Abstract 5551: The role of hypoxia-inducible factor-1 (HIF-1) protein on the radiosensitizing effect of gemcitabine, in vitro, under normoxic and anoxic conditions
Authors: Pauwels Bea , Wouters An , Ides Johan , Pattyn Greet , Lambrechts Hilde , Williams Kaye , Meijnders Paul , Vermorken Jan B. , Pauwels Patrick , Peeters Marc , Lardon Filip 
Published: February 2011

Introduction: Solid tumors often contain hypoxic regions. 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 tumours to survive in hypoxic conditions. HIF-1 is also thought to influence genes involved in resistance to therapy. In this study, we examined the role of HIF-1 in radiosensitization of gemcitabine under normoxic and anoxic conditions.

Materials & methods: Two isogenic breast cancer cell lines were used that differ in HIF-1 expression: MDA-MB-231 (HIF-1 wt) and MDA-MB-231 DN-HIF (HIF-1 deficient). Anoxic conditions (&lt;0.1% O2,) were achieved in a Bactron IV anaerobic chamber. Cytotoxicity of gemcitabine was examined by using the sulfordamine B test. To analyze the radiosensitizing effect of gemcitabine under normoxic versus anoxic conditions, the clonogenic assay was performed. Cells were exposed to normoxic or anoxic conditions and were simultaneously treated with 0-8 nM gemcitabine for 24h directly before radiation (0-8 Gy). Immediately following radiation, anoxic cells were reoxygenated and all cells were washed with drug-free medium. By mean of flow cytometry, we analyzed the influence of HIF-1 on the cell cycle effects of gemcitabine. Finally, we investigated the expression of VEGF at specific time points (0, 24 and 48 hours) in the different cell lines by using quantitative real-time PCR (qRT-PCR). All tests were done in normoxic and in anoxic conditions.

Results: The IC50 values of gemcitabine were comparable under normoxic and anoxic conditions: 14.1±2 and 15.0±5.2 nM for MDA-MB-231 and 6.3±1.3 and 7.3±1.1 nM for MDA-MB-231 DN-HIF cells (p=0.569). A concentration dependent radiosensitizing effect of gemcitabine was observed in both cell lines under normoxic and anoxic conditions; DEFs ranged from 1.1 to 2.0. Two-way ANOVA revealed that the radiosensitizing effect was not significantly influenced by the oxygen tension (p=0.698). Gemcitabine was able to overcome the anoxia-induced G0/1 phase block and established a significant S-phase block in both normoxic and anoxic cells. Using qRT-PCR, we showed an increase of VEGF expression in the wt MDA-MB-231 cells in anoxia. An increase of VEGF expression was seen with a longer incubation time under anoxia.

Conclusion: From these experiments, we can conclude that HIF-1 protein does not seem to play a role in the cytotoxic and radiosensitizing effect of gemcitabine in MDA-MB-231 breast cancer cells. In addition, HIF-1 protein did not influence the cell cycle effect of gemcitabine, declaring the comparable radiosensitizing effect in the different cell lines. There is no difference between normoxia and anoxia in the different experiments. Similar experiments will be performed with the metabolite of gemcitabine, difluorodeoxyuridine.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5551.