logy 564 (2007) 47–56www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Antioxidant effect of erythropoietin on 1-methyl-4-phenylpyridinium-induced neurotoxicity in PC12 cells

Yan Wu a,1, You Shang b,1, Shenggang Sun a,⁎, Rengang Liu c

a Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR Chinab Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan, PR China

c Department of Anatomy, Tongji Medical College, Huazhong University of Science and Technology. Wuhan, PR China

Received 25 October 2006; received in revised form 20 January 2007; accepted 2 February 2007Available online 17 February 2007

Abstract

The neuroprotective effects of erythropoietin on 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress and apoptosis in culturedPC12 cells as well as the underlying mechanism were investigated. Treatment of PC12 cells with MPP+ caused the loss of cell viability, which wasassociated with the elevation in apoptotic rate, the formation of reactive oxygen species and the disruption of mitochondrial transmembranepotential. It was also shown that MPP+ significantly induced upregulation of Bax/Bcl-2 ratio and activation of caspase-3. In contrast,erythropoietin reversed these phenotypes and had its maximum protective effect at 1 U/ml. The effect of erythropoietin was mediated by thephosphatidylinositol 3-kinase (PI3K) signaling pathway since erythropoietin failed to rescue cells from MPP+ insult in the presence of the PI3Kinhibitor, LY 294002. In addition, the downstream effector of PI3K, Akt, was activated by erythropoietin, and Akt activation was inhibited by LY294002. Furthermore, the effect of erythropoietin on reactive oxygen species levels was also blocked by LY 294002. These results show thaterythropoietin may provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative diseases such asParkinson disease.© 2007 Elsevier B.V. All rights reserved.

Keywords: 1-methyl-4-phenylpyridinium; PC12 cells; Erythropoietin; Oxidative stress; Apoptosis

1. Introduction

Oxidative stress has been implicated as a major role in theonset and progression of a vast variety of clinical abnormalitiesincluding neurodegenerative disorders. 1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine (MPTP) has been widely used as adopaminergic neurotoxin because it causes a severe parkinso-nian-like syndrome with loss of dopaminergic cells in animalsand humans (Lotharius and O'Malley, 2000). 1-methyl-4-phenylpyridinium ion (MPP+), which is the active metabolite ofMPTP, stimulates the production of the superoxide radicalin vitro and induces cell death in a rat adrenal glandpheochromocytoma cell line (PC12 cells) (Itano et al., 1994).

⁎ Corresponding author. Tel.: +86 27 83650719.E-mail address: sun_shenggang@163.com (S. Sun).

1 Equal contribution.

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.02.020

Moreover, MPTP generates hydroxyl radicals which causemembrane lipid peroxidation or DNA damage and this isthought to be the mechanism of degeneration of dopaminergicneurons (Alam et al., 1997; Chiueh and Rauhala, 1998;Dipasquale et al., 1991; Sriram et al., 1997). Excessive free-radical formation or antioxidant deficiency can result inoxidative stress, a possible mechanism of the toxicity ofMPTP (Lotharius and O'Malley, 2000). Hence, drugs thatreduce the oxidative stress induced by MPP+ may prove to beneuroprotective in Parkinson disease.

Erythropoietin is a cytokine that acts in erythroid progenitorproliferation and differentiation. Erythropoietin can amelioratethe oxidative stress occurs in several quite different disorders(Li et al., 2006; Nakamura et al., 2006; Kumral et al., 2005). Italso has been reported that erythropoietin displays efficientneuroprotective properties in a spectrum of different animalmodels. These extend from ischemia/hypoxia, excitotoxic

48 Y. Wu et al. / European Journal of Pharmacology 564 (2007) 47–56

paradigms, traumatic brain and spinal cord injury, and retina/optic nerve damage to inflammatory/auto-immunological dis-eases (Ehrenreich et al., 2004). Erythropoietin binds toerythropoietin receptor and leads to phosphorylation activationof the phosphatidylinositol 3-kinase (PI3K)/Akt pathway inneurons (Signore et al., 2006). PI3K and Akt are molecules thatpromote cell survival and prevent apoptosis. Recent studieshave revealed a possible protective role for erythropoietin inParkinson disease (Csete et al., 2004; Genc et al., 2001; Knabeet al., 2004; Signore et al., 2006).

But few data exist regarding the protective value oferythropoietin on MPP+ induced oxidative stress and thesubsequent apoptosis in PC12 cells, and the possible molecularmechanisms. In the present study, we demonstrated thaterythropoietin substantially reduced MPP+-induced cellulardeath in PC12 cells via its characteristics of an anti-oxidantand anti-apoptotic. The involvement of PI3K/Akt pathway wasalso demonstrated.

