RESUMEN
Cardio-oncology is a new discipline that focuses on understanding, detection, monitoring and treating cardiovascular disease during and after cancer treatment. The development of this emerging field is based on an interdisciplinary collaboration between cardiology and oncology researchers and clinicians. Cardio-oncology aims at identifying how cancer therapies impact cardiovascular homeostasis, particular risk factors, diagnostic biomarkers and novel therapeutic approaches to help to effectively detect, prevent and cure the cardiotoxicity.
Asunto(s)
Antineoplásicos/efectos adversos , Cardiología/tendencias , Cardiopatías/etiología , Oncología Médica/tendencias , Antineoplásicos/uso terapéutico , Cardiología/normas , Cardiotoxicidad , Certificación , Cardiopatías/inducido químicamente , Cardiopatías/terapia , Humanos , Oncología Médica/normas , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Grupo de Atención al PacienteRESUMEN
This corrects the article DOI: 10.1038/onc.2017.93.
RESUMEN
KRAS is one of the most frequently mutated oncogenes in human non-small cell lung cancers (NSCLCs). RAS proteins trigger multiple effector signalling pathways including the highly conserved RAF-MAPK pathway. CRAF, a direct RAS effector protein, is required for KRAS-mediated tumourigenesis. Thus, the molecular mechanisms driving the activation of CRAF are intensively studied. Prohibitin 1 (PHB1) is an evolutionarily conserved adaptor protein and interaction of CRAF with PHB1 at the plasma membrane is essential for CRAF activation. Here, we demonstrate that PHB1 is highly expressed in NSCLC patients and correlates with poor survival. Targeting of PHB1 with two chemical ligands (rocaglamide and fluorizoline) inhibits epidermal growth factor (EGF)/RAS-induced CRAF activation. Consistently, treatment with rocaglamide inhibited proliferation, migration and anchorage-independent growth of KRAS-mutated lung carcinoma cell lines. Surprisingly, rocaglamide treatment inhibited Ras-GTP loading in KRAS-mutated cells as well as in EGF-stimulated cells. Rocaglamide treatment further prevented the oncogenic growth of KRAS-driven lung cancer allografts and xenografts in mouse models. Our results suggest rocaglamide as a RAS inhibitor and that targeting plasma membrane-associated PHB1 with chemical ligands would be a viable therapeutic strategy to combat KRAS-mediated NSCLCs.
Asunto(s)
Benzofuranos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Animales , Benzofuranos/administración & dosificación , Benzofuranos/farmacología , Línea Celular Tumoral , Proliferación Celular , Familia de Proteínas EGF/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Ratones , Ratones Noqueados , Prohibitinas , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Factor 3 Asociado a Receptor de TNF/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/metabolismo , Proteínas ras/antagonistas & inhibidores , Proteínas ras/metabolismoRESUMEN
The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser(2). This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Flavanonas/toxicidad , Flavonas/toxicidad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antineoplásicos/uso terapéutico , Sitios de Unión , Línea Celular Tumoral , Simulación por Computador , Quinasa 9 Dependiente de la Ciclina/metabolismo , Flavanonas/uso terapéutico , Flavonas/uso terapéutico , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Scutellaria baicalensis/química , Transcripción GenéticaRESUMEN
Native and recombinant wild type and mutant forms of type I adenylyl cyclase, expressed in fall army worm ovarian cells (Sf9) cells, with mutations Lys-923-Ala, Lys-921-Ala, and Lys-350-Ala, retained the characteristic noncompetitive inhibition by adenine nucleoside 3'-polyphosphates, but exhibited substantially different sensitivities to inhibition by them. The type I K923A enzyme resulted in increased IC(50) values, e.g., >100-fold for 2'-deoxyadenosine-3'-monophosphate, but the shift diminished as the number of 3'-phosphates increased. The K921A mutation increased IC(50) values approximately 5-fold for all adenine nucleosides tested, whereas the K350A mutation increased IC(50) values approximately 6- to 8-fold for all adenine nucleosides tested except 2'-deoxyadenosine-3'-diphosphate, which was increased >/=2-fold. The data suggest that 3'-phosphates sufficiently increase binding affinity of these ligands to compensate for the reduced coordination of the adenine moiety induced by the K923A mutation. Moreover, the altered structures induced by both K350A and K921A mutations impair ligand binding in general, but paradoxically those resulting from the K350A change minimally affected nucleoside 3'-diphosphate binding, implying that selective changes in ligand binding can be induced by this site-specific mutation.
