TY - JOUR
T1 - NADPH oxidase-produced superoxide mediates EGFR transactivation by c-Src in arsenic trioxide-stimulated human keratinocytes
AU - Tseng, Hong Yu
AU - Liu, Zi Miao
AU - Huang, Huei Sheng
N1 - Funding Information:
ATO, nicotinamide adenine dinucleotide phosphate (NADPH), apocynin, xathine/xathine oxidase, dichlorofluorescin diacetate (DCFH-DA), and tiron were purchased from Sigma-Aldrich Co. (St. Louis, MI, USA). EGFR inhibitor PD153035 and Src inhibitor PP1 were purchased from Calbiochem (San Diego, CA, USA). Wizard Plus MiniPreps DNA purification system and luciferase assay system were from Promega (Madison, WI, USA). Qiagen-tip 100 was from Qiagen (Hilden, Germany). GENE-CLEAN III kit was from BIO 101 (La Jolla, CA, USA). SuperScriptTMIII and TRIzol RNA extraction kit reagent were from Invitrogen (Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, ribonuclease inhibitor, and Opti-MEM medium were from Gibco BRL (Grand Island, NY). Antibodies against phospho-Tyr phospho-EGFR-Y1173, phospho-ERK1/2, ERK2, c-Src and p21 were purchased from Santa Cruz Biotechnology. Antibody against phospho-EGFR-Y845 was purchased from Abcam (Cambridge, MA). Antibodies against phospho-c-Src-Y416 and p67Phox were purchased from Cell Signaling Technology (Beverly, MA, USA). The reporter of p21 promoter, p21-luc plasmid, was kindly provided by Dr. Bert Vogelstein of Johns Hopkins University Medical Institutions (Baltimore, MD, USA). The wild-type Src plasmid (wtSrc) was kindly provided by Dr. Y.R. Chen (National Health Research Institutes, Taiwan). The pLKO.1-p67phox-shRNA, which targets the human p67phox gene sequence 5′-GCTATCAAAGACCTTAAAGAA-3′, and a luciferase control (pLKO.1-shLuc) plasmid construct were kindly obtained from National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica (Taipei, Taiwan), supported by the National Research Program for Genomic Medicine Grants of the National Science Council (NSC97-3112-B-001-016). The cDNA encoding mutated Src kinase in the pUSE vector, pSrc (K297R), and antibody against cleaved PARP [poly(ADPribose) polymerase] were obtained from Upstate Biotechnology, Inc.
Funding Information:
Acknowledgments This work has been granted by the National Science Council of Taiwan (grant numbers: NSC95-2320-B-006-055-MY3 and NSC98-2320-B-006-008-MY3).
PY - 2012/6
Y1 - 2012/6
N2 - 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 p21WAF1/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 p67phox expression and translocation to membrane. In addition, knockdown of p67phox could abolish ATOinduced ROS production. Therefore, we suggest that NADPH oxidase-produced superoxide was a major source of ATO-induced ROS production. Conversely, ATOinduced 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 p67phox. Collectively, we suggest that NADPH oxidase was involved in the ATO-induced arrest/apoptosis of keratinocytes, which was regulated by c-Src activation.
AB - 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 p21WAF1/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 p67phox expression and translocation to membrane. In addition, knockdown of p67phox could abolish ATOinduced ROS production. Therefore, we suggest that NADPH oxidase-produced superoxide was a major source of ATO-induced ROS production. Conversely, ATOinduced 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 p67phox. Collectively, we suggest that NADPH oxidase was involved in the ATO-induced arrest/apoptosis of keratinocytes, which was regulated by c-Src activation.
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U2 - 10.1007/s00204-012-0856-9
DO - 10.1007/s00204-012-0856-9
M3 - Article
C2 - 22532027
AN - SCOPUS:84866044675
SN - 0340-5761
VL - 86
SP - 935
EP - 945
JO - Archives of Toxicology
JF - Archives of Toxicology
IS - 6
ER -
