ISSN
1916-6958

2010;
Volume 1 (Number 2): Pages 39-53

REVIEW
ARTICLE

Pharmacological strategies to
counteract doxorubicin-induced cardiotoxicity: the role of mitochondria

Gonçalo C. PEREIRA and Paulo J. OLIVEIRA*

Center for Neurosciences and Cell Biology, Department of
Zoology, University of Coimbra, Portugal

*Corresponding author:
[email-masked]

ABSTRACT

Since the discovery of doxorubicin (DOX) as
a potential anticancer agent, different types of human cancers have been
successfully treated. However, the use of the drug leads to an irreversible and
dose-dependent cumulative cardiotoxicity thus restraining the treatment
efficacy. Previous studies pointed out mitochondrial dysfunction as the main
mechanism for drug cardiotoxicity, although the story still remains incomplete.
Several approaches have been proposed in order to prevent DOX selective
cardiotoxicity, some of which specifically target mitochondria. The present
review performs a brief overview of the mitochondrial targets of DOX toxicity
and the pharmacological strategies designed to decrease it, with a special
focus on agents that act on mitochondria. This review is thus divided in to
three main sections: a) the effects of DOX on mitochondrial physiology and
bioenergetics as well as the consequences at the myocyte level; b)
non-pharmacological strategies already demonstrated to counteract DOX-induced
cardiotoxicity and, c) damage-preventing approaches involving mitochondria and
their mechanism. Our objective was to review the state-of-art regarding the
mechanisms of DOX-induced cardiotoxicity, especially focusing on mitochondria,
and to describe strategies aimed at preventing such toxicity. The present
review can help on the development of new strategies to counteract DOX toxicity
focusing on the mitochondria as the target.

Key words

Doxorubicin; Heart; Mechanism of action;
Mitochondria; Prevention; Toxic potential

RÉSUMÉ

La découverte de
la doxorubicine (DOX) comme agent anticancéreux potentiel a permis de traiter
avec succès différents types de cancers humains. L’utilisation répétée de cette
médication cause cependant une cardiotoxicité dose-dépendante et irréversible
qui limite son usage thérapeutique. Des études précédentes ont identifié une
dysfonction mitochondriale comme mécanisme principal de cette cardiotoxicité,
mais ce n’est qu’une partie de l’histoire. Différentes approches ont été
proposées pour prévenir cette toxicité sélective, certaines visant spécifiquement
les mitochondries. Cet article effectue une brève revue des cibles
mitochondriales de toxicité de la DOX et des stratégies pharmacologiques
conçues pour la réduire, avec un accent sur les molécules agissant sur les
mitochondries. Cette revue est divisée en trois sections: a) les effets de la
DOX sur la physiologie et la bioénergétique mitochondriale, et leurs
conséquences au niveau des myocytes; b) les stratégies non-pharmacologiques
contrecarrant la cardiotoxicité induite, et c) les approches préventives
impliquant les mitochondries et leur mécanisme. L’objectif était la revue du
“state-of-art” concernant les mécanismes de la cardiotoxicité induite par la
DOX avec un accent sur les mitochondries et la description des stratégies
visant à prévenir cette toxicité. Cette revue devrait aider à développer de
nouvelles stratégies pour contrecarrer cette toxicité en mettant l’accent sur
les mitochondries.

Mots clés

Doxorubicine;
Cœur; Mécanisme d’action; Mitochondries; Prévention; Potentiel toxique

RESUMEN

Desde el
descubrimiento de la doxorubicina (DOX) como un posible compuesto
anticancerígeno, diferentes tipos de cáncer han sido tratados exitosamente. Sin
embargo, el uso de este fármaco puede conducir a una cardiotoxicidad
irreversible, dosis-dependiente y acumulativa, limitando su eficacia
terapéutica. Estudios anteriores propusieron a la disfunción mitocondrial como
el principal mecanismo de cardiotoxicidad; sin embargo, aún son necesarios más
estudios. Se han propuesto varias alternativas para prevenir la cardiotoxicidad
selectiva de DOX, algunas dirigidas específicamente a la mitocondria. Este
artículo revisa en forma breve la toxicidad mitocondrial inducida por DOX y
propone estrategias farmacológicas orientadas a disminuirla, en especial con
agentes que actúan a nivel mitocondrial. Esta revisión se  divide en
tres secciones principales: a) los efectos de DOX en la fisiología
mitocondrial y su bioenergética  así como
las consecuencias a nivel del miocito; b) la descripción de estrategias no
farmacológicas para contrarrestar la cardiotoxicidad de la DOX, y c) acciones
preventivas del daño que involucran a la mitocondria en su mecanismo de acción.
Nuestro objetivo fue revisar el conocimiento actual de los mecanismos de
cardiotoxicidad inducida por DOX y su prevención, enfocados en forma particular
en la mitocondria. Esta revisión puede ayudar en el desarrollo de nuevas
estrategias para contrarrestar la toxicidad mitocondrial inducida por DOX.

Palabras clave

Doxoribicina;
Corazón; Mecanismo de acción; Mitocondria; Prevención; Toxicidad potencial

INTRODUCTION

Doxorubicin (DOX, Adriamycin) (Figure
1)
is an anthracycline antibiotic discovered and isolated from Streptomyces peucetius in the early
1960’s [1]. Presently, it is one of the most important
antineoplastic agents used against a broad spectrum of cancer types, such as
lymphomas, leukemias and solid tumors. The anticancer activity of DOX is explained
by its ability to bind and/or intercalate the DNA double strand causing a
stereochemical disorder and thus inhibiting the synthesis of DNA, RNA, and
proteins, eventually leading to cell death [2].

