Influence of Reduced Folate Carrier and Dihydrofolate Reductase Genes on Methotrexate-Induced Cytotoxicity

Abstract

The aim of this study is to investigate the effect of genetic variations and the expression of the reduced folate carrier (RFC) and dihydrofolate reductase (DHFR) on the drug sensitivity to methotrexate (MTX) in different cancer cell lines. We examined the six human cancer cell lines (MCF-7, AGS, A549, NCI-H23, HCT-116 and Saos-2). The cytotoxicity of MTX was measured by sulforhodamine B (SRB) assay. The expressions of the DHFR and RFC were evaluated by real-time PCR and western blotting. Four single nucleotide polymorphisms (SNPs) of the DHFR and two SNPs of the RFC were genotyped. The IC₅₀s of MTX was in an extensively broad range from 6.05±0.81 nM to>1,000 nM in the cell lines. The Saos-2 (>1,000 nM) and MCF-7 (114.31±5.34 nM) cells were most resistant to MTX; in contrast, the AGS and HCT-116 cells were highly sensitive to MTX with an IC(50) of 6.05±0.81 nM and 13.56±3.76 nM, respectively. A reciprocal change of the RFC and DHFR mRNA expression was found between the MTX-sensitive AGS and MTX-resistant Saos-2 cells. There was no significant difference in the expression levels of RFC protein in both the AGS and Saos-2 cells, whereas DHFR protein was more increased in the MTX-resistant Saos-2 cells treated with MTX. The genotype of the MTX-sensitive AGS cells were mutant variants of the DHFR; in contrast, the Saos-2 cells had the wild-type of the DHFR. In conclusion, this study showed that inverse change of the RFC and DHFR mRNA and protein expression was associated with RFC and DHFR polymorphisms and it is postulated that this phenomenon might play an important role in sensitivity of certain cancers to MTX.