RESUMO
Pulmonary fibrosis is a fatal condition characterized by fibroblast and myofibroblast proliferation and collagen deposition. TGF-ß plays a pivotal role in the development of pulmonary fibrosis. Therefore, modulation of TGF-ß signaling is a promising therapeutic strategy for treating pulmonary fibrosis. To date, however, interventions targeting TGF-ß have not shown consistent efficacy. CD109 is a GPI-anchored glycoprotein that binds to TGF-ß receptor I and negatively regulates TGF-ß signaling. However, no studies have examined the role and therapeutic potential of CD109 in pulmonary fibrosis. The purpose of this study was to determine the role and therapeutic value of CD109 in bleomycin-induced pulmonary fibrosis. CD109-transgenic mice overexpressing CD109 exhibited significantly attenuated pulmonary fibrosis, preserved lung function, and reduced lung fibroblasts and myofibroblasts compared with wild-type (WT) mice. CD109-/- mice exhibited pulmonary fibrosis comparable to WT mice. CD109 expression was induced in variety types of cells, including lung fibroblasts and macrophages, upon bleomycin exposure. Recombinant CD109 protein inhibited TGF-ß signaling and significantly decreased ACTA2 expression in human fetal lung fibroblast cells in vitro. Administration of recombinant CD109 protein markedly reduced pulmonary fibrosis in bleomycin-treated WT mice in vivo. Our results suggest that CD109 is not essential for the development of pulmonary fibrosis, but excess CD109 protein can inhibit pulmonary fibrosis development, possibly through suppression of TGF-ß signaling. CD109 is a novel therapeutic candidate for treating pulmonary fibrosis.
Assuntos
Fibrose Pulmonar , Humanos , Camundongos , Animais , Fibrose Pulmonar/metabolismo , Bleomicina/efeitos adversos , Fator de Crescimento Transformador beta/metabolismo , Pulmão/patologia , Fibroblastos/metabolismo , Camundongos Transgênicos , Fatores de Transcrição/metabolismo , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/metabolismo , Antígenos CD/metabolismo , Proteínas Ligadas por GPI/metabolismoRESUMO
REV7 is a multifunctional protein implicated in various biological processes, including DNA damage response. REV7 expression in human cancer cells affects their sensitivity to DNA-damaging agents. In the present study, we investigated the significance of REV7 in pancreatic ductal adenocarcinoma (PDAC). REV7 expression was immunohistochemically examined in 92 resected PDAC specimens and 60 endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) specimens of unresectable PDAC treated with platinum-based chemotherapy, and its association with clinicopathologic features was analyzed. Although REV7 expression was not significantly associated with the progression of primary tumors (T-factor and Stage) in either resected or unresectable PDAC, decreased levels of REV7 expression in EUS-FNAB specimens of unresectable PDAC were significantly associated with better outcomes of platinum-based chemotherapy and a favorable prognosis. REV7-deficient PDAC cell lines showed suppressed cell growth and enhanced sensitivity to cisplatin in vitro. Tumor-bearing mice generated using REV7-deficient PDAC cell lines also showed enhanced sensitivity to cisplatin in vivo. RNA sequencing analysis using WT and REV7-deficient PDAC cell lines revealed that REV7 inactivation promoted the downregulation of genes involved in the DNA repair and the upregulation of genes involved in apoptosis. Our results indicate that decreased expression of REV7 is associated with better outcomes of platinum-based chemotherapy in PDAC by suppressing the DNA damage response. It is also suggested that REV7 is a useful biomarker for predicting the outcome of platinum-based chemotherapy and the prognosis of unresectable PDAC and is a potential target for PDAC treatment.
