Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1

The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1 alpha protein that acts as a dominant-negative HIF-1 alpha (HIF-1 alpha-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1 alpha was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1 alpha-no-TAD or nontoxic concentrations (0.1 mu M; < IC10) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (< 0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC50 values in anoxia were 3-fold higher than those in air for HT1080 (2.2 +/- 0.3 versus 0.7 +/- 0.2 mu M) and HCT116 (9 +/- 4 versus 3 +/- 2 mu M) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC50 in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1 alpha-no-TAD( multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC50 values: HT1080, 0.7 +/- 0.04 mu M; HCT116, 3 +/- 1 mu M). HIF-1 alpha-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure.