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Sulfuretin inhibits 6-hydroxydopamine-induced neuronal cell death via reactive oxygen species-dependent mechanisms in human neuroblastoma SH-SY5Y cells

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Seung-Hwan Kwon, Shi-Xun Ma, Seok-Yong Lee, Choon-Gon Jang ⇑ Department of Pharmacology, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea

a r t i c l e

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Article history: Received 30 September 2013 Received in revised form 14 April 2014 Accepted 27 April 2014 Available online xxxx Keywords: Sulfuretin 6-Hydroxydopamine Reactive oxygen species Neuronal cell death Parkinson’s disease

a b s t r a c t Sulfuretin, a potent anti-oxidant, has been thought to provide health benefits by decreasing the risk of oxidative stress-related diseases. In this study, we investigated the mechanisms of sulfuretin protection of neuronal cells from cell death induced by the Parkinson’s disease (PD)-related neurotoxin 6-hydroxydopamine (6-OHDA). We examined whether sulfuretin acts as an anti-oxidant to reduce oxidative stress and mitochondrial-mediated apoptotic cascade events in 6-OHDA-induced neurotoxicity in SH-SY5Y cells. We also investigated whether sulfuretin specifically acts by inhibiting phosphorylation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3beta (GSK-3b) as well as activation of the nuclear factor-kappa B (NF-jB) pathway. Sulfuretin significantly inhibited neuronal cell death, neurotoxicity, apoptosis, and reactive oxygen species (ROS) production. Sulfuretin also strikingly attenuated 6-OHDA-induced mitochondrial dysfunction. Moreover, sulfuretin significantly attenuated 6-OHDA-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase 1/2 (ERK 1/2) MAPKs, PI3K/Akt, and GSK-3b. Eventually, sulfuretin inhibited 6-OHDA-induced NF-jB translocation to the nucleus induced by 6-OHDA. The results of the current study provide the first evidence that sulfuretin protects SH-SY5Y cells against 6-OHDA-induced neuronal cell death, possibly through inhibition of phosphorylation of MAPK, PI3K/Akt, and GSK-3b, which leads to mitochondrial protection, NF-jB modulations and subsequent suppression of apoptosis via ROS-dependent pathways. Thus, we conclude that sulfuretin may have a potential role for neuroprotection and, therefore, may be used as a therapeutic agent for PD. Ó 2014 Published by Elsevier Ltd.

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1. Introduction

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Parkinson’s disease (PD) is characterized by the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (Menendez et al., 2009), accompanied by a change in striatal dopamine concentration in the midbrain (Henning et al., 2008). PD

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Abbreviations: NAC, N-acetyl-L-cystein; PDTC, ammonium pyrrolidinedithiocarbamate; CAT, catalase; DCFH-DA, 2,70 -dichlorofluorescin diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; ERK 1/2, extracellular signal-regulated kinase 1/2; FBS, fetal bovine serum; GSH, glutathione; GSK-3b, glycogen synthase kinase-3beta; 6-OHDA, 6-hydroxydopamine; JNK, c-Jun N-terminal kinase; PD, Parkinson’s disease; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; NF-jB, nuclear factor-kappa B; MAPK, mitogen-activated protein kinase; MMP, mitochondria membrane potential; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SOD, superoxide dismutase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling. ⇑ Corresponding author. Tel.: +82 31 290 7780; fax: +82 31 292 8800. E-mail address: [email protected] (C.-G. Jang).

leads to tremors at rest, rigidity, slowness or absence of voluntary movement, postural instability, and freezing (Dauer and Przedborski, 2003). Although the etiology of PD is not completely understood, recent studies have suggested that strong oxidative stress, mitochondrial defects, and activation of apoptotic cascade events all known to induce apoptotic cell death in several cellular systems play an important role in its pathogenesis (Blum et al., 2001). The selective dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) has been found endogenously in the brain and urine of PD patients (Curtius et al., 1974). Because 6-OHDA selectively accumulates in dopaminergic neurons, it can be widely used as a selective dopaminergic neurotoxin to induce an experimental PD model in vitro and in vivo. Once inside the cell, 6-OHDA is oxidized quickly by intracellular oxygen to form reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, and hydroxyl radical, which cause selective neuronal death and cascade apoptosis (Gee and Davison, 1989; Kumar et al., 1995; Soto-Otero et al., 2000). These products cause lipid peroxidation, DNA damage,

http://dx.doi.org/10.1016/j.neuint.2014.04.016 0197-0186/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: Kwon, S.-H., et al. Sulfuretin inhibits 6-hydroxydopamine-induced neuronal cell death via reactive oxygen speciesdependent mechanisms in human neuroblastoma SH-SY5Y cells. Neurochem. Int. (2014), http://dx.doi.org/10.1016/j.neuint.2014.04.016

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disorganization of the cytoskeleton, mitochondrial dysfunction, and eventually cell death (Davison et al., 1986; Glinka and Youdim, 1995; Kumar et al., 1995). Particularly, in vivo and in vitro experiments have revealed that anti-oxidants prevent the loss of dopaminergic neurons caused by 6-OHDA, which has been used to establish experimental models of PD. Based on these facts, enhanced oxidative stress is thought to be responsible for the progression of dopaminergic neurodegeneration (Kim et al., 2010; Kwon et al., 2012). ROS formation is a naturally occurring process. Mammalian cells have developed several protective mechanisms to prevent ROS formation or to detoxify ROS. These mechanisms employ molecules called antioxidants as well as protective enzymes such as heme oxygenase-1, glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). Among the various cytoprotective enzymes, a chronic imbalance between formation of ROS with GSH, SOD, and CAT characterizes many pathological processes and disease conditions, including PD (Allen and Tresini, 2000; Kwon et al., 2012). The key molecular components of neuronal damage mechanisms include mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) (Kulich et al., 2007; Kwon et al., 2011a; Wilhelm et al., 2007). Such MAPKs are believed to be crucial to mitochondria-mediated neuronal cell death induced by the catecholaminergic neurotoxin 6-OHDA (Pan et al., 2007). In addition to MAPKs, many reports have suggested that phosphatidylinositol 3-kinase (PI3K)/Akt plays a key role in cell survival of the nervous system (Ha et al., 2003; Nakaso et al., 2008). The survival signaling kinase Akt has been shown to mediate striking neurotrophic and anti-apoptotic effects in a highly destructive neurotoxin model of PD (Ries et al., 2006). In addition, glycogen synthase kinase-3beta (GSK-3b) couples Akt-dependent signaling to regulate the activity of nuclear factor-kappaB (NF-jB) (Rossig et al., 2002). The activation of NF-jB, an important regulator of inflammation and adaptive immunity, is known to initiate apoptotic cell death related to oxidative stress (Mattson and Meffert, 2006; Lee et al., 2013). Activated NF-jB, a p50/p65 heterodimer, translocates to the nucleus where it induces expression of target genes. Moreover, the activation of NF-jB is involved in many neurodegenerative diseases affected by oxidative stress. NF-jB has been reported to be increased in dopaminergic neurons of patients with PD and has also been shown to play a prominent role in 6-OHDA-induced dopaminergic neuronal cell death. Therefore, considering that NF-jB would be an attractive target for the development of novel therapeutic approaches for a range of neurological disorders (Mattson, 2005), research into the activation of NF-jB induced by 6-OHDA in SH-SY5Y cells and the possible inhibitors of this action could contribute greatly to the understanding of the neurotoxicity to neurons and the neuroprotective mechanisms of some medicinal plants Q2 (Choi et al., 2011). Sulfuretin is a major flavonoid isolated from the stem bark of Q3 Albizzia julibrissin and heartwood of Rhus verniciflua (Jung et al., 2003; Kim et al., 2010). It has been used to reduce oxidative stress, platelet aggregation, anti-inflammatory activities, and mutagenesis (Jeon et al., 2006; Lee et al., 2002; Park et al., 2004; Song et al., 2010b). Thus, this compound is thought to provide health benefits by decreasing the risk of various diseases, particularly certain cancers, diabetes, and rheumatoid arthritis (Choi et al., 2003; Jang et al., 2003; Kim et al., 2009; Song et al., 2010b). Nevertheless, no information is available regarding the neuroprotective effects of sulfuretin against the pathogenesis of PD, although the bioactivities of sulfuretin are known to date. Therefore, we

