The Role of Human Equilibrative Nucleoside Transporter 1 on the Cellular Transport of the DNA Methyltransferase Inhibitors 5-Azacytidine and CP-4200 in Human Leukemia Cells

Abstract

The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [14 C]5-azacytidine demonstrated Na 1 -independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-59-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

Expression Levels of Human Equilibrative Nucleoside Transporter 1 and Deoxycytidine Kinase Enzyme as Prognostic Factors in Patients with Acute Myeloid Leukemia Treated with Cytarabine

“…Thereafter, the dCK enzyme produces a monophosphorylated product (dCMP), which is bi- and triphosphorylated to act as an antimetabolite. Resistance to Ara-C has been documented in AML cells with defective dCK or hENT1 expression [12,15,16]. Our results clearly show that recently diagnosed, untreated AML patients with higher hENT1 and dCK expression levels have a higher response rate and a better OS.…”

Differential expression of hENT1 and hENT2 in colon cancer cell lines

“…The application of an hENT1 inhibitor, nitrobenzylmercaptopurine ribonucleoside, significantly reduced the potency of this drug, indicating that hENT1 plays a role in cellular uptake (Schneider-Stock et al, 2005;Ueda et al, 2015). Furthermore, hENT1 was identified as a crucial transporter in cellular AZA uptake in human leukemia cells (Hummel-Eisenbeiss et al, 2013). DAC is also transported by hENT1; this uptake was one of the key determinants of DAC activity in the colon cancer cell line HCT116 (Ueda et al, 2015).…”

Lack of cross‐resistance to FF‐10501, an inhibitor of inosine‐5′‐monophosphate dehydrogenase, in azacitidine‐resistant cell lines selected from SKM‐1 and MOLM‐13 leukemia cell lines

“…2005; Hummel‐Eisenbeiss et al. 2013). ENT1 is involved in cellular uptake of azacitidine and decitabine, and is known to influence the inhibitory activities of azacitidine and decitabine.…”