Abstract 4774: Hypoxic bio-reduction of novel NBQ48 in treated tumor cells and the activity of cytochrome P450 reductase

Abstract

The main goal of this research was to evaluate the reduction activity of a novel Nitrobenzazolo[3,2-a]quinolinium (NBQ48) compound under hypoxic environment thru the formation of a fluorescent metabolite and further to evaluate the activity of the cytochrome P450 (CYP450) reducatase in the reduction of these compounds. To evaluate the reduction of NBQ48 and the formation of its fluorescent metabolite two (2) tumor cell lines in culture Toledo (lymphoma) and A431 (endothelial) where treated under nitrogen or argon saturated environments to create a hypoxic (low oxygen) environment. In a 15 ml conical tube 4×106 cells in RPMI 1640 media were treated with NBQ 48 (3mM) for a final volume of 5mL and incubated with argon or nitrogen saturated environment at 37ºC, 5%. Cells were exposed at different time periods (0 to 48 hours) to evaluate the effect of exposure time in the reduction of the NBQ48 by detecting the reduced fluorescent product, the amino-substituted ABQ48. In addition NBQ48 treated tumor cells were located inside a hypoxic chamber to evaluate the formation of the fluorescent metabolites indicative of a metabolically active cell. To determine if the CYP450 reductase enzyme is activated in the reduction of the NBQ towards the formation of the ABQ metabolite, an experiment containing the NBQs and the CYP reductases was designed. Four (4) samples were prepared: blank media hypoxic, NBQ experimental hypoxic, positive ABQ spiked and negative aerobic control NBQ. Samples with NBQ48 (0.4mM) were incubated for 24 hours. After the incubation period, fluorescence emissions indicating the reductive fluorescent product were measured using a Modulus fluorometer (blue filter). The results on tumor cells treated with NBQ48 under hypoxic conditions demonstrated reduction and the formation of a fluorescent product, amino-substituted ABQ48. The time dependent increase in fluorescence in comparison with non treated cells indicated that the reduction of the NBQ increased with time. Results on the determination of the activity of CYP450 in the reduction of the NBQ48 indicated that only samples containing the CYP450 showed the formation of a fluorescent species presumably, ABQ48. This study provides evidence of the reduction of NBQ48 and the formation of a fluorescent metabolite thru the activations of the CYP450 reductases. The implemented experimental design could also be used to determine activity of hypoxic tissues with clinical applications. Citation Format: Beatriz Zayas, Vivian Lebron, Juan P. Rivera, Christian Velez, Osvaldo Cox. Hypoxic bio-reduction of novel NBQ48 in treated tumor cells and the activity of cytochrome P450 reductase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4774.