RESUMO
Methotrexate (MTX) is known to induce serious lung diseases, such as pulmonary fibrosis. Although we demonstrated that MTX is associated with epithelial-mesenchymal transition (EMT), the underlying mechanism remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2), an oxidative stress response regulator, is related to EMT induction. In the present study, we examined the association of Nrf2 with the MTX-induced EMT in the alveolar epithelial cell line A549. MTX treatment decreased the mRNA expression of heme oxidase-1 (HO-1), a target of Nrf2, which was inhibited by co-treatment with diethyl maleate (DEM), an Nrf2 activator. Additionally, the MTX-induced increase in reactive oxygen species (ROS) production was significantly suppressed by DEM. Furthermore, DEM decreased mRNA/protein expression levels of α-smooth muscle actin (SMA), a representative EMT marker, which were upregulated by MTX. Nuclear expression and localization of Nrf2 were suppressed by MTX treatment, which led to a decrease in Nrf2 activity. Finally, in Nrf2 knockdown cells, the MTX-induced enhancement of α-SMA mRNA/protein expression was not observed, indicating that downregulation of Nrf2 may play a critical role in the MTX-induced EMT in A549 cells. These results suggest that Nrf2-regulated transcriptional activity would be associated with the MTX-induced EMT induction.
Assuntos
Transição Epitelial-Mesenquimal , Metotrexato , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais/metabolismo , Humanos , Metotrexato/farmacologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
In the lung alveolar region, the innate immune system serves as an important host defense system. We recently reported that peptide transporter 2 (PEPT2) has an essential role in the uptake of bacterial peptides and induction of innate immune response in alveolar epithelial cells. In this study, we aimed to clarify the effects of corticosteroids on PEPT2 function and PEPT2-dependent innate immune response. NCI-H441 (H441) cells were used as an in vitro model of human alveolar type II epithelial cells, and the effects of dexamethasone (DEX) and budesonide (BUD) on the transport function of PEPT2 and the innate immune response induced by bacterial peptides were examined. PEPT2 function, estimated by measuring ß-alanyl-Nε-(7-amino-4-methyl-2-oxo-2H-1-benzopyran-3-acetyl)-L-lysine (ß-Ala-Lys-AMCA) uptake in H441 cells, was suppressed by treatment with DEX and BUD in a concentration- and time-dependent manner. The suppression of PEPT2 function was partially recovered by a glucocorticoid receptor antagonist. The expression of PEPT2 and nucleotide-binding oligomerization domain 1 (NOD1) mRNAs was suppressed by treatment with DEX and BUD, while PEPT2 protein level was not changed by these treatment conditions. Additionally, the increased mRNA expression of interleukin (IL)-8 and the increased secretion of IL-8 into the culture medium induced by bacterial peptides were also suppressed by treatment with these corticosteroids. Taken together, these results clearly suggest that corticosteroids suppress PEPT2 function and bacterial peptide-induced innate immune response in alveolar epithelial cells. Therefore, PEPT2- and NOD1-dependent innate immune response induced by bacterial peptides in the lung alveolar region may be suppressed during the inhaled corticosteroid therapy.
Assuntos
Corticosteroides/farmacologia , Proteínas de Bactérias/farmacologia , Células Epiteliais/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Simportadores/metabolismo , Anti-Inflamatórios/farmacologia , Budesonida/farmacologia , Linhagem Celular , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Alvéolos Pulmonares/citologia , Simportadores/genéticaRESUMO
Molecular interaction analysis is an essential technique for the study of biomolecular functions and the development of new drugs. Most current methods generally require manipulation to immobilize or label molecules, and require advance identification of at least one of the two molecules in the reaction. In this study, we succeeded in detecting the interaction of low-molecular-weight (LMW) compounds with a membrane protein mixture derived from cultured cells expressing target membrane proteins by using the size exclusion chromatography-mass spectrometry (SEC-MS) method under the condition of 0.001% lauryl maltose neopentyl glycol as detergent and atmospheric pressure chemical ionization. This method allowed us to analyze the interaction of a mixture of medicinal herbal ingredients with a mixture of membrane proteins to identify the two interacting ingredients. As it does not require specialized equipment (e.g., a two-dimensional liquid chromatography system), this SEC-MS method enables the analysis of interactions between LMW compounds and relatively high-expressed membrane proteins without immobilization or derivatization of the molecules.
