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1.
J Mol Cell Cardiol ; 178: 36-50, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36963751

RESUMO

BACKGROUND: Dantrolene binds to the Leu601-Cys620 region of the N-terminal domain of cardiac ryanodine receptor (RyR2), which corresponds to the Leu590-Cys609 region of the skeletal ryanodine receptor, and suppresses diastolic Ca2+ leakage through RyR2. OBJECTIVE: We investigated whether the chronic administration of dantrolene prevented left ventricular (LV) remodeling and ventricular tachycardia (VT) after myocardial infarction (MI) by the same mechanism with the mutation V3599K of RyR2, which indicated that the inhibition of diastolic Ca2+ leakage occurred by enhancing the binding affinity of calmodulin (CaM) to RyR2. METHODS AND RESULTS: A left anterior descending coronary artery ligation MI model was developed in mice. Wild-type (WT) were divided into four groups: sham-operated mice (WT-Sham), sham-operated mice treated with dantrolene (WT-Sham-DAN), MI mice (WT-MI), and MI mice treated with dantrolene (WT-MI-DAN). Homozygous V3599K RyR2 knock-in (KI) mice were divided into two groups: sham-operated mice (KI-Sham) and MI mice (KI-MI). The mice were followed for 12 weeks. Survival was significantly higher in the WT-MI-DAN (73%) and KI-MI groups (70%) than the WT-MI group (40%). Echocardiography, pathological tissue, and epinephrine-induced VT studies showed that LV remodeling and VT were prevented in the WT-MI-DAN and KI-MI groups compared to the WT-MI group. An increase in diastolic Ca2+ spark frequency and a decrease in the binding affinity of CaM to the RyR2 were observed at 12 weeks after MI in the WT-MI group, although significant improvements in these values were observed in the WT-MI-DAN and KI-MI groups. CONCLUSIONS: Pharmacological or genetic stabilization of RyR2 tetrameric structure improves survival after MI by suppressing LV remodeling and proarrhythmia.


Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Taquicardia Ventricular , Camundongos , Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Dantroleno/farmacologia , Remodelação Ventricular , Miócitos Cardíacos/metabolismo , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/genética , Arritmias Cardíacas/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Calmodulina/metabolismo , Infarto do Miocárdio/complicações , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo
2.
Biochem Biophys Res Commun ; 521(1): 57-63, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31635807

RESUMO

Aberrant Ca2+ release from cardiac ryanodine receptors (RyR2) has been shown to be one of the most important causes of lethal arrhythmia in various types of failing hearts. We previously showed that dantrolene, a specific agent for the treatment of malignant hyperthermia, inhibits Ca2+ leakage from the RyR2 by correcting the defective inter-domain interaction between the N-terminal (1-619 amino acids) and central (2000-2500 amino acids) domains of the RyR2 and allosterically enhancing the binding affinity of calmodulin to the RyR2 in diseased hearts. In this study, we examined whether dantrolene inhibits this Ca2+ leakage, thereby preventing the pharmacologically inducible ventricular tachycardia in ventricular pressure-overloaded failing hearts. Ventricular tachycardia (VT) was easily induced after an injection of epinephrine in mice after 8 weeks of transverse aortic constriction-induced pressure-overload. Pretreatment with dantrolene almost completely inhibited the pharmacologically inducible VT. In the presence of dantrolene, the occurrence of both Ca2+ sparks and spontaneous Ca2+ transients was inhibited, which was associated with enhanced calmodulin binding affinity to the RyR2. These results suggest that dantrolene could be a new potent agent in the treatment of lethal arrhythmia in cases of acquired heart failure.


