled receptors kinase 2 (GRK2) plays a major role in receptor regulation and, as a consequence, in cell biology and physiology. GRK2-mediated receptor desensitization is performed by its kinase domain, which exerts receptor phosphorylation promoting G protein uncoupling and the cessation of signaling, and by its RGS homology (RH) domain, able to interrupt G protein signaling. Since GRK2 activity is exacerbated in several pathologies, many efforts to develop inhibitors have been conducted. Most of them were directed toward GRK2 kinase activity and showed encouraging results on in vitro systems and animal models. Nevertheless, limitations including unspecific effects or pharmacokinetics issues prevented them from advancing to clinical trials. Surprisingly, even though the RH domain demonstrated the ability to desensitize GPCRs, this domain has been less explored. Herein, we show in vitro activity of a series of compounds that, by inhibiting GRK2 RH domain, increase receptor cAMP response, avoid GRK2 translocation to the plasma membrane, inhibit coimmunoprecipitation of GRK2 with Gαs subunit of heterotrimeric G protein, and prevent receptor desensitization. Also, we preliminarily evaluated candidates' ADMET properties and observed suitable lipophilicity and cytotoxicity. These novel inhibitors of phosphorylation-independent actions of GRK2 might be useful in elucidating other RH domain roles and lay the foundation for the development of innovative pharmacologic therapy for diseases where GRK2 activity is exacerbated.


Asunto(s)
AMP Cíclico/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Línea Celular Tumoral , Desarrollo de Medicamentos , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HEK293 , Humanos , Fosforilación , Dominios Proteicos/efectos de los fármacos , Proteínas RGS/metabolismo , Transducción de Señal/efectos de los fármacos
5.
Cells ; 10(11)2021 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-34831249

RESUMEN

Antihistamines and glucocorticoids (GCs) are often used together in the clinic to treat several inflammation-related situations. Although there is no rationale for this association, clinical practice has assumed that, due to their concomitant anti-inflammatory effects, there should be an intrinsic benefit to their co-administration. In this work, we evaluated the effects of the co-treatment of several antihistamines on dexamethasone-induced glucocorticoid receptor transcriptional activity on the expression of various inflammation-related genes in A549 and U937 cell lines. Our results show that all antihistamines potentiate GCs' anti-inflammatory effects, presenting ligand-, cell- and gene-dependent effects. Given that treatment with GCs has strong adverse effects, particularly on bone metabolism, we also examined the impact of antihistamine co-treatment on the expression of bone metabolism markers. Using MC3T3-E1 pre-osteoblastic cells, we observed that, though the antihistamine azelastine reduces the expression of dexamethasone-induced bone loss molecular markers, it potentiates osteoblast apoptosis. Our results suggest that the synergistic effect could contribute to reducing GC clinical doses, ineffective by itself but effective in combination with an antihistamine. This could result in a therapeutic advantage, as the addition of an antihistamine may reinforce the wanted effects of GCs, while related adverse effects could be diminished or at least mitigated. By modulating the patterns of gene activation/repression mediated by GR, antihistamines could enhance only the desired effects of GCs, allowing their effective dose to be reduced. Further research is needed to correctly determine the clinical scope, benefits, and potential risks of this therapeutic strategy.


Asunto(s)
Antiinflamatorios/farmacología , Dexametasona/farmacología , Regulación de la Expresión Génica , Antagonistas de los Receptores Histamínicos/farmacología , Inflamación/genética , Receptores de Glucocorticoides/metabolismo , Animales , Biomarcadores/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Ratones , FN-kappa B/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Ftalazinas/farmacología , Factor de Necrosis Tumoral alfa/farmacología
6.
Pharmacol Res Perspect ; 9(5): e00868, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34609071

Asunto(s)
América del Sur
7.
Eur J Pharmacol ; 896: 173913, 2021 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-33508282

RESUMEN

Histamine H1 receptor ligands used clinically as antiallergics rank among the most widely prescribed and over-the-counter drugs in the world. They exert the therapeutic actions by blocking the effects of histamine, due to null or negative efficacy towards Gαq-phospholipase C (PLC)-inositol triphosphates (IP3)-Ca2+ and nuclear factor-kappa B cascades. However, there is no information regarding their ability to modulate other receptor responses. The aim of the present study was to investigate whether histamine H1 receptor ligands could display positive efficacy concerning receptor desensitization, internalization, signaling through Gαq independent pathways or even transcriptional regulation of proinflammatory genes. While diphenhydramine, triprolidine and chlorpheniramine activate ERK1/2 (extracellular signal-regulated kinase 1/2) pathway in A549 cells, pre-treatment with chlorpheniramine or triprolidine completely desensitize histamine H1 receptor mediated Ca2+ response, and both diphenhydramine and triprolidine lead to receptor internalization. Unlike histamine, histamine H1 receptor desensitization and internalization induced by antihistamines prove to be independent of G protein-coupled receptor kinase 2 (GRK2) phosphorylation. Also, unlike the reference agonist, the recovery of the number of cell-surface histamine H1 receptors is a consequence of de novo synthesis. On the other hand, all of the ligands lack efficacy regarding cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) mRNA regulation. However, a prolonged exposure with each of the antihistamines impaires the increase in COX-2 and IL-8 mRNA levels induced by histamine, even after ligand removal. Altogether, these findings demonstrate the biased nature of histamine H1 receptor ligands contributing to a more accurate classification, and providing evidence for a more rational and safe use of them.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Receptores Histamínicos H1/efectos de los fármacos , Células A549 , Señalización del Calcio/efectos de los fármacos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Agonismo Inverso de Drogas , Activación Enzimática , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ligandos , Fosforilación , Transporte de Proteínas , Receptores Histamínicos H1/metabolismo , Fosfolipasas de Tipo C/metabolismo
8.
FEBS J ; 288(1): 229-243, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32333821

RESUMEN

Intracellular cAMP (i-cAMP) levels play an important role in acute myeloid leukemia (AML) cell proliferation and differentiation. Its levels are the result of cAMP production, degradation, and exclusion. We have previously described histamine H2 receptors and MRP4/ABCC4 as two potential targets for AML therapy. Acting through histamine H2 receptors, histamine increases cAMP production/synthesis, while MRP4/ABCC4 is responsible for the exclusion of this cyclic nucleotide. In this study, we show that histamine treatment induces MRP4/ABCC4 expression, augmenting cAMP efflux, and that histamine, in combination with MRP inhibitors, is able to reduce AML cell proliferation. Histamine, through histamine H2 receptor, increases i-cAMP levels and induces MRP4 transcript and protein levels in U937, KG1a, and HL-60 cells. Moreover, histamine induces MRP4 promoter activity in HEK293T cells transfected with histamine H2 receptor (HEK293T-H2 R). Our results support that the cAMP/Epac-PKA pathway, and not MEK/ERK nor PI3K/AKT signaling cascades, is involved in histamine-mediated upregulation of MRP4 levels. Finally, the addition of histamine potentiates the inhibition of U937, KG1a, and HL-60 cell proliferation induced by MRP4 inhibitors. Our data highlight that the use of a poly-pharmacological approach aimed at different molecular targets would be beneficial in AML treatment.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Histamina/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Receptores Histamínicos H2/genética , Benzotiazoles/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Leucémica de la Expresión Génica , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Células HL-60 , Histamina/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Terapia Molecular Dirigida/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Probenecid/farmacología , Regiones Promotoras Genéticas , Propionatos/farmacología , Quinolinas/farmacología , Receptores Histamínicos H2/metabolismo , Transducción de Señal , Triazoles/farmacología , Células U937
9.
Biochem Pharmacol ; 175: 113904, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32156659