2. Materials and methods

2.1. Materials

Recombinant human erythropoietin injection was obtainedfrom Shenyang Sunshine Pharmaceutical Co. Ltd (China).1-methyl-4-phenylpyridinium ion (MPP+), 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorofluorescin diacetate (DCF-DA), rhodamine 123 andkinase inhibitor LY 294002 were from Sigma-Aldrich Inc. (St,Louis, MO, USA). Annexin V-fluorescein isothiocyanate (FITC)apoptosis detection kit was from Biosea BCL (Beijing, China).The in situ cell death detection kit was from the BoehringerMannheim, Co. (Mannheim, Germany). Bax and Bcl-2 anti-bodies were obtained from Santa Cruz (Santa Cruz, CA, USA).Phospho-Akt, and Akt antibodies were purchased from CellSignal Technology Inc. (Beverly, Massachusetts, USA). Thecaspase-3 fluorescent assay kit was from R&D systems(Minneapolis, MN, USA). Dulbecco's modified Eagle's medium(DMEM), heat-inactivated calf serum, fetal bovine serum werepurchased from Gibco-BRL-Life Technologies (Gaithersburg,MD). Bicinchoninic Acid Kit for protein determination (BCAkit) and Enhanced chemiluminescence (ECL) were purchasedfrom Pierce Chemical Company (Rockford, IL, USA).

2.2. Cell culture

Rat PC12 cells (adrenal gland; pheochromocytoma) wereobtained from Chinese Type Culture Collection. PC12 cellswere maintained in DMEM supplemented with 10% heat-inactivated calf serum, 5% fetal bovine serum, 100 U/mlpenicillin and 100 μg/ml streptomycin in a water-saturatedatmosphere of 5% CO2 at 37 °C. The culture medium waschanged every three days and cells were subcultured about oncea week. The medium was changed to serum-deprived mediumand cells were washed with serum-free DMEM 24 h beforeexperiments and replanted in the 96 and 24 well plates. The nextday MPP+ and/or erythropoietin were added to naive PC12

cells. The specific PI3K inhibitor LY 294002 was added tonaive PC12 cells preconditioning 1 h if necessary.

2.3. Measurement of cell viability

Cell viability was measured by using the MTT assay, whichis based on the conversion of MTT to formazan crystals bymitochondrial dehydrogenases (Yamamoto et al., 2000). PC12cells were seeded in 96-well plates at a density of 4×104 cellsper well and were treated with various concentrations oferythropoietin (0.1, 0.3, 1, 3 or 10 U/ml) and/or 500 μM MPP+

at 37 °C. LY 294002 was added to naive PC12 cellspreconditioning 1 h if necessary. After incubation for up to24 h, the medium was incubated with 10 μl of 5 mg/ml MTTsolution for 4 h. Then the culture medium with MTT wasremoved and 200 μl dimethyl sulphoxide (DMSO) was added toeach well to dissolve the formazan. Absorbance was measuredat 570 nm (540 nm as a reference) with a model 550-microplatereader. Cell viability was expressed as a percentage of the valuein control cultures.

2.4. Assessment of apoptosis

Flow cytometry was used to assess the membrane andnuclear events during apoptosis. The membrane events wereanalyzed by measuring the binding of FITC-Annexin V proteinto the phospholipid phosphatidylserine present on the externalsurface of the apoptotic cell membrane. The assay wasperformed with a two colour analysis of FITC-labelled AnnexinV binding and PI uptake using the Annexin V-FITC apoptosisdetection kit. Positioning of quadrants on Annexin V/PI dotplots was performed and live cells (Annexin V−/PI−), early/primary apoptotic cells (Annexin V+/PI−), late/secondaryapoptotic cells (Annexin V+/PI+) and necrotic cells (AnnexinV−/PI+) were distinguished (Vermes et al., 1995). Therefore,the total apoptotic proportion included the percentage of cellswith fluorescence Annexin V+/PI− and Annexin V+/PI+.Briefly, after the treatment period (24 h), the harvested cellswere resuspended in 1 ml binding buffer. An aliquot of 100 μlwas incubated with 5 μl Annexin V-FITC and 10 μl PI for15 min in dark at room temperature and 400 μl PBS was addedto each sample. The fluorescein-5-isothiocyanate (FITC) and PIfluorescence were measured through FL-1 filter (530 nm) andFL-2 filter (585 nm), respectively, on BD-LSR flow cytometerusing Cell Quest software and 10,000 events were acquired.

2.5. TUNEL assay for apoptotic DNA fragmentation

The DNA fragmentation of apoptotic cells were identified bythe TUNEL method which permits the specific labeling of the3′-OH end of DNA breaks with modified nucleotides by TdT.TUNEL assay was carried out as described previously withminor modification (Ohgoh et al., 1998). Briefly, PC12 cellswere seeded at a density of 1×105 cells/cm2 on coverslipscoated with poly-L-lysine. After washing with PBS, the cultureswere fixed with 4% paraformaldehyde in PBS (pH 7.4) for30 min at room temperature. Endogenous peroxidase was

49Y. Wu et al. / European Journal of Pharmacology 564 (2007) 47–56

quenched by incubation with 0.3% (v/v) hydrogen peroxide inmethanol for 30 min at room temperature and the cells furtherpermeabilized with 0.1% Triton X-100 in 0.1% sodium acetatefor 5 min at 4 °C. Thereafter, the cells were labeled byincubation with the TUNEL reaction mixture for 60 min at37 °C followed by labeling with peroxidase-conjugated anti-fluorescein anti-goat antibody (Fab fragment) for an additional30 min. Subsequently, cells were incubated with diaminobenzi-dine substrate (DAB) to produce a dark brown precipitate andslides were counterstained with hematoxylin stain. For eachexperiment, all treatments were performed in triplicate wells.Analyzed under light microscopy, the positive staining wasidentified under the light microscope as dark brown granules.

