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Hyoung Jin Kim, Sun Young Koo, Bong-Hyun Ahn, Oeuk Park, Doo Hoe Park, Dong Ook Seo, Jong Heon Won,. Hyeon Joo ..... analysis using Image J software (NIH, version 1.42), ..... and necroptosis as well as caspase-independent auto-.
Arch Pharm Res Vol 33, No 11, 1813-1823, 2010 DOI 10.1007/s12272-010-1114-4

NecroX as a Novel Class of Mitochondrial Reactive Oxygen Species and ONOO− Scavenger Hyoung Jin Kim, Sun Young Koo, Bong-Hyun Ahn, Oeuk Park, Doo Hoe Park, Dong Ook Seo, Jong Heon Won, Hyeon Joo Yim, Hyo-Shin Kwak, Heui Sul Park, Chul Woong Chung, Young Leem Oh, and Soon Ha Kim LG Life Sciences Ltd., R&D Park, Daejeon, 305-380, Korea (Received June 27, 2010/Revised July 29, 2010/Accepted August 3, 2010)

Mitochondrial reactive oxygen species and reactive nitrogen species are proven to be major sources of oxidative stress in the cell; they play a prominent role in a wide range of human disorders resulting from nonapoptotic cell death. The aim of this study is to examine the cytoprotective effect of the NecroX series against harmful stresses, including pro-oxidant (tertiarybutylhydroperoxide), doxorubicin, CCl4, and hypoxic injury. In this study, these novel chemical molecules inhibited caspase-independent cell death with necrotic morphology, which is distinctly different from apoptosis, autophagy, and necroptosis. In addition, they displayed strong mitochondrial reactive oxygen species and ONOO− scavenging activity. Further, oral administration of these molecules in C57BL/6 mice attenuated streptozotocin-induced pancreatic islet β-cell destruction as well as CCl4-induced hepatotoxicity in vivo. Taken together, these results demonstrate that the NecroX series are involved in the blockade of nonapoptotic cell death against mitochondrial oxidative stresses. Thus, these chemical molecules are potential therapeutic agents in mitochondria-related human diseases involving necrotic tissue injury. Key words: NecroX, Nonapoptotic cell death, Oxidative stress, Mitochondrial, Reactive oxygen species and reactive nitrogen species, Hepatotoxicity, Pancreatic islets

Selected by Editors INTRODUCTION Reactive oxygen species (ROS) such as superoxide, H2O2, hydroxyl radical and reactive nitrogen species (RNS) such as peroxynitrite (ONOO−), NO, NO2, HNO2, and NO3 are well known to be both deleterious and beneficial. These reactive species such as ROS and RNS cause potential biological damage, termed oxidative stress and nitrosative stress, respectively (Ridnour et al., 2004; Valko et al., 2006; Roberts et al., 2010). Oxidative stress caused by ROS and RNS has been implicated in various pathological conditions involving cardiovascular disease, cancer, neurological Correspondence to: Soon Ha Kim, LG Life Sciences Ltd., R&D Park, Daejeon, 305-380, Korea Tel: 82-42-866-4925, Fax: 82-42-861-2566 E-mail: [email protected]

disorders, diabetes, ischemia/reperfusion, and aging (Dalle-Donne et al., 2006; Valko et al., 2007). In vivo, peroxynitrite among RNS generation represents a crucial pathogenic mechanism in conditions of numerous diseases (Pacher et al., 2007). The mitochondrial respiratory chain is a major site of intracellular ROS generation and, at the same time, an important target for the damaging effects of ROS. Hence, any damage to the respiratory chain function might also affect cell viability. Accumulating evidence supports a direct link between mitochondrial oxidative stress and cell death; that is, mitochondrial oxidative stresses, such as ROS and RNS, contribute to the pathophysiology of cell death (Orrenius et al., 2007; Ott et al., 2007). Thus, ROS-induced mitochondrial structural modifications and permeability transition pore formation (mPTP) might be decisive events that lead to cell death, such as apoptosis, autophagy, and necrosis (Belizário et al., 2007; Gogvadze and Zhivotovsky, 2007; Kroemer et al., 2007). “Apoptosis,” or “programmed cell death,” is an in-

