TOXICOLOGICAL SCIENCES 97(2), 448–458 (2007)

doi:10.1093/toxsci/kfm040
Advance Access publication March 6, 2007

Inhibition of Paraquat-Induced Autophagy Accelerates the Apoptotic

Cell Death in Neuroblastoma SH-SY5Y Cells
Rosa A. González-Polo,*,1 Mireia Niso-Santano,* Miguel A. Ortı́z-Ortı́z,* Ana Gómez-Martı́n,* José M. Morán,*
Lourdes Garcı́a-Rubio,† Javier Francisco-Morcillo,‡ Concepción Zaragoza,§ Germán Soler,* and José M. Fuentes*

Downloaded from http://toxsci.oxfordjournals.org at Dokuz Eylul University Library (DEU) on August 20, 2010
*CIBER de Enfermedades Neurodegenerativas, Departamento de Bioquı´mica y Biologı´a Molecular y Gene´tica, E.U. Enfermerı´a y T.O., Universidad de
Extremadura, Avda. Universidad s/n 10071 Cáceres, Spain; †Departamento de Quı´mica Orgánica, ‡Departamento de Biologı´a Celular, §Departamento
de Medicina y Sanidad Animal, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain

Received January 12, 2007; accepted February 27, 2007

damaged organelles and cytoplasmic components) stress con-

Autophagy is a degradative mechanism involved in the recy-
cling and turnover of cytoplasmic constituents from eukaryotic
cells. This phenomenon of autophagy has been observed in
neurons from patients with Parkinson’s disease (PD), suggesting
a functional role for autophagy in neuronal cell death. On the
other hand, it has been demonstrated that exposure to pesticides
can be a risk factor in the incidence of PD. In this sense, paraquat
(PQ) (1,1#-dimethyl-4,4#-bipyridinium dichloride), a widely used
herbicide that is structurally similar to the known dopaminergic
neurotoxicant MPP+ (1-methyl-4-phenyl-pyridine), has been sug-
gested as a potential etiologic factor for the development of PD.
The current study shows, for the first time, that low concen-
trations of PQ induce several characteristics of autophagy in
human neuroblastoma SH-SY5Y cells. In this way, PQ induced
the accumulation of autophagic vacuoles (AVs) in the cytoplasm
and the recruitment of a LC3-GFP fusion protein to AVs.
Furthermore, the cells treated with PQ showed an increase of
the long-lived protein degradation which is blocked in the
presence of the autophagy inhibitor 3-methyladenine and regu-
lated by the mammalian target of rapamycin (mTOR) signaling.
Finally, the cells succumbed to cell death with hallmarks of
apoptosis such as phosphatidylserine exposure, caspase activation,
and chromatin condensation. While caspase inhibition retarded
cell death, autophagy inhibition accelerated the apoptotic cell
death induced by PQ. Altogether, these findings show the re-
lationship between autophagy and apoptotic cell death in human
neuroblastoma cells treated with PQ.
Key Words: paraquat; vacuoles; apoptosis; Parkinson.
Autophagy is an intracellular lysosome-mediated catabolic
mechanism that is responsible for the bulk degradation and
recycling of damaged or dysfunctional cytoplasmic compo-
nents and intracellular organelles (Klionsky et al., 2000).
Autophagy is a cellular response to both extracellular (nutrient
deprivation or hypoxia) and intracellular (accumulation of
1
To whom correspondence should be addressed. Fax: þ34-927-257-451.
E-mail: rosapolo@unex.es.

