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1.
Front Immunol ; 12: 613070, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815368

RESUMO

Lack of specific antiviral treatment for COVID-19 has resulted in long hospitalizations and high mortality rate. By harnessing the regulatory effects of adenosine on inflammatory mediators, we have instituted a new therapeutic treatment with inhaled adenosine in COVID-19 patients, with the aim of reducing inflammation, the onset of cytokine storm, and therefore to improve prognosis. The use of inhaled adenosine in COVID19 patients has allowed reduction of length of stay, on average 6 days. This result is strengthened by the decrease in SARS-CoV-2 positive days. In treated patients compared to control, a clear improvement in PaO2/FiO2 was observed together with a reduction in inflammation parameters, such as the decrease of CRP level. Furthermore, the efficacy of inhaled exogenous adenosine led to an improvement of the prognosis indices, NLR and PLR. The treatment seems to be safe and modulates the immune system, allowing an effective response against the viral infection progression, reducing length of stay and inflammation parameters.


Assuntos
Adenosina/farmacologia , /tratamento farmacológico , Adenosina/uso terapêutico , Adulto , Idoso , Antibacterianos/administração & dosagem , Azitromicina/administração & dosagem , /fisiopatologia , Estudos de Casos e Controles , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Síndrome da Liberação de Citocina/fisiopatologia , Inibidores Enzimáticos/administração & dosagem , Feminino , Heparina/administração & dosagem , Hospitalização , Humanos , Hidroxicloroquina/administração & dosagem , Inflamação/tratamento farmacológico , Lopinavir/administração & dosagem , Masculino , Pessoa de Meia-Idade , Prognóstico , Tomografia Computadorizada por Raios X
2.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557248

RESUMO

Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to a class of universally and commonly used anti-inflammatory analgesics worldwide. A diversity of drawbacks of NSAIDs have been reported including cellular oxidative stress, which in turn triggers the accumulation of unfolded proteins, enhancing endoplasmic reticulum stress, and finally resulting in renal cell damage. Cordyceps cicadae (CC) has been used as a traditional medicine for improving renal function via its anti-inflammatory effects. N6-(2-hydroxyethyl)adenosine (HEA), a physiologically active compound, has been reported from CC mycelia (CCM) with anti-inflammatory effects. We hypothesize that HEA could protect human proximal tubular cells (HK-2) from NSAID-mediated effects on differential gene expression at the mRNA and protein levels. To verify this, we first isolated HEA from CCM using Sephadex® LH-20 column chromatography. The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK-2 cells. The HK-2 cells were pretreated with HEA (10-20 µM) and then insulted with the NSAIDs diclofenac (DCF, 200 µM) and meloxicam (MXC, 400 µM) for 24 h. HEA (20 µM) effectively prevented ER stress by attenuating ROS production (p < 0.001) and gene expression of ATF-6, PERK, IRE1α, CDCFHOP, IL1ß, and NFκB within 24 h. Moreover, HEA reversed the increase of GRP78 and CHOP protein expression levels induced by DCF and MXC, and restored the ER homeostasis. These results demonstrated that HEA treatments effectively protect against DCF- and MXC-induced ER stress damage in human proximal tubular cells through regulation of the GRP78/ATF6/PERK/IRE1α/CHOP pathway.


Assuntos
Adenosina/análogos & derivados , Anti-Inflamatórios não Esteroides/farmacologia , Cordyceps/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Homeostase , Túbulos Renais Proximais/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Adenosina/farmacologia , Regulação da Expressão Gênica , Humanos , Túbulos Renais Proximais/metabolismo , Estresse Oxidativo
3.
Mol Immunol ; 132: 1-7, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33524770

