Cellular antioxidant adaptive survival response to 6-hydroxydopamine-induced nitrosative cell death in C6 glioma cells

Toxicology 283 (2011) 118–128

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Cellular antioxidant adaptive survival response to 6-hydroxydopamine-induced nitrosative cell death in C6 glioma cells Chan Lee a,1 , Gyu Hwan Park b,1 , Jung-Hee Jang c,∗ a b c

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

a r t i c l e

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Article history: Received 4 September 2010 Received in revised form 5 March 2011 Accepted 5 March 2011 Available online 11 March 2011 Keywords: Apoptosis C6 glioma cells Heme oxygenase-1 NF-E2-related factor 2 Nitrosative stress Peroxynitrite Self-defense

a b s t r a c t Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons in the substantia nigra. 6-Hydroxydopamine (6-OHDA) is a catecholaminergic neurotoxin widely used to produce experimental models of PD and has been reported to cause oxidative and/or nitrosative stress. In this study, we have investigated 6-OHDA-induced nitrosative cell death and its self-defense mechanism in C6 glioma cells. Treatment of C6 cells with 6-OHDA increased expression of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO). Furthermore 6-OHDA treatment led to peroxynitrite generation and nitrotyrosine formation. 6-OHDA-induced nitrosative stress ultimately caused apoptotic cell death as determined by decreased Bcl-2/Bax ratio, activation of c-Jun N-termianl kinase (JNK), and cleavage of caspase-3 and poly(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, exposure of C6 glioma cells to 6-OHDA resulted in an increased expression of heme oxygenase-1 (HO-1) and 6OHDA-induced cytotoxicity was effectively suppressed by the HO-1 inducer SnCl2 and aggravated by HO-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 possible involvement of NF-E2-related factor 2 (Nrf2), which plays an important role in the transcriptional regulation of phase II detoxifying and antioxidant enzymes. 6-OHDA treatment increased nuclear translocation and transcriptional activity of Nrf2, which seemed to be partly mediated by activation of upstream kinases such as Akt/protein kinase B (PKB). Taken together these findings suggest that HO-1 up-regulation via Nrf2 activation may mediate the cellular adaptive survival response to 6-OHDA-induced nitrosative cell death in C6 glioma cells. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Parkinson’s disease (PD) is mainly characterized by degeneration of dopaminergic neurons in the specific brain area such as substantia nigra pars compacta (SNpc). One of the widely used experimental models for PD involves treatment of in vitro cells and in vivo animals with 6-hydroxydopamine (6-OHDA). This compound has been responsible for the selective degeneration of dopaminergic neurons implicated in PD. Previous studies have suggested a possible role of 6-OHDA in the pathogenesis of PD, as concentration of 6-OHDA was increased in the brain (Curtius et al.,

∗ Corresponding author at: School of Medicine, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, South Korea. Tel.: +82 53 580 3866. E-mail addresses: [email protected], [email protected] (J.-H. Jang). 1 These authors equally contributed to this work. 0300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2011.03.004

1974) as well as urine (Andrew et al., 1993) of PD patients with l-3,4-dihydroxyphenylalanine (l-DOPA or Levodopa) therapy. The chronic administration of l-DOPA, often causes motor and psychiatric side effects which may be as debilitating as PD itself (Curtius et al., 1974; Andrew et al., 1993). Multiple lines of evidence indicate that oxidative stress is a critical pathogenic factor in PD. The crucial role of oxidative stress in the pathogenesis and progression of PD is supported by a wide array of oxidative markers. These include the high concentration of redox-active iron and diminished levels of antioxidants such as glutathione (GSH) and antioxidant enzymes in SNpc. Due to the oxidative metabolism of dopamine, oxidation of protein, formation of lipid peroxidation products including 4hydroxy-2-nonenal (HNE), and oxidative modification of DNA such as 8-hydroxyguanosine were markedly increased in the brains of patients with PD (Castellani et al., 2002; Zhang et al., 1999). The neurotoxic action of 6-OHDA has been reported to be mediated