Asunto(s)
Nucleótidos de Adenina/farmacología , Adenilil Ciclasas/metabolismo , Alanina , Inhibidores Enzimáticos/farmacología , Lisina , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Encéfalo/enzimología , Línea Celular , Secuencia Conservada , Femenino , Cinética , Datos de Secuencia Molecular , Ovario , Fragmentos de Péptidos/química , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecciónRESUMEN
A mild and efficient one-step method of thiophosphorylation was devised for acid-sensitive nucleosides. The procedure is based on thiophosphorylation of nucleoside magnesium alkoxide by 2-chloro-2-thio-1,3,2-dioxaphospholane. The utility and efficiency of this method combined with deprotection of the resulting cyclic triester were demonstrated by its application to the synthesis of both adenosine 3'- and 5'-thiophosphates. The procedure does not require protection of the exocyclic amino group and can be successfully used for the thiophosphorylation of nucleosides that are unusually sensitive to depurination.
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Nucleósidos/síntesis química , Animales , Compuestos Organofosforados/química , Fosforilación , Ratas , Análisis EspectralRESUMEN
Differentiation of Ob1771 preadipocytes to adipocytes was characterized by morphological changes and elevated expression of the specific marker enzyme, glycerol-3-phosphate dehydrogenase. A differentiation response substantially more complete and rapid than that obtained with insulin and 3,5,3'-triiodothyronine was observed with established inhibitors of adenylyl cyclases: 2', 5'-dideoxyadenosine (2',5'-dd-Ado), 9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or by N6-(2-phenylisopropyl)adenosine or 1, 3-diethyl-8-phenylxanthine, agonist and antagonist, respectively, for adenosine A1 receptors. Also ineffective were IBMX or forskolin, agents that can raise intracellular cAMP levels. Potency of the differentiation response followed the order 2',5'-dd-Ado (1-20 microM) > 9-CP-Ade (10-100 microM) = 9-Ara-Ade (10-100 microM) >> inosine, consistent with their potencies to inhibit adenylyl cyclases. The data suggest that inhibition of adenylyl cyclase via the P-site and the consequent reduction in cell cAMP levels facilitate the induction of differentiation in Ob1771 cells. The findings raise the question whether the known endogenous P-site ligands participate in the differentiation response induced by hormones.
Asunto(s)
Adenilil Ciclasas/metabolismo , Adipocitos/citología , Células Madre/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , AMP Cíclico/agonistas , AMP Cíclico/metabolismo , Estabilidad de Medicamentos , Inducción Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Glucosafosfato Deshidrogenasa/metabolismo , Ligandos , Ratones , Nucleósidos/farmacología , Receptores Purinérgicos P1/metabolismo , Células Madre/metabolismoRESUMEN
2'-Deoxyadenosine 3'-tetraphosphate (2'-deoxy-3'-A4P) and 2', 5'-dideoxyadenosine 3'-tetraphosphate (2',5'-dideoxy-3'-A4P) were synthesized, and their effects were tested on crude and purified forms of native adenylyl cyclases isolated from brain. Syntheses combined the method of alkoxide activation with the use of tribromoethyl phosphoromorpholino-chloridate as an initial phosphorylating agent. Inhibition of adenylyl cyclase was rapid in onset. With 2'-d-3'-A4P or 2',5'-dd-3'-A4P inhibition of a purified native enzyme conformed to a linear noncompetitive behavior with respect to substrate, metal-5'ATP. Order of potency was 2', 5'-dideoxy- > 2'-deoxyadenosine and 3'-tetraphosphate > 3'-triphosphate. Both mechanism of inhibition and rank order of potency were consistent with inhibition via the 3'-nucleotide-(P)-site on adenylyl cyclase. Neither 2',5'-dd-3'-ATP nor 2',5'-dd-3'-A4P had any effect on the activities of other adenosine nucleotide binding proteins such as Ca2+/calmodulin-sensitive cyclic nucleotide phosphodiesterase, Na+/K+-ATPase, or cAMP-dependent protein kinase. With purified adenylyl cyclase from bovine brain 2',5'-dd-3'-A4P and 2'-d-3'-A4P gave, respectively, IC50 values of 9.3 and 15 nM and Ki values of 23 and 53 nM. These 3'-nucleotides are the most potent regulators described for adenylyl cyclases.
Asunto(s)
Inhibidores de Adenilato Ciclasa , Encéfalo/enzimología , Nucleótidos de Desoxiadenina/farmacología , Hidrolasas Diéster Fosfóricas , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Adenilil Ciclasas/aislamiento & purificación , Animales , Sitios de Unión , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1 , Nucleótidos de Desoxiadenina/síntesis química , Nucleótidos de Desoxiadenina/química , Didesoxinucleótidos , Indicadores y Reactivos , Cinética , Ratas , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Relación Estructura-ActividadAsunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Adenina/análogos & derivados , Inhibidores Enzimáticos/síntesis química , Isoenzimas/antagonistas & inhibidores , Purinas/química , Purinas/farmacología , Adenina/síntesis química , Adenina/química , Adenina/farmacología , Inhibidores de la Adenosina Desaminasa , Anticonvulsivantes , AMP Cíclico/farmacología , GMP Cíclico/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Músculo Liso Vascular/enzimología , Relación Estructura-ActividadRESUMEN
Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3'-polyphosphates (Désaubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028-14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2',5'-dideoxyadenosine (2',5'-ddAdo) > beta-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) >/= 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3'-polyphosphates inhibitory potency followed the order Ado < 2'-dAdo < 2',5'-ddAdo and 3'-mono- < 3'-di- < 3'-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2',5'-dd-3'-ATP with IC50 values of 40-300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.
