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BACKGROUND/AIM: A genomic analysis based on next-generation sequencing is important for deciding cancer treatment strategies. Cancer tissue sometimes displays intratumor heterogeneity and a pathologic specimen may contain more than two tumor grades. Although tumor grades are very important for the cancer prognosis, the impact of higher tumor grade distribution in a specimen used for a genomic analysis is unknown. PATIENTS AND METHODS: We retrospectively analyzed the data of 61 clear cell carcinoma and 46 prostate cancer patients that were diagnosed between December 2018 and August 2022 using the GeneRead Human Comprehensive Cancer Panel or SureSelect PrePool custom Tier2. Genome annotation and curation were performed using the GenomeJack software. RESULTS: Tumor mutation burden (TMB) was increased in proportion to the higher tumor grade distribution in grade 2 clear cell renal cell carcinoma (ccRCC). In PC, Grade Group 3/4 specimens that included an increased distribution of Gleason pattern 4 had more frequent gene mutations. CONCLUSION: Our results suggest the importance of selecting the maximum distribution of higher tumor grade areas to obtain results on the precise gene alterations for genomics-focused treatments.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Neoplasias da Próstata , Masculino , Humanos , Carcinoma de Células Renais/genética , Estudos Retrospectivos , Neoplasias da Próstata/genética , Mutação , Neoplasias Renais/genéticaRESUMO
BACKGROUND/AIM: The Von Hippel-Lindau (VHL) gene encodes a protein (pVHL) that plays an important role in proteasome degradation of hypoxia inducible factor α (HIFα) through E3 activation. Accumulation of HIFα by loss of functional pVHL promotes tumorigenesis, thus, VHL has tumor suppressor gene capability in clear cell renal cell carcinoma (ccRCC). VHL is the most frequently mutated gene in ccRCC. The complete loss of VHL is mainly achieved by loss of chromosome 3p, which has a VHL coding region in combination with mutation or hypermethylation of the remaining copy of VHL. Given the risk of constitutional chromosome 3 translocation for RCC, it is important to detect the translocation and understand the mechanism underlying the development of multifocal ccRCC. CASE REPORT: A 67-year-old female patient diagnosed with multifocal RCC underwent robot-assisted partial nephrectomy (RAPN) for three kidney tumors. A cancer gene panel test using next generation sequencing (NGS) detected differential VHL mutations (c.533T>G; p.L178R, c.465_466insTA; p.T157Ifs*3, c.343C>A; p.H115N), while VHL mutation was not detected in peripheral blood DNA. A tendency toward copy number loss of genes on der(3) was also detected in all tumors, but not in the germline one. A karyotype analysis revealed a germline translocation between 3 and 6, t(3;6)(q12;q14). CONCLUSION: Chromosome 3 translocation and loss of derivative chromosome containing 3p and subsequent somatic differential VHL mutations in this case strongly support the previously proposed three-step model to explain the development of familial conventional ccRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Feminino , Humanos , Idoso , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Cromossomos Humanos Par 3/genética , Neoplasias Renais/patologia , Mutação , Translocação Genética , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
The environment and personnel are both exposed to powdered pharmaceuticals inside pharmacies. This makes developing new methods for rapidly determining such contaminants an important objective. In this study, we developed a liquid-chromatography tandem-mass-spectrometry (LC-MS/MS) method for the simultaneous qualitative and quantitative determination of powdered medicinal drugs, such as famotidine, risperidone, lansoprazole, olanzapine, haloperidol, clarithromycin, promethazine, levomepromazine, and chlorpromazine. The method involves the use of acetaminophen as the internal standard, an LC-MS/MS method with a core-shell column, and a 10 mM ammonium formate/acetonitrile gradient mobile phase. The analytes were separated within 14 min, and MS with an electrospray ionization source in positive-ion mode was used. The limits of detection for the 9 drugs were .1-8.4 ng/mL. Linear calibration curves in the 10-50 000 ng/mL range were constructed, and inter-day accuracies of 92.6-113.8% were determined for the 9 drugs. The coefficients of variation were less than 14.6%. These data suggest that the proposed method is applicable for the routine assaying of powdered-medicine contamination in pharmacies.
