Neurol Sci (2010) 31:307–313 DOI 10.1007/s10072-010-0216-6

ORIGINAL ARTICLE

Protective effects of heme oxygenase-1 against MPP+-induced cytotoxicity in PC-12 cells Jung-Woo Bae • Mi-Jeong Kim • Choon-Gon Jang Seok-Yong Lee

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Received: 9 May 2009 / Accepted: 7 January 2010 / Published online: 3 February 2010 Ó Springer-Verlag 2010

Abstract Heme oxygenase-1 (HO-1) catalyses the ratelimiting step of heme degradation to biliverdin, which is in turn reduced to bilirubin, CO and free iron. HO-1 can be induced by several harmful stimuli including oxidative stress, and it has a protective role against the cytotoxicity in different cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPP?) is a neurotoxic substance that induces the degeneration of dopaminergic neurons. This study examined whether HO-1 can be induced by MPP? and whether HO-1 has a protective role against the MPP?-induced cytotoxicity in PC-12 cells. MPP? triggered a relatively rapid induction of HO-1. The MPP?-induced cytotoxicity and reactive oxygen species (ROS) production markedly increased by HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX). The increase of ROS production by ZnPP-IX was completely abrogated by either two products of HO (biliverdin or bilirubin) while the increase of cytotoxicity by ZnPP-IX was attenuated partially. These suggest that HO-1 expression might have some cytoprotective effect against MPP?-induced cytotoxicity. Keywords Heme oxygenase-1 (HO-1)  Cytotoxicity  1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPP?)  PC-12 cell

Introduction The heme oxygenase (HO) system is the most effective mechanism for oxidative degradation of the heme

J.-W. Bae  M.-J. Kim  C.-G. Jang  S.-Y. Lee (&) Laboratory of Pharmacology, College of Pharmacy, Sungkyunkwan University, Chunchun-dong, Suwon 440-746, Republic of Korea e-mail: [email protected]

molecule, and consists of two isozymes, HO-1 and HO-2 [1]. HO-1 is inducible and distributed ubiquitously in mammalian tissue, while HO-2 is expressed constitutively and found mainly in the central nervous system [2]. The HO system is the only process that results in formation of an equimolar ratio of carbon monoxide (CO), chelated iron and biliverdin, which is subsequently reduced to bilirubin via biliverdin reductase. The discovery of links between the free radical quenching activity and function of CO as an activator of soluble guanylate cyclase (sGC) [3, 4] has led to considerable research in the cytoprotective and signaling activity of those entities. HO-1 is induced by a variety of stimuli, including nonheme inducers and various agents that cause oxidative stress, and prevents the oxidative DNA damage caused by heat shock and reactive oxygen species (ROS) [5]. There are some reports of the role of HO-1 in brain injury. HO-1 null mice showed lower resistance to oxidative stress [6], and HO-1 over-expressing mice were prone to decreased oxidative damage [7]. These reports suggest that HO-1 confers increased defense against cellular damage. However, the role of HO-1 in cerebral injury is unclear. Parkinsonism is characterized by dopaminergic neuronal degeneration in corpus striatum, and N-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a synthetic opiate contaminant, has been widely used to make animal model for Parkinsonism. MPTP induces acute permanent Parkinsonism through dopaminergic cell death by its active metabolite, 1-methyl-4-phenyl-2,3,-dihydropyridinium (MPP?), which is a mitochondrial complex I inhibitor [8, 9]. MPP? is accumulated actively in the mitochondria, where it blocks NADPH-CoQ10, which is a reductase (complex I) of the respiratory chain, by interacting with the rotenone binding site inside the cell [10]. In addition, the subsequent depletion of ATP by the

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inhibited electron transport system is believed to be the main reason for the observed the neurotoxicity [11]. Although the precise molecular mechanisms of MPP?induced cell death have not been established, some reports suggested the increase in free radical production [12, 13] as a reason for the neurotoxicity by MPP?. Because the oxidative stress is one of the major stimuli for HO-1 expression, there is quite a possibility that HO-1 is induced by MPP?. Thus, in this study, we studied whether HO-1 is induced by MPP?, a xenobiotic inducing oxidative stress, in dopaminergic PC-12 cells, and also whether the expressed HO-1 has a protective role against the MPP?-induced cytotoxicity.