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by excess production of reactive oxygen species (ROS), disruption of calcium homeostasis, and inhibition of mitochondrial complexes I and IV (Reichman et al., 1994; Glinka and Youdim, 1995). In addition to oxidative stress, 6-OHDA has been reported to produce reactive nitrogen species (RNS) such as nitric oxide (NO) by elevated expression of inducible nitric oxide synthase (iNOS) especially in neurons (Guo et al., 2005). Particularly, a representative ROS, superoxide anion can rapidly interact with NO and subsequently produce more powerful oxidant peroxynitrite (ONOO− ). Peroxynitrite is known to structurally and functionally modify critical cellular macromolecules and cause oxidative damages, which finally leads to apoptotic cell death (Liaudet et al., 2009). However, the molecular mechanisms underlying 6-OHDA-induced nitrosative cell death in astrocytes are still under investigation and need to be clarified. 6-OHDA selectively damages catecholaminergic neurons due to the presence of dopaminergic transporters as its reuptake carriers, while 6-OHDA at higher concentrations could exert extracellular toxicity towards various cell types that do not express dopaminergic transporters (Blum et al., 2000). Recently it has been reported that astrocytes took up dopamine by both Na+ -dependent and Na+ -independent transport system (Inazu et al., 1999). In normal conditions, astrocytes interact with neurons and play important roles in development, differentiation, maintenance and repair of the neurons by producing neurotrophic factors and eliminating neurotoxic molecules (Takuma et al., 2004). Particularly, astrocytes produced glial cell line-derived neurotrophic factor (GDNF) that can protect dopaminergic neurons from hydrogen peroxide-induced cytotoxicity (Saavedra et al., 2006), and continuous exposure to low levels of GDNF protected nigral dopaminergic neurons in the 6-OHDA-injected mouse model of PD (Cunningham and Su, 2002). Therefore, it is expected that when astrocytes are damaged by 6OHDA, neurons become more susceptible to 6-OHDA-induced cell death. In normal physiological conditions, an array of cellular defense systems exist to counteract oxidative and/or nitrosative damages. These include endogenous antioxidant enzymes such as superoxide dismutase, catalase, and glutathione-related enzymes and cellular antioxidants such as GSH. In this experiment, we have particularly been interested in the role of heme oxygenase-1 (HO-1) against 6-OHDA-induced nitrosative stress. HO is the rate-limiting enzyme in the oxidative degradation of heme and can prevent the heme-catalyzed production of hydroxyl radical from hydrogen peroxide (Schipper, 2000). While degrading and eliminating the potentially toxic free heme, HO produces equimolar biliverdin, ferrus ion, and carbon monoxide (Schipper, 2000). Biliverdin is further metabolized to bilirubin and these two molecules exhibit strong antioxidant properties. Expression of HO-1 is induced not only by its native substrate heme but also by a variety of exogenous non-heme and noxious stimuli such as heavy metals, ␤-amyloid, dopamine, kainic acid, cytokines, prostaglandins, ultraviolet (UV) light, lipopolysaccharide, GSH depletion, cardiac ischemia, reperfusion injury, and 3-morpholinosydnomine hydrochloride (SIN-1) that can provoke direct or indirect oxidative and/or nitrosative stress (Schipper, 2004; Li et al., 2006). Although the HO-1 expression by 6-OHDA-mediated oxidative stress was observed in neuronal cells, the possible involvement of HO-1 in the cellular defense against 6-OHDA-induced nitrosative stress in astrocytes has not been studied yet. Therefore, in the present study, we have investigated the molecular mechanisms of nitrosative stress and cell death induced by 6-OHDA and cellular defense against them through up-regulation of antioxidant enzymes, especially focusing on the role of HO-1 in C6 glioma cells.

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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-(4morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002) were the products of Cayman 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 bovine serum (FBS) and penicillin–streptomycin antibiotic were obtained from Gibco BRL (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 oxidoreductase 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 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-nitrotyrosine, anti-cleaved caspase-3, and anti-phospho-Akt (p-Akt) antibodies and 1,4-diamino-2,3-dicyano1,4-bis(2-aminophenylthio)butadiene (U0126) were supplied by Cell Signaling Technology (Beverly, MA, USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], anti-actin antibody, N-acetyl-l-cysteine (NAC), N-[[3(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), and other 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 incubator at 37 ◦ C. The media were changed every other day and cells were prepared at an appropriate density depending on each experimental scale. For primary astrocyteenriched cell cultures, brain cortices were obtained from 1 to 3 days old neonatal Sprague–Dawley rats. After removing the meninges, the cortices were treated with trypsin and DNase subsequently. The digested cortices were passed through a 100mm mesh. The isolated cells were grown in DMEM/F12 media containing 10% FBS. The purity of astrocytes was determined by immunofluorescence using antibody against glial fibrillary acidic protein (GFAP, Zymed, San Francisco, CA, USA), an astrocyte-specific marker.

2.3. Cytotoxicity assay Cells were plated at a density of 6 × 104 cells/300 ␮l in 48-well plates and cell viability was assessed by MTT reduction assay. After treatments of C6 cells and primary astrocyte-enriched cells with 6-OHDA in the presence or absence of other reagents, MTT solution was added and further incubated for 2 h. Then the formazan products 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 reduction in MTT to the vehicle-treated control cells.

