Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.231
Filtrar
1.
Int J Mol Sci ; 20(22)2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717457

RESUMO

Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8.


Assuntos
Benzodiazepinonas/farmacologia , Intestinos/patologia , Klebsiella oxytoca/efeitos dos fármacos , Junções Íntimas/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Humanos , Intestinos/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos
2.
Diabetes ; 68(11): 2107-2119, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31439645

RESUMO

The contribution of the sympathetic nervous system (SNS) versus the parasympathetic nervous system (PSNS) in mediating fatal cardiac arrhythmias during insulin-induced severe hypoglycemia is not well understood. Therefore, experimental protocols were performed in nondiabetic Sprague-Dawley rats to test the SNS with 1) adrenal demedullation and 2) chemical sympathectomy, and to test the PSNS with 3) surgical vagotomy, 4) nicotinic receptor (mecamylamine) and muscarinic receptor (AQ-RA 741) blockade, and 5) ex vivo heart perfusions with normal or low glucose, acetylcholine (ACh), and/or mecamylamine. In protocols 1-4, 3-h hyperinsulinemic (0.2 units/kg/min) and hypoglycemic (10-15 mg/dL) clamps were performed. Adrenal demedullation and chemical sympathectomy had no effect on mortality or arrhythmias during severe hypoglycemia compared with controls. Vagotomy led to a 6.9-fold decrease in mortality; reduced first- and second-degree heart block 4.6- and 4-fold, respectively; and prevented third-degree heart block compared with controls. Pharmacological blockade of nicotinic receptors, but not muscarinic receptors, prevented heart block and mortality versus controls. Ex vivo heart perfusions demonstrated that neither low glucose nor ACh alone caused arrhythmias, but their combination induced heart block that could be abrogated by nicotinic receptor blockade. Taken together, ACh activation of nicotinic receptors via the vagus nerve is the primary mediator of severe hypoglycemia-induced fatal cardiac arrhythmias.


Assuntos
Arritmias Cardíacas/fisiopatologia , Hipoglicemia/fisiopatologia , Sistema Nervoso Parassimpático/fisiopatologia , Animais , Arritmias Cardíacas/etiologia , Benzodiazepinonas/farmacologia , Modelos Animais de Doenças , Hipoglicemia/complicações , Masculino , Mecamilamina/farmacologia , Antagonistas Muscarínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Simpatectomia Química , Vagotomia
3.
EMBO Rep ; 20(8): e47026, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31379128

RESUMO

Checkpoint kinase 1 (CHK1) is critical for S-phase fidelity and preventing premature mitotic entry in the presence of DNA damage. Tumor cells have developed a strong dependence on CHK1 for survival, and hence, this kinase has developed into a promising drug target. Chk1 deficiency in mice results in blastocyst death due to G2/M checkpoint failure showing that it is an essential gene and may be difficult to target therapeutically. Here, we show that chemical inhibition of CHK1 kills murine and human hematopoietic stem and progenitor cells (HSPCs) by the induction of BCL2-regulated apoptosis. Cell death in HSPCs is independent of p53 but requires the BH3-only proteins BIM, PUMA, and NOXA. Moreover, Chk1 is essential for definitive hematopoiesis in the embryo. Noteworthy, cell death inhibition in HSPCs cannot restore blood cell formation as HSPCs lacking CHK1 accumulate DNA damage and stop dividing. Moreover, conditional deletion of Chk1 in hematopoietic cells of adult mice selects for blood cells retaining CHK1, suggesting an essential role in maintaining functional hematopoiesis. Our findings establish a previously unrecognized role for CHK1 in establishing and maintaining hematopoiesis.


Assuntos
Apoptose/genética , Células da Medula Óssea/metabolismo , Quinase 1 do Ponto de Checagem/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Benzodiazepinonas/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/deficiência , Embrião de Mamíferos , Feto , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Quinuclidinas/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
4.
Methods Mol Biol ; 2027: 49-59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31309471

RESUMO

Photochromic molecules can respond to external stimulations and undergo reversible conversion between different chemical structures, providing one photochromic molecule with multiple recognition states for targeting compounds. Here we design a facile sensor microchip with only one photochromic molecule (spirooxazine) to discriminate multiplex metal ions. The sensor chip performs in dark, ultraviolet, or visual stimulation, resulting in different molecular states of spirooxazine-metallic coordination and patterned fluorescent signals for analysis. By using this sensor microchip, 11 metal ions are discriminated. Furthermore, mineral water of 16 different brands and metal ions in human serum are distinguished.


