title: Proopiomelanocortin gene delivery induces apoptosis in melanoma through NADPH oxidase 4-mediated ROS generation.
abstract: 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.
authors.affiliation: Centre for Eye Research Australia, Melbourne, VIC 3002, Australia; Department of Ophthalmology, University of Melbourne, Melbourne, VIC 3002, Australia.
authors.forename: Guei-Sheung
authors.initials: GS
authors.lastname: Liu
authors.affiliation: Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan.
authors.forename: Jian-Ching
authors.initials: JC
authors.lastname: Wu
authors.affiliation: Institute of Biomedical Science, and National Sun Yat-sen University, Kaohsiung 804, Taiwan.
authors.forename: Han-En
authors.initials: HE
authors.lastname: Tsai
authors.affiliation: Centre for Eye Research Australia, Melbourne, VIC 3002, Australia; Department of Ophthalmology, University of Melbourne, Melbourne, VIC 3002, Australia.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Dusting
authors.affiliation: Centre for Eye Research Australia, Melbourne, VIC 3002, Australia; Department of Ophthalmology, University of Melbourne, Melbourne, VIC 3002, Australia.
authors.forename: Elsa C
authors.initials: EC
authors.lastname: Chan
authors.affiliation: Department of Dermatology, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan.
authors.forename: Chieh-Shan
authors.initials: CS
authors.lastname: Wu
authors.affiliation: Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan; Institute of Biomedical Science, and National Sun Yat-sen University, Kaohsiung 804, Taiwan; Center for Neuroscience, National Sun Yat-sen University, Kaohsiung 804, Taiwan. Electronic address: minghongtai@gmail.com.
authors.forename: Ming-Hong
authors.initials: MH
authors.lastname: Tai
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D051379:Mice; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D011333:Pro-Opiomelanocortin / Q000235:genetics* / Q000627:therapeutic use; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D017382:Reactive Oxygen Species; D011333:Pro-Opiomelanocortin; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D053148:Caspase 3
keywords: Apoptosis; Free radicals; Melanoma; NADPH oxidase; POMC; ROS