The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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ditions. Autophagy, being an evolutionarily ancient cellular
response to intra- and extracellular noxious stimuli, may
precede or coexist with apoptosis, and it may be induced by
apoptotic stimuli (Xue et al., 1999). Thus, autophagy and
apoptosis may be interconnected (Bursch et al., 2000). Cellular
autophagy is a physiological degradative process involved, like
apoptosis, in embryonic growth and development, cellular
remodeling, and the biogenesis of some subcellular organelles
(Filonova et al., 2000; Hariri et al., 2000; Sattler and Mayer,
2000). Autophagic cell death involves accumulation of auto-
phagic vacuoles (AVs) in the cytoplasm of dying cells as well
as mitochondria dilation and enlargement of the endoplasmic
reticulum and the Golgi apparatus. Autophagic cell death has
been described during the normal nervous system development
(Schweichel et al., 1973) and could be a consequence of
a pathological process such as those associated with neurode-
generative diseases (Petersen et al., 2001). Of note, autophagy
is highly enhanced in brain amyloidoses (Graeber et al., 2002),
Alzheimer disease (Stadelmann et al., 1999), Huntington’s
disease (Landles et al., 2004), and Parkinson’s disease (PD)
(Anglade et al., 1997).
PD is a chronic progressive neurodegenerative disease,
affecting at least 1% of the population over the age of 55.
Genetic forms of the disease represent less than 5% of current
cases, but the causes of the vast majority of sporadic cases of
PD are still unknown. Accumulating evidence strongly points
to environmental toxins as feasible triggers of neurodegenera-
tion of nigrostriatal dopaminergic neurons, and the common
use of pesticides in rural life has been correlated to parkinson-
ism in humans (Di Monte et al., 2000). In this sense, the
herbicide 1,1#-dimethyl-4,4#-bipyridinium dichloride (paraquat
[PQ]), widely used as a cationic nonselective bipyridyl
herbicide to control weeds and grasses in many agricultural
areas, has emerged as a putative risk factor on the basis of its
structural homology to 1-methyl-4-phenyl-pyridine (MPPþ),
the active metabolite of 1-methyl-4-phenyl-1,2,3,6 tetrahydro-
pyridine, a neurotoxicant that induces Parkinson’s-like features
 PARAQUAT INDUCES AUTOPHAGY
in rodents, nonhuman primates, and humans (Langston and
Ballard, 1984; Tanner and Ben Shlomo, 1999).
Occupational PQ exposures have been associated with
parkinsonism (Hertzman et al., 1990; Liou et al., 1997), although
the mechanism of PQ toxicity is yet poorly understood. Several
studies have suggested the involvement of reactive oxygen
species (ROS) in its effect (Gonzalez-Polo et al., 2004; Mollace
et al., 2003). Our group has demonstrated that free radicals play
an active role in the PQ-induced apoptotic events that culminate
with cell death (Gonzalez-Polo et al., 2004).
Here, we show for the first time that low concentrations of
PQ induce several characteristics of autophagy in human
neuroblastoma SH-SY5Y cells, which are commonly selected
as a cellular PD model (Lai et al., 1997; Lee et al., 2000;
Shavali et al., 2004). Finally, the cells succumbed to cell death
with hallmarks of apoptosis. Thus, we investigated the re-
lationship between autophagy and apoptosis in neurons treated
with PQ to propose a new and exciting strategy to the
knowledge of the PD mechanism and its possible treatment.
MATERIALS AND METHODS
Cell line and culture condition. Human neuroblastoma SH-SY5Y cells
were grown in Dulbecco’s modified Eagle medium (Gibco BRL, Paisley, U.K.)
containing 100 U/ml penicillin, 100 lg/ml streptomycin, and 10% fetal bovine
serum (Hyclone, Brevieres, France). Cells were seeded at 5 3 105 in a 75-cm2
tissue culture flask (TPP, Trasadingen, Switzerland) and incubated at 37C
under a saturating humidity atmosphere of 5% CO2/95% air.
Reagents and cell death induction. Confluent cells (~80%) in 75-cm2
tissue culture flasks were trypsinized and seeded in tissue culture dishes at
a concentration of 5 3 104 cells/cm2. Twenty-four hours later, the medium was
aspirated and replaced with fresh medium alone or containing the indicated
concentrations of PQ (Niso-Santano et al., 2006). Serum and amino acid
starvation of cells were performed using serum-free Earle’s balanced salt
solution medium (Sigma, St Louis, MO) (Boya et al., 2005). For caspase
inhibiton, a sublethal dose of N-benzyloxycarbonyl-Val-Ala-Asp fluorome-
thylketone (Z-VAD-fmk, 50lM, Bachem AG, Bubendorf, Switzerland) was
added 1 h before PQ. The antioxidants N-acetylcysteine (NAC 1mM), vitamin E
(100lM), glutathione (1mM), and the inihibitor of autophagy, 3-methyladenine
(3-MA, 10mM), were added before the addition of PQ.
Flow cytometry. To determine apoptosis-associated changes by cytofluor-
ometry, we used 3,3#-dihexyloxacarbocyanine iodide (DiOC6(3), 40nM) for the
mitochondrial transmembrane potential quantification, propidium iodide (PI,
1 lg/ml) for the determination of cell viability (Boya et al., 2003; Gonzalez-Polo
et al., 2005a), and Annexin-V labeled with fluorescein isothiocyanate (all of
them from Molecular Probes, Eugene, OR) for the assessment of phosphati-
dylserine exposure. For the determination of superoxide anion generation,