Assuntos
Preparações Farmacêuticas , Farmácias , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Humanos , Reprodutibilidade dos Testes , Extração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: Paclitaxel is a highly effective antitumor agent with notable adverse events, including hypersensitivity reactions, peripheral neuropathy, arthralgia, myalgias, and neutropenia. Solvent-based paclitaxel causes severe allergic, hypersensitivity, and anaphylactic reactions. Nanoparticle albumin-bound paclitaxel was recently developed and provides an advantage over solvent-based paclitaxel in avoiding solvent/surfactant-related adverse events. The aim of this study was to assess the adverse event profiles of solvent-based paclitaxel and nanoparticle albumin-bound paclitaxel formulations using data from the spontaneous adverse event reporting system of the US Food and Drug Administration Adverse Event Reporting System database. METHODS: This study relied on Medical Dictionary for Regulatory Activities preferred terms and standardized queries, and calculated the reporting ratio and reporting odds ratios of paclitaxel formulations. RESULTS: Of 8,867,135 reports recorded in the US Food and Drug Administration Adverse Event Reporting System database from January 2004 to December 2016, 3469 and 4447 adverse events corresponded to solvent-based paclitaxel and nanoparticle albumin-bound paclitaxel, respectively. Reporting odds ratios (95% confidence interval) for anaphylactic reaction (standardized MedDRA query code: 20000021) associated with the use of solvent-based paclitaxel and nanoparticle albumin-bound paclitaxel were 1.69 (1.56-1.84) and 0.75 (0.68-0.83), respectively. Reporting odds ratio signal for anaphylactic reaction was not detected for nanoparticle albumin-bound paclitaxel. Reporting odds ratios (95% confidence interval) for acute renal failure (standardized MedDRA query code: 20000003) associated with the use of solvent-based paclitaxel and nanoparticle albumin-bound paclitaxel were 0.75 (0.58-0.98) and 1.60 (1.37-1.89), respectively. CONCLUSION: This is the first study to evaluate the adverse event profile of nanoparticle albumin-bound paclitaxel using US Food and Drug Administration Adverse Event Reporting System data. Considering that the US Food and Drug Administration Adverse Event Reporting System database does not allow to infer causality or risk ranking, the different reporting frequencies of anaphylactic reaction and acute renal failure between solvent-based paclitaxel and nanoparticle albumin-bound paclitaxel must be further investigated via analytical observational research.
RESUMO
The major toxicities associated with paclitaxel(PTX)-based chemotherapy are myelosuppression, peripheral neuropathy and myalgia/arthralgia. In the present study, a retrospective study was carried out to compare the incidence of adverse events during PTX-based chemotherapy between 58 cases treated every 3 weeks(tri-weekly regimen)for breast cancer and non-small cell lung cancer and 47 cases with weekly regimen for breast cancer and gastric cancer. Hematological toxicity such as neutropenia and thrombocytopenia, peripheral neuropathy and myalgia/arthralgia occurred more frequently in patients with a tri-weekly regimen than in those with a weekly regimen(grade 3/4 neutropenia: 65.5% versus 12.8%, odds ratio; 12.98, grade 2/3 peripheral neuropathy: 24.1% versus 6.4%, odds ratio; 4.67, and grade 2/3 myalgia/arthralgia: 43.1% versus 4.3%, odds ratio; 17.05). The development of peripheral neuropathy and myalgia/arthralgia was dependent on the dose per injection, but not on the cumulative dose of PTX. The initial onset of peripheral neuropathy and myalgia/arthralgia was observed in more than 80% of patients during the first course of the tri-weekly regimen, while it was seen in any course of the weekly regimen. The incidence of other nonhematological adverse events was not different between the two regimens except for nausea. Our present findings suggest that the peripheral neuropathy and myalgia/arthralgia are highly frequent adverse events that depend on the dose per injection of PTX, in which the incidence was more frequent in the tri-weekly than in the weekly regimen.
