Laboratory Medical Oncology, Amsterdam UMC, location VU University Medical Center, 1081 HV Amsterdam, The Netherlands.
Department of Oncology, Wayne State University School of Medicine, Detroit, and Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201-1976, USA.
Int J Mol Sci. 2020 Apr 14;21(8):2717. doi: 10.3390/ijms21082717.
(1) Background: RX-3117 (fluorocyclopentenyl-cytosine) is a cytidine analog that inhibits DNA methyltransferase 1 (DNMT1). We investigated the mechanism and potential of RX-3117 as a demethylating agent in several in vitro models. (2) Methods: we used western blotting to measure expression of several proteins known to be down-regulated by DNA methylation: O-methylguanine-DNA methyltransferase (MGMT) and the tumor-suppressor genes, p16 and E-cadherin. Transport of methotrexate (MTX) mediated by the proton-coupled folate transporter (PCFT) was used as a functional assay. (3) Results: RX-3117 treatment decreased total DNA-cytosine-methylation in A549 non-small cell lung cancer (NSCLC) cells, and induced protein expression of MGMT, p16 and E-cadherin in A549 and SW1573 NSCLC cells. Leukemic CCRF-CEM cells and the MTX-resistant variant (CEM/MTX, with a deficient reduced folate carrier) have a very low expression of PCFT due to promoter hypermethylation. In CEM/MTX cells, pre-treatment with RX-3117 increased PCFT-mediated MTX uptake 8-fold, and in CEM cells 4-fold. With the reference hypomethylating agent 5-aza-2'-deoxycytidine similar values were obtained. RX-3117 also increased gene expression and PCFT protein. (4) Conclusion: RX-3117 down-regulates DNMT1, leading to hypomethylation of DNA. From the increased protein expression of tumor-suppressor genes and functional activation of PCFT, we concluded that RX-3117 might have induced hypomethylation of the promotor.
(1) 背景：RX-3117（氟环戊基胞嘧啶）是一种抑制 DNA 甲基转移酶 1（DNMT1）的胞嘧啶类似物。我们研究了 RX-3117 作为几种体外模型中去甲基化剂的机制和潜力。(2) 方法：我们使用 Western blot 来测量几个已知被 DNA 甲基化下调的蛋白质的表达：O-甲基鸟嘌呤-DNA 甲基转移酶（MGMT）和肿瘤抑制基因 p16 和 E-钙黏蛋白。质子偶联叶酸转运体（PCFT）介导的氨甲蝶呤（MTX）转运被用作功能测定。(3) 结果：RX-3117 处理降低了 A549 非小细胞肺癌（NSCLC）细胞中的总 DNA-胞嘧啶甲基化，并诱导了 A549 和 SW1573 NSCLC 细胞中 MGMT、p16 和 E-钙黏蛋白的蛋白表达。白血病 CCRF-CEM 细胞和 MTX 耐药变体（CEM/MTX，由于启动子超甲基化而缺乏还原叶酸载体）由于启动子超甲基化而表达极低的 PCFT。在 CEM/MTX 细胞中，用 RX-3117 预处理可使 PCFT 介导的 MTX 摄取增加 8 倍，在 CEM 细胞中增加 4 倍。参考低甲基化剂 5-氮杂-2'-脱氧胞苷也得到了类似的值。RX-3117 还增加了 基因表达和 PCFT 蛋白。(4) 结论：RX-3117 下调 DNMT1，导致 DNA 去甲基化。从肿瘤抑制基因的蛋白表达增加和 PCFT 的功能激活，我们得出结论，RX-3117 可能诱导了启动子的去甲基化。