hydroethidine (HE, 10lM) was used. After different experimental conditions,
cells were trypsinized and labeled with the fluorochromes at 37C, followed by
cytoflurometric analysis with a FACS Scan (Becton Dickinson, Frankin Lakes,
NJ) (Gonzalez-Polo et al., 2005a,b).
Immunofluorescence and light microscopy. Cells were cultured on
coverslips. After the experimental conditions, the cells were stained with
CMFDA (5-chloromethylfluorescein diacetate, 1lM) (Molecular Probes) for
30 min at 37C to visualize the vacuoles. Cells were fixed with para-
formaldehyde (4% wt:vol) for CMFDA staining and immunofluorescence
assays (Gonzalez-Polo et al., 2005a,2005b). Cells were stained for the detection
of activated caspase-3 with a polyclonal antibody from Cell Signaling
449
Technology, Inc. (Danvers, MA) developed with an anti-rabbit immunoglobulin
Alexa fluor conjugate (Molecular Probes) and counter-stained with Hoechst
33342, 2lM (Ho) (Sigma) before mounting. Fluorescence microscopy was
analyzed with an Olympus IX51 equipped with a DC300F camera.
Western blot analysis. Following experimental treatments, the cells
(cultured in 60 mm dishes) were rinsed twice with cold phosphate-buffered
saline (PBS) and removed by scrapping and then centrifuged at 900 3 g for 5 min
at 4C. Cells were lysed in a buffer containing 50mM N-2-hydroxyethyl-
piperazine-N#-2-ethanesulfonic acid pH 7.5, 100mM NaCl, 1% Triton X-100,
10% glycerol, 1mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 5mM
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MgCl2, 25mM NaF, and Protease Inhibitor Cocktail (Sigma). Cells were
centrifuged at 13,000 3 g 5 min at 4C. Supernatants were stored at
80C
until analysis by Western blot. Protein concentration was measured according to
Bradford (1976) using bovine serum albumin (BSA) as standard. Equal amounts
of proteins (20 lg/condition) were resolved in 12–15% sodium dodecyl sulfate
gel electrophoresis and transferred to polyvinylidene difluoride (PVDF)
membranes according to conventional partially modified methods (Fuentes
et al., 2000). Briefly, proteins were transferred (250 mA for 60 min) to PVDF
membranes using Mini Trans-Blot Cell apparatus (Bio-Rad, Hercules, CA). The
procedure for immunodetection includes the transfer and blocking of the
membrane (60 min at room temperature) with Tween–Tris-buffered saline
(TTBS) (10mM Tris/HCl pH 7.5, 150mM NaCl, and 0.2% Tween-20) containing
10% nonfat-dried milk. Membranes were then incubated overnight at 4C with
primary antibodies, p-mTORser2448, and cleaved caspase-3 from Cell Signal-
ling (Beverly, MA) and anti-LC3 from MBL Medical and Biological Labora-
tories Co., Ltd (Nagoya, Japan), all of them diluted 1:1000 in TTBS þ 10%
nonfat-dried milk or 5% BSA. After washing (for two 5-min periods with TTBS),
membranes were incubated (60 min at room temperature) with peroxidase-
conjugated secondary antibodies (1:5000 in TTBS with 10% nonfat-dried milk).
After washing (for two 5-min periods and one 10-min period), the detection of
bound antibodies was visualized by chemiluminiscence using the ECL-plus
reagent (GE Healthcare, Buckinghamshire, UK). Actin content was analyzed as
a control by means of a rabbit polyclonal antibody (from Sigma).
Analysis of protein degradation. Human neuroblastoma SH-SY5Y cells
were incubated with 0.2 mCi/ml. L-[14C]valine (GE Healthcare) in complete
medium (CM) for 24 h at 37C. Unincorporated radioisotope was removed by
washing the cells three times with PBS (pH 7.4). Cells were then incubated in
Earle’s balanced salt solution buffer plus 0.1% BSA (nutrient-free [NF]
medium) in the presence of 10mM cold valine, for 1 h (prechase period).
After this time, the medium was replaced by the appropriate fresh medium (NF
or CM) plus cold valine 10mM in the presence or absence of 10mM 3-MA and
10lM PQ for 4 h (chase period). Cells and radiolabeled proteins from the
medium were precipitated in trichloroacetic acid at the final concentration of
10% (vol/vol). The precipitated proteins were separated from the soluble
radioactivity by centrifugation at 600 3 g for 20 min and then dissolved in 1 ml
of 0.2 N NaOH. The rate of protein degradation was calculated by determining
the ratio of acid-soluble radioactivity recovered from both cells and medium to
the ratio of radioactivity in trichloroacetic acid-precipitated proteins obtained
from both cells and medium (Pattingre et al., 2003).
Electron microscopy. Scraped cells were spinned and immersed in
a mixture of 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer
(pH 7.4) for 1 h at 4C. Pellets were then rinsed in phosphate buffer, postfixed in
1% osmium tetroxide for 90 min, rinsed, dehydrated in a graded series of
acetone, and embedded in Araldite resin. Ultrathin 90-nm–thick sections were
obtained using a Leica Reichert Ultracut ultramicrotome equipped with a glass
knife. Sections were mounted on copper grids, stained with uranyl acetate and
lead citrate, examined, and photographed with a Jeol JEM-100 CX II
transmission electron microscope.
Transient transfections. SH-SY5Y cells were transiently transfected with
the Fugene procedure, with 1–2 lg DNA per 35-mm plate, according to the
manufacturer’s protocol (Roche Diagnostics, GmbH, Mannheim, Germany) to
label AVs with LC3-GFP plasmid (Kabeya et al., 2000).
450 GONZÁLEZ-POLO ET AL.