examined whether sulfuretin acts as an antioxidant to reduce oxidative stress and mitochondrial-mediated apoptotic cascade events in Parkinsonian mimetic 6-OHDA-induced neurotoxicity in an in vitro neurodegeneration model. In addition, we investigated whether sulfuretin specifically acts by inhibiting phosphorylation of MAPKs, PI3K/Akt, and GSK-3b as well as activation of the NFjB pathway.

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2. Materials and methods

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2.1. Chemicals and reagents

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Sulfuretin was purchased from Extrasynthese (Genay, France). N-acetyl-L-cystein (NAC), ammonium pyrrolidinedithiocarbamate (PDTC), 2,70 -dichlorofluorescin diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33258, 6-hydroxydopamine hydrochloride, propidium iodide, poly-D-lysine, rhodamine 123, Tween-20, and anti-b-actin antibody were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). U0126 (MEK inhibitor), LY294002 (PI3K/Akt inhibitor), TCS2002 (GSK-3b inhibitor), pifithrin-a-hydrobromide (PFT-a, p53 inhibitor) were purchased from Tocris Cookson, Ltd., (Briston, UK). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS), 0.25% trypsin–EDTA, and a penicillin/ streptomycin mixture were obtained from GIBCO–BRL (Grand Island, NY, USA). Rabbit anti-phospho-Akt (Ser473), rabbit anti-Akt (Ser473), rabbit anti-Bcl-2, rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-poly (ADP-ribose) polymerase (PARP), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204), rabbit anti-ERK1/2 (Thr202/Tyr204), rabbit anti-phospho-GSK-3a/b (Ser21/9), rabbit anti-GSK-3a/b (D75D3), rabbit anti-phospho-JNK (Thr183/ Tyr185), rabbit anti-JNK (Thr183/Tyr185), rabbit anti-NF-jB p65, and anti-rabbit horseradish peroxidase-linked IgG antibodies were purchased from Cell Signaling (Boston, MA, USA). Rabbit anti-Bax, rabbit anti-cytochrome c, rabbit anti-phospho-p38 MAPK (Thr180/ Tyr182), rabbit anti-p38 MAPK (Thr180/Tyr182), and anti-p53 antibodies were purchased from Epitomics (Burlingame, CA, USA). Texas redÒ-conjugated goat anti-rabbit IgG antibody was purchased from Invitrogen (Molecular Probes, Eugene, OR, USA). All other chemicals were of analytical grade.

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2.2. Cell culture and treatment

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SH-SY5Y cells were grown in DMEM supplemented with 10% heat-inactivated FBS and 0.1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells were fed every 3 days and sub-cultured once they reached 80–90% confluency in 100-mm2 cell culture dishes. 6-OHDA was prepared as a 20 mM stock immediately before use and diluted in PBS to the indicated final concentration. The test compounds were dissolved in DMSO and the stock solutions were added directly to the culture media to a final concentration of 0.1% (v/v) DMSO. The control cells were treated with DMSO only and no significant cytotoxicity was observed in any of the experiments (data not shown).

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2.3. Measurement of cell viability

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The MTT assay provides a sensitive measurement of the normal metabolic status of cells, particularly that of mitochondria, which reflects early cellular redox changes. SH-SY5Y cells (2.5  104 cells/well in 96-well plates) were incubated at 37 °C with 200 lM 6-OHDA for 24 h with or without pretreatment with sulfuretin or inhibitor and then treated with MTT solution (5 mg/

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ml) for 2 h. The dark-blue formazan crystals formed in intact cells were dissolved in DMSO, and the absorbance at 540 nm was measured with a microplate reader (SpectraMax 250, Molecular Device, Sunnyvale, CA, USA). The results were expressed as the percentage of MTT reduction relative to the absorbance of control cells.

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2.4. Measurement of the release of lactate dehydrogenase (LDH)

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Extracellular and intracellular LDH activities were spectrophotometrically measured using a Cytotoxicity Cell Death kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. SH-SY5Y cells (2.5  104 cells/well in 96-well plates) were incubated at 37 °C with 200 lM 6-OHDA for 24 h with or without sulfuretin pretreatment and the supernatant was then assayed. 100 ll of reaction mixture was added to each well and incubated for up to 30 min at room temperature. The absorbances of all samples were measured at 490 nm using a microplate reader. LDH release was expressed as the percentage (%) of the total LDH activity (LDH in the medium + LDH in the cell), according to the following equation: % LDH release = (LDH activity in the medium/total LDH activity)  100.