RESUMO

Macrophages perform the fundamental function of sensing cellular damage, initiating and mediating immune response and tissue repair. Adenine nucleotides are in relatively high abundance in cells and are released from cells during tissue damage that are converted to adenosine in the extracellular environment. The A1, A2A, A2B and A3 adenosine receptors serve to regulate immune function. Despite characterization of the adenosine receptors, a comprehensive examination of adenosine receptor signaling in THP-1 macrophage cells has not been done. Moreover, previous studies employed chemical agonists and antagonists that have the potential for off-target affects. Here we systematically knockdown each of the four known adenosine receptors in THP-1 macrophages using validated siRNA and investigated their function under LPS stimulation. We demonstrate that the A1 receptor is required for adenosine-stimulated IL-10 and IL-1ß secretion indicating an important role of this receptor during resolution of inflammation and tissue repair in these cells. The A1 and A3 receptor were required for IL-6 and IL-1ß secretion showing a net pro-inflammatory role for these receptors. Finally, we present the novel finding that THP-1 macrophages lacking the A2B receptor undergo pyroptosis when exposed to LPS, demonstrating a novel role of the A2B receptor in regulation of programmed cell death during inflammation. This work underscores the fundamental importance of adenosine signaling and provides insight into the independent roles of the adenosine receptors in modulating cytokine signaling.


Assuntos
Citocinas/metabolismo , Macrófagos/metabolismo , Piroptose/imunologia , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Adenosina/farmacologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Piroptose/efeitos dos fármacos , Piroptose/genética , RNA Interferente Pequeno , Receptor A1 de Adenosina/genética , Receptor A2A de Adenosina/genética , Receptor A2B de Adenosina/genética , Receptor A3 de Adenosina/genética , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/imunologia
4.
J Trauma Acute Care Surg ; 90(2): 369-375, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33502148

RESUMO

BACKGROUND: Uncontrolled hemorrhage is the leading cause of potentially survivable combat casualty mortality, with 86.5% of cases resulting from noncompressible torso hemorrhage. Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a minimally invasive technique used to stabilize patients with noncompressible torso hemorrhage; however, its application can take an average of 8 minutes to place. One therapeutic capable of bridging this gap is adenosine-lidocaine-magnesium (ALM), which at high doses induces a reversible cardioplegia. We hypothesize by using ALM as an adjunct to REBOA, the ALM-induced cardiac arrest will temporarily halt exsanguination and reduce blood loss, allowing for REBOA placement and control of bleeding. METHODS: Male Yorkshire swine (60-80 kg) were randomly assigned to REBOA only or ALM-REBOA (n = 8/group). At baseline, uncontrolled hemorrhage was induced via a 1.5-cm right femoral arteriotomy, and hemorrhaged blood was quantified. One minute after injury (S1), ALM was administered, and 7 minutes later (T0), zone 1 REBOA inflation occurred. If cardiac arrest ensued, cardiac function either recovered spontaneously or advanced life support was initiated. At T30, surgical hemostasis was obtained, and REBOA was deflated. Animals were resuscitated until they were humanely euthanized at T90. RESULTS: During field care phase, heart rate and end-tidal CO2 of the ALM-REBOA group were significantly lower than the REBOA only group. While mean arterial pressure significantly decreased from baseline, no significant differences between groups were observed throughout the field care phase. There was no significant difference in survival between the two groups (ALM-REBOA = 89% vs. REBOA only = 100%). Total blood loss was significantly decreased in the ALM-REBOA group (REBOA only = 24.32 ± 1.89 mL/kg vs. ALM-REBOA = 17.75 ± 2.04 mL/kg, p = 0.0499). CONCLUSION: Adenosine-lidocaine-magnesium is a novel therapeutic, which, when used with REBOA, can significantly decrease the amount of blood loss at initial presentation, without compromising survival. This study provides proof of concept for ALM and its ability to bridge the gap between patient presentation and REBOA placement.


Assuntos
Adenosina/farmacologia , Oclusão com Balão/métodos , Soluções Cardioplégicas/farmacologia , Fármacos Cardiovasculares/farmacologia , Exsanguinação/terapia , Parada Cardíaca Induzida/métodos , Lidocaína/farmacologia , Magnésio/farmacologia , Animais , Aorta , Modelos Animais de Doenças , Procedimentos Endovasculares/métodos , Hemostasia Cirúrgica/métodos , Soluções Farmacêuticas , Cuidados Pré-Operatórios/métodos , Ressuscitação/métodos , Suínos
5.
Mol Immunol ; 131: 60-67, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33358566