Statistical analysis. Data are representative of at least three independent
experiments each in triplicate determination. Statistical analysis of the data was
performed using one-way ANOVA. Significance (*) was defined at p < 0.05.
RESULTS
PQ Induces Apoptotic Death in Neuroblastoma Cells
As previously described (Chun et al., 2001; Gonzalez-Polo
including human neuroblastoma SH-SY5Y cells, are sensitive
to the PQ induced toxicity. We investigated, for the first time,
the cell death type in neuroblastoma cells exposed to the
bipiridinic pesticide PQ. As we show in Figure 1, the PQ
treatment caused phosphatidylserine exposure on the outer
leaflet of the plasma membrane, which can manifest before
plasma membrane permeabilization. This phenomenon was
increased in combination with PQ and starvation conditions.
The cells treated with 10–25lM of PQ exhibited a range of

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et al., 2004; Niso-Santano et al., 2006), several cell lines, 25–50% loss of cell viability in CM and NF medium. In parallel

FIG. 1. PQ-mediated sensitization of SH-SY5Y cells to starvation-induced cell death. The human neuroblastoma SH-SY5Y cells were cultured in the
indicated conditions (CM, NF), in the absence (Co) or in the presence of the indicated doses of PQ for 24 h, followed by simultaneous staining of the
phosphatidylserine exposure with Annexin-V fluorescein isothiocyanate (FITC) and viability with PI. Quantitative data (error bars are SE n ¼ 3) are shown in (A),
and representative FACS diagrams are shown in (B).
 PARAQUAT INDUCES AUTOPHAGY 451

experiments, we analyzed the involvement of caspases in the
apoptotic death induced by 10lM of PQ (Fig. 2). The maximum
level of caspase-3 activation was evident after 24 h of PQ
incubation, shown by Western blot detection of a 17-kDa
fragment (active form) and caspase-3 total, both in CM (Fig. 2A)
and NF medium (Fig. 2B). Morphological analysis of cells
subjected to the combined treatment of PQ 10lM and starvation
indicate a typical apoptotic chromatin condensation (as detect-
able with the nuclear stain Hoechst 33342) associated with
caspase inhibitor Z-VAD-fmk would prevent cell death in this
model. As shown in Figure 2E, Z-VAD-fmk retarded cell death
of PQ-treated, SH-SY5Y cells, both in CM and NF medium.
PQ Induces Reactive Oxygen Species Production in SH-SY5Y
Neuroblastoma Cells
The mechanism of action of PQ is as yet poorly understood.
However, it is well known that PQ acts as a reactive oxygen

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caspase-3 activation (Figs. 2C and 2D). Therefore, we won- species (ROS) producer (Bus et al., 1984; Suntres 2002). These
dered whether inhibition of caspases with the broad-spectrum ROS interact with unsaturated lipids of membranes (lipid

