title: NADPH oxidase-produced superoxide mediates EGFR transactivation by c-Src in arsenic trioxide-stimulated human keratinocytes.
abstract: Arsenic is a well-known poison and carcinogen in humans. However, it also has been used to effectively treat some human cancers and non-carcinogenic ailments. Previously, we demonstrated in keratinocytes that arsenic trioxide (ATO)-induced p21(WAF1/CIP1) (p21) expression leading to cellular cytotoxicity through the c-Src/EGFR/ERK pathway and generation of reactive oxygen species (ROS). In this study, we found that EGFR-Y845 and EGFR-Y1173 could be phosphorylated by ATO. Using confocal microscopy and flow cytometry, we found that pretreatment with apocynin, DPI, and tiron could remove ATO-induced ROS production. Furthermore, to increase NADPH oxidase activity, ATO could induce cytosolic p67(phox) expression and translocation to membrane. In addition, knockdown of p67(phox) could abolish ATO-induced ROS production. Therefore, we suggest that NADPH oxidase-produced superoxide was a major source of ATO-induced ROS production. Conversely, ATO-induced NADPH oxidase activation and superoxide generation could be inhibited by the c-Src inhibitor PP1, but not by the EGFR inhibitor PD153035. In addition, overexpression of c-Src as well as treatment with ATO could stimulate EGFR-Y845/ERK phosphorylation, p21 expression, and cellular arrest/apoptosis, which could be attenuated by pretreatment with apocynin or knockdown of p67(phox). Collectively, we suggest that NADPH oxidase was involved in the ATO-induced arrest/apoptosis of keratinocytes, which was regulated by c-Src activation.
authors.affiliation: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Tseng
authors.forename: Zi-Miao
authors.initials: ZM
authors.lastname: Liu
authors.forename: Huei-Sheng
authors.initials: HS
authors.lastname: Huang
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D010087:Oxides / Q000633:toxicity*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D001152:Arsenicals; D004791:Enzyme Inhibitors; D010087:Oxides; D017382:Reactive Oxygen Species; D013481:Superoxides; D004815:Epidermal Growth Factor; D019255:NADPH Oxidases; D019061:src-Family Kinases; D000077237:Arsenic Trioxide