Enhancing the efficacy of chemotherapy under hypoxic conditions using HIF-1 targeted approaches.

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Hypoxic cells are resistant to many forms of chemotherapy agents. This is achieved through multiple mechanisms that are poorly defined. We have investigated the role of hypoxia-inducible factor-1 (HIF-1) in hypoxic cell chemo-resistance. The sensitivity of cells to etoposide was monitored in air and hypoxia. Exposure of HT1080 cells to etoposide for 16h under hypoxic conditions resulted in an Ic50 value 4 -fold higher than in air (2.3±0.3 vs 0.6±0.1 μM). HCT116 exhibited a 2-fold increase in hypoxic conditions (5.2±1.7 vs 8.7±3.1 μM). Hepa-1 wt and HIF-1β deficient Hepa-1 c4 cells had identical sensitivities to etoposide in air. Hypoxia provoked a 2-fold resistance in wt cells whereas the c4 cells were more sensitive under these conditions, implicating a HIF-1-dependency for resistance. We transduced HT1080 and HCT116 cells with adenoviruses expressing either β-galactosidase (Ad β-gal) as a control or a dominant negative form of the HIF-1α protein (Ad HIF-neg). We have previously shown that the latter construct can inhibit HIF-1 mediated transactivation both in vitro and in vivo. 48h after infection, cells were exposed to etoposide under aerobic or hypoxic conditions for 16h. Ad-β-gal had no significant effect on the aerobic and hypoxic Ic50 values compared with uninfected cells. However, the hypoxic Ic50 values for etoposide decreased with increasing viral dose when cells were infected with Ad HIF-neg. For the HT1080 cells this achieved statistical significance after infection with 20 viral particles per cell (MOI 20), with an MOI 50 producing a further reduction in hypoxic Ic50, achieving a value resembling that seen in air (0.7±0.04 μM; p<0.005 vs uninfected control or Ad β-gal infected cells). A similar profile was seen in the HCT116 cells, although the data did not reach statistical significance. We then investigated whether similar sensitisation could be achieved using small molecule inhibitors of HIF-1. Cells were exposed to a sub-toxic dose (0.1μM) of quinocarmycin monocitrate KW2152 or its analogue DX-52-1 (NSC607097) for 1h prior to and during the 16h of etoposide exposure in air or hypoxia. In HT1080 cells, aerobic Ic50s for etoposide were not significantly altered but the hypoxic values were 0.6±0.08 and 1.0±0.2 μM for KW2152 and DX-52-1 treated cells respectively. The etoposide response in HCT116 cells was not significantly altered by KW2152 or DX-52-1 co-treatment. We are currently extending these observations to other drug types and to investigate whether HIF-1 inhibition can modify drug response in vivo.