RESUMO

BACKGROUND: Growing evidence shows that enhancer of zeste homolog 2 (EZH2) plays a role in various physiological functions and cancer pathogenesis. However, its contribution to allergic diseases remains controversial. We sought to investigate the role of EZH2 in the pathogenesis of allergic airway inflammation. METHODS: 3-Deazaneplanocin A (DZNep), an indirect inhibitor of EZH2, was administered via intraperitoneal injection in an ovalbumin (OVA)-induced murine model of allergic airway inflammation. The expression of EZH2 in the allergic airway tissues was examined by immunohistochemistry (IHC) and western blot. The inflammatory cell infiltration and the goblet cell hyperplasia in the murine nose and lung were detected by hematoxylin and eosin (H&E) staining and periodic acid-Schiff (PAS) staining. Levels of cytokines, including IL-4, IFN-γ, IL-6, and IL-10, were evaluated in the bronchoalveolar lavage fluid (BALF) using Enzyme-linked immune sorbent assay (ELISA). RESULTS: EZH2 expression was inhibited by DZNep treatment (P < 0.05). The administration of DZNep significantly inhibited the inflammatory cell infiltration (P < 0.0001) and goblet cell hyperplasia (P < 0.001). Moreover, it suppressed the secretion of IL-4 (P < 0.0001) and IL-6 (P < 0.01) in the BALF. CONCLUSIONS: Our findings demonstrate that DZNep attenuates allergic airway inflammation and could be a new therapeutic option for allergic rhinitis and asthma.


Assuntos
Adenosina/análogos & derivados , Anti-Inflamatórios/farmacologia , Hipersensibilidade/tratamento farmacológico , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Ovalbumina/farmacologia , Adenosina/farmacologia , Animais , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/metabolismo , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Hipersensibilidade/metabolismo , Inflamação/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
6.
J Med Chem ; 63(24): 16012-16027, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33325691

RESUMO

Following our report that A3 adenosine receptor (AR) antagonist 1 exhibited a polypharmacological profile as a dual modulator of peroxisome proliferator-activated receptor (PPAR)γ/δ, we discovered a new template, 1'-homologated adenosine analogues 4a-4t, as dual PPARγ/δ modulators without AR binding. Removal of binding affinity to A3AR was achieved by 1'-homologation, and PPARγ/δ dual modulation was derived from the structural similarity between the target nucleosides and PPAR modulator drug, rosiglitazone. All the final nucleosides were devoid of AR-binding affinity and exhibited high binding affinities to PPARγ/δ but lacked PPARα binding. 2-Cl derivatives exhibited dual receptor-binding affinity to PPARγ/δ, which was absent for the corresponding 2-H derivatives. 2-Propynyl substitution prevented PPARδ-binding affinity but preserved PPARγ affinity, indicating that the C2 position defines a pharmacophore for selective PPARγ ligand designs. PPARγ/δ dual modulators functioning as both PPARγ partial agonists and PPARδ antagonists promoted adiponectin production, suggesting their therapeutic potential against hypoadiponectinemia-associated cancer and metabolic diseases.


Assuntos
Adenosina/química , Adenosina/farmacologia , Adiponectina/metabolismo , Descoberta de Drogas , Obesidade/tratamento farmacológico , PPAR alfa/antagonistas & inibidores , PPAR gama/agonistas , Animais , Sítios de Ligação , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Simulação de Dinâmica Molecular , Obesidade/metabolismo , Obesidade/patologia , PPAR alfa/metabolismo , PPAR gama/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
7.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
8.
Nat Commun ; 11(1): 4153, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814769

RESUMO

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/genética , Receptores Androgênicos/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Compostos de Fenilureia/farmacologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Terapêutica com RNAi/métodos , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
9.
PLoS One ; 15(7): e0234103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645016

RESUMO

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546).


Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Animais , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Humanos , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia
10.
Nat Commun ; 11(1): 3717, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709887

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Assuntos
Betacoronavirus/enzimologia , Metiltransferases/química , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Domínio Catalítico , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Capuzes de RNA , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
11.
J Interv Cardiol ; 2020: 4829647, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32508541

RESUMO

Background: The index of microcirculatory resistance is an invasive measure of coronary microvascular function that has to be calculated during maximal hyperemia, classically achieved with intravenous adenosine (IV). The aim of this study was to evaluate the use of intracoronary (IC) adenosine for the calculation of IMR. Methods and Results: 31 patients with stable coronary artery disease were included in the study. Coronary pressure and thermodilution measurements were obtained at rest and during maximal hyperemia using a pressure-temperature sensor-tipped coronary guidewire. Duplicate measurements were performed using first IC and then IV adenosine. Dispersion of transit times was comparable for IC and IV adenosine. IMR values based on IC vs IV adenosine showed a high level of agreement and an intraclass correlation coefficient of 0.90. Applying an upper normal limit of 25, misclassification of IMR using IC adenosine was seen in just one patient in whom IC adenosine resulted in a lower value. A simplified procedure based on a single bolus dose of saline did not change the level of agreement or the rate of misclassification. Conclusions: We found an excellent agreement between IMR values measured during hyperemia induced by IC as compared to IV adenosine. The use of IC adenosine may facilitate invasive assessment of microvascular function and is potentially time- and cost-saving with less patient discomfort as compared to IV infusion. The trail is registered with NCT03369184.


Assuntos
Adenosina/farmacologia , Doença da Artéria Coronariana , Circulação Coronária , Injeções Intra-Arteriais/métodos , Microcirculação , Resistência Vascular , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/fisiopatologia , Feminino , Humanos , Hiperemia/induzido quimicamente , Hiperemia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Vasodilatadores/farmacologia
12.
Am J Physiol Cell Physiol ; 319(1): C183-C193, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432925

RESUMO

The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small-interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Vasa Vasorum/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Adenosina/farmacologia , Animais , Bovinos , Células Endoteliais/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Masculino , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vasa Vasorum/efeitos dos fármacos
13.
Mol Carcinog ; 59(4): 390-398, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32037613

RESUMO

Brain metastasis is a leading cause of death worldwide, but the mechanism involved remains unclear. Stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) signaling has been reported to induce the directed metastasis of cancers, and adenosine A2A receptor activation suppresses the SDF-1/CXCR4 interaction. However, whether A2A receptor activation implicates the SDF-1/CXCR4 signaling pathway and thus modulates brain metastasis remains unclear. In this study, Western blot was performed to evaluate the protein levels. Cell invasion and migration assays were used to estimate the metastasis ability of PC-9 cells. The viability of cells was demonstrated by lactate dehydrogenase and cell proliferation assays. And the findings in vitro were further identified in nude mice. Notably, adenosine A2A receptor activation inhibited the proliferation and viability of PC-9 cells and thus suppressed the brain metastasis. A2A receptor stimulation protected the function of blood-brain barrier (BBB). The suppression of brain metastasis and the protection of BBB by A2A receptor relied on SDF-1/CXCR4 signaling, and treatment using A2A receptor agonist and CXCR4 antagonist protected the nude mice from malignancy metastasis in vivo. Adenosine A2A receptor activation suppressed the brain metastasis by implicating the SDF-1/CXCR4 axis and protecting the BBB.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/prevenção & controle , Quimiocina CXCL12/metabolismo , Neoplasias Pulmonares/prevenção & controle , Receptor A2A de Adenosina/metabolismo , Receptores CXCR4/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Humanos , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenetilaminas/farmacologia , Receptores CXCR4/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
14.
Med Oncol ; 37(3): 17, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32030542

RESUMO

The epithelial-mesenchymal transition (EMT) is a molecular process connected to higher expression of vimentin and increased activity of transcription factors (Snail, Twist) which restrains E-cadherin. EMT has been linked to prostate cancer metastatic potential, therapy resistance, and poor outcomes. Kinetin riboside (9-(b-dribofuranosyl)-6-furfurylaminopurine, KR) is a naturally occurring cytokinin, which induces apoptosis and shows strong antiproliferative activity against various human cancer cell lines. To establish the effect of KR on human prostate cell lines, expression of, e.g. AR, E-, N-cadherins, Vimentin, Snail, Twist, and MMPs, was analysed at mRNA and protein levels using Western Blot and RT-PCR and/or RQ-PCR techniques. KR inhibited the growth of human prostate cancer cells, but also, to a small extent, of normal cells. This effect depended on the type of the cells and their androgen sensitivity. KR also decreased the level of p-Akt, which takes part in androgen signalling modulation. The antiapoptotic Bcl-2 protein was down-regulated in cancer cell lines, while that of Bax is up-regulated upon KR exposure. KR contributed to re-expression of the E-cadherin as well as to significant changes in cell migration. Taken together, our results indicate for the first time that KR can be proposed as a factor for signalling pathways regulation that participates in the inhibition of development of aggressive forms of prostate cancer, and may alter the approach to therapeutic interventions. We propose KR as a potent inhibitor of EMT in human prostate cells.