Assuntos
Proteínas de Membrana , Cromatografia em Gel , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peso MolecularRESUMO
Hydantoins, including the antiepileptic drug phenytoin, contain an amide nitrogen and an imide nitrogen, both of which can be alkylated. However, due to the higher acidity of its proton, N3 can be more easily alkylated than N1 under basic conditions. In this study, we explored methods for direct N1-selective methylation of phenytoin and found that conditions using potassium bases [potassium tert-butoxide (tBuOK) and potassium hexamethyldisilazide (KHMDS)] in tetrahydrofuran (THF) gave N1-monomethylated phenytoin in good yield. The applicable scope of this reaction system was found to include various hydantoins and alkyl halides. To explore the function of methylated hydantoins, the effects of a series of methylated phenytoins on P-glycoprotein were examined, but none of methylated products showed inhibitory activity toward rhodamine 123 efflux by P-glycoprotein.
Assuntos
Anticonvulsivantes/química , Hidantoínas/química , Fenitoína/química , Potássio/química , Anticonvulsivantes/síntese química , Azidas/química , Butanóis/química , Hidantoínas/síntese química , Metilação , Fenitoína/síntese químicaRESUMO
There is increasing evidence that epithelial-mesenchymal transition (EMT) contributes to the development of organ fibrosis. We demonstrated that methotrexate (MTX) clearly induced EMT through the transforming growth factor (TGF)-ß-related signaling pathway in human alveolar epithelial cell line, A549. However, critical factors associated with MTX-induced EMT have not yet been identified. In our study, we attempted to identify factors playing a crucial role in MTX-induced EMT in A549 cells. We focused on plasminogen activator inhibitor-1 (PAI-1) as the possible target for the prevention of MTX-induced EMT-related lung injury. Comprehensive gene expression analysis by microarray revealed that mRNA expression level of PAI-1 was clearly increased by MTX treatment. In addition, using several cloned A549 cells, we found a good correlation between MTX-induced increase in mRNA expression levels of α-smooth muscle actin (SMA), a representative EMT marker, and PAI-1. Furthermore, MTX upregulated mRNA and protein expression levels of PAI-1 in A549 cells; this upregulation was canceled by co-treatment with SB431542, a TGF-ß-related signaling pathway inhibitor. Notably, tiplaxtinin, a PAI-1 inhibitor, and knockdown of urokinase-type plasminogen activator receptor (uPAR) prevented MTX-induced EMT in A549 cells. These findings indicate that MTX may induce EMT via upregulation of PAI-1 expression and interaction of PAI-1 with uPAR in A549 cells.
Assuntos
Células Epiteliais Alveolares/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metotrexato/farmacologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Células A549 , Células Epiteliais Alveolares/efeitos dos fármacos , Ontologia Genética , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Drug-induced lung injury leads to serious lung diseases, such as pulmonary fibrosis. We demonstrated in an alveolar epithelial cell line A549/ABCA3 that certain microRNAs were associated with bleomycin induced epithelial-mesenchymal transition (EMT) which is closely related to pulmonary fibrosis. In this study, we focused on the role of miR-484 in drug-induced EMT using A549/ABCA3 cells and a mouse lung injury model. METHODS: The expression of EMT-related genes and miR-484 was detected by real-time polymerase chain reaction. miR-484-targeted proteins were analyzed by Western blot. Pulmonary fibrosis mouse model was prepared by the intratracheal administration of BLM. As miR-484 is known to target SMAD2 and zinc finger E-box binding homeobox 1 (ZEB1), which are the well-known EMT-related transcription factors, we assessed the effects of a miR-484 inhibitor or mimic on the mRNA/protein expression of both the factors. RESULTS: We found that bleomycin significantly suppressed