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Nitric oxide (NO) exerts pro-apoptotic and anti-apoptotic properties according to its local concentration, cellular target,as well as the presence of other radical species.The aim of the study was the in vitro and in vivo evaluation of the antitumoral properties of NO in liver cancer cells.The intracellular NO concentration was increased with NO donors (NONOate) or by stable nitric oxide synthase type 3 (NOS-3) overexpression in HepG2 cells using pcDNA4/TO expression vector containing NOS-3 eDNA sequence under the control of the cytomegalovirus (CMV) promoter.The tumor progression of NOS-3 overexpressing hepatoma cells and HepG2 cells after NO donor administration were also assessed in a mouse xenograft model.The activity of NOS-3, caspase-3,-8 and-9 were assessed by different approaches.The expression of p53, CD95, TNF-R1, TRAIL-R1, cFLIPs, cFLIPL and nitrated proteins was assessed by Western-blot analysis.CD95, TNF-R1 and TRAIL-R1 immunoprecipitation allowed the evaluation of cFLIP expression bound or unbound to cell death receptors.The mitochondrial complex Ⅰ/Ⅲ activities and oxygen consumption were determined by enzymatic assays and XF24 analyzer (Seahorse Biosciences Inc), respectively.NOS-3 overexpression increased p53, CD95, TNF-R1 and TRAIL-R1 expression, oxidative stress and reduced cell proliferation in HepG2 cells.These effects of NOS-3 were related to increased protein nitration, caspase-3 and-8 activities, and lactate dehydrogenase (LDH) release in cells.The subcutaneously administration of NOS-3 overexpressing HepG2 cell developed less tumors than control cells in a xenograft mouse model.The intratumoral administration of NO donor induces the expression of CD95, TNF-R1, TRAIL-R1 expression, and drastically reduced tumor growth in a xenograft mouse model.In conclusion, the overexpression of NOS-3 increased respiration and oxidative/nitrosative stress which was associated with reduced cell proliferation and increased cell death in HepG2.The overexpression of NOS-3 exerts antitumoral properties in a mouse xenograft model.