Am J Physiol Heart Circ Physiol 286: H2057–H2064, 2004.

First published Janaury 15, 2004; 10.1152/ajpheart.00778.2003.

Glutathione S-transferase overexpression protects against

anthracycline-induced H9C2 cell death
Thomas L’Ecuyer,1 Zuhair Allebban,2 Ronald Thomas,1 and Richard Vander Heide2,3
1
Department of Pediatrics and 2Department of Pathology, Wayne State University, and
3
Department of Pathology, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201
Submitted 13 August 2003; accepted in final form 13 January 2004

L’Ecuyer, Thomas, Zuhair Allebban, Ronald Thomas, and ACT is generally believed to be due to oxidative injury to
Richard Vander Heide. Glutathione S-transferase overexpression cardiac myocytes, a postreplicative cell type with limited defenses
protects against anthracycline-induced H9C2 cell death. Am J Physiol against oxidative stress (12, 37). Attempts to improve resistance to
Heart Circ Physiol 286: H2057–H2064, 2004. First published Janaury oxidative stress and thereby reduce AC cardiomyocyte injury

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15, 2004; 10.1152/ajpheart.00778.2003.—Anthracyclines (AC) are have been successful in some experimental model systems. For
antitumor antibiotics with significant activity against solid and hema-
tologic malignancies. One problem preventing more widespread use
example, in cultured cardiac myocytes, administration of the
has been the development of cardiac toxicity. Experimental evidence antioxidants amifostine, trolox, 5-aminosalicyclic acid, or ␣-phe-
supports oxidant stress as an important trigger and/or mediator of nyl-tert-butyl nitrone before AC exposure reduced indexes of
AC-induced cardiotoxicity (ACT). Therefore, reducing oxidant stress oxidative stress and myocyte injury (12, 13). In small animal
should be protective against ACT. To determine whether antioxidant models, the degree of AC-induced cardiac injury has also been
protein overexpression can reduce ACT, we developed a cell culture reduced by pretreatment with exogenous antioxidants, including
model system using the H9C2 cardiac cell line exhibiting controlled thymoquinone (3), butylated hydroxanisole (48), and the lipid-
overexpression of the ␣4-isoform of glutathione-S-transferase (GST). lowering antioxidant probucol (27). Probucol-induced protection
Treatment with the AC doxorubicin (DOX) produced both oncosis, was accompanied by an increase in activity of the antioxidant
manifested by an increase in the number of cells staining positive for enzymes catalase, glutathione peroxidase, and superoxide dis-
Trypan blue, and apoptosis, indicated by the presence of positive mutase (SOD) (41). Despite these encouraging results, no data in
terminal deoxynucleotidyl transferase-mediated dUTP nick-end label- large animal or human studies support the use of exogenous
ing (TUNEL) staining. In both cases, the loss of cell viability was
preceded by an AC-induced increase in fluorescence with carboxy-
antioxidants to reduce this problem.
2⬘,7⬘-dichlorofluorescein diacetate, demonstrating the presence of The primary antioxidant enzymes that protect cells from
high levels of reactive oxygen species (ROS). The DOX-induced oxidative stress are SOD, glutathione S-transferase (GST),
increase in ROS was reduced to control levels by maximal GST catalase, and glutathione peroxidase. Increasing the expression
overexpression. Coincident with this elimination of oxidative stress, level of SOD and catalase in the hearts of transgenic mice
there was a reduction in both Trypan blue and TUNEL-positive cells, protects against ACT (20, 50), as does overexpression of the
indicating that GST overexpression reduced both ROS and cell death redox-sensitive protein thioredoxin (40). However, in each of
in this model system. We conclude that GST overexpression may be these reports, the AC was given as a single large bolus rather
an important part of a protective strategy against ACT and that this than mimicking the human intermittent dosage schedule, so it
model system will aid in defining steps in the pathway(s) leading to is not clear whether improving oxidant defenses would protect
AC-induced cell death that can be therapeutically manipulated. against a more clinically relevant AC administration schedule.
apoptosis; oncosis; oxidative stress; antioxidant Iron chelation has appeal as a potential tool against ACT
because iron participates in the nonenzymatic generation of
free radicals. In isolated myocytes, iron chelation reduced
ANTHRACYCLINES (AC) are potent antibiotics used to treat a wide apoptosis induced by exposure to a doxorubicin (DOX) con-
variety of malignancies. The chief limitation to more wide- centration near the peak plasma level seen in human use but did
spread use of these highly effective agents has been the not protect from oncosis induced by exposure to high-dose
development of significant AC-induced cardiotoxicity (ACT), DOX (38). Small clinical trials, mostly in patients with ad-
which can progress to end-stage heart failure (8). The most vanced cancer with poor prognosis, showed that iron chelation
important determinant of ACT is the cumulative dose received; reduced short-term symptomatic ACT, permitting higher cu-
therefore, dose limitation is the only proven way to reduce or mulative AC dosages to be administered (42, 43). Because
avoid ACT (16). Despite the general relationship between the there is some relation between the cumulative AC dose and
cumulative dose received and incidence of congestive heart tumor response rate (7), it is possible that iron chelation will
failure (CHF), some patients have received ⬎1,000 mg/m2 improve long-term cancer survival. While effective in small
without developing CHF (36). In addition, some patients have clinical trials, the treatment has myeloid toxicity, has not been
developed CHF at much lower cumulative doses (24). A shown to influence delayed ACT, and does not influence
significant concern about arbitrary dose reduction as an ap- oxidative stress generated by AC via the enzymatic route (18).
proach to reducing ACT is that the probability of long-term It therefore may represent an important, but perhaps not ex-
cure from the underlying malignancy may be compromised. clusive component of a protection strategy from ACT.

