Effect of vitamin E on 24(S)-hydroxycholesterol-induced necroptosis-like cell death and apoptosis

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Journal of Steroid Biochemistry & Molecular Biology xxx (2015) xxx–xxx

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Effect of vitamin E on 24(S)-hydroxycholesterol-induced necroptosis-like cell death and apoptosis Takaya Nakazawa1, Yuta Miyanoki1, Yasuomi Urano, Madoka Uehara, Yoshiro Saito, Noriko Noguchi* Systems Life Sciences Laboratory, Department of Medical Life Systems, Faculty of Life and Medical Sciences, Doshisha University, 1-3 Miyakodani, Tatara, Kyotanabe, Kyoto 610-0394, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 November 2015 Received in revised form 29 February 2016 Accepted 1 March 2016 Available online xxx

24(S)-Hydroxycholesterol (24S-OHC) has diverse physiological and pathological functions. In particular, cytotoxic effects of 24S-OHC in neuronal cells are important in development of neurodegenerative diseases. 24S-OHC induces necroptosis-like cell death in SH-SY5Y cells expressing little caspase-8. In the present study, 24S-OHC was found to induce apoptosis as determined by caspase-3 activation in all-transretinoic acid (atRA)-treated SH-SY5Y cells in which expression of caspase-8 was induced. 24S-OHCinduced cell death was inhibited by a-tocopherol (a-Toc) but not by a-tocotrienol (a-Toc3) in SH-SY5Y cells regardless of whether cells were treated with atRA. In contrast, cumene hydroperoxide (CumOOH)induced cell death was significantly inhibited by a-Toc and a-Toc3. In atRA-treated SH-SY5Y cells, generation of reactive oxygen species (ROS) was induced by stimulation with CumOOH but was not induced by stimulation with 24S-OHC. These results suggest that inhibition of 24S-OHC-induced cell death by a-Toc cannot be explained by its radical scavenging antioxidant activity. Esterification of 24SOHC followed by lipid droplet (LD) formation due to acyl-CoA:cholesterol acyltransferase 1 (ACAT1) are key events in 24S-OHC-induced cell death in atRA-treated SH-SY5Y cells as demonstrated by inhibition of cell death by ACAT1 inhibitor. LD number was not changed by treatment with either a-Toc or a-Toc3. The different physical properties of a-Toc and a-Toc3 may account for their different inhibitory effects on 24S-OHC-induced cell death. ã 2016 Elsevier Ltd. All rights reserved.

Keywords: 24(S)-hydroxycholesterol Cell death a-Tocopherol a-Tocotrienol Caspase-8 Lipid droplet

1. Introduction 24(S)-Hydroxycholesterol (24S-OHC) has diverse functions [1,2]. 24S-OHC plays an important role in cholesterol metabolism in the brain, excess cholesterol being converted to 24S-OHC by cholesterol 24-hydroxylase (CYP46A1) [3], and this 24S-OHC being eliminated into the peripheral circulation across the blood-brain barrier [4]. In in vitro experiments using SH-SY5Y human neuroblastoma cells, it has been found that less than 10 mM

Abbreviations: ACAT1, acyl-CoA:cholesterol acyltransferase 1; AD, Alzheimer’s disease; PD, Parkinson’s disease; 24S-OHC, 24(S)-Hydroxycholesterol; atRA, alltrans-retinoic acid; a-Toc, a-tocopherol; a-Toc3, a-tocotrienol; CumOOH, cumene hydroperoxide; LD, lipid droplet; LDH, lactate dehydrogenase; MAP2, microtubuleassociated protein 2; MLKL, mixed lineage kinase domain-like; MS, multiple sclerosis; NB cells, neuroblastoma cell lines; Nec-1, necrostatin-1; RIPK1, receptorinteracting protein kinase 1; ROS, reactive oxygen species; STS, staurosporine; TNFa, tumor necrosis factor a. * Corresponding author. E-mail address: [email protected] (N. Noguchi). 1 These authors contributed equally to this work.

