Abstract 5722: Retention of the radiosensitizing effect of gemcitabine and its main metabolite dFdU under reduced oxygen conditions is not influenced by HIF-1 functionality

Abstract

Abstract Introduction: Regions within solid tumors often experience mild to severe oxygen deprivation, associated with resistance to chemotherapy and irradiation. The aim of this study was to evaluate the radiosensitizing effect of gemcitabine and its main metabolite dFdU under normal versus reduced oxygen conditions and to determine whether hypoxia-inducible factor 1 (HIF-1) is involved in the radiosensitizing mechanism. Materials & methods: The clonogenic assay was performed in three isogenic MDA-MB-231 breast cancer cell lines differing in HIF-1alpha proficiency (24h 0-8 nM gemcitabine or 0-4 microM dFdU, 0-8 Gy irradiation). Validation of the transfection with dominant negative HIF-1alpha was done by western blot and by assessment of HIF-1alpha activity. The relative expression of 84 genes related to the hypoxia signaling pathway was characterized by human hypoxia signaling pathway PCR array. Using radiosensitizing conditions, cells were collected for cell cycle analysis. Results: HIF-1 activity was significantly inhibited after transfection with a dominant negative protein. Furthermore, anoxia-induced VEGF secretion was significantly lower (p<0.05) in MDA-MB-231 cells stably expressing dnHIF (dominant negative HIF-1alpha) than in MDA-MB-231 EV (empty vector) cells. PCR array indicated that hypoxic conditions significantly influenced the expression of HIF-1alpha and ANGPTL4 (angiopoetin-like 4). A clear radiosensitizing effect under normoxic and anoxic conditions was observed for both gemcitabine and dFdU. The radiosensitizing effect was similar under normoxic and anoxic conditions (p=0.48 for gemcitabine, p=0.56 for dFdU) and the dose enhancement factor (DEF) was not significantly influenced by the cell line used. Under anoxia, DEFs for gemcitabine ranged from 1.26 to 1.59, from 1.11 to 1.77, and from 1.34 to 2.04 in MDA-MB-231 wt, EV and dnHIF cells, respectively. Statistical analysis revealed no significant differences in radiobiological parameters between HIF-1 proficient and HIF-1 deficient cells. Cell cycle analysis showed that, in contrast to previous findings in other cell lines, exposure of MDA-MB-231 cells to low oxygen conditions did not induce a significant increase in the percentage of G0/1 cells (p=0.21). Gemcitabine and dFdU caused a block of cells in the S phase of the cell cycle under both normoxic and anoxic conditions. Conclusion: For the first time, radiosensitization by dFdU, the main metabolite of gemcitabine, was demonstrated under low oxygen conditions. As dFdU has a prolonged half-life, the sustained presence of dFdU in the blood might induce radiosensitization despite the short half-life of the parent drug, gemcitabine. This might be highly relevant, especially considering delivery of the drug to hypoxic tumor regions. No major role for HIF-1 in radiosensitization by gemcitabine or dFdU could be shown. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5722. doi:1538-7445.AM2012-5722