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1.
Life Sci ; 259: 118180, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32758622

RESUMO

AIMS: Bufothionine had been used for gastric cancer (GC) treatment, and this study managed to uncover the underlying mechanisms. MATERIALS AND METHODS: Cell proliferation was determined by CCK-8 assay and colony formation assay. Flow cytometry (FCM) and TUNEL assay were used to measure cell apoptosis ratio. Intracellular ROS was measured by DCFH-DA probes. qRT-PCR was used to determine miRNAs levels. Western Blot was performed to probe proteins. Dual-luciferase reporter gene system was employed to validate the binding sites of miR-133a-3p and 3'UTR regions of IGF1R mRNA. Immunohistochemistry (IHC) was used to determine the expressions of Ki-67 in mice tumor tissues. KEY FINDINGS: Bufothionine inhibited cell viability, triggered ER stress and promoted ROS production in GC cells, and both ER stress inhibitor Salburinal (Sal) and ROS scavenger (NAC) abrogated Bufothionine induced GC cell death. Besides, miR-133a-3p was upregulated by Bufothionine, and Bufothionine-induced cell death was enhanced by miR-133a-3p overexpression while alleviated by miR-133a-3p knockdown. Furthermore, miR-133a-3p inactivated PI3K/Akt signal pathway by sponging IGF1R, and Bufothionine inhibited insulin-like growth factor 1 receptor (IGF1R) and inactivated PI3K/Akt cascade by upregulating miR-133a-3p. Notably, the promoting effects of overexpressed miR-133a-3p on Bufothionine-induced GC cell death were abrogated by overexpressing IGF1R, and aggravated by the PI3K/Akt cascade inhibitor (LY294002). SIGNIFICANCE: Bufothionine promoted GC cell death by triggering miR-133a-3p/IGF1R/PI3K/Akt axis mediated ER stress and ROS production.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Alcaloides Indólicos/farmacologia , MicroRNAs/genética , Compostos de Quinolínio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Gástricas/patologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Proliferação de Células , Cromonas/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/biossíntese , Morfolinas/farmacologia , Proteína Oncogênica v-akt/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor IGF Tipo 1/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Proc Natl Acad Sci U S A ; 117(34): 20803-20813, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32764148

RESUMO

Virus entry is a multistep process. It initiates when the virus attaches to the host cell and ends when the viral contents reach the cytosol. Genetically unrelated viruses can subvert analogous subcellular mechanisms and use similar trafficking pathways for successful entry. Antiviral strategies targeting early steps of infection are therefore appealing, particularly when the probability for successful interference through a common step is highest. We describe here potent inhibitory effects on content release and infection by chimeric vesicular stomatitis virus (VSV) containing the envelope proteins of Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (VSV-SARS-CoV-2) elicited by Apilimod and Vacuolin-1, small-molecule inhibitors of the main endosomal phosphatidylinositol-3-phosphate/phosphatidylinositol 5-kinase, PIKfyve. We also describe potent inhibition of SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 by Apilimod. These results define tools for studying the intracellular trafficking of pathogens elicited by inhibition of PIKfyve kinase and suggest the potential for targeting this kinase in developing small-molecule antivirals against SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Ebolavirus/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases , Triazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Betacoronavirus/fisiologia , Células Cultivadas , Infecções por Coronavirus , Ebolavirus/fisiologia , Edição de Genes , Humanos , Pandemias , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pneumonia Viral , Proteínas do Envelope Viral/genética
3.
Nat Commun ; 11(1): 3288, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620753

