Equilibrative-sensitive nucleoside transporter and its role in gemcitabine sensitivity

Salvage of preformed nucleosides requires transport across the plasma membrane by sodium-dependent (concentrative) and sodium-independent (equilibrative) mechanisms. These transport systems are also the route of cellular uptake for nucleoside analogues, including gemcitabine (2',2'-difluorodeoxycytidine), a deoxycytidine analogue used in the treatment of pancreatic cancer. To determine whether gemcitabine cytotoxicity is influenced by the equilibrative-sensitive nucleoside transporter (es-NT), basal levels of the es-NT were quantified in three human pancreatic cancer cell lines (PANC-1, HS-766T, and PK-8) and one human bladder cancer cell line (MGH-U1) by flow cytometric analysis, and the results were compared with gemcitabine cytotoxicity assessed by clonogenic assay. To determine whether the salvage pathway of DNA synthesis can be up-regulated by inhibiting de novo DNA synthesis, combination experiments were carried out using the thymidylate synthase (TS) inhibitors 5-fluorouracil or raltitrexed with gemcitabine in a concurrent and sequential fashion. No relationship between basal es-NT and gemcitabine cytotoxicity was demonstrated. For two pancreatic cell lines, sequence-dependent effects of the combination of TS inhibitors and gemcitabine were seen with maximum effect when the TS inhibitors preceded gemcitabine. This was also associated with a significant increase in es-NT levels caused by the TS inhibitors. Thus, modulation of the es-NT by pretreatment with TS inhibitors may have the potential to improve the therapeutic benefit of gemcitabine.