Despite the potent antineoplastic activity of
DOX, its clinical use is restrained by an irreversible and dose-dependent
cumulative cardiotoxicity. Acute effects may arise immediately after treatment
and are clinically characterized by arrhythmias, hypotension, pericarditis,
diastolic functional impairment (first) and systolic function alterations
(later) [3].

The reported effects are transient and
disappear as soon as the patient discontinues the treatment. However, chronic
effects, which are characterized by progressive left ventricular dysfunction,
lead to irreversible heart failure and are therefore more serious and difficult
to manage [3]. Morphological alterations usually include
myofibrillar loss, vacuolization of cytoplasm, dilatation of sarcoplasmic
reticulum and swollen mitochondria [4] [5].

DOX-cardiotoxicity markers generally include an
increase of cardiac-specific and non-specific protein levels in serum, such as
lactate dehydrogenase (LDH), cardiac troponin I (cTnI), creatine phosphokinase
(CPK), glutamate oxaloacetate transaminase (GOT) and glutamyltransferase (GTP) [6]. The correlation between DOX cumulative doses and the
risk of heart failure is well known. It has become widely accepted that
cumulative doses up to 550 mg m-2 exponentially increase the
probability of DOX-induced cardiomiopathy [7]. Dosages above this level are avoided in order to
prevent DOX-induced damage. Besides the cumulative effect of DOX, another
important issue is the peak plasma concentration of drug delivered to the heart
during treatment. It was hypothesized that DOX toxicity is primarily related to
the peak dose concentration while the antitumor efficacy is more related to
total drug exposure [4].

Although several intracellular targets of DOX
have been already indentified, the complete mechanism of DOX-induced
cardiotoxicity remains unclear. Over the years, different hypotheses were
proposed for such enhanced cardiotoxicity. Mitochondria have received
particular attention in this regard due to their role producing energy in heart
tissue by synthesizing ATP through oxidative phosphorylation; by producing
reactive oxygen species (ROS), which can act as second messengers, in some
cases, or be deleterious if in excess [8] [9]; and, by regulating cytosolic calcium [8]. DOX has been reported to inhibit some of the
respiratory complexes, to decrease the ATP content in cardiomyocytes, to
stimulate ROS production, and to decrease mitochondrial calcium-loading
capacity through induction of the mitochondrial permeability transition (MPT) [3] [4] [10] [11] [12]. Together with morphological alterations, the data
suggests mitochondrial dysfunction as one of the main causes for DOX-induced cardiomiopathy.

Due to the limitation imposed by the
DOX-cumulative effect, different approaches appeared over the years as an
attempt to prevent or to counteract DOX-induced cardiotoxicity. These
approaches rely on the different hypotheses explaining DOX toxicity and, in
fact, some were shown to be capable of decreasing the damage induced by DOX.
The different data obtained also suggests that the mechanism behind DOX
cardiotoxicity is most likely a multifactorial event rather than one isolated
effect.

Figure
1

Bioactivation of DOX by mitochondrial
complex I. DOX (A) can be activated/reduced by several oxyreductase enzymes,
here represented by the mitochondrial respiratory chain NADH dehydrogenase. NADH
acts as an electron donor for the reduction of DOX producing a semiquinone
radical (B) which can therefore be re-oxidized in the presence of molecular
oxygen, consequently regenerating DOX. This process is known as DOX redox
cycle and increases the production of superoxide anion which can directly
damage lipids and proteins or can also be converted to other ROS.
Alternatively, in the absence of molecular oxygen, DOX semiquinone can be
simply converted to secondary metabolite doxorubicinol (C) or can undergo
deglycosylation (D) forming a C7 radical (DNA alkylation) (E) which can form
dimers (F) or be converted to a 7-deoxyaglycone (G) (DNA intercalation).

It should be stressed, however, that
strategies should minimize the cardiotoxicity imparted by DOX while preserving
the antineoplastic efficacy of the drug. As mitochondria are a particular
target in DOX toxicity, new approaches should be focused on this particular
cellular structure in order to circumvent DOX cardiotoxicity.

CELLULAR
EFFECTS OF DOX-INDUCED TOXICITY – THE ROLE OF MITOCHONDRIA

Cardiomyocytes are terminally differentiated
muscle cells with little or no capacity to proliferate. As the endpoint of DOX
toxicity involves cardiomyocyte death, repetitive and cumulative induction of
cell death may lead to irreversible cardiotoxicity and consequently heart
failure. Over the years, different hypotheses have been proposed in order to
explain DOX-induced cardiotoxicity.

DOX-induced myocyte cell loss through
induction of apoptosis includes up-regulation of proapoptotic proteins (Bax,
caspases and cytochrome c) [13] [14] [15] with or without down-regulation of antiapoptotic
proteins (Bcl2, Akt) [13] [15], making mitochondrial-mediated apoptosis an
attractive mechanism.

However, it is not clear if apoptosis is a
cause or a consequence of drug effects. Since DOX binds to DNA, it should be
expected that this particular effect would be related to the cardiotoxicity
induced by the drug. However, cardiomyocytes are terminally differentiated
cells with low replication/division activity. Thus, DOX-induced impairment of
DNA replication is unlikely to trigger cardiomyopathy, although inhibition of
DNA transcription can also contribute to cardiac toxicity. In fact, other
anticancer agents that kill cancer cells through binding/intercalation in DNA,
such as cisplatin and actinomycin D, do not cause cardiotoxicity.

Another interesting fact is that p53, a DNA
damage-activated protein, is active in cardiomyocytes after treatment with DOX [16]. p53 activation may reflect a link between the redox
cycle/activation of DOX and the increase in oxidative stress with the transcriptional
activation of Bax by p53. Alternatively, DOX-stimulated oxidative stress may
also reflect the direct opening of the MPT pore, cytochrome c release, and
consequent apoptosis triggering.