RESUMEN

Breast cancer is the most common cancer type in females worldwide. Environmental exposure to pesticides affecting hormonal homeostasis does not necessarily induce DNA mutations but may influence gene expression by disturbances in epigenetic regulation. Expression of long interspersed nuclear element-1 (LINE-1) has been associated with tumorigenesis in several cancers. In nearly all somatic cells, LINE-1 is silenced by DNA methylation in the 5́'UTR and reactivated during disease initiation and/or progression. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-ß1 (TGF-ß1)/Smad pathway. Hexachlorobenzene (HCB) and chlorpyrifos (CPF), both weak AhR ligands, promote cell proliferation and migration in breast cancer cells, as well as tumor growth in rat models. In this context, our aim was to examine the effect of these pesticides on LINE-1 expression and ORF1p localization in the triple-negative breast cancer cell line MDA-MB-231 and the non-tumorigenic epithelial breast cell line NMuMG, and to evaluate the role of TGF-ß1 and AhR pathways. Results show that 0.5 µM CPF and 0.005 µM HCB increased LINE-1 mRNA expression through Smad and AhR signaling in MDA-MB-231. In addition, the methylation of the first sites in 5́'UTR of LINE-1 was reduced by pesticide exposure, although the farther sites remained unaffected. Pesticides modulated ORF1p localization in MDA-MB-231: 0.005 µM HCB and 50 µM CPF increased nuclear translocation, while both induced cytoplasmic retention at 0.5 and 5 µM. Moreover, both stimulated double-strand breaks, enhancing H2AX phosphorylation, coincidentally with ORF1p nuclear localization. In NMuMG similar results were observed, since they heighten LINE-1 mRNA levels. CPF effect was through AhR and TGF-ß1 signaling, whereas HCB action depends only of AhR. In addition, both pesticides increase ORF1p expression and nuclear localization. Our results provide experimental evidence that HCB and CPF exposure modify LINE-1 methylation levels and induce LINE-1 reactivation, suggesting that epigenetic mechanisms could contribute to pesticide-induced breast cancer progression.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Epiteliales/metabolismo , Elementos de Nucleótido Esparcido Largo/fisiología , Receptores de Hidrocarburo de Aril/metabolismo , Retroelementos/fisiología , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Hexaclorobenceno/metabolismo , Hexaclorobenceno/toxicidad , Humanos , Ligandos , Elementos de Nucleótido Esparcido Largo/efectos de los fármacos , Retroelementos/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología
10.
Front Pharmacol ; 11: 113, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32153413

RESUMEN

G protein coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR signaling. Canonical mechanism of GPCR desensitization involves receptor phosphorylation by GRKs followed by arrestin recruitment and uncoupling from heterotrimeric G protein. Although ß3-adrenergic receptor (ß3AR) lacks phosphorylation sites by GRKs, agonist treatment proved to induce ß3AR desensitization in many cell types. Here we show that GRK2 mediates short-term desensitization of ß3AR by a phosphorylation independent mechanism but mediated by its domain homologous to the regulator of G protein signaling (RGS). HEK293T cells overexpressing human ß3AR presented a short-term desensitization of cAMP response stimulated by the ß3AR agonist, BRL37344, and not by forskolin. We found that ß3AR desensitization was higher in cells co-transfected with GRK2. Similarly, overexpression of the RGS homology domain but not kinase domain of GRK2 increased ß3AR desensitization. Consistently, stimulation of ß3AR increased interaction between GRK2 and Gαs subunit. Furthermore, in rat cardiomyocytes endogenously expressing ß3AR, transfection with dominant negative mutant of RH domain of GRK2 (GRK2/D110A) increased cAMP response to BRL37344 and inhibited receptor desensitization. We expect our study to be a starting point for more sophisticated characterization of the consequences of GRK2 mediated desensitization of the ß3AR in heart function and disease.

11.
Pharmacol Res Perspect ; 7(6): e00530, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31859461

RESUMEN

Antihistamines and glucocorticoids (GCs) are often used together in the clinic, in several inflammatory-related situations. Even though there is no clear rationale for this drug association, the clinical practice is based on the assumption that due to their concomitant antiinflammatory effects, there should be an intrinsic benefit in their coadministration. Our group has studied the molecular interaction between the histamine H1 receptor and the glucocorticoid receptor (GR) signaling pathways, showing an enhancing effect on GC-induced GR transcriptional activity induced by antihistamines. We hypothesize that the existence of this synergistic effect could contribute in reducing the GCs clinical doses, ineffective by itself but effective in combination with an antihistamine. This could result in a therapeutic advantage as the GC-desired effects may be reinforced by the addition of an antihistamine and, as a consequence of the dose reduction, GC-related adverse effects could be reduced or at least mitigated. Here we discuss the potential therapeutic applications of this cotreatment seeking to evaluate its usefulness, especially in inflammatory-related conditions.