Methods and materials Materials Rat pheochromocytoma PC-12 cells were obtained from the American Type Culture Collection (ATCC). RPMI9-1640 medium was purchased from Gibco BRL (Gaithersburg, USA). Heat inactivated horse serum was from Biowittaker– Cambrex (Walkersville, USA). Heat inactivated fetal bovine serum (FBS) was from Gemini Bio (West Sacramento, USA). MPP?, zinc protophorphyrin-IX, bilirubin, biliverdin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluorescein diacetate (FDA), propidium iodide (PI) and dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma (St. Louis, USA). Rabbit anti-HO-1 polyclonal antibody was from StressMarq Biosciences (Victoria, Canada). Alkaline phosphate-conjugated goat anti-rabbit antibody and an enhanced amplified alkaline phosphatase immune-blot system were from BioRad (Hercules, USA). Cell culture PC-12 cells were cultured on poly-L-lysine coated plates (Costar, Cambridge, USA) in RPMI9-1640 medium containing 10% heat inactivated horse serum, 5% heat inactivated FBS, 2 g/L sodium bicarbonate, 100 U/mL penicillin, 100 lg/mL streptomycin, and 2.5 g/mL amphotericin at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Exponentially growing cells were harvested by centrifugation and resuspended in fresh medium. The cells were seeded in 24-well plates at 5 9 104 cells per well and used in experiments after 24 h. Drug treatment To measure the effects of MPP? on the cell viability of PC-12 cells, various concentrations of MPP? (final

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concentration of 100–800 lM) were added to the culture medium and the cells were incubated for desired times (0–48 h). To determine the protective effect of HO-1 against MPP? induced cell death, zinc protophorphyrin-IX (10 lM), a HO-1 inhibitor, or two products of HO-1, bilirubin (1 lM) and biliverdin (1 lM), was added to the culture medium. MTT assay of cell viability MTT assay was used to measure the cell viability using a quantitative calorimetric assay. The PC-12 cells were plated in 24 well plates at a density of 5 9 104 cells/well, and incubated for 24 h. The medium was changed to one containing 100–800 mM of MPP?. The culture medium was removed after treatment, and a MTT solution (final concentration 0.25 mg/mL) was added to each well. The plates were then incubated for 5 h at 37°C. The MTT solution was removed and a cell-lysing solution (400 lL DMSO) with 50 lL of a Sorenson’s buffer (100 mM glycine, pH 10.5) was added. The level of MTT reduction was determined from the change in absorbance at 550 nm. The results are expressed as the percentage of MTT reduction, assuming that the absorbance of the control cells was 100%. Assessment of membrane damage Fluorescein diacetate and PI dyes were used to assess the cell viability after MPP? incubation. FDA enters normal cells and emits a green fluorescence when it is cleaved by various esterases. Once cleaved, FDA can no longer permeate the cell membranes. PI is an intravital dye that is normally excluded from the cells. However, in a damaged cell membrane, PI penetrates the cells and binds to the DNA in the nucleus emitting a red fluorescence. The effect of MPP? on the morphological features of cell death was measured using these properties. The PC-12 cells were exposed to 400 lM MPP? for 24 h after treating them with each drug. The cells were incubated with 10 lg/mL of each FDA and PI for 5 min and examined by fluorescence microscopy. The percentage of surviving cells in 10 fields ([400 cells) of each monolayer was estimated by assessing the level of FDA/PI staining Detection of reactive oxygen species The level of intracellular ROS was quantified by fluorescence using DCF-DA. In viable cells, DCF-DA, a nonfluorescent compound, is deacetylated by ROS to fluorescent 20 ,70 -dichlorofluorescin. DCF-DA was dissolved in absolute ethanol at a concentration of 50 lg/0.3 mL DMSO immediately before use. The PC-12 cells, which had been cultured in 24 well plates, were washed with