Furthermore, it has been demonstrated that
the gene profile of cardiomyocytes can be altered by DOX treatment [17] [18]. Along with other cellular proteins, the levels of
some mitochondrial enzymes are decreased in cardiomyocytes exposed to DOX which
can partly explain the altered mitochondrial bioenergetics observed in vivo [17] [18] [19] [20] [21]. In fact, transcripts and/or protein levels of the
adenine nucleotide translocator and Rieske iron-sulfur protein are decreased
after DOX treatment [17] [22]. The results appear to indicate that although
DOX-induced cancer elimination and cardiomyopathy involve distinct mechanisms,
direct DOX effects on nuclear DNA, which has been proposed by some authors as
responsible for early events in cardiomyocyte death, is likely not to be the
major cause of toxicity [16].

The reason for the increased susceptibility
of the cardiac muscle for DOX toxicity is still under debate. Although a full
comparative study between different human organs is still to be performed, some
authors have proposed that DOX-induced oxidative stress may preferentially
affect the heart due to the lower antioxidant capacity of the cardiac muscle [23]. Another, very controversial, point of view is the
possibility of the existence of an organoselective mitochondrial NADH
dehydrogenase, located in the outer leaflet of the inner mitochondrial membrane
which would promote DOX-induced oxidative stress [24]. However, no further investigations have been
performed exploring this possibility.

Although the mechanism for such enhanced
toxicity in the cardiac muscle remains unclear, DOX affinity for some
macromolecules can explain the effects on different organelles. As already
referred, DOX has high affinity to DNA and therefore the nuclei are a particular
target. DOX has also a high affinity for cardiolipin (CL) [25] [26], a membrane phospholipid predominately present in mitochondrial
membranes.

The inner mitochondrial membrane is by far
the richest cellular structure containing CL and this phospholipid acts as a
co-factor for several enzymes [27]. CL-protein complexes may be crucial in several
mitochondrial processes including oxidative phosphorylation, since cytochrome c
oxidase (COX) requires the presence of CL [27] [28]. Interestingly, DOX is capable of inhibiting COX [29], most likely due to drug-lipid complexes rather than
direct drug-protein interactions [25]. DOX-interaction with mitochondrial membranes,
including lipid peroxidation, due to increases in oxidative stress are also
mechanisms in the toxicity induced by DOX.

More than 90% of the ATP utilized by
cardiomyocytes is produced by mitochondrial respiration and therefore any
alteration on mitochondrial function will have a strong impact in the energy of
cardiomyocytes subsequently inducing cardiotoxicity. DOX has been reported to
interfere with mitochondrial respiration decreasing both intracellular pools of
ATP and phosphocreatine (PCr) [3].

The inhibitory effect on mitochondrial
respiration is translated into a decrease in State 3 and sporadic stimulation
of State 4 respiration [19] [30]. Surprisingly, the decrease in State 3 respiration
was reversed by addition of dithiothreitol (DTT) suggesting that DOX induces
specific alterations in protein thiol groups [20]. Other effects observed on isolated mitochondrial
fractions include a decrease in the respiratory control ratio and ADP/O [31]. The increase in State 4 respiration is thought to be
due to proton leak from the inner membrane as a consequence of drug-lipid
interaction [25] [30]. Respiratory complexes are plausible targets in DOX
toxicity action, and inactivation of several mitochondrial enzymes was already
reported. DOX sensitive sites were mainly located in complex I, III and IV [25] [30] [32] [33]. NADH-dehydrogenase and COX activity were
particularly impaired after DOX treatment [31]. Although DOX inhibits ATP synthase activity, the
crucial targets of DOX seem to be peripheral proteolipid complexes involved in
the channeling of ATP and PCr from mitochondria to the cytosol (ANT, VDAC and
MtCK) [3] [31].

The large variability in the percentage of
inhibition in the activity of isolated respiratory complexes and/or intact
mitochondria respiration, reported in different in vivo studies, can be explained by different DOX whole body
distribution, binding specificity, and metabolism. Alternatively, the
differences may indicate that mitochondrial oxygen consumption is not a
sensitive method to evaluate mitochondrial dysfunction, as has already been
pointed out [12]. It is proposed instead that altering mitochondrial
calcium-loading capacity is a more sensitive and quantifiable indicator of
mitochondrial dysfunction upon DOX treatment [10] [12] [19] [20] [34]. DOX interferes with the sarcoplasmic reticulum,
increasing the open state probability of calcium release channels interfering
with intracellular calcium homeostasis [35].

The increase in cytoplasmatic calcium
concentration can be potentiated by inhibition of Na+/Ca2+
and Na+/K+ pumps [36] [37]. As calcium is the second most common messenger in
cells, DOX-induced increases in cytoplasmatic calcium concentrations can trigger
numerous cell responses affecting normal cell behavior. Cytosolic calcium
overload is more serious when calcium increases in microdomains between
mitochondria and the reticulum. Upon calcium dysregulation, mitochondria
accumulate an excess of calcium in the matrix. In fact, several in vitro and in vivo studies show that DOX, or even its metabolites, decrease
the capacity of mitochondria to accumulate and retain calcium [10] [19] [38].

Calcium over-accumulation on mitochondria has
been reported to increase the production of reactive oxygen species (ROS) [39] and can therefore be a link between oxidative stress
and alterations in calcium homeostasis. Mitochondrial calcium dysregulation
became an important component of DOX toxicity; in fact, ruthenium red, an
inhibitor of mitochondrial calcium uptake, not only decreased the production of
ROS but also increased cell viability [40]. Although the relationship between calcium release
from the sarcoplasmic reticulum and mitochondrial dysfunction has not yet been
fully explored, different studies have proposed that ROS production and
cytosolic calcium dysregulation are reciprocally regulated in cardiomyocytes
exposed to DOX.

