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1.
J Enzyme Inhib Med Chem ; 34(1): 1426-1438, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31401883

RESUMO

Anaplastic lymphoma kinase (ALK) has been recognised as a promising molecular target of targeted therapy for NSCLC. We performed SAR study of pyrazolo[3,4-b]pyridines to override crizotinib resistance caused by ALK-L1196M mutation and identified a novel and potent L1196M inhibitor, 10g. 10g displayed exceptional enzymatic activities (<0.5 nM of IC50) against ALK-L1196M as well as against ALK-wt. In addition, 10g is an extremely potent inhibitor of ROS1 (<0.5 nM of IC50) and displays excellent selectivity over c-Met. Moreover, 10g strongly suppresses proliferation of ALK-L1196M-Ba/F3 and H2228 cells harbouring EML4-ALK via apoptosis and the ALK signalling blockade. The results of molecular docking studies reveal that, in contrast to crizotinib, 10g engages in a favourable interaction with M1196 in the kinase domain of ALK-L1196M and hydrogen bonding with K1150 and E1210. This SAR study has provided a useful insight into the design of novel and potent inhibitors against ALK gatekeeper mutant.


Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Quinase do Linfoma Anaplásico/metabolismo , Apoptose/efeitos dos fármacos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Espectroscopia de Prótons por Ressonância Magnética , Pirazóis/química , Piridinas/química , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade
2.
Food Chem Toxicol ; 131: 110583, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31220533

RESUMO

We investigated the anti-inflammatory activity of protopine (PTP) and sought to determine its mechanism of action in LPS-stimulated BV2 cells and a carrageenan (CA)-induced mouse model. Treatment with PTP (5, 10, and 20 µM) significantly suppresses the secretion of NO and PGE2 in a concentration-dependent manner without affecting cell viability by downregulating iNOS and COX-2 expression in LPS-induced BV2 cells. PTP also attenuates the production of pro-inflammatory chemokines, such as MCP-1, and cytokines, including TNF-α, IL-1ß and IL-6, and augments the expression of the anti-inflammatory cytokine IL-10. In addition, PTP suppresses the nuclear translocation of NF-κB by hindering the degradation of IκB and downregulating the expression of mitogen-activated protein kinases (MAPKs), including p38, ERK1/2 and JNK protein. Furthermore, PTP treatment significantly suppresses CA-induced paw oedema in mice compared to that seen in untreated mice. Expression of iNOS and COX-2 proteins is also abrogated by PTP (50 mg/kg) treatment in CA-induced mice. PTP treatment also abolishes IκB phosphorylation, which hinders the activation of NF-κB. Collectively, these results suggest PTP has potential for attenuating CA- and LPS-induced inflammatory symptoms through modulation of MAPKs/NF-κB signaling cascades.


Assuntos
Anti-Inflamatórios/uso terapêutico , Benzofenantridinas/uso terapêutico , Alcaloides de Berberina/uso terapêutico , Inflamação/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Anti-Inflamatórios/toxicidade , Benzofenantridinas/toxicidade , Alcaloides de Berberina/toxicidade , Carragenina , Linhagem Celular Transformada , Quimiocinas/metabolismo , Inflamação/induzido quimicamente , Lipopolissacarídeos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos
3.
Genes Dev ; 33(11-12): 684-704, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048545

RESUMO

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
4.
Methods Mol Biol ; 1966: 175-192, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041747

RESUMO

Nuclear receptors act as ligand-activated transcription factors translating ligand signals into changes in gene expression. The 48 members of the superfamily of nuclear receptors identified so far are involved amongst others in maintenance of metabolic balance, inflammation, and cancer. Thus, they hold enormous potential for drug discovery and some nuclear receptors are experiencing considerable academic and industrial interest. Nuclear receptor modulator discovery requires reliable, robust, and economic test systems that allow for high throughput. In this chapter, we discuss the principle, strengths, and advantages of hybrid reporter gene assays for nuclear receptor focused drug discovery and describe how they can be developed, established, and validated.