Assuntos
Adenocarcinoma , Fenômenos Biológicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Platina/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Adenocarcinoma/tratamento farmacológico , Reparo do DNA/genéticaRESUMO
T315I mutation found in chronic myelogenous leukemia (CML) and Ph + ALL patients is the most serious one among resistance against BCR/ABL kinase inhibitors including imatinib and is only responsive to ponatinib (PNT). However, the novel strategy is required to reduce life-threatening adverse effects of PNT including ischemic cardiovascular disease. We examined the mechanism of PNT-induced cytotoxicity against a T315I(+) Ph + ALL cell line, TccY/Sr. PNT induced apoptosis (increased sub G1 cells, and cleaved caspase3 and PARP), and suppressed protein expression of MCL1, cyclin D2 and c-myc, which were reversed by a proteasome inhibitor, MG132, suggesting enhanced proteasomal degradation by PNT. Among BCL2 family inhibitors, MCL1 inhibitors (maritoclax and AZD5991) robustly induced cell death, showing the MCL1-dependent survival of TccY/Sr cells. Decreased MCL1 and c-myc expression by PNT was also observed in T315I(+) MEGA2/STIR cells. PNT suppressed PI3K activation followed by AKT inhibition and GSK3 dephosphorylation. PI3K/AKT inhibitors mimicked PNT, suggesting that PI3K/AKT signaling is important for survival of TccY/Sr cells. Moreover, GSK3 inhibitor (SB216763) reduced PNT-induced cytotoxicity and degradation of c-myc and MCL1. AZD5991 exhibited the synergistic action with PNT, anti-cancer drugs and venetoclax (BCL2 inhibitor), suggesting the utility of MCL1 inhibitor alone or in combination as a future clinical option for Ph + leukemia patients.
Assuntos
Antineoplásicos/farmacologia , Ciclina D2/metabolismo , Mesilato de Imatinib/farmacologia , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridazinas/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Ciclina D2/genética , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leupeptinas/farmacologia , Compostos Macrocíclicos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Wortmanina/farmacologiaRESUMO
BACKGROUND: Animal model studies show that reductive stress is involved in cardiomyopathy and myopathy, but the exact physiological relevance remains unknown. In addition, the microRNAs miR-143 and miR-145 have been shown to be upregulated in cardiac diseases, but the underlying mechanisms associated with these regulators have yet to be explored. METHODS: We developed transgenic mouse lines expressing exogenous miR-143 and miR-145 under the control of the alpha-myosin heavy chain (αMHC) promoter/enhancer. RESULTS: The two transgenic lines showed dilated cardiomyopathy-like characteristics and early lethality with markedly increased expression of miR-143. The expression of hexokinase 2 (HK2), a cardioprotective gene that is a target of miR-143, was strongly suppressed in the transgenic hearts, but the in vitro HK activity and adenosine triphosphate (ATP) content were comparable to those observed in wild-type mice. In addition, transgenic complementation of HK2 expression did not reduce mortality rates. Although HK2 is crucial for the pentose phosphate pathway (PPP) and glycolysis, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was unexpectedly higher in the hearts of transgenic mice. The expression of gamma-glutamylcysteine synthetase heavy subunit (γ-GCSc) and the in vitro activity of glutathione reductase (GR) were also higher, suggesting that the recycling of GSH and its de novo biosynthesis were augmented in transgenic hearts. Furthermore, the expression levels of glucose-6-phosphate dehydrogenase (G6PD, a rate-limiting enzyme for the PPP) and p62/SQSTM1 (a potent inducer of glycolysis and glutathione production) were elevated, while p62/SQSTM1 was upregulated at the mRNA level rather than as a result of autophagy inhibition. Consistent with this observation, nuclear factor erythroid-2 related factor 2 (Nrf2), Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) were activated, all of which are known to induce p62/SQSTM1 expression. CONCLUSIONS: Overexpression of miR-143 and miR-145 leads to a unique dilated cardiomyopathy phenotype with a reductive redox shift despite marked downregulation of HK2 expression. Reductive stress may be involved in a wider range of cardiomyopathies than previously thought.