Address for reprint requests and other correspondence: T. L’Ecuyer, Div. of The costs of publication of this article were defrayed in part by the payment
Cardiology, Children’s Hospital of Michigan, 3901 Beaubien Blvd., Detroit, of page charges. The article must therefore be hereby marked “advertisement”
MI 48201 (E-mail: thlecuye@med.wayne.edu). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

http://www.ajpheart.org H2057
H2058 GST OVEREXPRESSION AND ANTHRACYCLINE CARDIOTOXICITY

We have recently developed a cell culture model of AC fected with a second plasmid (pTK-Hyg) that permitted double stable
injury using cardiac-derived H9C2 myocytes, which differen- transfectants to be selected by inclusion of hygromycin in the culture
tiate and can express transfected foreign genes in a tightly medium. After individual clones were selected and expanded, they
regulated manner (25). We have suggested that this cell line were grown either in medium that contained or lacked Tet. Cell
extracts were harvested when the cells became subconfluent and were
represents a useful model in which to study the relationship
analyzed by immunoblotting using an antibody to ␣4-GST (a kind gift
between expression level of a putatively protective gene and of Dr. Piotr Zimniak, University of Arkansas) as described (26). The
the degree of protection from AC injury. In this report we first antibody was used at a 1:50,000 dilution, and chemiluminescence
describe using this cell line to overexpress the antioxidant with a second antibody at a dilution of 1:2,000 was used to detect
protein ␣4 GST and examine the effect on AC injury. This gene antibody binding (Amersham; Arlington Heights, IL). To define the
was selected because it is an antioxidant with an important role relation between the Tet dose and the GST expression level, the clone
in repair of oxidized lipid, DNA, and protein, all of which are with the highest induction on removal of Tet (GST-45) was plated in
generated by AC exposure. In addition, the observation that varying Tet concentrations, cell lysates were harvested and were
polymorphisms of GST genes have altered susceptibility to subjected to immunoblotting. Autoradiograms were scanned and den-
disease states (4, 23) raises the possibility that such polymor- sitometry was performed to quantify GST expression level.
Assay of oxidative stress. 2⬘,7⬘-Dichlorofluorescein diacetate
phisms may explain a portion of variability in susceptibility to