2.4. Immunoblotting After treatments of C6 cells or primary astrocyte-enriched cell with 6-OHDA and other reagents, the cells were collected and lysed with cell lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 mM NaF, 1 mM Na3 VO4 , and protease inhibitor cocktail tablet (Roche Diagnostics, IN, USA)] on ice for 20 min. After centrifugation at 14,000 × g for 15 min at 4 ◦ C, the protein contents were quantified by using the BCA reagent (Pierce, IL, USA). Protein samples (25–35 ␮g) were resolved on 10–12.5% polyacrylamide gels and transferred to a polyvinylidene fluoride (PVDF) membrane (Pall Co., MI, USA). After blocking the membranes with PBST solution [phosphate-buffered saline (PBS) with 0.05% Tween 20 containing 5% non-fat dried milk], the blots were further incubated with 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 (Stressgen, 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) was used to amplify the signal of primary antibodies binding specific target proteins. The signals were developed using the enhanced chemiluminescence (ECL) Western blotting 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 Griess reagent [0.1% N-(naphthyl)ethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid] was mixed and incubated with cell culture media for 30 min at RT. The absorbance at 540 nm was measured with an autonomic microplate reader (Emax, Molecular Devices Co.).

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2.6. Measurement of peroxynitrite generation: DHR 123 staining Formation of peroxynitrite from C6 glioma cells was quantitated by using DHR 123 dye. After treatment of 6-OHDA for indicated time periods, cells were further reacted with 50 ␮M DHR 123 in PBS for 20 min at 37 ◦ C. After three times washing with PBS, cells were solubilized with DMSO and then relative fluorescence intensity was determined by using an autonomic microplate reader (Gemini XS, Molecular Devices Co.) with excitation at 485 nm and emission at 535 nm. The values were expressed as a percentage of fluorescence intensity to the vehicle-treated control cells. 2.7. Immunocytochemistry For the detection of nitrotyrosine levels and nuclear translocation of Nrf2, C6 glioma cells were plated at a density of 1 × 105 cells/500 ␮l in 4-well chamber slide and treated with 6-OHDA in the presence or absence of other reagents. After washing 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% bovine serum albumin (BSA) in PBS] for additional 1 h at RT. The slides were further reacted with primary anti-nitrotyrosine or anti-Nrf2 antibody at 4 ◦ C overnight. After three times washing again with PBS, biotinylated secondary antibody (Santa Cruz Biotechnology) was added for 1 h, followed by incubation with avidin plus biotinylated HRP enzyme reagent (Santa Cruz) for 30 min at RT. The expression and location of specific proteins were determined by using 3,3 -diaminobenzidine (DAB, VECTOR Lab., CA, USA) as substrate. After mounting with 50% (v/v) glycerol, ntirotyrosine- or Nrf2stained images were obtained and recorded with a light microscopy (CXR, LABO AMERICA Inc., CA, USA). 2.8. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling: TUNEL staining For the detection of DNA fragmentation as a marker for apoptosis, TUNEL staining was performed. C6 glioma cells were exposed to 6-OHDA for 24 h in the presence or absence of FeTPPS and fixed with 10% neutral buffered formalin solution for 1 h at RT. After fixation, cells were incubated with 3% hydrogen peroxide in methanol for blocking and then 0.1% Triton X-100 in 0.1% sodium acetate for permeabilisation. After three times washing with PBS, slides were incubated with terminal deoxytransferase (TdT) and digoxigenin-labeled nucleotides for 1 h at 37 ◦ C (Roche Diagnostic GmbH). After additional reaction with anti-digoxigenin peroxidase for 30 min at 37 ◦ C, slides were developed by DAB solution. 2.9. Luciferase promoter assay For the measurement of transcriptional activity of antioxidant response element (ARE), C6 glioma cells were transiently transfected with the ARE-promoter luciferase construct using the DOTAP reagent (Roche Diagnostic GmbH) according to the protocol provided from the manufacturer. After transfection, cells were exposed to 6-OHDA for indicated times, and then lysed with reporter lysis buffer (Luciferase Assay System, Promega, Madison, WI, USA). The cell lysate was mixed with the luciferase assay reagent and relative luciferase activity was monitored by a luminometer (Tuner BioSystems, Sunnyvale, CA, USA). The ␤-galactosidase assay (␤-Galactosidase Enzyme Assay System, Promega) was performed to normalize the luciferase activity.

Fig. 1. 6-OHDA-induced iNOS expression and NO production. (A) C6 glioma cells were exposed to indicate concentrations of 6-OHDA for 24 h and cell viability was measured 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 periods and protein expression of iNOS was determined by Western blot analysis. (C) C6 glioma cells were treated with 250 ␮M 6-OHDA for indicated times and the amount of nitrite released into medium was measured by Griess assay. Data are presented as mean ± S.D. (n = 3). **Significantly different from the vehicle-treated control group (p < 0.01).