Assuntos
Bioensaio/métodos , Descoberta de Drogas/métodos , Genes Reporter , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular Transformada , Escherichia coli , Humanos , Ligantes
6.
Oxid Med Cell Longev ; 2019: 2715810, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31049129

RESUMO

Background: Hyperosmotic stress is an important pathophysiologic condition in diabetes, severe trauma, dehydration, infection, and ischemia. Furthermore, brain neuronal cells face hyperosmotic stress in ageing and Alzheimer's disease. Despite the enormous importance of knowing the homeostatic mechanisms underlying the responses of nerve cells to hyperosmotic stress, this topic has been underrepresented in the literature. Recent evidence points to autophagy induction as a hallmark of hyperosmotic stress, which has been proposed to be controlled by mTOR inhibition as a consequence of AMPK activation. We previously showed that methylglyoxal induced a decrease in the antioxidant proteins thioredoxin 1 (Trx1) and glyoxalase 2 (Glo2), which was mediated by AMPK-dependent autophagy. Thus, we hypothesized that hyperosmotic stress would have the same effect. Methods: HT22 hippocampal nerve cells were treated with NaCl (37, 75, or 150 mM), and the activation of the AMPK/mTOR pathway was investigated, as well as the levels of Trx1 and Glo2. To determine if autophagy was involved, the inhibitors bafilomycin (Baf) and chloroquine (CQ), as well as ATG5 siRNA, were used. To test for AMPK involvement, AMPK-deficient mouse embryonic fibroblasts (MEFs) were used. Results: Hyperosmotic stress induced a clear increase in autophagy, which was demonstrated by a decrease in p62 and an increase in LC3 lipidation. AMPK phosphorylation, linked to a decrease in mTOR and S6 ribosomal protein phosphorylation, was also observed. Deletion of AMPK in MEFs did not prevent autophagy induction by hyperosmotic stress, as detected by decreased p62 and increased LC3 II, or mTOR inhibition, inferred by decreased phosphorylation of P70 S6 kinase and S6 ribosomal protein. These data indicating that AMPK was not involved in autophagy activation by hyperosmotic stress were supported by a decrease in pS555-ULK1, an AMPK phosphorylation site. Trx1 and Glo2 levels were decreased at 6 and 18 h after treatment with 150 mM NaCl. However, this decrease in Trx1 and Glo2 in HT22 cells was not prevented by autophagy inhibition by Baf, CQ, or ATG5 siRNA. AMPK-deficient MEFs under hyperosmotic stress presented the same Trx1 and Glo2 decrease as wild-type cells. Conclusion: Hyperosmotic stress induced AMPK activation, but this was not responsible for its effects on mTOR activity or autophagy induction. Moreover, the decrease in Trx1 and Glo2 induced by hyperosmotic stress was independent of both autophagy and AMPK activation.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Neurônios/metabolismo , Pressão Osmótica , Transdução de Sinais , Tioléster Hidrolases/metabolismo , Tiorredoxinas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Linhagem Celular Transformada , Ativação Enzimática , Camundongos , Neurônios/citologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Tioléster Hidrolases/genética , Tiorredoxinas/genética
7.
Eur J Med Chem ; 174: 216-225, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31042617

RESUMO

The identification of a valid therapeutic treatment for Alzheimer's disease (AD) represents nowadays an urgent and still unmet medical need, since currently available anti-AD drugs only relieve symptoms and show a modest efficacy. Recent evidence indicates that multi-target-directed ligands (MTDLs) can potentially provide an effective strategy to develop innovative therapies directed towards the onset and progression of this multifactorial neurodegenerative disorder. In this work we designed, synthesized and evaluated a new series of MTDLs bearing the rivastigmine skeleton (ChE-inhibitor) linked to known metal-chelating moieties with linkers of different length. For all the novel derivatives, AChE/BuChE inhibitory activity, ROS scavenging activity and potential cytotoxicity have been assessed. For the best compound (4), copper chelating properties and neuroprotective effects were also evaluated. Our data demonstrated that hybrid derivative 4 is able to effectively inhibit AChE and BuChE and to chelate copper, showing a protective action on neurons. These results, although preliminary, indicate that compound 4 can be considered as a possible hit molecule for the development of new anti-AD MTDLs.


