RESUMO
Previously, we have reported that a multidrug-resistant, mitoxantrone (MX)-selected cell line, MCF7/MX, is highly cross-resistant to the antifolate methotrexate (MTX), because of enhanced ATP-dependent drug efflux (E. L. Volk et al., Cancer Res., 60: 3514-3521, 2000). These cells overexpress the breast cancer resistance protein (BCRP), and resistance to MTX as well as to MX was reversible by the BCRP inhibitor, GF120918. These data indicated that BCRP causes the multidrug-resistance phenotype. To further examine the role of this transporter in MTX resistance, and in particular the role of amino acid 482, we analyzed a number of BCRP-overexpressing cell lines. MTX resistance correlated with BCRP expression in all of the cell lines expressing the wild-type transporter, which contains an Arg at position 482. In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1-M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Concomitantly, the greatest reduction in MTX accumulation was observed in the MCF7/MX cells (BCRP(Arg)) as compared with cells expressing the Thr and Gly BCRP variants. Furthermore, the reduction in drug accumulation was sensitive to BCRP inhibition by GF120918. In conclusion, we have demonstrated a novel role for BCRP as a mediator of MTX resistance and have provided further evidence for the importance of amino acid 482 in substrate specificity.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Metotrexato/farmacologia , Proteínas de Neoplasias , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , Amplificação de Genes , Humanos , Metotrexato/farmacocinética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
BACKGROUND: Ribozymes are an effective tool to reduce the mRNA levels of specific target genes. Overexpression of the drug transport protein, ABCG2, has been associated with multidrug resistance in cancer cells. MATERIALS AND METHODS: An expression plasmid encoding a hammerhead ribozyme against the ABCG2 gene was stably transfected into multidrug-resistant MCF7/MX cells that express very high levels of the ABCG2 protein. The effect of the ribozyme was determined by quantitative real-time RT-PCR, Western blot and cytotoxicity assays. RESULTS: The ribozyme reduced ABCG2 mRNA levels to less than 10% of control values, which resulted in the concomitant reduction of ABCG2 protein levels and sensitization of the cells to mitoxantrone and methotrexate. CONCLUSION: The ribozyme used was highly effective in reducing the expression of its target gene, ABCG2, and was able to modulate the associated multidrug-resistant phenotype.