the intracellular expression and extracellular release of miR-484 in A549/ABCA3 cells. Moreover, the miR-484 mimic and inhibitor showed no drastic effects on the expression of the EMT-related transcription factors. In addition, the miR-484 mimic had no effect on the bleomycin-induced altered mRNA expression of the α-smooth muscle actin, a representative EMT marker. This suggested that miR-484 did not directly contribute to bleomycin-induced EMT in A549/ABCA3 cells. In contrast, the significant decrease in miR-484 expression in the lung tissue or plasma of bleomycin-administered mice suggested that miR-484 expression was closely correlated with bleomycin-induced lung injury. CONCLUSIONS: These findings indicate that miR-484 could be a novel diagnostic indicator for drug-induced pulmonary fibrosis.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Bleomicina/toxicidade , MicroRNAs/genética , Fibrose Pulmonar/induzido quimicamente , Células A549 , Actinas/genética , Animais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Humanos , Masculino , Camundongos , Fibrose Pulmonar/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE: Several anticancer drugs including bleomycin (BLM) and methotrexate (MTX) cause serious lung diseases such as pulmonary fibrosis. Although evidences showing the association of epithelial-mesenchymal transition (EMT) with pulmonary fibrosis are increasing, the mechanism underlying anticancer drug-induced EMT has been poorly understood. On the other hand, miR-34a, a non-coding small RNA, has been highlighted as a key factor to regulate EMT in lung. In this study, we elucidated the role of miR-34a in anticancer drug-induced EMT using A549/ABCA3 cells as a novel type II alveolar epithelium model. METHODS: Expression levels of α-smooth muscle actin (α-SMA) mRNA, miR-34a, and p53 were evaluated by real-time PCR and western blot analysis, respectively. RESULTS: BLM and MTX induced EMT-like morphological changes and increase in mRNA expression level of α-SMA, an EMT marker. Also, both drugs increased the expression level of miR-34a. Furthermore, mRNA expression level of α-SMA was enhanced by introduction of miR-34a mimic into A549/ABCA3 cells. To examine the mechanism underlying drug-induced enhancement of miR-34a expression, we focused on p53/miR-34a axis. Both drugs upregulated protein expression of p53, an inducer of miR-34a, as well as phosphorylation of Ser15 in p53. CONCLUSIONS: These findings indicated that p53/miR-34a axis may contribute to anticancer drug-induced EMT in type II alveolar epithelial cells.
Assuntos
Antineoplásicos/farmacologia , Bleomicina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metotrexato/farmacologia , MicroRNAs/genética , Proteína Supressora de Tumor p53/genética , Células A549 , Humanos , MicroRNAs/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Methotrexate (MTX) often induces serious lung diseases such as pulmonary fibrosis. Although MTX is known to be a folic acid (FA) antagonist, the effect of FA on MTX-induced lung injury remains unclear. Recent studies indicate that epithelial-mesenchymal transition (EMT) is involved in pulmonary fibrosis. Here, we aimed to clarify the effect of FA on MTX-induced EMT in human alveolar epithelial cell line A549 using conditioned medium (CM). CM was prepared from the supernatants of A549â¯cells treated with MTX in the absence (CMM) or presence (CMMF) of FA. FA suppressed EMT-like morphological changes and elevated mRNA/protein expression levels of α-smooth muscle actin induced by MTX in A549â¯cells. In addition, CMM induced EMT-like phenotypical changes, whereas CMMF had no effect on the phenotype of A549â¯cells, indicating that FA may suppress MTX-induced EMT via inhibiting the secretion of certain factors into the supernatant of the cells. Furthermore, FA also prevented CMM-induced EMT-like phenotypical changes in A549â¯cells. These findings indicate that FA may be a useful pharmaceutical for MTX-induced lung injury.
