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Toxicology 283 (2011) 118–128

Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

ellular antioxidant adaptive survival response to 6-hydroxydopamine-induceditrosative cell death in C6 glioma cells

han Leea,1, Gyu Hwan Parkb,1, Jung-Hee Jangc,∗

College of Oriental Medicine, Daegu Haany University, Daegu 706-828, South KoreaCollege of Pharmacy, CHA University, Seoul 135-080, South KoreaSchool of Medicine, Keimyung University, Daegu 704-701, South Korea

r t i c l e i n f o

rticle history:eceived 4 September 2010eceived in revised form 5 March 2011ccepted 5 March 2011vailable online 11 March 2011

eywords:poptosis6 glioma cellseme oxygenase-1F-E2-related factor 2itrosative stresseroxynitriteelf-defense

a b s t r a c t

Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized byselective loss of dopaminergic neurons in the substantia nigra. 6-Hydroxydopamine (6-OHDA) is a cate-cholaminergic neurotoxin widely used to produce experimental models of PD and has been reported tocause oxidative and/or nitrosative stress. In this study, we have investigated 6-OHDA-induced nitrosativecell death and its self-defense mechanism in C6 glioma cells. Treatment of C6 cells with 6-OHDAincreased expression of inducible nitric oxide synthase (iNOS) and subsequent production of nitricoxide (NO). Furthermore 6-OHDA treatment led to peroxynitrite generation and nitrotyrosine for-mation. 6-OHDA-induced nitrosative stress ultimately caused apoptotic cell death as determined bydecreased Bcl-2/Bax ratio, activation of c-Jun N-termianl kinase (JNK), and cleavage of caspase-3 andpoly(ADP-ribose)polymerase (PARP), which were attenuated by peroxynitrite decomposition catalyst,5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron(III) (FeTPPS). In another experiment, exposureof C6 glioma cells to 6-OHDA resulted in an increased expression of heme oxygenase-1 (HO-1) and 6-OHDA-induced cytotoxicity was effectively suppressed by the HO-1 inducer SnCl2 and aggravated byHO-1 inhibitor zinc protoporphyrin (ZnPP), supporting the cytoprotective role of HO-1. To elucidate the

molecular mechanism underlying 6-OHDA-mediated HO-1 induction, we have examined the possibleinvolvement of NF-E2-related factor 2 (Nrf2), which plays an important role in the transcriptional regula-tion of phase II detoxifying and antioxidant enzymes. 6-OHDA treatment increased nuclear translocationand transcriptional activity of Nrf2, which seemed to be partly mediated by activation of upstream kinasessuch as Akt/protein kinase B (PKB). Taken together these findings suggest that HO-1 up-regulation viaNrf2 activation may mediate the cellular adaptive survival response to 6-OHDA-induced nitrosative celldeath in C6 glioma cells.

. Introduction

Parkinson’s disease (PD) is mainly characterized by degener-tion of dopaminergic neurons in the specific brain area such asubstantia nigra pars compacta (SNpc). One of the widely usedxperimental models for PD involves treatment of in vitro cellsnd in vivo animals with 6-hydroxydopamine (6-OHDA). This com-

ound has been responsible for the selective degeneration ofopaminergic neurons implicated in PD. Previous studies have sug-ested a possible role of 6-OHDA in the pathogenesis of PD, asoncentration of 6-OHDA was increased in the brain (Curtius et al.,

∗ Corresponding author at: School of Medicine, Keimyung University, 2800 Dal-ubeoldaero, Dalseo-Gu, Daegu 704-701, South Korea. Tel.: +82 53 580 3866.

E-mail addresses: pamy202@kmu.ac.kr, pamy@dhu.ac.kr (J.-H. Jang).1 These authors equally contributed to this work.

300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2011.03.004

© 2011 Elsevier Ireland Ltd. All rights reserved.

1974) as well as urine (Andrew et al., 1993) of PD patients withl-3,4-dihydroxyphenylalanine (l-DOPA or Levodopa) therapy. Thechronic administration of l-DOPA, often causes motor and psychi-atric side effects which may be as debilitating as PD itself (Curtiuset al., 1974; Andrew et al., 1993).

Multiple lines of evidence indicate that oxidative stress isa critical pathogenic factor in PD. The crucial role of oxidativestress in the pathogenesis and progression of PD is supportedby a wide array of oxidative markers. These include the highconcentration of redox-active iron and diminished levels of antiox-idants such as glutathione (GSH) and antioxidant enzymes inSNpc. Due to the oxidative metabolism of dopamine, oxidation

of protein, formation of lipid peroxidation products including 4-hydroxy-2-nonenal (HNE), and oxidative modification of DNA suchas 8-hydroxyguanosine were markedly increased in the brains ofpatients with PD (Castellani et al., 2002; Zhang et al., 1999). Theneurotoxic action of 6-OHDA has been reported to be mediated

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C. Lee et al. / Toxico

y excess production of reactive oxygen species (ROS), disruptionf calcium homeostasis, and inhibition of mitochondrial com-lexes I and IV (Reichman et al., 1994; Glinka and Youdim,995).

In addition to oxidative stress, 6-OHDA has been reportedo produce reactive nitrogen species (RNS) such as nitricxide (NO) by elevated expression of inducible nitric oxideynthase (iNOS) especially in neurons (Guo et al., 2005). Par-icularly, a representative ROS, superoxide anion can rapidlynteract with NO and subsequently produce more powerfulxidant peroxynitrite (ONOO−). Peroxynitrite is known to struc-urally and functionally modify critical cellular macromoleculesnd cause oxidative damages, which finally leads to apo-totic cell death (Liaudet et al., 2009). However, the molecularechanisms underlying 6-OHDA-induced nitrosative cell death

n astrocytes are still under investigation and need to belarified.