24S-OHC has suppressive effect on amyloid b production [5] and oxidative damage [6]. In contrast to these physiological and protective functions of 24S-OHC, recent evidence has shown that 24S-OHC possesses a potent neurotoxicity which may contribute to the development of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [7–9]. Kölsh et al. reported that 24S-OHC caused cell death in SH-SY5Y cells [10], and that this 24S-OHCinduced cell death was apoptotic when SH-SY5Y cells were treated with all-trans-retinoic acid (atRA) [11]. They also showed that 24SOHC enhanced production of reactive oxygen species (ROS) and that cell death was suppressed by a-tocopherol (a-Toc). The protective effects of a-Toc have also been shown on cell death induced by other cytotoxic oxysterols such as 7-ketocholesterol and 7b-OHC [12,13]. We have recently reported that 24S-OHC elicits caspaseindependent programmed cell death in SH-SY5Y cells and in rat primary cortical neuronal cells [14]. We demonstrated that necroptosis, a type of programmed necrosis that exhibits dependency on receptor-interacting protein kinase 1 (RIPK1),

http://dx.doi.org/10.1016/j.jsbmb.2016.03.003 0960-0760/ ã 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: T. Nakazawa, et al., Effect of vitamin E on 24(S)-hydroxycholesterol-induced necroptosis-like cell death and apoptosis, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.03.003

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may account for 24S-OHC-induced cell death. Unlike apoptosis, in which the caspases play a central role in the cell death machinery, necroptosis occurs through a caspase-independent cell death mechanism [15]. In our earlier study, we found that caspase8 expression was at undetectable levels in the neuronal cells used, which we understood to be the reason why 24S-OHC induced necroptosis instead of apoptosis in that study [14]. We also showed that 24S-OHC induces apoptosis in human T lymphoma Jurkat cells which endogenously express caspase-8, and that 24S-OHC induces RIPK1-dependent non-apoptotic cell death in the presence of the pan-caspase inhibitor ZVAD [16]. These data indicate that 24S-OHC can induce either apoptosis or necroptosis in Jurkat cells, which of the two forms of cell death is induced being dependent on caspase activity in general, and on caspase-8 activity in particular. Jiang et al. showed that expression of caspase-8 could be induced in neuroblastoma cell lines (NB cells) by treatment with atRA or other RA [17]. We have hypothesized that 24S-OHC may cause apoptosis if caspase-8 expression is induced in SH-SY5Y cells by atRA treatment, which was not evaluated by Kölsh et al. Activation of RIPK3 and mixed lineage kinase domain-like (MLKL) as well as RIPK1 has been shown to be involved in the signaling pathway in necroptosis induced by tumor necrosis factor a (TNFa) in colorectal epithelial cells [18]. However, our recent finding that RIPK1 but neither RIPK3 nor MLKL is expressed in SH-SY5Y cells suggests that 24S-OHC-induced cell death in SH-SY5Y cells should be considered necroptosis-like [19]. Furthermore, we have shown that neither ROS generation nor lipid peroxidation occurs in 24SOHC-induced necroptosis-like cell death [2]. Many large-scale epidemiologic studies have supported the beneficial effects of vitamin E in fighting neurodegenerative diseases such as AD [20–23] and PD [24]. Vitamin E is the most abundant lipid-soluble radical-scavenging antioxidant in vivo and is composed of a chromanol ring and an aliphatic side chain [25,26]. Naturally occurring forms can be divided into two subgroups: tocopherols (Toc), which have a saturated phytyl side chain; and tocotrienols (Toc3), which have an unsaturated isoprenoid side chain. Toc and Toc3 each have four isomers, these being a-, b-, g-, and d-Toc; and a-, b-, g-, and d-Toc3 [27]. The radical scavenging activity in homogeneous solution of each isoform is determined by the position and number of methyl groups on the chromanol ring [28,29]. a-Toc and a-Toc3 therefore have the same radical scavenging antioxidant activity in homogeneous solution. In addition, since a-Toc and a-Toc3 display similar mobilities within the liposomal membrane, they show similar antioxidant activities in suppressing lipid peroxidation at the liposomal membrane. However, a-Toc gives more significant physical effect than a-Toc3 on the increase in rigidity at the membrane interior [28]. The side chain is known to affect mobility between liposomal membranes [28] as well as permeability into the cell, and it is known that a-Toc3 can move within the membrane and enter into cells with greater efficiency than a-Toc can [30,31]. The purpose of the present study is to characterize the type of cell death induced by 24S-OHC in SH-SY5Y cells with and without atRA treatment, and to determine the effect of a-Toc and a-Toc3 on 24S-OHC-induced cell death with and without atRA treatment. 2. Materials and methods 2.1. Materials 24S-OHC and the acyl-CoA:cholesterol acyltransferase 1 (ACAT1) inhibitor K-604 were kindly provided by Kowa Co., Ltd. (Aichi, Japan). a-Toc and a-Toc3 were kindly provided by Eizai Co. Ltd. (Tokyo, Japan) and Tama Biochemical Co., Ltd. (Tokyo, Japan). Dulbecco’s modified Eagle’s medium/nutrient mixture Ham’s F-12