Assuntos
Adenosina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Cinetina/farmacologia , Próstata/patologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , Próstata/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo
15.
Chem Biol Interact ; 317: 108965, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32001260

RESUMO

Endocrine therapies (e.g. tamoxifen and aromatase inhibitors) targeting estrogen action are effective in decreasing mortality of breast cancer. However, their efficacy is limited by intrinsic and acquired resistance. Our previous study demonstrated that overexpression of a histone methyltransferase NSD2 drives tamoxifen resistance in breast cancer cells and that NSD2 is a potential biomarker of tamoxifen resistant breast cancer. Here, we found that DZNep, an indirect inhibitor of histone methyltransferases, potently induces the degradation of NSD2 protein and inhibits the expression of NSD2 target genes (HK2, G6PD, GLUT1 and TIGAR) involved in the pentose phosphate pathway (PPP). DZNep treatment of tamoxifen-resistant breast cancer cells and xenograft tumors also strongly inhibits tumor growth and the cancer cell survival through decreasing cell production of NADPH and glutathione (GSH) and invoking elevated ROS to cause apoptosis. These findings suggest that DZNep-like agents can be developed to target NSD2 histone methyltransferase for effective treatment of tamoxifen-resistant breast cancer.


Assuntos
Adenosina/análogos & derivados , Histona-Lisina N-Metiltransferase/metabolismo , Metiltransferases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , S-Adenosil-Homocisteína/metabolismo , Adenosina/farmacologia , Antígenos Ly , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Homeostase , Humanos , Oxirredução , Proteólise/efeitos dos fármacos , Espécies Reativas de Oxigênio , Proteínas Repressoras/genética , Tamoxifeno , Ativador de Plasminogênio Tipo Uroquinase
16.
Int J Mol Sci ; 21(3)2020 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-32012688

RESUMO

Cholangiocarcinoma (CCA) is a lethal disease with increasing incidence worldwide. Previous study showed that CCA was sensitive to adenosine. Thereby, molecular mechanisms of CCA inhibition by adenosine were examined in this study. Our results showed that adenosine inhibited CCA cells via an uptake of adenosine through equilibrative nucleoside transporters (ENTs), instead of activation of adenosine receptors. The inhibition of ENTs by NBTI caused the inhibitory effect of adenosine to subside, while adenosine receptor antagonists, caffeine and CGS-15943, failed to do so. Intracellular adenosine level was increased after adenosine treatment. Also, a conversion of adenosine to AMP by adenosine kinase is required in this inhibition. On the other hand, inosine, which is a metabolic product of adenosine has very little inhibitory effect on CCA cells. This indicates that a conversion of adenosine to inosine may reduce adenosine inhibitory effect. Furthermore, there was no specific correlation between level of proinflammatory proteins and CCA responses to adenosine. A metabolic stable analog of adenosine, 2Cl-adenosine, exerted higher inhibition on CCA cell growth. The disturbance in intracellular AMP level also led to an activation of 5' AMP-activated protein kinase (AMPK). Accordingly, we proposed a novel adenosine-mediated cancer cell growth and invasion suppression via a receptor-independent mechanism in CCA.