Assuntos
Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Paclitaxel/uso terapêutico , Estudos Retrospectivos , Fatores de TempoRESUMO
BACKGROUND: Falls are a common but serious problem in older adults, and may lead to fractures and bleeding. As many factors, such as medication, aging, and comorbid diseases may simultaneously affect fall-related adverse events (AEs) in older adults, we evaluated the association between fall-related AEs and the use of medication, aging, and comorbid diseases using the Japanese Adverse Drug Event Report (JADER) database. METHODS: We analyzed reports of fall-related AEs associated with α-blockers, diuretics, calcium channel blockers, central nervous system (CNS)-active drugs (opioids, benzodiazepines, hypnotics and sedatives, non-selective monoamine reuptake inhibitors, and selective serotonin reuptake inhibitors (SSRI)) in the JADER database using the reporting odds ratio (ROR). For the definition of falls, we used the Preferred Terms of The Medical Dictionary for Regulatory Activities (MedDRA). We used the association rule mining technique to discover undetected associations, such as potential risk factors. RESULTS: The JADER database comprised 430,587 reports between April 2004 and November 2016. The RORs (95% CI) of α-blockers, diuretics, calcium channel blockers, opioids, benzodiazepines, hypnotics and sedatives, non-selective monoamine reuptake inhibitors, and SSRIs were 1.63 (1.27-2.09), 0.74 (0.63-0.86), 1.26 (1.15-1.38), 0.93 (0.80-1.07), 1.83 (1.68-2.01), 1.55 (1.12-2.14), 2.31 (1.82-2.95), and 2.86 (2.49-3.29), respectively. From the lift value in the association rule mining, the number of administered CNS-active drugs and patient age were associated with fall-related AEs. Furthermore, the scores of lift for patients with herpes zoster administered calcium channel blockers or benzodiazepines and patients with dementia administered benzodiazepines were high. CONCLUSION: Our results suggest that the number of administered CNS-active drugs and patient age are both associated with fall-related AEs. We recommend that patients with herpes zoster treated with calcium channel blockers and benzodiazepines be closely monitored for fall-related AEs.
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OTC combination cold remedies are widely used in Japan. In the present study, we aimed to evaluate the adverse event profiles of OTC combination cold remedy based on the components using the Japanese Adverse Drug Event Report (JADER) database. The JADER database contained 430587 reports between April 2004 and November 2016. 1084 adverse events associated with the use of OTC combination cold remedy were reported. Reporting odds ratio (ROR) was used to detect safety signals. The ROR values for "skin and subcutaneous tissue disorders", "hepatobiliary disorders", and "immune system disorders" stratified by system organ class of the Medical Dictionary for Regulatory Activities (MedDRA) were 9.82 (8.71-11.06), 2.63 (2.25-3.07), and 3.13 (2.63-3.74), respectively. OTC combination cold remedy containing acetaminophen exhibited a significantly higher reporting ratio for "hepatobiliary disorders" than OTC combination cold remedy without acetaminophen. We demonstrated the potential risk of OTC combination cold remedy in a real-life setting. Our results suggested that the monitoring of individuals using OTC combination cold remedy is important.