(DMEM/F-12) was obtained from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum was Hyclone fetal bovine serum from Thermo Scientific (Logan, UT, USA). Pan-caspase inhibitor ZVAD was from Medical and Biological Laboratories (Aichi, Japan). Cumene hydroperoxide (CumOOH), staurosporine (STS) and atRA were purchased from Wako (Osaka, Japan). Necrostatin-1 (Nec-1) and Nile red were from Sigma-Aldrich. 24S-OHC, a-Toc, a-Toc3, and atRA were dissolved in ethanol (EtOH) and stored at
20  C. K-604, ZVAD, Nec-1, and STS were dissolved in DMSO and stored at
20  C. Hoechst 33342 was from Dojindo (Kumamoto, Japan). The following antibodies were from commercial sources: caspase-8 (#M058-3), Medical and Biological Laboratories; caspase-3 (#9662) and microtubule-associated protein 2 (MAP2, #4542), Cell Signaling (Beverly, MA, USA); RIPK1 (#551041), BD Biosciences (San Jose, CA, USA); b-actin (#A5441), Sigma-Aldrich. All other chemicals, of analytical grade, were obtained from Sigma-Aldrich and Wako. 2.2. Cell lines and cell culture SH-SY5Y human neuroblastoma cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were routinely maintained in DMEM/F-12 medium containing 10% heatinactivated fetal bovine serum and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin; Life Technologies). Cells were grown at 37  C under an atmosphere of 95% air and 5% CO2. To induce caspase-8 in SH-SY5Y cells, cells were treated with 25 mM atRA for 7 days. Culture medium with 25 mM atRA was changed every two days. 2.3. Cell treatment To examine toxicity of 24S-OHC in SH-SY5Y cells, cells were treated with 50 mM 24S-OHC for the indicated periods. To evaluate the effects of vitamin E, cells were treated with a-Toc or a-Toc3 at the different concentrations in the presence or absence of 50 mM 24S-OHC or 20 mM CumOOH for 24 h. Control cells were treated with DMSO and/or ethanol, respectively. To evaluate the effects of other inhibitors, cells were pretreated with 20 mM ZVAD, 100 mM Nec-1, or 2 mM K-604 for 1 h before further stimulation with 50 mM 24S-OHC. 2.4. Determination of cell viability For determination of cell viability, WST-8 assay and lactate dehydrogenase (LDH) assay were used. WST-8 assay was performed using Cell Counting Kit-8 according to the manufacturer’s instructions (Dojindo). LDH activity assay was performed as described previously [16]. 2.5. Immunoblotting Whole cell lysates were prepared in lysis buffer (50 mM Tris– HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA) to which protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) had been added. Nuclei and cellular debris were removed by centrifugation at 16,000g for 10 min. The protein samples were subjected to SDSPAGE and were transferred to a PVDF membrane for 1 h at 100 V. Immunoblotting was visualized with enhanced chemiluminescence (Millipore, Billerica, MA, USA). 2.6. Immunofluorescence staining Cells grown on glass cover slips were fixed with 4% paraformaldehyde/PBS for 15 min, permeabilized with 0.5% Triton X-100/PBS for 10 min, and then blocked with 2% BSA/PBS for