Assuntos
Actinas/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ácido Fólico/administração & dosagem , Metotrexato/administração & dosagem , Células A549 , Interações Medicamentosas , HumanosRESUMO
PURPOSE: Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549. METHODS: FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively. RESULTS: FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation. CONCLUSION: AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Assuntos
Produtos da Oxidação Avançada de Proteínas/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Fluoresceína-5-Isotiocianato/análogos & derivados , Albumina Sérica/farmacocinética , Células A549 , Produtos da Oxidação Avançada de Proteínas/administração & dosagem , Células Epiteliais Alveolares/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacocinética , Humanos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Albumina Sérica/administração & dosagem , Albumina Sérica/metabolismo , Temperatura , Fatores de TempoRESUMO
The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Cobalto/farmacologia , Células Epiteliais/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Transportador de Glucose Tipo 1/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metotrexato/farmacologia , Mitoxantrona/farmacologia , Proteínas de Neoplasias/genética , RNA Mensageiro/metabolismoRESUMO
PURPOSE: Methotrexate (MTX) therapy of certain cancers and rheumatoid arthritis often induces serious interstitial lung complications including pulmonary fibrosis. In this study, we investigated the epithelial-mesenchymal transition (EMT) induced by MTX and by transforming growth factor (TGF)-ß1 in the human alveolar epithelial cell line A549 in order to develop new strategies for the prevention of EMT. METHODS: First, we examined the effect of TGF-ß1 and MTX on cell morphology and the expression of EMT-related mRNAs in A549 cells. Then, the effects of SB431542 (SB), a potent inhibitor of TGF-ß receptor kinase, and a neutralizing antibody for TGF-ß1 on the phenotypic changes of A549 cells induced by TGF-ß1 and MTX were examined. RESULTS: After incubation with TGF-ß1 and MTX, the mRNA expression of epithelial markers such as cytokeratin 19 was reduced, while that of mesenchymal markers such as α-smooth muscle actin was increased. SB suppressed the development of morphological changes and partially rescued alterations in mRNA expression of EMT markers induced by MTX. In addition, the enhancement of SMAD2 phosphorylation by MTX was also prevented by SB. On the other hand, EMT-related changes induced by MTX were not affected by a neutralizing antibody for TGF-ß1. CONCLUSION: We have demonstrated that phenotypic changes of A549 cells induced by MTX are partly mediated by a TGF-ß1-related intracellular signaling pathway, although TGF-ß1 itself is not directly involved in this process.
Assuntos
Células A549/efeitos dos fármacos , Antimetabólitos Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metotrexato/farmacologia , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Células A549/patologia , Actinas/genética , Anticorpos Neutralizantes/farmacologia , Benzamidas/farmacologia , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Fator de Crescimento do Tecido Conjuntivo/genética , Dioxóis/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Queratina-19/genética , Fenótipo , Fosforilação/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/imunologia , Vimentina/genéticaRESUMO
PURPOSE: The peptide transporter PEPT2 is expressed in alveolar type II epithelial cells. So far, however, no appropriate alveolar epithelial cell line for studying PEPT2 function has been known. In this study, we examined the functional expression of PEPT2 in the human distal lung epithelial cell line NCl-H441 (H441). METHODS: Expression of PEPT2 mRNA and protein was examined in H441 cells. Transport function of PEPT2 was studied using glycylsarcosine (Gly-Sar) as a substrate. RESULTS: Lamellar bodies were well developed in H441 cells and mRNA expression of type II cell markers and PEPT2 increased during time in culture. PEPT2 protein expression was confirmed in H441 cells, but not in A549 cells, by immunostaining and Western blotting. The uptake of Gly-Sar in H441 cells was inhibited by cefadroxil, and the cefadroxil-sensitive uptake was pH-dependent and peaked at pH 6.5. Gly-Sar uptake in H441 cells showed saturation kinetics with a Km value of 112.5 µM. In addition, apical-to-basal, but not basal-to-apical, transport of cephalexin across H441 cell monolayers was sensitive to cefadroxil. CONCLUSIONS: PEPT2 is functionally expressed in H441 cells, making the cell line a good in vitro model to study PEPT2 function and its regulation in human distal lung.