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The PC-12 cells were washed with PBS and lysed in 100 lL of the buffer (50 mM Tris–Cl, pH 7.5; 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 5 mM EGTA, 15 mM sodium pyrophosphate, 30 mM p-nitrophenyl phosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1% nonidet P-40 and 0.1% SDS) for 30 min on ice and centrifuged at 4°C for 10 min at 13,000 rpm. The supernatant fraction was obtained and 100 lg of aliquots were used for analysis. The nonspecific binding was blocked by incubation in TBS containing 10% nonfat dry milk, 5% BSA and 0.5% Tween 20 for 1 h at room temperature. The blots were probed with the rabbit anti-HO-1 polyclonal antibody. The secondary antibody was diluted 1:1,000 with the alkaline phosphate-conjugated goat anti-rabbit antibody. Detection was achieved by visualization using an enhanced amplified alkaline phosphatase immune-blot system. Statistical analysis All data are represented as the mean ± SEM and the significant levels between the groups were assessed by a Student’s t-test. A P value\0.05 was considered significant.

Results

Cell Viability (% of control)

Western blot analysis

MPP+ 100

75

50 100 uM 200 uM

25

400 uM 800 uM

0

0

12 24 36 Exposed Time (hour)

b

a

Membrane permeability

MTT assay

100

** Control

50

Control

MPP+

MPP?-induced cytotoxicity in PC-12 cells As shown in Fig. 1, the PC-12 cells exposed to MPP? (100–800 lM) for various times (0–48 h) showed a concentration and time dependent loss of cell viability. The percentage cell viability was approximately 60% after a 24 h treatment with 400 lM MPP?. This condition caused approximately 40% loss of cell viability (Fig. 2a). To rule out the possibility that MPP? directly inhibit MTT reduction by inhibiting mitochondrial function, percentage of membrane damage was also determined by FDA/PI fluorescence staining. As shown in Fig. 2b, for the most part, the control displayed green fluorescence and excluded PI. After exposure to 400 lM MPP? for 24 h, cells displaying red fluorescence were detected. Percentage of cells that displayed FDA fluorescence staining decreased to about 50% by MPP?. Therefore, all subsequent experiments to

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Fig. 1 Concentration- and time-response curve of MPP?-induced cytotoxicity. MPP? (100–800 lM) was added to culture medium and PC-12 cells were incubated with MPP? for indicated time. After incubation with MPP?, the cell viability was assessed by the level of MTT reduction and is expressed as a percentage of the viability of untreated control cells grown in a defined medium. The data is expressed as the mean ± SEM of 4–6 independent experiments. *p \ 0.05, **p \ 0.01, compared with the untreated control

Cell Viability (% of control)

modified Krebs–Ringer solution (20 mM HEPES, 10 mM glucose, 127 mM NaCl, 5.5 mM KCl, 1 mM CaCl2, and 2 mM MgSO4, pH 7.4). Three hours before detection, 100 lL of a Krebs–Ringer solution containing DCF-DA (1 lg/mL) was added to each well. The fluorescence by ROS was measured at excitation and emission wavelengths of 485 and 535 nm, respectively.

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+

MPP+

Fig. 2 Effect of MPP? on the cell viability and cell membrane damage. a MTT reduction in PC-12 cells was assessed after incubating the cells with 400 lM MPP? for 24 h. The data is reported as the mean ± SEM of four independent experiments. b 24 h after a treatment with 400 lM MPP?, the membrane integrity was assessed by measuring the percentage of cells with a green fluorescence after the FDA/PI treatment. The PI-stained colored cells indicate membrane damaged cells. **p \ 0.01

evaluate the cytotoxicity of MPP? were carried out using 400 lM MPP? over a 24 h period. Effect of MPP? on HO-1 expression The MPP?-induced HO-1 expression was identified by western blotting. Treatment with 400 lM MPP? markedly

