From the Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
Received for publication, October 23, 2000, and in revised form, January 10, 2001
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ABSTRACT |
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In the present study, we find that cyclopentenone
prostaglandins (PGs) of the J2 series,
naturally occurring derivatives of PGD2, are potential
inducers of intracellular oxidative stress that mediates cell
degeneration. Based on an extensive screening of diverse chemical
agents on induction of intracellular production of reactive oxygen
species (ROS), we found that the cyclopentenone PGs, such as
PGA2, PGJ2, Oxidative stress is increasingly seen as a major upstream
component in the signaling cascade involved in many of the cellular functions such as cell proliferation, inflammatory responses, stimulating adhesion molecule, and chemoattractant production (1). It
has been suggested that some level of oxidative stress may be required
in response to cytotoxic agents and converted into the redox regulatory
system as a downstream signaling pathway (2). However, excess oxidative
stress may be toxic, exerting cytostatic effects, causing membrane
damage, and activating pathways of cell death (apoptosis and/or
necrosis). Reactive oxygen species (ROS)1 generated during
oxidative stress may be responsible for these effects due to their
ability to damage cellular components, such as membrane lipids. Lipid
peroxidation mediated by a free radical chain reaction mechanism yields
lipid hydroperoxides as primary products, and subsequent decomposition
of the lipid hydroperoxides generates a large number of reactive
aldehydes, such as ketoaldehydes, 2-alkenals, and 4-hydroxy-2-alkenals
(3). There is increasing evidence that these aldehydes are causally
involved in most of the pathophysiological effects associated with
oxidative stress in cells and tissues.
The prostaglandins (PGs) are a family of structurally related molecules
that are produced by cells in response to a variety of extrinsic
stimuli and regulate cellular growth, differentiation, and homeostasis
(4, 5). PGs are derived from fatty acids, primarily arachidonate, that
are released from membrane phospholipids by the action of
phospholipases. Arachidonate is first converted to an unstable
endoperoxide intermediate by cyclooxygenases and subsequently converted
to one of several related products, including PGD2,
PGE2, PGF2 In the present study, as part of an effort to identify endogenous
inducer of intracellular oxidative stress and to elucidate the
molecular mechanism underlying the oxidative stress-mediated cell
degeneration, we examined the oxidized fatty acid metabolites for their
ability to induce intracellular ROS production in a human neuroblastoma
SH-SY5Y cell and found that the J2 series of the PGs
represent the most potent inducers. In addition, the intracellular ROS
production was accompanied by the alteration of cellular redox status
and the production of lipid peroxidation-derived highly cytotoxic
aldehydes, such as acrolein and 4-hydroxy-2-nonenal (HNE), which could
also induced the intracellular ROS production. These data suggest that
intracellular oxidative stress constitutes a pivotal step in the
pathway of cellular dysfunction induced by the PGs.
Materials--
PGs and several other lipid peroxidation products
were purchased from the Cayman Chemical Co. (Ann Arbor, MI).
Horseradish peroxidase-linked anti-goat and anti-mouse IgG
immunoglobulins and enhanced chemiluminescence (ECL) Western blotting
detection reagents were obtained from Amersham Pharmacia Biotech. The
protein concentration was measured using the BCA protein assay reagent obtained from Pierce. 3,3'-Dihexyloxacarbocyanine iodide (DiOC6(3)), carbonyl cyanide m-chlorophenylhydrazone (CCCP), and
N-acetylcysteine (NAC) were from Sigma.
2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was from Molecular
Probes Inc. (Eugene, OR).
Cell Culture--
SH-SY5Y cells were grown in Cosmedium-001
(Cosmo-Bio, Tokyo, Japan) containing 5% Nakashibetsu precolostrum
newborn calf serum, 100 µg/ml penicillin, and 100 units/ml
streptomycin. Cells were seeded in plates coated with polylysine and
cultured at 37 °C.
Cell Viability--
Cell viability was quantified by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Briefly, cells incubated with PGs or other chemicals were
treated with 10 µl of MTT solution (5 mg/ml) for 4 h. The cells
were then lysed with 0.04 N HCl in isopropyl
alcohol, and the absorbance was read at 570 nm.