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30 min. The cover slips were incubated with anti-MAP2 antibody and Alexa Fluor-conjugated secondary antibody. The nuclei were stained with Hoechst 33342. Confocal fluorescence images were obtained using a Zeiss LSM710 confocal microscope laser with oil objective lens and accompanying software (LSM Software ZEN 2009). 2.7. Detection of intracellular ROS by fluorescence microscopy Cells were incubated with 10 mM of DCFH-DA for 1 h, followed by treatment with 50 mM 24S-OHC for 6 h or 100 mM CumOOH for 3 h. Cells were fixed with 4% paraformaldehyde/PBS for 20 min and were washed with PBS twice. Fluorescence was detected using a fluorescence microscope (OLYMPUS IX71, Tokyo, Japan).

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determined by BCA assay kit (Thermo Scientific). Saponification was carried out by adding 300 mL of 1 M KOH, 3 mL of EtOH/benzene/H2O (80:20:5) and 50 mL of 100 mM stigmasterol as an internal standard followed by incubation at 80  C for 1 h. After saponification, lipids were extracted using chloroform/methanol (2:1, v/v) and the chloroform phase was evaporated under nitrogen gas. The dry residue was then dissolved in iso-propanol/acetonitrile (55/45, v/v), and was subjected to HPLC analysis. HPLC was carried out using an Inertsil ODS-3 column (5 mm, 4.6  250 mm; GL Science, Tokyo, Japan). The column temperature was set at 30  C. The flow rate of eluent (iso-propanol/acetonitrile/H2O, 47/37/16) was 1.0 mL/min. Sterols were detected by a UV detector set at 210 nm. 2.10. Statistical analysis

2.8. Nile red staining To confirm presence of lipid droplets (LD) in 24S-OHC-treated cells in the presence or absence of a-Toc, a-Toc3, or K-604, cells were fixed with 4% paraformaldehyde/PBS at room temperature for 15 min. After washing out fixing solution, cells were incubated with PBS containing 50 ng/ml Nile red for 5 min in the dark according to the literature with some modification [32]. Fluorescence images of Nile red staining were obtained using a fluorescence microscope (OLYMPUS IX71, Tokyo, Japan).

Data are reported as mean  SD of at least three independent experiments unless otherwise indicated. The statistical significance of the difference between determinations was calculated by analysis of variance (ANOVA) using the Tukey-Kramer multiple comparison test. The difference was considered significant when the p value was less than 0.05. 3. Results

2.9. Measurement of 24S-OHC

3.1. Effect of vitamin E on 24S-OHC-induced cell death in SH-SY5Y cells without atRA treatment

Cellular lipids were extracted and performed HPLC analysis as described previously [16] with some modifications. Briefly, the cells in 1 mL PBS were divided into three portions, 400 mL being for total sterol (with saponification), the other 400 mL being for free sterol (without saponification) and 200 mL being for the measurement of protein concentration. Protein concentration was

We previously reported that 10 mM a-Toc3 was observed not to suppress 24S-OHC-induced necroptosis-like cell death [14], and in the present study we decided to re-evaluate this finding using a variety of concentrations of a-Toc3. In the present study, we found that 50 mM a-Toc3 exhibited cytotoxicity, and we also found that, of various concentrations of a-Toc3 tested between 5 mM and

Fig. 1. a-Toc but not a-Toc3 significantly suppressed 24S-OHC-induced cell death in SH-SY5Y cells without treatment of atRA. (A and B) SH-SY5Y cells were treated with a-Toc3 (A) or a-Toc (B) at different concentrations in the presence or absence of 50 mM 24S-OHC for 24 h. Cell viability was measured by WST-8 assay. *P < 0.05, **P < 0.01, when compared with cells treated with 24S-OHC + vehicle; yyP < 0.01, when compared with cells treated with vehicle. (C) Cells were treated with 50 mM a-Toc or 10 mM a-Toc3 in the presence or absence of 20 mM CumOOH for 24 h. Cell viability was measured by WST-8 assay. **P < 0.01, when compared with cells treated with CumOOH + vehicle; yyP < 0.01, when compared with cells treated with CumOOH + a-Toc.

