Laboratory of Medical Oncology, Catholic Research Institute of Medical Science, The Catholic University of Korea School of Medicine, Seoul, Korea.
Cancer Res Treat. 2010 Sep;42(3):163-71. doi: 10.4143/crt.2010.42.3.163. Epub 2010 Sep 30.
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.
本研究旨在探讨遗传变异以及还原叶酸载体（RFC）和二氢叶酸还原酶（DHFR）的表达对不同癌细胞系中甲氨蝶呤（MTX）药物敏感性的影响。
我们检测了 6 个人类癌细胞系（MCF-7、AGS、A549、NCI-H23、HCT-116 和 Saos-2）。通过磺酰罗丹明 B（SRB）测定法测量 MTX 的细胞毒性。通过实时 PCR 和 Western 印迹法评估 DHFR 和 RFC 的表达。对 DHFR 的 4 个单核苷酸多态性（SNP）和 RFC 的 2 个 SNP 进行基因分型。
细胞系中 MTX 的 IC₅₀ 范围很广，从 6.05±0.81 nM 到>1000 nM。Saos-2（>1000 nM）和 MCF-7（114.31±5.34 nM）细胞对 MTX 的耐药性最强；相反，AGS 和 HCT-116 细胞对 MTX 非常敏感，IC（50）分别为 6.05±0.81 nM 和 13.56±3.76 nM。在 MTX 敏感的 AGS 和 MTX 耐药的 Saos-2 细胞之间发现了 RFC 和 DHFR mRNA 表达的相互变化。AGS 和 Saos-2 细胞中 RFC 蛋白的表达水平没有明显差异，而在用 MTX 处理的 MTX 耐药的 Saos-2 细胞中 DHFR 蛋白增加更多。MTX 敏感的 AGS 细胞的基因型为 DHFR 的突变变体；相反，Saos-2 细胞具有 DHFR 的野生型。
综上所述，本研究表明，RFC 和 DHFR mRNA 和蛋白表达的反向变化与 RFC 和 DHFR 多态性有关，并且推测这种现象可能在某些癌症对 MTX 的敏感性中起重要作用。