Flow Cytometry Analysis of ROS and Mitochondrial Membrane
Potential ( Glutathione Assay--
Measurement of GSH in the cells was
performed fluorometrically according to the method of Hissin and Hilf
(14). In brief, the cells incubated with HNE or NAC were washed twice
with PBS (pH 7.0) and extracted with the 25% (w/v) metaphosphoric acid solution containing 5 mM EDTA. After ultracentrifugation
(105,000 × g, 30 min), 1.8 ml of 0.1 M
phosphate solution (pH 8.0) containing 5 mM EDTA and 100 µl of the o-phthalaldehyde solution (1 mg/ml) were added
to the resulting supernatant (100 µl), and then the fluorescence
intensity at 420 nm was then determined with activation at 350 nm.
Glutathione Peroxidase Assay--
GSH peroxidase activity was
determined according to the method of Lawrence and Burk (15). One unit
was defined as the amount of enzyme required to oxidize 0.5 µmol of
NADPH (corresponding to 1 µmol of reduced GSH) per min.
Measurements of Acrolein and HNE Levels--
The levels of
acrolein and HNE were measured, as their protein-bound forms, by
competitive enzyme-linked immunosorbent assays (ELISA), using
anti-protein-bound HNE (mAbHNEJ2) (16) and anti-protein-bound acrolein
(mAb5F6) (17) monoclonal antibodies, as previously reported (18).
Immunoblot Analysis--
For detection of the ubiquitinated
proteins, whole cell lysates from SH-SY5Y cells treated with
15d-PGJ2 were incubated with SDS sample buffer for 5 min at
100 °C. The samples ware separated by 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. One gel was used for
staining with Coomassie Brilliant Blue; the other gel was transblotted
onto a nitrocellulose membrane, incubated with Blockace for blocking,
washed, and incubated with the anti-ubiquitin polyclonal antibody
(Biomeda Co., Foster City, CA). This procedure was followed by the
addition of horseradish peroxidase conjugated to rabbit anti-mouse IgG
and ECL reagents. The bands were visualized by Cool Saver AE-6955
(ATTO, Tokyo, Japan).
Immunocytochemistry--
For immunocytochemistry, cells were
fixed overnight in PBS containing 2% paraformaldehyde and 0.2% picric
acid at 4 °C. Membranes were permeabilized by exposing the fixed
cells to PBS containing 0.3% Triton X-100. The cells were then
sequentially incubated in PBS solutions containing blocking serum (5%
normal goat serum) and immunostained with anti-protein-bound acrolein
monoclonal antibody (mAb5F6) (17) or polyclonal antibodies that
specifically recognize protein-bound HNE (19). The cells were then
incubated for 1 h in the presence of fluorescein
isothiocyanate-labeled goat anti-rabbit and CyTM3-labeled
goat anti-mouse, rinsed with PBS containing 0.3% Triton X-100, and
covered with anti-fade solution. Images of cellular immunofluorescence
were acquired using a confocal laser microscope (Bio-Rad) with a 40×
objective (488-nm excitation and 518-nm emission).
Statistical Analysis--
The paired Student's t
test was used to compare the significance of the differences between data.
Cyclopentenone PGs as Potential Inducers of Intracellular ROS
Production--
To identify endogenous inducer of intracellular
oxidative stress, we screened a large number of lipophilic chemicals,
including oxidized fatty acid metabolites, on induction of
intracellular ROS production and found that some of the PG derivatives
showed potent pro-oxidant effects on human neuroblastoma SH-SY5Y cells. As shown in Fig. 1B, the
intracellular ROS production in SH-SY5Y cells was significantly induced
by PGA2 and by the PGD2 metabolites, such as
PGJ2, Correlation between ROS Production and Cytotoxicity--
To
examine the correlation between ROS production and cytotoxicity in
SH-SY5Y cells exposed to PGs, we examined the cytotoxic effects of PGs
by MTT assay. As shown in Fig.
2A, among the PGs tested, the
J series of PGs resulted in a rapid decrease in MTT reduction levels to
<40% of basal levels within 24 h of exposure. A significant
cytotoxicity was also observed when the cells were treated with
PGA2 and PGD2 at the concentration of 25 µM (data not shown). In contrast to these PGs, the MTT
reduction levels were maintained at 80-90% of basal levels in SH-SY5Y
cells exposed to other PGs, such as
13,14-dihydro-15-keto-PGA2, PGB2,
PGE2, and 15-keto-PGE2. As shown in Fig.
2B, 15-PGJ2 induced cell death in time- and
dose-dependent manners. Even 5 µM
15d-PGJ2 did cause a 40% decrease in the MTT reduction
level after 24 h of incubation, indicating that
15d-PGJ2, the terminal metabolite of PGD2, may represent the most potent cytotoxic metabolite. Similar results were
obtained from other cell viability assays, such as crystal violet and
trypan blue exclusion assays (data not shown). As shown in Fig.