2.6. Measurement of intracellular reactive oxygen speciesformation

Intracellular reactive oxygen species was monitored by usingthe fluorescent probe DCF-DA (Wang and Joseph, 1999;Lautraite et al, 2003). Intracellular H2O2 or low-molecular-weight peroxides oxidize DCF-DA to the highly fluorescentcompound dichlorofluorescin (DCF). PC12 cells (1×105 cells/ml) were treated with 500 μMMPP+ in the presence or absenceof indicated concentration erythropoietin for 24 h at 37 °C. LY294002 was added to naive PC12 cells preconditioning 1 h ifnecessary. Then the cells were incubated with 1 mM DCF-DAfor 1 h at 37 °C and were washed three times with PBS. Aftercentrifugation at 1000 ×g for 5 min, the supernatants wereremoved and the pellets were resolved with 1% Triton X-100,and fluorescence was measured at an excitation wavelength of480 nm and an emission wavelength of 540 nm using afluorescence microplate reader.

2.7. Measurement of mitochondrial transmembrane potential

Mitochondrial transmembrane potential was measured usingthe dye rhodamine 123 (Lee et al., 2003). It has been shownthat the uptake of rhodamine 123 into mitochondria is afunction of mitochondrial transmembrane potential. PC12 cells(4×104) were incubated with MPP+ for 4 h and then wereincubated for 20 min at 37 °C in DMEM containing 10 μMrhodamine 123. Cell suspension was centrifuged at 412 ×g for10 min, and medium was removed. Cells were dissolved with1% Triton X-100, and fluorescence was measured at anexcitation wavelength of 488 nm and an emission wavelengthof 510 nm using a fluorescence microplate reader.

2.8. Western blot analysis

After exposure to erythropoietin, and/or MPP+ for theindicated dosages and times, 5×106 cells were rinsed twicewith cold PBS and lysed in buffer (50 mmol/l Tris–HCl, pH 8.0,100 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l dithiothreitol, 1%Triton X-100, 0.1% sodium dodecyl sulfate, 50 mmol/l sodiumfluoride and 1 mmol/l sodium vanadate) containing a proteaseinhibitor cocktail to obtain whole cell protein. Lysates werecleared by centrifugation and protein concentration was deter-

mined by BCA kit. Equal amounts of proteins were fractionatedby sodium dodecyl sulfate–polyacrylamide gel electrophoresis,and transferred onto a nitrocellulose membrane. The membraneswere blocked with 5% defatted milk in TBS-Tween (TBS-T)(50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) andincubated with anti-phosphospecific Akt (1:1000), anti-Akt(1:1000), anti-Bcl-2 (1:500), or anti-Bax (1:500) antibodiesovernight at 4 °C. The signals were detected using goat anti-rabbitor anti-mouse horseradish peroxidase-conjugated secondaryantibody and enhanced chemiluminescence (ECL), then exposedto X-ray films (Fuji, Japan).

2.9. Measurement of caspase-3 activity

The activity of caspase-3 was detected with the caspase-3fluorometric assay kit (RnD) according to the instructionmanual. This kit uses synthetic tetrapeptide DEVD- 7-amino-4-trifluoromethyl coumarin (AFC) as the substrate. In thepresence of active caspase-3, the substrate is cleaved betweenDEVD and AFC, releasing the fluorogenic AFC, which is thendetected by spectrofluorometry. The fluorogenic AFC reflectsthe activity of caspase-3. PC12 cells were incubated in mediumcontaining serum-free medium with MPP+ and/or erythropoi-etin. Caspase activities were measured in PC12 cells aftererythropoietin and/or MPP+ treatment. After the incubation,cells were collected and lysed in a lysis buffer on ice for 10 min.The protein concentrations of the supernatant fluids wereascertained with the BCA kit. Samples containing 200 μgprotein were mixed with the reaction buffer and DEVD-AFCsubstrate, followed by 2 h incubation at 37 °C. The fluorescencewas measured at an excitation wavelength of 400 nm andemission wavelength of 505 nm with fluorometric reader. Acaspase-3-like activity was expressed as relative content againstthat in the cells incubated in the medium containing serumwithout MPP+.

2.10. Statistical analysis

Data are expressed as the mean±S.D. The significance ofinter-group difference was evaluated by one-way analyses ofvariance (ANOVA: Duncan's test for post hoc comparisons).Differences were considered significant at Pb0.05.

