Abstract 460: Influence of low oxygen conditions on the in vitro radiosensitizing effect of gemcitabine, in p53 wild type and p53 deficient non-small cell lung carcinoma 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. As the antimetabolite gemcitabine is a potent radiosensitizer, often used in the clinic, the influence of oxygen deficiency on the radiosensitizing effect of gemcitabine was investigated. Materials & methods: The human tumor cell lines included were A549 (lung carcinoma cell line, wt p53), A549-E6 (transduced with the HPV type 16 E6 gene, abrogating the p53 function) and A549-LXSN (vector transfected control, functional p53). 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 exposed to normoxic or anoxic conditions and were simultaneously treated with 0-15 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. Cell cycle analysis and determination of apoptotic cell death was performed using flow cytometry. P53 expression was studied by Western blot. Results: A clear concentration-dependent radiosensitizing effect of gemcitabine was observed under both normoxia and anoxia (dose enhancement factor 1.27-1.70 and 1.28-2.06 respectively). Combination index (CI) analysis showed an additive to strongly synergistic interaction between gemcitabine and radiation under both normal and reduced oxygen conditions (CI 0.672-1.083 and 0.587-1.090 respectively). Two-way ANOVA revealed that cell survival was significantly influenced by the concentration of gemcitabine, radiation dose, oxygen tension and cell line used. Post hoc analysis indicated a significant difference between A549-LXSN and A549-E6 cells, suggesting a potential role for p53 in radiosensitization by gemcitabine. Independent of the p53 functionality, 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. The percentage early and late apoptotic/necrotic cells increased significantly with the combination of gemcitabine and radiation, and was significantly influenced by the oxygen concentration. Interestingly, a significant difference between A549-LXSN and A549-E6 was observed. Conclusion: This study demonstrated, for the first time, that gemcitabine retains its radiosensitizing potential under low oxygen conditions. The p53 protein might be involved in this process, not by affecting cell cycle progression, but rather by acting on the induction of apoptotic cell death. 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 460.