2C, the PG-induced ROS production was well correlated with
the cytotoxicity. The correlation between ROS production and
cytotoxicity was also suggested by the observation that Redox Alteration Induced by Cyclopentenone PGs--
A potential
pathway that might mediate these effects of cyclopentenone PGs involves
alteration of cellular redox status. To examine whether cyclopentenone
PGs could influence the redox status, we measured the intracellular GSH
levels and GSH peroxidase activity. As shown in Fig.
3A, the GSH levels were
partially diminished by treatment with 15d-PGJ2. It was
also observed that the 15d-PGJ2 treatment of the cells
resulted in a significant decrease in the GSH peroxidase activity (Fig.
3B). To confirm whether the PG-induced redox alteration
plays a role in mediating the ROS production and cytotoxicity, SH-SY5Y
cells were pretreated with the thiol compound NAC prior to the exposure
to 15d-PGJ2, and then the ROS production and cell viability
were examined. As shown in Fig. 3, C and D, the
NAC pretreatment resulted in an increased survival as well as in the
inhibition of ROS production in the cells. These data suggest that
redox alteration may be closely related to the action of cyclopentenone
PGs.
Mitochondria as the Source of ROS--
It is believed that
mitochondrial oxidative phosphorylation is the major endogenous source
of the ROS and is involved in a wide variety of disorders (20).
Therefore, it was anticipated that the ROS detected in the cells
exposed to PGs may originate from the mitochondria, one of the major
ROS-producing organella. In this context, we measured the alteration of
the mitochondrial membrane potential ( Accumulation of Protein-bound Aldehydes and Ubiquitinated Proteins
in SH-SY5Y Cells Exposed to Cyclopentenone PGs--
In addition to the
ROS production, we found that the PG cytotoxicity was accompanied by
the production of lipid peroxidation-derived highly cytotoxic
aldehydes, such as acrolein and HNE (Fig.
5A), in the cells. The levels
of acrolein and HNE were measured, as the protein-bound forms, by
competitive ELISA and immunocytochemical assays. As shown in the Fig.
5B, 15d-PGJ2 enhanced the productions of the
protein-bound acrolein and HNE in a time-dependent manner. Maximum 3- and 8-fold increases in the production of acrolein and HNE,
respectively, was observed, and the production of both modified
proteins persisted for at least 24 h. Noncytotoxic PGs, such as
PGA2, PGB2, and PGE2, did not
produce the protein-bound aldehydes (Fig. 5, C and
D). Immunocytochemical analyses showed that exposure of the
cells to 15d-PGJ2 resulted in the appearance of acrolein
and HNE reactivity in essentially all cells (Fig. 6). Since these aldehydes are known to be
the most reactive electrophiles, it is likely that the cytotoxic effect
of 15d-PGJ2 was potentiated by these aldehydes.
On the other hand, it was anticipated that the PG-induced oxidative
stress leading to the formation of oxidatively modified proteins
provokes the misfolding of proteins, which may then be targeted for
degradation by the ubiquitin-dependent proteolytic pathway
(21). To examine whether the ubiquitin pathway is activated by the
PG-induced oxidative stress, ubiquitin-protein conjugates generated in
the cells were analyzed by an immunoblot analysis. As shown in Fig.
7A, the J series of PGs most
significantly induced the generation of ubiquitin-protein conjugates
with high molecular weights (>100 kDa); other PGs, including
PGA2, 15-keto-PGA2, PGB2, PGD2, PGE2, and 15-keto-PGE2, were
less effective or ineffective. Upon incubation with
15d-PGJ2, the ubiquitinated proteins were detected from 30 min to 24 h and returned to the level of the control at 48 h
(Fig. 7B). These data suggest that the PG-induced oxidative
stress may lead to the increased ubiquitination of aberrant proteins,
including oxidatively modified proteins.
Intracellular ROS Production and Cell Death Induced by Reactive
Aldehydes--
More strikingly, we found that the lipid
peroxidation-derived reactive aldehydes could be a second source of ROS
in the cells. As shown in Fig.
8A, when SH-SY5Y cells were
treated with a variety of reactive aldehydes, both acrolein and HNE
caused a significant increase in the ROS levels. Interestingly, the
PGD2 is known to be sequentially metabolized to
PGJ2, A characteristic of cyclopentenone PGs is that they contain
Although the PGs, such as PGA1 and PGE1, have
been reported to cause rapid degenerative changes in differentiated
murine neuroblastoma cells in culture (29, 30), the molecular mechanism
underlying the PG-induced cell degeneration had not been analyzed.