6-OHDA selectively damages catecholaminergic neurons due tohe presence of dopaminergic transporters as its reuptake carriers,hile 6-OHDA at higher concentrations could exert extracellular

oxicity towards various cell types that do not express dopaminer-ic transporters (Blum et al., 2000). Recently it has been reportedhat astrocytes took up dopamine by both Na+-dependent anda+-independent transport system (Inazu et al., 1999). In normalonditions, astrocytes interact with neurons and play importantoles in development, differentiation, maintenance and repair ofhe neurons by producing neurotrophic factors and eliminatingeurotoxic molecules (Takuma et al., 2004). Particularly, astrocytesroduced glial cell line-derived neurotrophic factor (GDNF) that canrotect dopaminergic neurons from hydrogen peroxide-inducedytotoxicity (Saavedra et al., 2006), and continuous exposure toow levels of GDNF protected nigral dopaminergic neurons in the-OHDA-injected mouse model of PD (Cunningham and Su, 2002).herefore, it is expected that when astrocytes are damaged by 6-HDA, neurons become more susceptible to 6-OHDA-induced celleath.

In normal physiological conditions, an array of cellular defenseystems exist to counteract oxidative and/or nitrosative damages.hese include endogenous antioxidant enzymes such as super-xide dismutase, catalase, and glutathione-related enzymes andellular antioxidants such as GSH. In this experiment, we have par-icularly been interested in the role of heme oxygenase-1 (HO-1)gainst 6-OHDA-induced nitrosative stress. HO is the rate-limitingnzyme in the oxidative degradation of heme and can preventhe heme-catalyzed production of hydroxyl radical from hydro-en peroxide (Schipper, 2000). While degrading and eliminatinghe potentially toxic free heme, HO produces equimolar biliverdin,errus ion, and carbon monoxide (Schipper, 2000). Biliverdin isurther metabolized to bilirubin and these two molecules exhibittrong antioxidant properties. Expression of HO-1 is inducedot only by its native substrate heme but also by a variety ofxogenous non-heme and noxious stimuli such as heavy met-ls, �-amyloid, dopamine, kainic acid, cytokines, prostaglandins,ltraviolet (UV) light, lipopolysaccharide, GSH depletion, car-iac ischemia, reperfusion injury, and 3-morpholinosydnomineydrochloride (SIN-1) that can provoke direct or indirect oxidativend/or nitrosative stress (Schipper, 2004; Li et al., 2006). Althoughhe HO-1 expression by 6-OHDA-mediated oxidative stress wasbserved in neuronal cells, the possible involvement of HO-1 inhe cellular defense against 6-OHDA-induced nitrosative stress instrocytes has not been studied yet.

Therefore, in the present study, we have investigated the molec-lar mechanisms of nitrosative stress and cell death induced by-OHDA and cellular defense against them through up-regulationf antioxidant enzymes, especially focusing on the role of HO-1 in6 glioma cells.

83 (2011) 118–128 119

2. Materials and methods

2.1. Materials

6-Hydroxydopamine was obtained from Sigma–Aldrich (St. Louis, MO, USA).5,10,15,20-Tetrakis(4-sulfonatophenyl)prophyrinato iron(III) (FeTPPS) and 2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002) were the products ofCayman Chemical (Ann Arbor, MI, USA) and Calbiochem (San Diego, CA, USA),respectively. Dihydrorhodamine 123 (DHR 123) was supplied from Invitrogen Co.(Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovineserum (FBS) and penicillin–streptomycin antibiotic were obtained from GibcoBRL (Grand Island, NY, USA). Anti-iNOS, anti-phospho-c-Jun N-terminal kinase(p-JNK), anti-JNK, anti-Bcl-2, anti-Bax, anti-cleaved poly(ADP-ribose)polymerase(PARP), anti-NF-E2-related factor 2 (Nrf2), anti-NAD(P)H:quinine oxidoreduc-tase 1 (NQO1), anti-phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2),anti-ERK, and anti-Akt/protein kinase B (PKB) antibodies were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA, USA). Anti-nitrotyrosine, anti-cleavedcaspase-3, and anti-phospho-Akt (p-Akt) antibodies and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) were supplied by Cell SignalingTechnology (Beverly, MA, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], anti-actin antibody, N-acetyl-l-cysteine (NAC), N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), andother chemicals were obtained from Sigma–Aldrich.

2.2. Cell culture

C6 glioma cells were grown in DMEM supplemented with 10% FBS, penicillin(100 units/ml), and streptomycin (0.1 mg/ml) in a humidified 5% CO2 incubatorat 37 ◦C. The media were changed every other day and cells were prepared at anappropriate density depending on each experimental scale. For primary astrocyte-enriched cell cultures, brain cortices were obtained from 1 to 3 days old neonatalSprague–Dawley rats. After removing the meninges, the cortices were treated withtrypsin and DNase subsequently. The digested cortices were passed through a 100-mm mesh. The isolated cells were grown in DMEM/F12 media containing 10% FBS.The purity of astrocytes was determined by immunofluorescence using antibodyagainst glial fibrillary acidic protein (GFAP, Zymed, San Francisco, CA, USA), anastrocyte-specific marker.

2.3. Cytotoxicity assay

Cells were plated at a density of 6 × 104 cells/300 �l in 48-well plates and cellviability was assessed by MTT reduction assay. After treatments of C6 cells andprimary astrocyte-enriched cells with 6-OHDA in the presence or absence of otherreagents, MTT solution was added and further incubated for 2 h. Then the formazanproducts formed in viable cells were solubilized with dimethyl sulfoxide (DMSO).The optical density at 540 nm was measured using an autonomic microplate reader(Emax, Molecular Device Co., CA, USA). Data were expressed as the percent reductionin MTT to the vehicle-treated control cells.

2.4. Immunoblotting

After treatments of C6 cells or primary astrocyte-enriched cell with 6-OHDAand other reagents, the cells were collected and lysed with cell lysis buffer [50 mMTris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 1 mMNa3VO4, and protease inhibitor cocktail tablet (Roche Diagnostics, IN, USA)] onice for 20 min. After centrifugation at 14,000 × g for 15 min at 4 ◦C, the proteincontents were quantified by using the BCA reagent (Pierce, IL, USA). Protein sam-ples (25–35 �g) were resolved on 10–12.5% polyacrylamide gels and transferredto a polyvinylidene fluoride (PVDF) membrane (Pall Co., MI, USA). After blockingthe membranes with PBST solution [phosphate-buffered saline (PBS) with 0.05%Tween 20 containing 5% non-fat dried milk], the blots were further incubatedwith specific primary antibodies such as anti-iNOS, anti-p-JNK, anti-JNK, anti-Bcl-2,anti-Bax, anti-cleaved-caspase-3, anti-cleaved-PARP, anti-Nrf2, anti-HO-1 (Stress-gen, Ann Arbor, MI, USA), anti-�-glutamylcysteine ligase (GCL, Thermo, Fremont,CA, USA), and anti-actin antibodies. Appropriate horseradish peroxidase (HRP)-conjugated secondary anti-rabbit or anti-mouse secondary antibody (Zymed) wasused to amplify the signal of primary antibodies binding specific target proteins.The signals were developed using the enhanced chemiluminescence (ECL) Westernblotting detection reagent (Amersham Biosciences, Piscataway, NJ, USA).

2.5. Measurement of nitric oxide production: Griess assay

For the determination of nitrite-nitrate derived from NO, equal volume of Griessreagent [0.1% N-(naphthyl)ethylenediamine dihydrochloride and 1% sulfanilamidein 5% phosphoric acid] was mixed and incubated with cell culture media for 30 minat RT. The absorbance at 540 nm was measured with an autonomic microplate reader(Emax, Molecular Devices Co.).

1 logy 283 (2011) 118–128

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Fig. 1. 6-OHDA-induced iNOS expression and NO production. (A) C6 glioma cellswere exposed to indicate concentrations of 6-OHDA for 24 h and cell viability wasmeasured by MTT dye reduction assay. Data are presented as mean ± S.D. (n = 3).(B) Treatment of C6 glioma cells with 250 �M 6-OHDA for indicated time periodsand protein expression of iNOS was determined by Western blot analysis. (C) C6

20 C. Lee et al. / Toxico

.6. Measurement of peroxynitrite generation: DHR 123 staining

Formation of peroxynitrite from C6 glioma cells was quantitated by using DHR23 dye. After treatment of 6-OHDA for indicated time periods, cells were furthereacted with 50 �M DHR 123 in PBS for 20 min at 37 ◦C. After three times washingith PBS, cells were solubilized with DMSO and then relative fluorescence intensityas determined by using an autonomic microplate reader (Gemini XS, Molecularevices Co.) with excitation at 485 nm and emission at 535 nm. The values werexpressed as a percentage of fluorescence intensity to the vehicle-treated controlells.

.7. Immunocytochemistry

For the detection of nitrotyrosine levels and nuclear translocation of Nrf2, C6lioma cells were plated at a density of 1 × 105 cells/500 �l in 4-well chamberlide and treated with 6-OHDA in the presence or absence of other reagents. Afterashing with PBS, cells were fixed with 10% neutral buffered formalin solution

Sigma–Aldrich) for 1 h at RT, and then incubated in blocking buffer [1% bovineerum albumin (BSA) in PBS] for additional 1 h at RT. The slides were further reactedith primary anti-nitrotyrosine or anti-Nrf2 antibody at 4 ◦C overnight. After three

imes washing again with PBS, biotinylated secondary antibody (Santa Cruz Biotech-ology) was added for 1 h, followed by incubation with avidin plus biotinylated HRPnzyme reagent (Santa Cruz) for 30 min at RT. The expression and location of specificroteins were determined by using 3,3′-diaminobenzidine (DAB, VECTOR Lab., CA,SA) as substrate. After mounting with 50% (v/v) glycerol, ntirotyrosine- or Nrf2-

tained images were obtained and recorded with a light microscopy (CXR, LABOMERICA Inc., CA, USA).

.8. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling:UNEL staining

For the detection of DNA fragmentation as a marker for apoptosis, TUNEL stain-ng was performed. C6 glioma cells were exposed to 6-OHDA for 24 h in the presencer absence of FeTPPS and fixed with 10% neutral buffered formalin solution for 1 ht RT. After fixation, cells were incubated with 3% hydrogen peroxide in methanolor blocking and then 0.1% Triton X-100 in 0.1% sodium acetate for permeabili-ation. After three times washing with PBS, slides were incubated with terminaleoxytransferase (TdT) and digoxigenin-labeled nucleotides for 1 h at 37 ◦C (Rocheiagnostic GmbH). After additional reaction with anti-digoxigenin peroxidase for0 min at 37 ◦C, slides were developed by DAB solution.

.9. Luciferase promoter assay

For the measurement of transcriptional activity of antioxidant response ele-ent (ARE), C6 glioma cells were transiently transfected with the ARE-promoter

uciferase construct using the DOTAP reagent (Roche Diagnostic GmbH) accord-ng to the protocol provided from the manufacturer. After transfection, cells werexposed to 6-OHDA for indicated times, and then lysed with reporter lysis bufferLuciferase Assay System, Promega, Madison, WI, USA). The cell lysate was mixedith the luciferase assay reagent and relative luciferase activity was monitored byluminometer (Tuner BioSystems, Sunnyvale, CA, USA). The �-galactosidase assay

�-Galactosidase Enzyme Assay System, Promega) was performed to normalize theuciferase activity.

