Elsevier

Biochemical Pharmacology

Volume 76, Issue 3, 1 August 2008, Pages 322-329
Biochemical Pharmacology

Adenoviral-mediated overexpression of human equilibrative nucleoside transporter 1 (hENT1) enhances gemcitabine response in human pancreatic cancer

https://doi.org/10.1016/j.bcp.2008.05.011Get rights and content

Abstract

Nucleoside-derived anticancer agents must be transported across the plasma membrane as a preliminary step to their conversion into active drugs. Hence, modulation of a specific nucleoside transporter may affect bioavailability and contribute significantly to sensitizing tumor cells to these anticancer agents. We have generated and functionally characterized a new recombinant adenovirus (Ad-hENT1) that has allowed us to overexpress the equilibrative nucleoside transporter hENT1 and to analyze its effects in human pancreatic tumor cells. Overexpression of hENT1 is associated with changes in cell cycle profile, in a variable manner depending on the particular cell type, thus suggesting a metabolic link between hENT1-mediated transport processes and the enzymatic machinery responsible for intracellular nucleoside metabolism. When assayed in vivo in a human pancreatic adenocarcinoma xenograft, intratumoral Ad-hENT1 injection improved the therapeutic response to gemcitabine. In summary, hENT1 overexpression is associated with alterations in nucleoside enzymatic machinery and cell cycle progression in cultured cells and enhances gemcitabine action in vivo.

Introduction

An increasing number of nucleoside analogues are being used to treat a wide variety of cancers. They are antimetabolites that compete with natural nucleosides and interact with intracellular targets to induce cytotoxicity. Nucleoside analogues exert their effects in a three-stage process: uptake, metabolism into the active drug, and action on the pharmacological targets. Alterations in any of these steps can impair drug bioavailability and induce resistance to treatment [1]. Most of these molecules require specialised transporter proteins to enter into cells. These proteins include the concentrative nucleoside transporters (hCNT) [2] and the equilibrative nucleoside transporters (hENT) [3], encoded by the gene families SLC28 and SLC29, respectively. hCNT carriers are secondary-active sodium-dependent transporters with selectivity for pyrimidine nucleosides (hCNT1), purine nucleosides plus uridine (hCNT2), and both pyrimidine and purine nucleosides (hCNT3) [4]. The equilibrative nucleoside transporters hENT1 and hENT2 are facilitative transporters found in plasma membranes, and exhibit similar nucleoside selectivity, although hENT2 is also able to transport some nucleobases. They can be functionally distinguished by either their sensitivity (hENT1) or resistance (hENT2) to inhibition by nitrobenzylmercaptopurine ribonucleoside (NBMPR).

Nucleoside transporter expression varies among different cell types and tissues and heterogeneity and selective loss of expression have been reported in neoplasias. Thus, hENT1 seems to be overexpressed in some tumors [5], while in rat hepatocarcinoma models CNT expression is impaired by transformation [6], [7]. A high variability in transporter expression among patients has also been reported for solid tumors [8], [9], [10].

In vitro studies show that the uptake of most chemotherapeutic nucleoside analogues is dependent on the activity of nucleoside transporters. Moreover, transporter function may determine the response to these drugs [11]. Nucleoside transport-deficient variants of several cell lines exhibit a higher resistance to nucleoside analogues than parental cell lines [12], [13] and the inhibition of equilibrative transporters confers resistance to gemcitabine [14]. In addition, a significant relationship between the levels of hENT1 mRNA and the IC50 values for gemcitabine has been found in non-small cell lung cancer cell lines [15], and in human pancreatic adenocarcinoma and biliary tract carcinoma cell lines [16]. In a panel of sixty cell lines, a positive correlation between hENT1 expression and activity and potency of the nucleoside analogues azacytidine and inosine-glycodialdehyde has also been reported [17]. However, contradictory results have also been described. For example, no correlation between basal levels of cell surface hENT1 and gemcitabine sensitivity was found in four cancer cell lines [18], and the mRNA levels of all cloned nucleoside transporters, in fifty cell lines, were found to display no correlation with sensitivity to nucleoside analogues [19].