3. Results

3.1. Effect of erythropoietin on MPP+-induced cell viabilityloss in PC12 cells

We tested the neurotoxicity of MPP+ in PC12 cells and cellviability was assessed by the MTT reduction assay. Comparedwith vehicle controls, exposure to 500 μM MPP+ for 24 hresulted in the loss of 52.5% of PC12 cells. The neurotoxic of500 μM MPP+ effects were significantly attenuated byerythropoietin, when the cells were co-incubated with differentconcentrations (0, 0.1, 0.3,1,3, or 10 U/ml) of erythropoietin andMPP+ for 24 h. Compared with the control group, the survivalrate of PC12 cells was 76.8±4.7%, 98.7±9.3%, 112.5±12.5%,

Fig. 1. Effect of erythropoietin on the MPP+-induced decrease in PC12 cellsviability. Cell viability was assessed by the MTT method as described inMaterials and methods. Cells were treated with 500 μM MPP+ for 24 h in theabsence or presence of erythropoietin. Data are expressed as percent of values inuntreated control cultures, and are means±S.D. of 3 replicate values in 4separate experiments. ⁎Pb0.05, ⁎⁎Pb0.01 compared with the group treated byMPP+ alone; ##Pb0.01, compared with control (untreated group).

50 Y. Wu et al. / European Journal of Pharmacology 564 (2007) 47–56

92.9±7.0% and 64.6±5.1% respectively when the cells weretreated with the indicated concentrations of erythropoietin for24 h (Fig. 1). The results in Fig. 1 showed that erythropoietinexhibited neuroprotective effects in a dose-dependent manner onthe cytotoxicity induced by MPP+ in PC12 cells, and maximalrescue occurred at a concentration of 1 U/ml erythropoietin.

Our data also demonstrated that erythropoietin can promotePC12 cell proliferation even when the cells were culturedwithout FBS or in the presence of small amounts of FBS in themedium (data not shown). Thus, it is possibly concluded thaterythropoietin is effective for the protection and viability ofPC12 cells.

3.2. Effect of erythropoietin against MPP+-induced apoptosisin PC12 cells

In the control (Fig. 2A), 87.2% cells excluded PI and werenegative for Annexin V-FITC binding, which is viable cells.PC12 cells exposed to 500 μM MPP+ for 24 h revealed that ofthe Annexin V positive cells (32.2%), 7.6% were PI negative(lower right quadrants), while 24.6% were PI positive (upperright quadrant), which indicated background early apoptosisand late apoptosis/necrosis, respectively (Fig. 2B). Simulta-neous incubation with 0.1, 0.3, 1, 3 or 10 U/ml erythropoietinsignificantly reduced the number of cells labeled with AnnexinV. The percentage of apoptotic cells was decreased to 16.7±

Fig. 2. Effect of erythropoietin against the MPP+-induced apoptosis in PC12cells measured by flow cytometry. Flow cytometric histograms of control PC12cells and PC12 cells after 24 h exposure to 500 μM MPP+ alone or associatedwith erythropoietin (0.1, 0.3, 1, 3 and 10 U/ml). After incubation, cells wereharvested and labeled with a combination of annexin V-FITC and propidiumiodide. The figure showed representative flow cytometric histogramsas follow:(A) Control; (B) 500 μM MPP+ treated alone; (C) 500 μM MPP++0.1 U/mlerythropoietin; (D) 500 μM MPP++0.3 U/ml erythropoietin; (E) 500 μMMPP++1 U/ml erythropoietin; (F) 500 μM MPP++3 U/ml erythropoietin;(G) 500 μMMPP++10 U/ml erythropoietin. Data are means±S.D of 3 replicatevalues in 4 separate experiments. ⁎⁎Pb0.01, compared with MPP+ alone;##Pb0.01, compared with control.

3.0%, 11.3±2.2%, 10.2±1.5%, 14.4±2.6%, and 19.1±2.5%respectively (Fig. 2C–G).

Consistent with the flow cytometric data, the results ofTUNEL assay further confirmed that erythropoietin-treatedgroup could protect MPP+-induced apoptosis in PC12 cells. In

Fig. 3. Photomicrograph of PC12 cells stained with TUNEL. PC12 cells were treated for 24 h exposure to 500 μM MPP+ alone or associated with erythropoietin(1 U/ml). After incubation, cells were fixed with 4% paraformaldehyde, and apoptotic cells were detected by the TUNEL staining as described in Materials andmethods. Hematoxylin was used to counterstain the cells. Apoptotic nuclei are those with dark brown precipitate. (A) Control; (B) 500 μM MPP+ treated alone;(C) 500 μM MPP+ with 1 U/ml erythropoietin; (D) 1 U/ml erythropoietin treated alone. The figures are representative for three different experiments.

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this histochemical analysis, the appearance of intensely stainednuclei is indicative of apoptotic nuclei. PC12 cells treated with500 μM MPP+ underwent apoptosis as determined by positiveTUNEL staining, which detects DNA fragmentation in situ.Erythropoietin, at 1 U/ml, lowered the proportion of TUNELpositive cells (Fig. 3).

