RX-3117 (Fluorocyclopentenyl-Cytosine)-Mediated Down-Regulation of DNA Methyltransferase 1 Leads to Protein Expression of Tumor-Suppressor Genes and Increased Functionality of the Proton-Coupled Folate Carrier

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.

Therapeutic Inhibitory Activities of N‐Hydroxy Derived Cytidines: A Patent Overview

“…It mainly regulates DNA methylation of tumour suppresser genes p16, stimulates the E‐cadherin and O 6 ‐methylguanine‐DNA methyltransferase, to increase proton‐coupled folate transporter for possible control of hypomethylation. It produced similar values when compared with standard drug 5‐aza‐2’‐deoxycytidine [44] …”

“…It produced similar values when compared with standard drug 5-aza-2'-deoxycytidine. [44] The US 2014/8624572 patent claims the structure of only one analogue of 1-(3-C-ethynyl-β-Dribopentofuranosyl)cytosine (ECyd) 16 (Figure 8) for the treatment of cancer by continuously administering it intravenously route in divided doses. The most prominent mechanism for inhibition of RNA synthesis is by the inhibition of RNA polymerases I, II, and III, from side to side phosphorylation of intracellular uridine/cytidine kinase and consequently to form triphosphate (ECTP), which becomes lethal to many essential cell lines by its high antitumor effect and reduced risk of expressing peripheral neurotoxicity.…”

Folic Acid–Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting

“…As for PCFT, this proton symporter contributes to the cellular uptake of folates and antifolates at acidic pH, usually fixed at 5.5 in published reports. 5 , 47 Therefore, its contribution is likely negligible at pH values 7.2 and 7.4, at which our uptake experiments were carried out. To further support this statement, we performed a few experiments on 2008, C13, A2780 and A2780/CP cells at pH 8 and found no significant change in the [ 3 H]FA uptake results.…”

DNA Methylation in Alcohol Use Disorder

“…DNA methylation inhibitors include the following categories: Cytidine analogs, DNA binders, oligonucleotides and polyphenols ( Table 1 ). Cytidine analogs include: 5-aza [ 34 ], RX-3117 [ 35 ] or other synthetic nucleoside analogues. They incorporate DNA during replication, competitively preempt DNMTs with cytosine, and covalently bind to sulfhydryl groups on cysteine residues of DNMTs, thus inactivating them [ 36 ].…”

“…DNA methylation inhibitors include the following categories: Cytidine analogs, DNA binders, oligonucleotides and polyphenols (Table 1). Cytidine analogs include: 5-aza [34], RX-3117 [35] or other synthetic nucleoside analogues. They incorporate DNA during replication, compet-…”