Anticancer nucleobase analogues 6-mercaptopurine and 6-thioguanine are novel substrates for equilibrative nucleoside transporter 2

doi:10.1016/j.ijpharm.2006.09.044

International Journal of Pharmaceutics

Volume 333, Issues 1–2, 21 March 2007, Pages 56-61

https://doi.org/10.1016/j.ijpharm.2006.09.044 Get rights and content

Abstract

Various antimetabolites of nucleobase analogues, such as 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 5-fluorouracil (5-FU), are used for cancer treatments. The first step in nucleobase analogue drug therapy is entry of these compounds into tumor cells. Equilibrative nucleoside transporter 2 (ENT2) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we investigated whether or not these nucleobase analogues are transported via ENT2, using mouse ENT2-overexpressing Cos-7 cells. The hypoxanthine uptake mediated by ENT2 was significantly reduced by the addition of 6-MP and 6-TG, and the inhibition of the hypoxanthine uptake by the 6-thiopurines was competitive. Transfection of ENT2 cDNA into Cos-7 cells resulted in an increase in 6-MP uptake. The 6-MP uptake via ENT2 showed clear time- and substrate concentration-dependent profiles, and was inhibited by 6-TG in an inhibitor concentration-dependent fashion. On the other hand, uracil was not a substrate for ENT2, and 5-FU had no effect on the hypoxanthine uptake via ENT2. Therefore, we concluded that 6-MP and 6-TG, but not 5-FU, are transported mediated by the same recognition site on ENT2 with hypoxanthine.

Introduction

Various structural analogues of nucleobase, such as 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 5-fluorouracil (5-FU), are cytotoxic and have found therapeutic use as antineoplastic agents (Spiegelman et al., 1980, Elgemeie, 2003). Once transported inside the cells, nucleobase analogue drugs interfere with DNA repair and replication. The efficient entry of these nucleobase analogue drugs into tumor cells, but not into normal cells, is thought to be necessary for rendering cytotoxic drug treatments more selective toward tumors while sparing the normal tissues. Therefore, an understanding of transport mechanisms of the nucleobase analogues is critical in the development of improved chemotherapeutic strategies.

Kinetic studies on nucleobase transport in a variety of mammalian cells have been vigorously performed. Mammalian cells simultaneously exhibit multiple nucleobase transport processes (Koning and Diallinas, 2000). However, no cDNA encoding a functional mammalian nucleobase transporter has been cloned. On the other hand, there are a few reports about nucleobase transport mediated by nucleoside transporters (NTs). Five mammalian NTs, which are expressed in the plasma membrane of cells, have been cloned, and they have been classified into two distinct unrelated families based on Na⁺ dependence (Griffith and Jarvis, 1996, Cass et al., 1998, Cass et al., 1999, Kong et al., 2004). The concentrative NTs (CNTs) are Na⁺-dependent, and three CNT subtypes (CNT1, pyrimidine-preferring; CNT2, purine-preferring; CNT3, broadly selective) have been characterized. The Na⁺-independent equilibrative NTs (ENTs) have broad substrate selectivity for purine and pyrimidine nucleosides. They have been classified, according to their sensitivity to the inhibitor nitrobenzylmercaptopurine riboside (NBMPR), into sensitive (ENT1) and insensitive (ENT2) systems. Previously, it was reported that ENT2, but not ENT1 and all CNTs, transported not only nucleosides but also several nucleobases (Ritzel et al., 2001, Yao et al., 2002). Overall, ENT2 is the only transporter identified as nucleobase transport system at the molecular level. However, the involvement of ENT2 in the uptake of 6-MP, 6-TG and 5-FU remains unclear.

In this study, therefore, we examined whether or not 6-MP, 6-TG and 5-FU are substrates for ENT2, using mouse ENT2-overexpressing Cos-7 cells (Cos-7/ENT2).

Section snippets

Chemicals

Nucleobases, 6-TG and 5-FU were from Wako Pure Chemical Ind. (Osaka, Japan), and 6-MP and NBMPR were purchased from Sigma Chemical Co. (MO, USA). Radioactive nucleobases and uridine were obtained from American Radiolabeled Chemicals Inc. (MO, USA), and [¹⁴C]6-MP was purchased from MORAVEK Biochem. Inc. (CA, USA). All other reagents were of commercial or analytical grade requiring no further purification.

Cell culture

Cos-7 cells were maintained in Dulbecco's modified Eagle's MEM (Nissui Pharmaceutical Co.,

Uptake of uridine and nucleobases by Cos-7/ENT2 and Cos-7/pCI-neo

In order to confirm the functional expression of recombinant ENT2 in Cos-7/ENT2, the uptake of uridine, a typical substrate of NTs, by Cos-7/ENT2 and Cos-7/pCI-neo with or without extracelluar Na⁺ was measured (Fig. 1A). The transfection of ENT2 cDNA into Cos-7 cells resulted in an increase in the uptake of uridine. The uridine uptake mediated by ENT2 was Na⁺-independent, which was consistent with the transport characteristic of ENT2.

Since 6-MP, 6-TG and 5-FU are analogues of hypoxanthine,

Discussion

The permeation through the plasma membrane is a key step for the cytotoxicity of 6-MP, 6-TG and 5-FU, because their molecular targets are expressed intracellularly (Spiegelman et al., 1980, Elion, 1989). In the present study, we examined whether or not 6-MP, 6-TG and 5-FU are substrates for ENT2, using ENT2-overexpressing Cos-7 cells.

6-MP was transported via ENT2 in time- and substrate concentration-dependent fashions. As shown in Fig. 3, Fig. 4, the uptake of hypoxanthine and 6-MP mediated by

Acknowledgement

This work was supported by the “Academic Frontier” Project organized by Kyoto Pharmaceutical University.

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Anticancer nucleobase analogues 6-mercaptopurine and 6-thioguanine are novel substrates for equilibrative nucleoside transporter 2

Abstract

Introduction

Section snippets

Chemicals

Cell culture

Uptake of uridine and nucleobases by Cos-7/ENT2 and Cos-7/pCI-neo

Discussion

Acknowledgement

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Int. J. Pharm.

J. Biol. Chem.

Recent advances in the molecular biology of nucleoside transporters of mammalian cells

Biochem. Cell Biol.

Nucleoside transporters of mammalian cells

Pharm. Biotechnol.

Thiopurine, mercaptopurine: their analogs and nucleosides as antimetabolites

Curr. Pharm. Des.

The purine path to chemotherapy

Science

Nucleotide metabolism and cell–cell interactions

Methods Mol. Biol.

Nitrobenzylthioinosine-binding protein overexpression in human breast, liver, stomach and colorectal tumor tissues

Anticancer Res.

Involvement of bidirectional adenosine transporters in the release of l-[3H]adenosine from rat brain synaptosomal preparations

J. Neurochem.

Involvement of bidirectional adenosine transporters in the release of l-[³H]adenosine from rat brain synaptosomal preparations