3.3. Effect of erythropoietin on MPP+-induced increase inintracellular reactive oxygen species level

MPP+ interacts with mitochondrial complex I, blocking ATPproduction and promoting oxygen free-radical formation(Adams et al., 1993). To examine whether the inhibitory effectof erythropoietin on the toxicity of MPP+ is mediated by the

Fig. 4. Erythropoietin reduced MPP+-induced accumulation of ROS. Thefluorescence intensity of DCF was measured after PC12 cells were exposed to500 μM MPP+ and /or erythropoietin at the indicated concentration for 24 h.Data are expressed as means±S.D. of 3 replicate values in 4 separateexperiments. ⁎Pb0.05, ⁎⁎Pb0.01, compared with MPP+ alone; ##Pb0.01,compared with control.

antioxidant ability, PC12 cells were treated with 500 μMMPP+

for 24 h, and the levels of reactive oxygen species weremeasured by DCF fluorescence. As shown in Fig. 4, when PC12cells were exposed to MPP+ , the intracellular reactive oxygenspecies level significantly increased from 100.0±7.9% (control)to 227.9±21.5% (Pb0.01), revealing that MPP+ enhancedreactive oxygen species concentration in PC12 cells. However,simultaneous treatment with erythropoietin effectively reducedreactive oxygen species generation, and the suppressing effectstrengthened with the increase of the concentration oferythropoietin. Erythropoietin inhibited the 500 μM MPP+-

Fig. 5. Effect of erythropoietin on MPP+-induced mitochondrial membranepotential alteration. PC12 cells were exposed to 500 μM MPP+ and/orerythropoietin at the indicated concentration for 24 h, then mitochondrialmembrane potential alteration was measured by fluorescence microplate readerusing rhodamine 123 (Rh 123) staining. Erythropoietin blocked MPP+-inducedmitochondrial membrane potential loss. Data are means±S.D of 3 replicatevalues in 4 separate experiments. ⁎⁎Pb0.01, compared with MPP+ alone;##Pb0.01, compared with control.

Fig. 7. Erythropoietin inhibited MPP+-induced increase in caspase-3 activity.Apoptosis in PC12 cells induced with MPP+ involves caspase-3 activation. Cellswere treated with 500 μM MPP+ for 24 h in the absence or presence oferythropoietin. Then lysateswere assayed for the cleavage of fluorogenic caspase-3substrates as detailed in Materials and methods. Values are means±S.D of 3replicate values in 4 separate experiments. ⁎Pb0.05, ⁎⁎Pb0.01, compared withMPP+ alone; ##Pb0.01, compared with control.

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induced increase in DCF fluorescence, and, at 1 U/ml, it showeda maximum inhibition.

3.4. Effect of erythropoietin on the loss of mitochondrialtransmembrane potential caused by MPP+

The disruption of mitochondrial transmembrane potentialand the subsequent cytochrome c release have been consideredas the early phenomena in the apoptotic process (Chandra et al.,2000). In this respect, we assessed the effect of MPP+ on themitochondrial transmembrane potential. Change in the mito-chondrial transmembrane potential in PC12 cells treated withMPP+ was quantified by measuring the cellular retention ofrhodamine 123. When PC12 cells were treated with 500 μMMPP+ for 24 h at 37 °C, a decrease in the retention of rhodamine123 was observed. Erythropoietin prevented the MPP+-induceddecrease in the retention of rhodamine 123 (Fig. 5).

3.5. Effect of erythropoietin on MPP+-induced downregulationof the Bax/Bcl-2 ratio

Extensive evidence suggests that Bcl-2 family shows acomplex network regulating apoptosis. Bcl-2 is an anti-

Fig. 6. Effect of erythropoietin on the expression of Bax and Bcl-2 in PC12 cells.Cells were treated with MPP+ (500 μM) and/or erythropoietin (1 U/ml) for 24 h,and then cell lysate were subject to Western blot analysis. The levels of Bax andBcl-2 were quantified by the densitometric analysis (A) and the Bax/Bcl-2 ratiowas determined (B). These results are representative of three independentexperiments. Data are means±S.D. ⁎Pb0.05, compared with MPP+ alone;#Pb0.05, compared with control.

apoptotic protein whereas Bax is pro-apoptotic. The balancebetween these proteins is critical to turning on and off thecellular apoptotic machinery (Cory and Adams, 2002).

In this study, we investigated whether erythropoietin has anyeffect on the expressions of Bax and Bcl-2 in MPP+-treated cellsusing Western blot analysis. As shown in Fig. 6A, Bax proteinexpression increased significantly in 500 μM MPP+-treatedgroup compared with that in control. However, erythropoietintreatment (1 U/ml) could decrease the Bax expression levelalmost to the normal values. In contrast, the level of Bcl-2 in theMPP+-treated group was significantly decreased compared withthat of the control group. Also, expression of Bcl-2 wasrecovered with erythropoietin treatment. After treatment ofPC12 cells for 24 h with erythropoietin at 1 U/ml, the proteinlevel of either Bcl-2 or Bax was similar to that of control group.The Bax/Bcl-2 ratio increased to 1.8-fold of control upontreatment with MPP+, while erythropoietin prevented theMPP+-induced increase of the Bax/Bcl-2 ratio (Fig. 6B).

The effect of erythropoietin on MPP+-induced apoptosismay be, at least in part, mediated by regulation of Bax and Bcl-2expression.

