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.

Reactive oxygen species (ROS) are involved in several cell death processes, including cerebral ischemic injury. We found that glutamate-induced ROS accumulation and the associated cell death in mouse hippocampal cell lines were delayed by pharmacological inhibition of autophagy or lysosomal activity. Glutamate, however, did not stimulate autophagy, which was assessed by a protein marker, LC3, and neither changes in organization of mitochondria nor lysosomal membrane permeabilization were observed. Fluorescent analyses by a redox probe PF-H 2 TMRos revealed that autophagosomes and/or lysosomes are the major sites for basal ROS generation in addition to mitochondria. Treatments with inhibitors for autophagy and lysosomes decreased their basal ROS production and caused a burst of mitochondrial ROS to be delayed. On the other hand, attenuation of mitochondrial activity by serum depletion or by high cell density culture resulted in the loss of both constitutive ROS production and an ROS burst in mitochondria. Thus, constitutive ROS production within mitochondria and lysosomes enables cells to be susceptible to glutamate-induced oxidative cytotoxicity. Likewise, inhibitors for autophagy and lysosomes reduced neural cell death in an ischemia model in rats. We suggest that cell injury during periods of ischemia is regulated by ROS-generating activity in autophagosomes and/or lysosomes as well as in mitochondria.Oxidative stress-induced cell death has been implicated in several diseases and in acute injury such as ischemia. Oxidative stress results from increased levels of reactive oxygen species (ROS), 3 which include free radicals such as superoxide and hydroxyl radical, and nonradical species such as hydrogen peroxide (1). Due to their high reactivity, ROS can oxidize cell constituents such as lipids, proteins, and DNA and thus damage cell structures and integrity. In addition to their potentially lethal effects, previous studies have shown that they are implicated in a variety of cell death processes (1). For example, ROS disrupt the integrity of the lysosomes, and this lysosomal membrane permeabilization then triggers caspase-mediated apoptosis or cathepsin-mediated necrotic cell death (2-4). Furthermore, ROS induce cell death associated with autophagosome accumulation (5), and this form of autophagic cell death is activated in the nervous system in response to oxidative stress (6). In mammalian cells, chaperone-mediated autophagy has been shown to act in the degradation of oxidized proteins during oxidative stress (7), and the selective degradation of mitochondria was proposed to decrease the potential oxidative damage from defective mitochondria (8). Thus, autophagy may have a dual role in regulating cell death in response to oxidative stress.The susceptibility of the brain to oxidative stress may be related to its high oxygen consumption rate, abundant lipid content, and relative paucity of antioxidant enzymes (9). The hippocampal cell line HT22 is an excellent model for studying the mechanism of oxidative glutam...

This new model of focal cerebral ischemia induces a reproducible amount of ischemic damage in both gray and white matter, and has significant utility for studies of the pathophysiology of ischemia in the gyrencephalic brain and for assessment of the therapeutic efficacy of drugs prior to the initiation of human clinical trials.

Background and Purpose-Lacunar infarction accounts for 25% of ischemic strokes, but the pathological characteristics have not been investigated systematically. A new experimental model of lacunar infarction in the miniature pig was developed to investigate the pathophysiological changes in the corticospinal tract from the acute to chronic phases. Methods-Thirty-five miniature pigs underwent transcranial surgery for permanent anterior choroidal artery occlusion.Animals recovered for 24 hours (nϭ7), 2 (nϭ5), 3 (nϭ2), 4 (nϭ2), 6 (nϭ1), 7 (nϭ7), 8 (nϭ2), and 9 days (nϭ1), 2 weeks (nϭ2), 4 weeks (nϭ3), and more than 4 weeks (nϭ3). Neurology, electrophysiology, histology, and MRI were performed. Seven additional miniature pigs underwent transient anterior choroidal artery occlusion to study muscle motor-evoked potentials and evaluate corticospinal tract function during transient anterior choroidal artery occlusion. Results-The protocol had a 91.4% success rate in induction of internal capsule infarction 286Ϯ153 mm 3 (meanϮSD). Motor-evoked potentials revealed the presence of penumbral tissue in the internal capsule after 6 to 15 minutes anterior choroidal artery occlusion. Total neurological deficit scores of 15.0 (95% CI, 13.5 to 16.4) and 3.4 (0.3 to 6.4) were recorded for permanent anterior choroidal artery occlusion and sham groups, respectively (PϽ0.001, maximum score 25) with motor deficit scores of 3.4 (95% CI, 2.9 to 4.0) and 0.0 (CI, 0.0 to 0.0), respectively (PϽ0.001, maximum score 9). Histology revealed that the internal capsule lesion expands gradually from acute to chronic phases. Conclusions-This