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tegral part of development and homeostasis. During some pathological conditions such as in some neurodegenerative diseases and stroke, the apoptotic program can be inappropriately implemented, resulting to detrimental cellular function (Ferri and Kroemer, 2001; Leist and Jäättelä, 2001) that requires energy and often even de novo macromolecular synthesis (Hengartner, 2000). Recently, the term necroptosis has been used to describe a particular type of programmed necrosis induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Necroptosis depends on the serine/threonine kinase activity of RIP1 as the primary cellular target responsible for the anti-necroptosis activity of necrostatin-1 (Degterev et al., 2008; Galluzzi and Kroemer, 2008; Hitomi et al., 2008). “Necrotic cell death” or “necrosis” is morphologically characterized by a gain in cell volume (oncosis), swelling of organelles, plasma membrane rupture, and subsequent loss of intracellular contents. Necrosis has been long considered merely a chaotic decadence process; that is, an accidental uncontrolled form of cell death. However, evidence is accumulating that necrotic cell death may be regulated by a set of signal transduction pathways and catabolic metabolism (Syntichaki and Tavernarakis, 2002; Festjens et al., 2006; Golstein and Kroemer, 2007). In humans, necrotic cell death occurs generally in response to severe physiological changes including hypoxia, ischemia, hypoglycemia, toxin exposure, and nutrient deprivation (Nicotera et al., 1999; Bhatia, 2004; Devarajan, 2005; Gasparini et al., 2010). In this study, we examined, using a variety of in vitro experimental models, whether members of NecroX have mitochondrial ROS and peroxynitrite scavenging activity as well as cytoprotective ability against various insults. Thus, the effects of NecroX were explored in various cellular systems, namely in primary rat hepatocyte and H9C2 cell death induced by doxorubicin and tertiary-butylhydroperoxide (tBHP), respectively. Furthermore, the influence of NecroX on streptozotocin (STZ)-induced pancreatic βcell injury and CCl4-induced acute hepatotoxicity in rodent models were studied. We report that NecroX can act on caspase-independent cell death pathway through mechanisms distinct from that of inhibitors of apoptosis, necroptosis, and autophagy. We suggest that NecroX should be therapeutic drug candidates in mitochondrial oxidative stress-related human diseases involving nonapoptotic tissue injury.

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MATERIALS AND METHODS Chemicals and reagents NecroX-1, -2, -5, and -18 (Fig. 1) were synthesized by LG Life Sciences. STZ, t-BHP, doxorubicin, and Trolox were from Sigma. DHR-123 and HepatoZYME-SEM were from Life Technologies. DPPH was from Fluka. ONOO− was from Cayman. Animals Adult male Hsd: CD-1 ICR mice and SpragueDawley rats, aged 6 - 7 weeks old upon arrival, were purchased from Koatech Co. All animals were kept under standard light, temperature, and feeding regimens, and were allowed to acclimate for at least 5 days before use. All protocols were approved by LG Life Sciences Institutional Animal Care and Use Committee. Cell culture and isolation of primary cells Rat cardiomyoblast H9C2 cells, LLC-PK1 renal cells, SK-N-MC neuronal cells, and RINm-5F pancreatic β cells were purchased from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640 medium containing 10% fetal bovine serum and 1 × penicillin/ streptomycin, and gassed with 5% CO2. Primary rat hepatocytes were isolated from SpragueDawley rats (200 - 300 g) by a 2-step collagenase perfusion method, and viable cells were collected by lowspeed centrifugation using Percoll solutions. Collected cells were washed 2 times with wash medium and counted for viability (90 - 95%). Primary rat hepatocytes (1.5 × 104 cells per well) were seeded into collagen-coated 96-well plates and attached for 3 - 4 h in HepatoZYME serum-free medium before any experiment. For primary rat chondrocyte isolation, the cartilage was collected from the knee bilaterally under sterile conditions, washed with calcium- and magnesium-free Dulbecco's phosphate-buffered saline, and minced into small pieces. Chondrocytes were released from articular cartilage after being digested by 0.2% collagenase type II (Worthington Biochemical) in DMEM/F-12 medium containing 10% fetal bovine serum and 1 × penicillin/streptomycin, overnight by gentle agitation at 37oC. Cells were seeded in 96 well tissue culture plates at a density of 1.5 × 104 per well. Assessment of cytoprotective effect All cells tested (1.5×104 - 2×104 per well) were seeded and grown for 24 h in 96-well plates. Each compound was pretreated 30 min before cellular injury from any insult. After 30 min, 10 µL of each toxin