Assuntos
Quelantes/farmacologia , Complexos de Coordenação/farmacologia , Cobre/química , Fármacos Neuroprotetores/farmacologia , Rivastigmina/análogos & derivados , Rivastigmina/farmacologia , Acetilcolinesterase/metabolismo , Animais , Butirilcolinesterase/metabolismo , Linhagem Celular Transformada , Quelantes/síntese química , Quelantes/toxicidade , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/toxicidade , Complexos de Coordenação/síntese química , Complexos de Coordenação/toxicidade , Depuradores de Radicais Livres/síntese química , Depuradores de Radicais Livres/farmacologia , Depuradores de Radicais Livres/toxicidade , Camundongos , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/toxicidade , Rivastigmina/síntese química , Rivastigmina/toxicidade
8.
Molecules ; 24(7)2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970577

RESUMO

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation of the epidermal cells and is clinically presented as thick, bright red to pink plaques with a silvery scale. Photodynamic therapy (PDT) using visible light has become of increasing interest in the treatment of inflammatory skin diseases. In this study, we demonstrate that a combination of curcumin-loaded chitosan/alginate nanoparticles (Cur-CS/Alg NPs) and blue light emitting diodes (LED) light irradiation effectively suppressed the hyperproliferation of tumor necrosis factor-alpha (TNF-α)-induced cultured human kerlatinocyte (HaCaT) cells. The Cur-CS/Alg NPs were fabricated by emulsification of curcumin in aqueous sodium alginate solution and ionotropic gelation with calcium chloride and chitosan using an optimized formulation derived from a Box-Behnken design. The fabricated Cur-CS/Alg NPs were characterized for their particle size, zeta potential, encapsulation efficiency, and loading capacity. The surrogate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, to measure the relative number of viable cells, showed that the CS/Alg NPs were nontoxic to normal HaCaT cells, while 0.05 µg/mL and 0.1 µg/mL of free curcumin and Cur-CS/Alg NPs inhibited the hyperproliferation of HaCaT cells induced by TNF-α. However, the Cur-CS/Alg NPs demonstrated a stronger effect than the free curcumin, especially when combined with blue light irradiation (10 J/cm²) from an LED-based illumination device. Therefore, the Cur-CS/Alg NPs with blue LED light could be potentially developed into an effective PDT system for the treatment of psoriasis.


Assuntos
Proliferação de Células , Sistemas de Liberação de Medicamentos , Queratinócitos/metabolismo , Luz , Nanopartículas/química , Psoríase/terapia , Fator de Necrose Tumoral alfa/farmacologia , Alginatos/química , Alginatos/farmacologia , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Quitosana/química , Quitosana/farmacologia , Curcumina/química , Curcumina/farmacologia , Humanos , Queratinócitos/fisiologia , Psoríase/metabolismo , Psoríase/patologia , Fator de Necrose Tumoral alfa/metabolismo
9.
J Neuroinflammation ; 16(1): 69, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940161

RESUMO

BACKGROUND: Acute liver failure resulting from drug-induced liver injury can lead to the development of neurological complications called hepatic encephalopathy (HE). Hepatic transforming growth factor beta 1 (TGFß1) is upregulated due to liver failure in mice and inhibiting circulating TGFß reduced HE progression. However, the specific contributions of TGFß1 on brain cell populations and neuroinflammation during HE are not known. Therefore, the aim of this study was to characterize hepatic and brain TGFß1 signaling during acute liver failure and its contribution to HE progression using a combination of pharmacological and genetic approaches. METHODS: C57Bl/6 or neuron-specific transforming growth factor beta receptor 2 (TGFßR2) null mice (TGFßR2ΔNeu) were treated with azoxymethane (AOM) to induce acute liver failure and HE. The activity of circulating TGFß1 was inhibited in C57Bl/6 mice via injection of a neutralizing antibody against TGFß1 (anti-TGFß1) prior to AOM injection. In all mouse treatment groups, liver damage, neuroinflammation, and neurological deficits were assessed. Inflammatory signaling between neurons and microglia were investigated in in vitro studies through the use of pharmacological inhibitors of TGFß1 signaling in HT-22 and EOC-20 cells. RESULTS: TGFß1 was expressed and upregulated in the liver following AOM injection. Pharmacological inhibition of TGFß1 after AOM injection attenuated neurological decline, microglia activation, and neuroinflammation with no significant changes in liver damage. TGFßR2ΔNeu mice administered AOM showed no effect on liver pathology but significantly reduced neurological decline compared to control mice. Microglia activation and neuroinflammation were attenuated in mice with pharmacological inhibition of TGFß1 or in TGFßR2ΔNeu mice. TGFß1 increased chemokine ligand 2 (CCL2) and decreased C-X3-C motif ligand 1 (CX3CL1) expression in HT-22 cells and reduced interleukin-1 beta (IL-1ß) expression, tumor necrosis factor alpha (TNFα) expression, and phagocytosis activity in EOC-20 cells. CONCLUSION: Increased circulating TGFß1 following acute liver failure results in activation of neuronal TGFßR2 signaling, driving neuroinflammation and neurological decline during AOM-induced HE.