RESUMO

The prognostic and therapeutic relevance of molecular subtypes for the most aggressive isocitrate dehydrogenase 1/2 (IDH) wild-type glioblastoma (GBM) is currently limited due to high molecular heterogeneity of the tumors that impedes patient stratification. Here, we describe a distinct binary classification of IDH wild-type GBM tumors derived from a quantitative proteomic analysis of 39 IDH wild-type GBMs as well as IDH mutant and low-grade glioma controls. Specifically, GBM proteomic cluster 1 (GPC1) tumors exhibit Warburg-like features, neural stem-cell markers, immune checkpoint ligands, and a poor prognostic biomarker, FKBP prolyl isomerase 9 (FKBP9). Meanwhile, GPC2 tumors show elevated oxidative phosphorylation-related proteins, differentiated oligodendrocyte and astrocyte markers, and a favorable prognostic biomarker, phosphoglycerate dehydrogenase (PHGDH). Integrating these proteomic features with the pharmacological profiles of matched patient-derived cells (PDCs) reveals that the mTORC1/2 dual inhibitor AZD2014 is cytotoxic to the poor prognostic PDCs. Our analyses will guide GBM prognosis and precision treatment strategies.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Isocitrato Desidrogenase/genética , Proteogenômica/métodos , Proteômica/métodos , Benzamidas/farmacologia , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Isocitrato Desidrogenase/classificação , Isocitrato Desidrogenase/metabolismo , Estimativa de Kaplan-Meier , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Morfolinas/farmacologia , Mutação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia
4.
Nature ; 586(7827): 113-119, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32707573

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.


Assuntos
Antivirais/análise , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Linhagem Celular , Inibidores de Cisteína Proteinase/análise , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Morfolinas/análise , Morfolinas/farmacologia , Pandemias , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Triazinas/análise , Triazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
5.
Am J Physiol Heart Circ Physiol ; 319(2): H507-H518, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32706268

RESUMO

The lymphatic system drains and propels lymph by extrinsic and intrinsic mechanisms. Intrinsic propulsion depends upon spontaneous rhythmic contractions of lymphatic muscles in the vessel walls and is critically affected by changes in the surrounding tissue like osmolarity and temperature. Lymphatics of the diaphragm display a steep change in contraction frequency in response to changes in temperature, and this, in turn, affects lymph flow. In the present work, we demonstrated in an ex vivo diaphragmatic tissue rat model that diaphragmatic lymphatics express transient receptor potential channels of the vanilloid 4 subfamily (TRPV4) and that their blockade by both the nonselective antagonist Ruthenium Red and the selective antagonist HC-067047 abolished the response of lymphatics to temperature changes. Moreover, the selective activation of TRPV4 channels by means of GSK1016790A mirrored the behavior of vessels exposed to increasing temperatures, pointing out the critical role played by these channels in sensing the temperature of the lymphatic vessels' environment and thus inducing a change in contraction frequency and lymph flow.NEW & NOTEWORTHY The present work addresses the putative receptor system that enables diaphragmatic lymphatics to change intrinsic contraction frequency and thus lymph flow according to the changes in temperature of the surrounding environment, showing that this role can be sustained by TRPV4 channels alone.


Assuntos
Linfa/fisiologia , Vasos Linfáticos/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Canais de Cátion TRPV/metabolismo , Temperatura , Animais , Diafragma , Feminino , Técnicas In Vitro , Vasos Linfáticos/efeitos dos fármacos , Masculino , Morfolinas/farmacologia , Músculo Liso/efeitos dos fármacos , Periodicidade , Pirróis/farmacologia , Ratos , Ratos Wistar , Rutênio Vermelho/farmacologia , Transdução de Sinais , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Fatores de Tempo
6.
Life Sci ; 256: 117972, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32544464

