RESUMO
Treatment of the mouse leukemic cell line RAW 264 with bafilomycin A1 or concanamycin A, inhibitors of vacuolar-type (H(+))-ATPases (V-ATPases), significantly increased the production of reactive oxygen species (ROS) and decreased cell viability. These effects were significantly suppressed by the presence of N-acetyl cysteine (NAC), an ROS scavenger. si-RNA mediated knockdown of the gene for the c subunit of the V0 domain of V-ATPase also resulted in an increase in ROS production and a decrease in cell viability. These results suggest that decreased cellular V-ATPase activity decreases cell viability by increasing ROS production in RAW 264 cells.
Assuntos
Inibidores Enzimáticos/toxicidade , Macrolídeos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , CamundongosRESUMO
We established a novel dermatitis model in mice earlobes and analyzed the roles of histamine using specific antagonists for histamine receptors. After sensitization with picryl chloride (PiCl) by painting it on the earlobes of cyclophosphamide-treated mice, 12-O-tetradecanoylphorbol 13-acetate (TPA) was painted twice at the same site, and then allergic inflammation was induced by painting with PiCl. Histamine antagonists and cyclosporin A were administered i.v. The application of TPA shifted the PiCl-induced allergic inflammation from a delayed-type response to a biphasic response and increased the infiltration of eosinophils and mast cells at the inflammatory site. In this model, the PiCl-induced increase in the thickness of the earlobe in the immediate phase was suppressed by the histamine H1 antagonist pyrilamine. In contrast, the increase in the swelling in the late phase and the infiltration of eosinophils were suppressed by the H3/H4 antagonist thioperamide. The inhibitory effect of the combined treatment with pyrilamine and thioperamide on TPA-modified contact dermatitis was as potent as that of cyclosporin A. Histamine plays significant roles in early-phase swelling via H1 receptors and in late-phase swelling via H3/H4 receptors in this TPA-modified allergic dermatitis model.
Assuntos
Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/etiologia , Histamina/fisiologia , Animais , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Quimioterapia Combinada , Antagonistas dos Receptores Histamínicos/uso terapêutico , Humanos , Camundongos , Cloreto de Picrila , Piperidinas/uso terapêutico , Pirilamina/uso terapêutico , Receptores Acoplados a Proteínas G , Receptores Histamínicos , Receptores Histamínicos H1 , Receptores Histamínicos H3 , Receptores Histamínicos H4 , Acetato de TetradecanoilforbolRESUMO
Pretreatment with parthenolide for 60 min inhibited the antigen-induced degranulation of RBL-2H3 mast cells; the IC(50) value being 4.5 ± 0.4 µM. The inhibition was not due to suppression of the phosphatidylinositol 3-kinase pathway because the antigen-induced phosphorylation of Akt was not inhibited by parthenolide. The antigen-induced increase in intracellular calcium levels was prevented by parthenolide, suggesting that parthenolide inhibited the antigen-induced degranulation by suppressing an increase in intracellular calcium levels. In support of this, parthenolide was found to prevent ionomycin-induced degranulation by inhibiting an increase in intracellular calcium levels. Therefore, parthenolide inhibits the degranulation of mast cells by preventing an increase in intracellular calcium levels.