Assuntos
Córtex Cerebral/patologia , Encefalopatia Hepática/etiologia , Falência Hepática Aguda/complicações , Falência Hepática Aguda/patologia , Neurônios/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/deficiência , Fator de Crescimento Transformador beta1/sangue , Animais , Anticorpos/uso terapêutico , Azoximetano/toxicidade , Benzamidas/farmacologia , Carcinógenos/toxicidade , Linhagem Celular Transformada , Modelos Animais de Doenças , Encefalopatia Hepática/tratamento farmacológico , Inflamação/tratamento farmacológico , Inflamação/etiologia , Isoquinolinas/farmacologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
10.
Food Chem Toxicol ; 128: 129-136, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30940595

RESUMO

Sulforaphane (SFN) has shown anti-cancer effects in cellular and animal studies but its effectiveness has been limited in human studies. Here, the effects of SFN were measured in both human hepatocytes (HHL5) and hepatoma (HepG2) cells. Results showed that SFN inhibited cell viability and induced DNA strand breaks at high doses (≥20 µM). It also activated the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and increased intracellular glutathione (GSH) levels at 24 h. Pre-treatment with a low dose SFN (≤5 µM) protected against hydrogen peroxide (H2O2)-induced cell damage. High doses of SFN were more toxic towards HHL5 compared to HepG2 cells; the difference is likely due to the disparity in the responses of Nrf2-driven enzymes and -GSH levels between the two cell lines. In addition, HepG2 cells hijacked the cytoprotective effect of SFN over a wider dose range (1.25-20 µM) compared to HHL5. Manipulation of levels of GSH and Nrf2 in HepG2 cells confirmed that both molecules mediate the protective effects of SFN against H2O2. The non-specific nature of SFN in the regulation of cell death and survival could present undesirable risks, i.e. be more toxic to normal cells, and cause chemo-resistance in tumor cells. These issues should be addressed in the context for cancer prevention and treatment before large scale clinical trials are undertaken.


Assuntos
Antioxidantes/farmacologia , Isotiocianatos/farmacologia , Antioxidantes/administração & dosagem , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Células Hep G2 , Humanos , Peróxido de Hidrogênio/toxicidade , Isotiocianatos/administração & dosagem , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Interferente Pequeno/genética
11.
Cell Physiol Biochem ; 52(5): 1139-1150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30990584

RESUMO

BACKGROUND/AIMS: Fabry disease (FD) is a lysosomal storage disorder characterized by impaired alpha-galactosidase A (α-Gal A) enzyme activity due to mutations in the GLA gene. While virtually all tissues are affected, renal damage is particularly critical for the patients' outcome. Currently, powerful diagnostic tools and in vivo research models to study FD in the kidney are lacking, which is a major obstacle for further improvements in diagnosis and therapy. The present study focuses on the effects of enzyme replacement therapy on a previously established podocyte cell culture model of Fabry disease. METHODS: We investigated the effect of in vitro application of α-Gal A on Fabry podocytes for 3 days, mimicking enzyme replacement therapy. We studied reduction of Gb3 levels and dysregulated molecular pathways such as autophagy, mTOR/AKT signaling and pro-fibrotic signaling by employing immunofluorescence, electron microscopy, tandem mass spectrometry and western blot. RESULTS: We detected complete resolution of Gb3 accumulation in Fabry podocytes upon α-Gal A treatment. Despite robust Gb3 clearance, dysregulation of the signaling pathways investigated was not reversed. CONCLUSION: This study presents first evidence for Gb3-independent effects regarding dysregulation of signal transduction mechanisms in FD not recovering upon α-Gal A treatment. We assume that intracellular alterations observed in FD may have a point of no return after which a reversal of dysregulated cellular signal transduction by α-Gal A treatment is not effective, despite Gb3 clearance. Our observations suggest further research on signal transduction mechanisms altered in Fabry podocytes and on determining the appropriate time for initiation of Fabry therapy.