Assuntos
Cardiomiopatias/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa Redutase/metabolismo , Glicólise/fisiologia , Hexoquinase/metabolismo , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , RNA Mensageiro/metabolismo , Regulação para Cima/fisiologiaRESUMO
PURPOSE: Hypertension is an important risk factor for death resulting from stroke, myocardial infarction, and end-stage renal failure. Hydrogen (H2) gas protects against many diseases, including ischemia-reperfusion injury and stroke. The effects of H2 on hypertension and its related left ventricular (LV) function have not been fully elucidated. The purpose of this study was to investigate the effects of H2 gas on hypertension and LV hypertrophy using echocardiography. METHODS: Dahl salt-sensitive (DS) rats were randomly divided into three groups: those fed an 8% NaCl diet until 12 weeks of age (8% NaCl group), those additionally treated with 2% H2 gas (8% NaCl + 2% H2 group), and control rats maintained on a diet containing 0.3% NaCl until 12 weeks of age (0.3% NaCl group). H2 gas was supplied through a gas flowmeter and delivered by room air (2% hydrogenated room air, flow rate of 10 L/min) into a cage surrounded by an acrylic chamber. We evaluated interventricular septal wall thickness (IVST), LV posterior wall thickness (LVPWT), and LV mass using echocardiography. RESULTS: IVST, LVPWT, and LV mass were significantly higher in the 8% NaCl group than the 0.3% NaCl group at 12 weeks of age, whereas they were significantly lower in the 8% NaCl + 2% H2 group than the 8% NaCl group. There was no significant difference in systolic blood pressure between the two groups. CONCLUSION: Our findings suggest that chronic H2 gas inhalation may help prevent LV hypertrophy in hypertensive DS rats.
Assuntos
Gases/uso terapêutico , Hidrogênio/uso terapêutico , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Animais , Pressão Sanguínea/efeitos dos fármacos , Ecocardiografia , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Masculino , Ratos , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta/administração & dosagem , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Molecular hydrogen (H2) is a biologically active gas that is used medically to ameliorate various systemic pathological conditions. H2 also regulates gene expression involved in intracellular signaling and metabolic pathways. However, it is unclear whether H2 affects gene expression directly or through indirect effects as a consequence of health improvement. Therefore, we attempted to identify genes that exhibit similar changes in expression in response to H2 by employing DNA microarrays and gene set enrichment analysis to analyze RNA from liver and lung of rats and mice with or without dietary stress. We found that H2 activated the expression of sets of genes regulated by histone H3K27 methylation status. H2 also modified the expression of many genes regulated by a wide variety of signaling pathways. RT-qPCR showed that H2 up-regulated expression of Kcnc3, a H3K27-regulated gene, in organs such as liver, lung, kidney and brain. Furthermore, using immunohistochemistry and immunoblot analysis, we observed changes in H3K27 methylation status in the liver of mice and rats administered H2. Moreover, we showed that H2 simultaneously induced the H3K27 demethylase, Jmjd3, and mitochondrial unfolded protein response (mtUPR)-related genes. Recently, alteration of mitochondrial function was shown to cause induction of H3K27 demethylase or chromatin restructuring, followed by mtUPR activation through the alteration of H3K27 or H3K9 methylation states. Taken together, our study suggests that H2 can induce beneficial effects through mtUPR activation via epigenetic histone modification and by modification of gene expression.
