Abstract
Purpose Solid tumors are highly heterogeneous in oxygenation and contain hypoxic regions resulting from imbalance between cell proliferation and angiogenesis. These hypoxic regions result in the higher possibility to survive after treatments than normoxic regions due to resistance to radiation and anticancer drug treatments. Under these circumstances, we found that tempol, one of nitoxides, strongly induced the expression of hypoxia inducible-factor (HIF)-1alpha with a combination of hypoxic conditions. This induction mechanism seems to enable us to enhance the hypoxic cell killing applying the vector bearing the suicide gene downstream of HIF-1 binding site, hypoxia responsive element (HRE). In order to realize this idea, we constructed the plasmids that regulate the expression of suicide gene with tempol and then evaluated the enhancement of the cell killing effect at in vitro level.
Materials and Methods The properties of enhancement of HIF-1alpha expression by the addition of tempol were determined by luciferase (Luc) assay using the plasmid inserted four copies of a HRE in promoter sequence and oxygen-dependent degradation domain upstream and downstream of the Luc gene, respectively. We then performed a colony assay to evaluate the enhancement of cell killing effect controlled by tempol after transfection of the plasmid replaced Luc gene with FcyFur fusion gene whose product converts the prodrug, 5-FC to the anticancer drug, 5-FU into MCF7 cells.
Results and Conclusion The Luc activity of cells with tempol addition at 1%O2 increased up to about 217-fold induction, compared to that without tempol addition at 20%O2 (about 10-fold induction compared to that without tempol addition at 1%O2). In addition, the transfected cells with the plasmid encoding FcyFur fusion gene indicated the highest cell killing effect after treatment of 5-FC, tempol and hypoxic mimic CoCl2. These results indicate the expression of the suicide gene can be enhanced specifically in hypoxic regions by the addition of tempol. Therefore, this strategy might become one of the candidates to eradicate hypoxic cancer cell.