Assuntos
Terapia de Reposição de Enzimas , Doença de Fabry , Modelos Biológicos , Podócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triexosilceramidas/metabolismo , alfa-Galactosidase/uso terapêutico , Técnicas de Cultura de Células , Linhagem Celular Transformada , Doença de Fabry/tratamento farmacológico , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Humanos , Podócitos/patologia
12.
Cancer Sci ; 110(6): 1959-1973, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31004547

RESUMO

Activation of transforming growth factor ß (TGF-ß) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial-mesenchymal transition (EMT). Hypoxia-inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF-1α expression by protein phosphatase activity on dissociation of the E-cadherin/ß-catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF-ß. By using lung cancer cells treated with HIF-1α stabilizers or carrying doxycycline-dependent HIF-1α deletion or point mutants, we investigated the role of stabilized HIF-1α expression on TGF-ß-induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF-ß and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF-1α expression was inhibited. Stabilized HIF-1α protein expression inhibited the TGF-ß-stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF-1α-induced repression of the TGF-ß-stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF-1α expression on inhibition of TGF-ß-induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fator de Crescimento Transformador beta1/farmacologia , Animais , Hipóxia Celular , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estabilidade Proteica , Interferência de RNA , Ratos
13.
Mol Cell ; 74(1): 101-117.e10, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30827740

RESUMO

During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/Polycomb spreading, silencing, and changes in chromosome architecture.


Assuntos
Fibroblastos/metabolismo , Deleção de Genes , Inativação Gênica , Células-Tronco Embrionárias Murinas/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 2/genética , RNA Longo não Codificante/genética , Inativação do Cromossomo X , Cromossomo X/genética , Animais , Linhagem Celular Transformada , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Motivos de Nucleotídeos , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica , RNA Longo não Codificante/metabolismo , Sequências Repetitivas de Ácido Nucleico , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Cromossomo X/metabolismo
14.
Viruses ; 11(3)2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836639

RESUMO

Rhinoviruses (RVs) are classified into three species: RV-A, B, and C. Unlike RV-A and -B, RV-C cannot be propagated using standard cell culture systems. In order to isolate RV-Cs from clinical specimens and gain a better understanding of their biological properties and pathogenesis, we established air⁻liquid-interface (ALI) culture methods using HBEC3-KT and HSAEC1-KT immortalized human airway epithelial cells. HBEC3- and HSAEC1-ALI cultures morphologically resembled pseudostratified epithelia with cilia and goblet cells. Two fully sequenced clinical RV-C isolates, RV-C9 and -C53, were propagated in HBEC3-ALI cultures, and increases in viral RNA ranging from 1.71 log10 to 7.06 log10 copies were observed. However, this propagation did not occur in HSAEC1-ALI cultures. Using the HBEC3-ALI culture system, 11 clinical strains of RV-C were isolated from 23 clinical specimens, and of them, nine were passaged and re-propagated. The 11 clinical isolates were classified as RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 types according to their VP1 sequences. Our stable HBEC3-ALI culture system is the first cultivable cell model that supports the growth of multiple RV-C virus types from clinical specimens. Thus, the HBEC3-ALI culture system provides a cheap and easy-to-use alternative to existing cell models for isolating and investigating RV-Cs.