Assuntos
Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Mastócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sesquiterpenos/farmacologia , Tanacetum/química , Animais , Antígenos/efeitos adversos , Linhagem Celular Tumoral , Concentração Inibidora 50 , Ionomicina , Mastócitos/fisiologia , RatosRESUMO
Human malignant pleural mesothelioma (HMPM) is an aggressive neoplasm that is highly resistant to conventional therapies. We established 3 HMPM cell lines (TCC-MESO-1, TCC-MESO-2 and TCC-MESO-3) from Japanese patients; the first 2 from the primary and metastatic tumors of a patient with the epithelioid type of HMPM, and the third from a patient with biphasic characteristics of the tumor (epithelioid and sarcomatous phenotypes). The 3 cell lines resembled the original HMPMs in their morphological and biological features, including the genetic alterations such as lack of p16 expression and mutation of p53. Their tumorigenicity was determined in SCID mice by orthotopic implantation (20-46%). The tumorigenicity of the HMPM cell lines, which was relatively low, was enhanced by repeated subcultures and orthotopic implantations, and 3 competent tumorigenic sublines were produced (Me1Tu, Me2Tu and Me3Tu sublines from the TCC-MESO-1, TCC-MESO-2 and TCC-MESO-3 cell lines, respectively). The resultant HMPM sublines efficiently generated tumors in the SCID mice (100%) following orthotopic implantation. SCID mice implanted with the competent sublines, into one of which the luciferase gene was introduced, displayed quantitative fluctuation of the bioluminescence for the tumor volume in vivo. Oral administration of S-1, an anticancer agent, suppressed the proliferation of the luciferase gene-expressing Me1Tu subline in the mouse models in vivo, with a treated-to-control ratio of the mean tumor volume of 0.2. The orthotopic implantation mouse model proved to be useful for quantitative evaluation of the efficacy of novel anticancer drugs and also for studying the biology of HMPMs in vivo.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Modelos Animais de Doenças , Mesotelioma/tratamento farmacológico , Ácido Oxônico/administração & dosagem , Fótons , Neoplasias Pleurais/tratamento farmacológico , Tegafur/administração & dosagem , Administração Oral , Animais , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Citocinas/metabolismo , Combinação de Medicamentos , Humanos , Técnicas Imunoenzimáticas , Luciferases/metabolismo , Masculino , Mesotelioma/metabolismo , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The irritancy of Nickel (Ni) ions has been well documented clinically. However, the chemical mediators involved in the acute inflammation induced by solid Ni are not fully understood. We used the Ni wire-implantation model in mice and examined roles of prostaglandins and histamine in plasma leakage in the acute phase. The subcutaneous implantation of a Ni wire into the back of mice induced plasma leakage from 8 to 24 h and tissue necrosis around the wire at 3 days, whereas the implantation of an aluminum wire induced no such inflammatory responses. An increase in the mRNA for cyclooxygenase (COX)-2 and HDC in cells around the Ni wire was detected 4 h after the implantation. The leakage of plasma at 8 h was inhibited by indomethacin in a dose-dependent manner. Dexamethasone and the p38 MAP kinase inhibitor SB203580 also inhibited the exudation of plasma consistent with the inhibition of the expression of COX-2 mRNA. Furthermore, plasma leakage was partially but siginificantly reduced in histamine H1 receptor knockout mice and histidine decarboxylase (HDC) knockout mice but not in H2 receptor knockout mice. These results suggested that the Ni ions released from the wire induced the expression of COX-2 and HDC, resulting in an increase in vascular permeability during the acute phase of inflammation.
Assuntos
Histamina/química , Inflamação , Níquel/química , Prostaglandinas/química , Animais , Ciclo-Oxigenase 2/metabolismo , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Knockout , Necrose , Permeabilidade , Piridinas/farmacologia , Receptores Histamínicos H1/genética , Pele/efeitos dos fármacos , Fatores de TempoRESUMO
We established a novel allergic dermatitis model in mouse ear lobes in which antigen-nonspecific inflammation was induced by painting 12-O-tetradecanoylphorbol 13-acetate (TPA) between sensitization and challenge with picryl chloride (PiCl). This model has an advantage for analyzing atopic dermatitis-like inflammation within a short period. Analysis of the time course changes in the PiCl-induced swelling showed that the allergic inflammation was shifted from a delayed-type response to a biphasic response consisting of a weak immediate-phase response and a late-phase response by painting with TPA. The application of TPA increased the PiCl-induced infiltration of eosinophils and mast cells at the inflammatory site and shifted the cytokine milieu from Th1 to Th2. The expression of the Th2-inducing cytokine thymic stromal lymphopoietin (TSLP) mRNA was also increased by TPA. These findings suggested that the induction of antigen-nonspecific inflammation by TPA before the antigen challenge enhanced the Th2 response and modified the PiCl-induced delayed type-hypersensitivity. Using this model, we clarified that histamine plays significant roles in the early-phase swelling via H(1) receptors and the late-phase swelling via H(3)/H(4) receptors. Thus, we suggested the usefulness of the combined treatment with an H(1) antagonist and an H(4) antagonist for the suppression of atopic dermatitis.