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Fig. 2. a-Toc but not a-Toc3 significantly suppressed 24S-OHC-induced cell death in SH-SY5Y cells treated with atRA. (A-D) SH-SY5Y cells were pretreated with 25 mM atRA for 7 days. (A) Cells were treated with 50 mM 24S-OHC for the indicated period or 1 mM STS for 7 h. Whole cell lysates were immunoblotted with appropriate antibodies as indicated. (B) Cells were pretreated with 20 mM ZVAD and/or 100 mM Nec-1 for 1 h and were then treated with 50 mM 24S-OHC for 24 h. Cell viability was measured by LDH activity assay. **P < 0.01, when compared with cells treated with 24S-OHC + vehicle. (C) Cells were treated with 50 mM a-Toc or 10 mM a-Toc3 in the presence or absence of 50 mM 24S-OHC for 24 h. Cell viability was measured by WST-8 assay. **P < 0.01, when compared with cells treated with 24S-OHC + vehicle. (D) Cells which were preincubated with 10 mM DCFH-DA for 1 h were treated with 50 mM 24S-OHC for 6 h or 100 mM CumOOH for 3 h. Fluorescence was detected using a fluorescence microscope. Representative blight-field (upper) or fluorescence (lower) images are shown. Bar, 100 mm.

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30 mM, 24S-OHC-induced cell death was suppressed only by 10 mM a-Toc3 (Fig. 1A). In contrast, we found that a-Toc suppressed 24S-OHC-induced cell death in a more strikingly dose-dependent manner (Fig. 1B). We also explored the inhibitory effect of 50 mM a-Toc and 10 mM a-Toc3 on CumOOH-induced cell death in which lipid peroxidation had been induced [2], as a result of which it was found that the inhibitory effect of a-Toc3 was greater than that of a-Toc but that CumOOH-induced cell death was significantly inhibited by both a-Toc and a-Toc3 (Fig. 1C), which suggested that the concentrations of a-Toc and a-Toc3 employed were sufficient to suppress lipid peroxidation-mediated cell death in SH-SY5Y cells. 3.2. Effect of vitamin E on 24S-OHC-induced cell death in SH-SY5Y cells with atRA treatment Expression of caspase-8 was evaluated by immunoblotting SHSY5Y cells that had been treated with and without 25 mM atRA for 7 days (Fig. 2A). As a result, it was found that SH-SY5Y cells that had been treated with atRA expressed pro-caspase-8 more strongly than SH-SY5Y cells that had not been treated with atRA. The procaspase-8 expressed in this way was then cleaved by stimulation with the apoptosis inducer STS to give the active form of caspase-8. This was then subjected to 6 h of 24S-OHC stimulation, as a result of which pro-caspase-8 was observed to have disappeared to a degree indistinguishable from that observed in STS-stimulated cells but no active form of caspase-8 was observed. After 24 h of 24S-OHC stimulation, a small amount of the active form of caspase-8 was observed, which though less prominent than was observed during STS stimulation, although 24S-OHC stimulation caused decrease in cell viability to 50% of control (Fig. 2B). Cleaved caspase-3 was observed after 24 h of stimulation with 24S-OHC in atRA-treated cells. It was found that RIPK1 protein was expressed in inverse fashion with respect to the protein levels of caspase8 and disappeared in response to STS stimulation in which caspase8 was activated. The effects of two inhibitors on 24S-OHC-induced cell death in atRA-treated cells were also evaluated under conditions similar to those described in the preceding paragraph (Fig. 2B). 24S-OHCinduced cell death was found to be suppressed slightly by the pancaspase inhibitor ZVAD but not at all by the RIPK1 inhibitor Nec-1, suggesting that 24S-OHC-induced cell death was at least partially caspase-dependent but RIPK1-independent, which we took to be indicative of apoptosis. Combined treatment with both ZVAD and Nec-1 did not produce synergistic inhibitory effect as compared with the sum of the respective inhibitory effects of these two inhibitors when each was used alone. Next, we compared the effects of a-Toc and a-Toc3 on 24SOHC-induced cell death in atRA-treated SH-SY5Y cells. We found that 24S-OHC-induced cell death in atRA-treated SH-SY5Y cells was inhibited by a-Toc but not by a-Toc3 (Fig. 2C), which was similar to what we had observed in cells without atRA-treatment (Fig. 1A and B). As we had previously observed in SH-SY5Y cells without atRA-treatment using the fluorescence probe DCFH-DA [2], our present results showed that reactive oxygen species (ROS) generation was induced in atRA-treated cells in response to CumOOH but not in response to 24S-OHC (Fig. 2D). These results suggest that the radical scavenging antioxidant activity of a-Toc does not play an important role in inhibiting 24S-OHC-induced cell death with or without atRA-treatment.