Mitochondrial ROS and ONOO− Scavenger

stock solutions was added to each well (final 0.1-0.2% DMSO concentration). For determination of cytoprotective effects against t-BHP, assays were performed from 2 to 5 h depending on the cell type. Cell viability was measured by the sulforhodamine B (SRB) assay or LDH assay. For SRB assay, each plate was fixed by 50 µL of formaldehyde solution for 10 min and washed 3 times by distilled water and then dried in a 50oC incubator. Each well was then stained with 50 µL SRB solution for 1 h and washed thoroughly by emerging the plate into 0.1% acetic acid solution. The plate was dried again and filled with 100 µL of 10 mM Tris buffer. The absorbance at 590 nm and 650 nm was read with a SpectraMax ELISA reader. LDH cytotoxicity assay was performed according to the manufacturer's protocol (Promega). This colorimetric assay quantifies the activity of LDH released from the cytosol of damaged cells into the supernatant. Primary rat hepatocytes were seeded as mentioned above. After attachment, each compound was treated for 30 min, doxorubicin (final 5 µM) was added to each well, and the compounds were further incubated for 2 days. At the end of the assay, 10 µL of WST-1 was added and compounds were incubated for 90 min. The absorbance at 440 nm was read with SpectraMax ELISA reader (Molecular Devices). For hypoxic injury, the plates with primary rat hepatocytes were placed in a hypoxic chamber gassed with 95% N2 + 5% CO2 for 10 min and incubated for 6 h under hypoxic condition in a 37oC incubator. The plate was taken out of the hypoxic chamber, returned to normal culture condition, and further cultured for 1 day. For viability, WST-1 assay (Takara) was used. Results were expressed relative to the control value specified in each experiment and were subjected to statistical analysis.

Determination of radical-scavenging activity Radical-scavenging activity was determined by the reduction of DPPH in methanol using Trolox as a reference antioxidant. In brief, 10 µL of each compound at the indicated concentrations was transferred to 96-well plates. DPPH stock solution (200 µM, 190 µL) was added to each well, the reaction mixture was kept in the dark at room temperature for 30 min, and the absorbance was monitored at 517 nm by using an ELISA reader. Detection and fluorescence microscopic analysis of mitochondrial H2O2 and ONOO− scavenging activity The ONOO− scavenging activity was detected by monitoring the oxidation of DHR-123 in vitro. Each compound was 3-fold diluted before use. One hundred

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seventy microliters of a DHR-123 mixture (5 mM DHR-123, 20 × PBS, 100 mM DTPA, distilled water) was added into each well. The plate was pre-read for background fluorescence. Twenty µL of peroxynitrite solution (10 µM in 0.3 N NaOH) was added, and fluorescence was read for 20 min with SpectraMax Gemini (Molecular Devices). For detection of mitochondrial ROS and RNS production, H9C2 cells (1.5 × 104 per well) were seeded into 96 well plates and then incubated for 1 day at 37oC in a CO2 incubator. Each compound was added into each well and then mixed with 1.25 µM of DHR123 probe. t-BHP (0.4 mM) was added to each well. After 2 h, 50 µL of formaldehyde solution was added for fixation for 10 min and washed twice with PBS. Each picture was taken, in the presence of 100 µL PBS buffer, by using Olympus I × 70 multiparameter fluorescence microscope (Olympus Optical Co., Ltd.) with NIBA fluorescence filter (ex/em, 470-495/510550). Fluorescence (ex/em, 485/530) was read with SpectraMax Gemini (Molecular Devices). IC25 value was assessed by calculating the compound concentration that inhibited 25% of fluorescence increase induced by t-BHP treatment.

Copper ion (Cu2+)-induced LDL oxidation assay LDL (50 µg/mL, Kale Biomedical) was incubated with final 10 µM copper sulfate in d-PBS (Rattan and Arad, 1998). Absorbance at 234 nm, of the reaction mixtures in UV-transparent 96-well plate (BD Falcon), was continuously monitored using SpectraMax 190 (Molecular Dynamics) at 25oC for 2 h. Compounds were added at the indicated concentrations. Animal studies Assessment of acute CCl4-induced hepatotoxicity For inducing hepatotoxicity in vivo, CCl4 was diluted in corn oil. Group of mice and rats (n = 5-7) were intraperitoneally administered with CCl4 (25 µL/kg for mice, 200 µL/kg for rats) or the respective vehicle. Serum samples were collected 24 h after CCl4 administration. Serum ALT and AST levels were measured as biochemical indicators of hepatotoxicity using Hitachi Instrument (Model 7180). Protection from STZ-induced pancreatic islet destruction Pancreatic islet cell death was induced by single intraperitoneal dosing of STZ in 50 mM citrate buffer (pH 4.5) at a dose level of 100 mg/kg. Animals were fasted for 16 h before STZ treatment, and otherwise received food and water ad libitum. Animals (12-13

