Abstract 4062: Nitric Oxide synthase dysregulation in solid tumors: The therapeutic potential of tetrahydrobiopterin

Abstract

Abstract Nitric Oxide (NO), in terms of cancer therapeutics, can be viewed as a double edge sword. Both anti-NO and NO-based anticancer treatments appear effective in several preclinical models. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2- instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO-), which is produced rapidly by the reaction of O2- with NO, and is a highly reactive species that can modify proteins, lipids and DNA. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7, Fadu, and A431 cells in vitro and Fadu and A431 cells in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is less than 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. Increasing the BH4:BH2 ratio leads to enhanced NO production as evidenced by an increase in cGMP formation. To directly examine the effects of manipulating the BH4:BH2 ratio, MCF7 and MDA cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. Current experiments focus on SP's effects in vivo and elucidating the mechanism of cell death and the signaling pathways involved. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2011-4062