Effect of Hypoxanthine on Functional Activity of Nucleoside Transporters ENT1 and ENT2 in Caco-2 Polar Epithelial Intestinal Cells

Senyavina, N. V.; Tonevitskaya, S. A.

doi:10.1007/s10517-015-3118-z

Cited by 10 publications

(16 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The vector controls did not exhibit downregulation of the other genes seen in the SLC43A3transfected cells. The downregulation of ENT2 and MRP4 may reflect compensation by the cells to the enhanced availability of nucleobases via ENBT1 to support intracellular metabolism (Senyavina and Tonevitskaya, 2015). Both of Fig.…”

Section: Discussionmentioning

confidence: 97%

Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter

et al. 2019

View full text Add to dashboard Cite

6-Mercaptopurine (6-MP) is a nucleobase analog used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disorders. However, the mechanisms underlying its transport into target cells have remained elusive. The protein encoded by SLC43A3_1 [equilibrative nucleobase transporter 1 (ENBT1)] has recently been shown to transport endogenous nucleobases. A splice variant (SLC43A3_2), encoding a protein with 13 additional amino acids in the first extracellular loop, is also expressed but its function is unknown. We hypothesized that 6-MP is a substrate for both variants of ENBT1. Human embryonic kidney 293 (HEK293) cells (lacking endogenous ENBT1 activity) were transfected with each of the coding region variants of SLC43A3. ENBT1 function was assessed via the rate of flux of [ 3 H]adenine and [ 14 C]6-MP across the plasma membrane. Both SLC43A3 variants encoded proteins with similar functional properties. [ 14 C]6-MP and [ 3 H]adenine had K m values (6S.D.) of 163 6 126 and 37 6 26 mM, respectively, for this system. Decynium-22, 6-thioguanine, and 6-methylmercaptopurine inhibited 6-MP uptake with K i values of 1.0 6 0.4, 67 6 30, and 73 6 20 mM, respectively. ENBT1 also mediated adenine-sensitive efflux of 6-MP from the SLC43A3-HEK293 cells. MRP4 also contributed to the efflux of 6-MP in this model, but was less efficient than ENBT1 in this regard. Furthermore, transfection of HEK293 cells with SLC43A3 increased the sensitivity of the cells to the cytotoxic effects of 6-MP by more than 7-fold. Thus, both variants of ENBT1 are key players in the transfer of 6-MP into and out of cells, and changes in SLC43A3 expression impact 6-MP cytotoxicity.

show abstract

Section: Discussionmentioning

confidence: 97%

Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Predominant apical localization of ENT2 in these cells was reported [11]. Previous studies of hypoxanthine transport fl uxes in Caco-2 cells showed high concentration of ENT transporters on the basolateral membrane of Caco-2 cells and their functional activity on the apical membrane [1]; however, the expression and localization of individual transporters remain unclear.…”

Section: Resultsmentioning

confidence: 94%

“…In view of the data on apical (16±1.3 nmol/mln cells) and basolateral (31.8±6.7 nmol/mln cells) hypoxanthine concentrations in different media in 24 h [1], the amount of transporter ENT2 (n 2 b ) on the basolateral membrane was calculated from (3): ENT-2 bas =(7.0±1.0)×10 -13 μmol/cell. Equation (4) refl ects the relationship between the apical content of transporters ENT1 and ENT2, and [8], which corresponds to 16×10 -13 μmol/ cell and qualitatively agrees with the results.…”

Section: Resultsmentioning

confidence: 99%

“…Monolayer of differentiated Caco-2 cells was created in Transwell membrane inserts (Corning), hypoxanthine concentration was determined in the culture medium, and calculation of absorption was performed as described previously [1]. RNA was extracted using commercial RNEasy Micro kit (Qiagen), the concentration and quality (RQI index) of samples were measured on a Nanodrop ND1000 spectrophotometer (Thermo Scientifi c) and an Experion capillary electrophoresis system (Bio-Rad), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…ENT1 and ENT2 transportes are characterized by the ability to transfer purine base hypoxanthine [15]. We have previously demonstrated [1] that functional activity of ENT1/ENT2 associated with hypoxanthine absorption is observed mainly on the basolateral membrane; however, the data was obtained on apical transport of hypoxanthine, which attested to the presence of at least one of the transporters of this fa mily on the apical membrane of Caco-2 cells.…”

mentioning

confidence: 94%

See 2 more Smart Citations

Localization and Expression of Nucleoside Transporters ENT1 and ENT2 in Polar Cells of Intestinal Epithelium

et al. 2016

Self Cite

View full text Add to dashboard Cite

Сасо-2 cell line is widely used in in vitro studies of the intestinal wall permeability for drugs, but transport activity of these cells has not been thoroughly studied. We analyzed localization and expression of nucleoside transporters of the ENT family transferring a number of anticancer and antiviral drugs. Immunocytochemical staining revealed apical localization of ENT1 and integral expression of ENT2 in Caco-2 cells. Based on these data and on the value of hypoxanthine uptake by these cells, we created a mathematical model allowing quantification of transporter expression on the apical and basolateral membranes of Caco-2 cells. The discrepancy between gene and protein expression of the transporter complicating prediction of patient's sensitivity to the drug upon individual therapy selection is discussed.

show abstract

Addressing the Clinical Importance of Equilibrative Nucleoside Transporters in Drug Discovery and Development

Hau

Wright

Cherrington

2023

Clin Pharma and Therapeutics

View full text Add to dashboard Cite

The US Food and Drug Administration (FDA), European Medicines Agency (EMA), and Pharmaceuticals and Medical Devices Agency (PMDA) guidances on small‐molecule drug–drug interactions (DDIs), with input from the International Transporter Consortium (ITC), recommend the evaluation of nine drug transporters. Although other clinically relevant drug uptake and efflux transporters have been discussed in ITC white papers, they have been excluded from further recommendation by the ITC and are not included in current regulatory guidances. These include the ubiquitously expressed equilibrative nucleoside transporters (ENT) 1 and ENT2, which have been recognized by the ITC for their potential role in clinically relevant nucleoside analog drug interactions for patients with cancer. Although there is comparatively limited clinical evidence supporting their role in DDI risk or other adverse drug reactions (ADRs) compared with the nine highlighted transporters, several in vitro and in vivo studies have identified ENT interactions with non‐nucleoside/non‐nucleotide drugs, in addition to nucleoside/nucleotide analogs. Some noteworthy examples of compounds that interact with ENTs include cannabidiol and selected protein kinase inhibitors, as well as the nucleoside analogs remdesivir, EIDD‐1931, gemcitabine, and fialuridine. Consequently, DDIs involving the ENTs may be responsible for therapeutic inefficacy or off‐target toxicity. Evidence suggests that ENT1 and ENT2 should be considered as transporters potentially involved in clinically relevant DDIs and ADRs, thereby warranting further investigation and regulatory consideration.

show abstract

Effect of Hypoxanthine on Functional Activity of Nucleoside Transporters ENT1 and ENT2 in Caco-2 Polar Epithelial Intestinal Cells

Cited by 10 publications

References 16 publications

Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter

Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter

Localization and Expression of Nucleoside Transporters ENT1 and ENT2 in Polar Cells of Intestinal Epithelium

Addressing the Clinical Importance of Equilibrative Nucleoside Transporters in Drug Discovery and Development

Contact Info

Product

Resources

About