Assuntos
Ensaios de Triagem em Larga Escala/instrumentação , Íons/sangue , Metais/sangue , Análise em Microsséries/instrumentação , Benzodiazepinonas , Corantes Fluorescentes/química , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , Íons/química , Metais/química , Águas Minerais/análise , Oxazinas/química , Compostos de Espiro/química
5.
ACS Chem Biol ; 14(9): 1913-1920, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31329413

RESUMO

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.


Assuntos
Receptor PAR-2/metabolismo , Receptores de GABA/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Aminoquinolinas/farmacologia , Benzamidas/farmacologia , Benzimidazóis/farmacologia , Benzodiazepinonas/farmacologia , Benzodioxóis/farmacologia , Álcoois Benzílicos/farmacologia , Bioensaio , Linhagem Celular Tumoral , Antagonistas GABAérgicos/farmacologia , Células HEK293 , Temperatura Alta , Humanos , Imidazóis/farmacologia , Transição de Fase/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Piridinas/farmacologia , Receptor PAR-2/antagonistas & inibidores , Receptor PAR-2/química , Receptores de GABA/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Tapsigargina/farmacologia
6.
Mol Carcinog ; 58(10): 1908-1918, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31313401

RESUMO

Gastrin signaling mediated through cholecystokinin-2 receptor (CCK2R) and its downstream molecules is altered in pancreatic cancer. CCK2R antagonists, YF476 (netazepide) and JNJ-26070109, were tested systematically for their effect on pancreatic intraepithelial neoplasia (PanIN) progression to pancreatic ductal adenocarcinoma (PDAC) in KrasG12D mice. After dose selection using wild-type mice, six-week-old p48Cre/+ -LSL-KrasG12D (22-24 per group) genetically engineered mice (GEM) were fed AIN-76A diets containing 0, 250, or 500 ppm JNJ-26070109 or YF-476 for 38 weeks. At termination, pancreata were collected, weighed, and evaluated for PanINs and PDAC. Results demonstrated that control-diet-fed mice showed 69% (males) and 33% (females) incidence of PDAC. Administration of low and high dose JNJ-26070109 inhibited the incidence of PDAC by 88% and 71% (P < .004) in male mice and by 100% and 24% (P > .05) in female mice, respectively. Low and high dose YF476 inhibited the incidence of PDAC by 74% (P < .02) and 69% (P < .02) in male mice and by 45% and 33% (P > .05) in female mice, respectively. Further, transcriptome analysis showed downregulation of Cldn1, Sstr1, Apod, Gkn1, Siglech, Cyp2c44, Bnc1, Fmo2, 623169, Kcne4, Slc27a6, Cma1, Rho GTPase activating protein 18, and Gpr85 genes in JNJ-26070109-treated mice compared with untreated mice. YF476-treated mouse pancreas showed downregulation of Riks, Zpbp, Ntf3, Lrrn4, Aass, Skint3, Kcnb1, Dgkb, Ddx60, and Aspn gene expressions compared with untreated mouse pancreas. Overall, JNJ-26070109 showed better chemopreventive efficacy than YF476. However, caution is recommended when selecting doses, as the agents appeared to exhibit gender-specific effects.


Assuntos
Adenocarcinoma/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Receptor de Colecistocinina B/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Benzodiazepinonas/farmacologia , Carcinoma in Situ , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Quimioprevenção/métodos , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinoxalinas/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia
7.
Expert Rev Anticancer Ther ; 19(6): 461-471, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31148500

