Abstract

β-l-Dioxolane-cytidine (l-OddC, Troxacitabine, BCH-4556), a novel l-configuration deoxycytidine analog, is under clinical trials for treating cancer. The cytotoxicity of l-OddC is dependent on the amount of the triphosphate form (l-OddCTP) in nuclear DNA. Phosphoglycerate kinase (PGK), a downstream protein of hypoxia-inducible-factor-1α (HIF-1α), is responsible for the phosphorylation of the diphosphate to the triphosphate of l-OddC. In this study, we studied the impact of hypoxia on the metabolism and the cytotoxicity of l-OddC and β-d-2′,2′-difluorodeoxycytidine (dFdC) in several human tumor cell lines including HepG2, Hep3B, A673, Panc-1, and RKO. Hypoxic treatment induced the protein expression of PGK 3-fold but had no effect on the protein expression of APE-1, dCK, CMPK, and nM23 H1. Hypoxic treatment increased l-OddCTP formation and incorporation of l-OddC into DNA, but it decreased the uptake and incorporation of dFdC, which correlated with the reduction of hENT1, hENT2, and hCNT2 expression. Using a clonogenic assay, hypoxic treatment of cells made them 2- to 3-fold more susceptible to l-OddC but not to dFdC after exposure to drugs for one generation. Dimethyloxallyl glycine enhanced the cytotoxicity of l-OddC but not dFdC in Panc-1 cells under normoxic conditions. Overexpression or down-regulation of PGK using transient transfection of pcDNA5-PGK or inducible shRNA in RKO cells affected the cytotoxicity of l-OddC but not that of dFdC. The knockdown of HIF-1α in inducible shRNA in RKO cells reduced the cytotoxicity of l-OddC but not dFdC under hypoxic conditions. In conclusion, hypoxia is an important factor that may potentiate the activity of l-OddC.