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(DFDA) is a cell-permeable compound, which is transformed into the
ACT that is not explained by cumulative dose received. fluorescent compound dichlorofluorescein on oxidation. DFDA is
MATERIALS AND METHODS capable of detecting oxidative stress in primary cardiac myocytes
induced by exposure to DOX (37). In the present experiments, control
Cell culture. The H9C2 cell line was originally derived from and GST-expressing cells were grown on chamber slides and were
embryonic rat heart tissue using selective serial passage (22) and was exposed for 4 – 6 h to 5 ␮g/ml DOX in culture medium before adding
purchased from the American Tissue Type Collection (Manassas, VA; DFDA (Molecular Probes; Eugene, OR) for 40 min in the dark at a
catalog no. CRL-1446). Cells were maintained in DMEM with 4 mM final concentration of 10 ␮M. In preliminary experiments, we found
l-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l that DOX alone generated a weak fluorescent signal in H9C2 cells,
glucose, and 1.0 mM sodium pyruvate, with 10% fetal bovine serum. although not to the degree as when DFDA was included. Similar
To prevent the loss of differentiation potential, cells were not allowed results were obtained using dihydrocalcein (Molecular Probes) as an
to become confluent. Cells were grown on tissue culture dishes for independent indicator of oxidative stress (data not shown). The cells
transfection, flow cytometry, Trypan blue (TB) staining, or to generate were washed several times with PBS and were viewed immediately
cell lysates, and were grown on Nunc Lab-Tech Permanox plastic with a Nikon TE 300 microscope equipped with a fluorescent attach-
chamber two-well slides (Fisher Scientific; Pittsburgh, PA) for the ment and a RT Color Spot Digital Camera. Continuous illumination of
fluorescent oxidative stress and terminal deoxynucleotidyl trans- cells loaded with DFDA slowly increases dye fluorescence. For this
ferase-mediated dUTP nick-end labeling (TUNEL) assays. In all reason, slides were scanned for several seconds to localize cells, and
experiments, the AC DOX was added to the complete culture medium a high-resolution image was obtained with a constant exposure time
and was washed off with PBS at the end of the defined incubation by a single scan of a new field using ImagePro Plus software (Media
period. In some studies, cells were incubated with the general caspase Cybernetics; Silver Spring, MD). Fluorescent intensity, which corre-
inhibitor benzylocarbonyl-valine-alanine-aspartate fluoromethyl lates with the degree of oxidant stress (37), was measured in cells
ketone (Z-VAD-fmk) (Calbiochem; La Jolla, CA) before treatment using ImagePro Plus software. The mean intensity of all fluorescent
with DOX. signals in a low-power field was compared between DOX-treated and
Transfection. H9C2 cells were plated onto 100 mm tissue culture control cells in the presence or absence of high-level GST expression.
dishes and were permitted to become 50% confluent in complete Preliminary experiments demonstrated that Tet had no consistent
medium. Cells were then transfected with the regulator plasmid influence on assays of oxidative stress and cytotoxicity (data not
pTet-Off (Clontech; Palo Alto, CA). Transfection utilized 50 ␮l of a shown).
reagent (Lipofectamine, GIBCO; Gaithersburg, MD) complexed with TB exclusion. TB is a vital dye excluded by viable cells with intact
12 ␮g of pTet-Off plasmid DNA in serum-free medium for 20 min at cell membranes. Cells dying via oncosis fail to exclude this dye
room temperature (25°C). After the cells were washed twice with indicating loss of membrane integrity. After membrane rupture, cells
serum-free medium, the transfection cocktail was added for 5 h, after release intracellular enzymes such as lactate dehydrogenase, which
