Abstract 2671: Study of the role of functional HIF-1 in the in vitro radiosensitizing effect of gemcitabine in normoxic and anoxic breast adenocarcinoma cells

Abstract

Abstract Introduction: It has been well documented that the efficacy of both chemotherapy and radiotherapy is directly linked with an adequate oxygen tension, and that hypoxic regions in solid tumors often contain viable cells that are intrinsically more resistant to anticancer treatment. Recently, it has been demonstrated that the cytotoxic drug gemcitabine retains its radiosensitizing potential under low oxygen conditions in lung adenocarcinoma cells. As the transcription factor ‘hypoxia inducible factor 1’ (HIF-1) plays a crucial role in regulating the adaptive responses of tumor cells to survive under hypoxic conditions, the present study investigated the potential influence of HIF-1 status on radiosensitization by gemcitabine. Materials & methods: Three isogenic human breast adenocarcinoma cell lines with different HIF-1 status were included in this study: MDA-MB-231 (breast adenocarcinoma cell line, wt HIF-1), MDA-MB-231 DN-HIF (transfected with dominant-negative HIF-1alpha, abrogating HIF-1 function) and MDA-MB-231 EV (empty vector transfected control, functional HIF-1). Anoxic conditions (<0.1% O2) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of gemcitabine under normoxic versus anoxic conditions, the clonogenic assay was performed. Cells were treated with 0-8 nM gemcitabine for 24h directly before radiation (0-8 Gy). Using radiosensitizing conditions, cell cycle distribution was monitored flow cytometrically according to the Vindelov method. Results: A clear concentration-dependent radiosensitizing effect of gemcitabine was observed under both normoxic and anoxic conditions (dose enhancement factor (DEF) under normoxia: 1.02-1.70; DEF under anoxia: 1.11-2.04). Combination index (CI) analysis showed an additive interaction between gemcitabine and radiation under normal oxygen conditions (CI 0.902-1.148), and a synergistic interaction under reduced oxygen conditions (CI 0.455-0.889). Statistical analysis using two-way ANOVA revealed no significant differences in radiobiological parameters (DEF, ID10, ID50, mean inactivation dose, surviving fraction at 2 Gy, oxygen enhancement ratio) between MDA-MB-231 EV and MDA-MB-231 DN-HIF cells, suggesting no influence of HIF-1 functionality on radiosensitivity. Considering the cell cycle distribution after treatment with gemcitabine, 24h treatment with 4 or 8 nM gemcitabine established a significant S-phase block in both normoxic and anoxic MDA-MB-231 wt and DN-HIF cells. Conclusion: This study showed that the retained radiosensitizing effect of gemcitabine under anoxic conditions was not tumor tissue specific and could be observed in MDA-MB-231 breast cancer cells. No major role for functional HIF-1 protein in radiosensitization by gemcitabine 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 2671. doi:10.1158/1538-7445.AM2011-2671