Assuntos
Dantroleno/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Relaxantes Musculares Centrais/farmacologia , Substâncias Protetoras/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/tratamento farmacológico , Animais , Insuficiência Cardíaca/patologia , Camundongos , Pressão , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologia
3.
Circ J ; 83(12): 2494-2504, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31631075

RESUMO

BACKGROUND: Little is known about the pattern of isotope accumulation in the heart on 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography in patients with cardiac sarcoidosis (CS) complicated by ventricular aneurysm (VA).Methods and Results:We prospectively enrolled 82 consecutive patients with CS; 54 patients with active CS (presence of abnormal 18F-FDG accumulation in the heart) were subdivided into VA (n=17) and non-VA groups (n=37). Strong 18F-FDG accumulation surrounding the VA and its disappearance in the VA center was observed in all patients with VA, probably because of scar formation at the VA. Peak standardized uptake value was higher around the VA than in the VA center (5.1±2.1 vs. 2.2±0.6, P=0.0003) and the VA center had no 18F-FDG accumulation (VA center: 2.2±0.6 vs. control area: 2.1±0.6, P=0.37). On the other hand, in non-VA patients with LV wall thinning (n=28), 18F-FDG accumulation was significantly high, even in the area of LV wall thinning (LV wall thinning area: 3.1±0.8 vs. control area: 2.0±0.6, P=0.00002). CONCLUSIONS: A pattern of strong 18F-FDG accumulation surrounding the VA and its disappearance in the VA center might be characteristic in patients with CS complicated by VA. Careful attention to FDG uptake would further elucidate CS pathophysiology and aid in the early treatment of VA.


Assuntos
Cardiomiopatias/diagnóstico por imagem , Fluordesoxiglucose F18/administração & dosagem , Aneurisma Cardíaco/diagnóstico por imagem , Miocardite/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/administração & dosagem , Sarcoidose/diagnóstico por imagem , Corticosteroides/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Cardiomiopatias/tratamento farmacológico , Feminino , Aneurisma Cardíaco/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Miocardite/tratamento farmacológico , Valor Preditivo dos Testes , Estudos Prospectivos , Sarcoidose/tratamento farmacológico , Resultado do Tratamento
4.
Cardiology ; 142(4): 195-202, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31137022

RESUMO

BACKGROUND: Tachycardia worsens cardiac performance in acute decompensated heart failure (ADHF). We investigated whether heart rate (HR) optimization by landiolol, an ultra-short-acting ß1-selective blocker, in combination with milrinone improved cardiac function in patients with ADHF and rapid atrial fibrillation (AF). METHODS AND RESULTS: We enrolled9 ADHF patients (New York Heart Association classification IV; HR, 138 ± 18 bpm; left ventricular [LV] ejection fraction, 28 ± 8%; cardiac index [CI], 2.1 ± 0.3 L/min-1/m-2; pulmonary capillary wedge pressure [PCWP], 24 ± 3 mm Hg), whose HRs could not be reduced using standard treatments, including diuretics, vasodilators, and milrinone. Landiolol (1.5-6.0 µg/kg-1/min-1, intravenous) was added to milrinone treatment to study its effect on hemodynamics. The addition of landiolol (1.5 µg/kg-1/min-1) significantly reduced HR by 11% without changing systolic blood pressure (BP) and resulted in a significant decrease in PCWP and a significant increase in stroke volume index (SVI), suggesting that HR reduction restores incomplete LV relaxation. Administration of more than 3.0 µg/kg-1/min-1 of landiolol decreased BP, CI, and SVI. CONCLUSION: The addition of landiolol at doses of <3.0 µg/kg/min to milrinone improved cardiac function in decompensated chronic heart failure with rapid atrial fibrillation by selectively reducing HR.


Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Cardiotônicos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Milrinona/uso terapêutico , Morfolinas/uso terapêutico , Ureia/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Feminino , Hemodinâmica/efeitos dos fármacos , Humanos , Japão , Masculino , Estudos Prospectivos , Taquicardia , Resultado do Tratamento , Ureia/uso terapêutico
5.
Biol Pharm Bull ; 42(12): 2069-2075, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31787721