RESUMO

Introduction: Small-cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumour, and its outcome is strongly conditioned by the rapid onset of resistance to conventional chemotherapeutics. First-line treatment with a combination of platinum agents and topoisomerase inhibitors has been the standard of care for over 30 years, with disappointing clinical outcome caused by early-acquired chemoresistance. In this disheartening scenario, novel treatment strategies are being implemented in order to either revert or bypass resistance mechanisms. Areas covered: The general mechanism of action of the standard frontline treatment regimens for SCLC, as well as the known resistance mechanisms to these drugs, is reviewed. Moreover, we focus on the current preclinical and clinical evidence on the potential role of PARP inhibitors and rovalpituzumab tesirine (Rova-T) to tackle chemoresistance in SCLC. Expert opinion: Preliminary evidence supports PARP inhibitors and Rova-T as two promising approaches to either revert or bypass chemoresistance in SCLC, respectively. The identification of potential predictive biomarkers of response to these innovative treatments (SLFN11 and DLL3) has shortened the gap between SCLC and personalized targeted therapy. Further large-scale clinical studies are urgently needed for a better designation of PARP inhibitors and Rova-T in the therapeutic algorithm of SCLC patients.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Benzodiazepinonas/administração & dosagem , Benzodiazepinonas/farmacologia , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacologia , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Carcinoma de Pequenas Células do Pulmão/patologia
8.
Pharmacol Res Perspect ; 7(3): e00484, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31149340

RESUMO

Mutations in leucine-rich repeat kinase 2 (LRRK2) gene have been pathogenically linked to Parkinson's disease, and pharmacological inhibition of LRRK2 is being pursued to tackle nigro-striatal dopaminergic neurodegeneration. However, LRRK2 kinase inhibitors may have manifold actions, affecting not only pathological mechanisms in dopaminergic neurons but also physiological functions in nondopaminergic neurons. Therefore, we investigated whether LRRK2 kinase inhibitors differentially modulate dopamine and glutamate release from the mouse striatum and cerebral cortex. Spontaneous and KCl-evoked [3H]-dopamine and glutamate release from superfused synaptosomes obtained from wild-type and LRRK2 knock-out, kinase-dead or G2019S knock-in mice was measured. Two structurally unrelated inhibitors, LRRK2-IN-1 and GSK2578215A, were tested. LRRK2, phosphoSerine1292 and phosphoSerine935 LRRK2 levels were measured in all genotypes, and target engagement was evaluated by monitoring phosphoSerine935 LRRK2. LRRK2-IN-1 inhibited striatal glutamate but not dopamine release; GSK2578215A inhibited striatal dopamine and cortical glutamate but enhanced striatal glutamate release. LRRK2-IN-1 reduced striatal and cortical phosphoSerine935 levels whereas GSK2578215A inhibited only the former. Neither LRRK2 inhibitor affected neurotransmitter release in LRRK2 knock-out and kinase-dead mice; however, they facilitated dopamine without affecting striatal glutamate in G2019S knock-in mice. GSK2578215A inhibited cortical glutamate release in G2019S knock-in mice. We conclude that LRRK2-IN-1 and GSK2578215A modulate exocytosis by blocking LRRK2 kinase activity, although their effects vary depending on the nerve terminal examined. The G2019S mutation unravels a dopamine-promoting action of LRRK2 inhibitors while blunting their effects on glutamate release, which highlights their positive potential for the treatment of PD, especially of LRRK2 mutation carriers.


Assuntos
Aminopiridinas/farmacologia , Benzamidas/farmacologia , Benzodiazepinonas/farmacologia , Corpo Estriado/citologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Pirimidinas/farmacologia , Córtex Visual/citologia , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Exocitose , Técnicas de Introdução de Genes , Ácido Glutâmico/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Masculino , Camundongos , Fosforilação , Serina/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Córtex Visual/efeitos dos fármacos , Córtex Visual/metabolismo
9.
Cell Oncol (Dordr) ; 42(3): 261-273, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30968324