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2.5. Hoechst 33258 and propidium iodide staining assay

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SH-SY5Y cells (5  10 cells/well in 6-well plates) were seeded on coverslips coated with poly-D-lysine for 24 h. Cells were then incubated at 37 °C with 200 lM 6-OHDA for 24 h with or without sulfuretin pretreatment and then washed with PBS and fixed with 4% paraformaldehyde for 15 min. The fixed cells were washed with PBS and stained with 5 lg/ml Hoechst 33258 and propidium iodide for 15 min. Following incubation, the cells were again washed with PBS and observed under a fluorescence microscope (20) (BX51, Olympus Optical Co., Ltd., Tokyo, Japan). 2.6. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay

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Apoptosis was detected by TUNEL analysis using the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. SH-SY5Y cells (5  105 cells/ well in 6-well plates) were seeded on coverslips coated with polyD-lysine for 24 h. The seeded cells were incubated at 37 °C with 200 lM 6-OHDA for another 24 h with or without sulfuretin pretreatment and then washed with PBS and fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, and permeabilized with 0.2% Triton X-100 for 5 min. After two more washes, each glass coverslip was covered with equilibration buffer for 10 min. The buffer was then aspirated and the glass coverslips were incubated with TdT buffer for 1 h at 37 °C. Following incubation, the cells were again washed with PBS and observed under a fluorescence microscope (20).

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2.7. Measurement of intracellular ROS accumulation

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DCFH-DA can be deacetylated in cells, where it can react quantitatively with intracellular radicals (mainly derived from H2O2) to be converted into its fluorescent product, DCF, which is retained within the cells. SH-SY5Y cells (2.5  104 cells/well in 96-well plates) were incubated at 37 °C with 200 lM 6-OHDA for 1 h with or without sulfuretin pretreatment. The cells were rinsed with PBS and 10 lM DCFH-DA was added. After 30 min incubation at 37 °C, cells were examined at 530 nm using a fluorescence spectrophotometer (Perkin Elmer, LS50B, Boston, MA, USA) with excitation at 488 nm. DCFH-DA fluorescence images were then observed using a fluorescence microscope (20).

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2.8. Measurement of intracellular mitochondria membrane potential (MMP)

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MMP was monitored using the fluorescent dye rhodamine 123, which is a cell-permeable cationic dye that preferentially partitions into mitochondria based on the highly negative MMP. SH-SY5Y cells (2.5  104 cells/well in 96-well plates) were incubated at 37 °C with 200 lM 6-OHDA for 6 h with or without sulfuretin pretreatment. The cells were rinsed with PBS and 10 lM rhodamine 123 was added. After 30 min of incubation at 37 °C, cells were examined at 530 nm using a fluorescence spectrophotometer with excitation at 480 nm. Rhodamine 123 fluorescence images were then observed using a fluorescence microscope (20).

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2.9. Measurement of GSH content, SOD, and CAT activities

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SOD and CAT activities as well as GSH content were determined using commercial assay kits (Biovision, Inc., Milpitas, CA, USA). SHSY5Y cells (1  106 cells/well in 6-well plates) were incubated at 37 °C with 200 lM 6-OHDA for 6 h with or without sulfuretin pretreatment. Briefly, at the end of the experiments, the medium was removed and cells were harvested by trypsinization. The cells were then washed with ice-cold PBS and centrifuged at 700g for 5 min. The cell pellet was resuspended in 500 ll of ice-cold assay buffer. The cells were lysed by strong vortex mixing and then the suspension was used for assays according to the manufacturer’s protocols. The absorbance of all samples was measured using a microplate reader (SOD activity, 450 nm; GSH content, 405 nm; CAT activity, 570 nm).

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2.10. Cytosolic and nuclear lysate preparation

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SH-SY5Y cells (1  106 cells/well in 6-well plates) were incubated at 37 °C with 200 lM 6-OHDA for 6 h with or without sulfuretin pretreatment. To measure the activation of NF-jB p65 translocation to the nucleus, nuclear and cytosolic fractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents for cultured cells (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. NF-jB p65 levels were determined by Western blot analysis as described below.

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2.11. Western blot analysis

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SH-SY5Y cells (5  105 cells/well in 6-well plates) were incubated at 37 °C with 200 lM 6-OHDA for the indicated time, with or without sulfuretin pretreatment, and then washed and harvested with ice-cold PBS and centrifuged at 400g for 3 min. The cell pellet was resuspended in 100 ll of ice-cold lysis T-per tissue protein extraction buffer (Thermo Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitor cocktails (Roche Diagnostics, GmbH, Germany) and incubated on ice for 30 min. After centrifugation at 10,000g for 15 min, the supernatant was separated and stored at 70 °C. The protein concentration was determined using a protein assay kit (Thermo Scientific). Proteins were separated on an 8–12% SDS–polyacrylamide gel and then transferred onto a polyvinylidene difluoride transfer membrane (Pall Corporation, Pensacola, FL, USA) that was blocked with 5% skim milk containing 0.5 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20 for 1 h at room temperature. The membrane was subsequently incubated with primary antibody overnight at 4 °C [each antibody at a dilution of 1:1000; caspase-3, caspase-9, PARP, Bcl-2, p53, cytochrome c, phospho-JNK (Thr183/Tyr185), JNK, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK 1/2 (Thr202/Tyr204), ERK 1/2, phospho-Akt (Ser473), Akt (Ser473), and NF-jB p65, except Bax (1:10,000) and cytochrome c (1:5000)]. After three washes with Tris-buffered saline containing

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Fig. 1. Chemical structure of sulfuretin. 311 312 313 314 315 316 317 318 319 320

0.1% Tween-20 (TBST), the blots were incubated with horseradishperoxidase-conjugated secondary antibodies in TBST with 5% nonfat milk at a 1:5000 dilution for 1 h at room temperature. The blots were then washed three times in TBST buffer. Blots were developed using the enhanced chemiluminescence (ECL) detection method by immersing them for 5 min in a mixture of ECL reagents (PerkinElmer, Boston, MA, USA) A and B at a 1:1 ratio and then exposing them to photographic film for a few minutes. Protein bands were quantified by densitometric analysis using Image Gauge 4.0 software (Fujifilm, Stamford, CT, USA).

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2.12. Immunocytochemistry

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SH-SY5Y cells (5  105 cells/well in 6-well plates) were seeded on coverslips coated with poly-D-lysine for 24 h. Cells were then incubated at 37 °C with 200 lM 6-OHDA for 6 h with or without pretreatment of sulfuretin and then washed with PBS and fixed with 4% paraformaldehyde for 15 min. After washing, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were blocked in 5% BSA solution in PBS for 1 h and incubated with anticytochrome c (1:500) and anti- NF-jB p65 (1:250) overnight at 4 °C. Cells were then washed with PBS and incubated for 1 h with Texas redÒ-conjugated goat anti-rabbit IgG antibody (1:500 for cytochrome c and 1:500 for NF-jB p65) and Hoechst 33258 (5 lg/ml). Cells were washed in PBS and mounted on glass slides in Permafluor aqueous mounting fluid. All the procedures were performed at room temperature. Cells were observed under a fluorescence microscope (100).