The fact that altered mitochondrial calcium
regulation is completely blocked by cyclosporin A (CsA), a specific inhibitor
of the MPT pore [41], implicates induction of MPT by DOX as another
potential cause for DOX-induced cardiotoxicity [20] [38]. The MPT pore can be defined as a voltage-dependent
high-conductance channel in the inner mitochondrial membrane. The pores are
suggested to be between 2-3 nm in diameter and to exclude molecules with
molecular weights higher than 145 kDa [42].

Although the identities of the individual
components of the MPT pore remain controversial, it is agreed that the adenine
nucleotide translocator (ANT), voltage-dependent anion channel (VDAC), and
cyclophilin-D are elements of the pore complex or at least are involved in its
formation or regulation. Matrix alkalinization, oxidative stress, and calcium
overload in the matrix are known to increase the susceptibility of mitochondria
to MPT pore opening [43] [44]. In the majority of in vitro studies, pore opening is accompanied by mitochondrial
depolarization, respiratory inhibition or stimulation, matrix swelling,
pyridine nucleotides depletion and release of intermembrane proteins, including
cytochrome c. In fact, all these effects were already reported in heart
mitochondria exposed in vitro or in vivo to DOX [10] [12] [19] [20].

In
vitro studies show that induction of MPT is
increased when DOX metabolites, such as aglycones and secondary alcohols, are
used instead of the parent compound [38]. DOX metabolites appear to be more deleterious to
mitochondria than DOX itself. MPT pore induction by DOX is thought to be due to
increasing production of ROS leading to the oxidation of specific critical
sulfhydryl groups [10] [31]. The model agrees with the ability of DOX to generate
free radicals through a redox cycle mechanism [11] [45].

Besides slower respiration, mitochondria from
DOX-treated rats exhibit an enhanced sensitivity to MPT and therefore the
calcium loading capacity is decreased [19] [30] [46] [47]. Curiously, this aspect of calcium dysregulation may
still persist after the end of the treatment [48]. It
was demonstrated that addition of CsA to a suspension of isolated cardiac mitochondrial
fractions from DOX-treated rats was able to reverse the DOX-induced decrease in
calcium loading capacity [20]; CsA was also able to revert the inhibition of
respiration induced by DOX. Both observations indicate that the decrease in
mitochondrial calcium-loading capacity is due to an increase in sensitivity to
the MPT and that the inhibition of oxidative phosphorylation by DOX is a
secondary effect of MPT induction. It was proposed that probable binding of CsA
to cyclophilin-D might cause a disassembly of pre-formed MPT pores allowing
ANT, which is crucial to oxidative phosphorylation, to be functional again.
Alternatively, it was also demonstrated that the ANT is depressed both in
content and function in DOX-treated rats, which can contribute to decreased
respiration and increased MPT occurrence [17].

DOX-induced oxidative stress appears to be
the main factor responsible for drug cardiotoxicity. DOX can stimulate the
production of ROS by two distinct mechanisms, one involving enzymatic reactions
and the other through an iron-dependent mechanism [4] [7]. In the first one, the DOX quinone moiety undergoes a
univalent reduction to yield a semiquinone free radical.

Figure
2

Doxorubicin-stimulated ROS production through
free-iron coordination. DOX can complex with free iron in a 1:3 ratio which
can then be reduced to a Fe2+-complex by the cellular reducing
system, or alternatively by another Fe2+-complex,
allowing the cycle to continue indefinitely, which can cause the formation of
ROS and cell oxidative stress.

The reduction process can be performed by
several oxyreductases; the mitochondrial NADH dehydrogenase (also known as
Complex I) is one important example [11]. After DOX activation, the semiquinone free radical
donates its unpaired electron to molecular oxygen (O2), which is
converted into a free radical – superoxide anion (O2.-) – and regenerates the
parent quinone in the process (Figure 1). The DOX
redox-cycle is probably the principal source of mitochondrial ROS production.

DOX also chelates free iron to form a stable
complex with a Fe3+ to DOX ratio of 1:3 (Figure 2). The complex can then be reduced to a Fe2+-DOX
complex by the cellular reducing system or even by another Fe2+-DOX
complex. The new Fe2+-DOX complex can univalently reduce O2,
increasing O2.-
levels and regenerating Fe3+-DOX complexes in the process. Alternatively,
Fe2+-DOX complexes can convert hydrogen peroxide (H2O2),
formed by dismutation of the O2.-
produced by previous Fe3+-DOX complexes, resulting in a far more
deleterious free radical – the highly reactive hydroxyl radical (HO.). In both mechanisms, the
increase in ROS can cause lipid peroxidation and DNA or protein oxidation
observed on cardiomyocytes exposed to DOX [25].

STRATEGIES TO COUNTERACT DOXORUBICIN-INDUCED
CARDIOTOXICITY

Strategies to prevent DOX toxicity are
necessary so higher doses and, therefore, enhanced therapeutic efficacy can be
achieved. Different strategies should be capable of minimizing DOX-induced
toxicity and should not interfere with antineoplastic activity.

Non-pharmacological strategies, including
decreasing the peak drug plasma concentration or decreasing the amount of drug
targeted to the heart, can prevent DOX-induced cardiomyopathy. A careful choice
on administration schedule showed that weekly administrations of DOX decrease
the incidence of heart failure compared to the standard schedule in which DOX
is administered every three weeks [4]. Another attempt to decrease DOX cardiotoxicity was
by administering DOX by continuous infusion over a 48 h period rather than the
standard 15 min bolus infusion [4]. This approach was capable of reducing the drug peak
plasma and also the damage in cardiac tissue as accessed by biopsy [49]. However, this procedure was later discarded when it
was found that continuous infusion might also reduce the antitumor efficacy
probably due to a decrease in the initial concentration at tumor level [50].