RESUMO

BACKGROUND: Small-cell lung cancer (SCLC) is an aggressive disease with still limited therapeutic options. Despite being both a chemo- and radiation-sensitive malignancy, SCLC recurrence occurs in most cases and negatively impacts patients' prognosis. Over the last few years, a deeper understanding of SCLC molecular aberrations has led to the identification of Notch pathway deregulation as a crucial event in SCLC tumorigenesis, disease progression and chemoresistance. In particular, the delta-like protein 3 (DLL3), a Notch inhibitory ligand whose expression is directly related to the key neuroendocrine transcription factor ASCL1, was found to be expressed in ~85% of SCLCs, while it exhibits minimal to absent surface expression in normal lungs. DLL3 thus represents an appealing novel biomarker as well as a potential target in SCLC. CONCLUSIONS: The first DLL3-targeted antibody-drug conjugate rovalpituzumab tesirine (Rova-T, SC16LD6.5) has shown promising results in terms of efficacy and safety for the management of extensive SCLC, supporting further studies on this novel therapeutic approach that combines specific SCLC targeting with the cell-killing ability of a pyrrolobenzodiazepine dimer. In the present review, we discuss currently available evidence on the biological role of Notch signaling in SCLC from early preclinical findings to current and future clinical implications.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Benzodiazepinonas/uso terapêutico , Humanos , Imunoconjugados/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteínas de Membrana/antagonistas & inibidores , Prognóstico , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia
10.
Psychoneuroendocrinology ; 106: 65-76, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30954920

RESUMO

The translocator protein 18 kDa (TSPO), initially characterized as peripheral benzodiazepine receptor, is a conserved outer mitochondrial membrane protein, implicated in cholesterol transport thereby affecting steroid hormone biosynthesis, as well as in general mitochondrial function related to bioenergetics, oxidative stress, and Ca2+ homeostasis. TSPO is highly expressed in steroidogenic tissues such as adrenal glands, but shows low expression in the central nervous system. During various disease states such as inflammation, neurodegeneration or cancer, the expression of mitochondrial TSPO in affected tissues is upregulated. The expression of TSPO can be traced for diagnostic purpose by high affinity radio-ligands. Moreover, the function of TSPO is modulated by synthetic as well as endogenous ligands with agonistic or antagonistic properties. Thus, TSPO ligands serve functions as both important biomarkers and putative therapeutic agents. In the present study, we aimed to characterize the effects of TSPO ligands on mouse BV-2 microglia cells, which express significant levels of TSPO, and analyzed the effect of XBD173, PK11195, and Ro5-4864, as well as the inflammatory reagent Lipopolysaccharides (LPS) on neurosteroid synthesis and on basic mitochondrial functions such as oxidative phosphorylation, mitochondrial membrane potential and Ca2+ homeostasis. Specific TSPO-dependent effects were separated from off-target effects by comparing lentiviral TSPO knockdown with shRNA scramble-controls and wild-type BV-2 cells. Our data demonstrate ligand-specific effects on different cellular functions in a TSPO-dependent or independent manner, providing evidence for both specific TSPO-mediated, as well as off-target effects.


Assuntos
Microglia/metabolismo , Mitocôndrias/metabolismo , Receptores de GABA/metabolismo , Animais , Benzodiazepinonas/farmacologia , Linhagem Celular , Inflamação/metabolismo , Isoquinolinas/farmacologia , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/fisiologia , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Neuroesteroides/metabolismo , Purinas/farmacologia , Receptores de GABA/fisiologia , Receptores de GABA-A/metabolismo , Esteroides/metabolismo
11.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30940732

RESUMO

Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here, we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop.


Assuntos
Células-Tronco Germinativas Adultas/fisiologia , Autorrenovação Celular/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Benzodiazepinonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica/fisiologia , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
12.
Sci Transl Med ; 11(484)2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894499

RESUMO

Histologic transformation to small cell neuroendocrine prostate cancer occurs in a subset of patients with advanced prostate cancer as a mechanism of treatment resistance. Rovalpituzumab tesirine (SC16LD6.5) is an antibody-drug conjugate that targets delta-like protein 3 (DLL3) and was initially developed for small cell lung cancer. We found that DLL3 is expressed in most of the castration-resistant neuroendocrine prostate cancer (CRPC-NE) (36 of 47, 76.6%) and in a subset of castration-resistant prostate adenocarcinomas (7 of 56, 12.5%). It shows minimal to no expression in localized prostate cancer (1 of 194) and benign prostate (0 of 103). DLL3 expression correlates with neuroendocrine marker expression, RB1 loss, and aggressive clinical features. DLL3 in circulating tumor cells was concordant with matched metastatic biopsy (87%). Treatment of DLL3-expressing prostate cancer xenografts with a single dose of SC16LD6.5 resulted in complete and durable responses, whereas DLL3-negative models were insensitive. We highlight a patient with neuroendocrine prostate cancer with a meaningful clinical and radiologic response to SC16LD6.5 when treated on a phase 1 trial. Overall, our findings indicate that DLL3 is preferentially expressed in CRPC-NE and provide rationale for targeting DLL3 in patients with DLL3-positive metastatic prostate cancer.