Asunto(s)
Glucocorticoides/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Inflamación/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Glucocorticoides/uso terapéutico , Antagonistas de los Receptores Histamínicos/uso terapéutico , Humanos , Inflamación/inmunología , Receptores de Glucocorticoides/metabolismo , Receptores Histamínicos H1/metabolismo , Transducción de Señal/inmunología
12.
Pharmacol Res Perspect ; 7(6): e00531, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31687162

RESUMEN

Glucocorticoids are among the most effective drugs to treat asthma. However, the severe adverse effects associated generate the need for its therapeutic optimization. Conversely, though histamine is undoubtedly related to asthma development, there is a lack of efficacy of antihistamines in controlling its symptoms, which prevents their clinical application. We have reported that antihistamines potentiate glucocorticoids' responses in vitro and recent observations have indicated that the coadministration of an antihistamine and a synthetic glucocorticoid has synergistic effects on a murine model of allergic rhinitis. Here, the aim of this work is to establish if this therapeutic combination could be beneficial in a murine model of asthma. We used an allergen-induced model of asthma (employing ovalbumin) to evaluate the effects of the synthetic glucocorticoid dexamethasone combined with the antihistamine azelastine. Our results indicate that the cotreatment with azelastine and a suboptimal dose of dexamethasone can improve allergic lung inflammation as shown by a decrease in eosinophils in bronchoalveolar lavage, fewer peribronchial and perivascular infiltrates, and mucin-producing cells. In addition, serum levels of allergen-specific IgE and IgG1 were also reduced, as well as the expression of lung inflammatory-related genes IL-4, IL-5, Muc5AC, and Arginase I. The potentiation of dexamethasone effects by azelastine could allow to reduce the effective glucocorticoid dose needed to achieve a therapeutic effect. These findings provide first new insights into the potential benefits of glucocorticoids and antihistamines combination for the treatment of asthma and grants further research to evaluate this approach in other related inflammatory conditions.


Asunto(s)
Antiasmáticos/farmacología , Asma/tratamiento farmacológico , Dexametasona/farmacología , Ftalazinas/farmacología , Administración Intranasal , Animales , Antiasmáticos/uso terapéutico , Asma/sangre , Asma/inmunología , Asma/patología , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Femenino , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Células HEK293 , Antagonistas de los Receptores Histamínicos H1 no Sedantes/farmacología , Antagonistas de los Receptores Histamínicos H1 no Sedantes/uso terapéutico , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Ratones , Ovalbúmina/inmunología , Ftalazinas/uso terapéutico , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/inmunología
13.
Mol Pharmacol ; 96(1): 13-25, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31043460

RESUMEN

Pancreatic cancer is one of the most lethal types of tumors with no effective therapy available; is currently the third leading cause of cancer in developed countries; and is predicted to become the second deadliest cancer in the United States by 2030. Due to the marginal benefits of current standard chemotherapy, the identification of new therapeutic targets is greatly required. Considering that cAMP pathway is commonly activated in pancreatic ductal adenocarcinoma (PDAC) and its premalignant lesions, we aim to investigate the multidrug resistance-associated protein 4 (MRP4)-dependent cAMP extrusion process as a cause of increased cell proliferation in human PDAC cell lines. Our results from in silico analysis indicate that MRP4 expression may influence PDAC patient outcome; thus, high MRP4 levels could be indicators of poor survival. In addition, we performed in vitro experiments and identified an association between higher MRP4 expression levels and more undifferentiated and malignant models of PDAC and cAMP extrusion capacity. We studied the antiproliferative effect and the overall cAMP response of three MRP4 inhibitors, probenecid, MK571, and ceefourin-1 in PDAC in vitro models. Moreover, MRP4-specific silencing in PANC-1 cells reduced cell proliferation (P < 0.05), whereas MRP4 overexpression in BxPC-3 cells significantly incremented their growth rate in culture (P < 0.05). MRP4 pharmacological inhibition or silencing abrogated cell proliferation through the activation of the cAMP/Epac/Rap1 signaling pathway. Also, extracellular cAMP reverted the antiproliferative effect of MRP4 blockade. Our data highlight the MRP4-dependent cAMP extrusion process as a key participant in cell proliferation, indicating that MRP4 could be an exploitable therapeutic target for PDAC. SIGNIFICANCE STATEMENT: ABCC4/MRP4 is the main transporter responsible for cAMP efflux. In this work, we show that MRP4 expression may influence PDAC patient outcome and identify an association between higher MRP4 expression levels and more undifferentiated and malignant in vitro models of PDAC. Findings prove the involvement of MRP4 in PDAC cell proliferation through a novel extracellular cAMP mitogenic pathway and further support MRP4 inhibition as a promising therapeutic strategy for PDAC treatment.


Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , AMP Cíclico/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Neoplasias Pancreáticas/metabolismo , Benzotiazoles/farmacología , Carcinoma Ductal Pancreático/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Células HEK293 , Humanos , Neoplasias Pancreáticas/genética , Probenecid/farmacología , Pronóstico , Propionatos/farmacología , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Triazoles/farmacología , Regulación hacia Arriba
14.
Front Pharmacol ; 10: 214, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30930776

RESUMEN

Regulatory factors that control gene transcription in multicellular organisms are assembled in multicomponent complexes by combinatorial interactions. In this context, nuclear receptors provide well-characterized and physiologically relevant systems to study ligand-induced transcription resulting from the integration of cellular and genomic information in a cell- and gene-specific manner. Here, we developed a mathematical model describing the interactions between the glucocorticoid receptor (GR) and other components of a multifactorial regulatory complex controlling the transcription of GR-target genes, such as coregulator peptides. We support the validity of the model in relation to gene-specific GR transactivation with gene transcription data from A549 cells and in vitro real time quantification of coregulator-GR interactions. The model accurately describes and helps to interpret ligand-specific and gene-specific transcriptional regulation by the GR. The comprehensive character of the model allows future insight into the function and relative contribution of the molecular species proposed in ligand- and gene-specific transcriptional regulation.

15.
Front Pharmacol ; 10: 146, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30853914

RESUMEN

The MasR receptor (MasR) is an orphan G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang-(1-7) protective axis of renin-angiotensin system. This receptor has been suggested to participate in several physiological processes including cardio- and reno-protection and regulation of the central nervous system function. Although the knowledge of the signaling mechanisms associated with MasR is essential for therapeutic purposes, these are still poorly understood. Accordingly, in the current study we aimed to characterize the signaling pathways triggered by the MasR. To do that, we measured cAMP and Ca2+ levels in both naïve and MasR transfected cells in basal conditions and upon incubation with putative MasR ligands. Besides, we evaluated activation of ERK1/2 by Ang-(1-7) in MasR transfected cells. Results indicated the existence of a high degree of MasR constitutive activity toward cAMP modulation. This effect was not mediated by the PDZ-binding motif of the MasR but by receptor coupling to Gαi-adenylyl cyclase signaling pathway. Incubation of MasR transfected cells with Ang-(1-7) or the synthetic ligand AVE 0991 amplified MasR negative modulation of cAMP levels. On the other hand, we provided evidence for lack of MasR-associated modulation of Ca2+ levels by Ang-(1-7). Finally, it was determined that the MasR attenuated Ang-(1-7)-induced ERK1/2 phosphorylation mediated by AT1R. We provided further characterization of MasR signaling mechanisms regarding its constitutive activity and response to putative ligands. This information could prove useful to better describe MasR physiological role and development of therapeutic agents that could modulate its action.