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

3. Results

Total RNA was extracted with TRI Reagent (Molecular Research Center, OH, USA) from C6 glioma cells. Total RNA isolated from each treatment was reverse transcribed for 60 min at 42 ◦ C using M-MLV reverse transcriptase (Promega) following the manufacturer’s instruction. Amplification of cDNA was conducted by polymerase chain reaction (PCR) using synthetic specific primers to GCL, NQO1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of primer sets are as follows: GCL, 5 -AGA CAC GGC ATC CTC CAG TT-3 (sense) and 5 -CTG ACA CGT 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 AAG GTC ATC CAT GAC AAC-3 (sense) and 5 -AGT GTA GCC CAG GAT GCC CTT-3 (antisense). Amplification was initiated by denaturation for 5 min at 95 ◦ C, then annealing of 33 cycle for 60 s at 53 ◦ C (GCL), 60 s at 48 ◦ C (NQO1) and 30 s at 57 ◦ C (GAPDH), and subsequent elongation for 60 s at 72 ◦ C. The amplified PCR products were analyzed by 1.5% agarose gel electrophoresis in Tris–borate–EDTA (TBE) buffer and stained with ethidium bromide. The agarose gels were examined under UV light using a gel documentation system (Vilber Lourmet, Marne-la-Vallaee Cedex, France).

3.1. 6-OHDA-induced nitrosative stress and damage

2.11. Statistical analysis The data were expressed as means ± S.D. (n = 3) and statistical analysis for multiple comparisons was performed by one way ANOVA followed by the Tukey’s test using SPSS software (SPSS 12.0 KO for windows). The criterion for statistical significance was p < 0.05.

C6 glioma cells were treated with various concentrations of 6OHDA (0, 100, 200 and 250 ␮M) for 24 h and cell viability was measured by conventional MTT reduction assay. The proportion of viable cells was decreased by 6-OHDA in a concentrationdependent manner (Fig. 1A). At 250 ␮M, 6-OHDA led to a 64.5% decrease in cell viability and this concentration was used for other experiments. To explore the possible involvement of nitrosative stress in 6-OHDA-induced cell death, protein expression of iNOS and subsequent production of NO were measured by Western blot analysis and Griess assay, respectively. The iNOS protein expression increased from 3 h after the 6-OHDA treatment and peaked at 12 h (Fig. 1B). 6-OHDA also elevated the amount of nitrite released into the medium in a time-related manner, which was sustained up to 24 h (Fig. 1C). It has been reported that superoxide anion and NO can rapidly interact to produce more potent oxidant peroxynitrite which plays a key role in mediating neuronal cell death in neurodegenerative

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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 and intracellular accumulation of peroxynitrite was assessed by using DHR 123 dye. Data are presented as mean ± S.D. (n = 3). **Significantly different from the vehicletreated control group (p < 0.01). (B) Nitrotyrosine formation in C6 glioma cells treated with 250 ␮M 6-OHDA for (a) 0 h, (b) 3 h, (c) 12 h, and (d) 24 h as measured by immunocytochemistry using anti-nitrotyrosine specific antibody. Image acquisition conditions are given in Section 2.

disorders. 6-OHDA treatment increased the production of peroxynitrite as measured by using DHR 123 dye which is rapidly oxidized by peroxynitrite to fluorescent rhodamine (Fig. 2A). In accordance with intracellular accumulation of peroxynitrite, formation of nitrotyrosine was also increased as observed by immunocytochemistry (Fig. 2B). Peroxynitrite is highly reactive to induce oxidative damages to critical cellular macromolecules such as proteins. Therefore, selective nitration of tyrosine residues occurs as a consequence of peroxynitrite production. As shown in Fig. 2B, increased cytosolic levels of nitrotyrosine were observed at 12 h after treatment with 6-OHDA. However, the nitrotyrosine immunoreactivity in nucleus was evident at 24 h. 3.2. Protective effect of FeTPPS on 6-OHDA-induced nitrosative cell death To examine the role of peroxynitrite in 6-OHDA-induced cell death, we have utilized a decomposition catalyst of peroxynitrite, FeTPPS. C6 glioma cells were pretreated with FeTPPS for 30 min before incubation with 6-OHDA (250 ␮M) for additional 24 h. FeTPPS effectively attenuated the 6-ODHA-induced cytotoxicity as determined by MTT reduction assay (Fig. 3A). The cells exposed to 250 ␮M 6-OHDA for 24 h exhibited DNA fragmentation, a typical hallmark of apoptosis as measured by TUNEL staining (Fig. 3B). However, 6-OHDA-elevated proportion