Assuntos
Carcinoma Neuroendócrino/tratamento farmacológico , Carcinoma Neuroendócrino/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Terapia de Alvo Molecular , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Idoso , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Benzodiazepinonas/farmacologia , Benzodiazepinonas/uso terapêutico , Carcinoma Neuroendócrino/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Fatores de Tempo , Resultado do Tratamento
13.
Life Sci ; 221: 72-82, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30738868

RESUMO

AIMS: The proliferation of VSMCs is the pathologic basis for intimal hyperplasia after angioplasty in diabetic patients. Translocator protein (TSPO), located in the outer mitochondrial membrane, has been found to regulate redox intermediate components in cell dysfunction. We hypothesized that TSPO may regulate VSMC proliferation and migration, and be involved in the intimal hyperplasia after angioplasty in diabetes. MATERIALS AND METHODS: Cell proliferation was measured by cell counting and MTT assays. Cell migration was measured by Transwell® and scratch-wound assays. TSPO expression in arteries of rats and high glucose-treated A10 cells were detected by immunoblotting and immunofluorescence staining. Neointimal formation of carotid artery was induced by balloon injury in type 2 diabetic rat. KEY FINDINGS: TSPO expression was increased in the arterial samples from diabetic rats and A10 cells treated with high glucose. Down-regulation of TSPO expression by siRNA decreased the high-glucose-induced VSMC proliferation and migration in A10 cells. This phenomenon could be simulated by using TSPO ligands, PK 11195 and Ro5-4864. cGMP/PKG signals were involved in the TSPO ligand action, since in the presence of cGMP or PKG inhibitor ODQ or KT5823 respectively, the effect of PK 11195 on VSMC proliferation was blocked. Furthermore, PK 11195 significantly inhibited neointimal formation by the inhibition of VSMC proliferation. SIGNIFICANCE: This study suggests that TSPO inhibition suppresses the proliferation and migration of VSMCs induced by hyperglycemia, consequently, preventing atherosclerosis and restenosis after angioplasty in diabetic conditions. TSPO may be a potential therapeutic target to reduce arterial remodeling induced by angioplasty in diabetes.


Assuntos
Proteínas de Transporte/metabolismo , Hiperplasia/metabolismo , Receptores de GABA-A/metabolismo , Animais , Benzodiazepinonas/farmacologia , Artérias Carótidas/patologia , Proteínas de Transporte/fisiologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Hiperplasia/prevenção & controle , Isoquinolinas/farmacologia , Ligantes , Masculino , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/fisiologia
14.
Proc Natl Acad Sci U S A ; 116(9): 3774-3783, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808763

RESUMO

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.


Assuntos
Enterocolite Pseudomembranosa/genética , Enterotoxinas/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Klebsiella oxytoca/genética , Animais , Benzodiazepinonas/metabolismo , Benzodiazepinonas/toxicidade , Dano ao DNA/efeitos dos fármacos , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/biossíntese , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Intestinos/microbiologia , Intestinos/patologia , Infecções por Klebsiella/genética , Infecções por Klebsiella/microbiologia , Klebsiella oxytoca/metabolismo , Klebsiella oxytoca/patogenicidade , Camundongos , Microtúbulos/efeitos dos fármacos , Oxiquinolina/análogos & derivados , Oxiquinolina/metabolismo , Oxiquinolina/toxicidade , Peptídeos/metabolismo , Peptídeos/toxicidade
15.
Hum Mol Genet ; 28(10): 1645-1660, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30629163