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2.13. Statistics

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Data were analyzed with Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA) and expressed as means ± S.E.M. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Newman–Keuls test. Statistical significance was set at p < 0.05 (See Fig. 1).

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3. Results

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3.1. Sulfuretin protects against 6-OHDA-induced cell death and cytotoxicity in SH-SY5Y cells

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The dose-dependent effects of sulfuretin on SH-SY5Y cells were assessed. Sulfuretin at concentrations of 2.5, 5, 10, 20, and 40 lM, for 30 min did not have cytotoxic effects on SH-SY5Y cells (Fig. 2A) and so, these concentrations of sulfuretin were used in subsequent experiments. In a previous study, 6-OHDA was found to significantly decrease cell viability in a dose-dependent manner (50–600 lM range; Kwon et al., 2012). Therefore, 200 lM 6-OHDA was used to injure SH-SY5Y cells in all subsequent experiments of the current study. To determine the neuroprotective effects of sulfuretin on 6-OHDA-induced cell death, SH-SY5Y cells were pretreated with sulfuretin at 5, 10, 20, and 40 lM for 30 min prior to exposure to 200 lM 6-OHDA for a further 24 h. As shown in Fig. 2B, the viability of cells incubated with 200 lM 6-OHDA for 24 h was 42.96 ± 4.26% of the control value (p < 0.001), and the viability significantly increased to 53.38 ± 5.66%, 73.86 ± 5.24%, 84.30 ± 8.84%, and 99.22 ± 4.01% when cells were pretreated with

sulfuretin at 5, 10, 20, and 40 lM, respectively (p < 0.05 and p < 0.001). In addition, we used an LDH assay to demonstrate the inhibitory effects of sulfuretin on 6-OHDA-induced cytotoxicity in SH-SY5Y cells. When cells were incubated with 200 lM 6-OHDA for 24 h, the LDH activity of 6-OHDA-treated cells increased significantly to 79.76 ± 4.99% of the LDH activity of control cells (Fig. 2C, p < 0.001). However, pretreatment with sulfuretin at 10, 20, and 40 lM, significantly decreased LDH activity to 52.82 ± 7.10%, 37.90 ± 1.49%, and 29.81 ± 2.03% of control cells, respectively (p < 0.001). To further investigate the neuroprotective effects of sulfuretin, we also performed staining with Hoechst 33258, propidium iodide, and TUNEL, which is another indicator of cell apoptosis and/or toxicity. As illustrated in the microphotographs, Hoechst 33258 and propidium iodide staining indicated nuclear necrosis or nuclear condensation after treatment with 200 lM 6-OHDA (Fig. 2D, upper and middle, respectively). Also, TUNEL staining revealed that DNA fragmentation occurred after treatment with 200 lM 6-OHDA (Fig. 2D, bottom). Interestingly, pretreatment with sulfuretin dramatically inhibited these apoptotic features.

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3.2. Sulfuretin protects against 6-OHDA-induced elevation of intracellular ROS and reduction of MMP in SH-SY5Y cells

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We then investigated intracellular ROS formation and MMP decreases using the fluorescent sensitive probes, DCFH-DA and rhodamine 123, respectively. Treatment with 200 lM 6-OHDA significantly increased intracellular ROS to 347.50 ± 5.26% of the control value (Fig. 3A, p < 0.001 and C, upper), but the 6-OHDAmediated increase in intracellular ROS was significantly inhibited to 226.40 ± 6.16%, 139.10 ± 16.66%, and 109.80 ± 3.23% of control values by sulfuretin pretreatment at 10, 20, and 40 lM, respectively (p < 0.001). On the other hand, treatment with 200 lM 6OHDA significantly decreased intracellular MMP to 50.25 ± 7.51% of the control value (Fig. 3B, p < 0.001 and C, bottom); however, pretreatment with sulfuretin at 10, 20, and 40 lM significantly increased intracellular MMP to 76.06 ± 9.04%, 81.90 ± 10.13%, and 91.54 ± 11.08% of control values, respectively (p < 0.01 and p < 0.001).

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3.3. Sulfuretin inhibits 6-OHDA-induced SOD, CAT, and GSH decreases in SH-SY5Y cells

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As shown in Fig. 4, treatment with 200 lM 6-OHDA significantly decreased SOD, CAT, and GSH activities to 58.50 ± 5.86%, 45.25 ± 2.39%, and 46.84 ± 2.54% of control values, respectively (Fig. 4A–C, p < 0.001). However, pretreatment with 5 lM sulfuretin significantly increased SOD and CAT activities to 64.96 ± 4.12% and 50.35 ± 2.19% of the control values, respectively (p < 0.05 and p < 0.01). Pretreatment with sulfuretin at 10 lM significantly increased SOD, CAT, and GSH activities to 71.82 ± 3.58%, 61.12 ± 3.21%, and 75.85 ± 6.32% of control values, respectively (p < 0.001). Pretreatment with sulfuretin at 20 lM significantly increased SOD, CAT, and GSH activities to 86.37 ± 7.61%, 81.18 ± 4.10%, and 79.52 ± 2.90% of control values, respectively (p < 0.001). Finally, pretreatment with sulfuretin at 40 lM considerably increased SOD, CAT, and GSH activities to 96.59 ± 3.95%, 83.05 ± 2.57%, and 89.75 ± 4.63% of control values, respectively (p < 0.001).

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3.4. Sulfuretin inhibits 6-OHDA-induced up- or down-regulation of p53, Bax, and Bcl-2 levels as well as release of cytochrome c in SHSY5Y cells

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Treatment with 6-OHDA (200 lM, 0–24 h) significantly decreased or increased expression levels of p53, Bax, and Bcl-2,

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6-OHDA

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Hoechst33258

Propidium iodide

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Fig. 2. Effects of sulfuretin on SH-SY5Y cells (A). Cells were treated with the indicated concentrations of sulfuretin for 30 min. Sulfuretin protects against 6-OHDA-induced cell death (B), cytotoxicity (C), and apoptosis ((D), upper and middle) as well as DNA fragmentation ((D), bottom) in SH-SY5Y cells. Cell viability and LDH release were determined by MTT and LDH assays, respectively, and are expressed as percentages of the corresponding values for the control group. In addition, nuclear condensation and fragmentation were assayed using Hoechst 33258, propidium iodide, and/or TUNEL fluorescent dyes. Cells were pretreated with indicated concentrations of sulfuretin for 30 min and then exposed to 200 lM 6-OHDA for 24 h. Representative nuclear morphology was visualized by a fluorescence microscope (20). Cells were pretreated with sulfuretin at the indicated concentrations for 30 min and then further treated with 200 lM 6-OHDA for 24 h. Data are presented as the means ± S.E.M. (n = 6). The images shown are representative of three experiments. ⁄⁄⁄p < 0.001 compared with the control group. #p < 0.05 and ###p < 0.001 compared with the 6-OHDA-treated group. Scale bar: 200 lm.