RESUMO

Acute kidney injury (AKI) has a high morbidity and mortality, and there is no targeted treatment yet. One of the main causes of AKI is ischemia-reperfusion (IR). Increased release of adenosine under stress and hypoxia exerts anti-inflammatory and antioxidant effects. Adenosine kinase (ADK) is an important enzyme that eliminates adenosine in cells, and can maintain low adenosine concentration in cells. Our previous studies have shown that pretreatment of adenosine kinase inhibitor ABT-702 could markedly attenuate cisplatin-induced nephrotoxicity both in vivo and in vitro. This study is designed to investigate the effect of ADK inhibition on IR-induced AKI. The results showed that ADK expression was positively correlated with the degree of renal tubular injury, which suggested that the degree of ADK inhibition reflected the severity of acute tubular necrosis. In vivo, ADK inhibitor could reduce IR-induced renal injury, which might play a protective role by increasing tissue adenosine level, inhibiting oxidative stress, and reducing cell apoptosis. In HK2 cells, cobaltous dichloride (CoCl2) increased the level of oxidative stress, up-regulated the production of pro-inflammatory factor, and induced apoptosis, ADK inhibition could alleviate the above damaging effects. Moreover, the anti-apoptotic effect exerted by ADK inhibition was independent of inosine. In summary, our results support the idea that ADK inhibition has protective effects on IR-induced AKI. Adenosine kinase inhibition might provide a new target for AKI prevention and treatment.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Lesão Renal Aguda/etiologia , Adenosina Quinase/antagonistas & inibidores , Morfolinas/uso terapêutico , Pirimidinas/uso terapêutico , Traumatismo por Reperfusão/complicações , Adenosina Quinase/metabolismo , Adulto , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Cobalto , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Inflamação/patologia , Inosina/farmacologia , Túbulos Renais/enzimologia , Túbulos Renais/patologia , Masculino , Camundongos Endogâmicos C57BL , Morfolinas/farmacologia , Necrose , Estresse Oxidativo/efeitos dos fármacos , Pirimidinas/farmacologia
7.
PLoS One ; 15(5): e0233020, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437355

RESUMO

Signaling through the endocannabinoid system is critical to proper functioning of the cerebellar circuit. However, most studies have focused on signaling through cannabinoid type 1 (CB1) receptors, while relatively little is known about signaling through type 2 (CB2) receptors. We show that functional CB2 receptors are expressed in Purkinje cells using a combination of immunohistochemistry and patch-clamp electrophysiology in juvenile mice. Pharmacological activation of CB2 receptors significantly reduces inhibitory synaptic responses and currents mediated by photolytic uncaging of RuBi-GABA in Purkinje cells. CB2 receptor activation does not change the paired-pulse ratio of inhibitory responses and its effects are blocked by inclusion of GDP-ß-S in the internal solution, indicating a postsynaptic mechanism of action. However, CB2 receptors do not contribute to depolarization induced suppression of inhibition (DSI), indicating they are not activated by endocannabinoids synthesized and released from Purkinje cells using this protocol. This work demonstrates that CB2 receptors inhibit postsynaptic GABAA receptors by a postsynaptic mechanism in Purkinje cells. This represents a novel mechanism by which CB2 receptors may modulate neuronal and circuit function in the central nervous system.


Assuntos
Células de Purkinje/fisiologia , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Receptores de GABA-A/metabolismo , Animais , Canabinoides/farmacologia , Cicloexanos/farmacologia , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Morfolinas/farmacologia , Técnicas de Patch-Clamp , Quinolinas/farmacologia , Receptor CB2 de Canabinoide/agonistas , Membranas Sinápticas/fisiologia , Transmissão Sináptica
8.
Life Sci ; 252: 117666, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32298737

RESUMO

AIMS: Euscaphic acid and Tormentic acid are aglycones of Kaji-ichigoside F1 and Rosamultin, respectively. These four compounds are pentacyclic triterpenoid, isolated from the subterranean root of the Potentilla anserina L. Based on the protective roles against hypoxia-induced apoptosis of Euscaphic acid and Tormentic acid in vascular endothelial cells, this study was designed to determine the mechanisms. MAIN METHODS: The model of hypoxic injuries in EA. hy926 cells was established. Through applications of PI3K/AKT inhibitor, LY294002 and ERK1/2 inhibitor, PD98059, we explored the relationships between pharmacodynamic mechanisms and PI3K/AKT or ERK 1/2 signaling pathway. The anti-hypoxic effects were studied by methyl-thiazolyl-tetrazolium (MTT) assay, Hematoxylin-Eosin (HE) staining, DAPI staining, and flow cytometry. The mechanisms of anti-mitochondrial apoptosis were explored by western blot. The expressions of p-ERK 1/2, ERK 1/2, p-AKT, AKT, p-NF-κB, NF-κB, Bcl-2, Bax, Cyt C, cleaved caspase-9 and cleaved caspase-3 were detected. KEY FINDINGS: Euscaphic acid protected vascular endothelial cells against hypoxia-induced apoptosis via ERK1/2 signaling pathway, and Tormentic acid brought its efficacy into full play via PI3K/AKT and ERK1/2 signaling pathways. In addition, PI3K/AKT signaling pathway positively regulated ERK1/2 pathway, and ERK1/2 pathway negatively regulated PI3K/AKT pathway. SIGNIFICANCE: This evidence provides theoretical and experimental basis for the following research on anti-hypoxic drugs of Potentilla anserina L.


Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Triterpenos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Cromonas/farmacologia , Células Endoteliais/metabolismo , Flavonoides/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Potentilla/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Proc Natl Acad Sci U S A ; 117(18): 9991-10002, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312805

RESUMO

The initial response to an addictive substance can facilitate repeated use: That is, individuals experiencing more positive effects are more likely to use that drug again. Increasing evidence suggests that psychoactive cannabinoid use in adolescence enhances the behavioral effects of cocaine. However, despite the behavioral data, there is no neurobiological evidence demonstrating that cannabinoids can also alter the brain's initial molecular and epigenetic response to cocaine. Here, we utilized a multiomics approach (epigenomics, transcriptomics, proteomics, and phosphoproteomics) to characterize how the rat brain responds to its first encounter with cocaine, with or without preexposure to the synthetic cannabinoid WIN 55,212-2 (WIN). We find that in adolescent (but not in adult) rats, preexposure to WIN results in cross-sensitization to cocaine, which correlates with histone hyperacetylation and decreased levels of HDAC6 in the prefrontal cortex (PFC). In the PFC, we also find that WIN preexposure blunts the typical mRNA response to cocaine and instead results in alternative splicing and chromatin accessibility events, involving genes such as Npas2 Moreover, preexposure to WIN enhances the effects of cocaine on protein phosphorylation, including ERK/MAPK-targets like gephyrin, and modulates the synaptic AMPAR/GluR composition both in the PFC and the nucleus accumbens (NAcc). PFC-NAcc gene network topological analyses, following cocaine exposure, reveal distinct top nodes in the WIN preexposed group, which include PACAP/ADCYAP1. These preclinical data demonstrate that adolescent cannabinoid exposure reprograms the initial behavioral, molecular, and epigenetic response to cocaine.


Assuntos
Comportamento Aditivo/genética , Comportamento Animal/efeitos dos fármacos , Canabinoides/efeitos adversos , Cocaína/efeitos adversos , Adolescente , Animais , Comportamento Aditivo/induzido quimicamente , Comportamento Aditivo/patologia , Benzoxazinas/efeitos adversos , Benzoxazinas/farmacologia , Canabinoides/farmacologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Cocaína/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Desacetilase 6 de Histona/genética , Humanos , Proteínas de Membrana/farmacologia , Morfolinas/efeitos adversos , Morfolinas/farmacologia , Naftalenos/efeitos adversos , Naftalenos/farmacologia , Fosfoproteínas/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Córtex Pré-Frontal/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Ratos , Transcriptoma/efeitos dos fármacos
10.
Arch Biochem Biophys ; 685: 108352, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32240637

RESUMO

Rab1A, a member of the Ras-like protein in rat brain (Rab) family, acts as an oncogene in a variety of malignant tumors. Previous studies reported that Rab1A was highly expressed in GC tissues. However, the function and molecular mechanism of Rab1A in gastric cancer (GC) development remain far from being addressed. Rab1A mRNA and protein levels were detected by qRT-PCR and western blot, respectively. Cell proliferation was evaluated by CCK-8 and BrdU incorporation assays. Apoptosis was estimated by flow cytometry analysis and western blot analysis of B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), Bcl-2 associated X (Bax), and Bcl-2 homologous antagonist/killer (Bak) expression. Alteration of the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway was detected by western blot. We found that Rab1A expression at both mRNA and protein was upregulated in GC cells. Rab1A knockdown significantly inhibited cell proliferation and induced apoptosis in GC cells. Rab1A overexpression promoted proliferation, inhibited cisplatin-induced apoptosis, and increased xenograft growth. In addition, we found that Rab1A knockdown suppressed the mTOR/p70S6K pathway in GC cells. Moreover, activation of mTOR/p70S6K pathway by MHY1485 abolished the effects of Rab1A knockdown on cell proliferation and apoptosis. In conclusion, Rab1A knockdown repressed proliferation and promoted apoptosis in GC cells by inhibition of the mTOR/p70S6K pathway.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Transdução de Sinais/fisiologia , Neoplasias Gástricas/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Morfolinas/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Triazinas/farmacologia , Regulação para Cima , Proteínas rab1 de Ligação ao GTP/genética
11.
Invest Ophthalmol Vis Sci ; 61(3): 3, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32150246

RESUMO

Purpose: In the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals. Methods: Using whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices. Results: Activation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs. Conclusions: CB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.