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3.3. Effect of a-Toc and a-Toc3 on 24S-OHC-induced lipid droplet formation in SH-SY5Y cells with atRA treatment We have identified LD formation by 24S-OHC as an initial key event which occurs upstream of both apoptosis and necroptosis in Jurkat cells [16]. In the present study, we confirmed that 24S-OHCinduced cell death in atRA-treated SH-SY5Y cells was inhibited dramatically by the ACAT1 inhibitor K-604 (Fig. 3A). We then investigated the effect of a-Toc and a-Toc3 as well as K-604 on LD formation induced by 24S-OHC in atRA-treated SH-SY5Y cells using fluorescence microscopy with Nile red staining, as a result of which we found that neither a-Toc nor a-Toc3 affected LD formation induced by 24S-OHC while K-604 significantly inhibited it (Fig. 3B). Furthermore, we measured 24S-OHC with and without saponification of cell lysates after treatment with 24S-OHC in the absence or presence of a-Toc, a-Toc3 or K-604 (Fig. 3C). 24S-OHC (without saponification, open bar) and total 24S-OHC (with saponification, solid bar) were both measured, the difference between these values being taken to be the amount of 24S-OHC esters. Accumulation of 24S-OHC and 24S-OHC esters was observed in 24S-OHC-treated cells, and it being possible to prevent this accumulation of 24S-OHC esters by treatment with K-604 but with neither a-Toc nor a-Toc3. 4. Discussion Whereas our previous studies had shown that 24S-OHC induces necroptosis-like cell death in neuronal cells [19] but induces both apoptosis and necroptosis-like cell death in lymphoma cells [16], the present study showed that 24S-OHC can under certain conditions induce apoptosis in atRA-treated SH-SY5Y neuronal cells. As previously reported in the literature using NB cells [17] and as confirmed by us in the present study, caspase-8 was not expressed in SH-SY5Y cells without atRA-treatment but was induced by atRA-treatment for 7 days. When atRA-treated cells were stimulated with STS in the present study, the cleaved form of caspase-8 was clearly observed along with disappearance of procaspase-8. We also found in the present study that in response to 24S-OHC stimulation, pro-caspase-8 disappeared after 6 h and 24 h but its cleaved form was observed only after 24 h. In the present study, we observed that apoptosis as confirmed by caspase3 cleavage was induced by 24S-OHC in atRA-treated SH-SY5Y cells but that the extent to which this induction of apoptosis occurred was limited as compared with that observed in STS-stimulated cells, which observation was supported by the fact that we found that ZVAD only partially suppressed 24S-OHC-induced cell death. In atRA-treated SH-SY5Y cells, treatment with Nec-1 did not suppress 24S-OHC-induced cell death, and since co-treatment with both ZVAD and Nec-1 did not increase cell viability as compared with treatment with ZVAD alone, this was taken as indication that RIPK1-dependent cell death was not induced in atRA-treated SH-SY5Y cells. Protein levels of RIPK1 in atRA-treated SH-SY5Y cells were lower than in non-treated cells (Fig. 2A), which may explain why RIPK1-dependent cell death did not occur. However, it remains unclear what mechanism might be responsible for the 24S-OHC-induced cell death which we observed in atRA-treated SH-SY5Y cells that was dependent on neither caspases nor RIPK1. It has been known for some time that atRA treatment can induce differentiation of NB cells [33]. Since the population of differentiated versus undifferentiated cells can be evaluated by MAP2 staining [34], we stained atRA-treated cells with an antibody