which an equal volume of complete medium with 20% serum was can be assayed as a parallel measure of cell death. In preliminary
added to bring the final concentration of serum to 10%. The pTet-Off studies, we found that lactate dehydrogenase release and TB exclusion
plasmid contains a neomycin-resistance cassette that permits selection yielded similar estimates of cell death in response to treatment with
of stably transfected clones by the addition of G418 to the culture DOX. Therefore, we utilized the simpler, more rapid procedure (TB)
medium. The plasmid directs the constitutive production of a tetracy- to assess the development of oncotic cell death. Wild-type (WT) and
cline transactivator protein that can activate high-level expression GST-expressing myocytes were plated in the absence of Tet and
from promoters with a Tet response element as long as Tet is not subjected to treatment with DOX (0.5 or 5 ␮g/ml for 7 h) or vehicle.
present in the culture medium (15). Clones surviving selection were After the cells were rinsed, they were incubated for an additional
expanded and screened by transient transfection with a plasmid 36 – 48 h in fresh medium to allow the injury to evolve (25). Cells
(pTreLuc) directing the production of luciferase. The clone with the were collected by gentle trypsinization, followed by neutralizing with
greatest induction of luciferase on Tet removal was transfected on a serum-containing medium. Subsequently, a small volume of 1% TB
100 mm dish with cells at 50% confluence using the same procedure was added to cells collected from each dish, and the number of cells
with a plasmid (pTre-GST) that expresses the ␣4 isoform of GST. The excluding or staining with the dye was counted on the Nikon micro-
pTre-GST plasmid was constructed by excising the SacII-BamH I scope as described (52). A cell was considered positive if the entire
fragment from pUbA4 plasmid (a generous gift of Dr. Philip Board, cytoplasm was diffusely stained with any shade of blue. Cells were
Australian National University) and ligating it into the SacII-BamH I counted immediately after addition of the vital dye to prevent eventual
site of a similarly digested pTre vector. pTre contains a promoter that staining of all cells as they became depleted of substrate. Data are
renders expression of cloned genes sensitive to the presence of Tet, presented as the percentage of cells staining with TB, i.e., nonviable.
permitting the expression level of GST to be controlled by the TUNEL assay. DNA fragmentation was visualized by use of the
concentration of Tet in the culture medium. pTre-GST was cotrans- ApopTag kit (Intergen; Norcross, GA). This system labels free 3⬘ OH
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 GST OVEREXPRESSION AND ANTHRACYCLINE CARDIOTOXICITY H2059
termini of DNA in cells with digoxigenin-tagged nucleotides with the
use of the enzyme terminal deoxynucleotidyl transferase. Binding of
a fluorescent antibody to digoxigenin in positive cells is visualized
using fluorescent microscopy. H9C2 and GST-45 cells were grown on
chamber slides in the absence of Tet to induce GST expression. DOX
was added at a concentration of 0.5 or 5 ␮g/ml for 8 h. One thousand
attached cells were scored as being TUNEL positive or negative from
each condition per experiment. Cells were only scored as being
positive under direct visualization if nuclear fluorescent staining was
detected at a level significantly above background, as described by
Akao et al. (2).
Flow cytometry. Annexin and propidium iodide staining was per-
formed in conjunction with flow cytometry to confirm TUNEL assay
data on a larger, unselected population of cells using the Annexin
V-FITC Apoptosis Detection Kit (Oncogene Research Products; San
Diego, CA). This assay takes advantage of the binding by annexin to