Assuntos
Citocinas/fisiologia , Dermatite Atópica/imunologia , Dermatite de Contato/imunologia , Modelos Animais de Doenças , Histamina/fisiologia , Acetato de Tetradecanoilforbol , Animais , Orelha , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Cloreto de Picrila/imunologia , Acetato de Tetradecanoilforbol/imunologia , Linfopoietina do Estroma do TimoRESUMO
Effects of artekeiskeanol A, a newly isolated coumarin derivative from Artemisa keiskeana Miq. (Compositae), the extract of which is used for treatment of rheumatoid arthritis as a folk medicine, on the antigen-induced activation of RBL-2H3 cells were examined. RBL-2H3 cells were sensitized with dinitrophenol (DNP)-specific IgE, and then stimulated with the antigen DNP-conjugated human serum albumin (DNP-HSA). Artekeiskeanol A at 10 to 100 microM inhibited the antigen-induced degranulation in a concentration-dependent manner, the IC(50) value being 38.0 + or - 0.2 microM. Degranulation induced by thapsigargin or A23187 also was inhibited by artekeiskeanol A at 10 to 100 microM. The antigen-induced increase in the levels of mRNA for tumor necrosis factor (TNF)-alpha and interleukin (IL)-13 and phosphorylations of Akt, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p44/42 MAPK were also suppressed by artekeiskeanol A. Our findings suggested that the effectiveness of the extract of A. keiskeana might partly be due to the inhibition of mast cell activation by artekeiskeanol A.
Assuntos
Artemisia/química , Degranulação Celular/efeitos dos fármacos , Cumarínicos/farmacologia , Fatores Imunológicos/farmacologia , Mastócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Terpenos/farmacologia , Animais , Antígenos/metabolismo , Calcimicina/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Imunoglobulina E , Concentração Inibidora 50 , Interleucina-13/genética , Interleucina-13/metabolismo , Mastócitos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Albumina Sérica , Tapsigargina/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Accessories, watches, coins and other items containing metal sometimes cause contact dermatitis and metal allergy. Among metals, nickel in alloys is ionized by sweat on the surface of the skin and exhibits particularly marked irritancy and allergenicity. Although eosinophils play important roles in allergy, the effects of nickel on eosinophils have not been elucidated. METHODS: Eosinophils were prepared from the peritoneal cavity in rats immunized with Ascaris suum extract. Purified rat eosinophils were incubated in the presence of various kinds of metals including nickel. The viability of eosinophils was analyzed using a flow cytometer. RESULTS: When rat eosinophils were incubated for 3 days in the presence of nickel chloride at 30-1,000 microM, the viability of eosinophils was decreased in a concentration-dependent manner. Nickel chloride at 300 muM significantly increased the percentage of annexin V+ PI- eosinophils. The population of annexin V+ PI- eosinophils was also increased by nickel sulfate, cobalt chloride and zinc sulfate. The binding of nickel ions to eosinophils was detected by flow cytometer. CONCLUSIONS: Nickel ions bind to eosinophils and decrease the viability of eosinophils through the induction of apoptosis. Nickel ions may exhibit activity which modifies the function of eosinophils in allergy.