Protection against DOX-induced heart damage
might also involve the development of new strategies to achieve tumor-targeted
delivery of DOX and thereby reduce systemic and cardiac toxicity. The
microvasculature in tumors is typically discontinuous and it contains pores big
enough for liposomes to move in from the blood to the space surrounding tumor
cells. The use of liposomal formulations has already been proven to be capable
of enhancing the treatment efficacy with improved pharmacokinetic indexes and
lower toxic profile [6] [7] .

The improvement can be explained by several
factors including prolonged half-life, prevention of metabolic conversion of
DOX to inactive forms or even to aglycones and secondary alcohol metabolites,
preferential distribution and prolonged release of the drug within the tumor
mass and, finally, limited accumulation in healthy tissues [4]. Currently, two different preparations of
DOX-liposomal formulations with superior action and fewer side effects when
compared with free DOX have been assessed in experimental models and clinical
trials. The first one is a polyethyleneglycol-coated (pegylated) liposomal DOX
that has more favorable pharmacokinetics than free DOX and is also capable of
overcoming the blood-brain barrier [4] [7]. The other preparation involves uncoated
citrate-containing liposomal DOX formulation, where citrate is included to
increase DOX encapsulation. The results showed less pronounced pharmacokinetics
indexes than pegylated liposomal DOX [51].

The search for novel DOX structure analogues
that may offer lower toxicity but have the same clinical efficacy is very
active. It was initially observed that different anthracyclines had similar
antitumor efficacy and only differed in a few functional groups. The knowledge
that the amino-sugar moiety plays a crucial role in DNA intercalation process
has enabled the development of new analogues [4] [6]. As a result of the search for ‘better
anthracyclines’, nearly 200 analogues have emerged. This fact should not be
surprising, if we consider the number of possible chemical modifications that
can be made on the tetracyclic ring or the amino-sugar moiety. However, only a
few of those analogues have reached the clinical approval. Epirubicin has been
one of the most extensively investigated DOX analogues and is now widely used
as an alternative to DOX [4] [7]. Epirubicin shows the same spectrum of antitumoral
efficacy although with different pharmacokinetics and metabolism. The toxicity
effects are the same in quality but quantitatively less when compared to DOX,
which enabled an increase in cumulative doses to almost double of those of DOX
(900 to 1000 mg m-2).

In the previous section it was mentioned that
apoptosis is one possible endpoint for DOX toxicity. Apoptosis can be initiated
by extrinsic or intrinsic signals [43]. However, in both pathways, cytochrome c released
from mitochondria seems to be a crucial step. For that reason, mitochondria
have a central role in the cell death process. The release of cytochrome c from
the mitochondrial intermembrane space allows this protein to interact with
monomeric APAF-1 leading to its oligomerization and recruitment of caspase-9 to
form the apoptosome [52]. The activated caspase-9 cleaves and activates
executioner caspases (caspase-3 and -7). If this event occurs without any
restraint the cell will inevitably be conducted to death [52] [53]. Usually, cytochrome c is involved in mitochondrial
oxidative phosphorylation and is located primarily in intercristae membrane
space, as well as in the mitochondrial intermembrane space [54]. Mitochondrial outer membrane permeabilization (MOMP)
is necessary so that the release of this protein to the cytosol can occur [55]. There are two possible mechanisms for this event.
The first involves the permeabilization of the inner membrane due to the
formation of MPT pore, leading to mitochondrial swelling and outer membrane
rupture [44]. The second event involves the oligomerization of Bax
within the outer mitochondrial membrane, forming small channels which allow
proteins, such as cytochrome c, to access the cytosol [56]. In this case, respiration, as well as ATP
production, is not primarily affected because the inner membrane is still
intact.

For all the reasons described so far,
strategies in which the objective is to maintain normal mitochondrial
homeostasis are plausible routes to counteract or at least diminish the extent
of apoptosis in cardiomyocytes exposed to DOX. Nevertheless, there are quite
few strategies that focus on apoptosis machinery itself. In vitro studies show that DOX-induced apoptosis of cardiac cells
is prevented by several compounds such as pifithrin-alpha, a p53 inhibitor;
survival factors signaled by phosphoinositide kinase; IGF-1; L-carnitine or metallothioneins
[4] [55] [57] [58] [59].  Antioxidant
compounds also show capacity to decrease apoptosis. This effect may be due to a
direct effect on oxidative stress rather than, contrarily to pifithrin-alpha, a
direct effect on the apoptosis machinery. Directly blocking DOX-induced cardiac
apoptosis with antioxidants may be a double-edged sword in clinical practice
since DOX-induced apoptosis of cancer cells would likely be inhibited as well.
However, different approaches aimed at preventing oxidative stress, cytosolic
calcium dysregulation and DNA damage, which can all trigger cell death,
demonstrate the capacity to increase cardiomyocyte viability after DOX
treatment [12] [19] [60]. In fact, a promising approach would consist in the
direct and selective inhibition of apoptosis in the heart of DOX treated
patients. Other approaches differ from what has been described so far because
they attempt to circumvent DOX-induced cardiotoxicity with a special focus on
mitochondrial-mediated events. Based on the data described before, prevention
of DOX toxicity can also be achieved by the use of iron chelators. Many agents
able to remove iron from a complex with DOX have been investigated as possible
cardioprotectors. The most effective in clinical trials is dexrazoxane (DEX)
which belongs to the bisdioxopiperazine family. The molecule is an analogue of
ethylene diamine tetraacetic acid (EDTA) and was initially designed as an
antitumor agent [7]. After diffusing to cardiomyocytes, DEX is hydrolyzed
to its open-ring form which can strongly chelate iron, thereby displacing Fe3+
from its complexes with DOX. Interestingly, the one-ring open intermediate demonstrates
the same capacity to remove Fe3+ from DOX complexes as DEX [4] [7]. However, DEX can also chelate Fe3+ from
transferrin, with ferritin depleting iron from storage and transport proteins [61]. Furthermore, DEX is also myelosuppressive and can
increase the myelosuppressive effects of DOX [62] [63]. Thus, despite the cardioprotective capacity of DEX,
its clinical use is not free of risks and other iron-chelating agents should be
developed and tested.