.10. Reverse transcription-polymerase chain reaction: RT-PCR

Total RNA was extracted with TRI Reagent (Molecular Research Center, OH,SA) from C6 glioma cells. Total RNA isolated from each treatment was reverse

ranscribed for 60 min at 42 ◦C using M-MLV reverse transcriptase (Promega) fol-owing the manufacturer’s instruction. Amplification of cDNA was conducted byolymerase chain reaction (PCR) using synthetic specific primers to GCL, NQO1, andlyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of primer setsre as follows: GCL, 5′-AGA CAC GGC ATC CTC CAG TT-3′ (sense) and 5′-CTG ACAGT AGC CTC GGT AA-3′ (antisense); NQO1, 5′-CAT TCT GAA AGG CTG GTT TGA-3′

sense) and 5′-TTT CTT CCA TCC TTC CAG GAT-3′ (antisense); GAPDH, 5′-GCC AAGTC ATC CAT GAC AAC-3′ (sense) and 5′-AGT GTA GCC CAG GAT GCC CTT-3′ (anti-ense). Amplification was initiated by denaturation for 5 min at 95 ◦C, then annealingf 33 cycle for 60 s at 53 ◦C (GCL), 60 s at 48 ◦C (NQO1) and 30 s at 57 ◦C (GAPDH), andubsequent elongation for 60 s at 72 ◦C. The amplified PCR products were analyzedy 1.5% agarose gel electrophoresis in Tris–borate–EDTA (TBE) buffer and stainedith ethidium bromide. The agarose gels were examined under UV light using a gelocumentation system (Vilber Lourmet, Marne-la-Vallaee Cedex, France).

.11. Statistical analysis

The data were expressed as means ± S.D. (n = 3) and statistical analysis for mul-iple comparisons was performed by one way ANOVA followed by the Tukey’s testsing SPSS software (SPSS 12.0 KO for windows). The criterion for statistical signif-

cance was p < 0.05.

glioma cells were treated with 250 �M 6-OHDA for indicated times and the amountof nitrite released into medium was measured by Griess assay. Data are presented asmean ± S.D. (n = 3). **Significantly different from the vehicle-treated control group(p < 0.01).

3. Results

3.1. 6-OHDA-induced nitrosative stress and damage

C6 glioma cells were treated with various concentrations of 6-OHDA (0, 100, 200 and 250 �M) for 24 h and cell viability wasmeasured by conventional MTT reduction assay. The proportionof viable cells was decreased by 6-OHDA in a concentration-dependent manner (Fig. 1A). At 250 �M, 6-OHDA led to a 64.5%decrease in cell viability and this concentration was used for otherexperiments. To explore the possible involvement of nitrosativestress in 6-OHDA-induced cell death, protein expression of iNOSand subsequent production of NO were measured by Western blotanalysis and Griess assay, respectively. The iNOS protein expres-sion increased from 3 h after the 6-OHDA treatment and peaked at12 h (Fig. 1B). 6-OHDA also elevated the amount of nitrite released

into the medium in a time-related manner, which was sustainedup to 24 h (Fig. 1C).

It has been reported that superoxide anion and NO can rapidlyinteract to produce more potent oxidant peroxynitrite which playsa key role in mediating neuronal cell death in neurodegenerative

C. Lee et al. / Toxicology 283 (2011) 118–128 121

Fig. 2. 6-OHDA-induced peroxynitrite generation and nitrotyrosine formation. (A)C6 glioma cells were exposed to 250 �M 6-ODHA for indicated time periods andintracellular accumulation of peroxynitrite was assessed by using DHR 123 dye.Data are presented as mean ± S.D. (n = 3). **Significantly different from the vehicle-ttic

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Fig. 3. Protective effect of FeTPPS on the 6-OHDA-induced cytotoxicity and apo-ptosis. (A) C6 cells were pretreated with 0, 10, 15, 20, and 25 �M FeTPPS for 30 minfollowed by exposure to 250 �M 6-OHDA for additional 24 h. Viable cells were deter-mined using the MTT reduction assay. Data are presented as mean ± S.D. (n = 3).Significantly different between the groups: **p < 0.01 compared with the vehicle-treated control group and ##p < 0.01 compared with 6-OHDA-treated alone group.(B) DNA fragmentation was determined by TUNEL staining. C6 glioma cells weretreated with 250 �M 6-OHDA for 24 h in the presence or absence of FeTPPS. (a)

reated control group (p < 0.01). (B) Nitrotyrosine formation in C6 glioma cellsreated with 250 �M 6-OHDA for (a) 0 h, (b) 3 h, (c) 12 h, and (d) 24 h as measured bymmunocytochemistry using anti-nitrotyrosine specific antibody. Image acquisitiononditions are given in Section 2.

isorders. 6-OHDA treatment increased the production of per-xynitrite as measured by using DHR 123 dye which is rapidlyxidized by peroxynitrite to fluorescent rhodamine (Fig. 2A).n accordance with intracellular accumulation of peroxynitrite,ormation of nitrotyrosine was also increased as observed bymmunocytochemistry (Fig. 2B). Peroxynitrite is highly reactiveo induce oxidative damages to critical cellular macromoleculesuch as proteins. Therefore, selective nitration of tyrosine residuesccurs as a consequence of peroxynitrite production. As shown inig. 2B, increased cytosolic levels of nitrotyrosine were observedt 12 h after treatment with 6-OHDA. However, the nitrotyrosinemmunoreactivity in nucleus was evident at 24 h.

.2. Protective effect of FeTPPS on 6-OHDA-induced nitrosativeell death

To examine the role of peroxynitrite in 6-OHDA-inducedell death, we have utilized a decomposition catalyst of perox-nitrite, FeTPPS. C6 glioma cells were pretreated with FeTPPSor 30 min before incubation with 6-OHDA (250 �M) for addi-

ional 24 h. FeTPPS effectively attenuated the 6-ODHA-inducedytotoxicity as determined by MTT reduction assay (Fig. 3A).he cells exposed to 250 �M 6-OHDA for 24 h exhibited DNAragmentation, a typical hallmark of apoptosis as measured byUNEL staining (Fig. 3B). However, 6-OHDA-elevated proportion

Vehicle-treated control; (b) 6-OHDA (250 �M) alone; (c) 6-OHDA (250 �M) + FeTPPS(25 �M). Quantitative data is shown in the right panel.

of TUNEL-positive cells was significantly decreased by FeTPPSpretreatment (Fig. 3B).

