RESUMO
OBJECTIVES: We had previously found that reduced folate carrier (RFC; SLC19A1) is mainly involved in an influx of transport of methotrexate (MTX), a folate analogue, using alveolar epithelial A549 cells. Therefore, we examined MTX uptake in NCl-H441 (H441) cells, another in vitro alveolar epithelial model, focusing on the localization of RFC in the present study. METHODS: Transport function of RFC in H441 cells was studied using [3 H]MTX. KEY FINDINGS: The uptake of MTX was increased remarkably after pretreatment of the cell monolayer with ethylenediaminetetraacetic acid (EDTA) in H441 cells but not in A549 cells, indicating the contribution of the basolaterally located transporter. In addition, folic acid and thiamine monophosphate, RFC inhibitors, inhibited the uptake of MTX from the basolateral side of the H441 cells. In order to compare the function of RFC on the apical and basolateral sides of the cells, the uptake of MTX from each side was examined using a Transwell chamber. Intracellular MTX amounts from the basolateral side were found to be significantly higher than those from the apical side. CONCLUSIONS: These findings suggest that the distribution of MTX in the lung alveolar epithelial cells may be mediated by basolaterally located RFC in alveolar epithelial cells.
Assuntos
Células Epiteliais Alveolares/metabolismo , Antimetabólitos Antineoplásicos/metabolismo , Metotrexato/metabolismo , Proteína Carregadora de Folato Reduzido/metabolismo , Células A549 , Transporte Biológico , Linhagem Celular Tumoral , Ácido Edético/farmacologia , Antagonistas do Ácido Fólico/metabolismo , HumanosRESUMO
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
Methotrexate (MTX), a drug used for the treatment of certain cancers as well as rheumatoid arthritis, sometimes induces serious interstitial lung injury. Although lung toxicity of MTX is related to its accumulation, the information concerning MTX transport in the lungs is lacking. In this study, we investigated the mechanisms underlying MTX influx into human alveolar epithelial cell line A549. MTX influx into A549 cells was time-, pH-, and temperature-dependent and showed saturation kinetics. The influx was inhibited by folic acid with IC50 values of 256.1 µM at pH 7.4 and 1.6 µM at pH 5.5, indicating that the mechanisms underlying MTX influx would be different at these pHs. We then examined the role of two folate transporters in MTX influx, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). The expression of RFC and PCFT mRNAs in A549 cells was confirmed by reverse transcription polymerase chain reaction. In addition, MTX influx was inhibited by thiamine monophosphate, an RFC inhibitor, at pH 7.4, and by sulfasalazine, a PCFT inhibitor, at pH 5.5. These results indicated that RFC and PCFT are predominantly involved in MTX influx into A549 cells at pH 7.4 and pH 5.5, respectively.