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Fig. 3. a-Toc and a-Toc 3 did not affect LD formation by 24S-OHC in SH-SY5Y cells with atRA treatment. (A, B) SH-SY5Y cells were pretreated with 25 mM atRA for 7 days. (A) Cells were pretreated with or without 2 mM K-604 for 15 min and were then treated with 50 mM 24S-OHC for 24 h. Cell viability was measured by LDH activity assay **, p < 0.01, when compared with cells treated with 24S-OHC alone. (B) Cells were treated with 50 mM 24S-OHC in the presence or absence of 2 mM K-604 or 50 mM a-Toc or 10 mM a-Toc3 for 6 h. Cells were stained with Nile red. Representative images are shown. Bar, 50 mm. (C) 24S-OHC (without saponification, open bar) and total 24S-OHC (with saponification, solid bar) were both measured under the same treatment conditions of cells shown in (B), the difference between these values being taken to be the amount of 24S-OHC esters.

specific to MAP2 and found that about 30% to 40% of the cells were MAP2 positive (Suppl. Fig. 1). While caspase-8 expression levels have been known to increase during differentiation and contribute to tumor regression by inducing apoptosis [35], it has also been observed that the expression levels of caspase-8 do not always correlate with differentiation levels. Wada et al., actually observed that caspase-8 expression levels increased as MAP2 expression levels decreased [36]. It is therefore reasonable to assume that several types of cell death may in general be occurring in response to 24S-OHC stimulation in atRA-treated SH-SY5Y cells, and that the relative importance of these different types of cell death may in addition change during differentiation. In this regard, caspase-8 is considered to be one among several determinants affecting 24SOHC-induced cell death signaling which should be explored in the near future. It should be of interest to investigate cell death signaling induced by 24S-OHC in differentiated neuronal cells by treatment with several kinds of differentiating inducer as well as atRA.

In the present study, we found that 24S-OHC-induced cell death in SH-SY5Y cells was significantly inhibited by a-Toc but not by a-Toc3. The fact that we observed such significant difference in the effects of a-Toc and a-Toc3 regardless of whether cells had or had not received atRA treatment indicated that cell conditions such as caspase-8 expression level and differentiation were not responsible for the different effects of a-Toc and a-Toc3 that we observed. Furthermore, since CumOOH-induced cell death was inhibited by both types of vitamin E, and since the effect of a-Toc3 was in fact observed to be more pronounced than that of a-Toc, this led us to theorize that the suppressive effect of a-Toc on 24S-OHC-induced cell death could not be due to radical scavenging activity, which thesis was supported by our observation that there was little ROS generation by 24S-OHC (Fig. 2D). Moreover, since cell death was not inhibited by either type of vitamin E when SH-SY5Y cells were pre-treated with either a-Toc or a-Toc3 for 24 h and then were washed to remove the a-Toc or a-Toc3 before being stimulated with 24S-OHC (Suppl. Fig. 2), this suggested to us the possibility