6-OHDA-induced cell death in C6 glioma cells was mediated viaapoptosis, which was further verified by some of the distinct mark-ers for apoptotic death. Activation of the JNK cascades has beenfrequently implicated in neuronal cell death induced by a widearray of toxicants. Treatment of C6 glioma cells with 6-OHDA ledto an increase in the phosphorylated form of JNK (p-JNK), while thetotal JNK expression remained constant (Fig. 4A). FeTPPS effectivelyblocked 6-OHDA-induced activation of JNK via phosphorylation.We also examined the expression of Bcl-2 family proteins. 6-OHDAtreatment increased the expression of proapoptotic Bax, whereasdecreased the expression of antiapoptotic Bcl-2 (Fig. 4B). 6-OHDA-induced alterations in the levels of Bcl-2 family proteins werereversed by FeTPPS pretreatment. To further analyze 6-OHDA-induced apoptotic cell death, we have examined the cleavage of

caspase-3 and PARP. Treatment of C6 glioma cells with 6-OHDAcaused an activation of caspase-3 (Fig. 4C) and PARP (Fig. 4D) viacleavage, which was inhibited by FeTPPS pretreatment.

122 C. Lee et al. / Toxicology 283 (2011) 118–128

Fig. 4. Effect of FeTPPS on the 6-OHDA-induced proapoptotic signals. C6 glioma cells were incubated with 250 �M 6-OHDA for 24 h in the presence or absence of FeTPPS(10 and 25 �M) and harvested for Western blot analysis. (A) Inhibitory effect of FeTPPS on the 6-OHDA-induced activation of JNK. The activation of JNK was determined byusing anti-phospho-JNK (upper panel) and anti-JNK (lower panel) antibodies. (B) Effect of FeTPPS on the expression of Bcl-2 family proteins. Protein levels of antiapoptoticBcl-2 (upper panel) and proapoptotic Bax (lower panel) were compared. FeTPPS attenuation of 6-OHDA-induced cleavage of caspase-3 (C) and PARP (D) was assessed byimmunoblot analysis using antibodies specifically detecting cleaved forms of caspase-3 (c-caspase3) and PARP (c-PARP), respectively. (E) Quantitative data on the relativee ± S.D.w HDA-t

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xpression of apoptosis-related signaling molecules. Values are represented as meanith the vehicle-treated control group, #p < 0.05 and ##p < 0.01 compared with 6-O

.3. HO-1 up-regulation as self-defense response to-OHDA-induced nitrosative cell death

To investigate cellular antioxidant defense molecule against-OHDA-induced nitrosative stress, C6 glioma cells were treatedith 250 �M 6-OHDA for indicated time periods and HO-1 protein

xpression was determined by Western blot analysis. The expres-ion of HO-1 started to increase from 3 h after treatment with-OHDA and peaked at 12 h (Fig. 5A). To determine the role ofO-1 up-regulation in 6-OHDA-triggered nitrosative cell death, C6lioma cells were pretreated with HO-1 inducer SnCl2 or inhibitornPP. Induction of HO-1 expression by SnCl2 attenuated the 6-HDA-induced cytotoxicity (Fig. 5B), which was aggravated by

nhibition of HO-1 activity by ZnPP (Fig. 5C) suggesting the cyto-rotective role of HO-1. In primary astrocyte-enriched cell cultures,

50 �M 6-OHDA also increased protein expression of HO-1 in aime-dependent manner (Fig. 5D). Furthermore, 6-OHDA-inducedytotoxicity in normal rat astrocytes was also ameliorated by induc-ion of HO-1 with SnCl2 and exacerbated by inhibition of HO-1 withnPP (Fig. 5E).

(n = 3). Significantly different between the groups: *p < 0.05 and **p < 0.01 comparedreated alone group.

To examine whether RNS and/or ROS are involved in 6-OHDA-mediated HO-1 expression, we have utilized the peroxynitritedecomposition catalyst FeTPPS, a selective iNOS inhibitor 1400W,and an ROS scavenger NAC. In this study, 6-OHDA-induced HO-1expression was effectively inhibited by decreasing peroxynitritelevels with FeTPPS (Fig. 6A) as well as suppressing the peroxyni-trite generating system such as expression of iNOS and productionof superoxide anion with 1400W (Fig. 6B) and NAC (Fig. 6C), respec-tively. These results suggest a possible involvement of RNS and/orROS in 6-OHDA-mediated up-regulation of HO-1 as self-defenseand adaptive cellular response. In another experiment, we con-firmed the protective effects of 1400W (Fig. 6D) and NAC (Fig. 6E)against 6-OHDA-induced cytotoxicity, which were less potent thandirect inhibition of peroxynitrite by FeTPPS.

3.4. Activation of Nrf2 as an upstream regulator for HO-1up-regulation

To elucidate an upstream regulator for HO-1 induction, we haveexamined 6-OHDA-induced activation of Nrf2, as shown in Fig. 7.

C. Lee et al. / Toxicology 283 (2011) 118–128 123

Fig. 5. 6-OHDA-induced expression of HO-1. (A, D) Effect of 6-OHDA on the protein expression of HO-1. C6 glioma cells (A) and primary astrocyte-enriched cells (D) weretreated with 250 �M 6-OHDA for indicated times and Western blot analysis was conducted to measure the HO-1 protein expression. Actin levels were monitored to ensuree ) and( for 1a ). Signc .