Another approach in the prevention of
DOX-increased oxidative stress would be increasing the activity of the cell
antioxidant system. The elimination of hydrogen peroxide is one critical step
in the detoxification process because it can lead to formation of the hydroxyl
radical, a highly reactive molecule. Hydrogen peroxide can be inactivated by
two enzymes, catalase, which converts it into water and oxygen, or by
glutathione peroxidase, which reduces hydrogen peroxide to water producing
oxidized glutathione in the process. As previously described, antioxidant
enzymes levels in the heart are relatively low comparative to liver or kidney,
which can in part explain the enhanced susceptibility of the heart to DOX
toxicity. One possible approach is to increase expression of specific
antioxidant enzymes. In fact, transgenic mice overexpressing catalase or
glutathione peroxidase, or even superoxide dismutase (SOD), were demonstrated
to decrease cardiac damage after DOX treatment [64]. Overexpression of the radical scavenger metallothionein
in the hearts of transgenic mice also prevented DOX-induced cardiotoxicity [64] [65], supporting the concept that detoxification by
overexpressing antioxidant proteins might be a plausible strategy to circumvent
DOX toxicity. However, this expression may not always be restricted to the
heart, thus making it necessary to first design a specific cardiac
overexpression system. Alternatively, the use of antioxidant agents
administered either in the diet or together with DOX may enhance protection of
the heart. These compounds can be precursors of antioxidant proteins due to
their high thiol content.

One important molecule responsible for the
removal of hydrogen peroxide is glutathione. Cellular levels of this protein
can be increased by thiol-containing agents such as N-acetylcysteine (NAC), a
molecule with high bioavailability which, upon hydrolysis, releases cysteine, a
precursor of glutathione biosynthesis [66] [67]. In the same way, cystamine or its reduced form
cysteamine, as well as metallothionein, are able to increase cellular
glutathione content [64] [68]. The use of these compounds both in vitro and in vivo has
decreased the toxicity of DOX [64]. Unfortunately, the use of NAC had no success in
clinical trials even when high doses were administered to patients [69]. The use of antioxidant nutrients have been shown to
prevent some damage on cardiac tissue caused by DOX treatment [70]. Vitamins (E, C and A), coenzyme Q, and
flavonoids/polyphenols normally found in the diet increased the cellular
contents of glutathione and SOD and also decreased malonyldialdehyde and lipid
peroxidation demonstrating the capacity to prevent DOX toxicity. In the case of
vitamin E (α-tocopherol), this antioxidant agent decreased oxidative
stress induced by DOX without preventing the mitochondrial dysfunction
associated with the treatment [19].

In one particular work, a diet enriched in
vitamin E did not afford protection against DOX-induced State 3 and State 4
inhibitions and decreased calcium loading capacity. However, protein carbonyl
content, a marker of oxidized proteins, was decreased in the DOX-treated group
fed with an α-tocopherol enriched diet. As reported in the previous case,
protection against DOX with antioxidants is not always achieved perhaps due to
difference between doses, pre-treatment times, and the type and schedule of
DOX-treatment. Also, the ineffective delivery of antioxidants to critical
subcellular compartments, such as mitochondrial membranes, might explain the
unsatisfactory results observed. Based on this, antioxidants alone might not be
a robust strategy to prevent DOX toxicity but when administered together with
other agents might improve the treatment efficacy. To overcome the problem of
target-specific subcellular compartments, new strategies involving the use of
compounds of interest attached to specific molecules (which can be recognized
by themselves or even accumulated within those structures) should be developed
allowing for specific accumulation of bio-active molecules. In fact, a similar
strategy has already been explored in the prevention of lipid peroxidation
taking the advantage of using triphenylphosphonium (TPP) cation linked to
α-tocopherol (MitoE) or ubiquinone (MitoQ) [71].
TPP is a positively charged lipophilic cation with high oral
bioavailability which can easily pass through phospholipid bilayers without a
specific uptake mechanism, and accumulate in the cell, driven by the plasma
membrane potential (Figure 3,
Panel B). Further substantial accumulation within mitochondria occurs, driven
by the negative-inside mitochondrial transmembrane potential. The approach
allows the accumulation of high concentrations of antioxidants within
mitochondria thus decreasing oxidative damage (Figure 3, Panel C). Furthermore, the antioxidant
can be recycled back to its active form through the respiratory chain [71].

Figure
3

Doxorubicin toxicity and a possible
strategy to counteract it. For the sake of simplicity, a single cell,
surrounded by a light colored bilayer, contains only one mitochondrial body,
surrounded by a dark lipid bilayer. A) Summary of DOX targets and effects.
DOX redox cycling at Complex I results in the generation of oxidative stress,
which contributes to the oxidation and decline in function of critical
mitochondrial proteins (a), DNA (b), membrane lipids (c) and to the formation
and opening of the MPT pore (d). B) Cellular accumulation of a bioactive
molecule (X) linked to TPP which is a lipophilic cation and can, therefore,
accumulate 5-fold in the cytoplasm due membrane potential. Moreover, the high
negative mitochondrial membrane potential allows for a 200-fold accumulation
inside the organelle. If the linked molecule behaves as an antioxidant, it is
possible to have it regenerated by the mitochondrial respiratory chain.
Adapted from Murphy et al. [71]. C) Hypothetical strategy to overcome DOX-increased
ROS production. If a construct TPP-antioxidant is actively accumulated in
mitochondria, it would be possible in theory to counteract DOX-induced
oxidative stress, by scavenging free radicals resulting from DOX redox cycle.