Assuntos
Código das Histonas/genética , Hidrogênio/administração & dosagem , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Resposta a Proteínas não Dobradas/genética , Animais , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Código das Histonas/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Especificidade da Espécie , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Taxoids are anti-cancer drugs frequently used to treat solid tumors, but they are sometimes ineffective and tumors may become resistant to their action. Here, we examined the involvement of sphingolipid metabolic enzymes in paclitaxel (PTX) resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. PTX (20 nM) suppressed cell proliferation and increased various ceramide species in PC3, but not PC3-PR, cells. PC3-PR contained higher S1P levels than did PC3, regardless of PTX treatment. Western blotting revealed that PC3-PR cells expressed higher levels of sphingosine kinase 1 (SPHK1) and glucosylceramide synthase (GCS) but lower levels of acid sphingomyelinase (ASMase) and neutral sphingomyelinase 2 than did PC3 cells. Inhibition of SPHK1 using siRNA or a pharmacological inhibitor decreased S1P levels in PC3-PR cells and inhibited proliferation in the presence or absence of PTX, suggesting that SPHK1 is at least partially responsible for PTX resistance. Similarly, GCS inhibitors (PDMP and PPMP) increased cellular ceramides and suppressed the proliferation of PC3-PR. However, inhibition of proteasome function or histone deacetylase activity increased SMase and ceramide levels and suppressed PC3-PR proliferation. These results suggest that modulation of metabolic enzyme expression and alteration of the sphingolipid rheostat protects cancer cells against PTX.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Paclitaxel/farmacologia , Esfingolipídeos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Células K562 , Masculino , Meperidina/análogos & derivados , Meperidina/farmacologia , Morfolinas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patient's prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of ßIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Paclitaxel/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Taxoides/farmacologia , Acetilação , Anilidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Estabilidade Proteica/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologia , VorinostatRESUMO
Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment.
Assuntos
Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Estilbenos/administração & dosagem , Ativação Transcricional/efeitos dos fármacos , Anticarcinógenos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Neoplasias Experimentais/patologia , Resveratrol , Esfingomielina Fosfodiesterase/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Attempts to establish a tissue bank from autopsy samples have led to uncovering of the secrets of many diseases. Here, we examined the length of time that the RNA from postmortem tissues is available for microarray analysis and reported the gene expression profile for up- and down-regulated genes during the postmortem interval. We extracted RNA from fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) brains and livers of three different groups of mice: 1) mice immediately after death, 2) mice that were stored at room temperature for 3h after death, and 3) mice that were stored at 4°C for 18h after death, as this storage resembles the human autopsy process in Japan. The RNA quality of the brain and the liver was maintained up to 18h during the postmortem interval. Based on the microarray analysis, we selected genes that were altered by >1.3-fold or <0.77-fold and classified these genes using hierarchical cluster analysis following DAVID gene ontology analysis. These studies revealed that cytoskeleton-related genes were enriched in the set of up-regulated genes, while serine protease inhibitors were enriched in the set of down-regulated genes. Interestingly, although the RNA quality was maintained due to high RNA integrity number (RIN) values, up-regulated genes were not validated by quantitative PCR, suggesting that these genes may become fragmented or modified by an unknown mechanism. Taken together, our findings suggest that under typical autopsy conditions, gene expression profiles that reflect disease pathology can be examined by understanding comprehensive recognition of postmortem fluctuation of gene expression.
Assuntos
Autopsia/métodos , Perfilação da Expressão Gênica/métodos , Mudanças Depois da Morte , Preservação de Tecido/métodos , Animais , Encéfalo/metabolismo , Análise por Conglomerados , Temperatura Baixa , Formaldeído , Ontologia Genética , Humanos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fixação de TecidosRESUMO
Molecular hydrogen (H2) is an agent with potential applications in oxidative stress-related and/or inflammatory disorders. H2 is usually administered by inhaling H2-containing air (HCA) or by oral intake of H2-rich water (HRW). Despite mounting evidence, the molecular mechanism underlying the therapeutic effects and the optimal method of H2 administration remain unclear. Here, we investigated whether H2 affects signaling pathways and gene expression in a dosage- or dose regimen-dependent manner. We first examined the H2 concentrations in blood and organs after its administration and found that oral intake of HRW rapidly but transiently increased H2 concentrations in the liver and atrial blood, while H2 concentrations in arterial blood and the kidney were one-tenth of those in the liver and atrial blood. In contrast, inhalation of HCA increased H2 equally in both atrial and arterial blood. We next examined whether H2 alters gene expression in normal mouse livers using DNA microarray analysis after administration of HCA and HRW. Ingenuity Pathway Analysis revealed that H2 suppressed the expression of nuclear factor-kappa B (NF-κB)-regulated genes. Western blot analysis showed that H2 attenuated ERK, p38 MAPK, and NF-κB signaling in mouse livers. Finally, we evaluated whether the changes in gene expression were influenced by the route of H2 administration and found that the combination of both HRW and HCA had the most potent effects on signaling pathways and gene expression in systemic organs, suggesting that H2 may act not only through a dose-dependent mechanism but also through a complex molecular network.