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Fig. 3. Protective effect of FeTPPS on the 6-OHDA-induced cytotoxicity and apoptosis. (A) C6 cells were pretreated with 0, 10, 15, 20, and 25 ␮M FeTPPS for 30 min followed by exposure to 250 ␮M 6-OHDA for additional 24 h. Viable cells were determined 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 vehicletreated control group and ## p < 0.01 compared with 6-OHDA-treated alone group. (B) DNA fragmentation was determined by TUNEL staining. C6 glioma cells were treated with 250 ␮M 6-OHDA for 24 h in the presence or absence of FeTPPS. (a) 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 FeTPPS pretreatment (Fig. 3B). 6-OHDA-induced cell death in C6 glioma cells was mediated via apoptosis, which was further verified by some of the distinct markers for apoptotic death. Activation of the JNK cascades has been frequently implicated in neuronal cell death induced by a wide array of toxicants. Treatment of C6 glioma cells with 6-OHDA led to an increase in the phosphorylated form of JNK (p-JNK), while the total JNK expression remained constant (Fig. 4A). FeTPPS effectively blocked 6-OHDA-induced activation of JNK via phosphorylation. We also examined the expression of Bcl-2 family proteins. 6-OHDA treatment increased the expression of proapoptotic Bax, whereas decreased the expression of antiapoptotic Bcl-2 (Fig. 4B). 6-OHDAinduced alterations in the levels of Bcl-2 family proteins were reversed by FeTPPS pretreatment. To further analyze 6-OHDAinduced apoptotic cell death, we have examined the cleavage of caspase-3 and PARP. Treatment of C6 glioma cells with 6-OHDA caused an activation of caspase-3 (Fig. 4C) and PARP (Fig. 4D) via cleavage, which was inhibited by FeTPPS pretreatment.

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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 by using 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 antiapoptotic Bcl-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 by immunoblot analysis using antibodies specifically detecting cleaved forms of caspase-3 (c-caspase3) and PARP (c-PARP), respectively. (E) Quantitative data on the relative expression of apoptosis-related signaling molecules. Values are represented as mean ± S.D. (n = 3). Significantly different between the groups: *p < 0.05 and **p < 0.01 compared with the vehicle-treated control group, # p < 0.05 and ## p < 0.01 compared with 6-OHDA-treated alone group.

3.3. HO-1 up-regulation as self-defense response to 6-OHDA-induced nitrosative cell death To investigate cellular antioxidant defense molecule against 6-OHDA-induced nitrosative stress, C6 glioma cells were treated with 250 ␮M 6-OHDA for indicated time periods and HO-1 protein expression was determined by Western blot analysis. The expression of HO-1 started to increase from 3 h after treatment with 6-OHDA and peaked at 12 h (Fig. 5A). To determine the role of HO-1 up-regulation in 6-OHDA-triggered nitrosative cell death, C6 glioma cells were pretreated with HO-1 inducer SnCl2 or inhibitor ZnPP. Induction of HO-1 expression by SnCl2 attenuated the 6OHDA-induced cytotoxicity (Fig. 5B), which was aggravated by inhibition of HO-1 activity by ZnPP (Fig. 5C) suggesting the cytoprotective role of HO-1. In primary astrocyte-enriched cell cultures, 250 ␮M 6-OHDA also increased protein expression of HO-1 in a time-dependent manner (Fig. 5D). Furthermore, 6-OHDA-induced cytotoxicity in normal rat astrocytes was also ameliorated by induction of HO-1 with SnCl2 and exacerbated by inhibition of HO-1 with ZnPP (Fig. 5E).

To examine whether RNS and/or ROS are involved in 6-OHDAmediated HO-1 expression, we have utilized the peroxynitrite decomposition catalyst FeTPPS, a selective iNOS inhibitor 1400W, and an ROS scavenger NAC. In this study, 6-OHDA-induced HO-1 expression was effectively inhibited by decreasing peroxynitrite levels with FeTPPS (Fig. 6A) as well as suppressing the peroxynitrite generating system such as expression of iNOS and production of superoxide anion with 1400W (Fig. 6B) and NAC (Fig. 6C), respectively. These results suggest a possible involvement of RNS and/or ROS in 6-OHDA-mediated up-regulation of HO-1 as self-defense and adaptive cellular response. In another experiment, we confirmed the protective effects of 1400W (Fig. 6D) and NAC (Fig. 6E) against 6-OHDA-induced cytotoxicity, which were less potent than direct inhibition of peroxynitrite by FeTPPS. 3.4. Activation of Nrf2 as an upstream regulator for HO-1 up-regulation To elucidate an upstream regulator for HO-1 induction, we have examined 6-OHDA-induced activation of Nrf2, as shown in Fig. 7.

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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) were treated 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 ensure equal amount of protein loading. (B, C, and E) Effects of HO-1 induction by SnCl2 (B, E) and HO-1 inhibition by ZnPP (C, E) on the 6-OHDA-induced cytotoxicity. C6 glioma cells (B, C) and primary astrocyte-enriched cells (E) were pretreated with SnCl2 and ZnPP for 1 h and 3 h, respectively. Cell viability was determined by MTT reduction assay after additional incubation with 6-OHDA for 24 h. Data are presented as mean ± S.D. (n = 3). Significantly different between the groups: **p < 0.01 compared with the vehicle-treated control group, # p < 0.05 and ## p < 0.01 compared with 6-OHDA-treated alone group.

