title: Endogenously generated hydrogen peroxide is required for execution of melphalan-induced apoptosis as well as oxidation and externalization of phosphatidylserine.
abstract: Hydrogen peroxide (H(2)O(2)) is generated endogenously during execution of both intrinsic as well as extrinsic apoptotic programs suggesting that it may function as a secondary messenger in apoptotic pathways. In the present study, we investigated the role of endogenously generated H(2)O(2) by using two cell lines-HL-60 cells and its subclone, H(2)O(2) resistant HP100 cells overexpressing catalase (CAT). With the exception of CAT, we found no differences in the expression of other primary antioxidant enzymes (Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase) or apoptosis-related proteins (Bcl-2 and Bax) in HP100 cells as compared with the parental HL-60 cells. Production of H(2)O(2) was readily detectable as early as 1 h after melphalan (Mel) exposure of HL-60 cells but not HP-100 cells. Biomarkers of apoptosis, such as release of cytochrome c, disruption of mitochondrial transmembrane potential, caspase-3 activation, and chromatin condensation, became apparent much later, 3 h and onward after Mel treatment of HL-60 cells. The emergence of essentially all biomarkers of apoptosis was dramatically delayed in HP100 cells as compared with HL-60 cells. A relatively minor phospholipid species, phosphatidylserine (PS), was markedly oxidized 3 h after Mel treatment in HL-60 cells (but not in HP100 cells) where it was significantly inhibited by exogenously added CAT. The two most abundant classes of membrane phospholipids, phosphatidylcholine and phosphatidyletanolamine, did not undergo any significant oxidation. PS oxidation took place 3 h after exposure of HL-60 cells to Mel and paralleled the appearance of cytochrome c in the cytosol. Neither cytochrome c release nor PS oxidation occurred in Mel-treated HP100 cells, indicating that both endogenous H(2)O(2) and cytochrome c were essential for selective PS oxidation detected in HL-60 cells. Mel-induced PS oxidation was also associated with externalization of PS on the surface of HL-60 cells. Given that 3-amino-1,2,4-triazole, a CAT inhibitor, suppressed the resistance of HP100 cells to apoptosis, production of reactive oxygen species, PS oxidation, and PS externalization induced by Mel, the results from the present study suggest that H(2)O(2) is critical for triggering the Mel-induced apoptotic program as well as PS oxidation and externalization.
authors.affiliation: Division of Medical Biochemistry, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago 683-8503, Japan. kagan@pitt.edu
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Matsura
authors.forename: Masachika
authors.initials: M
authors.lastname: Kai
authors.forename: Jianfei
authors.initials: J
authors.lastname: Jiang
authors.forename: Hareesh
authors.initials: H
authors.lastname: Babu
authors.forename: Vidisha
authors.initials: V
authors.lastname: Kini
authors.forename: Chiaki
authors.initials: C
authors.lastname: Kusumoto
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamada
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002374:Catalase / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008558:Melphalan / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects*; D010718:Phosphatidylserines / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
chemical_list: D018906:Antineoplastic Agents, Alkylating; C494826:BAX protein, human; D010718:Phosphatidylserines; D019253:Proto-Oncogene Proteins c-bcl-2; D014230:Triazoles; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D002374:Catalase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D008558:Melphalan