10 research outputs found
Nucleoside transporters and human organic cation transporter 1 determine the cellular handling of DNA-methyltransferase inhibitors
BACKGROUND AND PURPOSE
Inhibitors of DNA methyltransferases (DNMTs), such as azacytidine, decitabine and zebularine, are used for the epigenetic
treatment of cancer. Their action may depend upon their translocation across the plasma membrane. The aim of this study
was to identify transporter proteins contributing to DNMT inhibitor action.
EXPERIMENTAL APPROACH
Drug interactions with selected hCNT and hENT proteins were studied in transiently transfected HeLa and MDCK cells.
Interaction with human organic cation transporters (hOCTs) was assessed in transiently transfected HeLa cells and Xenopus
laevis oocytes.
KEY RESULTS
Zebularine uptake was mediated by hCNT1, hCNT3 and hENT2. Decitabine interacted with but was not translocated by any
nucleoside transporter (NT) type. hCNT expression at the apical domain of MDCK cells promoted net vectorial flux of
zebularine. Neither hOCT1 nor hOCT2 transported decitabine, but both were involved in the efflux of zebularine, suggesting
these proteins act as efflux transporters. hOCT1 polymorphic variants, known to alter function, decreased zebularine efflux.
CONCLUSIONS AND IMPLICATIONS
This study highlights the influence of human NTs and hOCTs on the pharmacokinetics and pharmacodynamics of selected
DNMT inhibitors. As hOCTs may also behave as efflux transporters, they could contribute either to chemoresistance or to
chemosensitivity, depending upon the nature of the drug or combination of drugs being used in cancer therapy
Identification and Characterization of a Secondary Sodium-Binding Site and the Main Selectivity Determinants in the Human Concentrative Nucleoside Transporter 3
The family of concentrative Na<sup>+</sup>/nucleoside cotransporters
in humans is constituted by three subtypes, namely, hCNT1, hCNT2,
and hCNT3. Besides their different nucleoside selectivity, hCNT1 and
hCNT2 have a Na<sup>+</sup>/nucleoside stoichiometry of 1:1, while
for hCNT3 it is 2:1. This distinct stoichiometry of subtype 3 might
hint the existence of a secondary sodium-binding site that is not
present in the other two subtypes, but to date their three-dimensional
structures remain unknown and the residues implicated in Na<sup>+</sup> binding are unclear. In this work, we have identified and characterized
the Na<sup>+</sup> binding sites of hCNT3 by combining molecular modeling
and mutagenesis studies. A model of the transporter was obtained by
homology modeling, and key residues of two sodium-binding sites were
identified and verified with a mutagenesis strategy. The structural
model explains the altered sodium-binding properties of the hCNT3C602R
polymorphic variant and supports previously generated data identifying
the determinant residues of nucleoside selectivity, paving the way
to understand how drugs can target this plasma membrane transporter