Assuntos
Dipeptídeos/metabolismo , Células Epiteliais/metabolismo , Pulmão/citologia , Simportadores/genética , Simportadores/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Expressão Gênica , Humanos , Pulmão/metabolismo , RNA Mensageiro/genética , Simportadores/análiseRESUMO
Nuclear factor-κB (NF-κB) is a key regulator of cancer progression and the inflammatory effects of disease. To identify inhibitors of DNA binding to NF-κB, we developed a new homogeneous method for detection of sequence-specific DNA-binding proteins. This method, which we refer to as DSE-FRET, is based on two phenomena: protein-dependent blocking of spontaneous DNA strand exchange (DSE) between partially double-stranded DNA probes, and fluorescence resonance energy transfer (FRET). If a probe labeled with a fluorophore and quencher is mixed with a non-labeled probe in the absence of a target protein, strand exchange occurs between the probes and results in fluorescence elevation. In contrast, blocking of strand exchange by a target protein results in lower fluorescence intensity. Recombinant human NF-κB (p50) suppressed the fluorescence elevation of a specific probe in a concentration-dependent manner, but had no effect on a non-specific probe. Competitors bearing a NF-κB binding site restored fluorescence, and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the NF-κB binding site of the competitor. Evaluation of two NF-κB inhibitors, Evans Blue and dehydroxymethylepoxyquinomicin ([-]-DHMEQ), was carried out using p50 and p52 (another form of NF-κB), and IC50 values were obtained. The DSE-FRET technique also detected the differential effect of (-)-DHMEQ on p50 and p52 inhibition. These data indicate that DSE-FRET can be used for high throughput screening of anticancer drugs targeted to DNA-binding proteins.
Assuntos
Proteínas de Ligação a DNA/análise , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Benzamidas/farmacologia , Sítios de Ligação , Cicloexanonas/farmacologia , Sondas de DNA , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Azul Evans/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Concentração Inibidora 50 , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Recently, we found that albumin overload induces expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) protein and several HIF-1 target genes in human renal proximal tubular epithelial cell line HK-2. In this study, the role of albumin-bound fatty acids in the albumin-induced HIF-1 activation was studied. The enhancing effect of fatty acid-bearing human serum albumin [FA(+)HSA] treatment on HIF-1α protein expression was much greater than that of fatty acid-depleted human serum albumin [FA(-)HSA] treatment. The FA(+)HSA treatment induced HIF-1 target gene mRNAs such as those of glucose transporter 1 (GLUT1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and breast cancer resistance protein (BCRP) in concentration-dependent manners, while FA(-)HSA caused no significant increases in these mRNAs. Consistent with increased GLUT1 mRNA, GLUT1 protein expression and GLUT inhibitor cytochalasin B-sensitive d-[(3)H]glucose uptake activity were significantly enhanced by treatment with FA(+)HSA, but not with FA(-)HSA. These findings indicate that fatty acids bound to albumin play a crucial role in albumin-induced HIF-1 activation followed by changes in HIF-1 target gene expression and protein product activity.
Assuntos
Células Epiteliais/metabolismo , Ácidos Graxos/administração & dosagem , Ácidos Graxos/sangue , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Túbulos Renais Proximais/metabolismo , Albumina Sérica/administração & dosagem , Albumina Sérica/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Ligação ProteicaRESUMO
Abemaciclib (ABM), a cyclin-dependent kinase 4/6 inhibitor, shows pharmacological effects in cell cycle arrest. Epithelial-mesenchymal transition is an important cellular event associated with pathophysiological states such as organ fibrosis and cancer progression. In the present study, we evaluated the contribution of factors associated with cell cycle arrest to ABM-induced epithelial-mesenchymal transition. Treatment with 0.6⯵M ABM induced both cell cycle arrest and epithelial-mesenchymal transition-related phenotypic changes. Interestingly, the knockdown of cyclin-dependent kinase 4/6, pharmacological targets of ABM or cyclin D1, which forms complexes with cyclin-dependent kinase 4/6, resulted in cell cycle arrest at the G1-phase and induction of epithelial-mesenchymal transition, indicating that downregulation of cyclin-dependent kinase 4/6-cyclin D1 complexes would mimic ABM. In contrast, knockdown of the Rb protein, which is phosphorylated by cyclin-dependent kinase 4/6, had no effect on the expression level of α-smooth muscle actin, an epithelial-mesenchymal transition marker. Furthermore, ABM-induced epithelial-mesenchymal transition was not affected by Rb knockdown, suggesting that Rb is not involved in the transition process. Our study is the first to suggest that cyclin-dependent kinase 4/6-cyclin D1 complexes, as pharmacological targets of ABM, may contribute to ABM-induced epithelial-mesenchymal transition, followed by clinical disorders such as organ fibrosis and cancer progression. This study suggests that blocking epithelial-mesenchymal transition might be a promising way to prevent negative side effects caused by a medication (ABM) without affecting its ability to treat the disease.