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qual amount of protein loading. (B, C, and E) Effects of HO-1 induction by SnCl2 (B, EB, C) and primary astrocyte-enriched cells (E) were pretreated with SnCl2 and ZnPPdditional incubation with 6-OHDA for 24 h. Data are presented as mean ± S.D. (n = 3ontrol group, #p < 0.05 and ##p < 0.01 compared with 6-OHDA-treated alone group

he transcription factor Nrf2 regulates the expression of phaseI detoxifying and antioxidant enzymes, and contributes to pre-erve redox homeostasis and cell viability in response to varioustress stimuli. Treatment of C6 glioma cells with 6-OHDA causedn increase in the expression of Nrf2 which started from 3 h afterhe treatment of 6-OHDA (Fig. 7A). We also verified activation ofrf2 by examining translocation of Nrf2 from cytosol to nucleussing immunocytochemistry (Fig. 7B) and by monitoring tran-criptional activation of Nrf2 using ARE-luciferase promoter assayFig. 7C). The nuclear accumulation of Nrf2 (Fig. 7B) and the ele-ated ARE-luciferase promoter activity (Fig. 7C) were evident afterh treatment of 6-OHDA. The 6-OHDA-induced Nrf2 activation waslso indirectly confirmed by measuring up-regulation of other Nrf2ownstream target enzymes such as GCL and NQO1. The protein as

ell as mRNA expression of GCL and NQO1 was increased by 6-HDA (250 �M) treatment with similar kinetic patterns (Fig. 8). Aaximal induction of GCL and NQO1 was achieved at 24 h after

reatment with 6-OHDA and the protein expression was directlyorrelated with mRNA levels (Fig. 8).

HO-1 inhibition by ZnPP (C, E) on the 6-OHDA-induced cytotoxicity. C6 glioma cellsh and 3 h, respectively. Cell viability was determined by MTT reduction assay after

ificantly different between the groups: **p < 0.01 compared with the vehicle-treated

3.5. A molecular mechanism for 6-ODHA-induced transientactivation of Nrf2

To elucidate the upstream kinases regulating 6-OHDA-inducedNrf2 activation, we have focused on the role of Akt/PKB and ERK1/2.Activation of Akt/PKB and ERK1/2 was assessed by immunoblotanalysis with specific antibodies against the phosphorylated formsof these kinases. The time course experiment showed the activa-tion of Akt/PKB and ERK1/2 peaked at 6 h and 3 h after 6-OHDAtreatment, respectively (Fig. 9A and B). To properly assess the roleof Akt/PKB and ERK1/2, LY294002 [a pharmacological inhibitor ofphosphoinositide-3-kinase (PI3K), an upstream of Akt/PKB] andU0126 [a pharmacological inhibitor of mitogen-activated proteinkinase kinase 1/2 (MEK1/2), an upstream of ERK1/2] compounds

were added to C6 glioma cells 1 h before the addition of 6-OHDA. In this study, 6-OHDA-induced expression (Fig. 9C) andnuclear translocation (Fig. 9D) of Nrf2 were selectively inhibitedby LY294002 compound. In addition, pretreatment with LY294002effectively suppressed the 6-OHDA-induced protein expression

124 C. Lee et al. / Toxicology 283 (2011) 118–128

F C6 gliop HO-1a s are mw ed alo

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ig. 6. Possible involvement of ROS/RNS in the 6-OHDA-induced HO-1 expression.rior to 6-OHDA (250 �M) treatment for additional 12 h (A–C) or 24 h (D, E). (A–C)ntibody. (D, E) The cell viability was measured by MTT dye reduction assay. Valueith the vehicle-treated control group and ##p < 0.01 compared with 6-OHDA-treat

f HO-1 which was moderately decreased by U0126 (Fig. 9E).herefore, we suggest that 6-OHDA-induced Nrf2 activation andubsequent HO-1 expression is likely to be mainly mediated viaI3K signaling pathway.

. Discussion

In this study we have investigated 6-OHDA-induced nitrosativetress and cell death in astrocytes and elucidate a possible molec-lar mechanism for the intracellular self-defense against them.-OHDA is one of the most common neurotoxins used to induce

n vitro and in vivo experimental models of PD in neurons and gliancluding astrocytes. Astrocytes normally regulate the synthesisnd release of a variety of neurotransmitters, neurotropic peptidesnd growth factors (Takuma et al., 2004), which protect and supporthe functions of neural or other glial cells. Astrocytes are known toe not only less susceptible than neurons to the cytotoxicity causedy ROS and/or RNS but also utilize an effective antioxidant systemo protect themselves and other adjacent neuronal cells from ROSnd/or RNS-mediated injuries (Takuma et al., 2004). Particularly,

strocytes exhibit a high concentration of GSH, a representativentracellular antioxidant (Shimizu et al., 2002; Zhang et al., 2005).n accordance with their higher capacity for antioxidant defense,strocytes were less sensitive to 6-OHDA toxicity in comparisonith neurons. In the present study, 6-OHDA at relatively high

ma cells were pretreated with FeTPPS (A), 1400W (B, D), and NAC (C, E) for 30 minprotein expression was assessed by Western blot analysis using anti-HO-1 specific

ean ± S.D. (n = 3). Significantly different between the groups: **p < 0.01 comparedne group.

concentrations caused cytotoxicity in a concentration-dependentmanner in C6 glioma cells, as a model system for astrocytes. How-ever, when astrocytes were affected by neurotoxic insults, neuronsbecome more susceptible to apoptotic cell death.

Under certain conditions, glial cells could be also harmful byactivating pro-inflammatory mechanisms such as induction ofiNOS and cyclooxygenase-2 expression and subsequent produc-tion of NO, peroxynitrite, prostaglandins, and pro-inflammatorycytokines, which possibly propagate and accelerate the neurode-generative process in PD (Teismann et al., 2003). In this study,treatment of C6 glioma cells with 6-OHDA increased iNOS expres-sion, NO generation, peroxynitrite production, and nitrotyrosineformation in a time-related manner.