Abbreviations: RC: respiratory chain;
MPTP: MPT pore.

Several lipophilic cations, such as
rhodamine, JC-1 and MitoTracker compounds, share the membrane permeabilization
and mitochondrial accumulation capacity of TPP. However, TPP-modified cations
are actually the most synthesized and evaluated, probably because TPP is a well
known molecule in terms of bioavailability and toxicology and also because the
interactions with mitochondria are well understood. Theoretically, any molecule
can be linked to TPP moiety to satisfy different targeting strategies. Several
antioxidants targeted to mitochondria have been developed, including MitoSOD (O2.-), MitoPeroxidase (H2O2),
MitoTEMPOL (.OH), MitoE2
and MitoQ10 (lipid peroxidation) [71].

Besides their beneficial effects, the
toxicity of these compounds should be evaluated as higher concentrations of
mitochondrial-targeted compounds can disrupt membrane integrity or inhibit
respiration and ATP synthesis [71]. It is clear that the compound of interest should
display protective effects at low concentrations in order to prevent cell
damaging effects.

A particular compound that can be
co-administered with DOX is carvedilol [Kredex, Coreg,
(1-[carbazolyl-(4)-oxy]-3-[(2-methoxyphenoxyethyl)amino] -propanol-(2)].
Carvedilol is a β-adrenergic receptor antagonist which blocks β1,
β2 and α1 receptors and consequently slows the
heart rhythm without increasing blood pressure [72]. The drug is used to treat cases of hypertension and
has also demonstrated to be helpful in the management of cardiomyopathies.
Carvedilol is primarily used to prevent heart failure, one endpoint of DOX
toxicity [72]. In fact, DOX-treated patients showed significant
improvement of left ventricular dysfunction after carvedilol treatment [73]. However, not all generic β-blockers are equally
helpful against DOX toxicity. In fact, the β-blockers propanolol and
atenolol are not as efficient as carvedilol in circumventing DOX-toxicity [12]. Although both compounds are equally effective in
reducing blood pressure, they were not capable of preventing mitochondrial
dysfunction [12]. Different reports suggest that the greater
cardioprotection achieved by carvedilol may be due to its potent antioxidant
activity which is unshared by other β-blockers [72]. The antioxidant mechanism of carvedilol was
attributed to free radical scavenging through its carbazole moiety [74]. In addition it was also demonstrated that carvedilol
can sequester ferric ions, thereby preventing ferric ion-induced oxidation [75].

As mentioned above, an exogenous
NADH-dehydrogenase was proposed as the main site of DOX activation in the heart
[24]. Interestingly, carvedilol has been shown to inhibit
the so-called external NADH-dehydrogenase [76], with the pure β-blocker propanolol showing
reduced effects [77]. Although some discussion may arise on the particular
site of DOX-activation at complex I level, the existence of a specific
inhibitor of DOX activation site can be of great value in the prevention of DOX
side effects on the heart. At the cellular level, carvedilol has shown the
capacity to prevent most of the toxic effects of DOX in a sub-chronic model of
mitochondrionopathy. Carvedilol decreases the extent of cellular vacuolization,
and prevents the inhibition of cardiac mitochondrial respiration, the
inhibition of complex I of heart mitochondria, and the decrease in calcium
loading capacity, all associated with DOX treatment [30]. In other models of pro-oxidant-induced mitochondrial
damage, carvedilol was able to inhibit the MPT and reduce cytochrome c release
due to inhibition of the oxidation of mitochondrial thiol groups [34]. However, carvedilol was only capable of inhibiting
the high-conductance form of the MPT; a sub-form of the MPT is still active
after carvedilol action [34] [78].

The fact that carvedilol was only effective
against the MPT induced by oxidative stress [78] demonstrates not only that the antioxidant properties
of carvedilol are crucial for its cardioprotective effect but also that this
action might be due, in part, to a selective inhibition of a deleterious high-conductance
state of the MPT pore [79]. In a sub-chronic model of DOX toxicity (2 mg Kg-1
per week to a total of 14 mg Kg-1), carvedilol prevented oxidative
damage to mitochondrial complexes and increased mitochondrial calcium loading
capacity in DOX-treated animals [30]. Carvedilol possesses a protonophore activity, which
is translated into a decrease of mitochondrial transmembrane potential, not
shared by propanolol [80]. The protonophoretic mechanism of carvedilol was
independent of potassium fluxes across mitochondrial membranes [77]. Raman and theoretical ab-initio calculations indicated that the amino side chain group
was responsible for protonation/deprotonation cycles [81] [82]. Carvedilol protonophoretic activity might be of
extreme importance since a ‘mild uncoupling’ can induce a reduction on ROS
production [83] or prevent mitochondrial calcium overload.
Nonetheless, it has been described that carvedilol has no effect on total
calcium accumulation by mitochondria but may induce a decrease of ROS produced
by mitochondrial respiratory chain [72]. As mentioned elsewhere [84], carvedilol increases the cytotoxicity of DOX as a
consequence of inhibition of multidrug resistance protein (MDR). Consequently, the
antineoplastic activity of DOX on tumor cells is not impaired, indicating that
carvedilol antioxidant and β-receptor antagonism activity could be a very
useful tool to counteract DOX-induced cardiotoxicity.

