Since human Nucleoside Transporters (hNTs) were identified by their activity as transport systems, extensive work has been done to fully characterize them at the molecular and physiological level. Many efforts have been addressed to the identification of their selectivity for natural substrates and nucleoside analogs used to treat several diseases. hNTs belong to two different gene families, SLC28 and SLC29, encoding human Concentrative Nucleoside Transporters (hCNTs) and human Equilibrative Nucleoside Transporters (hENTs), respectively. hCNTs and hENTs are integral membrane proteins, albeit structurally unrelated. Both families share common features as substrate selectivity and often tissue localization. This apparent biological redundancy may anticipate some different roles for hCNTs and hENTs in cell physiology. Thus, hENTs may have a major role in maintaining nucleoside homeostasis, whereas hCNTs could contribute to nucleoside sensing and signal transduction. In this sense, the ascription of hCNT1 to a transceptor reinforces this hypothesis. Moreover, some evidences could suggest a putative role of hCNT2 and hCNT3 as transceptors. The interacting proteins identified for hCNT2 suggest a link to energy metabolism. Moreover, the ability of hCNT2 and hCNT3 to transport adenosine links both proteins to purinergic signaling. On the other hand, the broad selectivity transporters hENTs have a crucial role in salvage pathways and purinergic signaling by means of nucleoside pools regulation. In particular, the two new hENT2 isoforms recently described together with hENT2 seem to be key elements controlling nucleoside and nucleotide pools for DNA synthesis. This review focuses on all these NTs functions beyond their mere translocation ability.

Extracellular adenosine concentrations are regulated by a panel of membrane transporters which, in most cases, mediate its uptake into cells. Adenosine transporters belong to two gene families encoding Equilibrative and Concentrative Nucleoside Transporter proteins (ENTs and CNTs, respectively). The lack of appropriate pharmacological tools targeting every transporter subtype has introduced some bias on the current knowledge of the role of these transporters in modulating adenosine levels. In this regard, ENT1, for which pharmacology is relatively well-developed, has often been identified as a major player in purinergic signaling. Nevertheless, other transporters such as CNT2 and CNT3 can also contribute to purinergic modulation based on their high affinity for adenosine and concentrative capacity. Moreover, both transporter proteins have also been shown to be under purinergic regulation via P1 receptors in different cell types, which further supports its relevance in purinergic signaling. Thus, several transporter proteins regulate extracellular adenosine levels. Moreover, CNT and ENT proteins are differentially expressed in tissues but also in particular cell types. Accordingly, transporter-mediated fine tuning of adenosine levels is cell and tissue specific. Future developments focusing on CNT pharmacology are needed to unveil transporter subtype-specific events.

Nucleoside and nucleobase analogs are currently used in the treatment of solid tumors, lymphoproliferative diseases, viral infections such as hepatitis and AIDS, and some inflammatory diseases such as Crohn. Two gene families are implicated in the uptake of nucleosides and nucleoside analogs into cells, SCL28 and SLC29. The former encodes hCNT1, hCNT2, and hCNT3 proteins. They translocate nucleosides in a Na+ coupled manner with high affinity and some substrate selectivity, being hCNT1 and hCNT2 pyrimidine- and purine-preferring respectively, and hCNT3 a broad selectivity transporter. SLC29 genes encode four members, being hENT1 and hENT2 the only two which are unequivocally implicated in the translocation of nucleosides and nucleobases (the latter mostly via hENT2) at the cell plasma membrane. Some nucleoside-derived drugs can also interact with and be translocated by members of the SLC22 gene family, particularly hOCT and hOAT proteins. Inter-individual differences in transporter function and perhaps, more importantly, altered expression associated with the disease itself might modulate the transporter profile of target cells, thereby determining drug bioavailability and action. Drug transporter pharmacology has been periodically reviewed. Thus, with this contribution we aim at providing a state-of-the-art overview of the clinical evidence generated so far supporting the concept that these membrane proteins can indeed be biomarkers suitable for diagnosis and/or prognosis. Last but not least, some of these transporter proteins can also be envisaged as drug targets, as long as they can show “transceptor” functions, in some cases related to their role as modulators of extracellular adenosine levels, thereby providing a functional link between P1 receptors and transporters.

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.