Assuntos
Hidrogênio/administração & dosagem , Hidrogênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Administração por Inalação , Administração Oral , Ar , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hidrogênio/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Especificidade de Órgãos/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/genética , Fatores de Tempo , Água/administração & dosagem , Água/farmacologiaRESUMO
Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.
Assuntos
Interleucina-6/imunologia , Queratinócitos/efeitos da radiação , Melanócitos/enzimologia , Melanoma/enzimologia , Sialiltransferases/genética , Fator de Necrose Tumoral alfa/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinócitos/imunologia , Melanócitos/imunologia , Melanócitos/efeitos da radiação , Melanoma/genética , Melanoma/imunologia , Sialiltransferases/imunologia , Raios Ultravioleta , Regulação para CimaRESUMO
CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-ß signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-ß signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.
Assuntos
Antígenos CD/metabolismo , Epiderme/anormalidades , Epiderme/patologia , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/metabolismo , Animais , Epiderme/metabolismo , Técnicas de Introdução de Genes , Marcação de Genes , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Humanos , Hiperplasia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Testículo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta-Galactosidase/metabolismoRESUMO
Growth-inhibitory effects of mimosine, a plant amino acid, on rat C6 glioma cells were analyzed. Mimosine markedly inhibited proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent manner. Mimosine-mediated apoptosis was accompanied by promoting reactive oxygen species (ROS) generation in mitochondria, and by decreased mitochondrial membrane potential (Δψ), and release of cytochrome c from mitochondria, followed by caspase 3 activation. Furthermore, mimosine increased the phosphorylation level of c-Jun-N-terminal protein kinase and p38, which was the downstream effect of ROS accumulation. Mimosine was confirmed to show profound effects on apoptosis of C6 glioma cells by ROS-regulated mitochondria pathway, and these results bear on the hypothesized potential for mimosine as promising agents in the treatment of malignant gliomas.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Glioma/patologia , MAP Quinase Quinase 4/metabolismo , Mimosina/farmacologia , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Western Blotting , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Ensaio Cometa , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Imunofluorescência , Glioma/enzimologia , Glioma/metabolismo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismoRESUMO
Invasive diseases such as toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) are re-emerging infectious diseases. The mechanism of pathogenesis is not completely understood although the virulence of this organism has been analyzed using animal model systems, particularly using mice. The analysis of the progression of infection, however, is difficult. Computed tomography (CT) scanning is an extremely powerful technique that we applied to the mouse model of cutaneous infection with S. pyogenes. Two or three days after subcutaneous administration of bacteria, high density reticular areas were detected in the lung by CT. Histopathological examination of the lung was performed to examine the results of CT. Increased numbers of cytokeratin-positive epithelial cells, probably alveolar type II epithelial cells, were detected but no remarkable increase of inflammatory cell infiltrates was observed. Our results show that the pathological lesions of the lung in this model, wherein relatively few numbers of neutrophils were in the alveoli, are well correlated with the lung of a part of streptococcal toxic shock syndrome patients. Therefore, CT may be useful in assessing the progression of S. pyogenes infection, particularly in the pathological lesions of the lung in this model.