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Effect of HO-1 on MPP?-induced cell death PC-12 cells were incubated with 10 lM ZnPP-IX, a HO-1 inhibitor, and 400 lM MPP? for 24 h. Co-treatment with HO-1 inhibitor further aggravated MPP?-induced cell death by 60% compared to the MPP? only treatment group (Fig. 4, p \ 0.01). A similar result was obtained by FDA/PI fluorescence staining, as shown by the stronger red fluorescence, than that observed in the MPP? treatment group (Fig. 5). Either co-administered biliverdine (1 lM) or bilirubin (1 lM), the products of HO-1, abolished partially the increase of cell toxicity by ZnPP-IX (Figs. 4, 5). Effect of HO-1 on cellular ROS production The level of ROS in the MPP?-exposed PC-12 cells was measured (Fig. 6). The 400 lM MPP? treated cells showed a significant increase in the production of ROS, up to sixfold, compared to the untreated control (p \ 0.01). HO1 inhibitor (10 lM ZnPP-IX) potentiated the 400 lM MPP?-induced ROS production (p \ 0.05), and either biliverdin (1 lM) or bilirubin (1 lM) completely abrogated the 400 lM ZnPP-IX-induced increase in ROS production.

HO-1 density (% of control)

HO-1

400

200

C

3

6

12

24

48

Exposed Time (hour) Fig. 3 Effect of MPP? on the cellular level of HO-1 protein. Western blot analysis demonstrated an increase in the HO-1 protein level in the PC-12 cells after incubation with 400 lM MPP? for 3–48 h. Each lane contained 50 lg of protein. The data is expressed as the mean ± SEM of three independent experiments

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MTT Assay

Cell Viability (% of control)

increased the expression of HO-1 (Fig. 3). MPP? triggered a relatively rapid increase in HO-1 production after the treatment for 3 h and maximum increment was detected after the treatment for 6 h. Subsequently, the levels of expressed HO-1 were maintained until 24 h.

100

*

*

50

** MPP

+

MPP + + ZnPP IX

MPP + + ZnPP IX + Biliverdin

MPP + + ZnPP IX + Bilirubin

Fig. 4 Effect of the HO-1 inhibitor on MPP? induced cell death. The changes in MTT reduction were measured in PC-12 cells treated with 400 lM MPP? for 24 h. A HO-1 inhibitor (10 lM ZnPP-IX) or a product of HO-1 (1 lM biliverdine or 1 lM bilirubin) was added in culture medium with 400 lM MPP?. The data is expressed as the mean ± SEM of five independent experiments. *p \ 0.05, **p \ 0.01

Discussion There are many reports implicating a neuroprotective role for HO-1 in intact animals as well as in tissue culture. Following exposure to a variety of noxious stimuli, HO-1 induction occurs in neuronal and non-neuronal brain cells [14]. Cerebellar granule cells derived from transgenic mice designed to over-express HO-1 in neurons appear to be relatively resistant to glutamate- and H2O2-mediated oxidative damage in vitro [15]. Similarly, neuroblastoma cell lines transfected with HO-1 cDNA were less prone than control cells to oxidative damage [16, 17]. Astrocytes derived from HO-1 knockout mice exhibit enhanced vulnerability to hemin toxicity relative to wild type cells [18]. In an in vivo study, HO-1 transgenic mice subjected to cerebral ischemia manifested decreased lipid peroxidation, enhanced expression of the anti-apoptotic factor, B-cell CLL/lymphoma 2 and smaller infarct volumes relative to normal littermates [19]. HO-1 may also confer neuroprotection in animal models of traumatic [20, 21] and excitotoxic [22, 23] brain damage and spinal cord injury [24]. Rapid heme/hemoprotein degradation and the intracellular accumulation of antioxidant bile pigments, biliverdin and bilirubin, may be responsible, at least in part, for the neuroprotection associated with the induction of HO-1 [25, 26]. CO released in the course of heme catabolism may also mediate some of the cytoprotective effects of HO-1 [27]. While HO-1 induction may provide neuroprotection under certain conditions, the action of HO-1 in other models of CNS injury and disease was reported to be