3.6. Effect of erythropoietin on MPP+-induced activation ofcaspase-3

Caspases are the crucial mediators of the complex biochem-ical events associated with apoptosis. As the activation ofcaspase-3 has been shown to be final effector for the apoptosis(Hartmann et al., 2000), its activity was examined in this study.Following 24 h treatment of PC12 cells with 500 μMMPP+, wedetected a 240.0±14.6% increase of caspase-3-like activitycompared with the control cells. In contrast, PC12 cells whichwere simultaneously treated with erythropoietin (0.1, 0.3, 1, 3,10 U/ml) showed a significant decrease in caspase-3 activitycompared with the MPP+-treated cells (210.0±20.0%, 150.0±12.5%, 130.0±14.4%, 170.0±13.1%, 220.0±20.5%, respec-tively) (Fig. 7). Erythropoietin alone did not show a significanteffect on the caspase-3 activity in PC12 cells. The results show

Fig. 8. Effect of PI3K inhibitor on the protective effect of erythropoietin. PC12cell viability was measured using the MTT assay. Pre-incubation with thespecific PI3K inhibitor LY 294002 (10 μM) resulted in the complete eliminationof the erythropoietin (1 U/ml)-induced neuroprotection. LY294002 alone did notalter cell viability in control cells. Data are means±S.D. of 3 replicate values in 4separate experiments. ##Pb0.01, compared with control; ⁎⁎Pb0.01, comparedwith MPP+ alone;++Pb0.01, compared with MPP+ plus erythropoietin.

Fig. 10. Effect of PI3K inhibitor on erythropoietin-mediated reduction inreactive oxygen species levels. Cells were pretreated with or without 10 μM LY294002 for 1 h and then treated with or without 500 μMMPP+ or erythropoietin(1 U/ml) for additional 24 h as indicated. The data are presented as percentage ofrelative DCF fluorescence compared with control untreated cells and are themean±S.D. of three determinations. Each experiment was repeated at least threetimes with similar results. ##Pb0.01, compared with control; ⁎⁎Pb0.01, comparedwith MPP+ alone; ++Pb0.01, compared with MPP+ plus erythropoietin.

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that treatment with erythropoietin resulted in the inhibition ofMPP+-induced activation of caspase-3.

3.7. The PI3K/Akt pathway was activated by erythropoietinand mediated erythropoietin-induced neuroprotection

Akt is a pro-survival kinase and is activated by thephosphorylation at Ser 473 via the PI3K pathway, whichpromotes cell survival and prevents apoptosis (Dudek et al.,1997). To evaluate a possible mechanism by which erythropoi-etin prevents cellular death in our model, we measured thechanges in phosphorylation levels of Akt in the PC12 cellsexposed to erythropoietin.

This effect of rescuing the cells from death induced byerythropoietin was lost with addition of the specific inhibitor ofPI3K, LY 294002 (10 μM) (Fig. 8). These results indicated thatPI3K activity may be required for the erythropoietin protectionfrom MPP+-induced cell death and encouraged us to furtherinvestigate the involvement of PI3K activation induced byerythropoietin.

Fig. 9. Effect of erythropoietin on the phosphorylation of Akt in PC12 cells.(A). Representative Western blots showing the protein levels of phosphorylatedand total Akt in PC12 cells at serial time points.Western blot analysis of PC12 cellsstained for activated phospho-Akt (p-Akt) shows that erythropoietin quicklyinduces a sustained activation of Akt, lasting at least 12 h. (B). Erythropoietin-induced phosphophorylation of Akt in PC12 cells was suppressed by PI3Kinhibitor LY294002 3 h after MPP+ and/or EPO exposure. The blots presented arerepresentatives of three independent experiments with similar results in all blots,staining for total Akt was used as a loading control.

As shown in Fig. 9, the level of phospho-Akt was low inPC12 cells under unchallenged condition. Enhanced levels ofphospho-Akt were detected 30 min after erythropoietin wasadded to the culture, and levels remained elevated for at least12 h (Fig. 9A). To confirm the involvement of Akt further, weinhibited the upstream pathway that controls Akt activation. LY294002 (10 μM) was added to PC12 cells 1 h beforeerythropoietin. LY 294002 thoroughly abolished erythropoie-tin-induced phosphorylation of Akt, indicating that erythropoi-etin activates Akt likely through PI3K (Fig. 9B). Total Aktlevels in each experiment remained constant (Fig. 9, Akt lanes).

We next examined the effect of LY 294002 on reactiveoxygen species generation. As shown in Fig. 10, MPP+

treatment induced a dramatic increase in reactive oxygenspecies levels. Erythropoietin blocked the MPP+-inducedelevation in reactive oxygen species. Consistent with theinvolvement of PI3K in the protection induced by erythropoi-etin, LY 294002 blocked the erythropoietin-mediated reductionin reactive oxygen species levels. These results indicated thaterythropoietin can abolish the MPP+-induced production ofreactive oxygen species via the PI3K/Akt signaling pathway.