Assuntos
Pulmão/patologia , Dermatopatias Infecciosas/patologia , Pele/patologia , Infecções Estreptocócicas/patologia , Animais , Modelos Animais de Doenças , Pulmão/microbiologia , Camundongos , Pele/microbiologia , Dermatopatias Infecciosas/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenesRESUMO
Glial cell line-derived neurotrophic factor (GDNF) is important for the development and maintenance of dopamine neurons (Lin et al. [1993] Science 260: 1130-1132). GDNF is neuroprotective in animal models of Parkinson disease, where dopamine neurons show selective degeneration. We previously reported GDNF-induced SPHK1 gene expression in a neuroblastoma cell line, TGW (Murakami et al. [2007] J Neurochem 102: 1585-1594). In the present study, we focused on the regulatory mechanism of GAP43 (GDNF-induced neuronal phenotype) transcription to further elucidate physiological roles of GDNF-induced SPHK1 expression and activity. Stable wild-type (SPHK1-WT) but not dominant-negative SPHK1 (SPHK1-DN) overexpression increased both control- and GDNF-induced GAP43 expression. SPHK1-WT cells showed enhanced GDNF-induced sphingosine 1-phosphate (S1P) secretion compared with mock- and SPHK1-DN cells. Exogenous S1P also increased GAP43 expression. In TGW cells, PD98059, a MEK inhibitor, but not SB203580 (a p38 MAPK inhibitor) and LY294002 (a PI3K inhibitor) inhibited GDNF-induced GAP43 expression, suggesting the MEK/ERK pathway has a major role in GDNF-induced GAP43 transcription. A G-protein-coupled receptor inhibitor, pertussis toxin, and S1P(1) and S1P(3) receptor antagonists (VPC23019 and CAY10444) also inhibited ERK activation. Moreover, both S1P1 and S1P3 were serine-phosphorylated by GDNF, suggesting their activated states. C/EBPß transcription factor was induced by GDNF, and DNA pull-down and chromatin immunoprecipitation assays revealed the C/EBP binding site between -131 bp and -98 bp from the first exon of GAP43. Taken together, our results showed that in TGW cells, GDNF increased SPHK1 transcription, leading to the production and secretion of S1P. Through MEK/ERK pathway, S1P stimulates GAP43 transcription with increased binding of C/EBPß to the 5'-promoter.
Assuntos
Proteína GAP-43/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Transcrição Gênica , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Clonagem Molecular , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Transdução de Sinais , Esfingosina/metabolismoRESUMO
Molecular hydrogen has been reported to be effective for a variety of disorders and its effects have been ascribed to the reduction of oxidative stress. However, we have recently demonstrated that hydrogen inhibits type I allergy through modulating intracellular signal transduction. In the present study, we examined the hydrogen effects on lipopolysaccharide/interferon γ LPS/IFNγ-induced nitric oxide (NO) production in murine macrophage RAW264 cells. Treatment with hydrogen reduced LPS/IFNγ-induced NO release, which was associated with a diminished induction of inducible isoform of nitric oxide synthase (iNOS). Hydrogen treatment inhibited LPS/IFNγ-induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream signaling molecules, p38 MAP kinase and JNK, as well as IκBα, but did not affect activation of NADPH oxidase and production of reactive oxygen species (ROS). As ROS is an upstream activator of ASK1, inhibition of ASK1 by hydrogen without suppressing ROS implies that a potential target molecule of hydrogen should be located at the receptor or immediately downstream of it. These results suggested a role for molecular hydrogen as a signal modulator. Finally, oral intake of hydrogen-rich water alleviated anti-type II collagen antibody-induced arthritis in mice, a model for human rheumatoid arthritis. Taken together, our studies indicate that hydrogen inhibits LPS/IFNγ-induced NO production through modulation of signal transduction in macrophages and ameliorates inflammatory arthritis in mice, providing the molecular basis for hydrogen effects on inflammation and a functional interaction between two gaseous signaling molecules, NO and molecular hydrogen.