RESUMO

Proteome profiling based on two-dimensional (2D)-DIGE might be a useful tool for investigating drug-like compounds and the mode of action of drugs. However, obtaining data for profiling requires high labor costs, and it is difficult to control the reproducibility of spot positions because 2D-DIGE usually requires large-size glass plates and spot alignments are greatly affected by the quality of DryStrips and polyacrylamide gels (PAGs). Therefore, we have developed a novel platform by employing small size DryStrips and PAGs, and an image analysis strategy based on dual correction of spot alignment and volume. Our system can automatically detect a large number of consistent spots through all images. Cytosol fractions of HeLa cells treated with dimethyl sulfoxide (DMSO) or bortezomib were analyzed, 1697 consistent spots were detected, and 775 of them were significantly changed with the treatment. Deviations between different days and lot sets of DryStrips and PAGs were investigated by calculating the correlation coefficients. The mean values of the correlation between days and lot sets were 0.96 and 0.94, respectively. Clustering analysis of all the treatment data clearly separated the DMSO or bortezomib treated groups beyond day deviations. Thus, we have succeeded in developing an easy-to-handle 2D-DIGE system that can be a novel proteome profiling platform.


Assuntos
Proteoma , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional , Animais , Bortezomib/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dimetil Sulfóxido/farmacologia , Células HeLa , Humanos , Inibidores de Proteassoma/farmacologia , Proteoma/efeitos dos fármacos , Células Sf9
6.
Am J Physiol Endocrinol Metab ; 310(8): E643-E651, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26860984

RESUMO

Extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance associated with obesity and type 2 diabetes mellitus. We have now examined the potential of pharmacological targeting of the ERK pathway with MEK (ERK kinase) inhibitors (PD184352 and PD0325901) for the treatment of obesity-associated insulin resistance. The effects of PD184352 and PD0325901 on the expression of adipocytokines and lipolysis activity were thus examined in 3T3-L1 adipocytes maintained in long-term culture as a model of adipocyte hypertrophy. Leptin receptor-deficient (db/db) mice and high-fat diet-fed KKAy mice, both of which are models of type 2 diabetes, were also treated orally with PD184352 to examine its effects on the diabetic condition. ERK activity was increased in hypertrophic 3T3-L1 adipocytes as well as in adipose tissue of db/db mice and high-fat diet-fed KKAy mice, and this enhanced ERK signaling was associated with dysregulation of adipocytokine expression and increased lipolysis activity. Specific blockade of the ERK pathway in hypertrophic 3T3-L1 adipocytes by MEK inhibitors ameliorated the dysregulation of adipocytokine expression and suppressed the enhanced lipolysis activity. Furthermore, repeated oral administration of PD184352 normalized hyperglycemia and hyperlipidemia and improved insulin sensitivity and glucose tolerance in the diabetic mice. These results suggest that sustained activation of the ERK pathway in adipocytes is associated with the pathogenesis of type 2 diabetes and that selective blockade of this pathway with MEK inhibitors warrants further study as a promising approach to the treatment of insulin resistance and type 2 diabetes.


Assuntos
Adipócitos/efeitos dos fármacos , Benzamidas/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Difenilamina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Resistência à Insulina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Células 3T3-L1 , Adipócitos/metabolismo , Adipocinas/metabolismo , Adiponectina/metabolismo , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Dieta Hiperlipídica , Difenilamina/farmacologia , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/metabolismo , Teste de Tolerância a Glucose , Hiperlipidemias/metabolismo , Immunoblotting , Técnicas In Vitro , Insulina/metabolismo , Interleucina-6/metabolismo , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Receptores para Leptina/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
7.
J Biol Chem ; 287(13): 10289-10300, 2012 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-22270368