FIG. 2. Involvement of caspases in PQ-induced apoptosis in SH-SY5Y cells. Caspase-3 activation induced by PQ, (A–C). (A, B) Activation of caspase-3 was
determined by Western blot as indicated in ‘‘Materials and Methods’’ section. Actin expression was determined as a loading control. Cells were incubated in CM
(A) or NF medium (B) at the times indicated with 10lM of PQ. Blots are representative of at least three independent experiments. (C, D) SH-SY5Y cells cultured in
poly-L-lysine–treated coverslips were incubated in the absence (Co) or in the presence of PQ (10lM) in the indicated medium for 24 h. Cells were fixed and
immunostained for the detection of active caspase-3 (green fluorescence) and nuclear chromatin condensation (Hoechst 33324, blue fluorescence). Representative
microphotographs are depicted in (C), and the frequency of the measured changes is analyzed in (D) (*p < 0.05 compared with control). (E) Effect of the caspase
inhibitor Z-VAD-fmk on cell killing. Cells were cultured in the absence or presence of nutrients, PQ 10lM and/or Z-VAD-fmk 50lM for 24 h, and the frequency of
cells with high Annexin-V fluorescein isothiocyanate (FITC) or PI incorporation was determined as in Figure 1A (*p < 0.05 compared with PQ treatment without
Z-VAD-fmk).
452 GONZÁLEZ-POLO ET AL.
peroxidation) and destroy organelles subsequently leading to
cell death (Dodge, 1971). In this sense, several studies have
suggested the involvement of ROS in the toxicity induced by
PQ, including studies in vitro in several cell lines as in human
lung epithelial cells (Cappelletti et al., 1998) or in cerebellar
granule cells (Bus et al., 1984; Gonzalez-Polo et al., 2004),
as well as studies in vivo (Mollace et al., 2003). In our study, we
showed that exposure to low concentrations of PQ (10lM) for
24 h caused an increase of intracellular levels of ROS in human
neuroblastoma SH-SY5Y cells (as measured by the oxidation
of the nonfluorescent dye HE into its fluorescent product
ethidium [Eth]) (Fig. 3). This production was enhanced when
the cells were treated with a combination of nutrient depletion
conditions and PQ. The ROS generation was abolished in
the presence of several antioxidants as N-acetylcysteine as well
as the ROS scavenger vitamin E (a-tocopherol) (data not
shown).
The Treatment of SH-SY5Y Neuroblastoma Cells with
Low Concentrations of PQ Produces Characteristics
of Autophagy
It has been described that autophagy is a cellular response to
extracellular (nutrient deprivation, hypoxia) and intracellular
stress conditions (accumulation of damaged organelles and
cytoplasmic components). This phenomenon is characterized
morphologically by the accumulation of AVs in the cytoplasm.
FIG. 3. PQ-induced formation of superoxide. SH-SY5Y cells were
incubated in the indicated medium, CM or NF, in the absence (Co) or in the
presence of PQ (10lM) for 24 h followed by quantitation of the frequency of
cells that exhibit oxidation of HE into the fluorescent Eth. All results are
representative of at least three independent determinations. Numbers in FACS
diagrams refer to the percentage of cells that exhibit oxidation of HE into the
fluorescent Eth.
We have tested in our model that, in NF medium, that is, in the
absence of serum and amino acids, as well as in CM, control
cells did not develop any discernible vacuoles, while cells
treated with PQ 10lM exhibited an increase in cytoplasmic
vacuolization visible as ‘‘holes’’ in CMFDA-stained cells of
a time-dependent way (Figs. 4A and 4B). Moreover, these
vacuoles induced in the presence of PQ showed, clearly, the
characteristic double membranes of AVs as visible by trans-
mission electron microscopy (Fig. 4C). Figures 4A and 4C
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show images in CM conditions. Microphotographs in NF
conditions are equivalents (data not shown).
Similarly, after treatment of serum and amino acid starva-
tion, 10lM of PQ induced the recruitment of a LC3-GFP fusion
protein to AV in discrete foci (Kabeya et al., 2000; Mizushima
2004) (Figs. 5A and 5B). In agreement with the microscopic
observations, we detected by Western blotting the accumula-
tion of the autophagosome marker LC3-II (Boya et al., 2005;
Kabeya et al., 2000) both in CM (Fig. 5C) and under the
conditions of starvation (data not shown) in SH-SY5Y treated
with PQ 10lM. Furthermore, as compared to the prototypic
autophagy inhibitor 3-MA, PQ (10lM) induced the autophagy
(as determined by quantifying the turnover of proteins in
starved cells) as shown in Figure 6A. In this sense, exposure of
human neuroblastoma cells with 10lM of PQ produced
a marked decrease in the phosphorylation (Ser2448) status of
mTOR (Figs. 6B and 6C), protein involved in the autophagy
signaling, with a maximal effect after 30 min of herbicide
exposure in CM (Fig. 6B). In starved cells incubated with PQ,
the activation of phosphorylated mTOR was invaluable to all
the tested times (Fig. 6C).
The Inhibition of PQ-Induced Autophagy Accelerates
the Apoptotic Cell Death in SH-SY5Y Cells
We investigated the possible connection between the accu-
mulation of autophagic phenomenon and the apoptotic cell
death induced by PQ in SH-SY5Y cells. The morphological
analysis (Figure 7A) showed that the prototypic autophagy
inhibitor 3-MA inhibited the PQ-induced autophagic vacuoli-
zation as well as protein degradation, already previously
mentioned in Figure 6A. Moreover, cytofluorometric quantifi-
cation revealed that a significant fraction of cells treated with
3-MA and/or PQ lost the capacity to retain the dye DiOC6(3)
and hence dissipated the mitochondrial transmembrane poten-
tial (Dwm). Among this Dwm low population, a fraction of cells
incorporated the vital dye PI and thus lost the barrier function
of their plasma membrane (Fig. 7B). This event was more
evident in combination of 3-MA and the herbicide PQ. In the
same way, the caspase-3 activation, as measured by Western
blot detection of 17-kDa fragment (Fig. 7C), was more
increased with PQ and 3-MA together that each one separately.
Therefore, the combination of these results clearly suggests
that the inhibition of PQ-induced autophagy accelerates the
apoptotic cell death in SH-SY5Y cells.
 PARAQUAT INDUCES AUTOPHAGY 453