Assuntos
Hidrogênio/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico/antagonistas & inibidores , Animais , Artrite Experimental/tratamento farmacológico , Linhagem Celular , Feminino , Hidrogênio/administração & dosagem , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Transdução de Sinais/efeitos dos fármacosRESUMO
Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RARalpha, and RXRalpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.
Assuntos
Fator I de Transcrição COUP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Sítios de Ligação/efeitos dos fármacos , Fator I de Transcrição COUP/genética , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Éxons/efeitos dos fármacos , Éxons/fisiologia , Humanos , Imunoprecipitação/métodos , Dados de Sequência Molecular , Neuritos/efeitos dos fármacos , Neuroblastoma , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides/metabolismo , Transfecção/métodosRESUMO
The various beneficial effects of the intake of molecular hydrogen (H2) have been demonstrated in the field of sports science. Although supplementation of H2 has been reported to increase mitochondrial metabolism in animal studies, the effects of the administration of H2 on aerobic capacity during exercise in humans are still not clear. We investigated whether a single or 2-week continuous intake of H2-rich water (HW) enhanced the aerobic capacity during incremental exercise in healthy humans. In this randomized, single-blinded, placebo-controlled experimental study, the participants performed an incremental cycling exercise to measure peak oxygen uptake and peak load before and after a single (500 mL) or a 2-week supplementation (total 5 L) of HW. In the latter experiment, the participants drank the 500 mL of HW on all weekdays (i.e., 10 times). The single intake of HW did not significantly increase peak oxygen uptake and peak load, and did not significantly alter the responses in oxidative stress, antioxidant activity, and lactate levels. However, importantly, the 2-week continuous consumption of HW significantly augmented peak oxygen uptake and tended to increase the peak load without any significant changes in lactate levels, oxidative stress, and antioxidant responses. In conclusion, the continuous supplementation of HW potentially augments the aerobic capacity, implying that continuous supplementation of H2 might help improve aerobic exercise performance and physical health. This study protocol was approved by the Ethical Committee of Chubu University (approval No. 260086-2) on March 29, 2018.
Assuntos
Ciclismo/fisiologia , Exercício Físico , Hidrogênio/análise , Oxigênio/metabolismo , Água/química , Água/farmacologia , Adulto , Transporte Biológico/efeitos dos fármacos , Método Duplo-Cego , Voluntários Saudáveis , Humanos , Masculino , Placebos , Adulto JovemRESUMO
Aerobic exercise is widely accepted as a beneficial option for reducing fat in humans. Recently, it has been suggested that molecular hydrogen (H2) augments mitochondrial oxidative phosphorylation. Therefore, the hypothesis that inhaling H2 could facilitate lipid metabolism during aerobic exercise was investigated in the current study by measuring the breath acetone levels, which could be used as non-invasive indicators of lipid metabolism. This study aimed to investigate the effect of inhaling H2 on breath acetone output during submaximal exercise using a randomized, single-blinded, placebo-controlled, and cross-over experimental design. After taking a 20-minute baseline measurement, breath acetone levels were measured in ten male subjects who performed a 60% peak oxygen uptake-intensity cycling exercise for 20 minutes while inhaling either 1% H2 or a control gas. In another experiment, six male subjects remained in a sitting position for 45 minutes while inhaling either 1% H2 or a control gas. H2 significantly augmented breath acetone and enhanced oxygen uptake during exercise (P < 0.01). However, it did not significantly change oxidative stress or antioxidant activity responses to exercise, nor did it significantly alter the breath acetone or oxygen uptake during prolonged resting states. These results suggest that inhaling H2 gas promotes an exercise-induced increase in hepatic lipid metabolism. The study was approved by the Ethical Committee of Chubu University, Japan (approved No. 260086-2) on March 29, 2018.