Assuntos
Apoptose , Eosinófilos/efeitos dos fármacos , Níquel/farmacologia , Animais , Asma/induzido quimicamente , Asma/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Eosinófilos/imunologia , Níquel/imunologia , RatosRESUMO
BACKGROUND: In atopic dermatitis, inflammation induced by antigen-nonspecific stimuli further enhances the allergic inflammation. However, there is no experimental model in which allergic dermatitis is evoked where the inflammation has been induced by antigen-nonspecific stimuli. Here, we established a novel dermatitis model in mice and analyzed the role of histamine. METHODS: After sensitization with picryl chloride (PiCl) by painting on ear lobes of cyclophosphamide-treated mice, 12-O-tetradecanoylphorbol 13-acetate (TPA) was painted twice at the same site, and then allergic inflammation was induced by painting PiCl. Histamine antagonists and cyclosporine A (CsA) were administered intravenously. RESULTS: The application of TPA shifted the PiCl-induced allergic inflammation from a delayed-type response to a biphasic response, increased the infiltration of eosinophils and mast cells at the inflammatory site, shifted the cytokine milieu from Th1 to Th2 and induced the expression of thymic stromal lymphopoietin in the ear lobes. The PiCl-induced increase in the thickness of the ear lobe in the immediate phase was suppressed by the H1 antagonist pyrilamine. In contrast, the increase in the swelling in the late phase and the infiltration of eosinophils were suppressed by the H3/H4 antagonist thioperamide. The inhibitory effect of the combined treatment with pyrilamine and thioperamide on the TPA-modified contact dermatitis was as potent as that of CsA. CONCLUSION: Induction of the antigen-nonspecific inflammation by TPA enhanced the PiCl-induced allergic inflammation. Histamine plays significant roles in the early-phase swelling via H1 receptors, and the late-phase swelling via H3/H4 receptors in this TPA-modified allergic dermatitis model.
Assuntos
Dermatite Alérgica de Contato/imunologia , Modelos Animais de Doenças , Pavilhão Auricular/imunologia , Histamina/imunologia , Cloreto de Picrila/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Contagem de Células , Cimetidina/farmacologia , Ciclofosfamida/farmacologia , Ciclosporina/farmacologia , Citocinas/genética , Dermatite Alérgica de Contato/tratamento farmacológico , Dermatite Alérgica de Contato/metabolismo , Dermatite Alérgica de Contato/patologia , Pavilhão Auricular/efeitos dos fármacos , Pavilhão Auricular/metabolismo , Pavilhão Auricular/patologia , Peroxidase de Eosinófilo/metabolismo , Eosinófilos/citologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Antagonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/uso terapêutico , Imunoglobulina E/sangue , Interferon gama/genética , Interleucina-4/genética , Masculino , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Pirilamina/farmacologia , Pirilamina/uso terapêutico , Linfopoietina do Estroma do TimoRESUMO
To characterize active principles for prevention and treatment of diabetic complications, the isolation of protein glycation inhibitors from the fruiting body of Phellinus linteus was conducted in vitro using the model systems of hemoglobin-delta-gluconolactone (early stage), bovine serum albumin-methylglyoxal (middle stage), and N(alpha)-acetyl-glycyl-lysine methyl ester-D-ribose (last stage) assays. Nine compounds were isolated from the active ethylacetate fraction of the fruiting body and identified as protocatechuic acid (1), protocatechualdehyde (2), caffeic acid (3), ellagic acid (4), hispidin (5), davallialactone (6), hypholomine B (7), interfungins A (8), and inoscavin A (9) by spectroscopic analyses. At the early stage of protein glycation, compounds 6, 8, and 9 exhibited inhibitory activity on hemoglobin A(1C) formation. For the middle stage, compounds 2, 6, and 9 showed a significant inhibitory effect on methylglyoxal-medicated protein modification and their IC(50) values were 144.28, 213.15, and 158.66 muM, respectively. At the last stage of glycation, compound 8 was found to be a potent inhibitor of the cross-linking of proteins, which was more effective than that of aminoguanidine, a well-known inhibitor for advanced glycation end products. Consequently, compound 8 showed the most potent inhibitory effects at each stage of protein glycation. This mechanism may help to provide a protective effect against hyperglycemia-mediated protein damage.
