Abstract
(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: O6-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 PCFT 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.
Highlights
RX-3117 is a novel cytidine analog modified in the sugar ring moiety [1] (Figure 1), which has shown promising anti-tumor activity against gemcitabine resistant xenografts from different pathologies [2]and is currently in clinical trial (NCT02030067) [3]
Since hypermethylation of tumor-suppressor gene (TSG) is cell line dependent we chose A549 cells to study the effect of RX-3117 on protein expression of O-6-methylguanine-DNA methyltransferase (MGMT) and E-cadherin, while SW1573 cells were chosen to study the effect on p16
Since DNA methyl transferase 1 (DNMT1) down-regulation is time-dependent, as shown for A549 cells (Figure 2A), we chose a 24 h exposure to determine the effect of RX-3117 on total hypomethylation and protein expression of various TSGs (Figure 2B–D)
Summary
RX-3117 is a novel cytidine analog modified in the sugar ring moiety [1] (Figure 1), which has shown promising anti-tumor activity against gemcitabine resistant xenografts from different pathologies [2]and is currently in clinical trial (NCT02030067) [3]. RX-3117 is a novel cytidine analog modified in the sugar ring moiety [1] (Figure 1), which has shown promising anti-tumor activity against gemcitabine resistant xenografts from different pathologies [2]. RX-3117 was shown to down-regulate DNA methyl transferase 1 (DNMT1) [1,3,4,5,6]. DNMT1 acts as a maintenance methylation enzyme for newly synthesized DNA and ensures methylation patterns in newly dividing cells, using already existing hemi-methylation patterns [7]. Cytidine rich regions of the DNA are substrates for DNMT1, so-called CpG islands [8]. Promoters of genes are enriched with CpG islands and methylation of gene promoters has a silencing effect on gene transcription [5]. Many inhibitors of DNMT1 are base-modified cytidine analogs such as
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