Assuntos
Receptor CB1 de Canabinoide/fisiologia , Células Bipolares da Retina/fisiologia , Células Amácrinas/metabolismo , Células Amácrinas/fisiologia , Animais , Benzoxazinas/farmacologia , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Endocanabinoides/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Retroalimentação Fisiológica/fisiologia , Feminino , Antagonistas de Receptores de GABA-A/farmacologia , Ácido Glutâmico/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Morfolinas/farmacologia , Naftalenos/farmacologia , Técnicas de Patch-Clamp/métodos , Ácidos Fosfínicos/farmacologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/efeitos dos fármacos , Retina , Células Bipolares da Retina/efeitos dos fármacos , Transdução de Sinais/fisiologia
12.
World Neurosurg ; 138: e659-e664, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32179193

RESUMO

OBJECTIVE: To investigate mechanism of endoplasmic reticulum (ER) stress-mediated autophagy in spinal cord injury (SCI). METHODS: An in vitro model of spinal cord injury (SCI) was established by recombinant human beta nerve growth factor (NGF)-induced PC12 cells. Immunofluorescence was used to detect properties of PC12 cells induced by NGF. Western blot assay was used to detect expressions of the autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3)I/II, the ER stress-related protein (HSPA5/GRP78), as well as the PI3K/AKT/mTOR signaling pathway-related proteins after mechanical injury at different time points. Then the sample assigned into sham, SCI, LY294002, SCI+LY294002, 4-PBA (4-phenylbutyric acid), and SCI+4-PBA groups. The expressions of the LC3I/II and PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blot assay. RESULTS: NGF-induced PC12 cells have neurophysiological characteristics. After administration of the PI3K-specific inhibitor LY294002, phosphorylation levels of AKT and mTOR decreased, and the ratio of LC3II/I was higher in the inhibitor-treated injury group than the simple-injury group. After administration of the ER stress inhibitor 4-PBA, the results were similar to LY294002 group's results compared with SCI group. CONCLUSIONS: Our study showed that NGF-induced PC12 cells can induce autophagy and ER stress after mechanical injury. ER stress inhibitor 4-PBA obtained similar effects to PI3K inhibitor LY294002, enhanced autophagy via PI3K/AKT/mTOR signaling pathway.


Assuntos
Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Células PC12 , Fenilbutiratos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Cromonas/farmacologia , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Morfolinas/farmacologia , Proteína Oncogênica v-akt/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosforilação , Ratos , Proteínas Recombinantes/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Serina-Treonina Quinases TOR/efeitos dos fármacos
13.
Life Sci ; 248: 117474, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32112869

RESUMO

BACKGROUND/OBJECTIVES: Nicotinamide N-methyltransferase (NNMT) is a novel regulator of energy homeostasis in adipocytes. NNMT expression in adipose tissue is increased in obesity and diabetes. Knockdown of NNMT prevents mice from developing diet-induced obesity, which is closely linked to insulin resistance. An early sign of systemic insulin resistance is reduced expression of glucose transporter 4 (GLUT4) selectively in adipose tissue. Adipose tissue-specific knockout and overexpression of GLUT4 cause reciprocal changes in NNMT expression. The aim of the current study was to elucidate the mechanism that regulates NNMT expression in adipocytes. METHODS: 3T3-L1 adipocytes were cultured in media with varying glucose concentrations or activators and inhibitors of intracellular pathways. NNMT mRNA and protein levels were measured with quantitative polymerase chain reaction and Western blotting. RESULTS: Glucose deprivation of 3T3-L1 adipocytes induced a 2-fold increase in NNMT mRNA and protein expression. This effect was mimicked by inhibition of glucose transport with phloretin, and by inhibition of glycolysis with the phosphoglucose isomerase inhibitor 2-deoxyglucose. Conversely, inhibition of the pentose phosphate pathway did not affect NNMT expression. Pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin (mTOR) pathway caused an increase in NNMT levels that was similar to the effect of glucose deprivation. Activation of mTOR with MHY1485 prevented the effect of glucose deprivation on NNMT expression. Furthermore, upregulation of NNMT levels depended on functional autophagy and protein translation. CONCLUSION: Glucose availability regulates NNMT expression via an mTOR-dependent mechanism.