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phosphatidyl serine residues exposed on the cell surface soon after
induction of apoptosis before nuclear breakdown occurs (when pro-
pidium iodide staining is present). H9C2 cells or GST-45 cells were
plated on 60 mm dishes until 70% confluent, at which time they were
treated with vehicle or DOX at doses of 0.5 or 5 ␮g/ml. Twenty-four
hours later, cells were collected by trypsinization and were suspended
at a concentration of 106 cells/ml in PBS. All subsequent steps were
Fig. 1. Glutathione S-transferase (GST) expression is regulated by the concen-
performed with the cells and reagents on ice. Cells were pelleted and tration of tetracycline (Tet) in the culture medium of stably transfected H9C2
resuspended in binding buffer before the addition of 1.25 ␮l annexin cells. A: representative immunoblot showing GST expression from lysates of
V-FITC and incubating for 15 min in the dark. Cells were pelleted a single stably transfected clone grown for at least 1 wk in the indicated
again and resuspended in a second binding buffer before the addition concentrations of Tet (in ␮g/ml). A lysate from an untransfected [wild type
of 10 ␮l propidium iodide. Cells were assayed by flow cytometry (WT)] H9C2 culture is included. B: bar graph summarizing densitometry
using a Beckman Flow Cytometer, with results positive or negative averaged from five immunoblots of GST expression as a function of Tet
for both propidium iodide and annexin. Viable cells do not stain with concentration, expressed as fold elevation above the GST level in 1 ␮g/ml Tet.
either reagent, early apoptotic cells stain with annexin only, and either
late apoptotic or oncotic cells stain with both annexin and propidium
iodide. Because we were mainly interested in the number of cells that medium. Although not shown, higher concentrations of Tet did
were clearly apoptotic, cells positive for annexin alone were compared not further suppress GST expression. Therefore, the GST
as a function of GST expression and treatment condition. expression level was reproducibly regulated by the concentra-
Data analysis. SPSS software for Windows 98 version was used for tion of Tet in the culture medium, particularly between 0.01
all statistical comparisons. The means ⫾ SD of each measure (per- and 0.001 ␮g/ml. This cell line (GST-45) was subsequently
centage of cells positive for TB, fluorescent intensity, number of
treated with DOX and assayed for extent of injury and oxida-
TUNEL positive or annexin-positive cells) was obtained from at least
three experiments. Observations are presented as means ⫾ SE, and tive stress as a function of GST expression compared with WT
one-way ANOVA was used to compare means, followed by the post H9C2 cells.
hoc Sidak test (P ⬍ 0.05 was considered significant). Figure 2 schematically illustrates experiments subsequently
performed on GST overexpressing and control H9C2 cells.
RESULTS DOX-induced oxidative stress is prevented by GST overex-
pression. Oxidative stress precedes the development of irre-
Regulated expression of GST by stable transfectants. We versible cell injury after DOX exposure in H9C2 cells (25). To
previously described the generation of multiple H9C2 cell lines determine whether GST overexpression mitigated DOX-in-
stably transfected with the pTet-Off plasmid (25). Each trans- duced early oxidative stress, control cells were compared with
fected clone constitutively expresses a tetracycline transactiva- GST-45 cells induced to maximally express GST. Cells were
tor protein at high level (15). The H9C2 clone with the greatest loaded with DFDA after exposure to 5 ␮g/ml DOX or vehicle
luciferase induction on Tet removal was subsequently trans- for 4 h. A representative set of fluorescent micrographs from
fected with the pTre-GST plasmid cotransfected with a plasmid such an experiment is shown in Fig. 3. Control cells had a low
conferring hygromycin resistance, allowing double stable baseline level of fluorescence (Fig. 3A). Exposure to DOX
transfectants to be selected. The clone from this second trans- without GST induction (Fig. 3B) significantly increased fluo-
fection with the highest GST induction by immunoblotting was rescence, indicating that DOX generated oxidative stress.
subsequently grown in varying Tet concentrations and cell DFDA fluorescence in DOX-treated GST overexpressing cells
lysates were analyzed by immunoblotting to determine the (Fig. 3C) was reduced compared with treated cells without
dose-response relationship. Figure 1A shows a representative GST expression (Fig. 3B). Figure 4 shows the mean fluorescent
immunoblot, whereas Fig. 1B shows the average GST expres- intensity from 8 independent experiments as a percentage of
sion level as a function of the Tet concentration from five fluorescent intensity in control (vehicle treated) cells. DOX
separate experiments. GST was not detectable in untransfected treatment in H9C2 cells without high-level GST expression
H9C2 cells (WT), was expressed at a very low level in the increased the level of oxidative stress ⬎100-fold above vehicle
presence of 1 ␮g/ml Tet, was induced 2-fold when the Tet treatment (P ⬍ 0.001). GST induction reduced DOX-induced
concentration was reduced to 0.01 ␮g/ml, and reached maxi- oxidative stress to the level seen in cells exposed to vehicle
mal induction of 13-fold when Tet was removed from the alone.
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H2060 GST OVEREXPRESSION AND ANTHRACYCLINE CARDIOTOXICITY