Assuntos
Diferenciação Celular , Enterovirus/crescimento & desenvolvimento , Células Epiteliais/virologia , Cultura de Vírus , Contagem de Células , Linhagem Celular Transformada , Enterovirus/genética , Enterovirus/fisiologia , Humanos , Sistema Respiratório/citologia
15.
Proc Natl Acad Sci U S A ; 116(14): 6858-6867, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894482

RESUMO

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Corpos Multivesiculares/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Transformada , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Edição de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Corpos Multivesiculares/genética , Transporte Proteico/fisiologia
16.
Biochim Biophys Acta Mol Cell Res ; 1866(5): 945-956, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30826331

RESUMO

The maintenance of stem cells often requires the support of feeder cells. Primary mouse embryonic fibroblasts (MEFs) have traditionally been used as feeder cells, and although these MEF-derived feeder cells have exhibited a reasonable performance, they require repeated cell isolation, since MEFs cannot expand indefinitely. To overcome this limitation, immortalized cells, such as STO cells, have been used. However, one major disadvantage is that previously reported immortalized cells can only support stem cell cultures for a relatively short period, typically 4 to 7 days. In this study, we found that our newly established rat-derived fibroblasts immortalized by the expression of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase, can function as feeder cells for relatively long cell culture periods of approximately 14 days. The rat-derived immortalized cells developed in this study should be a useful source of feeder cells to support stem cell research.


Assuntos
Ciclina D/biossíntese , Quinase 4 Dependente de Ciclina/biossíntese , Células Alimentadoras/metabolismo , Fibroblastos/metabolismo , Mutação , Células-Tronco/metabolismo , Telomerase/biossíntese , Animais , Linhagem Celular Transformada , Técnicas de Cocultura , Ciclina D/genética , Quinase 4 Dependente de Ciclina/genética , Células Alimentadoras/citologia , Fibroblastos/citologia , Camundongos , Ratos , Células-Tronco/citologia , Telomerase/genética
17.
J Neuroinflammation ; 16(1): 57, 2019 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-30851734

RESUMO

BACKGROUND: Neuromyelitis optica spectrum disorder (herein called NMO) is an inflammatory demyelinating disease that can be initiated by binding of immunoglobulin G autoantibodies (AQP4-IgG) to aquaporin-4 on astrocytes, causing complement-dependent cytotoxicity (CDC) and downstream inflammation. The increased NMO pathology in rodents deficient in complement regulator protein CD59 following passive transfer of AQP4-IgG has suggested the potential therapeutic utility of increasing the expression of complement regulator proteins. METHODS: A cell-based ELISA was developed to screen for pharmacological upregulators of endogenous CD55 and CD59 in a human astrocyte cell line. A statin identified from the screen was characterized in cell culture models and rodents for its action on complement regulator protein expression and its efficacy in models of seropositive NMO. RESULTS: Screening of ~ 11,500 approved and investigational drugs and nutraceuticals identified transcriptional upregulators of CD55 but not of CD59. Several statins, including atorvastatin, simvastatin, lovastatin, and fluvastatin, increased CD55 protein expression in astrocytes, including primary cultures, by three- to four-fold at 24 h, conferring significant protection against AQP4-IgG-induced CDC. Mechanistic studies revealed that CD55 upregulation involves inhibition of the geranylgeranyl transferase pathway rather than inhibition of cholesterol biosynthesis. Oral atorvastatin at 10-20 mg/kg/day for 3 days strongly increased CD55 immunofluorescence in mouse brain and spinal cord and reduced NMO pathology following intracerebral AQP4-IgG injection. CONCLUSION: Atorvastatin or other statins may thus have therapeutic benefit in AQP4-IgG seropositive NMO by increasing CD55 expression, in addition to their previously described anti-inflammatory and immunomodulatory actions.


Assuntos
Aquaporina 4/imunologia , Astrócitos/metabolismo , Antígenos CD55/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Imunoglobulina G/administração & dosagem , Neuromielite Óptica/tratamento farmacológico , Regulação para Cima/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações de Medicamentos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Camundongos , Neuromielite Óptica/metabolismo , Neuromielite Óptica/patologia , RNA Mensageiro/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Ativação Transcricional/efeitos dos fármacos , Receptor fas/metabolismo
18.
J Neuroinflammation ; 16(1): 65, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898121

RESUMO

BACKGROUND: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. METHODS: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. RESULTS: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. CONCLUSIONS: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.


