The Ohio State University, James Cancer Hospital and Solove Research Center Columbus, OH, USA.
Biochem Biophys Res Commun. 2010 Jan 15;391(3):1465-70. doi: 10.1016/j.bbrc.2009.12.095. Epub 2009 Dec 24.
Mesothelioma is an uncommon malignancy whose global incidence continues to rise. The therapeutic standard for advanced disease is intravenous pemetrexed and cisplatin. The anti-folate capecitabine is significantly less effective than pemetrexed. The balance between thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) is critical to the efficacy of capecitabine. DNA from mesothelioma cell lines was bisulfite treated and examined by MS-PCR, RNA was obtained for real-time PCR analysis, and protein lysates were obtained for Western immunoblot analysis. Cytotoxicity was assessed by MTT assay, comparing 5-aza-CdR pretreated or untreated cells with 5'-deoxy-5-fluorouridine (DFUR), 5-FU, and pemetrexed. Finally bisulfite sequencing of the extracellular growth factor-1 (ECGF-1) gene was performed on 4 mesothelioma samples and pericardial tissue. One of the four cell lines tested (H290) was methylated for ECGF-1. This corresponded to a lack of TP expression by real-time PCR and Western immunoblot. Treatment with 1muM 5-aza-CdR increased TP mRNA and protein expression in H290. DFUR, the substrate for TP, showed increased cytotoxicity when delivered after 5-aza-CdR exposure in the methylated cell line. There was no difference in any of the unmethylated cell lines when cells were exposed to 5-FU or pemetrexed with or without 5-aza-CdR. Patient tumor samples revealed an increased number of methylated CpG sites in ECGF-1 compared to normal pericardium. Methylation of ECGF-1, leads to transcriptional silencing of TP and may explain the lack of any effect of capecitabine, especially when compared to pemetrexed.
间皮瘤是一种罕见的恶性肿瘤，其全球发病率持续上升。晚期疾病的治疗标准是静脉注射培美曲塞和顺铂。抗叶酸卡培他滨的疗效明显不如培美曲塞。胸苷酸合成酶（TS）、二氢嘧啶脱氢酶（DPD）和胸苷磷酸化酶（TP）之间的平衡对卡培他滨的疗效至关重要。用亚硫酸氢盐处理间皮瘤细胞系的 DNA，并通过 MS-PCR 进行检查，获得 RNA 进行实时 PCR 分析，并获得蛋白质裂解物进行 Western 免疫印迹分析。通过 MTT 测定评估细胞毒性，比较用 5-氮杂胞苷预处理或未处理的细胞与 5'-脱氧-5-氟尿苷（DFUR）、5-FU 和培美曲塞的细胞毒性。最后，对 4 个间皮瘤样本和心包组织的细胞外生长因子-1（ECGF-1）基因进行亚硫酸氢盐测序。在测试的四个细胞系之一（H290）中，ECGF-1 被甲基化。这对应于实时 PCR 和 Western 免疫印迹中缺乏 TP 表达。用 1μM 5-氮杂胞苷处理后，H290 中的 TP mRNA 和蛋白表达增加。DFUR 是 TP 的底物，在甲基化细胞系中暴露于 5-氮杂胞苷后，其细胞毒性增加。在用或不用 5-氮杂胞苷处理时，未甲基化的细胞系在暴露于 5-FU 或培美曲塞时没有任何差异。患者肿瘤样本显示，与正常心包相比，ECGF-1 中有更多的甲基化 CpG 位点。ECGF-1 的甲基化导致 TP 的转录沉默，这可能解释了卡培他滨，尤其是与培美曲塞相比，没有任何效果的原因。