Fig. 4. DFDA intensity in control, untreated cells, DOX-treated control cells,

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and DOX-treated GST-expressing cells, expressed on logarithmic scale as a
percentage of control. Signal intensity from eight independent experiments was
averaged for each condition. *P ⬍ 0.001, significantly different from control
untreated cells.
Fig. 2. Diagram of experimental design with WT and GST-overexpressing
H9C2 cells. Z-VAD, benzylcarbonyl valine-alanine-aspartate-fluoromethyl ke-
tone (Z-VAD-fmk); DOX, doxorubicin; DFDA, 2⬘,7⬘-dichlorofluorescein di-
acetate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick- in primary cardiac myocytes that apoptosis is induced by
end labeling.
exposure to low concentrations of DOX (e.g., 1 ␮M or less),
whereas oncosis is induced by exposure to higher concentra-
Cell membrane injury is reduced by GST overexpression. In tions (e.g., 10 ␮M or higher) (38). Consistent with this obser-
a separate series of experiments, H9C2 and GST-45 cells were vation, we observed extensive TB staining on exposure to
treated for 7 h with 0.5 or 5 ␮g/ml DOX, followed by a DOX concentrations near 10 ␮M (⬃5 ␮g/ml), but less so at 1
medium change and incubation for an additional 36 – 48 h to ␮M (⬃0.5 ␮g/ml, Fig. 5). To examine whether apoptosis
allow for the development of injury. The percentage of nonvi- occurs in response to DOX exposure in our system, H9C2 or
able cells in each condition (i.e., that stained with TB) is shown GST-45 cells were subjected to a TUNEL assay immediately
in Fig. 5 as a function of DOX dose and cell type. High-dose after exposure to 0.5 or 5 ␮g/ml DOX for 8 h, and the number
treatment of control H9C2 cells with 5 ␮g/ml DOX increased of TUNEL-positive cells per 1,000 cells was counted. (After
the proportion of TB-positive cells, from 5 to 50%, indicative longer DOX treatment, cells begin to lift off the slides, pre-
of oncotic cell death (P ⬍ 0.001 compared with untreated cluding evaluation by TUNEL staining.) A representative
control cells). When GST-45 cells were treated with the same TUNEL micrograph of H9C2 cells exposed to 0.5 ␮g/ml DOX
DOX dose, 39% of cells became TB positive, indicating that is shown in Fig. 6. Cells containing brightly fluorescing nuclei
increased GST expression provided protection against cell were scored as positive (arrows). When H9C2 or GST-45 cells
death (P ⬍ 0.006), albeit not to control levels. Low-dose were exposed to vehicle alone, no TUNEL-positive cells were
treatment of control H9C2 cells with 0.5 ␮g/ml DOX induced detected. After exposure of H9C2 cells to 0.5 ␮g/ml DOX,
a slight increase in TB-positive cells above control, from 5 to TUNEL-positive cells developed at a rate of 2.9% (29 cells per
15%, a significant increase (P ⬍ 0.001). Treatment of GST-45 1,000). Exposure to a DOX concentration of 5 ␮g/ml produced
cells with the same DOX dose resulted in 8% TB-positive only 1.4% positive cells (14 per 1,000) suggesting that apop-
cells; significantly less than treated H9C2 cells (15%, P ⬍ totic cell death predominates at the lower DOX dose and that
0.001) and not significantly different from untreated control an additional mechanism is operative when cells are treated
cells (5%, P ⫽ 1.0), indicating that GST expression eliminates with high-dose DOX. The number of TUNEL-positive cells
cell death at this DOX dose. was significantly higher at both doses compared with untreated
GST overexpression reduces apoptosis in response to DOX control cells (P ⬍ 0.001). When GST-45 cells were similarly
treatment. When complete protection from high-dose DOX treated, the prevalence of TUNEL-positive cells in response to
injury was not observed by GST expression, we considered the treatment with 0.5 or 5 ␮g/ml DOX was significantly below
possibility that GST may protect against apoptotic rather than that in H9C2 cells treated with the same doses (1.5% and 0.8%,
oncotic cell death. In support of this concept, it has been shown respectively; P ⬍ 0.002 for both groups).

Fig. 3. DOX exposure produces oxidative

stress, which is reduced by GST overexpres-
sion. Micrographs from DFDA staining of
control (A), untreated cells, control cells
treated with DOX for 4 h (B), and GST-
overexpressing cells treated with DOX for
4 h (C).

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 GST OVEREXPRESSION AND ANTHRACYCLINE CARDIOTOXICITY H2061
stress, likely, however, plays an etiologic role at serum con-
centrations of DOX encountered clinically.
The GST family of proteins is important in detoxification of
lipid peroxidation products generated by exposure to oxidating
agents by conjugating glutathione to these products, repairing
oxidative damage (54). We found a tight correlation between
extent of oxidative stress and cell death after DOX treatment
that was reduced by GST overexpression, suggesting that
oxidative stress is important in both apoptotic and oncotic cell
death. This observation is consistent with a recent report
treating neonatal rat cardiomyocytes with DOX, where oxida-
tive stress was clearly documented after exposure to a dose of
DOX producing primarily apoptosis and where elimination of
oxidant stress protected against apoptosis (49). Our results
Fig. 5. DOX exposure increases extent of lethal injury to a greater extent in
differ, however, from another recent study (38) showing that

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H9C2 than GST-45 cells [mean percent Trypan blue (TB)-positive cells from
at least 5 experiments]. H9C2 or GST overexpressing cells were exposed to 0.5 addition of exogenous antioxidants (i.e., ascorbic acid, tocoph-
or 5 ␮g/ml DOX or vehicle for 8 h. The percentage of cells 36 – 48 h after erol, and N-acetylcysteine) reduced oxidant stress but failed to
treatment that were TB positive is indicated. DOX produced cell death in both
cell types (*P ⬍ 0.001 compared with untreated H9C2 cells). GST overex-
pressing cells had fewer TB-positive cells at both DOX doses (⫹P ⬍ 0.001
compared with treated H9C2 cells).

