The radiosensitising effect of difluorodeoxyuridine, a metabolite of gemcitabine, in vitro

Purpose: Gemcitabine is an active antitumour agent with radiosensitising properties. Gemcitabine is rapidly metabolised, intracellularly as well as extracellularly, by deoxycytidine deaminase to difluorodeoxyuridine (dFdU), a compound with little antitumour activity. However, plasma concentrations are maintained for a prolonged period (>24 h) at levels known to cause growth inhibition. This is the first study that investigates the radiosensitising potential of dFdU in vitro.

Methods: ECV304 and H292, human Cancer cells, were treated with different concentrations dFdU (0-100 microM) during 24 h before radiation treatment (RT). The schedule dependency of the radiosensitising effect was studied by varying the interval between dFdU and radiation treatment. In addition, the cell cycle effect of dFdU was investigated with flow cytometry, and the induction of Apoptosis under radiosensitising conditions was determined by Annexin V staining and Caspase 3 cleavage.

Results: dFdU caused a clear concentration-dependent radiosensitising effect in both ECV304 and H292 cells. Dose enhancement factor (DEF) increased with an increasing concentration of dFdU: DEFs were 1.10, 1.60 and 2.17 after treatment with 10, 25 and 50 microM dFdU, respectively, in ECV304 cells and 1.08, 1.31 and 1.60 after treatment with 25, 50 and 100 microM, respectively, in H292 cells. DEFs decreased with an increasing interval of 0-24 h between dFdU treatment and radiation. Under radiosensitising conditions, the combination dFdU and radiation resulted in an increased induction of Apoptosis. In addition, the cell cycle effect of dFdU, an arrest at the early S phase, is comparable with the cell cycle effect of gemcitabine.

Conclusions: dFdU, the main metabolite of gemcitabine, causes a concentration- and schedule- dependent radiosensitising effect in vitro. Since the metabolite is present in plasma for a long period (>24 h) after treatment with gemcitabine, it might be partly responsible for the interaction between radiotherapy and gemcitabine. This observation might have important consequences for the optimal schedules of the combination gemcitabine and radiation therapy.