Targeting Nitric Oxide Signaling with nNOS Inhibitors As a Novel Strategy for the Therapy and Prevention of Human Melanoma

Abstract

Aims: Our previous studies have shown that nitric oxide (NO) plays an important role in increasing the invasion and proliferation of human melanoma cells, suggesting that targeting NO signaling may facilitate therapy and prevention. Neuronal nitric oxide synthase (nNOS) is present in melanocytes, a cell type that originates from the neural crest. The aims of this study were to determine the role of nNOS in melanoma progression and the potential antitumor effects of novel synthesized nNOS inhibitors. Results: In vitro studies demonstrated abundant expression of nNOS in melanoma compared to melanocytes, which was inducible by ultraviolet radiation and was associated with increased NO generation. nNOS was also detected in melanoma biopsies that increased with disease stage. Knockdown of nNOS in melanoma cells diminished L-arginine-induced NO production; the metastatic capacity was also reduced as well as the levels of MMP-1, Bcl-2, JunD, and APE/Ref-1. Similar inhibition of NO and invasion potential was observed utilizing novel, highly selective nNOS inhibitors. In three-dimensional human skin reconstructs, the nNOS inhibitor cpd8 effectively reversed the melanoma overgrowth stimulated by NO stress. Innovation: Our work lays the foundation for development of clinical ''drug-like'' nNOS inhibitors as a new and promising strategy for the chemoprevention of early melanoma progression and the inhibition of secondary melanoma in high-risk individuals. Conclusion: Based on our observations, we propose that nNOS in melanoma results in constitutive overproduction of NO, which stimulates proliferation and increases invasion potential, leading to subsequent development of metastases.

Updates of reactive oxygen species in melanoma etiology and progression

“…iNOS has been well characterized in melanoma: its expression promotes cell proliferation; is associated with poor patient survival; and increases resistance to cisplatin [104–109]. Although an early study demonstrated nNOS expression in melanocytes and melanoma cells [110], these investigations were not followed up until we recently discovered that nNOS played a role in melanoma invasion and proliferation [111, 112]. The eNOS uncoupling is also an important source of superoxide in melanoma which drives the malignant transformation [113].…”

Developing an Irreversible Inhibitor of Human DDAH‐1, an Enzyme Upregulated in Melanoma

“…Enhanced production of nitric oxide has previously been shown to play a promoting role in melanomas. [15] So, we chose to screen melanoma cell lines for DDAH-1 levels. Using immunohistochemistry as a semi-quantitative gauge of DDAH-1, DDAH-2 and iNOS concentrations, we assayed a panel consisting of two control melanocyte lines and 64 melanoma cell lines that encompass different stages of malignancy, metastases and mutational status.…”

Melanocytes and Oxidative Stress

“…Melanoma risk was also found to be increased by MC-1R variant alleles [31,70] suggesting that ROS would be a driver of melanomagenesis [71]. Higher sensitivity of melanocytes from melanoma lesions to oxidizing agents was long demonstrated [71] as well as increased levels of markers of oxidative stress such as O 2 - [72] or NOS [73].…”