Tumor cells, especially pancreatic tumors, adapt to grow in a hypoxic environment. Hypoxia-inducing factor-1 (HIF-1) helps these tumor cells adapt to hypoxia by up-regulating target genes responsible for angiogenesis, nutrient uptake, and anti-apoptotic processes. Thus, blocking HIF-1 has emerged as an attractive anti-cancer strategy. Imexon is a thiol-binding small molecule which causes the accumulation of reactive oxygen species (ROS), leading to mitochondrial disruption and apoptosis. Gene expression studies in tumor cells and in patients revealed up-regulation of several anti-oxidant genes including many containing the anti-oxidant response element. We evaluated the effects of Imexon, a novel cyanoaziridine, on HIF-1α levels in two human pancreatic tumor cell lines in vitro. Levels of HIF-1α were measured by Western blot analyses. Levels of ROS were determined by hydroethidium and MitoSOX Red fluorescence. Imexon induced a concentration-dependant increase in ROS levels and reduced the expression of HIF-1α levels in both Panc-1 and MiaPaca-2 cells. Effects were noted after 24 hours of exposure to > 300 uM, a concentration that has been achieved in patients treated in a Phase I clinical trial. Since it is known that imexon causes the accumulation of superoxide anions (O2.-), we used two cell permeable superoxide dismutase (SOD) scavengers, TempolR, and PEG-SOD, to decrease cellular O2.- levels. We found that both TempolR and PEG-SOD raised HIF-1α levels when used in combination with imexon, suggesting that HIF-1α levels may be regulated by cellular O2.-. The addition of menadione sodium bisulfite, a mitochondrial O2.- generator, attenuated hypoxia induced HIF-1α expression, further suggesting that HIF-1α levels in pancreatic tumor cells are regulated by O2.-. Since HIF-1α is known to be degraded by the ubiquitin-proteasome pathway, we evaluated whether the imexon induced loss of HIF-1α protein could be due to increased proteasomal destruction of HIF-1α protein. We confirmed that the loss of HIF-1α due to imexon could be reversed by the addition of MG132, a proteasome inhibitor. This suggests that the reduced HIF-1α levels could be mediated at least in part by the ubiquitin-proteasome system. There was no effect of imexon on transcription of HIF-1α mRNA, but an effect on the translation of HIF-1α mRNA has not been studied. In summary, we’ve shown that HIF-1α levels in pancreatic cancer cells are reduced by imexon, possibly by O2.- mediated degradation via the ubiquitin-proteasome pathway. Imexon is currently undergoing clinical evaluation in patients with pancreatic cancer (combined with gemcitabine), malignant melanoma (combined with dacarbazine), and multiple myeloma.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]