Changes in the expression of 5-fluorouracil metabolism associated enzyme genes by regulation of methylation

我们使用MTT法研究了在总共7种胃肠癌细胞系（MKN-1、MKN-45、MKN-74、C-1、Panc-1、MIaPaCa-2、HuH-7）中加入5-氮杂-2 '-脱氧胞苷（5-aza）（2 μM）后对5-氟尿嘧啶的药物敏感性的变化，发现MKN-1、MKN-74、Panc-1、MIaPaCa-2对5-FU的敏感性显著降低。采用实时荧光定量RT-PCR技术检测了5-FU代谢相关酶基因（胸苷酸合成酶（TS）、二氢嘧啶脱氢酶（DPD）、乳清酸磷酸核糖转移酶（OPRT）、胸苷磷酸化酶（TP）、核糖核苷酸还原酶（RNR））在加入2 μM和10μM 5-aza后的变化。5-aza可剂量依赖性上调除DPD外的其他基因的表达水平。结果提示，5-FU代谢相关酶基因在人胃肠道癌细胞系中存在高度甲基化。我们进一步研究了基因表达水平的变化与药物敏感性之间的相关性。TS表达上调与药物敏感性降低显著相关。

We investigated the changes in drug sensitivity against 5-fluorouracil by adding 5-aza-2'-deoxycitidine (5-aza) (2 μM) in a total of 7 gastrointestinal cancer cell lines (MKN-1, MKN-45, MKN-74, C-1, Panc-1, MIaPaCa-2, HuH-7) using MTT assay and found that sensitivity to 5-FU significantly decreased in MKN-1, MKN-74, Panc-1, MIaPaCa-2. We also investigated the changes in 5-FU metabolism associated enzyme gene (thymidylate synthase (TS), dihydropyrimidine dehydroohgenase (DPD), orotatephosphoribosyl transferase (OPRT), thymidine phospholyrsase (TP), ribonucleotide reductase (RNR)) by adding 2 μM or 10μM of 5-aza using real-time RT-PCR. The expression levels were upregulated by 5-aza in a dose dependent manner in most of genes except for DPD. From these results, it is suggested that 5-FU metabolism associated enzyme gene are highly methylated in human gastrointestinal cancer cell lines. We further investigated the correlation between the changes in gene expression level and drug sensitivity. It is found that up-regulation of TS significantly correlated with the decreased drug sensitivity.