Assuntos
Basidiomycota/química , Carpóforos/química , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Hemoglobinas Glicadas/química , Glicosilação/efeitos dos fármacos , Hemoglobinas/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas/química , Aldeído Pirúvico/química , Soroalbumina Bovina/químicaRESUMO
The extract of the root of Acanthopanax chiisanensis Nakai is used for the treatment of inflammation. To analyse the action mechanism of this extract, the effect of hyperin (quercetin-3-O-beta-d-galactose) isolated from the ethyl acetate fraction of the root of A. chiisanensis on nitrite production and induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS, 1 microg/mL)-stimulated rat peritoneal macrophages were examined. The effect of the structurally related compounds, isoquercitrin (quercetin-3-O-beta-d-glucose) and quercetin (an aglycone of the two compounds) isolated from the extract of the leaves of Vaccinium koreanum Nakai was also examined to compare the effect. It was shown that hyperin inhibited the LPS-induced iNOS expression and nitrite production. Of the three compounds, quercetin showed the most potent inhibitory activity. The phosphorylation of p44/42 mitogen activated protein kinase (MAPK), p38 MAPK and c-Jun N-terminal kinase (JNK) were also inhibited by these compounds. These findings suggested that hyperin in the extract of the root of A. chiisanensis inhibits nitric oxide (NO) production through inhibition of the expression of iNOS by attenuation of p44/p42 MAPK, p38 MAPK and JNK, and thus participates in the antiinflammatory activity of the extract.
Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Nitritos/metabolismo , Quercetina/análogos & derivados , Animais , Eleutherococcus/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos Peritoneais/metabolismo , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Quercetina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Effects of compounds isolated from medicinal plants in Korea on prostaglandin E2 (PGE2) production in rat peritoneal macrophages were examined, and mechanism of action of the active constituents was analyzed. The active constituents were as follows; tectorigenin and tectoridin isolated from the rhizomes of Belamcanda chinensis, platycodin D isolated from the roots of Platycodon grandiflorum, imperatorin isolated from the roots of Angelica dahurica, and hyperin isolated from the roots of Acanthopanax chiisanensis. These compounds inhibit the induction of cyclooxygenase-2 (COX-2), thus inhibiting PGE2 production. The chemically synthesized chalcone derivative, 2'-hydroxy-4'-methoxychalcone, also inhibits PGE2 production by suppressing COX-2 induction. In contrast, taiwanin C isolated from the roots of Acanthopanax chiisanensis inhibited PGE2 production by direct inhibition of COX-1 and COX-2.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/biossíntese , Fitoterapia , Animais , Anti-Inflamatórios não Esteroides/química , Inibidores de Ciclo-Oxigenase/química , Avaliação Pré-Clínica de Medicamentos , Furocumarinas/química , Furocumarinas/farmacologia , Isoflavonas/química , Isoflavonas/imunologia , Isoflavonas/metabolismo , Isoflavonas/farmacologia , Coreia (Geográfico) , Lactonas/química , Lactonas/farmacologia , Lignanas/química , Lignanas/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Medicina Tradicional do Leste Asiático , Quercetina/análogos & derivados , Quercetina/química , Quercetina/farmacologia , Ratos , Saponinas/química , Saponinas/farmacologia , Triterpenos/química , Triterpenos/farmacologiaRESUMO
BACKGROUND: Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate. METHODS: EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein. RESULTS: When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days. CONCLUSIONS: The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA.
Assuntos
Butiratos/farmacologia , Inibidores Enzimáticos/farmacologia , Eosinófilos/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Inibidores de Histona Desacetilases , Síndrome Hipereosinofílica/tratamento farmacológico , Proteínas de Fusão Oncogênica/análise , Peptídeos Cíclicos/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/análise , Fatores de Poliadenilação e Clivagem de mRNA/análise , Acetilação/efeitos dos fármacos , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Eosinófilos/metabolismo , Fator de Iniciação 2 em Eucariotos/análise , Fator de Iniciação 2 em Eucariotos/efeitos dos fármacos , Histona Desacetilases/metabolismo , Humanos , Síndrome Hipereosinofílica/metabolismo , Proteínas de Fusão Oncogênica/efeitos dos fármacos , Proteínas de Fusão Oncogênica/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/efeitos dos fármacos , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
The constitutively activated tyrosine kinase Fip1-like 1 (FIP1L1)-platelet-derived growth factor receptor alpha (PDGFRalpha) causes eosinophilic leukemia EoL-1 cells to proliferate. Recently, we demonstrated that histone deacetylase inhibitors suppressed this proliferation and induced the differentiation of EoL-1 cells into eosinophils in parallel with a decrease in the level of FIP1L1-PDGFRalpha. In this study, we analyzed the mechanism by which FIP1L1-PDGFRalpha induces the proliferation and whether the suppression of cell proliferation triggers the differentiation into eosinophils. The FIP1L1-PDGFRalpha inhibitor imatinib inhibited the proliferation of EoL-1 cells and decreased the level of the oncoprotein c-Myc as well as the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK). The proliferation of EoL-1 cells and expression of c-Myc were also inhibited by the MEK inhibitor U0126 and JNK inhibitor SP600125. The expression of the eosinophilic differentiation marker CCR3 was not induced by imatinib. These findings suggest that FIP1L1-PDGFRalpha induces the proliferation of EoL-1 cells through the induction of c-Myc expression via ERK and JNK signaling pathways, but is not involved in the inhibition of differentiation toward mature eosinophils.