Based on the observations that (i) 15d-PGJ2 partially
reduced intracellular GSH levels (Fig. 3A), (ii) the
15d-PGJ2 treatment of the cells resulted in a significant
decrease in the GSH peroxidase activity (Fig. 3B), and (iii)
the NAC pretreatment significantly inhibited both ROS production and
cytotoxicity by 15d-PGJ2 (Fig. 3, C and D), intracellular redox status appeared to represent a
critical parameter for the PG-induced ROS production and cytotoxicity. The fact that cyclopentenone PGs are susceptible to nucleophilic addition reactions with thiols (22, 23) suggests that the action of
cyclopentenone PGs is closely related to direct reaction with GSH
and/or other thiol compounds. However,
L-buthionine-(SR)-sulfoximine, a specific
inhibitor of GSH biosynthesis, itself did not so effectively induce
intracellular ROS production and cell
death,2 indicating that the
effects of cyclopentenone PGs may not merely result from the GSH
depletion alone and suggesting an involvement of other redox
regulators. From this standpoint, it is noteworthy that the
cyclopentenone PG dramatically induced a depletion of GSH peroxidase
activity (Fig. 3). GSH peroxidase contains a selenocysteine residue,
which is essential for peroxidase activity (31, 32). This
selenocysteine residue resembles a cysteine residue in terms of
chemical properties but has a higher reactivity (33, 34). ROS and
electrophiles, such as cyclopentenone PGs and reactive aldehydes, are
likely to react with the selenocysteine residue of GSH peroxidase via a
Michael-type addition reaction, resulting in the depletion of GSH
peroxidase activity. The redox alteration, represented by the depletion
of the antioxidant defenses, may be closely associated with the
induction of ROS production, leading to the acceleration of oxidative
stress, and may be crucial for the PG-induced cell death (Fig. 9).
On the other hand, the findings that (i) the production of ROS and
reactive aldehydes represents the early cellular event observed within
30 min, (ii) the PG-induced ROS production was well correlated with the
cytotoxicity (Fig. 2C), and (iii) It is also noticeable that the increased intracellular ROS production
induced by cyclopentenone PGs was accompanied by the production of
lipid peroxidation products, such as acrolein and HNE, in the cells
(Figs. 5 and 6). Among all the It has recently been shown that cyclopentenone PG-like compounds,
A2/J2-isoprostanes, are formed in
vivo as the products of isoprostane pathway (38, 45). As the
reactive PGs are physiologically present in body fluids in
picomolar-to-nanomolar concentrations (46); however, arachidonate
metabolism is highly increased in several pathological conditions,
including hyperthermia, infection, and inflammation (47), and local PG concentrations in the micromolar range have been detected at sites of
acute inflammation (48). In addition, elevated cyclopentenone PG
synthesis has also been detected in the late phases of inflammation (49). Therefore, up-regulation of PG biosynthesis is suggested to be
involved in the pathophysiological processes relevant to inflammatory
responses. The findings that (i) PGD2, the precursor of
cyclopentenone PGs, is one of the most abundantly produced PGs in
several tissues, (ii) PGD2 can be converted readily to J2 PGs in the presence of plasma in vitro (8),
and (iii) the cyclopentenone PGs, such as 15d-PGJ2, have
been shown to be formed from PGD2 in vivo (9)
strongly suggest that levels of PGD2 derivatives may reach
functionally significant levels in inflammation and its related disorders.
In conclusion, these experiments demonstrate that the J2
series of the PGs represent the most potent inducers of intracellular oxidative stress and that the production of ROS in the cells is closely
associated with the excitotoxic effect of PGs. Although the present
data were obtained from the in vitro experiments using a
cell line, it is suggested that the intracellular oxidative stress may
underlie the well documented antiproliferative and antitumor effects of
cyclopentenone PGs. The present study may therefore represent a first
step in establishing a link between the intracellular oxidative stress
and cell degeneration induced by the PGs.
12-PGJ2,
and 15-deoxy-
12,14-PGJ2, showed the most
potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the
intracellular events that mediate the PG cytotoxicity, we observed (i)
the cellular redox alteration represented by depletion of antioxidant
defenses, such as glutathione and glutathione peroxidase; (ii) a
transient decrease in the mitochondrial membrane potential (
);
(iii) the production of protein-bound lipid peroxidation products, such
as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of
ubiquitinated proteins. These events correlated well with the reduction
in cell viability. In addition, the thiol compound,
N-acetylcysteine, could significantly inhibit the
PG-induced ROS production, thereby preventing cytotoxicity, suggesting
that the redox alteration is closely related to the pro-oxidant effect
of cyclopentenone PGs. More strikingly, the lipid peroxidation end
products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated
cells potently induced the ROS production, which was accompanied by the
accumulation of ubiquitinated proteins and cell death, suggesting that
the membrane lipid peroxidation products may represent one of the
causative factors that potentiate the cytotoxic effect of
cyclopentenone PGs by accelerating intracellular oxidative stress.
