Abstract 2057: Tumor suppressor effects of tetrahydrobiopterin are mediated through beta-catenin in breast cancer cells

Nitric Oxide (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 O 2 − instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO − ), which is produced rapidly by the reaction of O 2 − with NO. 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 and MDA-231 cells in tissue culture and in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is around 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. To ensure that increasing the BH4:BH2 ratio leads to enhanced NOS coupling, the production of NO was measured by cGMP formation and O 2 − levels were assessed by HPLC. As hypothesized NO levels went up and O 2 − levels went down. To directly examine the effects of manipulating the BH4:BH2 ratio 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. To assess the effects of SP on xenografts, we used an ex-vivo clonogenic assay. This assay also showed that colony formation is significantly reduced upon treatment of the animals with SP. Previous studies in colon cancer cells have shown that when PKG, the major pathway activated by NO, is activated β-catenin levels are reduced. In our experiments MCF7 xenografts showed a significant decrease in β-catenin levels after treatment with SP. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2057. doi:1538-7445.AM2012-2057