Flow cytometry studies. Annexin staining, indicative of

exposure of phosphoserine residues, is an additional hallmark
of early apoptotic cell death. Flow cytometry confirmed that
exposure to DOX caused cells to stain positive for annexin,
consistent with apoptosis (data not shown). The extent of
apoptosis was abrogated by GST expression at both DOX
doses, although the differences did not reach statistical signif-
icance. Preincubation of H9C2 cells with a general caspase
inhibitor (Z-VAD-fmk) significantly reduced the number of
TB-positive cells after treatment with 5 ␮g/ml DOX from 45 to
31% and after treatment with 0.5 ␮g/ml DOX from 21 to 14%
(both P ⬍ 0.01) (Fig. 7A) albeit not to untreated control levels.
Caspase inhibition also reduced the number of TUNEL-posi-
tive cells after 5 ␮g/ml DOX treatment from 1.5 to 0.5%, and
after 0.5 ␮g/ml DOX treatment from 3.6 to 1.5% (Fig. 7B, P ⬍
0.01). The sum of these results indicate that apoptosis and
oncosis occur in response to DOX treatment at high and low
doses and that high-level GST expression reduces both types of
cell death.
DISCUSSION

This report demonstrates for the first time that overexpres-

sion of the ␣4-isoform of GST markedly reduces fluorescent
indexes of oxidative stress induced by DOX exposure and
significantly reduces apoptotic and oncotic cell death caused by
this agent. GST may conjugate glutathione directly to oxidized
derivatives of DOX or alternatively may exert its protective
effect by sequestration of DOX from cellular elements neces-
sary to generate reactive oxygen species. Indeed, a sequestra-
tion role has been demonstrated for members of the ␣-class of
GST enzymes and may be especially prominent at high cellular
GST levels (29). Apoptosis induced by low concentrations of Fig. 6. DOX exposure induces apoptosis to a greater extent in H9C2 cells than
DOX is almost completely eliminated by GST overexpression, GST overexpressing cells. A: representative micrograph of TUNEL stain of
whereas oncosis induced by high concentrations of DOX is less H9C2 cells exposed to 0.5 ␮g/ml DOX for 8 h. Arrows indicate cells
extensively reduced by GST overexpression. Because GST interpreted as apoptotic. B: average number of TUNEL-positive cells per 1,000
counted (from at least four separate experiments) for H9C2 and GST overex-
overexpression eliminates oxidative stress generated by DOX, pressing cells under control conditions, or treated with 0.5 or 5 ␮g/ml DOX.
this suggests that other factors in addition to oxidative stress *P ⬍ 0.001, significantly different from control. ⫹P ⬍ 0.001, difference
play a role in DOX-induced cell death at high doses. Oxidative between similarly treated H9C2 and GST overexpressing cells.

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H2062 GST OVEREXPRESSION AND ANTHRACYCLINE CARDIOTOXICITY

the dominant mechanism of cell death, at higher doses apop-

tosis does occur, as supported by positive TUNEL staining and
by a decrease in extent of cell death when a caspase inhibitor
was added before DOX exposure. A previous report using
primary cardiac myocytes also demonstrated that low-dose
DOX exposure induced apoptosis whereas high-dose exposure
primarily induced oncosis (38). With the use of a targeting
dosage similar to that used in humans, the peak plasma con-
centration of DOX after bolus infusion in experimental animals
(i.e., 1 to 2 ␮g/ml) was observed to be closer to the level
producing apoptosis than oncosis (11). Typical indexes of
cardiomyocyte necrosis (i.e., creatine kinase or troponin re-
lease) are not detected in patients after treatment with AC,
further suggesting that apoptosis may be the more relevant cell
death mechanism associated with clinical ACT (14). In leuke-