These data suggest that the intracellular oxidative stress, represented
by ROS production/lipid peroxidation and redox alteration, may underlie
the well documented biological effects, such as antiproliferative and
antitumor activities, of cyclopentenone PGs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, prostacyclin (PGI2),
and thromboxane A2, through the action of specific PG
synthetases. PGD2 is a major cyclooxygenase product in a
variety of tissues and cells and has marked effects on a number of
biological processes, including platelet aggregation, relaxation of
vascular and nonvascular smooth muscles, and nerve cell functions (6).
PGD2 readily undergoes dehydration in vivo and
in vitro to yield additional biologically active PGs of the J2 series (Fig. 1A) (7-9). Members of
J2 series of PGs, characterized by the presence of a
reactive
,
-unsaturated ketone in the cyclopentenone ring
(cyclopentenone PGs), have their own unique spectrum of biological effects, including antitumor activity, the inhibition of cell cycle
progression, the suppression of viral replication, the induction of
heat shock protein expression, and the stimulation of osteogenesis (10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)--
DCFH-DA was employed to measure ROS (11, 12).
Cells were incubated with 10 µM 2',7'-dichlorofluorescein
diacetate (dissolved in dimethyl sulfoxide) for 30 min at 37 °C and
then treated with different agents for an additional 30 min at
37 °C. After chilling on ice, the cells were washed with ice-cold
PBS, scraped from the plate, and resuspended at 1 × 106 cells/ml in PBS containing 10 mM EDTA. For
the detection of
, 40 nM DiOC6(3) (13) in the absence
or presence of 15-deoxy-
12,14-PGJ2
(15d-PGJ2) was added and incubated for 15 min at 37 °C. The fluorescence was measured using a flow cytometer (Epics XL, Beckman Coulter).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12-PGJ2, and
15d-PGJ2. Among the J2 series of PGs, the
intracellular ROS production was most potently induced by
15d-PGJ2, which was followed by it precursors,
PGJ2 and
12-PGJ2. The level of
ROS in the cells exposed to 15d-PGJ2 (10 µM) was ~10-fold higher than that of the control. Other oxidized fatty acids, including
(9R)-hydroxy-(10E,12Z)-octadecadienoic
acid, (+)-13-hydroxy-(9Z,11E)-octadecadienoic
acid,
(9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid, (13S)-hydroperoxy-(9IZ,11IE)-octadecadienoic acid,
9-oxo-(10E,12Z)-octadecadienoic acid,
13-oxo-(9Z,11E)-octadecadienoic acid,
(+)-13-hydroxy-(9Z,11E)-octadecadienoic acid
cholesteryl ester, and (+)-9(10)-epoxy-(12Z)-octadecenoic acid, had no significant effects on the ROS production (data not shown).
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Fig. 1.
Intracellular ROS production induced by
PGs. A, metabolism of PGD2. B,
SH-SY5Y cells were incubated with DCFH-DA (10 µM) for 30 min and then treated with 10 µM PGs for 1 h. The
fluorescence intensity of more than 10,000 cells was analyzed using a
flow cytometer.
-tocopherol, a lipophilic antioxidant, significantly inhibited the PG cytotoxicity (Fig. 2D).
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Fig. 2.
Correlation between intracellular ROS
production and cytotoxicity. A, viability of SH-SY5Y
cells exposed to PGs. Cells were exposed to 10 (hatched bar)
or 25 µM (closed bar) of PGs for 24 h.
The cell viability was measured by the MTT assay. Data are expressed as
percent of control culture conditions. B, viability of
SH-SY5Y cells exposed to 15d-PGJ2. Time- and
dose-dependent reduction of cell viability induced by 5 (open circle), 10 (open square), 25 (closed
triangle), and 50 µM (closed circle) of
15d-PGJ2. The cell viability was measured by the MTT assay.
Data are expressed as percent of control culture conditions.