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as well as release of cytochrome c protein in a time-dependent manner (Fig. S2A–D). Treatment with 200 lM 6-OHDA significantly up-regulated p53 and Bax levels to 172.80 ± 15.38% and 171.50 ± 22.65% of control values, respectively (Fig. 5A and B, p < 0.001). In contrast, treatment with 200 lM 6-OHDA significantly down-regulated Bcl-2 levels to 57.72 ± 5.09% of the control value (Fig. 5C, p < 0.001). However, the up-regulation of Bax was significantly inhibited to 148.50 ± 13.80% of the control value by pretreatment with sulfuretin at 10 lM (p < 0.001), and the downregulation of Bcl-2 was inhibited to 71.88 ± 6.79% of the control value (p < 0.001). Also, pretreatment with sulfuretin at 20 lM significantly inhibited up-regulation of p53 and Bax to 135.50 ± 9.62% and 103.20 ± 4.96% of control values, respectively (p < 0.05 and p < 0.001), while the down-regulation of Bcl-2 was inhibited to 98.23 ± 1.99% of the control value (p < 0.001). Moreover, pretreat-

ment with 40 lM sulfuretin significantly inhibited the 6-OHDAmediated up- or down-regulation of p53, Bax, and Bcl-2 to 106.80 ± 8.82%, 100.20 ± 5.85%, and 98.23 ± 1.99% of control values, respectively (p < 0.001). Furthermore, treatment with 200 lM 6OHDA significantly increased the release of cytochrome c to 172.80 ± 15.38% of the control value (Fig. 5D, p < 0.001). However, the increase of cytochrome c release was significantly inhibited to 118.10 ± 1.06% of the control value by pretreatment with sulfuretin at 10 lM (p < 0.001). Also, pretreatment with sulfuretin at 20 lM and 40 lM significantly inhibited the release of cytochrome c to 106.00 ± 8.56% and 99.24 ± 5.70% of control values, respectively (p < 0.001). In a parallel experiment, immunocytochemistry data for cytochrome c distribution showed a rod-like network in control cells but a diffuse distribution in cells treated with 6-OHDA, which was abolished by sulfuretin (Fig. 5E).

Please cite this article in press as: Kwon, S.-H., et al. Sulfuretin inhibits 6-hydroxydopamine-induced neuronal cell death via reactive oxygen speciesdependent mechanisms in human neuroblastoma SH-SY5Y cells. Neurochem. Int. (2014), http://dx.doi.org/10.1016/j.neuint.2014.04.016

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Fig. 3. Sulfuretin protects against 6-OHDA-induced elevation of intracellular ROS ((A) and (C), upper) and reduction of MMP ((B) and (C), bottom) in SH-SY5Y cells. Representative pictures were taken by a fluorescence microscope (20). In addition, intracellular ROS and MMP abnormalities were assayed using DCFH-DA or rhodamine 123 fluorescent dyes, respectively. Cells were pretreated with the indicated concentrations of sulfuretin for 30 min and then exposed to 200 lM 6-OHDA for 1 or 6 h. Data are presented as the means ± S.E.M. (n = 6). The images shown are representative of three experiments. ⁄⁄⁄p < 0.001 compared with the control group. ##p < 0.01 and ###p < 0.001 compared with the 6-OHDA-treated group. Scale bar: 200 lm.

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Fig. 4. Sulfuretin inhibits 6-OHDA-induced SOD (A), CAT (B), and GSH (C) decreases in SH-SY5Y cells. Cells were pretreated with the indicated concentrations of sulfuretin for 30 min and then exposed to 200 lM 6-OHDA for 6 h. SOD, GSH, and CAT assays were determined by commercially available assay kits and expressed as percentages of the corresponding values for the control group. Data are presented as the means ± S.E.M. (n = 6). ⁄⁄⁄p < 0.001 compared with the control group. ##p < 0.01 and ###p < 0.001 compared with the 6-OHDA-treated group.

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3.5. Sulfuretin inhibits 6-OHDA-induced up-regulation of cleaved caspase-9, cleaved caspase-3, and cleaved PARP levels in SH-SY5Y cells We also examined the preliminary effects of 6-OHDA on apoptosis signaling in SH-SY5Y cells. As shown in Fig. S3A–C, treatment with 6-OHDA increased expression levels of cleaved caspase-9, cleaved caspase-3, and cleaved PARP protein. Treatment with 200 lM 6-OHDA significantly increased cleaved caspase-9, cleaved caspase-3, and cleaved PARP to 220.90 ± 14.03%, 536.60 ± 129.60%,

and 645.10 ± 26.52% of control values, respectively (Fig. 6A–C, p < 0.01 and p < 0.001). However, the increase in cleaved caspase9 expression was significantly suppressed to 171.60 ± 46.78% of the control value by pretreatment with sulfuretin at 10 lM (p < 0.05). Furthermore, pretreatment with 20 lM sulfuretin significantly inhibited up-regulation of cleaved caspase-9 and cleaved PARP to 131.00 ± 18.29% and 433.90 ± 7.86% of control values, respectively, (p < 0.01 and p < 0.001). In contrast, increases in expression levels of cleaved caspase-9, cleaved caspase-3, and

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Fig. 5. Sulfuretin inhibits 6-OHDA-induced up-or down-regulation of p53 (A), Bax (B), and Bcl-2 (C) as well as release of cytochrome c (D) in SH-SY5Y cells. Release of cytochrome c was visualized by immunocytochemistry ((E), 100). Cells were pretreated with the indicated concentrations of sulfuretin for 30 min and then exposed to 200 lM 6-OHDA for 6 h. Levels of p53, Bax, Bcl-2, cytochrome c, and b-actin were evaluated by Western blot analysis. Densitometric results are presented as means ± S.E.M. (n = 3). The images shown are representative of three experiments. ⁄⁄⁄p < 0.001 compared with the control group. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the 6OHDA-treated group. Scale bar: 50 lm.

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Fig. 6. Sulfuretin inhibits 6-OHDA-induced up-regulation of cleaved caspase-9 (A), cleaved caspase-3 (B), and cleaved PARP (C) levels in SH-SY5Y cells. Cells were pretreated with the indicated concentrations of sulfuretin for 30 min and then exposed to 200 lM 6-OHDA for 6 h. Levels of caspase-9, caspase-3, PARP, and b-actin were evaluated by Western blot analysis. Densitometric results are presented as means ± S.E.M. (n = 3). ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 compared with the control group. #p < 0.05, ##p < 0.01, and ### p < 0.001 compared with the 6-OHDA-treated group.