Assuntos
Síndrome Hipereosinofílica/patologia , Proteínas de Fusão Oncogênica/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Benzamidas , Butadienos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Síndrome Hipereosinofílica/enzimologia , Mesilato de Imatinib , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirimidinas/farmacologia , Receptores CCR3/metabolismoRESUMO
In the mouse macrophage-like cell line RAW 264, vacuolar-type (H(+))-ATPase (V-ATPase) inhibitors, bafilomycin A(1) and concanamycin A, increased the level of cyclooxygenase (COX)-2 protein and its mRNA. The V-ATPase inhibitor-induced expression of COX-2 was suppressed by inhibitors of c-jun N-terminal kinase (JNK) and nuclear factor-kappaB, and by inhibitors of Na(+)/H(+) exchangers (NHEs). The bafilomycin A(1)-induced activation of JNK but not degradation of IkappaB-alpha was suppressed by NHE inhibitors and by an inhibitor of Na(+)/Ca(2+) exchanger SN-6. These results suggested that V-ATPase inhibitors induce the expression of COX-2 via NHE-dependent and -independent pathways.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Trocadores de Sódio-Hidrogênio/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Antracenos/farmacologia , Compostos de Benzil/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrolídeos/farmacologia , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Tiazolidinas/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
BACKGROUND: EoL-1 cells have a FIP1L1-PDGFRA fusion gene which causes the transformation of eosinophilic precursor cells into leukemia cells. Recently, we suggested that the induction of differentiation of EoL-1 cells into eosinophils by the HDAC inhibitors apicidin and n-butyrate is due to the continuous inhibition of HDACs. However, neither apicidin nor n-butyrate inhibited the expression of FIP1L1-PDGFRA mRNA, although both these inhibitors suppressed cell proliferation. Therefore, in this study, we analyzed whether the levels of FIP1L1-PDGFRalpha protein and phosphorylated-Stat5 involved in the signaling for the proliferation of EoL-1 cells are attenuated by HDAC inhibitors. METHODS: EoL-1 cells were incubated in the presence of apicidin, TSA or n-butyrate. FIP1L1-PDGFRalpha and phosphorylated-Stat5 were detected by Western blotting. RESULTS: Treatment of EoL-1 cells with apicidin at 100 nM or n-butyrate at 500 microM decreased the levels of FIP1L1-PDGFRalpha protein and phosphorylated-Stat5, while that with trichostatin A at 30 nM did not. CONCLUSIONS: The decrease in the level of FIP1L1-PDGFRalpha protein caused by apicidin and n-butyrate might be one of the mechanisms by which EoL-1 cells are induced to differentiate into eosinophils by these HDAC inhibitors.