Assuntos
Adipócitos/efeitos dos fármacos , Transportador de Glucose Tipo 4/genética , Glucose/farmacologia , Nicotinamida N-Metiltransferase/genética , Serina-Treonina Quinases TOR/genética , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Desoxiglucose/farmacologia , Metabolismo Energético/genética , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Homeostase/genética , Camundongos , Morfolinas/farmacologia , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Floretina/farmacologia , Biossíntese de Proteínas , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Triazinas/farmacologia
14.
Proc Natl Acad Sci U S A ; 117(14): 8013-8021, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193335

RESUMO

AMP-activated protein kinase (AMPK) functions as an energy sensor and is pivotal in maintaining cellular metabolic homeostasis. Numerous studies have shown that down-regulation of AMPK kinase activity or protein stability not only lead to abnormality of metabolism but also contribute to tumor development. However, whether transcription regulation of AMPK plays a critical role in cancer metastasis remains unknown. In this study, we demonstrate that AMPKα1 expression is down-regulated in advanced human breast cancer and is associated with poor clinical outcomes. Transcription of AMPKα1 is inhibited on activation of PI3K and HER2 through ΔNp63α. Ablation of AMPKα1 expression or inhibition of AMPK kinase activity leads to disruption of E-cadherin-mediated cell-cell adhesion in vitro and increased tumor metastasis in vivo. Furthermore, restoration of AMPKα1 expression significantly rescues PI3K/HER2-induced disruption of cell-cell adhesion, cell invasion, and cancer metastasis. Together, these results demonstrate that the transcription control is another layer of AMPK regulation and suggest a critical role for AMPK in regulating cell-cell adhesion and cancer metastasis.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Mama/patologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Cromonas/farmacologia , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lapatinib/farmacologia , Camundongos , Morfolinas/farmacologia , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Prognóstico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Análise Serial de Tecidos , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 110-118, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32027262

RESUMO

OBJECTIVE: To investigate the proliferation inhibition and pro-apoptosis effect of LY294002 (PI3K/AKT inhibtor) combined with daunorubicin (DNR) on the chronic myeloid leurenia cell line K562 and its possible mechanisms. METHODS: The effect of LY294002 and DNR on the proliferation of K562 cells in different treating time and concentration were measured by MTT assay. The cell cycle was determined by flow cytometry, the mRNA and protein expression of SKP2 , P27, BCL-2 and BAX were determined by RT-PCR and Western blot. RESULTS: LY294002 and DNR were able to inhibit the growth of K562 cells and promote apoptosis in time- and concentration-dependent manner (P<0.05), both the cell proliferation-inhibiting rate and apoptosis rate in combination therapy group were higher than that in DNR-monotherapy group (P<0.05). After K562 cells treated by LY294002 combined DNR for 36 h, the cells were statistically significantly reduced in G2/M phase (P<0.05), as compared with control group and DNR group. Compared with DNR group, the cell level of G0/G1 phase rased (P<0.05) and cell level of S phase decreased (P>0.05). Compared with DNR group, the expresson of SKP2 and BCL-2 mRNA decreased, and the expression of P27 mRNA increased in the combination therapy group (P<0.05). The expression of BAX mRNA was not significantly different between different groups. The same result was found in the protein expression. CONCLUSION: LY294002 has the sensibilizative effect on DNR chemotherapy, which may be relative with blocking the cell cycle and inducing cell apoptosis.