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cleaved PARP were suppressed to 111.50 ± 21.56%, 189.20 ± 68.82%, and 216.40 ± 53.01% of control values, respectively, by pretreatment with sulfuretin at 40 lM (p < 0.01 and p < 0.001). 3.6. Sulfuretin inhibits 6-OHDA-induced phosphorylation of JNK, p38, PI3K/Akt, and GSK-3b levels in SH-SY5Y cells As shown in Fig. S4A–E, evaluation of the phosphorylation statuses of JNK, p38, ERK 1/2, PI3K/Akt, and GSK-3b revealed that maximum phosphorylation levels occurred at 1 h after treatment with 6-OHDA. Treatment with 200 lM 6-OHDA dramatically and/

or rapidly increased phosphorylation of JNK, p38, ERK 1/2, Akt, and GSK-3b to 611.90 ± 75.74%, 155.20 ± 12.96%, 164.40 ± 40.03%, 181.90 ± 30.76%, and 194.00 ± 8.33% of control values, respectively (Fig. 7A–E, p < 0.001). However, the 6-OHDA-mediated increases in phosphorylation of JNK and Akt were significantly inhibited to 296.80 ± 64.47% and 88.74 ± 4.30% of control values, respectively, by pretreatment with sulfuretin at 10 lM (p < 0.001). Also, pretreatment with 20 lM sulfuretin significantly suppressed phosphorylation of JNK, p38, Akt, and GSK-3b to 229.80 ± 21.04%, 115.90 ± 9.57%, 71.43 ± 6.11%, and 102.70 ± 5.21% of control values, respectively (p < 0.01 and p < 0.001). Furthermore, pretreatment

Please cite this article in press as: Kwon, S.-H., et al. Sulfuretin inhibits 6-hydroxydopamine-induced neuronal cell death via reactive oxygen speciesdependent mechanisms in human neuroblastoma SH-SY5Y cells. Neurochem. Int. (2014), http://dx.doi.org/10.1016/j.neuint.2014.04.016

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Fig. 7. Sulfuretin inhibits 6-OHDA-induced phosphorylation of JNK (A), p38 (B), ERK 1/2 (C), PI3K/Akt (D), and GSK-3b (E) in SH-SY5Y cells. Cells were pretreated with indicated concentrations of sulfuretin for 30 min and then exposed to 200 lM 6-OHDA for 1 h. Levels of JNK, p38, ERK 1/2, PI3K/Akt, and GSK-3b were evaluated by Western blot analysis. Densitometric results are presented as means ± S.E.M. (n = 3). ⁄p < 0.05 and ⁄⁄⁄p < 0.001 compared with the control group. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the 6-OHDA-treated group.

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with 40 lM sulfuretin decreased phosphorylation of JNK, p38, Akt, and GSK-3b to 111.90 ± 6.51%, 100.90 ± 7.96%, 56.72 ± 6.79%, and 102.70 ± 5.21% of control values, respectively (p < 0.001). In contrast, phosphorylation levels of ERK 1/2 were not affected. 3.7. Sulfuretin inhibits 6-OHDA-induced activation of NF-jB p65 translocation from the cytosol to the nucleus in SH-SY5Y cells As shown in Fig. S5A, treatment with 6-OHDA significantly activated nuclear translocation of NF-jB p65 and maximum translocation expression levels occurred 6 h after treatment with 6-OHDA. Western blot data revealed that treatment with 200 lM 6-OHDA significantly induced translocation of NF-jB p65 from the cytosol to the nucleus to 57.62 ± 3.96% and 165.3 ± 6.51% of control values, respectively (Fig. 8A, p < 0.001). However, pretreatment with 10 lM sulfuretin significantly inhibited 6-OHDA-induced activation of NF-jB p65 translocation from the cytosol to the nucleus to 86.29 ± 3.75% and 149.40 ± 4.21% of control values, respectively (p < 0.05 and p < 0.001). Furthermore, pretreatment with 20 lM sulfuretin inhibited activation of NF-jB p65 translocation from the cytosol to the nucleus to 94.93 ± 2.53% and 120.40 ± 9.43% of control values, respectively (p < 0.001), whereas the activation of NF-jB p65 translocation from the cytosol to the nucleus was even more strongly inhibited to 98.84 ± 5.34% and 103.90 ± 3.28% of control values at a 40 lM concentration of sulfuretin, indicating a dose-dependent effect (p < 0.001). Simultaneously, these results were confirmed by immunocytochemistry of NF-jB p65 in

SH-SY5Y cells, showing that sulfuretin dramatically attenuated 6-OHDA-induced activation of NF-jB p65 translocation from the cytosol to the nucleus (Fig. 8B).

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In the present study, we demonstrated for the first time that sulfuretin exerted protective effects in a cellular model of PD induced by 6-OHDA. Neuroprotection of sulfuretin has not been investigated in any models of neurodegenerative diseases until now. Therefore, we investigated the possible molecular mechanisms underlying the neuroprotective effects of sulfuretin against 6-OHDA-induced neuronal cell death mediated by elevation of intracellular ROS in human neuroblastoma SH-SY5Y cells. Suppressants or inhibitors of 6-OHDA-induced apoptosis are considered as potential agents for chemopreventive and chemotherapeutic strategies; however, there have been only a few studies performed regarding the protective effects of natural products on 6-OHDA-induced apoptotic cell death. Thus, we initially examined the neuroprotective effects of sulfuretin on 6-OHDAinduced cytotoxicity and/or apoptosis in SH-SY5Y cells using MTT and LDH assays as well as Hoechst 33258, propidium iodide, and TUNEL staining. The results of the present study showed that sulfuretin had protective effects against 6-OHDA-induced cytotoxicity and apoptosis in SH-SY5Y cells and elucidated some of the underlying molecular mechanisms. Several lines of evidence have demonstrated that oxidative stress injury contributes to apoptotic

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Fig. 8. Sulfuretin inhibits 6-OHDA-induced activation of NF-jB p65 translocation from the cytosol to the nucleus in SH-SY5Y cells (A). NF-jB translocation was visualized by immunocytochemistry (B, 100). Cells were pretreated with the indicated concentrations of sulfuretin for 30 min and then exposed to 200 lM 6-OHDA for 6 h. Levels of NF-jB p65 and b-actin were evaluated by Western blot analysis. Densitometric results are presented as means ± S.E.M. (n = 3). The images shown are representative of three experiments. ⁄⁄⁄p < 0.001 compared with the control group. #p < 0.05 and ###p < 0.001 compared with the 6-OHDA-treated group. Scale bar: 50 lm.