4. Discussion

Research during the past years has clearly demonstrated thaterythropoietin is a potent promoter of neuronal survival. Studiesin vitro providedmost of the information related to the molecularpathways involved in erythropoietin action. These data showedthat erythropoietin might have a direct protective role against avariety of neurotoxic insults, such as hypoxic conditions (Martiet al., 1996), glutamate toxicity (Morishita et al., 1997), free-radical injury (Chong et al., 2003b), and exposure toneurotoxicants (Villa et al., 2003). In addition, erythropoietinreceptor is abundantly expressed on adult dopaminergic neurons(Csete et al., 2004), suggesting a direct effect of erythropoietinon neurons. Erythropoietin also can protect dopaminergicneurons against MPTP and 6-hydroxydopamine neurotoxicityand significantly reverse behavioral deficits in mice (Genc et al.,2001; Signore et al., 2006). Protection by erythropoietin in CNS

54 Y. Wu et al. / European Journal of Pharmacology 564 (2007) 47–56

is mediated through a series of cellular pathways that maintainintricate links between one another.

However, up to date, the mechanisms that underlie theprotective effect of erythropoietin on the neurotoxicity of MPP+

have not fully been understood. In order to explore this subject,we observed the effects of MPP+ on PC12 cells which retaindopaminergic characteristics. It was shown that MPP+ attenu-ated the cell viability and induced apoptosis in PC12 cells. Toevaluate erythropoietin as neuroprotective agent in this paper,the protective roles of erythropoietin were investigated by MTTassay measurements, TUNEL assay and flow cytometricanalysis. According to these studies, erythropoietin couldexert a protective effect against MPP+-induced cell viabilityloss. In cellular ultrastructure, erythropoietin blocked apoptosisin the morphological changes. Similarly, the flow cytometricanalysis showed that erythropoietin decreased the apoptotic rateduring the whole experimental process.

Our study showed that erythropoietin exerted a concentrationdependent neuroprotective effect against the loss of cellviability and apoptosis induced by MPP+ in PC12 cells. Butour results slightly deviated from the previous report (Signoreet al., 2006). The maximum neuroprotection occurred when1 U/ml erythropoietin concomitant with 500 μM MPP+, whilethe neuroprotective effect of larger erythropoietin doses wereobviously depressed. Possible explanations for these conflictingresults lie in the use of different cell lines and differentneurotoxins in these experiments. Accordingly, the cellularresponses may be not all the same.

The subsequent experiments explored the mechanisms of theneuroprotection of erythropoietin on MPP+-induced cell deathin PC12 cells. Here, our results demonstrated that severalmechanisms, separately or in association, may be involved inthe neuroprotective effects of erythropoietin. A body of workhas been generated to support the premise that MPP+ candirectly lead to the formation of reactive oxygen species andmitochondrial dysfunction (Cleeter et al., 1992; Sriram et al.,1997; Cassarino et al., 1999; Lotharius et al., 1999; Lee et al.,2000). The production of reactive oxygen species can lead tocell injury through cell membrane lipid destruction andcleavage of DNA (Vincent and Maiese, 1999; Wang et al.,2003), which plays a critical role in apoptosis. Oxidative stresscaused by MPP+ might be, at least in part, responsible for theopening of mitochondrial permeability transition pore and thecollapse of mitochondrial membrane potential (Cassarino et al.,1999). Antioxidants that modulate reactive oxygen species havebeen shown to prevent the age-related increase in the proteincarbonyl content in mouse synaptic mitochondria (Renzi et al.,2002) and reduce apoptotic cell death in the dopaminergic celllines and PC12 cells treated with MPP+ (Blum et al., 2001).Erythropoietin has been shown to afford its neuroprotection byits antioxidant effects (Liu et al., 2006; Kumral et al., 2005;Ozturk et al., 2005; Genc et al., 2002) which is furtherconfirmed by our data that erythropoietin highly effective oninhibiting the MPP+-induced reactive oxygen species formationand mitochondrial membrane potential loss in PC12 cells. Thepresent results have suggested that erythropoietin attenuates themitochondrial damage and cell death in PC12 cells caused by

oxidative stress. Based on these findings, we postulated that theanti-oxidative properties of erythropoietin may contribute to theprotection of PC12 cells from damage by MPP+.

Consequently, some other mechanisms could also bepertinent in the erythropoietin protective mechanism. Bcl-2family plays a key role in the mitochondrial apoptotic pathway(Cory and Adams, 2002). Bcl-2 is localized the mitochondria(Akao et al., 1994) which are active sources of reactive oxygenspecies, it could prevent the depolarization of mitochondrialmembrane potential and the production of reactive oxygenspecies from mitochondria, therefore preventing apoptosis (LudCadet et al., 2000; Kane et al., 1993). Bcl-2 over expressionmay be involved in the inhibition of erythropoietin on theincreased reactive oxygen species concentration induced byMPP+ in the present study. In contrast, Bax has beendemonstrated to increase the production of reactive oxygenspecies from mitochondria (Kirkland et al., 2002). Cell survivalin the early phases of apoptotic cascade depends mostly on thebalance between the Bax and Bcl-2. The Bax/Bcl-2 ratio maybetter predict the apoptotic fate of the cell than the absoluteconcentrations of either (Cory and Adams, 2002).