Assuntos
Dor Crônica/diagnóstico , Regulação da Expressão Gênica/fisiologia , Hibridização In Situ/métodos , Elementos da Série dos Lantanídeos/metabolismo , RNA Mensageiro/metabolismo , Receptor 4 Toll-Like/genética , Animais , Linhagem Celular Transformada , Dor Crônica/etiologia , Modelos Animais de Doenças , Fluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Luminescência , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microglia/efeitos dos fármacos , Microglia/metabolismo , Medição da Dor , Neuropatia Ciática/complicações , Neuropatia Ciática/diagnóstico , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Receptor 4 Toll-Like/metabolismo
19.
Methods Mol Biol ; 1951: 75-85, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825145

RESUMO

Macrophages are professional phagocytic cells that play key roles in innate and adaptive immunity, metabolism, and tissue homeostasis. Lipid metabolism is tightly controlled at the transcriptional level, and one of the key players of this regulation in macrophages and other cell types is the LXR subfamily of nuclear receptors (LXRα and LXRß). The use of LXR double knockout (LXR-DKO) macrophages in vitro has yielded extensive benefits in metabolism research, but this technique is hindered by primary macrophage cell expansion capability, which diminishes along terminal cell differentiation process. Here we detail a method to immortalize LXR double knockout bone marrow-derived macrophage cells at an early stage of differentiation, using a retroviral delivery of a combination of murine v-myc and v-raf oncogenes. This methodology enables the generation of autonomous self-renewing macrophages bearing an LXR-DKO genetic background, as a valuable tool for research in lipid metabolism and other LXR receptor-mediated effects.


Assuntos
Receptores X do Fígado/deficiência , Macrófagos/metabolismo , Animais , Biomarcadores , Linhagem Celular Transformada , Vetores Genéticos/genética , Receptores X do Fígado/metabolismo , Macrófagos/imunologia , Camundongos , Retroviridae/genética , Transdução Genética , Transgenes
20.
Cell Physiol Biochem ; 52(2): 198-211, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816668

RESUMO

BACKGROUND/AIMS: Directional migration of corneal epithelial cells is essential for healing of corneal wounds, which is a robust response mediated by biochemical and bioelectrical cues. Naturally occurring electric fields at corneal wounds provide a powerful guidance cue for directional cell migration, as does extracellular ATP. Our recent large-scale siRNA library screening identified a role for purinergic signaling in the electric field-guided migration (galvanotaxis/electrotaxis) of human corneal epithelial (hTCEpi) cells. METHODS: We examined the effect of extracellular ATP on galvanotaxis of hTCEpi cells. Galvanotactic cell migration was recorded by video microscopy, and directedness and migration speed was calculated. The role of purinergic receptors in galvanotaxis regulation was evaluated by pharmacological inhibition or knocking down of P2X and P2Y receptors. RESULTS: Addition of ATP enhanced galvanotaxis, and most remarkably sensitized galvanotaxis response to very low level of electric fields in the physiological range (10-30 mV/mm). The stimulatory effect of extracellular ATP was diminished by apyrase treatment. Importantly, cells stimulated with extracellular ATP migrated with significantly increased directedness and speed, which were diminished by knocking down or pharmacological inhibition of P2X and P2Y receptors. Inhibition of pannexin-1 (ATP permeable channel) significantly impaired galvanotaxis. Moreover, pharmacological inhibition of ectoATPase enhanced galvanotaxis. CONCLUSION: Extracellular ATP and physiological electric fields synergistically enhanced the galvanotaxis response of hTCEpi cells. hTCEpi cells are likely to secrete ATP actively, and purinergic signaling is down-regulated by ecto-ATPases. Both P2X and P2Y receptors coordinately play a role for galvanotaxis of hTCEpi cells.


Assuntos
Trifosfato de Adenosina/metabolismo , Movimento Celular , Campos Eletromagnéticos , Células Epiteliais/metabolismo , Epitélio Anterior/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Transdução de Sinais , Linhagem Celular Transformada , Humanos
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