Nitric Oxide Synthase Activity and its Modulation in the Treatment of Colorectal Cancer

The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under non-inflammatory conditions NOS generates Nitric Oxide. However, in an inflammatory environment, such as the oxidative tumor microenvironment, NOS’s cofactor tetrahydrobiopterin (BH4) is oxidized to dihydrobiopterin (BH2). NOS bound to BH2 is said to be uncoupled and produces superoxide O2-and peroxynitrite (ONOO-). Previous work in our and other’s labs have shown that increased production of ROS/RNS leads to the activation of pro-inflammatory/proliferative molecules such as NFκB, Stat3, β-Catenin and Akt. NOS can be re-coupled by supplementing cells and animals with BH4 or its precursor Sepiapterin (SP). Herein we show that recoupling NOS with SP in HCT116, Caco-2 and HT29 cells, decreased tumor cell proliferation, increased β-Catenin degradation and decreased Akt activity. We also see increased tumor cell death measured by in vitro clonogenic assay, as well as decreased metabolic uptake in Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) induced colorectal cancer in vivo measured by [18F]-fluorodeoxyglucose ([18F]-FDG) positron emitted topography (PET) imaging. We believe by recoupling NOS both in vivo and in vitro we are modulating Wnt signaling via Akt and GSK-3β. Lastly, we conducted studies to determine a mechanistic explanation of how tumor cells maintain a decreased BH4:BH2 ratio