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that the inhibitory effect of a-Toc on 24S-OHC-induced cell death might be attributable to suppression of the mechanism by which 24S-OHC is incorporated into cells at the plasma membrane. Royer et al. reported that unlike g-Toc, a-Toc displayed a specific distribution pattern in plasma membrane, with a propensity to associate with the lipid raft domains, which might account for a clear difference between a-Toc and g-Toc in inhibitory effect on 7ketocholesterol-induced cell death [37]. They showed that a-Toc needed to be added to cells before treatment with 7-ketocholesterol to cause the redistribution of 7-ketocholesterol out of lipid raft domain, and its anti-apoptotic effect was reduced when added together. In contrast, a-Toc needed to be added together with 24SOHC and its pretreatment did not inhibit 24S-OHC-induced cell death, suggesting that 24S-OHC may enter through plasma membrane out of lipid rafts domains. The further investigation using g-Toc should give more information. Similar to our previous observations made in SH-SY5Y cells without atRA treatment [16], we found in the present study as well that ACAT1 inhibitor was effective in suppressing 24S-OHCinduced cell death in atRA-treated cells (Fig. 3A), indicating that ACAT1-mediated esterification of 24S-OHC followed by formation of LD may be initial key events in 24S-OHC-induced cell death regardless of presence or absence of atRA treatment. It is noteworthy that LD formation in SH-SY5Y cells is a specific event which is induced by 24S-OHC but not by other oxysterols such as 7ketocholesterol, 7a-OHC, 7b-OHC, and 22R-OHC [16]. Takahashi et al. have reported that a-Toc can decrease fluidity of phospholipid membranes [38]. It has also been reported that presence of the phytyl side chain in a-Toc increases rigidity of the lipid membrane interior and limits mobility of a-Toc between liposomal membranes, but that presence of the isoprenoid side chain in a-Toc3 does not have similar rigidity-increasing or mobility-reducing effect (Suppl. Fig. 3) [28,39]. In the present study, LD staining with a fluorescent probe showed that neither a-Toc nor a-Toc3 changed the number of LD while ACAT1 inhibitor K-604 diminished it. Furthermore, the quantitative study by using an HPLC showed similar results (Fig. 3B and C). We therefore speculate that a-Toc might inhibit 24S-OHC-induced cell death by affecting the structure of LD or by affecting cell death signaling downstream of LD formation. Further investigation is needed to understand the detailed mechanism by which a-Toc inhibits 24SOHC-induced cell death in neuronal cells. Vitamin E can be expected to prevent demyelinating neurodegenerative diseases such as multiple sclerosis (MS) as well as nondemyelinating neurodegenerative diseases [40]. It is important to protect oligodendrocytes from cytotoxic effects of oxysterols in MS pathogenesis. Nury et al. showed that a-Toc prevented oligodendrocytes from cell death induced by 7-ketocholesterol, 7b-OHC, and 24S-OHC [13]. In conclusion, we report that 24S-OHC induces apoptosis and necroptosis-like cell death in SH-SY5Y cells with and without atRA treatment, respectively. ROS generation is not involved in 24SOHC-induced cell death which is inhibited significantly by a-Toc but not by a-Toc3, indicating that radical scavenging antioxidant activity of a-Toc does not play an important role in the mechanism by which a-Toc inhibits 24S-OHC-induced cell death in SH-SY5Y cells regardless of cells were treated with atRA. Acknowledgements We thank Kowa Co., Ltd. (Aichi, Japan), Eisai Co., Ltd. (Tokyo, Japan), and Tama Biochemical Co., Ltd. (Tokyo, Japan) for providing valuable reagents. We also thank Gerry Peters (JTT K.K., Takatsuki, Japan) for his careful editing of the manuscript. This work was supported in part by Adaptable and Seamless Technology Transfer Program through target-driven R&D, JST to Y.U., the Foundation of

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Please cite this article in press as: T. Nakazawa, et al., Effect of vitamin E on 24(S)-hydroxycholesterol-induced necroptosis-like cell death and apoptosis, J. Steroid Biochem. Mol. Biol. (2016), http://dx.doi.org/10.1016/j.jsbmb.2016.03.003