The role of nucleoside transporters in vivo is less known mainly due to the lack, until recently, of suitable antibodies. A positive correlation between cytarabine sensitivity and hENT1 expression has been found in acute myeloblastic and lymphoblastic leukaemias [20], [21] and equilibrative transport activity correlates with ex vivo fludarabine cytotoxicity in chronic lymphocytic leukaemia cells [22], particularly with regard to hENT2 protein levels [23]. Patients with pancreatic adenocarcinoma with uniformly detectable hENT1 immunostaining and high hENT1-encoding mRNA levels have a significantly longer survival time after gemcitabine chemotherapy than those bearing tumors with low or undetectable hENT1 [24], [25]; this relationship has recently also been suggested for non-small cell lung cancer [26].

The particular role NT proteins play in nucleoside-derived drug uptake and chemosensitivity has been recently reviewed [27]. Nevertheless, this issue is still far from being completely elucidated, since nucleoside-metabolizing enzymes can also modulate chemosensitivity. For most nucleoside-derived drugs, the rate-limiting step in their metabolism is the phosphorylation step catalyzed by deoxycytidine kinase (dCK), and dCK activity has been shown to be decreased in various resistant cell lines [28]. The triphosphorylated forms are required for cytotoxic effects, either by direct incorporation into DNA and RNA or by interfering with enzymes involved in nucleic acid synthesis (DNA polymerases, ribonucleotide reductase), thus eliciting apoptotic cell death.

In this study, we have generated a recombinant adenovirus (Ad5CMV-hENT1) to overexpress hENT1, in an attempt to study in depth the specific role of hENT1 in nucleoside-derived cytotoxicity. This has allowed us to analyze in vitro the effect of hENT1 overexpression on nucleoside transport capacity, and on some enzymes involved in nucleic acid metabolism and the cell cycle, as well as to evaluate its impact on gemcitabine sensitivity in subcutaneous tumors established from human pancreatic cancer cells.

Section snippets

Cell lines

NP-9, NP-18 and NP-29 cell lines were derived from human pancreatic adenocarcinomas, which had been perpetuated as xenografts in nude mice. NP-9 and NP-29 were maintained in Dulbecco's modified Eagle's medium and F12 mixture (1:1) and NP-18 in RPMI 1640 medium (Gibco, Grand Island, NY), both supplemented with 10% foetal bovine serum (FBS) and antibiotics at 37 °C, in a humidified atmosphere containing 5% CO2 and subcultured every 3–4 days.

Construction of Ad5CMV-hENT1 (Ad-hENT1) and infection conditions

hENT1 cDNA was fused to a simian virus 40 polyadenylation

Uridine uptake versus gemcitabine sensitivity into tumor pancreatic cell lines

In order to examine the relationship between gemcitabine sensitivity and hENT1 transport, 1 μM uridine uptake was determined at 1 min, either in the presence or in the absence of NBTI (1 μM), to assess the NBTI-sensitive component of equilibrative nucleoside transport (hENT1). Fig. 1 shows hENT1-mediated uridine uptake into cells of the three cell lines, and basal hENT1 mRNA levels together with IC50 values for the cytotoxicity triggered by 24-hour gemcitabine treatment determined by MTT assays 72 

Discussion

Human equilibrative nucleoside transporter 1 (hENT1) is broadly expressed in human tissues, probably playing a major role in nucleoside salvage processes, particularly following cell proliferation [30]. Under selected physiological conditions, hENT1 can mediate the unidirectional uptake of nucleosides whenever tight coupling between their transport across the plasma membrane and their subsequent metabolism occurs. hENT1-mediated channelling of nucleosides into DNA [30] might be relevant in

Acknowledgements

This research was supported by Lilly S.A., Spanish Ministry of Education and Science (BIO2005-08682-C03-03, SAF2005-01259, PTR1995-0837-OP, CIBER EHD), and Catalunya Government (2005SGR00315). We thank Jaume Comas, who heads the flow cytometry section at the Scientific-technique Services at the University of Barcelona and Neil Davies for editorial help.

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