RESUMO

Blockade of the ERK signaling pathway by ERK kinase (MEK) inhibitors selectively enhances the induction of apoptosis by microtubule inhibitors in tumor cells in which this pathway is constitutively activated. We examined the mechanism by which such drug combinations induce enhanced cell death by applying time-lapse microscopy to track the fate of individual cells. MEK inhibitors did not affect the first mitosis after drug exposure, but most cells remained arrested in interphase without entering a second mitosis. Low concentrations of microtubule inhibitors induced prolonged mitotic arrest followed by exit of cells from mitosis without division, with most cells remaining viable. However, the combination of a MEK inhibitor and a microtubule inhibitor induced massive cell death during prolonged mitosis. Impairment of spindle assembly checkpoint function by RNAi-mediated depletion of Mad2 or BubR1 markedly suppressed such prolonged mitotic arrest and cell death. The cell death was accompanied by up-regulation of the pro-apoptotic protein Bim (to which MEK inhibitors contributed) and by down-regulation of the anti-apoptotic protein Mcl-1 (to which microtubule and MEK inhibitors contributed synergistically). Whereas RNAi-mediated knockdown of Bim suppressed cell death, stabilization of Mcl-1 by RNAi-mediated depletion of Mule slowed its onset. Depletion of Mcl-1 sensitized tumor cells to MEK inhibitor-induced cell death, an effect that was antagonized by knockdown of Bim. The combination of MEK and microtubule inhibitors thus targets Bim and Mcl-1 in a cooperative manner to induce massive cell death in tumor cells with aberrant ERK pathway activation.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Mad2 , Proteínas de Membrana/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
8.
Biochem Biophys Res Commun ; 433(4): 456-62, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23501104

RESUMO

The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients.


Assuntos
Benzamidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Sistema de Sinalização das MAP Quinases , Piridinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Benzimidazóis/farmacologia , Sinergismo Farmacológico , Feminino , Células HT29 , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Estresse Oxidativo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Genes Cells ; 16(5): 514-26, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21501342

RESUMO

Although the extracellular signal-regulated kinase (ERK) pathway functions downstream of Ras in induction of the cell motility response, the detailed molecular mechanism by which this pathway regulates cell motility has remained elusive. The application of a functional expression cloning strategy to discover proteins that regulate cell motility has resulted in the identification of an SH3 domain-containing protein, SH3P2. Overexpression of SH3P2 in HeLa S3 cells inhibited cell motility, whereas RNA interference-mediated depletion of SH3P2 enhanced motility in various tumor cell lines, suggesting that SH3P2 functions as a negative regulator of cell motility. The expression level of SH3P2 alone did not correlate well with the motility of tumor cells, however. SH3P2 was phosphorylated on Ser(202) by ribosomal S6 kinase (RSK) in an ERK pathway-dependent manner, and such phosphorylation inhibited the ability of SH3P2 to suppress cell motility. The RSK inhibitor BI-D1870 suppressed SH3P2 phosphorylation and tumor cell motility as effectively as did the MEK inhibitor PD184352. Furthermore, expression of the unphosphorylatable SH3P2 mutant SH3P2(S202A) inhibited tumor cell motility, indicating that phosphorylation of SH3P2 at Ser(202) is a key determinant of such motility. These results suggest that SH3P2 is an essential molecule that functions downstream of the ERK pathway to modulate cell motility.


Assuntos
Movimento Celular , Proteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Serina/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Proteínas/genética , Pteridinas/farmacologia , Interferência de RNA , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Serina/genética
10.
Nephrol Dial Transplant ; 27(4): 1351-8, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22076433

RESUMO

BACKGROUND: Nephronophthisis (NPHP), the most frequent genetic cause of end-stage kidney disease in children and young adults, is characterized by a variable number of renal cysts associated with cortical tubular atrophy and interstitial fibrosis. The p38 mitogen-activated protein kinase (MAPK) pathway is an important intracellular signaling pathway involved in the production of profibrotic mediators. The relationship between p38 MAPK and renal fibrosis in NPHP2 is unknown. METHODS: We administered a selective p38 MAPK inhibitor, FR167653, in a NPHP2 mouse model (inv/inv, invΔC mice) from 3 to 6 weeks old, and the kidneys were examined at 6 weeks of age. Phosphorylation of p38 MAPK (p-p38 MAPK) protein levels, the degree of renal fibrosis, messenger RNA (mRNA) levels for extracellular matrix genes and mRNA levels for transforming growth factor in the kidneys were studied. Effect of an extracellular signal-regulated protein kinase (ERK) kinase (MEK) inhibitor on renal fibrosis was also evaluated. RESULTS: Expression of extracellular matrix genes and p-p38 MAPK were increased in the NPHP2 mouse model kidney. FR167653 successfully decreased p-p38 MAPK levels, the degree of fibrosis and extracellular matrix gene expressions. However, the FR167653 did not prevent cyst expansion, abnormal cell proliferation and acceleration of apoptosis and did not influence ERK activation. In contrast, MEK inhibition reduced both cyst expansion and fibrosis without affecting p38 MAPK activation. CONCLUSIONS: These results suggest that inhibition of p38 MAPK reduced renal fibrosis but not cyst expansion, cell proliferation and apoptosis in NPHP2 model mice. Our results suggest that p38 MAPK and ERK signaling pathways independently affect renal fibrosis in inv mutant mice.