16.
Eur J Pharmacol ; 847: 42-52, 2019 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-30685431

RESUMEN

Histamine [2-(4-Imidazolyl)-ethylamine] modulates different biological processes, through histamine H1 and H2 receptors, and their respective blockers are widely used in treating allergic and gastric acid-related disorders. Histamine H1 and H2 receptor crossdesensitization and cointernalization induced by its agonists have been previously described. In this study, we show how this crosstalk determines the response to histamine H1 and H2 receptor inverse agonists and how histamine H1 and H2 receptor inverse agonists interfere with the other receptor's response to agonists. By desensitization assays we demonstrate that histamine H1 and H2 receptor inverse agonists induce a crossregulation between both receptors. In this sense, the histamine H1 receptor inverse agonists desensitize the cAMP response to amthamine, a histamine H2 receptor agonist. In turn, histamine H2 receptor inverse agonists interfere with histamine H1 receptor signaling. We also determine that the crossdesensitization induced by histamine H1 or H2 receptor agonists alters the histamine inverse agonists receptor response: activation of histamine H1 receptor affects cAMP response induced by histamine H2 receptor inverse agonists, whereas histamine H2 receptor agonist induces a negative regulation on the anti-inflammatory response of histamine H1 receptor inverse agonists. Binding studies revealed that histamine H1 and H2 receptors cointernalize after stimulus with histamine receptor inverse agonists. In addition, the inhibition of the internalization process prevents receptor crossregulation. Our study provides new insights in the mechanisms of action of histamine H1 and H2 receptors that explain the effect of histamine H1 and H2 receptor inverse agonists and opens up new venues for novel therapeutic applications.


Asunto(s)
Agonistas de los Receptores Histamínicos/metabolismo , Antagonistas de los Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/metabolismo , Línea Celular , Línea Celular Tumoral , Células HEK293 , Histamina/metabolismo , Humanos , Transducción de Señal/fisiología , Células U937
17.
Biochem Pharmacol ; 146: 117-126, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-28962836

RESUMEN

Despite the pivotal role GPCRs play in cellular signaling, it is only in the recent years that structural biology has begun to elucidate how GPCRs function and to provide a platform for structure-based drug design. It is postulated that GPCR activation involves the movement of transmembrane helices. The finding that many residues, which have been shown to be critical for receptor activation and are highly conserved among different GPCRs, are clustered in particular positions of transmembrane helices suggests that activation of GPCRs may involve common molecular mechanisms. In particular, phenylalanine 6.44, located in the upper half of TMVI, is highly conserved among almost all GPCRs. We generated Phe 2436.44 Ala/Ser mutants of histamine H2 receptor and found that while the substitutions do not affect receptor expression or ligand signaling, are able to specifically alter cimetidine and ranitidine mechanisms of action from simply inactivating the receptor to produce a ligand-induced G-protein sequestering conformation, that interferes with the signaling of ß2-adrenoceptor. Taking advantage of the cubic ternary complex model, and mathematically modeling our results, we hypothesize that this alteration in ligand mechanism of action is consequence of a change in ligand-induced conformational rearrangement of receptor and its effect on G-protein coupling. Our results show that receptor point mutations can not only alter receptor behavior, as shown for activating/inactivating mutations, but also can have more subtle effects changing ligand mechanism of action.


Asunto(s)
Cimetidina/farmacología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Ranitidina/farmacología , Receptores Histamínicos H2/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutación , Conformación Proteica
18.
Handb Exp Pharmacol ; 241: 141-160, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27316911

RESUMEN

Acute myeloid leukemia (AML) consists in a cancer of early hematopoietic cells arising in the bone marrow, most often of those cells that would turn into white blood cells (except lymphocytes). Chemotherapy is the treatment of choice for AML but one of the major complications is that current drugs are highly toxic and poorly tolerated. In general, treatment for AML consists of induction chemotherapy and post-remission therapy. If no further post-remission is given, almost all patients will eventually relapse. Histamine, acting at histamine type-2 (H2) receptors on phagocytes and AML blast cells, helps prevent the production and release of oxygen-free radicals, thereby protecting NK and cytotoxic T cells. This protection allows immune-stimulating agents, such as interleukin-2 (IL-2), to activate cytotoxic cells more effectively, enhancing the killing of tumor cells. Based on this mechanism, post-remission therapy with histamine and IL-2 was found to significantly prevent relapse of AML. Alternatively, another potentially less toxic approach to treat AML employs drugs to induce differentiation of malignant cells. It is based on the assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment results in tumor reprogramming and the induction of terminal differentiation. There are promissory results showing that an elevated and sustained signaling through H2 receptors is able to differentiate leukemia-derived cell lines, opening the door for the use of H2 agonists for specific differentiation therapies. In both situations, histamine acting through H2 receptors constitutes an eligible treatment to induce leukemic cell differentiation, improving combined therapies.