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mice per group) were treated with vehicle, 30 mg/kg NecroX-1, or 100 mg/kg Necro X-1 through oral gavage, 2 h before STZ treatment and then 24 h after the first dosing. Pancreas was collected 24 h after the second dosing. The β-cell fractions in pancreatic islets were determined by immunohistochemistry on pancreatic islets using polyclonal guinea pig anti-swine insulin antibody (Dakocytomation) and were calculated as the positive area for insulin vs total area of the islets. The β-cell fractions were determined by morphometric analysis using Image J software (NIH, version 1.42), analyzing 3 slides per pancreas and roughly 10 islets per slide, 150 µm apart from each section. Islet numbers of each animal were counted by observing each slide with an optical microscope using 3 levels of size discrimination. Small, medium, and large islets were in the size of 300 µm2 to 5,000 µm2, 5,000 µm2 to 20,000 µm2, and >20,000 µm2, respectively.

Statistical analysis In vitro study Where necessary, data are expressed as mean ± S.D. p < 0.05 was used as the criterion for statistical significance. In vivo study All values are expressed as mean ± S.D. Comparison between any 2 groups was performed using 1-way analysis of variance followed by Tukey’s multiple comparison test using the computer program SPSS. Statistically significant differences between groups were defined as p < 0.05.

RESULTS NecroX is a mitochondrial ROS and ONOO− scavenger NecroX-1, -2, -5, and -18 were synthesized by LG Life Sciences, Ltd. (Fig. 1). By screening chemical compounds that protect rat primary hepatocytes from drug-induced cell death, we identified a series of small molecules that inhibited cell death with necrotic morphology. To further unravel the cellular cascades contributing to necrosis-like morphology, we developed tools to specifically interface with cellular events through key mediators at certain molecular levels. First, we used small molecules to define antioxidant activity against oxidative stress such as mitochondrial ROS and RNS. We determined whether NecroX might also regulate oxidative stress. NecroX-1, -2, -5 and -18 compounds showed in vitro antioxidant activity (Table I), which was confirmed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ONOO− scavenging dihydroxyr-

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Fig. 1. Structure of the NecroX series: NecroX-1, -2, -5, and -18. We report a novel class of NecroX compounds with inhibitory potential activity for caspase-independent nonapoptotic cell death and mitochondrial ROS/RNS scavenging activities.

hodamine (DHR)-123 assays. Trolox was used as reference antioxidant in DPPH assay using DHR-123 probe (Trolox IC50 = 15.5 µM). These results indicate that NecroX might be able to attenuate oxidative stress-induced cell death through antioxidant activity. Low-density lipoprotein (LDL) cholesterol, which is known as “bad” cholesterol, becomes even more dangerous when oxidized. Oxidized LDL can cause inflammation in the arteries, which supply blood to organs and other tissues, thus promoting atherosclerosis and increasing the risk of heart attack or stroke. Oxidation of LDL occurs when LDL particles react with free radicals. Oxidized LDL itself then becomes more reactive with the surrounding tissues, which can result to tissue damage. Based on the above data on antioxidant activity, we examined whether NecroX chemicals can inhibit the generation of oxidized LDL. The absence of NecroX-1 did not affect LDL oxidation, whereas NecroX-1, -2, -5, and -18 effectively inhibited the generation of oxidized LDL (Fig. 2). Thus, we suggest NecroX to be novel therapeutic agents of atherosclerosis. Oxidative stress is a condition induced by proTable I. In vitro antioxidant activity in DPPH assay (IC50, µM)

a

Compound

In vitro DDPH assaya

In vitro ONOO− scavenging activity

NecroX-1 NecroX-2 NecroX-5 NecroX-18

20 17 22 19

3.40 0.90 NDb 0.21

Trolox was used as the reference antioxidant in DPPH assay (Trolox IC50 = 15.5 µM). b not determined.