Assuntos
Acetaminofen/efeitos adversos , Sistemas de Notificação de Reações Adversas a Medicamentos , Mineração de Dados , Bases de Dados Factuais , Uso de Medicamentos/estatística & dados numéricos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Medicamentos sem Prescrição/efeitos adversos , Acetaminofen/administração & dosagem , Sistemas de Notificação de Reações Adversas a Medicamentos/estatística & dados numéricos , Doenças Biliares/induzido quimicamente , Doenças Biliares/epidemiologia , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Combinação de Medicamentos , Doenças do Sistema Imunitário/induzido quimicamente , Doenças do Sistema Imunitário/epidemiologia , Japão/epidemiologia , Medicamentos sem Prescrição/administração & dosagem , Razão de Chances , Risco , Dermatopatias/induzido quimicamente , Dermatopatias/epidemiologia , Fatores de TempoRESUMO
Combined estrogen-progestin preparations (CEPs) are associated with thromboembolic (TE) side effects. The aim of this study was to evaluate the incidence of TE using the Japanese Adverse Drug Event Report (JADER) database. Adverse events recorded from April 2004 to November 2014 in the JADER database were obtained from the Pharmaceuticals and Medical Devices Agency (PMDA) website (www.pmda.go.jp). We calculated the reporting odds ratios (RORs) of suspected CEPs, analyzed the time-to-onset profile, and assessed the hazard type using Weibull shape parameter (WSP). Furthermore, we used the applied association rule mining technique to discover undetected relationships such as the possible risk factors. The total number of reported cases in the JADER contained was 338,224. The RORs (95% confidential interval, CI) of drospirenone combined with ethinyl estradiol (EE, Dro-EE), norethisterone with EE (Ne-EE), levonorgestrel with EE (Lev-EE), desogestrel with EE (Des-EE), and norgestrel with EE (Nor-EE) were 56.2 (44.3-71.4), 29.1 (23.5-35.9), 42.9 (32.3-57.0), 44.7 (32.7-61.1), and 38.6 (26.3-56.7), respectively. The medians (25%-75%) of the time-to-onset of Dro-EE, Ne-EE, Lev-EE, Des-EE, and Nor-EE were 150.0 (75.3-314.0), 128.0 (27.0-279.0), 204.0 (44.0-660.0), 142.0 (41.3-344.0), and 16.5 (8.8-32.0) days, respectively. The 95% CIs of the WSP-ß for Ne-EE, Lev-EE, and Nor-EE were lower and excluded 1. Association rule mining indicated that patients with anemia had a potential risk of developing a TE when using CEPs. Our results suggest that it is important to monitor patients administered CEP for TE. Careful observation is recommended, especially for those using Nor-EE, and this information may be useful for efficient therapeutic planning.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Estrogênios/administração & dosagem , Estrogênios/efeitos adversos , Progestinas/administração & dosagem , Progestinas/efeitos adversos , Tromboembolia/induzido quimicamente , Adolescente , Adulto , Sistemas de Notificação de Reações Adversas a Medicamentos , Androstenos/administração & dosagem , Androstenos/efeitos adversos , Criança , Anticoncepcionais Orais Combinados/administração & dosagem , Anticoncepcionais Orais Combinados/efeitos adversos , Bases de Dados Factuais , Desogestrel/administração & dosagem , Desogestrel/efeitos adversos , Etinilestradiol/administração & dosagem , Etinilestradiol/efeitos adversos , Feminino , Humanos , Japão , Levanogestrel/administração & dosagem , Levanogestrel/efeitos adversos , Masculino , Pessoa de Meia-Idade , Noretindrona/administração & dosagem , Noretindrona/efeitos adversos , Norgestrel/administração & dosagem , Norgestrel/efeitos adversos , Razão de Chances , Adulto JovemRESUMO
Statins, specific inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are now widely used for treatment of patients with hypercholesterolemia. In addition to the reduction of cholesterol biosynthesis, accumulating evidence indicates that statins have several pleiotropic effects especially on cardiovascular system. However, the exact role of statin in cardiac myocytes remains unclear. In the present study, we investigated whether atorvastatin induces vascular endothelial growth factor (VEGF) release in cardiac myocytes, and the underlying mechanism. We observed that atorvastatin significantly stimulated VEGF release in a dose-dependent manner. It induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The atorvastatin-induced VEGF release was enhanced by PD98059, which is a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Further, it was significantly reduced by SB203580, a specific inhibitor of p38 MAP kinase. Furthermore, the atorvastatin-induced phosphorylation of p38 MAP kinase was attenuated by SB203580, whereas it was enhanced by PD98059. Taken together, these results suggest that the atorvastatin-induced VEGF release in cardiac myocytes is positively regulated by p38 MAP kinase and negatively regulated byp44/p42 MAP kinase and that the atorvastatin-induced phosphorylation of p38 MAP kinase is regulated by p44/p42 MAP kinase in these cells.
