Hypoxia in the tumor microenvironment triggers differential signaling pathways for
tumor survival. In this study, we characterize the involvement of
hypoxia and
reactive oxygen species (ROS) generation in the
antineoplastic mechanism of
proopiomelanocortin (
POMC) gene delivery in a mouse B16-F10
melanoma model in vivo and in vitro. Histological analysis revealed increased TUNEL-positive cells and enhanced hypoxic activities in
melanoma treated with adenovirus encoding
POMC (Ad-
POMC) but not control vector. Because the apoptotic cells were detected mainly in regions distant from blood vessels, it was hypothesized that
POMC therapy might render
melanoma cells vulnerable to hypoxic insult. Using a hypoxic chamber or
cobalt chloride (CoCl2), we showed that
POMC gene delivery elicited apoptosis and
caspase-3 activation in cultured B16-F10 cells only under hypoxic conditions. The apoptosis induced by
POMC gene delivery was associated with elevated ROS generation in vitro and in vivo. Blocking ROS generation using the
antioxidant N-acetyl-l-cysteine abolished the apoptosis and
caspase-3 activities induced by
POMC gene delivery and
hypoxia. We further showed that
POMC-derived
melanocortins, including α-
MSH, β-
MSH, and
ACTH, but not γ-
MSH, contributed to
POMC-induced apoptosis and ROS generation during
hypoxia. To elucidate the source of ROS generation, application of the
NADPH oxidase inhibitor
diphenyleneiodonium attenuated α-
MSH-induced apoptosis and ROS generation, implicating the proapoptotic role of
NADPH oxidase in
POMC action. Of the
NADPH oxidase isoforms, only Nox4 was expressed in B16-F10 cells, and Nox4 was also elevated in Ad-
POMC-treated
melanoma tissues. Silencing Nox4 gene expression with Nox4
siRNA suppressed the stimulatory effect of α-
MSH-induced ROS generation and cell apoptosis during
hypoxia. In summary, we demonstrate that
POMC gene delivery suppressed
melanoma growth by inducing apoptosis, which was at least partly dependent on Nox4 upregulation.