Assuntos
Modelos Animais de Doenças , Fibrose/prevenção & controle , Nefropatias/prevenção & controle , Pirazóis/farmacologia , Piridinas/farmacologia , Fatores de Transcrição/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Apoptose , Western Blotting , Proliferação de Células , Cistos/tratamento farmacológico , Cistos/enzimologia , Cistos/prevenção & controle , Fibrose/tratamento farmacológico , Fibrose/enzimologia , Inibidores do Crescimento/farmacologia , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/enzimologia , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Biol Pharm Bull ; 34(12): 1781-4, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22130230

RESUMO

The extracellular signal-regulated kinase (ERK) pathway is a major determinant in the control of diverse cellular processes such as proliferation, survival, and motility. This pathway is often upregulated in human cancers and as such represents an attractive target for mechanism-based approaches to cancer treatment. However, specific blockade of the ERK pathway alone induces mostly cytostatic rather than proapoptotic effects, resulting in limited therapeutic efficacy. Blockade of the constitutively activated ERK pathway by an ERK kinase (MEK) inhibitor sensitizes tumor cells to apoptotic cell death induced by several cytotoxic anticancer agents including microtubule-destabilizing agents and histone deacetylase inhibitors, not only in vitro but also in tumor zenografts in vivo. Thus, low concentrations of these anticancer drugs that by themselves show little cytotoxicity effectively kill tumor cells in which the ERK pathway is constitutively activated when co-administrated with a MEK inhibitor. The combination of a cytostatic signaling pathway inhibitor (MEK inhibitors) and conventional anticancer drugs (microtubule-destabilizing agents or histone deacetylase inhibitors) provides an excellent basis for the development of safer anticancer chemotherapies with enhanced efficacy through lowering the required dose of the latter cytotoxic drugs.


Assuntos
Antineoplásicos/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Inibidores de Histona Desacetilases/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Animais , Apoptose , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/metabolismo
12.
Biochem Biophys Res Commun ; 391(4): 1610-5, 2010 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-20026060

RESUMO

Deregulated activation of protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and Abl, is associated with human cancers including non-small cell lung cancer (NSCLC) and chronic myeloid leukemia (CML). Although inhibitors of such activated kinases have proved to be of therapeutic benefit in individuals with NSCLC or CML, some patients manifest intrinsic or acquired resistance to these drugs. We now show that, whereas blockade of either the extracellular signal-regulated kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway alone induced only a low level of cell death, it markedly sensitized NSCLC or CML cells to the induction of apoptosis by histone deacetylase (HDAC) inhibitors. Such enhanced cell death induced by the respective drug combinations was apparent even in NSCLC or CML cells exhibiting resistance to EGFR or Abl tyrosine kinase inhibitors, respectively. Co-administration of a cytostatic signaling pathway inhibitor may contribute to the development of safer anticancer strategies by lowering the required dose of cytotoxic HDAC inhibitors for a variety of cancers.