Assuntos
Anticolesterolemiantes/farmacologia , Ácidos Heptanoicos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Miócitos Cardíacos/metabolismo , Pirróis/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Atorvastatina , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imidazóis/farmacologia , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Fosforilação , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Treatment of PBMCs with TNF-alpha decreased the levels of heat shock protein (HSP) 27, but had little effect on the level of HSP70. Parallel to the decrease of HSP27, TNF-alpha increased the level of HSP27 in the incubation medium of the cells. The decrease of HSP27 induced by TNF-alpha was suppressed by the pretreatment of PBMCs with the specific protein kinase C (PKC) inhibitor, GF109203X. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, decreased the levels of HSP27. To investigate the effect of TNF-alpha on the oligomerization state of HSP27 in PBMCs, we performed sucrose density gradient centrifugation with subsequent fractionation and immunoassay. Extract of vehicle-treated PBMCs contained mainly dissociated forms of HSP27. The amounts of dissociated forms of HSP27 in PBMCs was decreased by TNF-alpha, while the amounts of aggregated form of HSP27 was little changed. In intact PBMCs, HSP27 is constitutively phosphorylated at Ser78, but not at Ser15 or at Ser82. The amount of phosphorylated HSP27 at Ser78 was decreased by TNF-alpha. These results indicate that TNF-alpha reduces HSP27 in PBMCs through PKC activation. This decrease may be due to efflux of dissociated form of HSP27, phosphorylated HSP27 at Ser78, from the cells.
Assuntos
Proteínas de Choque Térmico/metabolismo , Leucócitos Mononucleares/enzimologia , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Carcinógenos/farmacologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Indóis/farmacologia , Leucócitos Mononucleares/citologia , Maleimidas/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.
Assuntos
Proteínas de Choque Térmico/metabolismo , Músculo Liso Vascular/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Trombina/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/fisiologia , Animais , Aorta/citologia , Bucladesina/farmacologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/farmacologia , Imidazóis/farmacologia , Músculo Liso Vascular/citologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Piridinas/farmacologia , RatosRESUMO
Retinoic acid modulates cell growth and differentiation of the vascular system. Vascular endothelial growth factor (VEGF) is known as a vascular permeability factor and a potent mitogen for vascular endothelial cells. In the present study, we investigated whether retinoic acid induces VEGF release in aortic smooth muscle A10 cells and if so, the mechanism of VEGF release. Retinoic acid stimulated VEGF release dose-dependently over the range 0.1 nM-0.1 microM. The retinoic acid-stimulated VEGF release was significantly reduced by actinomycin D. Retinoic acid induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase but not p38 MAP kinase or stress-activated protein kinase/c-Jun N-terminal kinase among the MAP kinase superfamily. This effect of retinoic acid was dose-dependent (30 nM-5 microM) and the maximum effect was observed at 0.3 microM. The retinoic acid-stimulated release of VEGF was significantly reduced by PD98059 and U0126, specific MEK inhibitors, which attenuated the retinoic acid-induced phosphorylation of p44/p42 MAP kinase. These results strongly suggest that retinoic acid stimulates the release of VEGF in a p44/p42 MAP kinase-dependent manner in aortic smooth muscle cells.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Tretinoína/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Técnicas de Cultura de Células , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosforilação/efeitos dos fármacos , RatosRESUMO
As for the pathogenesis of rheumatoid arthritis (RA), prostaglandins (PGs) act as important mediators of inflammation and joint destruction. Among them, PGD2 is well recognized as a potent regulator of osteoblastic functions. We previously showed that PGD2 stimulates the induction of heat shock protein 27 (HSP27) via protein kinase C (PKC)-dependent p38 mitogen-activated protein (MAP) kinase and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Therefore, it is a current topic to clarify how HSP27 plays a role for regulating osteoblastic functions in the lesion of RA. On the other hand, methotrexate (MTX) is one of the most effective medicines for the treatment of RA. Here, we examined the effect of MTX on PGD2-stimulated HSP27 induction in MC3T3-E1 cells. The cells were pretreated with various doses of MTX including therapeutic dosage for RA, and then stimulated by PGD2. MTX significantly enhanced the PGD2- increased levels of HSP27 in a dose-dependent manner, although MTX alone had no effect on the levels of HSP27. In addition, MTX amplified the PGD2-increased levels of HSP27 mRNA. On the contrary, MTX had little effect on PGD2-induced formation of inositol phosphates, PKC activation and phosphorylations of MAP kinases. Our results strongly suggest that MTX enhances PGD2-stimulated HSP27 induction at a point downstream from MAP kinases in osteoblasts.