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A

B

100

MPP+

MPP+ + ZnPPIX + Bilirubin

Cell Viability (% of control)

* * 50

* * ** MPP+

MPP+ + ZnPPIX

MPP+ + ZnPPIX + Biliverdin

Fig. 5 Effect of the HO-1 inhibitor on MPP?-induced cell membrane damage. a Morphological features of the cell death induced by the treatment with MPP? (400 lM) for 24 h. ZnPP-IX (10 lM), biliverdine (1 lM), or bilirubin (1 lM) was treated with MPP? for 24 h. b The membrane integrity was assessed by measuring the percentage of

∗ ROS (% of control)

1500

∗

∗

1000

500

MPP+

MPP + + ZnPPIX

MPP+ + ZnPPIX + Bilirubin

MPP+ + ZnPPIX + Biliverdin

Fig. 6 Effect of the HO-1 inhibitor on MPP?-induced reactive oxygen species (ROS) production. Changes in the ROS level were measured 24 h after the MPP? (400 lM) treatment. ZnPP-IX (10 lM), biliverdine (1 lM), or bilirubin (1 lM) was treated with MPP? for 24 h. The data is reported as the mean ± SEM of seven independent experiments. *p \ 0.05

detrimental. Metalloporphyrin suppression of HO activity has been shown to diminish tissue necrosis and edema formation following focal cerebral ischemia in rats [28], confer neuroprotection in an experimental model of

MPP+ + ZnPPIX

MPP+ MPP+ + + ZnPPIX ZnPPIX + + Biliverdin Bilirubin

cells with a green fluorescence after the FDA/PI treatment. The data is expressed as the mean ± SEM of four independent experiments. **p \ 0.01, compared with the MPP? only treatment; *p \ 0.05, compared with the MPP? ? ZnPP-IX treatment

intracerebral hemorrhage [29, 30] and alleviate traumatic cornu ammonis 1 insults in rat hippocampal slices [31]. Suppression of hippocampal HO-1 expression has also been suggested as a mechanism by which nimodipine treatment ameliorates aluminum neurotoxicity in mice [32]. In addition, HO-1 enhanced dopaminergic cell injury following exposure to polychlorinated biphenyls [33]. This study examined the effect of HO-1 expression against the MPP?-induced cytotoxicity in dopaminergic PC-12 cells. The impact of HO-1 expression on MPP? exposure was demonstrated by Western blot analysis. The induction of the HO-1 protein reached a peak after 6 h exposure to 400 lM MPP?, and was maintained for 24 h. The half-lives of HO-1 mRNA and protein have been estimated to be 3 and 15–21 h, respectively [34]. The oxidative stress is one of the major stimuli for HO-1 expression and the accumulation and/or formation of ROS was markedly increased over sixfold after exposure with 400 lM MPP?. Thus, the enhanced production of ROS by MPP? might be responsible for the HO-1 induction. In this study, MTT assay was used to determine the cytotoxicity of PC-12 cells. Because there is a possibility that MPP? directly inhibit MTT reduction by inhibiting mitochondrial function, the membrane damage of cells was also measured by FDA/PI fluorescence staining. Data from FDA/PI staining were well correlated with data from MTT assay. So, the possibility that MPP? directly inhibit MTT reduction can be excluded. The cell viability was decreased

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to approximately 60% after a 24 h treatment with 400 lM MPP?. The MPP?-induced cytotoxicity and ROS production were increased significantly when incubated with ZnPP-IX, a metalloporphyrin inhibitor of the HO isozymes. While the enhanced effects of HO-1 inhibitor on the MPP?-induced ROS production was completely attenuated by a product of HO, biliverdin or bilirubin, the enhanced effects of HO-1 inhibitor on the MPP?–induced cytotoxicity was partially attenuated by a product of HO, biliverdin or bilirubin. These results suggest that increased HO-1 expression plays a role in the cytoprotective effect against MPP? exposure and that although antioxidant bile pigments produced by HO-1 may be partially responsible for the protective effects of HO-1, there might be another mechanism responsible for the protection of HO-1 against MPP? neurotoxicity.

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