Assuntos
Adenosina/metabolismo , Colangiocarcinoma/metabolismo , Proteínas de Transporte de Nucleosídeos/metabolismo , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais , Adenosina/farmacologia , Monofosfato de Adenosina/metabolismo , Ductos Biliares Intra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/metabolismo , Biomarcadores , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Relação Dose-Resposta a Droga , Humanos , Mediadores da Inflamação/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Ann Biol Clin (Paris) ; 78(1): 27-34, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32108577

RESUMO

Unfractionated heparin (UFH) is the main anticoagulante used in intensive care unit. The anticoagulant effect is monitored by activated partial thrombin time (aPTT) and anti-Xa activity (anti-Xa) measurement. However, delayed centrifugation induces platelet factor 4 (PF4) release and anti-Xa decrease. Several studies have concluded that aPTT and anti-Xa measurement should be performed within 2 hours in citrated anticoagulant but may be delayed longer in citrate theophylline adenosine and dypiridamol (CTAD) anticoagulant. The objective of this study was to compare the stability of both aPTT and anti-Xa in citrate and CTAD samples, and to determine the effect of delayed centrifugation on both aPTT, anti-Xa results, and PF4 release in citrate samples only. METHODS: aPTT and anti-Xa were measured in citrate and CTAD anticoagulant samples from 93 patients. Delayed centrifugation was performed in citrate samples from 31 additional patients, with hourly aPTT and anti-Xa measurement from 1 to 6 hours. In 14 of these last patients, PF4 release was also evaluated with Human CXCL4/PF4 Quantikine ELISA Kit. RESULTS: We observed a significant correlation between citrate and CTAD anticoagulant for aPTT (r2=0.94) and anti-Xa (r2=0.95). With Bland-Altman correlation, a minor bias was observed for anti-Xa (-0.025±0.041). Delayed centrifugation in citrated anticoagulant showed an excellent concordance from 1 to 4 hours for aPTT (-4.0±5.3 s) and anti-Xa (1.10-9±0.058 UI/mL) measurements. Moreover, PF4 release was not different between 1 hour (31.5±14.7 ng/mL) and 4 hours (33.8±11.8 ng/mL). CONCLUSION: We have demonstrated that anti-Xa measurement for unfractionated heparin should be done 4 hours in citrated plasma and that CTAD was not better than citrate. However, these initial findings require confirmation using other aPTT and calibrated anti-Xa assays.


Assuntos
Anticoagulantes/farmacologia , Coleta de Amostras Sanguíneas/métodos , Monitoramento de Medicamentos/métodos , Heparina/uso terapêutico , Adenosina/química , Adenosina/farmacologia , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Testes de Coagulação Sanguínea/métodos , Preservação de Sangue/métodos , Centrifugação/métodos , Fracionamento Químico/métodos , Ácido Cítrico/química , Ácido Cítrico/farmacologia , Dipiridamol/química , Dipiridamol/farmacologia , Fator Xa/metabolismo , Inibidores do Fator Xa/análise , Inibidores do Fator Xa/sangue , Heparina/análise , Humanos , Tempo de Tromboplastina Parcial , Teofilina/química , Teofilina/farmacologia , Tempo de Trombina , Fatores de Tempo
18.
Yakugaku Zasshi ; 140(1): 15-22, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-31902879

RESUMO

The development of serious lung diseases, such as pulmonary fibrosis, is associated with several drugs. A recent study has shown that the epithelial-mesenchymal transition (EMT) plays an essential role in the development of pulmonary fibrosis. However, the mechanisms underlying drug-induced EMT in alveolar epithelial cells have not been characterized. The present study showed that methotrexate (MTX), a drug known to cause lung injury, induced EMT-like phenotypic changes in an A549 cell model of the alveolar epithelium. We also found that the transforming growth factor (TGF)-ß1-mediated signaling pathway was associated with MTX-induced EMT. In addition, our results showed that certain secreted factors and microRNAs, a class of small noncoding RNAs, may be involved in MTX-induced EMT. The effects of COA-Cl, a novel synthetic small molecule, on TGF-ß1-induced EMT were evaluated to determine the therapeutic potential of COA-Cl against drug-induced lung injury. COA-Cl significantly suppressed TGF-ß1-induced EMT-like phenotypic changes, as evidenced by the inhibition of EMT-related transcription factors. Furthermore, MTX-induced EMT was completely suppressed by co-treatment with folic acid. Thus, these compounds may be promising therapeutic agents against drug-induced lung injury. In conclusion, the present study elucidated mechanisms underlying drug-induced EMT and highlighted a potential novel therapeutic approach to drug-induced lung diseases.