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neuronal cell death, and the preventive effects of anti-oxidant natural products on these causative cellular events conclusively blocks cell death (Muller et al., 1997). Although the etiology of PD is still ill-defined, there is growing interest in the phenomena underlying the degeneration process. In particular, oxidative stress has often been put forward as one of the major causes of substantia nigra degeneration. It is worth noting that the general term oxidative stress is commonly and intimately associated with substantia nigral cell death in PD (Abou-Sleiman et al., 2006). Oxidative stress-induced ROS reacts with biological target molecules, induces lipid peroxidation, decreases levels of GSH, SOD, and CAT, and damages the mitochondrial membrane, eventually resulting in the collapse of MMP and cell death (Andersen, 2004; Perry et al., 1982; Saggu et al., 1989). Several independent groups have reported that flavonoids inhibit 6-OHDA-induced apoptosis via the attenuation of intracellular ROS accumulation, decreases in SOD and CAT activities as well as GSH content, and a reduction in MMP, which presumably results in its anti-oxidant function in SH-SY5Y cells (Ikeda et al., 2008; Kwon et al., 2012). Thus, the initial blockade of intracellular ROS generation, anti-oxidant enzyme degradation, and MMP dysfunction might be a very important factor for the protection of neurons. To determine whether the suppression of intracellular ROS production and MMP reduction were effective in preventing apoptosis, anti-oxidant enzymes (SOD, CAT, and GSH) were employed to examine the effects of sulfuretin on oxidative stress induced by 6-OHDA in SH-SY5Y cells. In this study, sulfuretin significantly blocked intracellular ROS production and anti-oxidant enzyme degradation in SH-SY5Y cells exposed to 6-OHDA. Sulfuretin also reduced the decrease in MMP and cell death induced by 6-OHDA. Furthermore, the antioxidant, NAC, significantly inhibited the cell death induced by 6-OHDA in a dose-dependent manner at concentrations of 0.5 mM and above, with complete protection at the concentration of 5 mM (Fig. S1). Therefore, prevention of neuronal cell death by sulfuretin might be partly mediated by a decrease in intracellular ROS production, recovery of anti-oxidant enzymes, and an increase in MMP abnormality. Moreover, our findings that 6-OHDA-induced cell death was blocked by NAC in SH-SY5Y cells strongly support the results of previous studies (Ouyang and Shen, 2006; Yamamuro et al., 2006). Mitochondrial membrane dysfunction appears to be a common event in in oxidative stress and cell signaling, ultimately leading to dopaminergic cell death through the production of ROS in PD (Green and Kroemer, 2004). It is well known that many apoptogenic and/or oxidative stress inducers, such as 6-OHDA, cause MMP dysfunction (Gomez-Lazaro et al., 2008). The Bcl-2 family

consists of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) members. Bcl-2, which resides in the outer mitochondrial membrane, inhibits cytochrome c release (Hengartner, 2000). In contrast, Bax activation and translocation to the mitochondrial membrane might lead to loss of MMP and an increase in mitochondrial permeability (Borner, 2003; Hengartner, 2000). In addition, the release of cytochrome c from damaged mitochondria triggers activation of cleaved caspase-3 through up-regulation of cleaved caspase-9, eventually leading to apoptosis (Junn and Mouradian, 2001; Kwon et al., 2011b). The p53 protein has also been identified as a Q4 critical mediator of neuronal apoptosis (Duan et al., 2002; Tieu et al., 2001). In most cases, p53-mediated apoptosis proceeds through activation of Bax and mitochondrial release of cytochrome c (Schuler et al., 2000), and apoptosis-inducing factor occurs predominantly as a consequence of mitochondrial membrane dysfunction. From our data, we speculated that, at least in part, sulfuretin potentially inhibited 6-OHDA-induced apoptosis through mitochondrial protection. Moreover, our study provides evidence for the activation of the mitochondrial apoptosis pathway in response to 6-OHDA, with a reduction in MMP, release of cytochrome c, up- or down-regulation of p53, Bax, and Bcl-2 levels, as well as activation of cleaved caspase-9, cleaved caspase-3, and cleaved PARP. MAPKs control many cellular events, including differentiation, proliferation, and apoptosis, and to date at least three major MAPK subfamilies have been characterized, JNK, ERK 1/2, and p38 (Lewis et al., 1998). Also, a growing body of evidence suggests that phosphorylation of PI3K/Akt and GSK-3b pathways is a key step in diverse biological processes, including proliferation, growth, survival, and apoptosis (Chen et al., 2004; Ikeda et al., 2008; Li et al., 2011; Schroeter et al., 2002). In particular, MAPK, PI3K/Akt, and GSK-3b signaling pathways have been suggested to play pathological roles in PD. Moreover, phosphorylation of MAPKs, PI3K/Akt, and GSK-3b were enhanced in the post-mortem brain of PD patients. As mentioned in the introduction, MAPKs increase the level of p53, which is a crucial target for modulation of cell death. Subsequently, p53 activation leads to an increase in expression of Bax, which promotes neuronal cell death by apoptosis. The proapoptotic protein Bax induces cytochrome c release from the mitochondria to the cytoplasm, and subsequent activation of cleaved caspase-3 and caspase-9 leads to apoptosis (Li et al., 1997). Moreover, phosphorylation of PI3K/Akt and GSK-3b signaling pathways is important in the inhibition of oxidative stress-induced apoptosis activated by downstream signaling through mitochondrial damage, up-regulation of p53, and activation of cleaved caspase-3 and cleaved PARP. However, it has also been suggested that PI3K/Akt