Asunto(s)
Células Sanguíneas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Receptores Histamínicos H2/sangre , Receptores Histamínicos H2/metabolismo , Histamina/metabolismo , Humanos , Interleucina-2/metabolismo , Leucemia Mieloide Aguda/sangre , Masculino
19.
Mol Pharmacol ; 90(5): 640-648, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27625037

RESUMEN

H1 and H2 histamine receptor antagonists, although developed many decades ago, are still effective for the treatment of allergic and gastric acid-related conditions. This article focuses on novel aspects of the pharmacology and molecular mechanisms of histamine receptors that should be contemplated for optimizing current therapies, repositioning histaminergic ligands for new therapeutic uses, or even including agonists of the histaminergic system in the treatment of different pathologies such as leukemia or neurodegenerative disorders. In recent years, new signaling phenomena related to H1 and H2 receptors have been described that make them suitable for novel therapeutic approaches. Crosstalk between histamine receptors and other membrane or nuclear receptors can be envisaged as a way to modulate other signaling pathways and to potentiate the efficacy of drugs acting on different receptors. Likewise, biased signaling at histamine receptors seems to be a pharmacological feature that can be exploited to investigate nontraditional therapeutic uses for H1 and H2 biased agonists in malignancies such as acute myeloid leukemia and to avoid undesired side effects when used in standard treatments. It is hoped that the molecular mechanisms discussed in this review contribute to a better understanding of the different aspects involved in histamine receptor pharmacology, which in turn will contribute to increased drug efficacy, avoidance of adverse effects, or repositioning of histaminergic ligands.


Asunto(s)
Histamina/metabolismo , Receptor Cross-Talk , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/metabolismo , Animales , Humanos , Ligandos , Modelos Biológicos
20.
Biochim Biophys Acta ; 1860(9): 1998-2007, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27316323

RESUMEN

BACKGROUND: Histamine, through histamine H2 receptor (H2R), modulates different biological processes, involving the modulation of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Many evidences have demonstrated the existence and importance of the crossregulation between these two signaling pathways. The aim of the present work was to determine the molecular mechanisms leading to PI3K and ERK pathways modulation induced by the H2R agonist amthamine and to evaluate the possible interplay between them. METHODS: Phosphorylation levels of ERK and Akt were examined by Western blot in HEK293T cells expressing the human H2R, in the presence of H2R agonist and dominant negative mutants or pharmacological inhibitors of different proteins/pathways. Transcriptional activity assays were assessed to determine SRE activity. Amthamine-mediated cellular proliferation was investigated in MA-10A cells in the presence of PI3K inhibitor. RESULTS: H2R agonist inhibits PI3K/Akt/mTOR and stimulates Ras/MEK/ERK pathways. Moreover, PI3K/Akt/mTOR signaling inhibition is necessary to achieve H2R mediated ERK activation. In the presence of a constitutive active mutant of Akt, amthamine is not able to mediate ERK activation. This crosstalk affects classical ERK downstream targets such as Elk1 phosphorylation and the transcriptional activity of the SRE, classically associated to proliferation. We further demonstrate that amthamine-induced proliferation in Leydig MA-10 tumor cells, is enhanced by LY294002, a PI3K inhibitor. CONCLUSIONS: These results describe a crosstalk between PI3K/AKT/mTOR and Ras/MEK/ERK pathways induced by H2R stimulation with implications in cell proliferation. GENERAL SIGNIFICANCE: This work indicates that the modulation of PI3K/AKT/mTOR pathway by H2R in turn regulates Ras/MEK/ERK activation conditioning the proliferative capacity of the cells.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Histamina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Células HEK293 , Humanos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Tiazoles/farmacología
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