C, correlation between ROS production and cytotoxicity in
the cells exposed to PGs. Cells were exposed to 10 µM of
PGs for 1 h for measuring ROS production and for 24 h for
measuring cell viability. Symbol numbers: 1,
15-keto-PGE2; 2, PGE2; 3,
PGB2; 4, PGD2; 5,
15-keto-PGA2; 6, PGA2; 7,
PGJ2; 8, 12-PGJ2;
9, 15d-PGJ2. D, effect of
-tocopherol (
-Toc) on cell death induced by
15d-PGJ2. The cells were treated with
-tocopherol
(0-100 µM) for 4 h, washed twice with PBS, and then
exposed to 10 µM 15d-PGJ2 or vehicle for
24 h. A, B, and D, the data represent
mean ± S.D. of triplicate determinations.
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Fig. 3.
15d-PGJ2-induced redox alteration
and effect of NAC pretreatment on ROS production and cell death induced
by 15d-PGJ2. A, changes in intracellular
GSH level. Cells were exposed to 10 µM of
15d-PGJ2 for different time intervals as indicated in the
figure. Intracellular GSH levels were fluorometrically measured as
described under "Experimental Procedures." B, changes in
intracellular GSH peroxidase activity. Cells were exposed to 10 µM of 15d-PGJ2 for different time intervals
as indicated in the figure. *, statistical difference between pre- and
post-15d-PGJ2 treatment (p < 0.05).
C, effect of NAC pretreatment on ROS production induced by
15d-PGJ2. The cells pretreated with NAC (0-400
µM) for 4 h were incubated with 10 µM
DCFH-DA for 30 min, washed twice with PBS, and then treated with 10 µM 15d-PGJ2 for 1 h. After washing with
PBS, the cells were resuspended in PBS containing 10 µM
EDTA, and then the fluorescence intensity of more than 10,000 cells was
analyzed using a flow cytometer. D, effect of NAC
pretreatment on cell death induced by 15d-PGJ2. The cells
were treated with NAC (0-500 µM) for 4 h, washed
twice with PBS, and then exposed to 20 µM
15d-PGJ2 or vehicle for 24 h. Cell viability was then
measured by the MTT assay. Data are expressed as percent of control
culture conditions. A, B, and D, the data
represent means ± S.D. of triplicate determinations.
), which is a component of
the overall proton motive force that drives the ATP production in the
mitochondria. As shown in Fig.
4A, 15d-PGJ2
induced a significant decrease in mitochondrial
, suggesting that
the PG acted on the process of oxidative phosphorylation. We then
examined the effect of CCCP treatment on the
15d-PGJ2-induced ROS production. CCCP has a dissociable proton and acts by carrying protons across the inner mitochondrial membrane, resulting in depletion of mitochondrial electrochemical gradient (
) by dissipating the proton gradient. As shown in Fig.
4B, CCCP alone did not cause ROS production, whereas the pretreatment of CCCP led to a dose-dependent inhibition of
intracellular ROS production induced by 15d-PGJ2. These
results suggest that mitochondrial electron transport chain is involved
in the 15d-PGJ2-induced ROS production.
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Fig. 4.
Mitochondria as the source of intracellular
ROS. A, time-dependent alterations of
induced by 15d-PGJ2. The cells were incubated with
40 nM DiOC6(3) in the absence or presence of
15d-PGJ2 for 15 min at 37 °C. B, effect of
CCCP pretreatment on the 15d-PGJ2-induced ROS production.
The cells incubated with 10 µM DCFH-DA for 30 min were
pretreated with CCCP (0-100 µM) for 30 min and then
treated with 10 µM 15d-PGJ2 for 30 min.
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Fig. 5.
Detection of protein-bound reactive aldehydes
in SH-SY5Y cells exposed to 15d-PGJ2. A,
chemical structures of lipid peroxidation products, acrolein, and HNE.
B, competitive ELISA analysis of protein-bound acrolein
(open square) and protein-bound HNE (closed
square) in the cells exposed to 10 µM
15d-PGJ2. C, competitive ELISA analysis of
protein-bound HNE in the cells exposed to 10 µM PGs.
D, competitive ELISA analysis of protein-bound acrolein in
the cells exposed to 10 µM PGs. B, C, and
D, the data represent means ± S.D. of triplicate
determinations.
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Fig. 6.
Immunocytochemical detection of
protein-bound HNE and protein-bound acrolein in SH-SY5Y cells exposed
to 15d-PGJ2. The cells were incubated with 10 µM 15d-PGJ2 at 37 °C. The digitized images
were colorized and combined using Adobe Photoshop, 3.0. Fluorescein
isothiocyanate fluorescence (protein-bound HNE, green) is
shown in the left column of panels (a-d);
CyTM3 fluorescence (protein-bound acrolein, red)
is shown in the center column of panels (e-h),
and the corresponding combined (superimposed) images are shown in the
right column of panels (i-l) (yellow
represents colocalization).