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FIG. 4. PQ induces accumulation of AVs. (A, B) SH-SY5Y cells were cultured in CM or NF in the presence of PQ 10lM the times indicated. The presence of
cytoplasmic vacuoles was determined by staining with CFMDA as indicated in ‘‘Materials and Methods’’ section. Cytoplasmic vacuoles are visualized as holes.
Representative microphotographs in CM are depicted in (A), and the percentage of vacuolated cell area is analyzed in (B) (error bars are SE n ¼ 3).
(C) Ultrastructure of PQ-treated SH-SY5Y cells. The cells were treated as in (A). Electron microscopy of representative cells are shown.

DISCUSSION development of nongenetic PD (Cory-Slechta et al., 2005;

Landrigan et al., 2005; Norris et al., 2004; Ritz et al., 2000;
Several studies have shown the possibility that environmen- Sherer et al., 2001). PQ is one of the possible pesticides
tal neurotoxicants such as pesticides may be related to the involved in PD genesis because of the similar chemical
454 GONZÁLEZ-POLO ET AL.

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FIG. 5. Effect of PQ on the subcellular localization and status of the AV marker LC3. (A, B) Redistribution of LC3-GFP. The cells were transfected with an
LC3-GFP fusion construct as indicated in ‘‘Materials and Methods’’ section. Twenty-four hours after transient transfection, cells were incubated in the indicated
medium, CM or NF, treated in the absence (Co) or presence of PQ 10lM for 15 h and fixed. Representative fluorescence microphotographs are shown in (A), and
the frequency of cells exhibiting the accumulation of LC3-GFP in vacuoles was quantified (*p < 0.05 compared with control) in (B). (C) Immunoblot analyses of
accumulating LC3-II protein in cells treated with PQ 10lM the indicated times. Actin expression was determined as a loading control in the same conditions. Blots
are representative of at least three independent experiments.