RESUMO

Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Benzodiazepinonas/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/análogos & derivados , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/patologia , Mitofagia/efeitos dos fármacos , Mutação , Doença de Parkinson/patologia , Fosforilação , Cultura Primária de Células , Pirimidinas/farmacologia
16.
Clin Cancer Res ; 25(4): 1261-1271, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30397180

RESUMO

PURPOSE: Isocitrate dehydrogenase (IDH)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%-90% IDH-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody-drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH-mutant glioma. EXPERIMENTAL DESIGN: We evaluated DLL3 expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known IDH wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH-mutant glioma tumorspheres was determined by cell viability assay. RESULTS: Compared to IDH wild-type glioblastoma, IDH-mutant gliomas have significantly higher DLL3 RNA (P < 1 × 10-15) and protein by IHC (P = 0.0014 and P < 4.3 × 10-6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived IDH-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. CONCLUSIONS: DLL3 is selectively and homogeneously expressed in IDH-mutant gliomas and can be targeted with Rova-T in patient-derived IDH-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.


Assuntos
Glioma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Isocitrato Desidrogenase/genética , Proteínas de Membrana/genética , Terapia de Alvo Molecular , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Benzodiazepinonas/uso terapêutico , Encéfalo/patologia , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genótipo , Glioma/genética , Glioma/patologia , Humanos , Imunoconjugados/genética , Imunoconjugados/uso terapêutico , Ligantes , Masculino , Mutação , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , RNA/genética , Receptores Notch/genética
17.
J Biol Chem ; 294(1): 116-129, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30413535

RESUMO

Latency-reversing agents (LRAs) are considered a potential strategy for curing cells of HIV-1 infection. Certain protein kinase C (PKC) activators have been previously reported to be LRAs because they can reverse HIV latency. In the present study, we examined the activities of a panel of benzolactam derivatives against cells latently infected with HIV. Using determination of p24 antigen in cell supernatants or altered intracellular GFP expression to measure HIV reactivation from latently infected cells along with a cytotoxicity assay, we found that some of the compounds exhibited latency-reversing activity, which was followed by enhanced release of HIV particles from the cells. One derivative, BL-V8-310, displayed activity in ACH-2 and J-Lat cells latently infected with HIV at a concentration of 10 nm or higher, which was superior to the activity of another highly active PKC activator, prostratin. These results were confirmed with peripheral blood cells from HIV-infected patients. We also found that these drugs up-regulate the expression of caspase 3 and enhance apoptosis specifically in latently HIV-infected cells. Moreover, combining BL-V8-310 with a bromodomain-containing 4 (BRD4) inhibitor, JQ1, not only enhanced HIV latency-reversing activity, but also reduced the effect on cytotoxic cytokine secretion from CD4+ T-cells induced by BL-V8-310 alone. Our results suggest that BL-V8-310 and its related benzolactam derivatives are potential LRA lead compounds that are effective in reversing HIV latency and reducing viral reservoirs in HIV-positive individuals with few adverse effects.


Assuntos
Apoptose/efeitos dos fármacos , Benzodiazepinonas/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Proteína Quinase C/metabolismo , Latência Viral/efeitos dos fármacos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Caspase 3/biossíntese , Caspase 3/genética , Proteínas de Ciclo Celular , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/genética , Infecções por HIV/patologia , Humanos , Masculino , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Quinase C/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Neurosci Lett ; 690: 219-224, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30366010

RESUMO

P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC50 of 0.75 µM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 µM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 µM concentration but not 0.75 µM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.


Assuntos
Benzodiazepinonas/farmacologia , Receptores Purinérgicos P2X/fisiologia , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Hipocampo/fisiologia , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Antagonistas do Receptor Purinérgico P2X/farmacologia , Ratos , Receptores Purinérgicos P2X/genética
20.
Drug Test Anal ; 11(3): 541-549, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30578721

RESUMO

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC MS/MS), liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4'-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.


Assuntos
Benzodiazepinas/metabolismo , Benzodiazepinonas/metabolismo , Drogas Desenhadas/metabolismo , Flurazepam/análogos & derivados , Desentoxicação Metabólica Fase I , Microssomos Hepáticos/metabolismo , Biotransformação , Cromatografia Líquida , Flurazepam/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Técnicas In Vitro , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA