In cancer cells the small compounds erastin and RSL3 promote a novel type of cell death called ferroptosis, which requires iron‐dependent accumulation of lipid reactive oxygen species. Here we assessed the contribution of lipid peroxidation activity of lipoxygenases (LOX) to ferroptosis in oncogenic Ras‐expressing cancer cells. Several 12/15‐LOX inhibitors prevented cell death induced by erastin and RSL3. Furthermore, siRNA‐mediated silencing of ALOX15 significantly decreased both erastin‐induced and RSL3‐induced ferroptotic cell death, whereas exogenous overexpression of ALOX15 enhanced the effect of these compounds. Immunofluorescence analyses revealed that the ALOX15 protein consistently localizes to cell membrane during the course of ferroptosis. Importantly, treatments of cells with ALOX15‐activating compounds accelerated cell death at low, but not high doses of erastin and RSL3. These observations suggest that tumor ferroptosis is promoted by LOX‐catalyzed lipid hydroperoxide generation in cellular membranes.

Pharmacological challenges to oncogenic Ras-expressing cancer cells have shown a novel type of cell death, ferroptosis, which requires intracellular iron. In the present study, we assessed ferroptosis following treatment of human fibrosarcoma HT1080 cells with several inhibitors of lysosomal activity and found that they prevented cell death induced by the ferroptosis-inducing compounds erastin and RSL3. Fluorescent analyses with a reactive oxygen species (ROS) sensor revealed constitutive generation of ROS in lysosomes, and treatment with lysosome inhibitors decreased both lysosomal ROS and a ferroptotic cell-death-associated ROS burst. These inhibitors partially prevented intracellular iron provision by attenuating intracellular transport of transferrin or autophagic degradation of ferritin. Furthermore, analyses with a fluorescent sensor that detects oxidative changes in cell membranes revealed that formation of lipid ROS in perinuclear compartments probably represented an early event in ferroptosis. These results suggest that lysosomal activity is involved in lipid ROS-mediated ferroptotic cell death through regulation of cellular iron equilibria and ROS generation.

Background and Purpose-Systemic inflammatory response syndrome (SIRS) without infection is a well-known phenomenon that accompanies various acute cerebral insults. We sought to determine whether the initial SIRS score was associated with outcome in subarachnoid hemorrhage (SAH). Methods-In 103 consecutive patients with SAH, the occurrence of SIRS was assessed according to the presence of Ն2 of the following: temperature of Ͻ36°C or Ͼ38°C, heart rate of Ͼ90 bpm, respiratory rate of Ͼ20 breaths/min, and white blood cell count of Ͻ4000/mm 3 or Ͼ12 000/mm 3 . SIRS criteria and other prognostic parameters were evaluated as predictors of dichotomous Glasgow Outcome Scale score. Results-SIRS was highly related to poor clinical grade (Hunt and Hess clinical grading scale), a large amount of SAH on CT (Fisher CT group), and high plasma glucose concentration on admission. By univariate analysis, the occurrence of SIRS was associated with higher mortality and morbidity rates than was the nonoccurrence (PϽ0.001). Among individual SIRS criteria, heart rate (Pϭ0.003), respiration rate (Pϭ0.003), and white blood cell count (Pϭ0.03) were significant outcome predictors. By multivariate logistic regression analysis, the presence of SIRS independently predicted outcome. SIRS carried an increased risk of subsequent intracranial complications such as vasospasm and normal pressure hydrocephalus, as well as systemic complications. Conclusions-In SAH patients, SIRS on admission reflected the extent of tissue damage at onset and predicted further tissue disruption, producing clinical worsening and, ultimately, a poor outcome.

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATPbinding cassette transporter A1 (ABCA1). Astrocytes from Abca1)/) mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1. Keywords: apolipoprotein E, astrocyte, ATP-binding cassette transporter A1, high-density lipoprotein, sphingosine 1-phosphate. Sphingosine 1-phosphate (S1P), a sphingolipid metabolite, is a pleiotropic lipid mediator involved in a variety of cell activities, including morphology, motility, and growth. Five subtypes of S1P-specific receptors, S1P 1-5 , have been isolated so far (Ishii et al. 2004). In neural cells, these receptors are expressed in a cell-specific manner (Van Brocklyn et al. 1999;Sato et al. 2000) and involved in the regulation of neural cell functions; for example, a rapid process retraction through S1P 5 in oligodendrocytes (Jaillard et al. 2005), rounding of the cell body in PC12 and N1E115 cells (Postma et al. 1996;Sato et al. 1997) possibly through S1P 2 (Van Brocklyn et al. 1999), and regulation of motility in astrocytes and glioma cells . In addition, S1P induces the proliferation in astroglial cells, which was associated with the activation of extracellular signal-regulated kinase (ERK), activation of the phospholipase C, and Ca 2+ mobilization (Tas and Koschel 1998;Sato et al. 1999;Pebay et al. 2001). S1P 1 is more important than S1P 2 for the stimulation of ERK, while S1P 2 may be responsible for the activation of phospholipase C and Ca 2+ mobilization (Sato et al. 2000;Malchinkhuu et al. 2003). Thus, S1P and its receptors may play important roles in maintaining the functions of the CNS.We have recently demonstrated that S1P is concentrated in lipoproteins, such as high-density lipoprotein (HDL), in circulating blood (Murata et al. 2000b), and that the lipid mediates lipoprotein-induced anti-atherogenic actions, including cell survival, cell migration, and inhibition of adhesion molecule expression through S1P receptors in endothelial cells Okajima 2002). Thus, lipoproteins seem to serve as carriers for extracellular S1P in circulating blood. In astrocytes as well, plasma HDL ...