The transcription factor Nrf2 regulates the expression of phase II detoxifying and antioxidant enzymes, and contributes to preserve redox homeostasis and cell viability in response to various stress stimuli. Treatment of C6 glioma cells with 6-OHDA caused an increase in the expression of Nrf2 which started from 3 h after the treatment of 6-OHDA (Fig. 7A). We also verified activation of Nrf2 by examining translocation of Nrf2 from cytosol to nucleus using immunocytochemistry (Fig. 7B) and by monitoring transcriptional activation of Nrf2 using ARE-luciferase promoter assay (Fig. 7C). The nuclear accumulation of Nrf2 (Fig. 7B) and the elevated ARE-luciferase promoter activity (Fig. 7C) were evident after 6 h treatment of 6-OHDA. The 6-OHDA-induced Nrf2 activation was also indirectly confirmed by measuring up-regulation of other Nrf2 downstream target enzymes such as GCL and NQO1. The protein as well as mRNA expression of GCL and NQO1 was increased by 6OHDA (250 ␮M) treatment with similar kinetic patterns (Fig. 8). A maximal induction of GCL and NQO1 was achieved at 24 h after treatment with 6-OHDA and the protein expression was directly correlated with mRNA levels (Fig. 8).

3.5. A molecular mechanism for 6-ODHA-induced transient activation of Nrf2 To elucidate the upstream kinases regulating 6-OHDA-induced Nrf2 activation, we have focused on the role of Akt/PKB and ERK1/2. Activation of Akt/PKB and ERK1/2 was assessed by immunoblot analysis with specific antibodies against the phosphorylated forms of these kinases. The time course experiment showed the activation of Akt/PKB and ERK1/2 peaked at 6 h and 3 h after 6-OHDA treatment, respectively (Fig. 9A and B). To properly assess the role of Akt/PKB and ERK1/2, LY294002 [a pharmacological inhibitor of phosphoinositide-3-kinase (PI3K), an upstream of Akt/PKB] and U0126 [a pharmacological inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK1/2), an upstream of ERK1/2] compounds were added to C6 glioma cells 1 h before the addition of 6OHDA. In this study, 6-OHDA-induced expression (Fig. 9C) and nuclear translocation (Fig. 9D) of Nrf2 were selectively inhibited by LY294002 compound. In addition, pretreatment with LY294002 effectively suppressed the 6-OHDA-induced protein expression

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Fig. 6. Possible involvement of ROS/RNS in the 6-OHDA-induced HO-1 expression. C6 glioma cells were pretreated with FeTPPS (A), 1400W (B, D), and NAC (C, E) for 30 min prior to 6-OHDA (250 ␮M) treatment for additional 12 h (A–C) or 24 h (D, E). (A–C) HO-1 protein expression was assessed by Western blot analysis using anti-HO-1 specific antibody. (D, E) The cell viability was measured by MTT dye reduction assay. Values are 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.

of HO-1 which was moderately decreased by U0126 (Fig. 9E). Therefore, we suggest that 6-OHDA-induced Nrf2 activation and subsequent HO-1 expression is likely to be mainly mediated via PI3K signaling pathway. 4. Discussion In this study we have investigated 6-OHDA-induced nitrosative stress and cell death in astrocytes and elucidate a possible molecular mechanism for the intracellular self-defense against them. 6-OHDA is one of the most common neurotoxins used to induce in vitro and in vivo experimental models of PD in neurons and glia including astrocytes. Astrocytes normally regulate the synthesis and release of a variety of neurotransmitters, neurotropic peptides and growth factors (Takuma et al., 2004), which protect and support the functions of neural or other glial cells. Astrocytes are known to be not only less susceptible than neurons to the cytotoxicity caused by ROS and/or RNS but also utilize an effective antioxidant system to protect themselves and other adjacent neuronal cells from ROS and/or RNS-mediated injuries (Takuma et al., 2004). Particularly, astrocytes exhibit a high concentration of GSH, a representative intracellular antioxidant (Shimizu et al., 2002; Zhang et al., 2005). In accordance with their higher capacity for antioxidant defense, astrocytes were less sensitive to 6-OHDA toxicity in comparison with neurons. In the present study, 6-OHDA at relatively high