Asunto(s)
Adenosina/análogos & derivados , Inhibidores de Adenilato Ciclasa , Isoenzimas/antagonistas & inhibidores , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Adenosina/farmacología , Adenilil Ciclasas , Animales , Sitios de Unión , Encéfalo/enzimología , Calcio/metabolismo , Calmodulina/metabolismo , Bovinos , Didesoxiadenosina/análogos & derivados , Didesoxiadenosina/farmacología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas Oncogénicas/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The synthesis of a number of adenine nucleoside 3'-polyphosphates has been devised via a phosphotriester approach that combines the method of alkoxide activation with the use of 2,2,2-tribromoethyl phosphoromorpholinochloridate as a phosphorylating agent. The family of compounds included 3'ADP, 3'ATP, 2'-deoxy-3'ADP, 2'-deoxy-3'ATP, 2',5'-dideoxy-3'ADP, and 2',5'-dideoxy-3'ATP. Potency as inhibitors of adenylyl cyclases followed the order: 3'-mono- < 3'-di- < 3'-triphosphate and adenosine (Ado) < 2'-d-Ado < 2',5'-dd-Ado derivatives, with 2',5'-dideoxy-3'ATP exhibiting an IC50 of approximately 40 nM. This order was maintained with purified and recombinant forms of the type I enzyme. The nucleoside 3'-phosphates caused noncompetitive inhibition of the type I adenylyl cyclase from bovine brain, consistent with inhibition via the P-site. Inhibition was not due to hydrolytic products because this was minimal and inhibition kinetics by inorganic polyphosphates were inconsistent with those caused by the nucleoside 3'-polyphosphates. Only 3'ATP underwent cation-catalyzed, nonenzymatic hydrolysis, with the primary product being 2':3'-cAMP. Because 3'-ADP and 3'-ATP are naturally occurring, this class of compounds may physiologically regulate adenylyl cyclases and possibly other enzymes, mediating responses that include a reduction in 3':5'-cAMP levels and consequent reductions in protein kinase A-activated pathways.
Asunto(s)
Nucleótidos de Adenina/síntesis química , Nucleótidos de Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Inhibidores Enzimáticos/síntesis química , Nucleótidos de Adenina/química , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/síntesis química , Adenosina Difosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/síntesis química , Adenosina Trifosfato/farmacología , Adenilil Ciclasas/aislamiento & purificación , Animales , Encéfalo/enzimología , Bovinos , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cinética , Espectroscopía de Resonancia Magnética , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
2',5'-Dideoxyadenosine 3'-di- and triphosphates were tested as inhibitors of brain adenylyl cyclases. With an IC50 approximately 40 nM, 2',5'-dideoxy-3'-ATP is the most potent nonprotein synthetic regulator of adenylyl cyclases thus far described. Neither 2',5'-dideoxy-3'-ADP nor 2',5'-dideoxy-3'-ATP inhibited activity by competition with substrate, and the linear noncompetitive inhibition observed was consistent with interaction via a distinct domain. The availability of this ligand will permit the development of a variety of probes that will be extremely useful in investigating adenylyl cyclase structure and the role(s) that this class of compound may play in physiologically regulating cell function.
Asunto(s)
Inhibidores de Adenilato Ciclasa , Nucleótidos de Desoxiadenina/farmacología , Inhibidores Enzimáticos/farmacología , Animales , Encéfalo/enzimología , Bovinos , Didesoxinucleótidos , RatasRESUMEN
The anticonvulsant BW A78U, tested in a free mouse exploratory situation, reduced in a dose-dependent fashion the locomotion and the number of rearings, this sedative effect being significant up to a dose of 15 mg/kg (IP, 20 min before testing). In an unconditioned conflict test, the light/dark box choice situation, specific for anxiolytics, low doses of BW A78U increased the time spent by mice in the lit box as well as the number of transitions between the two boxes. Finally, we demonstrated that this drug was able to protect mice against pentylenetetrazole-induced convulsions. Our data show that BW A78U possesses some of the characteristic properties of the minor tranquilizers. However, since this compound binds to the benzodiazepine receptor with a very low affinity (IC50 = 13.6 microM), it can be assumed that this drug does not exert its behavioral effects through these receptors. It may interfere with other targets involving adenosine, another potent physiological regulator of neuronal excitability.