Assuntos
Lesão Pulmonar/induzido quimicamente , Metotrexato/efeitos adversos , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina/uso terapêutico , Animais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ácido Fólico/administração & dosagem , Ácido Fólico/uso terapêutico , Humanos , Lesão Pulmonar/tratamento farmacológico , MicroRNAs , Terapia de Alvo Molecular , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/fisiologia
19.
Am J Physiol Renal Physiol ; 318(4): F870-F877, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984792

RESUMO

Adenosine plays an important role in various aspects of kidney physiology, but the specific targets and mechanisms of actions are not completely understood. The collecting duct has the highest expression of adenosine receptors, particularly adenosine A1 receptors (A1Rs). Interstitial adenosine levels are greatly increased up to a micromolar range in response to dietary salt loading. We have previously shown that the basolateral membrane of principal cells has primarily K+ conductance mediated by Kir4.1/5.1 channels to mediate K+ recycling and to set up a favorable driving force for Na+/K+ exchange (47). Intercalated cells express the Cl- ClC-K2/b channel mediating transcellular Cl- reabsorption. Using patch-clamp electrophysiology in freshly isolated mouse collecting ducts, we found that acute application of adenosine reversely inhibits ClC-K2/b open probability from 0.31 ± 0.04 to 0.17 ± 0.06 and to 0.10 ± 0.05 for 1 and 10 µM, respectively. In contrast, adenosine (10 µM) had no measureable effect on Kir4.1/5.1 channel activity in principal cells. The inhibitory effect of adenosine on ClC-K2/b was abolished in the presence of the A1R blocker 8-cyclopentyl-1,3-dipropylxanthine (10 µM). Consistently, application of the A1R agonist N6-cyclohexyladenosine (1 µM) recapitulated the inhibitory action of adenosine on ClC-K2/b open probability. The effects of adenosine signaling in the collecting duct were independent from its purinergic counterpartner, ATP, having no measurable actions on ClC-K2/b and Kir4.1/5.1. Overall, we demonstrated that adenosine selectively inhibits ClC-K2/b activity in intercalated cells by targeting A1Rs. We propose that inhibition of transcellular Cl- reabsorption in the collecting duct by adenosine would aid in augmenting NaCl excretion during high salt intake.


Assuntos
Agonistas do Receptor A1 de Adenosina/farmacologia , Adenosina/farmacologia , Proteínas de Transporte de Ânions/antagonistas & inibidores , Canais de Cloreto/antagonistas & inibidores , Cloretos/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Receptor A1 de Adenosina/efeitos dos fármacos , Reabsorção Renal/efeitos dos fármacos , Animais , Proteínas de Transporte de Ânions/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Receptor A1 de Adenosina/metabolismo
20.
Am J Physiol Renal Physiol ; 318(3): F732-F740, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984796

RESUMO

Renal autoregulation is mediated by the myogenic response and tubuloglomerular feedback (TGF) working in concert to maintain renal blood flow and glomerular filtration rate despite fluctuations in renal perfusion pressure. Intercellular communication through gap junctions may play a role in renal autoregulation. We examine if one of the building blocks in gap junctions, connexin45 (Cx45), which is expressed in vascular smooth muscle cells, has an influence on renal autoregulatory efficiency. The isolated perfused juxtamedullary nephron preparation was used to measure afferent arteriolar diameter changes in response to acute changes in renal perfusion pressure. In segmental arteries, pressure myography was used to study diameter changes in response to pressure changes. Wire myography was used to study vasoconstrictor and vasodilator responses. A mathematical model of the vascular wall was applied to interpret experimental data. We found a significant reduction in the afferent arteriolar constriction in response to acute pressure increases in Cx45 knockout (KO) mice compared with wild-type (WT) mice. Abolition of TGF caused a parallel upward shift in the autoregulation curve of WT animals but had no effect in KO animals, which is compatible with TGF providing a basal tonic contribution in afferent arterioles whereas Cx45 KO animals were functionally papillectomized. Analysis showed a shift toward lower stress sensitivity in afferent arterioles from Cx45 KO animals, indicating that the absence of Cx45 may also affect myogenic properties. Finally, loss of Cx45 in vascular smooth muscle cells appeared to associate with a change in both structure and passive properties of the vascular wall.


Assuntos
Conexinas/metabolismo , Homeostase/fisiologia , Rim/fisiologia , Adenosina/farmacologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos
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