Effect of its deaminated metabolite, 2′,2′-difluorodeoxyuridine, on the transport and toxicity of gemcitabine in HeLa cells

Abstract

Gemcitabine is a pyrimidine analog effective against many solid tumors. Following intravenous administration, deaminases in the plasma rapidly convert the parent compound, gemcitabine, to its deaminated metabolite, 2′,2′-difluorodeoxyuridine (dFdU), resulting in an elimination half-life for gemcitabine of 8 min. The half-life of dFdU, however, is upwards of 14 h, yielding plasma concentrations that are frequently 10–20-fold higher than that of gemcitabine. The uptake of gemcitabine into tumor cells is facilitated by both concentrative (hCNT) and equilibrative (hENT) nucleoside transporters. Recently, it was observed that dFdU is a substrate for hCNT as well. The purpose of this study was to investigate the effects of dFdU on gemcitabine uptake and efflux via hENT1 and hENT2 in HeLa cells. Our results suggest that dFdU is a substrate for both hENT1 and hENT2 as well as a competitive inhibitor of gemcitabine transport at concentrations >100-fold lower than those typically achieved in plasma (IC₅₀ = 0.45 and 1.2 μM for hENT1/2 and hENT2, respectively). However, inhibition of gemcitabine uptake is time-dependent, as dFdU limits gemcitabine uptake into HeLa cells by more than 80% during short (<20 s) incubation periods but increases net gemcitabine retention as incubation length increases. While dFdU enhances the accumulation of gemcitabine by up to 1.5-fold following a 60 min incubation, dFdU did not enhance gemcitabine cytotoxicity. In conclusion, this is the first report of an interaction between dFdU and gemcitabine suggesting that the deaminated metabolite may play an important role in the disposition of gemcitabine in tumor cells.

Introduction

Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC), is an analog of deoxycytidine with high activity against many types of solid tumors including pancreatic, cervical, ovarian, breast, bladder, and non-small cell lung cancers [1], [2], [3], [4]. As a hydrophilic nucleoside analog, gemcitabine utilizes nucleoside transporters to cross plasma membranes. Once inside the cell, this prodrug is quickly phosphorylated to its active di- and triphosphate moieties (dFdCDP, dFdCTP) [5]. dFdCDP and dFdCTP are then incorporated into nascent DNA and RNA strands, eventually leading to inhibition of DNA polymerases, chain termination and cessation of DNA replication [6].

In addition to phosphorylation, gemcitabine may also undergo intra- and extracellular deamination to the much less active form, 2′,2′-difluorodeoxyuridine (dFdU), via cytidine deaminase (CDA), which is present at high concentrations in many types of normal and malignant tissues as well as in the plasma [7], [8]. In fact, after intravenous administration, cytidine deaminases in the plasma rapidly convert gemcitabine to dFdU, resulting in a gemcitabine plasma elimination half-life of only 8 min [9]. The long half-life of dFdU (14 h), leads to plasma concentrations of the dFdU that are frequently 10–20-fold higher than those of the parent compound when measured shortly after gemcitabine administration [10], [11], [12]. Additionally, deamination of the monophosphate form of gemcitabine may also occur intracellularly via deoxycytidine monophosphate deaminase (dCMPD) to yield dFdUMP, which may then be further phosphorylated to dFdUDP and dFdUTP or dephosphorylated to dFdU [5]. As with gemcitabine, dFdU may also be directly phosphorylated intracellularly to its mono-, di-, and triphosphate metabolites by deoxycytidine kinase. However, the majority of dFdU nucleotides are thought to be formed by the breakdown of gemcitabine nucleotides as dFdU is predicted to have a low affinity for deoxycytidine kinase (dCK) [13], [14], [15].

Gemcitabine is transported into cells by both the concentrative (hCNT) and equilibrative (hENT) nucleoside transporters. Human concentrative nucleoside transporter 1 (hCNT1) is the most efficient transporter of gemcitabine, with a K_m of around 18 μM, but the distribution of this protein throughout the body is limited when compared to the more ubiquitously expressed ENT family [16], [17]. hENT1 and hENT2 also facilitate gemcitabine uptake into cells, and clinical studies have demonstrated a correlation between the expression of these transporters and response to gemcitabine therapy [18], [19], [20].

Recently, it has been reported that dFdU is transported by both hCNT1 and hENT1 into hCNT1-transfected MDCK cells [21]. The extent of dFdU uptake was nearly the same as that of gemcitabine, suggesting that dFdU is a high affinity substrate for these transporters as well. Additionally, it was determined in the same study that dFdU undergoes biphasic efflux from HepG2 and A549 cells preloaded with gemcitabine, presumably via the same nucleoside transporters.

When administered alone, dFdU displays minimal cytotoxicity when compared with gemcitabine. Yet, with both the parent and metabolite competing for the same transporters, high plasma concentrations of dFdU may affect the efficacy of gemcitabine against solid tumors by decreasing its cellular uptake. Therefore, the objective of this study was to investigate the effects of dFdU on gemcitabine uptake and efflux via hENT1 and hENT2 in HeLa cells, a cell line which endogenously expresses these two nucleoside transporters. Based on our results, we have developed a mechanistic model whereby dFdU limits the uptake of gemcitabine into the cell, yet ultimately results in an increased intracellular sequestration of gemcitabine within cells.