Assuntos
Analgésicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Neoplasias Pulmonares/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Benzamidas/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gefitinibe , Humanos , Mesilato de Imatinib , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Quinazolinas/farmacologia
13.
Commun Biol ; 3(1): 714, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33244105

RESUMO

Cardiac hypertrophy is a well-known major risk factor for poor prognosis in patients with cardiovascular diseases. Dysregulation of intracellular Ca2+ is involved in the pathogenesis of cardiac hypertrophy. However, the precise mechanism underlying cardiac hypertrophy remains elusive. Here, we investigate whether pressure-overload induced hypertrophy can be induced by destabilization of cardiac ryanodine receptor (RyR2) through calmodulin (CaM) dissociation and subsequent Ca2+ leakage, and whether it can be genetically rescued by enhancing the binding affinity of CaM to RyR2. In the very initial phase of pressure-overload induced cardiac hypertrophy, when cardiac contractile function is preserved, reactive oxygen species (ROS)-mediated RyR2 destabilization already occurs in association with relaxation dysfunction. Further, stabilizing RyR2 by enhancing the binding affinity of CaM to RyR2 completely inhibits hypertrophic signaling and improves survival. Our study uncovers a critical missing link between RyR2 destabilization and cardiac hypertrophy.


Assuntos
Sinalização do Cálcio , Calmodulina/metabolismo , Cardiomegalia , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Feminino , Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Pressão , Ligação Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
14.
Biochem Biophys Res Commun ; 378(3): 650-5, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19059207

RESUMO

The extracellular signal-regulated kinase (ERK) signaling pathway is constitutively activated in many human tumor cell types. Given the cytoprotective role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although blockade of ERK signaling alone did not induce substantial cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the ERK pathway is constitutively activated. The synergistic activation of c-Jun NH(2)-terminal kinase by the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent appeared to be responsible, at least in part, for this effect. These results suggest that administration of the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells with constitutive activation of the ERK pathway.


Assuntos
Apoptose , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Microtúbulos/efeitos dos fármacos , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Moduladores de Tubulina/farmacologia , Linhagem Celular Tumoral , Flavonoides/farmacologia , Humanos , Vincristina/farmacologia
15.
Pulse (Basel) ; 6(3-4): 161-168, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31049316

RESUMO

BACKGROUND: Recently, new devices using oscillometric cuff have been developed to derive central blood pressure (BP) waveform from brachial BP waveform. Late systolic pressure augmentation of central BP waveform is a marker of central hemodynamics. Mobil-O-Graph, a cuff-based oscillometric device, can assess central augmentation pressure (AP) together with central BP. Central AP measurement using the Mobil-O-Graph in the European population was reported to be associated with age and sex. However, factors influencing central AP in the Asian population have not been shown. OBJECTIVES AND METHODS: We enrolled 110 normotensive volunteers (50 men; age range, 21‒76 years). Central BP and AP were measured using the Mobil-O-Graph on the left arm with the subjects in the seated position after resting for at least 5 min. We compared central hemodynamics between the sexes. We investigated factors influencing central AP in uni- and multivariate linear regression analyses and age-related change in central AP using the Mobil-O-Graph in healthy Japanese individuals. RESULTS: Central AP were lower in men than in women (5.5 ± 2.8 vs. 11.0 ± 4.7 mm Hg, p < 0.001). The central AP in the total cohort was positively correlated with age (r = 0.325, p < 0.001) and inversely correlated with height (r = -0.601, p < 0.001) in the Pearson correlations. In multivariate regression analysis, the parameters influencing central AP (R>sup<2>/sup< = 0.467) were age (ß = 0.097, p < 0.001), sex (ß = -2.890, p = 0.010), and height (ß = -0.153, p = 0.031). The central AP (10.0 ± 4.8 mm Hg) in the ≥50-year-old group significantly increased compared with those in the 20- to 39-year-old group (6.7 ± 4.2 mm Hg, p < 0.05). CONCLUSIONS: Age, sex, and height influenced central AP, as assessed using the Mobil-O-Graph. Age-related increase in central AP was observed in normotensive Japanese individuals. Brachial cuff-based waveform recordings using the Mobil-O-Graph are feasible for the estimation of central AP in the Asian population.