Erythropoietin may also exert its neuroprotective effect viathe differential regulation of gene expression ruling the balancebetween these molecules (Renzi et al., 2002; Wen et al., 2002;Chong et al., 2003a; Maiese et al., 2005). A more recent studyin vivo has reported that erythropoietin significantly preventedBax mRNA upregulation and reversed downregulation in Bcl-2transcription induced by hypoxia–ischemia in brain tissue(Kumral et al., 2006). Our present study showed that MPP+

significantly increased the ratio of the pro-apoptotic Bax to theanti-apoptotic Bcl-2, which consisted with the previous studies(Blum et al., 2001; Wang and Xu, 2005). However, treatmentwith erythropoietin reduced the expression of Bax and increasedthe expression of Bcl-2, thereby ameliorated the MPP+-inducedBax/Bcl-2 ratio elevation in PC12 cells. The results furthersupport the anti-oxidative effect of erythropoietin on MPP+-induced apoptosis maybe, at least partly, mediated by regulatingthe expression of Bax and Bcl-2.

It has been reported that PC12 cells cultured with highconcentration of erythropoietin (10 U/ml or 25 nM) showedsignificant changes in gene expression by 3 h with a return tobasal expression levels for the vast majority of genes by 24 h,whereas lower concentration of erythropoietin (10 pM) wasobserved to be effective at mediating the expression of the Bcl-xl and bak and these changes in gene expression required alonger pretreatment time of 24 h (Renzi et al., 2002). Onepossible explanation for this phenomenon is that lowerconcentration of erythropoietin results in lower levels ofreceptor occupancy and decreases activation of signalingpathways, therefore requiring a longer period of time to havea full effect on gene expression. The Bcl-2 and Bax mRNAlevel may reach their largest change in response to moderateconcentration of erythropoietin (1 U/ml) at a period of timemuch shorter than 24 h and the protein level may return to basallevels by 24 h. In the present study, after treatment of PC12 cellsfor 24 h with erythropoietin at 1 U/ml, the protein level of eitherBcl-2 or Bax was similar to that of control group.

55Y. Wu et al. / European Journal of Pharmacology 564 (2007) 47–56

The increasing findings have shown that the activation ofcaspase-3 during reactive oxygen species exposure occurs in theMPP+-induced neurodegeneration (Blum et al., 2001). Howev-er, erythropoietin effectively suppressed MPP+-induced activa-tion of caspase-3, suggesting that erythropoietin may actupstream of caspase-3 to block apoptosis. It is hypothesizedthat a compensatory induction of Bcl-2 was unable to counteractthe apoptotic action of Bax, resulting in caspase-3 activationand cell death (O'Malley et al., 2003), which has been closelyassociated to the pathogenesis of Parkinson disease. From ourown observation, a decrease in caspase-3 activity correlates wellwith a decrease in the Bax/Bcl-2 ratio, as pro-apoptotic Bax isthought to be upstream of the caspase in the mitochondria-mediated apoptotic death pathway (Cory and Adams, 2002),suggesting that the mechanisms by which erythropoietininhibits MPP+-triggered activation of caspase-3 might includeboth its anti-oxidative activity and its regulatory function in Bcl-2 family.

PI3K is a well known signaling pathway involved in cellprotection under various stresses, including oxidative stress.One of the downstream effectors of PI3K is the serine/threoninekinase PKB/Akt, which plays a role in PI3K-mediated neuronalcell survival (Dudek et al., 1997). PI3K/Akt pathway promotescardiomyocytes survival against oxidative stress-induced apo-ptosis (Hong et al., 2001) and also delivers an anti-apoptoticsignal in PC12-ErbB4 cells against oxidative stress induced byMPP+ (Di Segni et al., 2006). Our experiments demonstratedthat the protection conveyed by erythropoietin was mediated viathe PI3K pathway because LY 294002 inhibited this effect. Inaddition, erythropoietin can activate Akt. Also, LY 294002inhibited erythropoietin-induced Akt activation. Our resultsfurther demonstrated that erythropoietin can decrease theMPP+-mediated elevation in reactive oxygen species levelsthrough the PI3K signaling pathway. Erythropoietin also has theability to activate the extracellular signal-regulated kinases(ERKs) pathway, which are important for neuron survival (Xiaet al., 1995). However, PD98059, a specific inhibitor of theactivation of ERKs did not affect the survival effect oferythropoietin in MPP+-induced cell death (data not shown),which indicated that the survival signal pathway of erythropoi-etin was restricted by the PI3K/Akt pathway in MPP+-inducedcell death.

In this study, our results show that erythropoietin amelioratesMPP+-induced reactive oxygen species production, attenuatesthe mitochondrial transmembrane potential loss, downregulatesthe Bax/Bcl-2 ratio, prevents the activation of caspase-3.Erythropoietin affords significant neuroprotection againstMPP+-induced injury in PC12 cells via the PI3K/Akt-mediatedsignaling pathway. These findings, taken together, support thetheory that cytoprotection mediated by erythropoietin is due, inpart, to inhibition of the oxidative stress resulting from themitochondrial apoptotic pathway.

Acknowledgments

The authors gratefully acknowledge Jieping Zhou fortechnical assistance. This study was supported by a grant from

the Program of National Natural Science Foundation of China(NO. 30570627/C030307).

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