Oncotarget 10650 www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ Oncotarget, Vol. 5, No. 21 Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue Florian Rouaud 1 , Miguel Romero-Perez 2 , Huan Wang 3 , Irina Lobysheva 2 , Booma Ramassamy 4 , Etienne Henry 3 , Patrick Tauc 3 , Damien Giacchero 5 , Jean-Luc Boucher 4 , Eric Deprez 3 , Stéphane Rocchi 1 and Anny Slama-Schwok 6 1 INSERM U1065 team 1, Université de Nice Sophia Antipolis et Centre Méditerranéen de Médecine Moléculaire, Nice, France 2 Pole of Pharmacology and Therapeutics, FATH5349, IREC, UCL Medical Sector, Brussels, Belgium 3 Laboratoire de Biologie et Pharmacologie Appliquée (LBPA), ENS-Cachan, CNRS UMR 8113, IDA FR3242, Cachan, France 4 CNRS UMR 8601, Université Paris Descartes, 45 rue des Saints Pères, Paris, France 5 Service de Dermatologie, Hôpital Archet II, CHU Nice, France; 6 Virologie et Immunologie Moléculaires, UR 892, INRA, Jouy en Josas, France Correspondence to: Anny Slama-Schwok, email: Anny.Schwok@gmail.com Correspondence to: Stéphane Rocchi, email: stephane.rocchi@unice.fr Keywords: Cellular signaling; Angiogenesis; NADPH analogue; cell proliferation; ROS; melanoma, endothelium Received: August 04, 2014 Accepted: September 24, 2014 Published: September 25, 2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 frst induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX 4 and ROS levels that could lead to a specifc proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX 4 – associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis. INTRODUCTION A critical pathogenic factor in the development of endothelial dysfunction is redox stress involving generation of reactive oxygen and nitrogen species, RNOS. Identifying sources of excessive or unbalanced RNOS levels and developing alternative strategies to regulate their respective levels should help in designing novel rational therapies. Previous studies identifed NADPH oxidases of the NOX family, the respiratory chain in mitochondria and uncoupled endothelial NO-synthase (eNOS) as major sources of reactive oxygen species (ROS) in endothelial dysfunction [1-3]. Superoxide ions and H 2 O 2 share with nitric oxide (NO) a dual role in cytotoxicity and signalling at high and low concentrations, respectively [4]. The signalling pathways regulated by NO formed by eNOS are linked with activation of soluble guanylate cyclase and vasodilation [5, 6]. NO is also involved in pathways regulating cell survival in response to lipid or oncogenes (KRas) (PI3K/ Akt/ eNOS