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Fig. 2. In vitro inhibitory effect of NecroX on low-density lipoprotein (LDL) oxidation. LDL (50 µg/mL, Kale Biomedical) was incubated with final 10 µM copper sulfate in d-PBS. NecroX series compounds were added at the indicated concentrations. The reaction mixture was incubated in a UV-transparent 96-well plate (BD Falcon) at 25oC for 9 h. The absorbance at 234 nm was monitored continuously in SpectraMAX plus (Molecular Devices).

oxidant compounds such as t-BHP and can also be induced in vivo by ischemia/reperfusion injury, which is very common in cardiac tissues. The myoblast cell line H9C2 has been used as an in vitro cellular model to address the hypothesis that the cytoprotective effect of NecroX-5 upon exposure of cells to t-BHP could be due to the mitochondrial ROS/RNS scavenging

activity. NecroX-5 effectively inhibited the t-BHPinduced mitochondrial H2O2 and ONOO− generation (Fig. 3 and Table II), which was confirmed by DHR123 oxidation assay. NecroX-5 displayed no effect of scavenging t-BHP-induced cytosolic ROS using DCFDA (data not shown). From these results, we suggest NecroX as a novel class of mitochondria-targeting

Fig. 3. In vitro mitochondrial H2O2 and ONOO− scavenging activity of NecroX-5 in t-BHP-treated H9C2 cells by DHR-123 assay. Effect of NecroX-5 on accumulation of mitochondrial ROS and RNS induced by t-BHP. H9C2 cells were incubated with 400 µM t-BHP for 90 min in the presence or absence of NecroX-5. The levels of mitochondrial H2O2 and ONOO- were measured by DHR-123 assay, as described in Materials and Methods.

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Table II. In vitro mitochondrial ROS (H2O2) and RNS (ONOO−) scavenging activity of NecroX in t-BHP-treated H9C2 cells (IC25, µM)

a

NecroX series

DHR-123 oxidation assay in H9C2 cellsa

NecroX-1 NecroX-2 NecroX-5 NecroX-18

25 30

0.45 >30 >30 >30

30 16 >30

30 6.1 NDc

and b were assessed by SRB and LDH method, respectively. not determined. IDN-6556: pan-caspase inhibitor; 3-MA: 3-methyladenine, autophgy inhibitor; Necrostatin-1: necroptosis inhibitor; SNK-MC: human neuroblastoma cell line. c

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Fig. 6. Protective effect of NecroX-1 on the CCl4-induced acute hepatotoxicity in rodent models. NecroX protects the liver in CCl4-induced in vivo models. (A) Protective effect of Necrox-1 in CCl4-induced severe acute hepatotoxicity in the mice model. (B) Protective effect of Necrox-5 in CCl4-induced severe acute hepatotoxicity in the rat model. Values are mean ± S.D., n = 10. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively.

animals from a well-known type II diabetes model. NecroX-1 protected pancreatic β cells against STZinduced cell death when administered to animals orally 2 h before and 22 h after STZ treatment (Fig. 7). β-cell fractions in pancreatic islets analyzed by a morphometric method were significantly higher in animals treated with 100 mg/kg NecroX-1 (0.38 ± 0.04) than those with vehicle (0.18 ± 0.03). Our data indicate that NecroX-1 would help protect pancreatic islets from cellular injury when exposed to STZ in vivo because of their ROS/RNS scavenging and inhibitory activities.

DISCUSSION The findings presented here demonstrate that NecroX chemicals and their structurally related compounds can afford protection against various insults in vitro as well as in vivo, and support the hypothesis that NecroX plays a role in protecting cells from oxidative damage agents such as mitochondrial ROS and RNS (peroxynitrite) that lead to caspase-independent nonapoptotic cell death with necrotic morphology. Our results indicate that a key step in the nonapopto-

tic cell death pathway would be highly amenable to inhibition by NecroX. Considering the emerging importance of nonapoptotic cell death in the studies of pathologic injury and disease in vivo as well as the control of cell viability in vitro (Hetz et al., 2005), our results suggest that NecroX would provide a strong basis for a better understanding of nonapoptotic cell death mechanisms, which may be translated into the development of strategies for treatment of human inflammatory disorders. To summarize our findings, we have shown that tBHP-induced necrosis in various cells was attenuated by NecroX, accompanied by stabilization of mitochondrial membrane potential depolarization, delayed opening of the mPTP, and preservation of intracellular ATP levels (unpublished data), which are proposed key determinants of nonapoptotic cell death. Our data demonstrate that NecroX effectively inhibits cellular damage against mitochondrial oxidative stresses caused by ROS and peroxynitrite, indicating that these molecules act on the nonapoptotic cell death pathway, which is distinct from caspase-dependent apoptosis and necroptosis as well as caspase-independent autophagy. Accordingly, mitochondria-targeted antioxi-