structure to the dopaminergic neurotoxicant MPPþ and the
strong correlation between the incidence of the disease and the
amount of PQ used (Ritz et al., 2000). In the present study we
report, for the first time, that low concentrations of PQ induce
molecular events compatible with autophagy in neuroblastoma
SH-SY5Y cells. We also show that the inhibition of autophagy
exacerbates PQ-induced apoptosis.
The human dopaminergic cell line SH-SY5Y, which has
many qualities of nervous system neurons, was used as target
neuronal cells. First, we confirmed that PQ induced neurotox-
icity in SH-SY5Y cells in a dose-dependent manner both in
CM and in starvation conditions (Fig. 1). This result was
similar to the results reported by Gonzalez-Polo et al. (2004)
and Yang et al. (2005). Our group found that PQ induced dose-
dependent cell death and apoptosis in SH-SY5Y cells within
a period of 24 h. PQ is a well-known oxidative-stress inducer
(Gonzalez-Polo et al., 2004; Mollace et al., 2003), so most of
the reports about it are focused on ROS production. In this
sense, we show in our study that low concentrations of PQ
(10lM) induce a robust ROS generation (Fig. 3), which is more
evident in NF conditions. However, the major finding of this
study is that low PQ concentrations produce characteristics of
autophagy.
Autophagy was described for the first time by Schweichel
et al. (1973). It is characterized by the appearance of numerous
cytoplasmic AVs of lysosomal origin, followed by mitochon-
drial dilation and enlargement of the endoplasmic reticulum
and the Golgi apparatus. Dysfunction of cellular degradation
has been implicated in PD pathogenesis. Macroautophagy is
the primary mechanism of degrading long-lived proteins and
damaged organelles. The autophagic process begins with the
remodeling of subcellular membranes into double-membrane
vesicles that sequester cytoplasm and organelles in AVs. These
vacuoles are fused with the lysosome to form the autophago-
lysosome. The content of the autophagolysosome are ulti-
mately degraded by lysosomal degradative enzymes (Klionsky
et al., 2000). The electron microscopy images in Figure 4 show
that PQ induces the accumulation of double-membrane AVs in
the cytoplasm. This event is accompanied by other autophagic
characteristics as accumulation of the autophagosome marker
LC3-II (Fig. 5) or protein degradation regulated negatively by
the mTOR kinase signaling (Fig. 6). In this sense, it has been
demonstrated that the inactivation of mTOR in yeast or
mammalian cells leads to induction of autophagy, even in
nutrient-rich medium, indicating that mTOR negatively con-
trols starvation-induced autophagy (Blommaart et al., 1995).
So, under unfavorable conditions, mTOR is inactive, leading to
a reduction in protein synthesis and an upregulation of protein
degradation (Dennis et al., 1999). Thus, we report here that PQ
is involved in the control of autophagy in neuroblastoma
SH-SY5Y cells. So, one possibility is that the cell treated with
PQ triggers autophagy as a defense mechanism to degrade the
oxidized proteins by ROS and damaged or dysfunctional
cytoplasmic components.
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FIG. 6. Study of the protein degradation and changes of mTOR phosphorylation in the PQ-mediated neurotoxicity. (A) The protein half-life was determined by
pulse chasing with radioactive valine as indicated in ‘‘Materials and Methods’’ section. The cells were incubated in CM or NF and treated with PQ 10lM in the
presence or absence of 3-MA 10mM for 6 h. *p < 0.05 compared with control. (B, C) mTOR dephosphorylation induced by PQ. The SH-SY5Y cells were
incubated with 10lM of PQ the indicated times in CM (B) or in NF medium (C). Cell lysates were analyzed for mTOR activation by Western blotting using
a phospho-specific mTOR (ser2448) antibody as indicated in ‘‘Materials and Methods’’ section. Actin expression was determined as a loading control in the same
conditions. Blots are representative of at least three independent experiments.

The pesticide induced, in the first place, the morphological
appearance of autophagy (Figs. 4–6). But finally the cells
succumbed in morphological and biochemical changes that are
typical of apoptotic cell death, as phosphatidylserine exposure
(Fig. 1), Dwm dissipation (Fig. 7), caspase-3 activation (Fig. 2),
and chromatin condensation (Fig. 2). Interestingly, inhibition
of caspases retarded cell death (Fig. 2), yet had no influence on
autophagic vacuolization (data not shown). In this sense, there
is some controversy in the bibliography regarding the precise
role that autophagy might play in programed cell death. There
are many reports describing situations in which autophagy
activation accompanies apoptosis, but there are also cases in
which autophagy enhancement leads to cell death in the
absence of caspase activation (Bursch et al., 1996; Butler
et al., 2000; Chi et al., 1999; Xiang et al., 1996). Moreover,
autophagic cell death has been described in association with
several neurodegenerative diseases, including PD (Anglade
et al., 1997; Marino et al., 2004; Petersen et al., 2001).
Increased levels of autophagy were observed in neuronal cell
lines expressing mutant proteins associated with such diseases.
Expression of PD-associated A53T, but not wild-type,
a-synuclein in PC12 cells induced massive AV formation
(Stefanis et al., 2001). In addition, dopaminergic neurons with
AVs have been observed in the substantia nigra of PD patients,
along with neurons displaying apoptotic features (Anglade
et al., 1997). However, the functional consequence of increased
autophagic activity for neurodegeneration is not clear but has
been suggested that excessive autophagy may directly lead to
neuronal cell death (Yuan et al., 2003).
In the literature, concentrations of high micromolar or
millimolar range of PQ are normally used to study its
neurotoxicity and apoptosis induction (Gonzalez-Polo et al.,
2004; Kim et al., 2004; Peng et al., 2004). The evidence that
PQ triggers apoptotic cell death has been demonstrated in other
cellular models, as in human lung epithelial cells (Cappelletti
et al., 1998) as well as in PC12 cells (Li et al., 1999). Our
experiments show that PQ induces apoptosis in SH-SY5Y cells
at low concentrations (10lM) at the same concentrations that
456 GONZÁLEZ-POLO ET AL.

Downloaded from http://toxsci.oxfordjournals.org at Dokuz Eylul University Library (DEU) on August 20, 2010
FIG. 7. Inhibition of PQ-induced autophagy accelerates the apoptotic cell death. (A, B) 3-MA inhibits the cytoplasmic vacuolization induced by PQ. The
human neuroblastoma SH-SY5Y cells were cultured in CM or NF and treated with PQ 10lM in the presence or absence of 3-MA 10mM for 24 h. The cells then
were stained with CFMDA and fixed to visualize the presence of cytoplasmatic vacuoles as holes. Representative microphotographs in CM are depicted in A, and
the percentage of vacuolated cell area is analyzed in B (*p < 0.05 compared with PQ-treated cells without 3-MA). (C) Quantitative assessment of synergic cell
death induction. Cells cultured in CM or NF in the presence or absence of 3-MA 10mM and/or PQ 10lM for 24 h were stained to determine the loss of
mitochondrial transmembrane potential (with DiOC6(3)) and viability (with PI). *p < 0.05 compared with PQ-treated cells without 3-MA. (D) Synergic caspase-3
activation induced in combination of PQ and 3-MA treatment. Activation of caspase-3 was determined by Western blot as indicated in ‘‘Materials and Methods’’
section. Actin expression was determined as a loading control. Cells were incubated in complete medium for 24 h with 10lM of PQ in the presence or absence of
3-MA 10mM. Blot is representative of at least three independent experiments.

produces autophagic events. These low concentrations are not
normally used in the PQ toxicity study, and nobody in the
present has described evidences that relate PQ-induced apo-
ptosis and autophagy.
In our study, we have shown for the first time that PQ
initially induces a morphological appearance of autophagy but
it finally succumbed to a typical apoptotic cell death caspase
dependent. Hereby, we demonstrated that, with the prototypic
autophagy inhibitor 3-MA 10mM, not only the vacuoles
formation and the protein degradation induced by PQ are
inhibited but also the apoptotic cell death was accelerated and
the caspase-3 activation increased. In this regard, recent studies
(Boya et al., 2005; Gonzalez-Polo et al., 2005a) already have
demonstrated that the inhibition of autophagy triggers apopto-
sis, so in this case autophagy would be considered as a defense
mechanism and not as a type of cell death; moreover, recent
evidence (Kiffin et al., 2006) supports a protective role of the
lysosomal system, which can eliminate altered intracellular
components through autophagy, at least in the first stages of
oxidative injury. So, we suggest that the early activation of
autophagy induced by low concentrations of PQ in neuroblas-
toma SH-SY5Y can be attributed to the cell response like
 PARAQUAT INDUCES AUTOPHAGY
a defense mechanism against the rapid oxidative stress induced
after PQ exposure. Our results indicated that the systems of
defense of the cell seemed not to be sufficient because the cell
finally succumbed to the apoptotic cell death. In addition, we
have seen for the first time in our study that when we inhibited
PQ-induced autophagy in presence of 3-MA, we accelerated
the apoptotic death process.
In conclusion, our results help to suggest PQ neurotoxicity as
an emerging PD model system. This study also provides new
molecular bases for the knowledge of the relationship between
autophagy and the cell response to stress and apoptosis cell
death. However, further studies are required to elucidate the
exact mechanism involved in PQ-induced autophagy and its
relation with neuronal cell death.
ACKNOWLEDGMENTS
Supported by grants 2PR04B002, 2PR06B124, and 3PR05C015 (Junta de
Extremadura, Spain) and PI040828 (Fondo de Investigación Sanitaria, Ministerio
de Sanidad y Consumo, Spain). R.A.G.P. was supported by the Junta de
Extremadura re-incorporation fellowship. M.N.S. was supported by the Valhon-
do-Calaff Foundation fellowship. M.A.O. was supported by the Junta of
Extremadura predoctoral fellowship. The authors would like to thank P. Delgado,
R. Tarazona, and V. Llorente Vera for their invaluable technical assistance. We
also thank Dr Guido Kroemer (Institute Gustave Roussy, Villejuif, France) and
Tamotsu Yoshimori (National Institute of Genetics, Mishima, Japan) for regeants.
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