concentrations caused cytotoxicity in a concentration-dependent manner in C6 glioma cells, as a model system for astrocytes. However, when astrocytes were affected by neurotoxic insults, neurons become more susceptible to apoptotic cell death. Under certain conditions, glial cells could be also harmful by activating pro-inflammatory mechanisms such as induction of iNOS and cyclooxygenase-2 expression and subsequent production of NO, peroxynitrite, prostaglandins, and pro-inflammatory cytokines, which possibly propagate and accelerate the neurodegenerative process in PD (Teismann et al., 2003). In this study, treatment of C6 glioma cells with 6-OHDA increased iNOS expression, NO generation, peroxynitrite production, and nitrotyrosine formation in a time-related manner. Besides oxidative stress, nitrosative stress induced by RNS including peroxynitrite has been reported to play a pivotal role in the pathogenesis of PD (Ebadi and Sharma, 2003). In the cerebrospinal fluid (CSF) with PD patients, the concentration of nitrite was elevated compared with control patients without dopaminergic dysfunction (Qureshi et al., 1995). In the animal models of PD, 6-OHDA treatment increased the levels of hydroxylation and nitration products in specific brain regions such as striatum and substantia nigra (Henze et al., 2005). Peroxynitrite easily nitrates a free and bound tyrosine to form 3-nitrotyrosine, a typical marker of peroxynitrite production. Free 3-nitrotyrosine treatment in mice caused behavioral abnormalities and significantly reduced

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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 by Western blot analysis. Proteins from cell lysates were analyzed by using Nrf2 specific antibody. Actin levels were monitored to ensure equal amount of protein loading. (B) Nrf2 nuclear translocation was assessed by immunocytochemistry using specific anti-Nrf2 antibody. C6 glioma cells were treated with 250 ␮M 6-OHDA for 0 h (a), 3 h (b), and 6 h (c), respectively. Image acquisition procedures are given in Section 2. (C) Relative transcriptional activity of ARE was measured by ARE-luciferase promoter assay as demonstrated in Section 2.

the expression of tyrosine hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of dopamine (Mihm et al., 2001). Furthermore, the two representative proteins related with PD, parkin and synuclein have been reported to be nitrosylated or nitrated by RNS (Chung et al., 2005; Paxinou et al., 2001), which causes functional alterations in these molecules. Moreover, peroxynitrite has been implicated in the apoptosis of dopaminergic neurons in PD (Szabó, 2003). Peroxynitrite is a powerful oxidant that can readily react with a variety of biological target molecules including DNA, protein, and lipid and deplete endogenous protective antioxidant system such as GSH, which ultimately leads to apoptotic cell death (Szabó, 2003). In C6 glioma cells, we also have found that 6-OHDA-induced nitrosative stress subsequently triggered the proapoptotic signals such as phosphorylation of JNK, increased Bax to Bcl-2 ratio, activation of caspase-3, cleavage of PARP, and DNA fragmentation, which were effectively attenuated by pretreatment with FeTPPS, a decomposition catalyst of peroxynitrite.

Although oxidative and/or nitrosative stress can cause neuronal cell death, moderate amounts of ROS and/or RNS may mediate the intracellular signal transduction leading to transcriptional activation of the adaptive genes. The antioxidant defense pathway is one mechanism by which the cells can respond to oxidative and/or nitrosative stress. In agreement with this notion, our present study demonstrates that alterations of the cellular redox status by 6OHDA immediately turn on the cellular signaling cascades in such a way activating HO-1 to rescue the C6 glioma cells from subsequent nitrosative stress. HO-1, stress-inducible type of HO, catalyzes the breakdown 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 finding that pretreatment with HO-1 inducer, SnCl2 attenuated and HO-1 inhibitor, ZnPP aggravated the 6-OHDA-mediated cytotoxicity. In line with this notion, pharmacological induction of HO-1

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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 expression of 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 analysis for 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 mRNA loaded, 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 with 6-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 expression was 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 pretreated with 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-1 protein levels were assessed by Western blot analysis using anti-HO-1 antibody.

