Abstract 2661: The role of HIF-1 protein on the radiosensitizing effect of difluorodeoxyuridine, the main metabolite of gemcitabine, under normoxic and anoxic conditions
Authors: Pauwels Bea , Wouters An , Vermorken Jan B. , Ides Johan , Deschoolmeester Vanessa , Lambrechts Hilde AJ , Pattyn Greet GO , Meijnders Paul , Pauwels Patrick , Williams Kaye J. , Peeters Marc , Lardon Filip 
Published: June 2012

It is known that gemcitabine and difluorodeoxyuridine (dFdU) have radiosensitizing properties. It has become evident that solid tumors often contain hypoxic regions and that this is one of the causes of resistance or decreased sensitivity to cancer therapy. In hypoxic conditions, the transcription factor, hypoxia-inducible factor-1 (HIF-1), is upregulated. HIF-1 is responsible for the cellular and adaptive responses of tumors to survive in hypoxic conditions. Recently, it has been demonstrated that gemcitabine retains its radiosensitizing potential under hypoxia. In the present study, the radiosensitizing potential of dFdU under anoxic conditions is investigated as well as the role of HIF-1 protein.

In order to explore the role of HIF-1, human tumor cell lines included in this study were MDA-MB-231 (breast adenocarcinoma cell line, wt HIF-1), MDA-MB-231 DN-HIF (transfected with dominant-negative HIF-1α) and MDA-MB-231 EV (empty vector transfected control). Anoxic conditions (&lt;0.1% O2,) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of dFdU, the clonogenic assay was performed. Cells were exposed to normoxic or anoxic conditions and were simultaneously treated with 0, 2 or 4 microM dFdU for 24h immediately before irradiation (0-8 Gy, room temperature, linear accelerator). Immediately following radiation, anoxic cells were reoxygenated and cells were washed with drug-free medium. Using radiosensitizing conditions, cells were collected for cell cycle analysis by flow cytometry.

A clear concentration-dependent radiosensitizing effect of dFdU was observed under both normoxic and anoxic conditions (dose enhancement factor (DEF) under normoxia: 1.74-3.38; under anoxia: 2.08-3.81). Combination index (CI) analysis showed a synergistic to additive interaction between dFdU and radiation under normoxia (CI 0.651 ± 0.175), yet an additive interaction under anoxia (CI 0.784 ± 0.120). Statistical analysis using two-way ANOVA revealed that cell survival was significantly influenced by the concentration of dFdU, the dose of radiation, the oxygen tension and the cell line. Post hoc analysis indicated no significant difference between the three cell lines for DEF, ID50, mean inactivation dose and survival fraction at 2 Gy, suggesting that the functionality of HIF-1 protein has no impact on the radiosensitizing effect of dFdU. Considering the cell cycle distribution after treatment with dFdU, 24h treatment with 2 or 4 microM dFdU established a significant S-phase block in both normoxic and anoxic MDA-MB-231 wt and DN-HIF cells.

In conclusion, this study showed that dFdU has a clear concentration-dependent radiosensitizing effect in MDA-MB-231 breast cancer cells under normoxic as well as anoxic conditions. No major role for functional HIF-1 protein in radiosensitization by dFdU could be demonstrated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2011-2661