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Fig. 7. Continued

Fig. 7. In vivo protective effect of NecroX-1 on the streptozotocin (STZ)-induced pancreatic islet destruction in type 2 diabetic mice model. Protective effect of NecroX-1 on STZ-induced pancreatic β-cell death in type 2 diabetes mice model. (A) Histological representation of insulin immunopositive islet cells of vehicle-treated animals (left) and NecroX-1-treated animals at the dose level of 100 mg/kg (right). These are representatives of insulin immunohistochemistry obtained from animals for each group. β-cell fraction (B) and islet size count (C) comparison from each group.

dants such as mitoquinone have now been used in a wide range in vivo studies such as rats and mice, which indicates a potential clinical benefit in phase II trials for parkinson’s disease, hepatitis C (Smith et al., 2008; Gane et al., 2010; Smith and Murphy, 2010; Snow et al., 2010). Data obtained from our in vitro experiments were

superior to those of other studies that examined wellknown antioxidants, such as the nonapoptotic cell death inhibitors IM-54 (Dodo et al., 2005) and melatonin (Reiter et al., 2003). Thus, we intend to carry out further investigations using in vivo models to establish whether mitochondrial ROS/RNS and nonapoptotic cell death are key features of the changes in 2 different animal models, such as type 2 diabetes and drug-induced acute hepatotoxicity in rodents, since mitochondrial oxidative stress caused by ROS and RNS has been proven to be associated with cellular injury induced by STZ and CCl4. We have shown that NecroX affords cytoprotection in 2 different in vivo models such as STZ-induced pancreatic β-cell death and CCl4-induced hepatotoxicity. NecroX administered in vivo and in vitro possibly resides in the mitochondrial matrix and then scavenges mitochondrial ROS such as superoxide, hydrogen peroxide, and peroxynitrite during cellular damage. Our results are consistent with previous studies showing that chemical compounds containing a key moiety of indole ring, such as melatonin, have antioxidant activity in in vitro (Poeggeler et al., 1996; Millán-Plano et al., 2010) and in vivo models (Jaworek et al., 2003) through attenuation of mitochondrial dysfunction (Acuna-Castroviejo et al., 2007; Escames et al., 2007). Further studies are needed to verify the antioxidant behavior of NecroX in terms of target site, intracellular localization, tissue distribution, and electron spin resonance (ESR) study. Another interesting observation on the in vitro effective dose in cell-based assay was that 0.1 µM of

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NecroX protected against nonapoptotic cell death, potentiating strong efficacy of NecroX at lower concentrations. As regards the finding that ischemic reperfusion injury (Theruvath et al., 2008; Baines, 2009) may be linked to apoptosis, our study claims that the effects of NecroX are mediated by a mechanism distinct from apoptosis, necroptosis, and autophagy since pancaspase inhibitors (IDN-6556, z-VAD-fmk), a necroptosis inhibitor (necrostatin-1), and an autophagy inhibitor (3-MA) did not effectively prevent nonapoptotic cell death and stabilize mitochondrial membrane permeability transition in t-BHP-induced in vitro cellular injury model (unpublished data). In conclusion, the identification of chemical small molecule family such as NecroX series targeting the proposed nonapoptotic cell death pathway through their mitochondrial ROS and peroxynitrite scavenging activity may lead to the development of more effective anti-nonapoptotic therapies. Our study is the first to indicate that NecroX can protect against nonapoptotic insults in both in vitro and in vivo systems, possibly through mechanisms other than those that are generally accepted as being involved (i.e., apoptosis and necroptosis). To have insights into the significance of NecroX in the field of nonapoptotic cell death and its related pathophysiology, the molecular targets and potential cellular mechanisms should be elucidated.

ACKNOWLEDGEMENTS We apologize to the authors who have made contributions to the field but have not been cited due to space limitations.

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