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with CoCl2 and moderate overexpression of HO-1 with a retroviral expression vector attenuated 6-OHDA-induced oxidative cell death in PC12 cells (Salinas et al., 2003). Furthermore, the transgenic mice overexpressing HO-1 are less vulnerable to cellular injury caused by ischemic stroke (Panahian et al., 1999) and cerebellar granule cells harvested from HO-1 transgenic mice seemed to be relatively resistant to glutamate- and hydrogen peroxide-induced oxidative injury (Chen et al., 2000). Conversely, pretreatment of SN56 cells with HO-1 antisense oligonucleotides exacerbated the hydrogen peroxide-induced cytotoxicity, which was attenuated by hemin, a HO-1 inducer (Le et al., 1999). The molecular mechanisms of HO-1 up-regulation are not fully clarified but it has been reported that the activated Nrf2 can induce HO-1 expression. Putative Nrf2 binding sites have been found in the 5 -flanking regions of the mouse and human HO-1 genes (Itoh et al., 1997). Hara et al. have reported that apomorphine, a dopamine agonist-induced HO-1 expression was mediated via the translocation of Nrf2 to nucleus and activation of ARE in SHSY5Y human neuroblastoma cells (Hara et al., 2006). Increasing Nrf2 activity by various methods including chemical induction and Nrf2 overexpression or Keap1 siRNA knockdown protected cells from specific type of oxidative damage such as 1-methyl4-phenylpyridinium (MPP+ ), 6-OHDA or SIN-1 (Cao et al., 2005). Conversely, Nrf2 knockout mice are more vulnerable to toxicity induced by MPP+ or rotenone (Lee et al., 2003). In addition, dominant-negative mutation of Nrf2 decreased the HO-1 mRNA levels in response to heme, cadmium, zinc, arsenite, and tertbutylhydroquinone (t-BHQ) (Alam et al., 1999). In this study, we have shown that 6-OHDA treatment increased nuclear translocation and transcriptional activity of Nrf2 and subsequent expression of HO-1. Furthermore, 6-OHDA elevated the mRNA and protein levels of GCL and NQO1, the two other antioxidant target genes of Nrf2 activation. In the next experiment, to elucidate whether ROS and/or RNS could mediate 6-OHDAinduced Nrf2 activation, we have utilized the NAC, a precursor of GSH, 1400W, an inhibitor of iNOS, and FeTPPS, a peroxynitrite decomposition catalyst. The 6-OHDA-elevated induction of HO-1 was effectively suppressed by increasing concentrations of FeTPPS, 1400W, and NAC, supporting the possible involvement of RNS as well as ROS in the 6-OHDA-induced up-regulation of HO-1. However, the inhibitory effect on the expression of HO-1 was most evident when cells were pretreated with FeTPPS. In another experiment, to verify the upstream kinases regulating 6-OHDA-induced Nrf2 activation, we have focused on the role of Akt/PKB and ERK1/2. In this work, we noted that 6-OHDA treatment can mediate Nrf2 activation in C6 glioma cells mainly through Akt/PKB. The PI3K, the upstream of Akt/PKB has emerged as one of the critical factors in antiapoptotic signal transduction against oxidative and/or nitrosative stress (Song et al., 2005). Akt/PKB is a serine/threonine protein kinase that mediates cell survival signals and is fully activated by phosphorylation at Thr 308 and Ser 473 in response to a vast variety of extracellular stimuli (Alessi and Cohen, 1998). Under oxidative stress condition, the activation of PI3K results in depolymerization of actin microfilaments thereby facilitating Nrf2 translocation to the nucleus (Kang et al., 2002). In this study, the phosphorylation of Akt/PKB and ERK1/2 occurred after treatment of C6 glioma cells with 6-OHDA. Moreover, pretreatment of C6 glioma cells with LY294002, a pharmacological inhibitor of PI3K effectively suppressed 6OHDA-induced Nrf2 activation and subsequent HO-1 expression. Therefore, we suggest that 6-OHDA-induced Nrf2 activation and HO-1 expression are likely to be mediated largely through activation of PI3K-Akt/PKB signaling pathway in C6 glioma cells. U0126 partially suppressed the induction of HO-1 by 6-OHDA, while it had little effect on the activation of Nrf2. The discrepancy between inhibition of Nrf2 and HO-1 by U0126 might be due to a possi-

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

ble involvement of other transcription factors in the up-regulation of HO-1. In the promoter region of HO-1, besides ARE, additional binding sites for other transcription factors including activator protein-1 (AP-1) have been identified, which also can be regulated by ERK1/2 as an upstream kinase. In RBA-1 astrocytes, bradykinin up-regulated HO-1 expression via ROS-dependent AP-1 induction as well as Nrf2 activation (Hsieh et al., 2010). The role of PI3K in up-regulation of HO-1 against diverse stimuli has been well documented in other studies. Salinas et al. showed that nerve growth factor (NGF) modulated expression of HO-1 through the PI3K-Akt/PKB survival pathway in 6-OHDA-induced oxidative cell death (Salinas et al., 2003). PI3K-Akt/PKB pathway, but not ERK1/2 was involved in the acquired neuroprotection in SH-SY5Y cells against 6-OHDA following cell–cell interaction with astrocytes (Jiang and Yu, 2005). In SIN-1-treated PC12 cells, PI3K was a key signal-transducing enzyme responsible for the nuclear translocation and enhanced ARE binding of Nrf2 that causes the up-regulation of HO-1 (Li et al., 2006). In summary, 6-OHDA induced nitrosative stress in C6 glioma cells which was determined by increased production of RNS, nitrosative damage, and expression of proapoptotic signaling markers. RNS and/or ROS generated by 6-OHDA treatment can induce the up-regulation of HO-1 expression through the activation of Nrf2 conferring adaptive survival response to 6-OHDA-induced apoptosis in C6 glioma cells. The pharmacological inhibitors of PI3K effectively suppressed Nrf2 activation and subsequent HO-1 expression, suggesting the potential role of this kinase in 6-OHDAmediated 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 for the adaptive survival response to 6-OHDA-induced nitrostative damage and cell death in C6 glioma cells is schematically represented in Fig. 10.

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