Cytochrome c release is required for phosphatidylserine peroxidation during Fas-triggered apoptosis in lung epithelial A549 cells.

Oxidation of phosphatidylserine (PtdSer) has been shown to play a pivotal role in signaling during cell apoptosis and subsequent recognition of apoptotic cells by phagocytes. However, the redox catalytic mechanisms involved in selective PtdSer oxidation during apoptosis remain poorly understood. Here we employed anti-Fas antibody CH-11-treated A549 cells as a physiologically relevant model to investigate the involvement of PtdSer oxidation and its potential mechanism during apoptosis. We demonstrated that ligation of CH-11 with its cognate receptor initiated execution of apoptotic program in interferon gamma-pretreated A549 cells as evidenced by activation of caspase and DNA fragmentation. A significant increase of cytochrome c (cyt c) content in the cytosol as early as 2 h after CH-11 exposure was detected indicating that Fas-induced apoptosis in A549 cells proceeds via extrinsic type II pathway and includes mitochondrial signaling. PtdSer was selectively oxidized 3 h after anti-Fas triggering while two more abundant phospholipids--phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn)--and the major intracellular antioxidant, glutathione, remained nonoxidized. A pan-caspase inhibitor, z-VAD, fully blocked cyt c release and oxidation of PtdSer in Fas-treated A549 cells. On the other hand, z-DQMD, a caspase-3 inhibitor, completely inhibited caspase-3 activity but did not fully block caspase-8 activation and release of cyt c. Importantly, z-DQMD failed to protect PtdSer from oxidation. In addition, in a model system, we demonstrated that peroxidase activity of cyt c was greatly enhanced in the presence of dioleoylphosphatidylserine containing liposomes by monitoring oxidation of 2',7'-dichlorodihydrofluorescein to 2',7'-dichlorofluorescein. We further showed that peroxidase activity of cyt c catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine using a newly developed HPLC assay. MS analysis of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine revealed that in addition to its mono- and dihydroperoxides, several different PtdSer oxidation products can be formed. Overall, we concluded that cyt c acts as a catalyst of PtdSer oxidation during Fas-triggered A549 cell apoptosis.