Lipid Antioxidant, Etoposide, Inhibits Phosphatidylserine Externalization and Macrophage Clearance of Apoptotic Cells by Preventing Phosphatidylserine Oxidation

Abstract

A common feature of the apoptotic program is phospholipid signaling aimed at the generation of "eat me" signals on the surface of the apoptotic cell that make it recognizable by phagocytes (1). This apoptotic signaling is mediated through the loss of plasma membrane phospholipid asymmetry and the concomitant externalization of phosphatidylserine (PS) 1 (2). PS-dependent signaling is coupled to the final common pathway of apoptosis, i.e. the caspase-driven dismantling of the cell, thus allowing for effective phagocytosis and clearance of cell corpses. The importance of phagocytosis of apoptotic cells for prevention of spillage of cellular contents and resultant tissue disruption and inflammation has been emphasized in numerous studies in recent years (3, 4); however, the specific mechanisms that govern PS externalization and recognition during apoptosis remain to be elucidated. We have recently shown that PS externalization during apoptosis is preceded by its selective oxidation, likely catalyzed by cytochrome c released from mitochondria (5, 6). We have also demonstrated that oxidized PS (PS-OX) may serve as an "eat me" signal for macrophage receptor(s), thus facilitating recognition and PS-dependent engulfment of apoptotic cells. (7,8) Based on these observations, we hypothesized that PS oxidation acts as an essential component of the signaling pathway that is required for PS externalization and the safe clearance of apoptotic cells by macrophages (5, 8, 9, 10). Our hypothesis predicts that lipid antioxidants capable of blocking PS oxidation will inhibit PS externalization and/or recognition of apoptotic cells by phagocytes. An important feature of such a lipid antioxidant, however, is that it should block phospholipid oxidation without affecting other redox-sensitive mechanisms.Our previous work has established that a phenolic antitumor drug, etoposide (a topoisomerase II inhibitor), acts as a powerful lipid antioxidant but does not protect thiols against oxidation because of a relatively high reactivity of etoposide phenoxyl radicals toward SH-groups (11). Etoposide has been reported to cause DNA damage and induce apoptosis accompanied by ROS generation (12, 13). It is conceivable that the fastidious lipid antioxidant traits of etoposide may dissociate PS-dependent signaling pathways of apoptosis from the final common pathway for apoptosis by inhibiting PS oxidation. However, etoposide effects on phospholipid peroxidation, as * This work was supported by National Institutes of Health Grants HL70755, CA90787, and GM64610 and the Swedish Society for Medical Research (to B. F.). J. C. Y. has a significant equity interest, as defined by the U. S. Public Health Service, in Bristol-Myers Squibb Co., a manufacturer of etoposide, used in this research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ ‡ To whom correspondence should be addre...

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

“…Oxidative conditions can contribute to lipid peroxidation products, HNE and acrolein, within the bilayer of apoptotic cells (Butterfield et al, 2006a;Lovell et al, 2001;Markesbery and Lovell, 1998) that could interfere with PtdSer asymmetry by initiating further lipid peroxidation via free radical mechanisms (Prasad et al, 1998). By diffusing from their formation sites, these neurotoxic alkenals could react via Michael addition with flippase by covalently binding a critical cysteine residue of this transporter (Daleke and Lyles, 2000;Daleke, 2003;Paulusma and Oude Elferink, 2005;Tyurina et al, 2004a), suggesting a definite relation between PtdSer asymmetry and lipid peroxidation.…”

“…These reactive alkenals have been found to interfere with PtdSer asymmetry via redox dependent flippase (Castegna et al, 2004;Daleke, 2003;Tyurina et al, 2004b). Furthermore, much evidence indicates that oxidative modification of flippase by reactive alkenals and/or apoptotic proteins, resulting in asymmetric collapse (Castegna et al, 2004;Kagan et al, 2002;Mandal et al, 2005;Mohmmad Abdul and Butterfield, 2005;Tyurina et al, 2004a), is greatly elevated in the early apoptotic phenotype of AD models (Herrmann and Devaux, 1990;Kagan et al, 2000).…”

The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

“…8 Furthermore, the flippase ATP11C was also identified as a key regulator of PS exposure on apoptotic cells, in which caspase-mediated inactivation of ATP11C promoted the distribution of PS on the outer leaflet of the plasma membrane during apoptosis 9 ( Figure 2a). It is also worth noting that PS can be modified by reactive oxygen species (ROS; a form of 'phospholipid modifier') to generate oxidized PS in certain cell types, a process that could aid PS externalization 10,11 and recognition by phagocytes. 12 The exposure of PS on the surface of apoptotic cells can function as a key 'eat-me' signal to facilitate apoptotic cell recognition by phagocytes via a variety of 'phospholipid detectors' (Table 1).…”

Nitrosative Stress Inhibits the Aminophospholipid Translocase Resulting in Phosphatidylserine Externalization and Macrophage Engulfment

“…Measurement of Thiol Contents-Low molecular weight thiols and protein thiol contents in the cells were determined fluorometrically using ThioGlo TM -1 as described previously (18). Briefly, cells treated with either SNCEE (50 -300 M) or GSNO (50 -300 M) for 30 min at 37°C were collected by centrifugation, washed, and resuspended in PBS.…”

“…During apoptosis, APLT is inactivated causing egress of PS from the inner to the outer leaflet of the plasma membrane (17)(18)(19). The mechanisms of apoptotic APLT inactivation remain to be elucidated.…”