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Fig. 7.
Immunoblot analysis of ubiquitin-protein
conjugates. A, generation of ubiquitin-protein
conjugates in the cells exposed to 10 µM PGs for 8 h. B, time-dependent generation of
ubiquitin-protein conjugates in the cells exposed to
15d-PGJ2. The cells were incubated with 10 µM
15d-PGJ2 at 37 °C.
,
-unsaturated aldehydes, such as crotonaldehyde and 2-nonenal,
possessing an analogous functionality to acrolein and HNE, were all
inactive. We also found that both acrolein and HNE showed the most
potent cytotoxicity (Fig. 8B). In addition, generation of
ubiquitinated protein was observed in the cells exposed to the aldehyde
(HNE) (Fig. 8C). These results are consistent with the
observation (Fig. 2) that the ROS production in the cells exposed to
PGs was closely associated with the cytotoxicity. These data suggest
that the reactive aldehydes, such as acrolein and HNE, may potentiate
the effect of 15d-PGJ2 by accelerating the ROS production
and redox alteration in the cells (Fig.
9).
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Fig. 8.
Cell death, intracellular ROS production, and
accumulation of ubiquitinated proteins induced by lipid peroxidation
products. A, cytotoxicity of lipid peroxidation
products. SH-SY5Y cells were exposed to lipid peroxidation products (50 µM) for 12 h, and cell viability was measured by the
MTT assay. Data are expressed as percent of control culture conditions.
B, changes in intracellular ROS level in SH-SY5Y cells
exposed to lipid peroxidation products (50 µM). The cells
were incubated with DCFH-DA (10 µM) for 30 min and then
treated with lipid peroxidation products (50 µM) for
1 h. After washing with PBS, the cells were resuspended in PBS
containing 10 µM EDTA and then the fluorescence intensity
of more than 10,000 cells was analyzed using a flow cytometer.
Abbreviations: MDA, malondialdehydes; MG,
methylglyoxal; CRA, crotonaldehyde. The data represent
means ± S.D. of triplicate determinations. C,
immunoblot analysis of ubiquitin-protein conjugates in the cells
exposed to HNE. The cells were incubated with 50 µM HNE
at 37 °C.
View larger version (20K):
[in a new window]
Fig. 9.
Model for mechanisms by which
15d-PGJ2 exerts cytotoxicity mediated by productions of ROS
and reactive aldehydes, such as acrolein and HNE, and by redox
alteration.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12-PGJ2, and
15d-PGJ2 (Fig. 1A) (7-9). A comparison of the
PG biosynthetic pathway with the pro-oxidant profile reveals that
induction of intracellular oxidative stress is mediated mainly by the
metabolites of PGD2, the most active of which is the
terminal metabolite 15d-PGJ2 (Fig. 1B). The
induction potency was 15d-PGJ2 >
12-PGJ2 > PGJ2
PGD2, indicating a gain in biological potency as the
catabolism of PGD2 proceeds. Since PGJ2 is
easily converted to
12-PGJ2, the activity of
PGJ2 may be mediated by
12-PGJ2.
If this is the case, the dienone structure of
12-PGJ2 and 15d-PGJ2 may be
critical for the ROS production. As far as we know, this is the first
report that demonstrated the intracellular ROS production by PGs.
,
-unsaturated ketones, which are very susceptible to nucleophilic addition reactions with thiols, and are essential for the actions of
the PGs (22, 23). It has been shown that PGA1 forms a
monoconjugate with thiols, whereas
12-PGJ2
forms a bisconjugate with two thiols (24, 25). It has also been shown
that the binding of PGA1 to synthetic polymer-supported thiols as a model of thiol-containing proteins is reversible but that
of
12-PGJ2 is irreversible (26). Several
previous observations have also indicated that PGD2 and its
J-ring metabolites might exert effects through interactions with
intracellular proteins as follows: (i) Narumiya et al. (27)
have shown that radiolabeled
12-PGJ2 is
actively incorporated into cells and transferred to the nucleus, where
it is associated with proteins; and (ii) some PGs, including
PGD2, PGJ2, and
12-PGJ2, have been shown to bind with high
affinity to liver fatty acid-binding protein and intracellular protein
involved in the uptake, intracellular transport, and metabolism of free
fatty acids and their acyl-CoA esters (28).