Besides oxidative stress, nitrosative stress induced by RNSincluding peroxynitrite has been reported to play a pivotal rolein the pathogenesis of PD (Ebadi and Sharma, 2003). In the cere-brospinal fluid (CSF) with PD patients, the concentration of nitritewas elevated compared with control patients without dopamin-ergic dysfunction (Qureshi et al., 1995). In the animal modelsof PD, 6-OHDA treatment increased the levels of hydroxylation

and nitration products in specific brain regions such as stria-tum and substantia nigra (Henze et al., 2005). Peroxynitrite easilynitrates a free and bound tyrosine to form 3-nitrotyrosine, a typicalmarker of peroxynitrite production. Free 3-nitrotyrosine treatmentin mice caused behavioral abnormalities and significantly reduced

C. Lee et al. / Toxicology 283 (2011) 118–128 125

Fig. 7. 6-OHDA treatment-induced activation of Nrf2. C6 glioma cells were treated with 250 �M 6-OHDA for indicated time periods. (A) Nrf2 levels were determined byW fic antN nti-Nra latived

tetabf

opcemcsrcao

estern blot analysis. Proteins from cell lysates were analyzed by using Nrf2 specirf2 nuclear translocation was assessed by immunocytochemistry using specific and 6 h (c), respectively. Image acquisition procedures are given in Section 2. (C) Reemonstrated in Section 2.

he expression of tyrosine hydroxylase, the initial and rate-limitingnzyme in the biosynthesis of dopamine (Mihm et al., 2001). Fur-hermore, the two representative proteins related with PD, parkinnd synuclein have been reported to be nitrosylated or nitratedy RNS (Chung et al., 2005; Paxinou et al., 2001), which causesunctional alterations in these molecules.

Moreover, peroxynitrite has been implicated in the apoptosisf dopaminergic neurons in PD (Szabó, 2003). Peroxynitrite is aowerful oxidant that can readily react with a variety of biologi-al target molecules including DNA, protein, and lipid and depletendogenous protective antioxidant system such as GSH, which ulti-ately leads to apoptotic cell death (Szabó, 2003). In C6 glioma

ells, we also have found that 6-OHDA-induced nitrosative stressubsequently triggered the proapoptotic signals such as phospho-

ylation of JNK, increased Bax to Bcl-2 ratio, activation of caspase-3,leavage of PARP, and DNA fragmentation, which were effectivelyttenuated by pretreatment with FeTPPS, a decomposition catalystf peroxynitrite.

ibody. Actin levels were monitored to ensure equal amount of protein loading. (B)f2 antibody. C6 glioma cells were treated with 250 �M 6-OHDA for 0 h (a), 3 h (b),transcriptional activity of ARE was measured by ARE-luciferase promoter assay as

Although oxidative and/or nitrosative stress can cause neuronalcell death, moderate amounts of ROS and/or RNS may mediate theintracellular signal transduction leading to transcriptional activa-tion of the adaptive genes. The antioxidant defense pathway is onemechanism by which the cells can respond to oxidative and/ornitrosative stress. In agreement with this notion, our present studydemonstrates that alterations of the cellular redox status by 6-OHDA immediately turn on the cellular signaling cascades in such away activating HO-1 to rescue the C6 glioma cells from subsequentnitrosative stress. HO-1, stress-inducible type of HO, catalyzes thebreakdown of heme leading to formation of biliverdin/bilirubin,carbon monoxide and ferrous ion and has putative cytoprotective,antiapoptotic, and anti-inflammatory properties (Schipper, 2000).

The present study reveals that HO-1 has a cytoprotective effect

against 6-OHDA-induced nitrosative cell death, based on the find-ing that pretreatment with HO-1 inducer, SnCl2 attenuated andHO-1 inhibitor, ZnPP aggravated the 6-OHDA-mediated cytotox-icity. In line with this notion, pharmacological induction of HO-1

126 C. Lee et al. / Toxicology 283 (2011) 118–128

Fig. 8. 6-OHDA elevated protein and mRNA levels of GCS and/or NQO1. C6 glioma cells incubated 250 �M 6-OHDA for indicated time periods and mRNA and protein expressionof GCL and NQO1 was determined by Western blot analysis and RT-PCR using specific anti-GCL or anti-NQO1 antibody and GCL or NQO1 primer. (A, B) Immunoblot analysisfor GCL (A) and NQO1 (B). (C, D) RT-PCR analysis for GCL (C) and NQO1 (D). Actin and GAPDH levels were measured for the confirmation of equal amount of protein and mRNAloaded, respectively.

Fig. 9. Effects of pharmacological inhibitors of PI3K and MEK1/2 on the 6-OHDA-induced Nrf2 activation and HO-1 expression. (A, B) After treatment of C6 glioma cells with6-OHDA (250 �M) for indicated time periods, the expression of both phosphorylated and total forms of Akt/PKB (A) and ERK1/2 (B) were measured by Western blot analysis.(C) C6 glioma cells were pretreated with LY294002 (10 and 25 �M) or U0126 (10 and 25 �M) for 1 h before 6-OHDA treatment (250 �M) for additional 6 h. Nrf2 expressionwas determined by immunoblot analysis using Nrf2 specific antibody. (D) Nuclear translocation of Nrf2 was verified by immunocytochemistry using anti-Nrf2 antibody. (a)Vehicle-treated control; (b) 6-OHDA (250 �M) alone; (c) 6-OHDA (250 �M) + LY294002 (25 �M); (d) 6-OHDA (250 �M) + U0126 (25 �M). (E) C6 glioma cells were pretreatedwith LY294002 (10 and 25 �M) and U0126 (10 and 25 �M) for 1 h before the 6-OHDA treatment (250 �M) for additional 12 h to measure the expression of HO-1. The HO-1protein levels were assessed by Western blot analysis using anti-HO-1 antibody.

logy 283 (2011) 118–128 127

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ith CoCl2 and moderate overexpression of HO-1 with a retroviralxpression vector attenuated 6-OHDA-induced oxidative cell deathn PC12 cells (Salinas et al., 2003). Furthermore, the transgenic miceverexpressing HO-1 are less vulnerable to cellular injury causedy ischemic stroke (Panahian et al., 1999) and cerebellar granuleells harvested from HO-1 transgenic mice seemed to be relativelyesistant to glutamate- and hydrogen peroxide-induced oxidativenjury (Chen et al., 2000). Conversely, pretreatment of SN56 cells

ith HO-1 antisense oligonucleotides exacerbated the hydrogeneroxide-induced cytotoxicity, which was attenuated by hemin, aO-1 inducer (Le et al., 1999).