Another important end-point of DOX-induced mitochondrionopathy
is calcium homeostasis dysregulation, particularly the stimulation of MPT.
Therefore, one plausible approach is the use of a specific inhibitor of the
pore – cyclosporin A (CsA) – in a similar fashion to in vitro experiments. CsA inhibits the MPT pore through the binding
of cyclophilin (CyP) D, a matrix peptidyl-prolyl cis-trans isomerase [42]; the role of CyP-D on the MPT pore physiology is
thought to be as a regulator rather than structural. Taken together, CyP-D
normally binds to the adenine nucleotide translocator regulating the opening of
the pore which explains why CsA inhibits the MPT. In vivo experiments showed that CsA is capable of preventing
DOX-induced toxicity end-points, including increased serum LDH and
transaminases and decreased calcium loading capacity [60]; tracolimus (FK506) prevented DOX cardiotoxicity as
well. FK506 and CsA are both immunosuppressants, although the former does not
bind CyP-D, thus not affecting MPT opening. The immunosuppressive effects are
caused by the Ca2+-calmodulin-dependent inhibition of calcineurin
through the complex between CsA or FK506 and CyP-A [42]. Although direct effects on calcineurin activity are
not usually involved on MPT inhibition, calcineurin may affect mitochondrial
functions through dephosphorylation of BAD releasing pro-apoptotic factors [42].

Taken together, the immunosuppressive effects
of the drugs could prevent cardiomyocyte death through interaction with CyP-A,
without explaining the prevention by FK506 of DOX-induced loss calcium loading
capacity. More research is necessary to understand the effects of FK506 in the
prevention of DOX-induced calcium homeostasis dysregulation, as well as to
understand the systemic effects due to the use of immunosuppressants as
cardio-protection therapy. It would also be of interest to develop and to
evaluate the protective effects of CsA derivatives, without immunosuppressive
activity but with the capacity to bind to CyP-D, against DOX-induced
cardiotoxicity.

Although some caution should be taken,
ischemic events and DOX-induced cardiomyopathy are fairly similar. For example,
in both processes increased ROS production and calcium accumulation are
observed, resulting (in some cases) in the stimulation of MPT. Previous studies
have suggested that mitochondrial ATP-sensitive K+ channels (mitoKATP) play a
role in the mechanism of ischemic preconditioning mediated cardioprotection [85]. This mechanism is probably due to mild uncoupling
and increased matrix volume after mitoKATP opening which leads to a decrease in
ROS production and ATP hydrolysis. A decrease in transmembrane membrane
potential, which decreases matrix calcium accumulation, also contributes to
energy conservation and prevention of the MPT. Based on this data, it is
acceptable to think that mitoKATP opening may attenuate DOX-induced toxicity
and, in fact, this approach was already explored showing promising preliminary
results [86] [87].

However, most mitoKATP agonists and
antagonists also present other effects on mitochondrial and cellular function,
even for micromolar concentrations [85]. One of the strategies used combined sildenafil and a
selective inhibitor of cGMP specific phosphodiesterase-5, which protected
against oxidative stress injury through opening mitoKATP in a chronic model of
DOX cardiotoxicity [86]. In this model, sildenafil was capable of attenuating
cardiomyocyte apoptosis, of maintaining mitochondrial membrane potential, and
preventing left ventricular dysfunction and ST prolongation. All protective
effects were abolished in the presence of mitoKATP inhibitors. As also referred
by the authors, sildenafil has proved to be a safe drug for different pathologies,
including erectile dysfunction and pulmonary hypotension, which makes it
acceptable to counteract DOX-induced cardiotoxicity. Although its effects on
DOX neoplastic activity were not evaluated, sildenafil would allow the use of
increased doses of DOX improving its treatment efficacy.

CONCLUDING REMARKS

Doxorubicin is a powerful antineoplastic
agent and therefore it is prescribed for several human and animal cancer
treatments. However, together with its high potential efficacy the treatment is
accompanied by a selective and cumulative cardiotoxicity which, ultimately, may
escalate into heart failure. Since its discovery, efforts to counteract
DOX-induced cardiotoxicity have been made. However, none of the preventative
measures are fully efficient, possibly because the full extent of DOX toxicity
mechanism still remains to be discovered. One particular target of DOX is the
mitochondrial compartment. DOX is activated in mitochondria by undergoing a
redox-cycle in mitochondrial Complex I, which results into ROS production.
Although increased ROS production upon DOX treatment is thought to be one of
the major events responsible for DOX toxicity, calcium homeostasis
dysregulation has also received particular attention.

Since the mitochondrial function is extremely
important for the cardiac tissue due to a high energy demand, preventing
mitochondrial dysfunction would have exceptional importance to prevent
toxicity. The most important strategies which have mitochondria as their main
target involve antioxidants or inhibitors of the MPT pore. However, few of
those have shown high efficacy within in
vivo models, probably because mitochondria targeting was not effective. It
is therefore necessary to develop strategies involving molecules such as MitoQ
or MitoE which have lipophilic cations attached to molecules of interest and
are selectively accumulated within mitochondria.

CONCLUSION

Mitochondrial-targeted strategies have shown
capacity to prevent DOX-induced cardiotoxicity and might become the most
efficient means to decrease DOX cardiotoxicity. The discovery of every
mechanism of DOX toxicity and the development of new strategies to counteract
them should coincide.

ACKNOWLEDGEMENTS

Gonçalo C. Pereira is supported by a
Portuguese PhD fellowship from Foundation for Science and Technology (FCT) -
SFRH/BD/36938/2007. Research in the authors’ laboratory is supported by grant
PTDC-SAU-OSM-64084-2006 from the Portuguese Foundation for Science and
Technology.

CONFLICT OF INTERESTS/DISCLAIMERS

PJ Oliveira is member of the Editorial Board
of the journal. There are no other conflicts of interest to declare.

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