Department of Clinical Pharmacy and Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Department of Pharmacy, University of Miyazaki Hospital, Miyazaki, Japan.
Anticancer Res. 2019 Aug;39(8):4129-4136. doi: 10.21873/anticanres.13571.
BACKGROUND/AIM: 5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP).
The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively.
Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU.
Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.
背景/目的：5-氮杂-2-脱氧胞苷（5-Aza-CdR）可增强 5-氟尿嘧啶（5-FU）的敏感性，但分子机制尚不完全清楚。本研究旨在通过胸苷磷酸化酶（TP）探讨 5-Aza-CdR 增强 5-FU 敏感性的分子机制。
通过 MTT 法在几种癌细胞系中测定药物敏感性。通过免疫印迹和 RT-PCR 分别检测蛋白和 mRNA 水平。通过 siRNA、ChIP 测定和亚硫酸氢钠基因组测序分别进行基因沉默、Sp1 与 DNA 的结合和 DNA 甲基化。
表皮样癌细胞中 TP 启动子中的 Sp1 结合位点发生甲基化。5-Aza-CdR 去甲基化 Sp1 结合位点并增强了对 5-FU 的敏感性。
5-Aza-CdR 对 Sp1 结合位点的去甲基化是增强 5-FU 敏感性的关键因素，这可能使更多癌症患者通过 5-Aza-CdR 和 5-FU 的联合治疗获得更有效的治疗效果。