Assuntos
Cromonas/farmacologia , Morfolinas/farmacologia , Apoptose , Daunorrubicina , Humanos , Células K562 , Fosfatidilinositol 3-Quinases
16.
Life Sci ; 246: 117428, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057901

RESUMO

PURPOSE: Arl4c is overexpressed in several cancer tissues and is involved in cancer development. Nevertheless, the exact mechanism that regulates Arl4c expression in lung cancer has not been fully elucidated. The aim of this study was to investigate the regulatory mechanism of Arl4c and to explore potential chemotherapeutic drugs targeting Arl4c. METHODS: Immunohistochemistry was used to examine Arl4c expression levels in human lung adenocarcinoma cancer specimens. Protein expression was detected by western blot. Overexpression of Arl4c-Flag protein was used to detect the ubiquitination of Arl4c. A short interfering RNA against Arl4c was used for gene silencing. RESULTS: Arl4c was overexpressed in lung cancer tissues, and knockdown of Arl4c expression by siRNA decreased lung cancer A549 and 95-D cell proliferation. In addition, Arl4c expression was downregulated via inhibition of the AKT pathway in A549 and 95-D cells, whereas exposure to benzo (a) pyrene (a carcinogen in smoke) increased Arl4c expression in 16HBE cells via AKT activation. Finally, we found that chemotherapy drug hydroxycamptothecin (HCPT) could decrease Arl4c expression levels by inhibiting the activation of the AKT pathway in A549 and 95-D cells. Moreover, accumulation of ubiquitinated Arl4c protein was increased by HCPT and LY294002 (an AKT inhibitor) treatment whereas the proteasome inhibitor MG-132 attenuated the inhibitory effect of HCPT and LY294002 on Arl4c expression. CONCLUSION: Here, we highlighted the AKT pathway as an important regulatory pathway for Arl4c expression in lung cancer cells and identified HCPT as a promising drug for lung adenocarcinoma treatment that functioned by targeting Arl4c expression.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células A549 , Western Blotting , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Cromonas/farmacologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Leupeptinas/farmacologia , Morfolinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fumar/efeitos adversos
17.
Clin Sci (Lond) ; 134(5): 439-458, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32091078

RESUMO

Sphingolipids have been implicated in the etiology of atherosclerosis. The commonly used sphingolipid inhibitors, myriocin (a ceramide inhibitor) and d-PDMP (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glycosphingolipid inhibitor), have shown therapeutic potential but their efficacy and their underlying mechanisms remain unclear. Here, apolipoprotein E-deficient (apoE-/-) mice were fed a high-fat diet (HFD) and treated with a control, myriocin, d-PDMP, or atorvastatin for 12 weeks. We analyzed the effects of these drugs on the size and detailed composition of atherosclerotic plaques. Molecular biological approaches were used to explore how the inhibitors affect lipid metabolism and foam-cell formation. Treatment with myriocin or d-PDMP led to smaller and less vulnerable atherosclerotic lesions and was almost as effective as atorvastatin. Sphingolipid inhibitors down-regulated the expression of monocyte chemotactic protein 1 (MCP-1) and its receptor chemoattractant cytokine receptor 2 (CCR2), which play a key role in monocyte recruitment. They also decreased pro-inflammatory Ly-6chigh monocytes and influenced the uptake of modified LDL by down-regulating the expression of cluster of differentiation 36 (CD36) and lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1). The inhibitors exhibited the advantage of maintaining normal glucose homeostasis compared with atorvastatin. These findings reveal for the first time that the modulation of sphingolipid synthesis can effectively alleviate atherosclerosis progression by preventing lipid uptake and reducing inflammatory responses in the arterial walls.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Ácidos Graxos Monoinsaturados/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Morfolinas/farmacologia , Vasculite/prevenção & controle , Animais , Anticolesterolemiantes/farmacologia , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Atorvastatina/farmacologia , Transporte Biológico/efeitos dos fármacos , Ceramidas/antagonistas & inibidores , Ceramidas/metabolismo , Glicoesfingolipídeos/antagonistas & inibidores , Glicoesfingolipídeos/metabolismo , Imunossupressores/farmacologia , Lipídeos/sangue , Lipídeos/farmacocinética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Placa Aterosclerótica/prevenção & controle , Vasculite/metabolismo
18.
Horm Cancer ; 11(2): 87-96, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32037484

