Involvement of reactive oxygen species in arsenite-induced downregulation of phospholipid hydroperoxide glutathione peroxidase in human epidermoid carcinoma A431 cells.
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is unique in the substrate specificity among the glutathione peroxidase family because it can interact with lipophilic substrates, including the peroxidized phospholipids and cholesterol, and reduce these hydroperoxide to hydroxide compounds. However, what kinds of ligand can regulate the PHGPx expression is still unknown. In the present study, we found that sodium arsenite induced downregulation of mRNA, protein expression, and enzyme activity of PHGPx in time- and dose-dependent manners. At the same time, it upregulated mRNA and protein expression of p21(WAF1/CIP1). With the aid of agarose gel electrophoresis, and propidium iodide and annexin-V staining, we found that treatment of 30 microM sodium arsenite for 24 h induced apoptosis in human epidermoid carcinoma A431 cells and EA.hy926 cells. An increase of intracellular peroxide levels was measured by flow cytometry using 2',7'-dichlorofluorescin diacetate (DCFH-DA) after treatment of arsenite. Overexpression of PHGPx prevented arsenite-induced increase of intracellular peroxide levels, downregulation of PHGPx, upregulation of p21(WAF1/CIP1), and apoptosis in A431 cells. N-Acetyl-L-cysteine also significantly prevented arsenite-induced effects in A431 cells. Therefore, we concluded that reactive oxygen species were involved in arsenite-induced downregulation of PHGPx, upregulation of p21(WAF1/CIP1), and apoptosis in A431 cells.
Huang
Huei-Sheng
HS
Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. huanghs@mail.ncku.edu.tw
Chang
Wen Chang
WC
Chen
Ching Jiunn
CJ
D017209:Apoptosis; D018053:Arsenites / Q000494:pharmacology*; D015153:Blotting, Western; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D005609:Free Radicals; D017930:Genes, Reporter; D005822:Genetic Vectors; D005979:Glutathione Peroxidase / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D010743:Phospholipids / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D018053:Arsenites; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D005609:Free Radicals; D010743:Phospholipids; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012313:RNA; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase; D008156:Luciferases; C015001:arsenite