The molecular mechanisms of HO-1 up-regulation are not fullylarified but it has been reported that the activated Nrf2 can induceO-1 expression. Putative Nrf2 binding sites have been found

n the 5′-flanking regions of the mouse and human HO-1 genesItoh et al., 1997). Hara et al. have reported that apomorphine,

dopamine agonist-induced HO-1 expression was mediated viahe translocation of Nrf2 to nucleus and activation of ARE in SH-Y5Y human neuroblastoma cells (Hara et al., 2006). Increasingrf2 activity by various methods including chemical inductionnd Nrf2 overexpression or Keap1 siRNA knockdown protectedells from specific type of oxidative damage such as 1-methyl--phenylpyridinium (MPP+), 6-OHDA or SIN-1 (Cao et al., 2005).onversely, Nrf2 knockout mice are more vulnerable to toxic-

ty induced by MPP+ or rotenone (Lee et al., 2003). In addition,ominant-negative mutation of Nrf2 decreased the HO-1 mRNA

evels in response to heme, cadmium, zinc, arsenite, and tert-utylhydroquinone (t-BHQ) (Alam et al., 1999).

In this study, we have shown that 6-OHDA treatment increaseduclear translocation and transcriptional activity of Nrf2 and sub-equent expression of HO-1. Furthermore, 6-OHDA elevated theRNA and protein levels of GCL and NQO1, the two other antiox-

dant target genes of Nrf2 activation. In the next experiment,o elucidate whether ROS and/or RNS could mediate 6-OHDA-nduced Nrf2 activation, we have utilized the NAC, a precursorf GSH, 1400W, an inhibitor of iNOS, and FeTPPS, a peroxynitriteecomposition catalyst. The 6-OHDA-elevated induction of HO-1as effectively suppressed by increasing concentrations of FeTPPS,

400W, and NAC, supporting the possible involvement of RNS asell as ROS in the 6-OHDA-induced up-regulation of HO-1. How-

ver, the inhibitory effect on the expression of HO-1 was mostvident when cells were pretreated with FeTPPS.

In another experiment, to verify the upstream kinases regulat-ng 6-OHDA-induced Nrf2 activation, we have focused on the rolef Akt/PKB and ERK1/2. In this work, we noted that 6-OHDA treat-ent can mediate Nrf2 activation in C6 glioma cells mainly throughkt/PKB. The PI3K, the upstream of Akt/PKB has emerged as onef the critical factors in antiapoptotic signal transduction againstxidative and/or nitrosative stress (Song et al., 2005). Akt/PKB isserine/threonine protein kinase that mediates cell survival sig-als and is fully activated by phosphorylation at Thr 308 and Ser73 in response to a vast variety of extracellular stimuli (Alessi andohen, 1998). Under oxidative stress condition, the activation ofI3K results in depolymerization of actin microfilaments therebyacilitating Nrf2 translocation to the nucleus (Kang et al., 2002).

In this study, the phosphorylation of Akt/PKB and ERK1/2ccurred after treatment of C6 glioma cells with 6-OHDA.oreover, pretreatment of C6 glioma cells with LY294002,pharmacological inhibitor of PI3K effectively suppressed 6-

HDA-induced Nrf2 activation and subsequent HO-1 expression.herefore, we suggest that 6-OHDA-induced Nrf2 activation and

O-1 expression are likely to be mediated largely through activa-

ion of PI3K-Akt/PKB signaling pathway in C6 glioma cells. U0126artially suppressed the induction of HO-1 by 6-OHDA, while itad little effect on the activation of Nrf2. The discrepancy between

nhibition of Nrf2 and HO-1 by U0126 might be due to a possi-

Fig. 10. A schematic diagram of the cellular adaptive survival response to 6-OHDA-induced nitrostative cell death in C6 glioma cells.

ble involvement of other transcription factors in the up-regulationof HO-1. In the promoter region of HO-1, besides ARE, additionalbinding sites for other transcription factors including activatorprotein-1 (AP-1) have been identified, which also can be regulatedby ERK1/2 as an upstream kinase. In RBA-1 astrocytes, bradykininup-regulated HO-1 expression via ROS-dependent AP-1 inductionas well as Nrf2 activation (Hsieh et al., 2010).

The role of PI3K in up-regulation of HO-1 against diverse stimulihas been well documented in other studies. Salinas et al. showedthat nerve growth factor (NGF) modulated expression of HO-1through the PI3K-Akt/PKB survival pathway in 6-OHDA-inducedoxidative cell death (Salinas et al., 2003). PI3K-Akt/PKB pathway,but not ERK1/2 was involved in the acquired neuroprotection inSH-SY5Y cells against 6-OHDA following cell–cell interaction withastrocytes (Jiang and Yu, 2005). In SIN-1-treated PC12 cells, PI3Kwas a key signal-transducing enzyme responsible for the nucleartranslocation and enhanced ARE binding of Nrf2 that causes theup-regulation of HO-1 (Li et al., 2006).

In summary, 6-OHDA induced nitrosative stress in C6 gliomacells which was determined by increased production of RNS,nitrosative damage, and expression of proapoptotic signalingmarkers. RNS and/or ROS generated by 6-OHDA treatment caninduce the up-regulation of HO-1 expression through the activationof Nrf2 conferring adaptive survival response to 6-OHDA-inducedapoptosis in C6 glioma cells. The pharmacological inhibitors ofPI3K effectively suppressed Nrf2 activation and subsequent HO-1expression, suggesting the potential role of this kinase in 6-OHDA-mediated HO-1 up-regulation as well as Nrf2 activation. However,the complete molecular mechanism that coordinates all these

events needs to be clarified. A putative molecular mechanism forthe adaptive survival response to 6-OHDA-induced nitrostativedamage and cell death in C6 glioma cells is schematically repre-sented in Fig. 10.

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onflict of interest statement

There is no conflict of interest.

cknowledgements

This work was supported by the National Research Foundationf Korea (NRF) grant funded by the Korea Government (MEST:inistry of Education, Science and Technology) (Grant Number:

31-2007-1-E00042).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.tox.2011.03.004.

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