RESUMO

Proline-, glutamic acid-, leucine-rich protein 1 (PELP1) is a novel estrogen receptor (ER) coregulator, demonstrated distinctive characters from other ERα coregulators, and has been suggested to be involved in metastasis of several cancers. In ERα-positive breast cancer, PELP1 overexpression enhanced ruffles and filopodium-like structure stimulated by estradiol (E2) through extranuclear cell signaling transduction hereby increased cell motility. However, whether PELP1 is also involved in extracellular matrix remodeling of ERα-positive breast cancer cells is still unknown. In this study, we investigated the role of PELP1 in E2-induced MMP-9 expression and the underlined mechanism. The results demonstrated the following: E2-induced ERα-positive MCF-7 breast cancer cell MMP-9 mRNA and protein expression in a rapid response and concentration-dependent manner. Knocked down PELP1 significantly suppressed E2-induced MMP-9 expression. E2-bovine serum albumin (BSA), a large molecular membrane-impenetrable conjugate of E2, can also upregulate MMP-9 protein expression in MCF-7, and the action of E2-BSA can be abolished by PI3K inhibitor LY294002; treating MCF-7 simultaneously with PELP1-shRNA and LY294002 did not show synergetic inhibitory effect on E2-BSA-induced MMP-9 expression. Our results indicated that estrogen-induced MMP-9 expression in ER-positive breast cancer cells may be through PELP1-mediated PI3K/Akt signaling pathway.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas Correpressoras/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Fatores de Transcrição/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Células MCF-7 , Metaloproteinase 9 da Matriz/genética , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
19.
Nat Commun ; 11(1): 414, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964872

RESUMO

WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.


Assuntos
Proteínas Desgrenhadas/metabolismo , Descoberta de Drogas/métodos , Receptores Frizzled/agonistas , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Piridinas/química , Tiofenos/química , Via de Sinalização Wnt/efeitos dos fármacos , Membrana Celular/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular/métodos , Morfolinas/farmacologia , Estudo de Prova de Conceito , Purinas/farmacologia , Piridinas/farmacologia , Receptor Smoothened/agonistas , Relação Estrutura-Atividade , Tiofenos/farmacologia
20.
Cancer Chemother Pharmacol ; 85(2): 401-412, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31901955

RESUMO

PURPOSE: ABL tyrosine kinase inhibitors (TKIs) have demonstrated potency in the treatment of chronic myeloid leukemia (CML) patients. However, resistance to ABL TKIs can develop in CML patients due to BCR-ABL point mutations. Furthermore, CUDC-907 is an oral inhibitor of class I phosphoinositide 3-kinase (PI3K) as well as class I and II histone deacetylase (HDAC) enzymes. METHODS: In this study, we evaluated the effect of combination therapy of CUDC-907 and ABL TKIs, using BCR-ABL-positive cell lines and primary samples. RESULTS: CUDC-907 treatment for 72 h resulted in cell growth inhibition. Over the same period, an increase in histone acetylation and both caspase three and poly (ADP-ribose) polymerase (PARP) enzyme activity was observed. When ABL TKI treatment and CUDC-907 treatment were combined, significantly greater cytotoxicity was observed. Moreover, combined oral therapy with ponatinib (20 mg/kg/day) and CUDC-907 (30 mg/kg/day) greatly inhibited tumor growth compared to each drug alone. Lastly, CUDC-907 treatment also inhibited the growth of Ba/F3 ponatinib-resistant cells, K562 nilotinib-resistant cells, and T315I mutant primary samples. CONCLUSION: Taken together, our results indicate that administration of CUDC-907, a dual PI3K and HDAC inhibitor, may be an effective strategy against ABL TKI-resistant cells, including cells harboring the T315I mutation. Moreover, CUDC-907 may enhance the cytotoxic effects of ABL TKI when a combined treatment strategy is used against Philadelphia chromosome-positive leukemia cells.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Cromossomo Filadélfia/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Imidazóis/farmacologia , Células K562 , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Pirimidinas/farmacologia
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