Assuntos
Aminopiridinas , Benzimidazóis , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Transição Epitelial-Mesenquimal , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Benzimidazóis/farmacologia , Humanos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Aminopiridinas/farmacologia , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina D1/genéticaRESUMO
The aim of this study was to investigate the effect of human serum albumin (HSA) overload on the expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) in human renal proximal tubular cell line HK-2. First, the cell viability and cytotoxic activity were examined to assess the cellular conditions in HK-2 cells with HSA treatment employed in this study. HSA treatment for 48h decreased the cell viability and increased the leakage of lactate dehydrogenase (LDH) into the medium in a concentration-dependent manner, but the toxicity was relatively mild. Western Blot analysis revealed that HSA treatment induced the expression of HIF-1α protein in a concentration-dependent manner without a change in ß-actin protein expression. Confocal microscopy analysis revealed that HIF-1α protein was predominantly localized in the nucleus but was also observed in the cytoplasm. The HIF-1 target gene mRNAs, glucose transporter 1 and glyceraldehyde 3-phosphate dehydrogenase, were up-regulated by HSA treatment, leading to the increases in the protein expression levels. In addition, the mRNA of HIF-1α was increased by HSA treatment. In conclusion, albumin loading induces HIF-1α in HK-2 cells, resulting in the increases in the expression of proteins of its target genes.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Túbulos Renais Proximais/metabolismo , Albumina Sérica/metabolismo , Western Blotting , Linhagem Celular , Imunofluorescência , Humanos , Túbulos Renais Proximais/citologia , Microscopia Confocal , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Protamine, a mixture of polypeptides that is rich in arginine, has been used clinically as an antidote to heparin overdoses and a complexing agent in a long-acting insulin preparation. When protamine is administered intravenously, its abundant accumulation in the kidneys has been reported. However, the renal uptake mechanism for protamine is not clear. In this study, we examined the transport mechanism for protamine in opossum kidney (OK) cells, a suitable in vitro model for renal proximal tubular epithelial cells. Flow cytometric analysis revealed that the association of fluorescein isothiocyanate (FITC)-labeled protamine from salmon (FITC-protamine) by OK cells was inhibited by unlabeled protamine in a concentration-dependent manner. The association of FITC-protamine was temperature- and energy-dependent. Confocal microscopy analysis showed that the fluorescence was localized in the cytoplasm and nucleus of OK cells. In addition, FITC-protamine association was inhibited by cationic drugs such as polycationic gentamicin and polymixin B, but it was increased by a basic amino acid, arginine. Inhibitors for clathrin- and caveolin-dependent endocytosis showed inhibitory effects on FITC-protamine association. Pretreatment with heparinase III partially but significantly decreased the association of FITC-protamine. These results suggest that protamine may be taken up by OK cells via receptor-mediated endocytosis, which may result in its localization in the cytoplasm and nucleus of the cells.