16.
Cancer Sci ; 99(2): 376-84, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18201278

RESUMO

Although DNA-damaging agents are among the most effective anticancer drugs in clinical use, their overall effectiveness is limited by the development of cross-resistance to these drugs. Given that histone deacetylase (HDAC) inhibitors increase the acetylation of core histones, resulting in an open chromatin configuration that is more accessible to DNA-targeting agents, we examined whether HDAC inhibitors might enhance the cytotoxicity of DNA-damaging drugs in six human ovarian tumor cell lines that exhibit different cisplatin sensitivities. Low concentrations of HDAC inhibitors, which alone exhibited little cytotoxicity, markedly enhanced the induction of apoptotic cell death not only by cisplatin but also by a wide variety of DNA-targeting anticancer drugs in these tumor cell lines, irrespective of their sensitivities to the respective drugs. In contrast, HDAC inhibitors did not increase the cytotoxicity of metabolic antagonists or microtubule-targeting agents. HDAC inhibitors potentiated both the phosphorylation of histone H2AX on serine-139 (a marker of DNA double-strand breaks) as well as the accumulation of reactive oxygen species induced by DNA-damaging agents in tumor cells. The enhanced generation of reactive oxygen species appeared to be responsible for the enhanced apoptotic cell death induced by the combination of these drugs. These results indicate that the combination of an HDAC inhibitor with a wide variety of DNA-damaging agents is a promising chemotherapeutic strategy for the eradication of tumor cells, regardless of whether the cells are sensitive or resistant to the DNA-damaging anticancer drugs.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Apoptose , Morte Celular , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Quebras de DNA de Cadeia Dupla , DNA de Neoplasias/metabolismo , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Imunofluorescência , Histona Desacetilases/metabolismo , Humanos , Espécies Reativas de Oxigênio
17.
Biochem Biophys Res Commun ; 368(1): 162-7, 2008 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-18211802

RESUMO

Rho GTPases play an essential role in the regulation of many cellular processes. Although various guanine nucleotide exchange factors (GEFs) are involved in the activation of Rho GTPases, the precise mechanism regulating such activity remains unclear. We have examined whether ERK1/2 are involved in the phosphorylation of GEF-H1, a GEF toward RhoA, to modulate its activity. Expression of GEF-H1 in HT1080 cells with constitutive ERK1/2 activation induced its phosphorylation at Thr(678), which was totally abolished by treating the cells with PD184352, an ERK pathway inhibitor. Stimulation of HeLa S3 cells with 12-O-tetradecanoyl-phorbol-13-acetate induced the phosphorylation of GEF-H1 in an ERK-dependent manner. ERK1/2-mediated Thr(678)-phosphorylation enhanced the guanine nucleotide exchange activity of GEF-H1 toward RhoA. These results suggest that the ERK pathway, by enhancing the GEF-H1 activity, contributes to the activation of RhoA to regulate the actin assembly, a necessary event for the induction of cellular responses including proliferation and motility.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Nucleotídeos de Guanina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho
18.
Mol Cancer Ther ; 6(3): 1133-42, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17363506

RESUMO

Constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway is associated with the neoplastic phenotype in many human tumor cell types. Given the antiapoptotic role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although specific blockade of the PI3K-Akt pathway alone with inhibitors such as LY294002 did not induce cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents such as vincristine. This effect was apparent only in tumor cells in which the PI3K-Akt pathway is constitutively activated. Blockade of the PI3K-Akt pathway induced the activation of glycogen synthase kinase-3beta, which phosphorylates microtubule-associated proteins such as tau and thereby reduces their ability to bind and stabilize microtubules. The consequent destabilization of microtubules induced by the inhibition of PI3K-Akt signaling appeared to increase their sensitivity to low concentrations of microtubule-destabilizing agents that alone do not lead to the disruption of cytoplasmic microtubules in tumor cells. Such a synergistic effect on microtubule integrity was not apparent for stable microtubules in the neurites of neuronal cells. These results suggest that the administration of a combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells in which this signaling pathway is constitutively activated.