-tocopherol, a
lipophilic antioxidant, significantly inhibited the PG cytotoxicity (Fig. 2D) suggest that intracellular ROS production may be
involved, at least in part, in the cell death induced by the
cyclopentenone PGs (Fig. 9). Although the detailed mechanism for the
PG-induced ROS production is currently unknown, the accumulating data
suggest the involvement of mitochondria, one of the major ROS-producing organella. It is believed that inhibition of electron transport chain
components involved in oxidative phosphorylation is the major pathway
for generation of ROS (35). The findings that cyclopentenone PGs could
directly modify cellular proteins, such as I
B kinase
(36, 37)
and human serum albumin (38), suggest that reversible or irreversible
modification of intracellular proteins with cyclopentenone PGs is
involved in the mitochondrial ROS production followed by cell death.
Cyclopentenone PGs may therefore inhibit the process of oxidative
phosphorylation by direct modification of electron transport chain
components, leading to the accumulation of electrons in the early
stages of electron transport chain, where they can be donated directly
to molecular oxygen to give ROS. ROS may further inactivate the
iron-sulfur (Fe-S) centers of electron transfer chain complexes I, II,
and III, resulting in shutdown of mitochondrial energy production and
dysfunction of mitochondrial oxidative phosphorylation (35). ROS could
also react with the thiol groups of GSH localized at the mitochondrial
membrane level (39) and contribute to the lower GSH level. A low level
of GSH could favor the decrease in
, which would subsequently
activate the opening of permeability transition pores to finally induce
the release of cell death-promoting factors, including cytochrome
c, the apoptosis-inducing factor, and latent forms of
specialized proteases called caspases (20). Indeed, the
15d-PGJ2-induced reduction in cell viability was
morphologically characterized by rounded cells and condensed nuclei. In
addition, we have observed that 15d-PGJ2 induced DNA
fragmentation in SH-SY5Y cells. These observations suggest that
cyclopentenone PGs may induce apoptotic cell death.2 Thus,
oxidative stress leads to a marked reduction in mitochondrial energy
production, and an increase in oxidative stress could activate the
mitochondrial permeability transition pore and initiate apoptosis.
,
-unsaturated aldehydes, acrolein
and HNE represent the strongest electrophiles and show the highest
reactivity with nucleophiles, such as proteins and DNA (3). These
aldehydes have been shown to be toxic to cultured cells (40-42) and
have received considerable attention as endogenous cytotoxic agents
that potentially could play a role in the pathogenesis of many
degenerative diseases, including cardiovascular and neurodegenerative
disorders (43, 44). The observations in the present study are in line
with the accumulating body of literature supporting the role of lipid
peroxidation at some point in the pathogenesis of these disorders. We
also found that these reactive aldehydes further exerted an increased
production of intracellular ROS (Fig. 8). The intracellular oxidative
stress results in the production of reactive aldehydes, which may
trigger the production of ROS and accelerate oxidative stress (Fig. 9). These sequential events may lead to the delayed onset of cell death.
Due to the fact that cell death depends mainly on the extent of stress
and involves multiple cellular processes, chronic exposure to ROS and
reactive aldehydes may be required for the phenotypic expression of
cell death.
,
-unsaturated carbonyl seems essential for the
cyclopentenone PG-induced ROS production and cytotoxicity, it is
anticipated that all of the cyclopentenone isoprostanes may exert
similar effects. In addition, the observations that the cyclopentenone
isoprostanes are detected in abundant quantities following induction of
oxidative injury (38, 45) suggest that not only cyclopentenone PGs but
also cyclopentenone isoprostanes may exert biological effects relevant to the pathobiology of oxidative stress.
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Dr. W. Maruyama (National Institute of Longevity Sciences) for supplying the SH-SY5Y cells.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of Food and
Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya
University, Nagoya 464-8601, Japan. Tel.: 81-52-789-4127; Fax:
81-52-789-5741; E-mail: uchidak@agr.nagoya-u.ac.jp.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M009630200
2 M. Kondo, T. Kumagai, and K. Uchida, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
ROS, reactive oxygen
species;
PGs, prostaglandins;
15d-PGJ2, 15-deoxy-12,14-PGJ2;
PGD2, prostaglandin D2;
HNE, 4-hydroxy-2-nonenal;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
ELISA, enzyme-linked immunosorbent assay;
DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide;
CCCP, carbonyl cyanide
m-chlorophenylhydrazone;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody;
NAC, N-acetylcysteine;
DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate;
, mitochondrial
membrane potential.
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