Assuntos
Células Epiteliais/metabolismo , Antagonistas de Heparina/metabolismo , Rim/citologia , Protaminas/metabolismo , Animais , Células Cultivadas , Endocitose , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Masculino , Microvilosidades/metabolismo , Gambás , Ratos , Ratos WistarRESUMO
OBJECTIVES: Linagliptin is a novel, highly selective and long acting DPP-4 inhibitor for the treatment of type 2 diabetes mellitus (T2DM). Linagliptin exhibits non-linear pharmacokinetics (PK) due to saturable binding to plasma and tissue DPP-4. The aim of this study was to characterize the PK and PK/DPP-4 inhibition relationship of linagliptin in Japanese patients with T2DM using a population PK/DPP-4 model and to support the rationale for the therapeutic dose in Japanese patients by simulation. METHODS: Linagliptin plasma concentration and DPP-4 inhibition measurements from a placebo-controlled, parallel group multiple (28 days) dose trial that included 36 T2DM patients (18 patients each in 2.5 mg and 10 mg dose group) were used for analysis. Modeling was performed using FOCE INTERACTION estimation method implemented in NONMEM V. The linagliptin plasma concentration- and DPP-4 inhibition- time profiles were simulated for Japanese patients receiving 5 mg linagliptin once daily by the model established. RESULTS: Nonlinear PK of linagliptin in T2DM patients were well described by a 2-compartment model assuming concentration-dependent binding to DPP-4 in the central and peripheral compartment. Plasma DPP-4 inhibition was integrated in the model by relating the model-predicted DPP-4 occupancy with linagliptin linearly to DPP-4 inhibition. The simulation predicted that for the 5 mg dose group the trough DPP-4 inhibition at steady-state was 84.2%, which is higher than the target inhibition (≥80%) for an effective dose of DPP-4 inhibitor. In 2.5 mg dose group, steady-state DPP-4 inhibition of >80% was not maintained over 24 hours (observed and simulated). CONCLUSIONS: The nonlinear PK of linagliptin and its plasma DPP-4 inhibition in patients were well characterized by a target-mediated drug disposition model relating DPP-4 occupancy with linagliptin to DPP-4 inhibition. Simulations of plasma DPP-4 inhibition suggest that 5 mg linagliptin once daily is an appropriate therapeutic dose for Japanese patients with T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacocinética , Hipoglicemiantes/farmacocinética , Modelos Biológicos , Purinas/farmacocinética , Quinazolinas/farmacocinética , Adulto , Idoso , Povo Asiático , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/sangue , Inibidores da Dipeptidil Peptidase IV/farmacologia , Método Duplo-Cego , Feminino , Humanos , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacologia , Linagliptina , Masculino , Pessoa de Meia-Idade , Purinas/sangue , Purinas/farmacologia , Quinazolinas/sangue , Quinazolinas/farmacologiaRESUMO
BACKGROUND: The main purpose of this study was to evaluate the effect of cigarette smoke extract (CSE) on insulin transport in alveolar epithelial cells. METHODS: We first examined the effect of CSE pretreatment on cell viability, mRNA expression, and lamellar body structures in A549 cells. Then the effect of CSE pretreatment on FITC-insulin transport was examined. RESULTS: When A549 cells were treated with 30 µg/ml of CSE for 48 h, the expression of some mRNAs abundantly expressed in type II alveolar epithelial cells such as surfactant protein B was significantly increased. Lamellar bodylike structures became more evident with CSE treatment. FITC-insulin uptake from the apical side and subsequent efflux to the basal side was enhanced by CSE treatment in A549 cells. The enhancing effect of CSE on FITC-insulin uptake was concentration-dependent and reversible. A concentration-dependent enhancing effect of CSE on FITC-insulin uptake was also observed in normal, primary cultured alveolar type II epithelial cells isolated from rats. CONCLUSIONS: Treatment of A549 cells by CSE may direct the cells to a more type II-like phenotype. In accordance with this observation, FITC-insulin uptake was enhanced by CSE treatment. These results may partly explain the higher insulin absorption from the lung in smokers than in nonsmokers.
Assuntos
Insulina/metabolismo , Nicotiana/química , Alvéolos Pulmonares/metabolismo , Fumaça , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Humanos , Microscopia Confocal , Alvéolos Pulmonares/ultraestrutura , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fumaça/análiseRESUMO
To improve the dissolution and oral absorption properties of probucol, a novel wet-milling process using the ULTRA APEX MILL was investigated. The particle size of bulk probucol powder was 17.1 µm. However, after wet-milling with dispersing agents such as Gelucire 44/14, Gelucire 50/13, vitamin E-TPGS, and Pluronic F-108, the probucol particle sizes decreased to about 77-176 nm. Scanning electron microscopy (SEM) analysis also suggested that the probucol particles were successfully milled into the nanometer range. An in vitro dissolution study showed that the dissolution rates of all nanopowders were several folds higher than those of the corresponding mixed powders. When orally administered to rats, the AUC values of probucol nanopowders treated with Gelucire 44/14 and 50/13, and vitamin E-TPGS were about 3.06-3.54-folds greater than that of the bulk powder. Therefore, through this study, we have developed a new pharmaceutical technique to improve the dissolution rate and oral absorption of probucol using the ULTRA APEX MILL by wet-milling with various dispersing agents.