Assuntos
Butiratos/farmacologia , Eosinófilos/citologia , Inibidores de Histona Desacetilases , Síndrome Hipereosinofílica/patologia , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Peptídeos Cíclicos/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/fisiologia , Fatores de Poliadenilação e Clivagem de mRNA/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Síndrome Hipereosinofílica/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fator de Transcrição STAT5/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/biossíntese , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Apicularens A and B were isolated from the myxobacterial genus Chondromyces apiculatus JW184. Apicularen A inhibited bafilomycin A1-sensitive ATP-dependent proton transport into microsome vesicles more potently than apicularen B. Bone resorption in cultures of mouse calvariae induced by human parathyroid hormone (PTH) or interleukin-1beta (IL-1beta) was inhibited by apicularen A at 10 and 100 nM, while apicularen B had no effect. The bisphosphonate incadronate inhibited bone resorption at 100 nM, being less effective than apicularen A. Our findings indicate that apicularen A inhibits bone resorption induced by PTH or IL-1beta more potently than apicularen B, probably due to inhibition of the V-ATPase.
Assuntos
Reabsorção Óssea , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Myxococcales/química , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/isolamento & purificação , Difosfonatos/química , Difosfonatos/farmacologia , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Macrolídeos/farmacologia , Camundongos , Hormônio Paratireóideo/farmacologia , Crânio/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
We analyzed the effects of the Na+/H+ exchanger (NHE) inhibitor 3,5-diamino-6-chloro-N-(diaminomethylidene)pyrazine-2-carboxamide hydrochloride (amiloride) and its analogs 5-(N,N-dimethyl)-amiloride (DMA) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the lipopolysaccharide (LPS)-induced production of prostaglandin (PG) E2 in vitro and in vivo. In the mouse macrophage-like cell line RAW 264, these inhibitors suppressed the LPS (1 microg/ml)-induced production of PGE2 at 8 h in a concentration-dependent manner. They also reduced the LPS-induced release of arachidonic acid from membrane phospholipids at 4 h and the LPS-induced increase in the level of cyclooxygenase (COX)-2 protein at 6 h, but not the level of COX-2 mRNA at 3 h. The LPS-induced phosphorylation of mitogen-activated protein kinases and degradation of inhibitor of kappaB-alpha were not inhibited by these drugs. In an air pouch-type LPS-induced inflammation model in mice 30 mg/kg amiloride and 10 mg/kg EIPA as well as the COX inhibitor indomethacin (10 mg/kg), significantly reduced the level of PGE2 in the pouch fluid at 8 h and the vascular permeability from 4 to 8 h. The accumulation of pouch fluid and leukocytes in the pouch fluid at 8 h was significantly inhibited by amiloride and EIPA but not by indomethacin. These findings suggested that the NHE inhibitors suppress the production of PGE2 through inhibiting the release of arachidonic acid and the increase in COX-2 protein levels and thus induce anti-inflammatory activity.
Assuntos
Amilorida/análogos & derivados , Amilorida/farmacologia , Dinoprostona/biossíntese , Inflamação/prevenção & controle , Lipopolissacarídeos/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Ácido Araquidônico/metabolismo , Western Blotting , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase/farmacologia , Proteínas I-kappa B/metabolismo , Indometacina/farmacologia , Inflamação/induzido quimicamente , Leucócitos/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidor de NF-kappaB alfa , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
EoL-1 cells differentiate into eosinophils in the presence of n-butyrate, but the mechanism has remained to be elucidated. Because n-butyrate can inhibit histone deacetylases, we hypothesized that the inhibition of histone deacetylases induces the differentiation of EoL-1 cells into eosinophils. In this study, using n-butyrate and two other histone deacetylase inhibitors, apicidin and trichostatin A, we have analyzed the relationship between the inhibition of histone deacetylases and the differentiation into eosinophils in EoL-1 cells. It was demonstrated that apicidin and n-butyrate induced a continuous acetylation of histones H4 and H3, inhibited the proliferation of EoL-1 cells without attenuating the level of FIP1L1-PDGFRA mRNA, and induced the expression of markers for mature eosinophils such as integrin beta7, CCR1, and CCR3 on EoL-1 cells, while trichostatin A evoked a transient acetylation of histones and induced no differentiation into eosinophils. These findings suggest that the continuous inhibition of histone deacetylases in EoL-1 cells induces the differentiation into mature eosinophils.