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mic cells, the concentration of DOX also determines the
mechanism of cell death, with apoptosis occurring over a
concentration range of 1–3 ␮M, and necrosis at higher doses,
possibly because the apoptotic program requires ongoing RNA
synthesis, which is inhibited at high concentrations of DOX.
Thus the apoptotic pathway may be initiated but not executed
at higher DOX concentrations (31). It seems plausible to
suggest similar events occur in cardiomyocytes, but this will
require further experimental verification.
Potential signaling pathways. Oxidative stress, such as ex-
posure to hydrogen peroxide, causes apoptosis in a number of
cell types, including cardiac myocytes. The signal transduction
pathway connecting oxidant exposure to apoptosis may involve
Fig. 7. Caspase inhibition reduces DOX effect on cell viability and apoptosis p53 (47), p53 and p66Shc (44), c-Jun NH2-terminal kinase (6,
in H9C2 cells. Cells were exposed to 0.5 or 5 ␮g/ml DOX or vehicle for 8 h 45, 51), or the mitochondrial death pathway (1). It seems
in the presence or absence of the caspase inhibitor Z-VAD-fmk and either
processed for TUNEL staining immediately or incubated in fresh medium for reasonable to assume that because DOX causes oxidative stress
36 h before TB staining. A: percentage of TB-positive cells 36 h after treatment in cells and oxidative stress leads to apoptosis, a pathway
is indicated. DOX caused cell death at both doses that was reduced by caspase would connect the two events. In addition to the involvement
inhibition. *P ⬍ 0.02, percentage of TB positive in treated cells is greater than of members of the MAP kinase family (35, 53), other pathways
untreated cells; ⫹P ⬍ 0.02, reduced cell death on DOX treatment in presence
of inhibitor compared with without inhibitor. B: number of TUNEL-positive connecting DOX exposure to apoptosis include p53 (28), erb
cells after 0.5 and 5 ␮g/ml DOX treatment is reduced by the presence of receptors 2 and 4 (39), phosphatidyl inositol 3-kinase/Akt (34),
general caspase inhibitor Z-VAD-fmk (*⫹P ⬍ 0.007). and the Fas/Fas ligand system (33). Our model system provides
a valuable environment in which to define signaling pathways
reduce apoptosis induced by low-dose DOX exposure. This involved in DOX-induced apoptosis. Furthermore, the GST-
discrepancy may reflect the ability of GST to repair oxidative overexpressing cells described in this report should prove a
damage, a property not seen with exogenous antioxidants. useful reagent in the study of the specific role of oxidant stress
Whether AC directly cause cardiotoxicity or whether the tox- in triggering apoptotic pathways as well as other oxidative
icity is mediated by an intermediate compound has not been disease states that affect cardiac myocytes, including ischemia-
conclusively demonstrated. The alcohol metabolite of the AC reperfusion and septic shock.
daunorubicin, for example, is concentrated in heart tissue after Mechanism of DOX-induced cardiomyocyte death. Other
bolus administration, persists for at least 3 days after the parent mechanisms have been put forth to explain the molecular basis
compound has disappeared, and can directly reduce cardiac of AC toxicity to cardiomyocytes. One explanation is that AC
function (10). This metabolite may produce irreversible cardiac disrupt expression and activity of the transcription factor
myocyte injury by generation of reactive oxygen species by GATA-4 (21). Because GATA-4 activity induces expression of
delocalizing iron from aconitase, a protein critical in normal genes such as glutathione peroxidase and Bcl-x that protect
cellular iron homeostasis (30). This disruption of iron metab- cardiomyocytes from the apoptotic program, interference with
olism may be as or more important than lipid peroxidation as its activity might reduce cardiac gene transcription, impair
a mediator of AC-induced myocyte injury and provides a antioxidant defenses, and thereby induce apoptosis. In the
possible mechanistic explanation for the observed clinical current report, we observed apoptosis after a shorter exposure
benefit of iron chelation as a prophylactic measure against ACT. to DOX than was described as necessary to reduce GATA-4
Dosage of DOX and mechanism of cell death. Our results protein levels in the cited study. The steps upstream of inhibi-
confirm previous reports that the mechanism of DOX toxicity tion of GATA-4 activity need to be elucidated to further define
is dependent on the concentration of the compound experi- the contribution of this mechanism to ACT. Alternatively,
enced by the target cell, with apoptosis predominating at low disruption of myocyte expression of genes important in energy
doses and oncosis predominating at high doses. Although not production has also been suggested to be important in the
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 GST OVEREXPRESSION AND ANTHRACYCLINE CARDIOTOXICITY H2063
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