Functional expression and characterization in Xenopus laevis oocytes of the ABCG2 transporter derived from A549 human lung adenocarcinoma cells.
Oncol Rep
; 27(2): 499-503, 2012 Feb.
Article
em En
| MEDLINE
| ID: mdl-21971989
ABSTRACT
We cloned the ATP-binding cassette sub-family G member 2 (ABCG2) transporter, the most recently identified among several major human multidrug-resistance pumps, from A549 human lung adenocarcinoma cells in order to characterize its function and substrate specificity. In a previous report, we confirmed that a stem cell-like side population of A549 cells highly expressed the ABCG2 gene and had a unique ability to resist the anticancer drug methotrexate (MTX). In this study, ABCG2 cDNA was cloned by RT-PCR and converted into cRNA by an in vitro transcription system for expression in Xenopus laevis (X. laevis) oocytes. The transcribed cRNA of the ABCG2 gene was injected into the oocytes under the absence of cofactors or heterologous partner proteins or some lipids from the media. A high expression of ABCG2 was observed on the oocyte surface by immunofluorescence and confocal laser microscopy. We tested the functional effect of ABCG2 expression on drug efflux by directly injecting MTX into X. laevis oocytes. The drug concentration within the oocytes was quantified with LC-MS/MS; the analysis showed that the accumulation of MTX was significantly decreased in the X. laevis oocytes expressing ABCG2 compared with the control oocytes not expressing ABCG2. These findings show that the ABCG2 protein has an important role in the efflux of MTX through the cell membrane of X. laevis oocytes. Therefore, it might be that ABCG2, abundantly expressed in the stem cell population of A549 cells, can modulate resistance to MTX in lung cancer therapy.
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Base de dados:
MEDLINE
Assunto principal:
Adenocarcinoma
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Transportadores de Cassetes de Ligação de ATP
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Neoplasias Pulmonares
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Proteínas de Neoplasias
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article