Hypoxia: Induction of stress-caused resistance to the topoisomerase II inhibitor etoposide in human cancer cells

Conditions within solid tumor microenvironments can activate stress response pathways leading to the development of resistance to anti-cancer drugs. To elucidate pathways by which stress-induced drug resistance occurs, human cell lines under normoxic and hypoxic conditions were evaluated. Exposure to hypoxia or Brefeldin A (BFA) led to the development of resistance to the topoisomerase II inhibitor etoposide in MDA-MB-231 and MCF-7 human breast cancer cells, PC-3 human prostate cancer cells and H1299 (p53-/-) human lung cancer cells. Experiments using an NF-kappaB-regulated luciferase reporter construct demonstrated that hypoxia or BFA induce NF-kappaB activation in MCF-7, PC-3 and H1299 (p53-/-) cells. Further, inhibition of constitutive NF-kappaB sensitized all cell lines to etoposide. Experiments using H1299 cells transfected with wild-type p53 in a tetracycline inducible system showed wild-type p53 expressing cells to be similarly resistant to etoposide as their p53-/- counterparts, showing that the stress-induced resistance pathway is independent of p53.; The effects of repetitive hypoxic insults on the development of resistance to etoposide in human cancer cells were investigated by creating 5 subpopulations using H1299 cells, which were: non-treated (C); treated with 25muM etoposide 25muM E) in oxygen; hypoxia (H); hypoxia and 25muM etoposide (H+25muM E); and hypoxia and 100muM etoposide (H+100muM E). H+25pM E represented the equidose and H+100muM E the equitoxic dose subpopulation to the 25muM E treated population. Hypoxia pretreatment resulted in an exaggerated response of H1299 cells to subsequent stress. This event has been successfully reproduced with another set of H1299 subpopulations. Furthermore, hypoxic stress also suppressed the acquired resistance phenotype seen in the 25muM E cells. NF-kappaB activation upon hypoxia treatment in the C and hypoxia-pretreated subpopulations was similar, suggesting that the exaggerated resistance phenotype induced by hypoxia pretreatment is not mediated by NF-kappaB. Constitutive expression of Hypoxia inducible factor-1alpha (HIF-1alpha) appeared to be higher in hypoxia-pretreated subpopulations than in C cells, but hypoxia-induced activation was similar in all subpopulations. No difference in the levels of production of HIF-1alpha-regulated Vascular Endothelial Growth Factor (VEGF) was observed. Finally, hypoxia-pretreated H1299 cells appeared to lose their exaggerated resistance phenotype after 8 passages under normal cell culture conditions.