Assuntos
Apoptose/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Moduladores de Tubulina/farmacologia , Antineoplásicos/farmacologia , Comunicação Celular , Diferenciação Celular/efeitos dos fármacos , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Imunofluorescência , Humanos , Morfolinas/farmacologia , Neoplasias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Vincristina/farmacologia
19.
Heart Rhythm ; 15(6): 905-914, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29427818

RESUMO

BACKGROUND: Ryanodine receptor (RyR2) is known to be a causal gene of catecholaminergic polymorphic ventricular tachycardia (CPVT), an important inherited disease. Some of the human CPVT-associated mutations have been found in a domain (4026-4172) that has EF hand motifs, the so-called calmodulin (CaM)-like domain (CaMLD). OBJECTIVE: The purpose of this study was to investigate the underlying mechanism by which CPVT is induced by a mutation at CaMLD. METHODS: A new N4103K/+ knock-in (KI) mice model was generated. RESULTS: Sustained ventricular tachycardia was frequently observed after infusion of caffeine plus epinephrine in KI mice. Endogenous CaM bound to RyR2 decreased even at baseline in isolated KI cardiomyocytes. Ca2+ spark frequency (CaSpF) was much higher in KI cells than in wild-type cells. Addition of GSH-CaM (higher affinity CaM to RyR2) significantly decreased CaSpF. In response to isoproterenol, spontaneous Ca2+ transient (SCaT) was frequently observed in intact KI cells. Incorporation of GSH-CaM into intact KI cells using a protein delivery kit decreased SCaT significantly. An assay using a quartz crystal microbalance technique revealed that mutated CaMLD peptide showed higher binding affinity to CaM binding domain (CaMBD) peptide. CONCLUSION: In the N4103K mutant, CaM binding affinity to RyR2 was significantly reduced regardless of beta-adrenergic stimulation. We found that this was caused by an abnormally tight interaction between CaMBD and mutated CaM-like domain (N4103K-CaMBD). Thus, CaMBD-CaMLD interaction may be a novel therapeutic target for treatment of lethal arrhythmia.


Assuntos
Cálcio/metabolismo , DNA/genética , Mutação , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Animais , Sinalização do Cálcio , Células Cultivadas , Análise Mutacional de DNA , Modelos Animais de Doenças , Camundongos , Miócitos Cardíacos/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologia
20.
Curr Drug Discov Technol ; 4(2): 69-76, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17691911

RESUMO

Diethyl-4,4'-methylenebis(N-phenylcarbamate) (MDU) is a urethane compound that we originally synthesized, along with three other compounds, to investigate how polyurethane is hydrolysed. We tested the four compounds for cytotoxicity in two Chinese hamster cell lines (CHL and V79) and a human cancer cell line (HeLa S3). MDU showed the strongest cytotoxicity in all the cell lines with an IC50 of around 0.1 microg/ml. We further investigated MDU for its ability to induce chromosome aberrations (CAs) and micronuclei (MN) in CHL cells. MDU induced around 100% polyploid cells at 0.5 microg/ml after 24- and 48-h treatment in the CA test and a significantly increased frequency of micronuclei, polynuclear cells, and mitotic cells in the MN test, suggesting that it may induce numerical CAs. MDU's ability to cause mitotic arrest in CHL cells was greater than that of taxol and colchicine. Based on a COMPARE analysis using JFCR39, a panel of cancer cell lines, we predicted MDU to be a tubulin inhibitor. We confirmed this possibility in nerve growth factor-stimulated PC12 cells as well as in HT1080 cells, in which MDU exhibited the activity to inhibit tubulin polymerization. MDU is simpler in structure than existing anticancer drugs taxol and vincristine and can be synthesized relatively easily. Here we offer MDU as a potential new type of anticancer drug, stable even at room temperature, and inexpensive.


Assuntos
Antineoplásicos/química , Uretana/química , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Testes de Mutagenicidade
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