Assuntos
Proteínas de Choque Térmico/metabolismo , Metotrexato/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Prostaglandina D2/metabolismo , Animais , Antracenos/farmacologia , Northern Blotting , Células Cultivadas , Cicloeximida/farmacologia , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dactinomicina/farmacologia , Ativação Enzimática , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Fosfatos de Inositol/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/metabolismoRESUMO
We investigated whether tumor necrosis factor-alpha (TNF-alpha) stimulates the induction of heat shock protein 27 (HSP27) in human neutrophils and the mechanism underlying this induction. In intact neutrophils, almost no HSP27 was detected. Stimulation of neutrophils by TNF-alpha increased the levels of HSP27 in the presence, but not in the absence, of cycloheximide. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that TNF-alpha also induced HSP27 mRNA in the presence of cycloheximide. TNF-alpha induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TNF-alpha was significantly suppressed by 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) or 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole (PD169316); both are specific inhibitors of p38 MAP kinase, but not by 2'-amino-3'-methoxyflavone (PD098059, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase). The accumulation of HSP27 induced by TNF-alpha plus cycloheximide was also suppressed by pretreatment with a specific protein kinase C (PKC) inhibitor. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, stimulated the accumulation of HSP27. Interestingly, SB203580 did not inhibit PMA-stimulated HSP27 induction. These results strongly suggest that TNF-alpha may act as the regulator of HSP27 induction in neutrophils. p38 MAP kinase (but not p44/p42 MAP kinase) and PKC take part in TNF-alpha-stimulated HSP27 induction in human neutrophils.
Assuntos
Proteínas de Choque Térmico/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neutrófilos/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Humanos , Imidazóis/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Inibidores da Síntese de Proteínas , Piridinas/farmacologia , Pirrolidinas/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiocarbamatos/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
We previously reported that transforming growth factor-beta (TGF-beta) stimulates the release of vascular endothelial growth factor (VEGF) from aortic smooth muscle A10 cells via activation of p38 mitogen-activated protein (MAP) kinase. In the present study, we investigated whether nuclear hormone receptor superfamily members affect TGF-beta-stimulated VEGF release from A10 cells. Retinoic acid or 1,25-dihydroxyvitamin D3 enhanced TGF-beta-induced VEGF release in a concentration-dependent manner, whereas dexamethasone or corticosterone suppressed TGF-beta-induced VEGF release. 1,25-Dihydroxyvitamin D3 and TGF-beta stimulated phosphorylation of p38 MAP kinase in an additive manner. SB203580, an inhibitor of p38 MAP kinase, decreased the VEGF release induced by TGF-beta or 1,25-dihydroxyvitamin D3. However, retinoic acid, dexamethasone, or corticosterone did not affect phosphorylation of p38 MAP kinase. These results indicate that retinoic acid, 1,25-dihydroxyvitamin D3, and glucocorticoids affect TGF-beta-stimulated VEGF release from aortic smooth muscle cells. The stimulatory effect of 1,25-dihydroxyvitamin D3 occurs, in part, via modification of TGF-beta-induced activation of p38 MAP kinase.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Esteroides/farmacologia , Hormônios Tireóideos/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Calcitriol/farmacologia , Células Cultivadas , Colecalciferol/farmacologia , Corticosterona/farmacologia , Dexametasona/farmacologia , Inibidores Enzimáticos/farmacologia , Hormônios Esteroides Gonadais/farmacologia , Imidazóis , Músculo Liso Vascular/metabolismo , Fosforilação , Piridinas , Ratos , Tretinoína/farmacologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Interleukin 6 (IL-6) is a pleiotropic cytokine that plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). In bone metabolism, it is known that IL-6 is produced and secreted by osteoblasts, and that IL-6 induces osteoclast formation and stimulates bone resorption. Various bone inflammatory agonists such as tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, prostaglandin D2 (PGD2), PGE2, and PGF2alpha, which play important roles in the pathogenesis of RA, induce IL-6 synthesis in osteoblast-like MC3T3-E1 cells. Low dose methotrexate (MTX) is currently used for treatment of patients with RA. We investigated the effect of MTX on IL-6 synthesis induced by these agents in MC3T3-E1 cells. METHODS: Cultured cells were pretreated with various doses of MTX, and then stimulated by these inflammatory agonists. The IL-6 in the conditioned medium was measured by IL-6 enzyme immunoassay. RESULTS: MTX significantly suppressed IL-6 synthesis stimulated by these agonists in a dose-dependent manner, although MTX alone had no effect on the levels of IL-6. In addition, MTX significantly inhibited the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. MTX reduced the levels of IL-6 induced by 12-O-tetradecanoylphorbol 13-acetate, a direct activator of protein kinase C (PKC), suggesting that MTX inhibits PKC signals for IL-6 synthesis. CONCLUSION: MTX suppresses IL-6 synthesis stimulated by various inflammatory agonists in osteoblasts.
Assuntos
Antirreumáticos/farmacologia , Interleucina-6/metabolismo , Metotrexato/farmacologia , Osteoblastos/efeitos dos fármacos , Células 3T3 , Animais , Carcinógenos/farmacologia , Células Cultivadas , Dinoprosta/farmacologia , Dinoprostona/farmacologia , Interações Medicamentosas , Interleucina-1/farmacologia , Interleucina-17/farmacologia , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Prostaglandina D2/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We previously reported that p38 mitogen-activated protein (MAP) kinase takes a part in arginine vasopressin (AVP)-induced heat shock protein 27 (HSP27) phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the phosphorylation of HSP27 in these cells. AVP time-dependently induced the phosphorylation of PI3K and Akt. Akt inhibitor, 1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, partially suppressed the phosphorylation of HSP27. The AVP-induced HSP27 phosphorylation was attenuated by LY294002, a PI3K inhibitor. The combination of Akt inhibitor and SB203580, a p38 MAP kinase inhibitor, completely suppressed the AVP-induced phosphorylation of HSP27. Furthermore, LY294002 or Akt inhibitor did not affect the AVP-induced phosphorylation of p38 MAP kinase and SB203580 did not affect the phosphorylation of PI3K or Akt. These results suggest that PI3K/Akt plays a part in the AVP-induced phosphorylation of HSP27, maybe independently of p38 MAP kinase, in aortic smooth muscle A10 cells.
Assuntos
Aorta/metabolismo , Proteínas de Choque Térmico/metabolismo , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vasopressinas/farmacologia , Animais , Western Blotting , Linhagem Celular , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP27 , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases , Chaperonas Moleculares , Morfolinas/farmacologia , Músculo Liso Vascular/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-akt , Piridinas/farmacologia , Quinolonas/farmacologia , Ratos , Fatores de TempoRESUMO
We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release. In the present study, we investigated the effects of ciglitazone and pioglitazone, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, on the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ciglitazone. The amplifying effect of ciglitazone was dose-dependent between 0.1 and 10 microM. Pioglitazone had a similar effect on the VEGF release. GW9662, an antagonist of PPAR-gamma, reduced the effects of ciglitazone and pioglitazone. Ciglitazone or pioglitazone markedly enhanced the phosphorylation of SAPK/JNK induced by FGF-2 without affecting both the FGF-2-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. GW9662 markedly reduced the amplification by ciglitazone of the SAPK/JNK phosphorylation. Taken together, these results strongly suggest that PPAR-gamma ligands up-regulate FGF-2-stimulated VEGF release resulting from amplifying activation of SAPK/JNK in osteoblasts.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , PPAR gama/metabolismo , Tiazolidinedionas/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células 3T3 , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Ligantes , Camundongos , Pioglitazona , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.