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and GSK-3b promote activation of pro-apoptotic Bax, which upon aggregation and mitochondrial localization induces cytochrome c release. With respect to sulfuretin, the molecular mechanisms by which this compound inhibits 6-OHDA-induced apoptosis have not been fully understood until now. Thus, the current study investigated whether MAPK, PI3K/Akt, and GSK-3b pathways are involved in the protective effects of sulfuretin on 6-OHDA-induced apoptosis. The results obtained from Western blot analysis strongly suggested that sulfuretin significantly suppressed 6-OHDA-induced oxidative stress and mitochondrial dysfunction by inhibiting the phosphorylation of MAPK, PI3K/Akt, and GSK-3b pathways; however the activity of the ERK 1/2 MAPKs was not affected. In support of these findings, several reports demonstrated that 6-OHDA activated mitochondrial apoptosis pathways via MAPK-, PI3K/Akt-, and GSK-3b-mediated up- or down-regulation of p53, Bax, and Bcl-2 levels, release of cytochrome c, and activation of cleaved caspase-9, cleaved caspase-3, and cleaved PARP (Chen et al., 2004; Gomez-Lazaro et al., 2008; Ikeda et al., 2008; Koh et al., 2003; Kwon et al., 2012; Li et al., 2011; Xiang et al., 2011). NF-jB proteins are ubiquitous transcription factors that are activated in response to oxidative stress and mitochondrial membrane dysfunction (Li et al., 1996). Abundant evidence has shown that NF-jB plays a key and highly complex role in cell survival of the nervous system. NF-jB has also been reported to be increased in dopaminergic neurons of patients with PD (Hunot et al., 1996) and plays a prominent role in 6-OHDA-induced dopaminergic neuronal cell death (Song et al., 2010a). Thus, modulating NF-jB activation could be an important strategy for reducing neuronal injury, and research on the activation of NF-jB induced by 6-OHDA in SH-SY5Y cells and possible inhibitors of NF-jB could contribute to studies on neuronal cell death and neuroprotective mechanisms. In the current study, our Western blot results confirmed significant activation and expression of NF-jB p65 in the nucleus by

treatment with 6-OHDA in SH-SY5Y cells. Furthermore, by immunocytochemistry, we also found that 6-OHDA induced activation of NF-jB p65 translocation from the cytosol to the nucleus and demonstrated for the first time that sulfuretin exerted significant inhibition on the activation and nuclear translocation of NF-jB p65 by 6-OHDA in SH-SY5Y cells. However, in the presence of an NF-jB inhibitor, PDTC, neuronal cell death induced by 6-OHDA in SH-SY5Y cells was significantly ameliorated (Fig. S5B). This data may point to a drug-specific inhibition of NF-jB as a survival determinant for neuronal cells. Recently, 6-OHDA-induced activation of NF-jB translocation was shown to be directly and/or indirectly regulated through the activation of p53, MAPKs, PI3K/Akt, and GSK-3b, which had a significant role in PD (Cunha et al., 2013; Kwon et al., 2012; Liang et al., 2007). Consistent with these previous studies, we found in the current study that 6-OHDA-induced activation of NF-jB translocation was strongly down-regulated by specific inhibitors, U0126 (MEK inhibitor), LY294002 (PI3K/Akt inhibitor), TCS2002 (GSK-3b inhibitor), PFT-a (p53 inhibitor) (Fig. S5C). These results indicate that NF-jB activation may contribute, at least in part, to 6-OHDA-induced degeneration of neuronal cells through a p53, MAPK, PI3K/Akt, and GSK-3b-dependent NFjB signaling pathway. Taken together, our findings indicated that sulfuretin blocked the partially modulation of NF-jB by inhibiting phosphorylation of MAPKs, PI3K/Akt, and GSK-3b, as well as activation of p53. In conclusion, findings from our research indicated that sulfuretin can effectively attenuate mitochondrial dysfunction-mediated cell death induced by 6-OHDA, as summarized in Fig. 9. Considering the importance of our results, sulfuretin markedly attenuated 6-OHDA-induced cell death in SH-SY5Y cells, presumably through inhibition of intracellular ROS accumulation, by decreasing the loss of anti-oxidant molecules such as SOD, GSH, and CAT, as well as activation of MAPKs, PI3K/Akt, GSK-3b, and p53. Furthermore,

Fig. 9. Proposed schematic of the molecular mechanisms for the effects of sulfuretin on 6-OHDA-induced cell death in SH-SY5Y cells. 6-OHDA induces intracellular ROS accumulation, triggering phosphorylation of MAPKs, PI3K/Akt, and GSK-3b, as well as activation of p53. Phosphorylation and activation of these proteins induces mitochondrial dysfunction leading to MMP reduction, up- or down-regulation of Bax and Bcl-2 levels, and subsequent release of cytochrome c, which initiates activation of NF-jB translocation from the cytosol to the nucleus. Moreover, 6-OHDA sequentially triggers increases in the levels of cleaved capase-9, cleaved caspase-3, and cleaved PARP, and finally induces apoptosis. Initially, sulfuretin inhibits intracellular ROS generated from 6-OHDA and sequentially blocks the activation of downstream target molecules such as the phosphorylation of MAPKs, PI3K/Akt, and GSK-3b, activation of p53, up-regulation of cleaved caspase-9, cleaved caspase-3, and cleaved PARP, and the activation of NF-jB translocation, thereby promoting neuronal cell survival and/or inhibiting neuronal cell death.

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the protective effects of sulfuretin against 6-OHDA-induced mitochondrial dysfunction in SH-SY5Y cells was associated with inhibition of decreased MMP, increased cytochrome c release, activation of cleaved caspase-9, cleaved caspase-3, and cleaved PARP, and up- or down-regulation of Bax and Bcl-2. In addition, sulfuretin blocked activation of the NF-jB pathway through possible upstream target molecules such as phosphorylation of MAPKs, PI3K/Akt, and GSK-3b, and activation of p53 to attenuate oxidative stress induced by 6-OHDA in SH-SY5Y cells. To the best of our knowledge, this is the first report demonstrating the neuroprotective effects of sulfuretin on ROS-mediated cell death and/or apoptosis induced by 6-OHDA in SH-SY5Y cells. However, to confirm the involvement of multiple signaling pathways in the mechanism mediating the protective effects of sulfuretin against neurotoxicity, we are currently working to better understand the role of sulfuretin in neurodegenerative diseases such as Alzherimer’s disease and stroke. These further studies will require extensive evaluations regarding the neuroprotective effects of sulfuretin in other animal models involving neurotoxins, such as 1-methyl-4-phenyl-pyridium-induced PD. Our elucidation of the mechanisms underlying the neuroprotective effects of sulfuretin on ROS-mediated cell death induced by 6-OHDA will provide additional insights into the molecular basis of the effects of this compound in clinical settings. Therefore, sulfuretin may provide potential therapeutic compound for the prevention and treatment of PD.

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Conflict of interest statement

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The authors declare that there are no conflicts of interest. 5. Uncited references Burke (2007), Kim et al. (xxxx), Schuler and Green (2001), Song Q5 et al. (xxxx) and Tiong et al. (2010).

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This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A6A3A01027711 and MRC-2012-0009851).

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuint.2014.04. 016.

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Sulfuretin inhibits 6-hydroxydopamine-induced neuronal cell death via reactive oxygen species-dependent mechanisms in human neuroblastoma SH-SY5Y cells.

Sulfuretin, a potent anti-oxidant, has been thought to provide health benefits by decreasing the risk of oxidative stress-related diseases. In this st...
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